CN114920838A - anti-IL-17A single-domain antibody and application thereof - Google Patents

anti-IL-17A single-domain antibody and application thereof Download PDF

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CN114920838A
CN114920838A CN202210489565.5A CN202210489565A CN114920838A CN 114920838 A CN114920838 A CN 114920838A CN 202210489565 A CN202210489565 A CN 202210489565A CN 114920838 A CN114920838 A CN 114920838A
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CN114920838B (en
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苏志鹏
王乐飞
张云
孟巾果
谢维
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Nanjing Rongjiekang Biotechnology Co ltd
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Nanjing Rongjiekang Biotechnology Co ltd
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Abstract

The invention belongs to the field of immunology, and relates to an anti-IL-17A single domain antibody and application thereof. The single domain antibody is composed of a heavy chain comprising heavy chain CDRs 1, 2, and 3. The amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are one of (2) - (4) or (6) - (9). Compared with the prior art, the invention has the beneficial effects that: the invention screens out the single-domain antibody specific to the IL-17A by using a biological gene engineering technology, has better antibody affinity, can block specific cells from releasing cytokines, has good binding activity through prokaryotic expression and eukaryotic expression, and has certain druggability.

Description

anti-IL-17A single-domain antibody and application thereof
Technical Field
The present invention relates to a single domain antibody capable of specifically binding to IL-17A (hereinafter, abbreviated as "IL-17A single domain antibody"), and a pharmaceutical composition containing the single domain antibody as an active ingredient, and a pharmaceutical therapeutic use thereof.
Background
Psoriasis is a common chronic inflammatory skin disease, commonly called psoriasis. The psoriasis is easy to relapse or aggravate in winter and is mostly relieved in spring and autumn, the global prevalence rate is about 2% -3%, wherein 1/3 patients with psoriasis have psoriatic arthritis (PsA), the psoriatic arthritis is often accompanied by joint swelling and pain, stiffness and dyskinesia, part of the psoriatic arthritis can affect the spine, serious patients can cause disability, and the physical and mental health of the patients is seriously affected. Psoriasis is classified according to its clinical features, mainly in the following categories: the psoriasis is more than 90% of the psoriasis of the vulgaris, the joint type, the pustule type and the erythrodermic type, and other types of the psoriasis are caused by sudden withdrawal of the patient during the treatment period by using external irritant drugs, overusing glucocorticoids and immunosuppressants.
The pathogenesis of psoriasis is unclear. At present, the treatment scheme about PsA mainly comprises non-steroidal anti-inflammatory drugs, glucocorticoids, antirheumatic drugs, azathioprine, tretinoin and the like, and the treatment methods of physical treatment, traditional Chinese medicine treatment and the like are used for relieving symptoms and controlling the disease condition. The traditional therapy is gradually inclined to the research of biological preparations clinically due to poor curative effect and adverse reaction, such as various side effects (blood pressure rise, blood sugar rise, osteoporosis, peptic ulcer, skin atrophy and the like) caused by long-term use of hormone, and tumor necrosis factor inhibitors are often selected as the first biological therapy of PsA patients and anti-IL-17 biological preparations.
Psoriasis has keratinocyte hyperproliferation, inflammatory cell infiltration and neovascularization as three elements of histopathological changes. The patients have a certain latent period of onset of the dysfunction of various immune cells, immune molecules, intracellular signal transduction systems and the like, and the onset of the disease can be induced by taking antimalarials, antipsychotic lithium preparations, antihypertensive beta-blockers and angiotensin converting enzyme inhibitors in the period. Seriously affecting the life quality and even the physical and mental health of the patients.
The pathogenesis of psoriasis is unclear, and it is currently considered to be an autoimmune disorder in a polygenic genetic background. The pathogenesis of the cancer is related to T lymphocyte, mainly CD4+ Th1 lymphocyte mediated immunity, and the pathogenesis process comprises the steps that initial T lymphocyte is activated into memory-effect T lymphocyte, the memory-effect T lymphocyte enters circulation to migrate to the skin, is gathered at a pathological part, and secretes various cytokines to play various biological functions to cause diseases.
IL-17A is the first discovered member of the IL-17 cytokine family and is secreted predominantly by Th17 cells. In 1986, Mosmann, coffman and related panelists found two subsets of helper T cells-Th 1 and Th2 (mosmanet, 1986). Besides Th17 cells, other T cells (including CD8+ T cells, gamma delta T cells, NKT cells) and innate immune cells (including NK cells, ILC3 cells, etc.) are capable of secreting IL-17A. In recent years, a number of studies on IL-17A and Th17 cells have shown that IL-17A is involved in the pathological progression of many autoimmune diseases (e.g., rheumatoid arthritis and encephalomyelitis) and plays an important protective role in immunity against bacterial and fungal infections.
Psoriasis is an autoimmune skin disease with hyperproliferation of epidermal cells, and studies have shown that IL-17A deficient mice have reduced epidermal proliferation (rizzoet al, 2011); clinical data also show important pathological roles for Th17 cells and IL-17A in psoriasis (hueberetal, 2010; wilsonet al, 2007). Based on the above findings regarding IL-17A in autoimmune diseases, the treatment of autoimmune diseases by inhibiting the differentiation of Th17 cells and blocking IL-17A signaling pathway has become a current focus.
The psoriasis is stubborn and difficult to treat, is listed as an important research subject in the field of dermatology in the world at present, and is one of important diseases for preventing and treating the dermatology in the world. With the advent of a range of new biologic drugs on the market, psoriasis patients have more and better medication options. TNFa antagonists and Interleukin (IL) class drugs are currently the focus of research and development in the field of autoimmunity, with the IL-12 family and IL-17 being of greatest interest.
At present, the prior art still lacks a single-domain antibody product with strong affinity and medicinal value for resisting IL-17A.
Disclosure of Invention
The invention of this patent aims to provide a single domain antibody capable of specifically binding to IL-17A and uses thereof.
In a first aspect, the invention provides an anti-IL-17A single domain antibody, said single domain antibody consisting of a heavy chain comprising the heavy chain CDR1 of any one of SEQ ID NO 19-25, the heavy chain CDR2 of any one of SEQ ID NO 26-32 and the heavy chain CDR3 of any one of SEQ ID NO 33-41. anti-IL-17A single domain antibodies, i.e., single domain antibodies directed against IL-17A.
Preferably, the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are one of the following (1) to (9):
(1) CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 28, CDR3 shown in SEQ ID NO. 36;
(2) CDR1 shown in SEQ ID NO. 21, CDR2 shown in SEQ ID NO. 29, CDR3 shown in SEQ ID NO. 35;
(3) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26, CDR3 shown in SEQ ID NO. 41;
(4) CDR1 shown in SEQ ID NO. 23, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 37;
(5) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26, CDR3 shown in SEQ ID NO. 40;
(6) CDR1 shown in SEQ ID NO. 20, CDR2 shown in SEQ ID NO. 30, CDR3 shown in SEQ ID NO. 33;
(7) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 38;
(8) CDR1 shown in SEQ ID NO. 24, CDR2 shown in SEQ ID NO. 31, CDR3 shown in SEQ ID NO. 34;
(9) CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 32, and CDR3 shown in SEQ ID NO. 39.
The above 9 CDR combinations (1) to (9) correspond to SEQ ID NO.1 to 9 in order.
All the above sequences may be replaced with a sequence having "at least 80% homology" with the sequence or a sequence having only one or a few amino acid substitutions; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".
In one embodiment, wherein any one to five of any one or more CDRs of the heavy chain CDRs 1, CDR2 and CDR3 may be substituted with their conserved amino acids, respectively. Specifically, 1 to 5 amino acid residues in the heavy chain CDR1 may be replaced by their conserved amino acids; 1 to 5 amino acid residues in the heavy chain CDR2 may be replaced by conserved amino acids thereof; in the heavy chain CDR3, 1 to 5 amino acid residues may be replaced by their conserved amino acids.
As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies of the exact same sequence have 100% homology.
In some embodiments, sequences that replace only one or a few amino acids compared to the aforementioned sequences, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, may also achieve the objectives of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. Indeed, the skilled person may consider so-called "conservative" amino acid substitutions, in which case the substitution will preferably be a conservative amino acid substitution, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combination in a single domain antibody. The conservative amino acid, which may be generally described as an amino acid substitution in which an amino acid residue is replaced with another amino acid residue having a similar chemical structure, has little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are common in the art, for example conservative amino acid substitutions are those in which one or a few amino acids within the following groups (a) - (d) are replaced by another or a few amino acids within the same group: (a) polar negatively charged residues and their uncharged amides: asp, Asn, Glu, Gln; (b) polar positively charged residues: his, Arg, Lys; (c) aromatic residue: phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val and Cys. Particularly preferred conservative amino acid substitutions are as follows: asp substituted by Glu; asn is replaced by Gln or His; glu is substituted with Asp; gln is substituted by Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys substituted by Arg, Gln; phe is replaced by Met, Leu, Tyr; trp is substituted by Tyr; tyr is substituted by Phe, Trp; ala by Gly or Ser; ser substituted by Thr; thr is substituted by Ser; gly by Ala or Pro; met is substituted by Leu, Tyr or Ile; leu is substituted by Ile or Val; ile is substituted by Leu or Val; val is substituted by Ile or Leu; cys is replaced by Ser. In addition, the skilled person knows that the creativity of single domain antibodies is found in the CDR1-3 region, whereas the framework region sequence FR1-4 is not unalterable and the sequence of FR1-4 may take the form of conservative sequence variants of the sequences disclosed in the present invention.
The meaning of "anti-IL-17A single domain antibody" in the present invention includes not only the whole single domain antibody but also fragments, derivatives and analogs of said anti-IL-17A single domain antibody. As used herein, the terms "fragment," "derivative," and "analog" are synonymous and all refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with an Fc tag). Such fragments, derivatives and analogs are well within the skill of those in the art in light of the teachings herein.
In a preferred embodiment, the antibody sequence further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR 4; the amino acid sequences of the FR regions of the framework regions are:
a variant of FR1 or FR1 represented by any one of SEQ ID NOS as 42-44, said variant of FR1 comprising a substitution of up to 5 amino acids in said FR 1;
a variant of FR2 or FR2 as set forth in any one of SEQ ID NOs 45-52, said variant of FR2 comprising a substitution of up to 5 amino acids in said FR 2;
a variant of FR3 or FR3 as set forth in any one of SEQ ID NOs 53-59, said variant of FR3 comprising a substitution of up to 5 amino acids in said FR 3;
a variant of FR4 or FR4 as set forth in any one of SEQ ID NOS: 60-62, said variant of FR4 comprising a substitution of up to 5 amino acids in said FR 4.
In a second aspect, the invention provides the amino acid sequence of a single domain antibody against IL-17A, said single domain antibody having the amino acid sequence shown in SEQ ID No.1-9, respectively, or said single domain antibody having at least 80% sequence homology with the amino acid sequence of SEQ ID No.1-9, and being capable of specifically binding to IL-17A protein.
In one embodiment, the anti-IL-17A single domain antibody binds to a polypeptide selected from the group consisting of SEQ ID NO: 1-9 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology, and are capable of specifically binding to IL-17A protein.
A third aspect of the invention provides a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, the first antigen-binding portion being a single domain antibody, the amino acid sequence of said single domain antibody being as shown in SEQ ID No.1 to 9 or having at least 80% homology with SEQ ID No.1 to 9, respectively. The second antigen binding portion is another antibody, for example, can be for TNF alpha, IL-6R, IL-6, IL-23R antibody (can be monoclonal antibody, polyclonal antibody, single domain antibody or antibody of any other form); the second antigen-binding portion may also be an antibody to another antigen for use in the diagnosis, prevention or treatment of disease or detection of an antigen.
A fourth aspect of the invention provides the use of an anti-IL-17A single domain antibody of any one of the preceding claims in the preparation of a bispecific antibody.
A fifth aspect of the invention provides an Fc fusion antibody or a humanized antibody of the anti-IL-17A single domain antibody of any one of the preceding.
In a sixth aspect of the present invention, there is provided a nucleotide molecule encoding the aforementioned anti-IL-17A single domain antibody or the aforementioned Fc fusion antibody or the aforementioned humanized antibody, whose nucleotide sequences are set forth in SEQ ID NOs: 10-18, or a variant of SEQ ID NO:10-18 has at least 95% sequence homology.
In one embodiment, the nucleic acid molecule encoding said anti-IL-17A single domain antibody hybridizes with a sequence selected from the group consisting of SEQ ID NO:10-18 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology, and encodes a single domain antibody against IL-17A that is capable of specifically binding to IL-17A protein.
In a seventh aspect of the invention, there is provided an expression vector comprising a nucleotide molecule encoding a single domain antibody or Fc fusion antibody or humanized antibody against IL-17A, the nucleotide sequences of which are set forth in SEQ ID NOs: 10-18.
In a preferred embodiment, the expression vector used is RJK-V4-3 (the nucleotide molecule encoding the anti-IL-17A single domain antibody or Fc fusion antibody or humanized antibody thereof is integrated into RJK-V4-3 by genetic engineering means), and other general expression vectors may be selected as necessary.
An eighth aspect of the present invention is to provide a host cell capable of expressing the aforementioned anti-IL-17A single domain antibody, Fc fusion antibody or humanized antibody, or an expression vector comprising the same. Preferably the host cell is a bacterial cell, a fungal cell or a mammalian cell.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell, including bacteria, fungi.
In another preferred embodiment, the host cell is selected from the group consisting of: coli, yeast cells, mammalian cells, phage, or combinations thereof.
In another preferred embodiment, the prokaryotic cell is selected from the group consisting of: escherichia coli, Bacillus subtilis, Lactobacillus, Streptomyces, Proteus mirabilis, or a combination thereof.
In another preferred embodiment, the eukaryotic cell is selected from the group consisting of: pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, or a combination thereof.
In another preferred embodiment, the eukaryotic cell is selected from the group consisting of: insect cells such as Spodoptera frugiperda, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.
In another preferred embodiment, the host cell is a suspension ExpCHO-S cell.
In another preferred embodiment, the host cell is a suspension 293F cell.
In a ninth aspect, the invention provides a recombinant protein comprising the aforementioned single domain antibody against IL-17A. The recombinant protein can be the single-domain antibody shown by the SEQ ID NO.1-9, can also be a single-domain antibody with at least 80% homology with the SEQ ID NO.1-9, and can also be a multi-epitope antibody, a multi-specific antibody and a multivalent antibody; for example, the multi-epitope antibody may consist of more than one sequence of SEQ ID No. 1-9; the multivalent antibody can be formed by repeatedly arranging one sequence in SEQ ID NO.1-9 for a plurality of times; such multispecific antibodies include, but are not limited to, bispecific antibodies as described above, and trispecific antibodies; in addition, the recombinant protein may be a fragment, derivative and analog of the aforementioned antibody.
A tenth aspect of the invention provides a pharmaceutical composition comprising the aforementioned anti-IL-17A single domain antibody and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically determined based on the isoelectric point of the antibody (the pH of the aqueous carrier medium is offset from and differs by about 2 from the isoelectric point of the antibody). The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous, transdermal (direct application or plaster application to the affected part).
The pharmaceutical compositions of the invention can be used directly to bind to IL-17A protein molecules and thus can be used to treat psoriasis. In addition, other psoriasis therapeutic agents can be used in combination.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the aforementioned single domain antibody and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions.
An eleventh aspect of the present invention provides an agent for treating psoriasis, which comprises the aforementioned anti-IL-17A single domain antibody as an active ingredient.
In a twelfth aspect of the invention, there is provided a kit for detecting IL-17A levels, which comprises the aforementioned anti-IL-17A single domain antibody. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, and the like.
In a preferred embodiment, the kit comprises an antibody recognizing IL-17A protein, a lysis medium for solubilizing a sample, general-purpose reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, and the like. The test kit may be an in vitro diagnostic device.
In a preferred embodiment, the kit further comprises a second antibody and an enzyme for detection or a fluorescent or radioactive label, and a buffer.
In a preferred embodiment, the second antibody of the kit may be the aforementioned antibody against a single domain antibody of IL-17A (as an anti-antibody), and may be a single domain antibody, a monoclonal antibody, a polyclonal antibody, or any other form of antibody.
In a thirteenth aspect of the invention, there is provided a method of producing a single domain antibody against IL-17A, comprising the steps of:
(a) culturing the host cell of the eighth aspect of the invention under conditions suitable for the production of single domain antibodies, thereby
Obtaining a culture comprising said anti-IL-17A single domain antibody; and
(b) isolating or recovering said anti-IL-17A single domain antibody from said culture; and
(c) optionally, purifying and/or modifying the single domain antibody to IL-17A obtained in step (b).
The fourteenth aspect of the invention provides the use of a single domain antibody or pharmaceutical composition against the aforementioned IL-17A in the manufacture of a medicament for inhibiting IL-17A gene expression or an anti-psoriasis medicament.
A fifteenth aspect of the invention provides the use of the aforementioned anti-IL-17A single domain antibody or the aforementioned pharmaceutical composition in the manufacture of a medicament for the treatment of a disease.
In a preferred embodiment, the disease is an autoimmune disease, or the disease is a disorder associated with aberrant expression of IL-17A.
In a preferred embodiment, the disease is psoriasis.
In a preferred embodiment, the disease is arthritis.
A sixteenth aspect of the invention provides the use of a single domain antibody directed against the aforementioned anti-IL-17A for the manufacture of a medicament for blocking the binding of IL-17A to IL-17R.
Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) the single domain antibodies of the invention are specific for IL-17A proteins with the correct spatial structure.
(2) The single domain antibody has better specificity, can block the interaction of the IL-17A and the IL-17R binding site, has blocking activity higher than that of the existing medicament, and has great application prospect in preparing the medicament for treating autoimmune diseases.
(3) The single domain antibody obtained by the invention has flexible selection of an expression system, can be expressed in a prokaryotic system and also can be expressed in a eukaryotic system of a yeast cell or a mammalian cell, and has low expression cost in the prokaryotic expression system, thereby reducing the later-stage production cost.
(4) The single domain antibody obtained by the invention has simple multi-combination form modification, can obtain a multivalent and multi-specific antibody by simple series connection in a genetic engineering mode, has low immune heterogeneity, and can not generate stronger immune reaction under the condition of not carrying out humanized modification.
(5) The single-domain antibody obtained by the invention has a wider affinity range, and before affinity maturation is not carried out, the affinity range can be from nM level to pM level, so that multiple selections are provided for later-stage antibodies with different purposes.
Drawings
In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a library enrichment of the targeted IL-17A antibody screen in example 3;
FIG. 2 is a graph showing the measurement of the antigen-binding capacity-effect curve of the antibody in example 13 (a part of clones);
FIG. 3 is a graph showing the measurement of the antigen-binding capacity and effect curve of the antibody in example 13 (another part of clone, Tab1, hIgG);
FIG. 4 is a graph of an assay of the antibody of example 14 blocking the binding of IL-17A to IL-17R (a subset of clones);
FIG. 5 is a graph of an assay for the antibody blocking the binding of IL-17A to IL-17R in example 14 (another portion of clones, hIgG);
FIG. 6 is a graph showing the results of the experiment in example 15 in which antibody-neutralizing human IL-17A (eukaryotic sample) induced the release of IL-6 from Hela cells (a part of clones, Tab, hIgG);
FIG. 7 is a graph showing the results of the experiment in which antibody-neutralizing human IL-17A (a eukaryotic sample) induced the release of IL-6 from Hela cells in example 15 (another part of clones);
FIG. 8 is a graph showing the results of IL-6 release from Hela cells induced by antibody neutralization of human IL-17A (humanized sample) in example 16 (Tab, hIgG, 2G 3);
FIG. 9 is a graph showing the results of experiments in example 16 in which antibody-neutralizing human IL-17A (humanized sample) induced the release of IL-6 from Hela cells (2G3, 1A 10);
FIG. 10 is a graph showing the results of the experiment in which antibody-neutralizing human IL-17A (humanized sample) induced the release of IL-6 from Hela cells in example 16 (1A 10).
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
As used herein, "single domain antibodies" (sdabs, also referred to by the developer Ablynx as nanobodies or VHHs) are well known to those skilled in the art. A single domain antibody is an antibody whose complementarity determining regions are part of a single domain polypeptide. Thus, single domain antibodies comprise a single complementarity determining region (single CDR1, single CDR2, and single CDR 3). Examples of single domain antibodies are heavy chain-only antibodies (which do not naturally contain a light chain), single domain antibodies derived from conventional antibodies, and engineered antibodies.
Single domain antibodies may be derived from any species, including mouse, human, camel, llama, goat, rabbit and cow. For example, naturally occurring VHH molecules may be derived from antibodies provided by species in the family camelidae (e.g. camel, dromedary, llama and guanaco). Like intact antibodies, single domain antibodies are capable of selectively binding to a particular antigen. Single domain antibodies may contain only the variable domains of immunoglobulin chains, with CDR1, CDR2 and CDR3, and the framework regions.
As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is often expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies of an identical sequence have 100% homology.
As used herein, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc region of an antibody of interest to a molecule of a functional protein having biological activity using genetic engineering techniques.
The term "humanized antibody" refers to an antibody obtained by fusing the variable region of the heavy chain of a target antibody (e.g., an animal antibody) with the constant region of a human antibody, an antibody obtained by grafting the complementarity determining regions (CDR 1-3 sequences) of a target antibody into the variable region of a human antibody, or an antibody obtained by mutating the amino acids of a target antibody according to the characteristics of the framework regions (FR 1-4) of a human antibody. Humanized antibodies can be synthesized or mutated at a site.
In the present invention, a single domain antibody against IL-17A can also be obtained from a sequence having high sequence homology to CDR1-3 disclosed in the present invention. In some embodiments, sequences having "at least 80% homology" to the sequences in SEQ ID Nos. 1-9, or sequences having "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" are all suitable for the purposes of the present invention.
In some embodiments, the polypeptide of SEQ ID NO: sequences 1-9 that are substituted for only one or a few amino acids compared to sequences containing, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions may also serve the purpose of the invention. Indeed, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2, and CDR3 combination in a single domain antibody, the skilled person may consider so-called "conservative" amino acid substitutions, in which case the substitution will preferably be a conservative amino acid substitution, which may generally be described as the substitution of an amino acid residue with another amino acid residue having a similar chemical structure, and which has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are common in the art, for example conservative amino acid substitutions are those in which one or a few amino acids within the following groups (a) - (d) are replaced by another or a few amino acids within the same group: (a) polar negatively charged residues and their uncharged amides: asp, Asn, Glu, Gln; (b) polar positively charged residues: his, Arg, Lys; (c) aromatic residue: phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val and Cys. Particularly preferred conservative amino acid substitutions are as follows: asp substituted by Glu; asn is replaced by Gln or His; glu is substituted with Asp; gln is substituted by Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys substituted by Arg, Gln; phe is replaced by Met, Leu, Tyr; trp is substituted by Tyr; tyr is substituted by Phe, Trp; ala substituted by Gly or Ser; ser substituted by Thr; thr is substituted by Ser; gly by Ala or Pro; met is substituted by Leu, Tyr or Ile; leu is substituted by Ile or Val; ile is substituted by Leu or Val; val is substituted by Ile or Leu; cys is replaced by Ser. In addition, the skilled person knows that the creativity of single domain antibodies is found in the CDR1-3 region, while the framework region sequence FR1-4 is not unalterable, and the sequence of FR1-4 can take the conservative sequence variants of the sequences disclosed in the present invention.
Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.
The method is characterized in that a target protein and a truncated form of the target protein are prepared through a genetic engineering technology, then the obtained antigen protein is used for immunizing an inner Mongolia Alaga bactrian camel, after multiple immunizations, peripheral blood lymphocytes or spleen cells of the camel are obtained, a camel source antibody variable region coding sequence is recombined into a phage display carrier through a genetic engineering mode, a specific antibody aiming at the antigen protein is screened out through a phage display technology, and the binding capacity of the camel source antibody variable region coding sequence and the application of the camel source antibody in treatment of autoimmune diseases are further detected.
The above technical solution is now split and described in detail by way of specific examples:
example 1: preparation of human IL-17A recombinant extracellular domain protein:
the humanized recombinant extracellular domain protein used in the patent is obtained by expression and purification of a company, and the design scheme of the humanized IL-17A recombinant extracellular domain protein expression vector is as follows:
(1) the coding sequence for IL-17A was retrieved at NCBI and was deposited under NM-002190.2, the amino acid sequence generated by this sequence was deposited under accession number NP-002181.1 and the Uniprot ID was Q16552.
(2) The amino acid sequence corresponding to NP-002181.1 was analyzed for the transmembrane region and extracellular end of the protein by TMHMM and SMART websites, respectively.
(3) The analysis result shows that IL-17A is a secretory protein, and the 1-23 sites are signal peptides of the protein.
(4) The nucleotide sequence of 24-155 amino acids of the protein coding IL-17A is cloned into the vector pcDNA3.4 by means of gene synthesis.
(5) Performing Sanger sequencing on the constructed vector, comparing the original sequences, extracting the recombinant plasmid in batches after confirming no errors, removing endotoxin, transfecting and suspending 293F to express and purify target protein, wherein the purity of the purified protein is up to 90 percent, and the requirement of animal immunity is met.
Example 2: construction of a library of single domain antibodies directed against IL-17A protein:
1mg of the human recombinant IL-17A protein obtained by purification in example 1 is mixed with equal volume of Freund complete adjuvant, one inner Mongolia Alexanthema is immunized, the immunization is carried out once a week for 7 times continuously, and the rest six times except the first immunization are animal immunization by mixing 1mg of the IL-17A protein with equal volume of Freund incomplete adjuvant, wherein the immunization process is to intensively stimulate the camel to generate antibodies aiming at the IL-17A protein.
After animal immunization is finished, 150mL of camel peripheral blood lymphocytes are extracted, and RNA of the cells is extracted. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified by nested PCR reaction using the cDNA as a template.
Then, respectively carrying out enzyme digestion on the pMECS vector and the VHH fragment by utilizing restriction enzymes, and then linking the enzyme-digested fragment with the vector. The ligated fragments were electrically transformed into competent cells TG1, a phage display library of IL-17A protein was constructed and the library capacity was determined, the size of the library capacity being about 1X 10 9 Meanwhile, the correct insertion rate of the detection library in the target fragment is identified through colony PCR.
The results showed that after PCR amplification of 40 colonies randomly selected from the library, 38 clones amplified a band of the predicted size, and 2 clones amplified a band of the incorrect size, so the correct insertion rate was 38 ÷ 40X 100% ≈ 95%.
Example 3: single domain antibody screening against IL-17A protein:
200 μ L of recombinant TG1 cells from example 2 were cultured in 2 XTY medium during which TG1 cells were infected with 40 μ L of helper phage VCSM13 and cultured overnight to amplify phages, the next day phages were pelleted with PEG/NaCl and the amplified phages were collected by centrifugation.
NaHCO diluted at 100 mM pH8.3 3 500 mu g of IL-17A protein in the reagent is coupled on an enzyme label plate, is placed at 4 ℃ overnight, and is simultaneously provided with a negative control hole (culture medium control); adding 200 μ L of 3% skimmed milk the next day, sealing at room temperature for 2 hr; after the end of blocking, 100. mu.l of the amplified phage library (approx.2X 10) was added 11 Individual phage particles), and reacting for 1h at room temperature; after 1 hour of action, the unbound phage were washed off 15 times with PBS + 0.05% Tween-20.
The phage specifically bound to the IL-17A protein was dissociated with trypsin at a final concentration of 25mg/mL and infected with E.coli TG1 cells in logarithmic growth phase, cultured at 37 ℃ for 1h, phage was generated and collected for the next round of screening, and the same screening procedure was repeated for 1 round to gradually obtain enrichment.
When the enrichment factor reaches more than 10 times, the enrichment effect is shown in figure 1.
In fig. 1, P/N is the number of monoclonal bacteria grown after phage eluted from positive well was infected with TG1 bacteria in biopanning/the number of monoclonal bacteria grown after phage eluted from positive well was infected with TG1 bacteria, which parameter gradually increased after enrichment occurred; I/E ═ total number of phage added to positive wells per round of biopanning/total number of phage eluted from positive wells per round of biopanning, this parameter will gradually approach 1 after enrichment has occurred.
Example 4: screening of specific positive clones for IL-17A by phage enzyme-linked immunosorbent assay (ELISA):
after 3 rounds of screening against a single domain antibody against IL-17A protein according to the screening method described in example 3 above, the phage enrichment factor against IL-17A protein reached 10 or more, 384 single colonies from the positive clones obtained by screening were individually inoculated into 96-well plates of 2 XTY medium containing 100. mu.g/mL ampicillin, and a blank was set, and after incubation at 37 ℃ until a logarithmic phase, IPTG was added at a final concentration of 1 mM, and incubation at 28 ℃ was carried out overnight.
Obtaining a crude antibody by using an osmotic bursting method; the IL-17A recombinant protein was released separately into 100 mM NaHCO pH8.3 3 And 100. mu.g of protein was coated overnight at 4 ℃ in an ELISA plate (ELISA plate). Transferring 100 mu L of the obtained antibody crude extract to an ELISA plate added with an antigen, and incubating for 1h at room temperature; unbound Antibody was washed away with PBST, 100. mu.l of Mouse Anti-HA tag Antibody (HRP) (Mouse Anti-HA horseradish peroxidase labeled Antibody, Thermo Fisher) diluted 1:2000 was added, and incubated at room temperature for 1 h; washing away unbound antibody with PBST, adding horseradish peroxidase developing solution, reacting at 37 deg.C for 15min, adding stop solution, and reading the absorbance at 450nm wavelength on an enzyme-labeling instrument.
When the OD value of the sample hole is more than 5 times of that of the control hole, judging the sample hole as a positive cloning hole; the positive colony well was transferred into LB medium containing 100. mu.g/mL ampicillin to extract plasmids and sequenced.
The gene sequences of the respective clones were analyzed according to the sequence alignment software VectorNTI, and strains having the same CDR1, CDR2 and CDR3 sequences were regarded as the same clones, and strains having different sequences were regarded as different clones, and finally single domain antibodies specific to the IL-17A protein were obtained (SEQ ID nos. 1 to 9 and other single domain antibodies having no sequence shown, including 1C9, 1D1, etc. in the drawing).
The amino acid sequence of the antibody is the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the whole VHH is formed. The obtained single domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally single domain antibody protein is obtained.
The CDR and FR sequences of the 9 single-domain antibodies are shown in tables 1-6. The amino acid sequences of the 9 single-domain antibodies are respectively shown as SEQ ID NO.1-9, and the DNA sequences of the coded 9 single-domain antibodies are respectively shown as SEQ ID NO. 10-18.
CDR1 sequences of the Table 19 single domain antibodies
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CDR2 sequences of Table 29 single domain antibodies
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CDR3 sequences of the Table 39 single domain antibodies
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FR1 sequences of the 49 single domain antibodies in Table 49
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FR2 sequences of Table 59 Single Domain antibodies
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FR3 sequences of 69 single-domain antibodies
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FR4 sequences of the 79 single domain antibodies in Table
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The amino acid sequences SEQ ID NO.1-9 of the single domain antibody correspond to the single domain antibodies 1A10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2 and 4A12 one by one. The nucleotide sequences SEQ ID NO.10-18 of the single domain antibodies also correspond one-to-one to the single domain antibodies 1A10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2, 4A12 in this order.
Example 5: purification and expression of specific single domain antibody of IL-17A protein in host bacterium escherichia coli
Plasmids of the different clones obtained in the sequencing analysis of example 4 (pMECS-VHH) were electrotransformed into E.coli HB2151 and spread on LB + amp + glucose, i.e.culture plates containing ampicillin and glucose, and incubated overnight at 37 ℃; individual colonies were picked and inoculated in 5mL of LB medium containing ampicillin and shake-cultured overnight at 37 ℃.
Inoculating 1mL of overnight culture strain into 330mL of LTB culture solution, shake culturing at 37 deg.C until OD600nm value reaches 0.6-0.9, adding 1M IPTG, and shake culturing at 28 deg.C overnight; centrifuging, collecting Escherichia coli, and obtaining crude antibody extractive solution by permeation cracking method;
purifying the antibody by a nickel column affinity chromatography, wherein the purified single domain antibody comprises VHH 1-9.
VHH1-9 corresponds to the single domain antibodies of amino acid sequence SEQ ID No.1-9 in sequence, i.e. VHH1-9 corresponds to: 1a10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2, 4a 12.
Example 6: humanization of Single Domain antibodies to IL-17A
The humanization method was performed by high throughput screening of antibody framework region mutation libraries constructed based on the results of big data analysis. The detailed steps are as follows:
(1) sequence analysis of human/camel antibody data: carrying out amino acid preference analysis on 13873 Nb (human) sequences downloaded from an NCBI website in batches, and simultaneously carrying out amino acid preference analysis on 2000 nano-antibody sequences of the company to obtain amino terminal proportion data of each site of a framework region;
(2) comprehensive weighting analysis of human camel source: the source/camel source antibody sequences are numbered uniformly according to an IMGT numbering rule and correspond one to one, the analysis results of the proportion of the amino acids in the two species are combined, the weighted analysis is carried out according to the weight of 90 percent of camel source 10 percent of human source, the weighted proportion of the amino acids at each site is counted, and the amino acids are sequenced from high to low; according to the final weighting result, only reserving amino acid types accounting for more than 10% of a single site in the framework region, and calculating the final weight of the amino acid accounting for more than 10% of the single site according to the standard that the reserved ratio is integrated into 1 to be used as the design basis of a subsequent amino acid customized library;
(3) scheme design of amino acid custom libraries: specifying the number of amino acids of which the percentage is more than 10% as n and the ratio of the highest value to the lowest value of which the percentage is more than 10% as V for the single site to be mutated, and judging the properties of the site to be mutated: if V is more than or equal to 3 and n is less than or equal to 2, the site is considered to belong to a high-concentration site, otherwise, the site is considered to belong to a low-concentration site. According to the method, the customized amino acid library is divided into two high/medium/low concentration ratio libraries, and the amino acid customized libraries are constructed respectively, wherein the final weight in the step (2) is a reference basis of the types and proportions of the amino acids at the positions in the library.
(4) High throughput screening of amino acid custom libraries:
humanized antibody libraries are respectively constructed for antibody strains, 1A10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2 and 4A12, corresponding antigens are respectively used for panning aiming at the constructed libraries, and finally, antibody sequences with high affinity and high humanized degree are obtained.
For example, the following humanized antibodies can be obtained: 8 humanized antibodies (2G-V, the sequences of the 8 humanized antibodies of 2G are all different) obtained by performing humanized modification on the 2G single domain antibody, and 8 humanized antibodies (1A-V, 1A-V or 1A-V, the sequences of the 8 humanized antibodies of 1A are all different) obtained by performing humanized modification on the 1A single domain antibody.
Example 7: construction of Fc fusion antibody eukaryotic expression vector of anti-IL-17A single domain antibody
(1) The target sequence obtained in example 4 was subcloned into eukaryotic expression vectors: the antibody screened out in the example 4 is subjected to Sanger sequencing to obtain a nucleotide sequence;
(2) the nucleotide sequence (SEQ ID NO: 10-18) is synthesized into a vector RJK-V4-3 designed and modified by the company to obtain a recombinant eukaryotic expression vector by a sequence synthesis mode, and the modification method of the vector is described in example 11;
(3) transforming the recombinant eukaryotic expression vector constructed in the step (2) into DH5 alpha escherichia coli, culturing, performing plasmid extraction, and removing endotoxin;
(4) carrying out sequencing identification on the extracted plasmid;
(5) the recombinant vector determined to be free of errors is prepared for subsequent eukaryotic cell transfection and expression, and after expression of the Fc protein of VHH by the method of example 8 or 9 and purification of the above antibody by the method of example 10.
Example 8: expression of single domain antibodies against IL-17A protein in suspension ExpicHO-S cells
(1) 3 days before transfection at 2.5X 10 5 (ii) passaging and expanding the cells in/mL expiCHO-S-cells, and transferring the calculated desired cell volume to a 500mL shake flask containing fresh pre-warmed 120mL (final volume) expiCHO-cell expression medium; to achieve a cell concentration of about 4X 10 6 -6×10 6 Viable cells/mL;
(2) one day before transfection, the ExpCHO-S.cell was diluted to a concentration of 3.5X 10 6 Viable cells/mL, cells were cultured overnight;
(3) on the day of transfection, cell density and percentage of viable cells were determined. The cell density before transfection should reach about 7X 10 6 -10×10 6 Viable cells/mL;
(4) cells were diluted to 6X 10 with fresh ExpiCHO ™ expression medium preheated to 37 ℃ 6 Viable cells/mL. The calculated required cell volume was transferred to a 500mL shake flask containing fresh pre-warmed 100mL (final volume) of ExpCHO cell expression medium;
(5) gently inverting and mixing Expifectamine ™ CHO reagent, diluting Expifectamine ™ CHO reagent with 3.7mL OptiPRO ™ medium, and swirling or mixing;
(6) diluting plasmid DNA with refrigerated 4mL OptiPRO ™ culture medium, and mixing;
(7) incubating Expifactamine CHO/plasmid DNA (plasmid DNA is Fc fusion antibody eukaryotic expression vector of anti-IL-17A single domain antibody prepared in example 7) complex for 1-5 min at room temperature, then adding gently into the prepared cell suspension, and gently swirling the flask during the addition process;
(8) cells were incubated at 37 ℃ with 8% CO 2 In humidified airPerforming shake culture;
(9) on day 1 post-transfection (18-22 h later), 600ul Expifeacamine ™ CHO Enhancer and 24mL ExpicHO feed were added.
(10) Supernatants were collected at about 8 days post transfection (cell viability below 70%).
Example 9: expression of Single-Domain antibodies against IL-17A protein in 293F cells in suspension
Recombinant single domain antibody expression experimental protocol (taking 500mL shake flask as an example):
(1) 3 days before transfection at 2.5X 10 5 The 293F cells were passaged and expanded and the calculated required cell volume was transferred to 500mL shake flasks containing fresh pre-warmed 120mL OPM-293 CD05 Medium (final volume). The cell concentration is about 2X 10 6 -3×10 6 Viable cells/mL.
(2) On the day of transfection, cell density and percentage of viable cells were determined. The cell density before transfection should reach about 2X 10 6 -3×10 6 Viable cells/mL.
(3) Cells were diluted to 1X 10 with preheated OPM-293 CD05 Medium 6 Viable cells/mL. The required cell volume was calculated and transferred to a 500mL shake flask containing fresh pre-warmed 100mL (final volume) of medium.
(4) Diluting PEI (1 mg/mL) reagent with 4mL of Opti-MEM medium, and swirling or blowing to mix evenly; plasmid DNA (plasmid DNA is the Fc fusion antibody eukaryotic expression vector for the single domain antibody against IL-17A prepared in example 7) was diluted with 4mL of Opt-MEM medium, vortexed, mixed well, and filtered through a 0.22um filter head. Incubate at room temperature for 5 min.
(5) Diluted PEI reagent was added to the diluted DNA and mixed by inversion. The PEI/plasmid DNA complex was incubated at room temperature for 15-20 minutes and then gently added to the prepared cell suspension, gently swirling the flask during the addition.
(6) Cells were incubated at 37 ℃ in 5% CO 2 And shake culture at 120 rpm.
(7) 5mL OPM-CHO PFF05 feed was added at 24h, 72h post transfection.
(8) Supernatants were collected at about 7 days post transfection (cell viability below 70%).
Example 10: purification of Single Domain antibodies against IL-17A protein
(1) Filtering the protein expression supernatant obtained in example 8 or 9 with a 0.45 μm disposable filter to remove insoluble impurities;
(2) performing affinity chromatography purification on the filtrate by using a Protein purifier, and purifying by using agarose filler coupled with Protein A by utilizing the binding capacity of human-derived Fc and Protein A;
(3) passing the filtrate through a Protein A pre-packed column at a flow rate of 1 mL/min, wherein the target Protein in the filtrate is bound to the packing material;
(4) washing the impurity protein bound on the column by low-salt and high-salt buffer solutions;
(5) performing a system of target proteins bound to the column with a low pH buffer;
(6) adding the eluent into Tris-HCl solution with pH9.0 rapidly for neutralization;
(7) dialyzing the neutralized protein solution, performing SDS-PAGE analysis to determine that the protein has a purity of 95% or more and a concentration of 0.5mg/mL or more, and storing at low temperature for later use.
Example 11: construction of Single Domain antibody eukaryotic expression vector RJK-V4-3
The general target vector RJK-V4-3 for the nano-antibody is obtained by fusing the Fc segment in the heavy chain coding sequence of human IgG4 on the basis of an invitrogen commercial vector pCDNA3.4 (vector data link: https:// Assets. thermofisher. com/TFS-Assets/LSG/vitamins/pcdna 3_4_ topo _ ta _ cloning _ kit _ man.pdf), namely the vector comprises a Hinge region (Hinge) CH2 and a CH3 region of IgG4 heavy chain. The specific modification scheme is as follows:
(1) selecting restriction sites XbaI and AgeI on pcDNA3.4;
(2) introducing a Multiple Cloning Site (MCS) and a6 × His tag at the 5 'end and the 3' end of the Fc fragment coding sequence respectively by means of overlapping PCR;
(3) amplifying the fragment by using a pair of primers with XbaI and AgeI enzyme cutting sites respectively in a PCR mode;
(4) the recombinant DNA fragments in pcDNA3.4 and (3) are respectively cut by restriction enzymes XbaI and AgeI;
(5) and (3) connecting the vector and the insert after enzyme digestion under the action of T4 ligase, then transforming the connection product into escherichia coli, amplifying, sequencing and verifying to obtain the recombinant plasmid.
Example 12: expression and purification of a Tool antibody (Tool antibody, Tab 1) targeting human IL-17A
Herein, Tab1 is Tab. Tab1 (Secukinumab) is Secukinumab, with the sequence from IMGT.
The searched sequence was subjected to codon optimization of mammalian cell expression system by general biosystems (Anhui) Ltd and cloned into pcDNA3.1 vector. After resistance selection, plasmid positive bacteria were selected and amplified, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat # 740412.50). Transient Expression in 293F cells (medium: FreeStyle 293 Expression medium, Thermo, Cat #12338026 + F-68, Thermo, Cat # 24040032) using PEI was performed as per 100mL of cells with 100. mu.g of plasmid (40. mu.g of heavy chain + 60. mu.g of light chain); after transfection for 6-24 h, 5% volume of 10% Peptone (Sigma, Cat # P0521-100G) and 8% CO are added 2 Culturing at 130rpm for about 7-8 days; when the cell viability is reduced to 50%, collecting the expression supernatant, and purifying by using a ProteinA (GE, Cat # 17-5438-02) gravity column; after PBS dialysis, the concentration is measured by using Nanodrop, the purity is identified by SEC, and the binding capacity is verified by indirect ELISA;
the Tab obtained by the method has the concentration not less than 2 mg/ml and the purity more than 95 percent.
Example 13: antigen binding dose-response curve assay for antibodies
This example was carried out using a standard enzyme-linked immunosorbent assay (ELISA) protocol.
(1) Coating 50. mu.L of 1. mu.g/mL IL-17A & F (Acro, Cat # ILF-H52WF, Lot # G41-88ZF1-RD) overnight at 4 ℃.
(2) Washing the plate; add 200. mu.L of 5% milk and block for 2h at 37 ℃.
(3) VHH-hFc was diluted to 2ug/mL, followed by a 5-fold gradient dilution of the antibody for 8 concentration gradients. Here, VHH-hFc refers to the Fc fusion antibody (expressed in 293F cells) of the single domain antibody against IL-17A protein prepared in example 9, which was purified in example 10. Besides, hIgG and Tab controls are also set respectively; tab1 was obtained from example 12;
(4) washing the plate; add 50. mu.L of the single domain antibody diluted in step (3), duplicate wells, incubate for 1h at 37 ℃.
(5) Washing the plate; mu.L of HRP-Goat anti hIgG secondary antibody was added and incubated at 37 ℃ for 30 min.
(6) Washing the plate (washing for several times); adding 50 μ L of TMB recovered to normal temperature in advance, and reacting for 15min at normal temperature in the dark.
(7) Add 50. mu.L of stop buffer (1N HCl) and read by microplate reader.
(8) Plotting the curves and calculating the EC50, as shown in fig. 2-3, where hIgG refers to isotype control, immunoglobulin molecules that do not bind to any target, were obtained by commercial purchase; it can be seen that the single domain antibodies 1a10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2, 4a12 all have excellent binding potency and specificity for IL-17A protein.
In FIG. 2, 2A shows the binding capacity of single domain antibodies 1A10, 1C9, 1D1, 1D4, 1D7, 1E2 to IL-17A protein, and 2B shows the binding capacity of single domain antibodies 1E6, 1H2, 2D8, 2E2, 2F5, 2G3 to IL-17A protein. In FIG. 3, 3A shows the binding capacity of single domain antibodies 3A6, 3A8, 3B11, 3D1, 3E1, 3E2 to IL-17A protein, 3B shows the binding capacity of single domain antibodies 3F8, 3H2, 4A1, 4A12, 4F4, 1H3 to IL-17A protein, and 3C shows the binding capacity of single domain antibodies 3B10, 4A1, 4G4, Tab1, hIgG to IL-17A protein.
Example 14: assay for blocking IL-17A binding to IL-17R by antibodies
This example was carried out using a standard enzyme-linked immunosorbent assay (ELISA) protocol.
(1) Coating 50. mu.L of 1. mu.g/mL IL-17RA & IL-17RC (Acro, Cat # ILC-H5257, Lot # G173a-2085F1-TE) at 4 ℃ overnight.
(2) Washing the plate; add 200. mu.L of 5% milk and block for 2h at 37 ℃.
(3) Biotin-IL-17A (Acro, Cat # ILA-H82Q1, Lot # CBV296P1-97RF 1-QJ)) was mixed with the VHH-hFc diluted in a gradient to obtain a mixture (the concentration of Biotin-IL-17A in the mixture was 3.8 ng/ml). Here, VHH-hFc refers to the Fc fusion antibody (expressed in 293F cells) of the single domain antibody against IL-17A protein prepared in example 9, which was purified in example 10. Furthermore, a hIgG control was also set; hIgG refers to isotype control, immunoglobulin molecules that do not bind to any target, and are obtained by commercial purchase;
(4) washing the plate; add 50. mu.L of the above mixture, duplicate wells, incubate for 1h at 37 ℃.
(5) Washing the plate; add 50 u L Streptavidin-HRP, 37 degrees C were incubated for 30 min.
(6) Washing the plate (washing for several times); adding 50 μ L of TMB which is recovered to normal temperature in advance, and reacting for 15min at normal temperature in the dark.
(7) Add 50. mu.L of stop buffer (1N HCl) and read by microplate reader.
(8) Curves were drawn and EC50 was calculated. The results are shown in FIGS. 4-5, where the abscissa in FIGS. 4-5 represents the specific concentration of VHH-hFc diluted in a gradient. As can be seen, the single domain antibodies 1A10, 1D7, 1E6, 2E2, 2G3, 3D1, 3E1, 3H2, 4A12 all effectively blocked the binding of IL-17A to IL-17R.
In fig. 4, 4A shows the measurement results of 1a10, 1C9, 1D7, 1E2, 1D1, and 1D4, 4B shows the measurement results of single domain antibodies 3a6, 3A8, 3E1, 3E2, 3B11, and 3D1, and 4C shows the measurement results of 3F8, 3H2, 4F4, 1H3, 4A1, and 4A 12. In fig. 5, 5A shows the assay results of 3B10, 4a1, hIgG, 4G4, and 5B shows the assay results of single domain antibodies 1E6, 1H2, 2F5, 2G3, 2D8, 2E 2.
Example 15: antibody (eukaryotic sample) neutralization human IL-17A induced Hela cell to release IL-6 experiment
The following operations are carried out according to methods common to those skilled in the art:
(1) spreading 10000 Hela cells with 50 mul per well after recovery for 3 times or more into a 96-well plate;
(2) the gradient diluted antibodies were mixed with 4 x 55ng/ml of human IL-17A (Novo, Cat # C774, Lot #0331872-4147) in equal volumes and 50. mu.l of this mixture was added to the cell culture wells per well; the antibody herein was the Fc fusion antibody (expressed in 293F cells) of the single domain antibody against IL-17A protein prepared in example 9, purified as described in example 10; besides, hIgG and Tab controls are also set respectively; hIgG refers to isotype control, immunoglobulin molecules that do not bind to any target, and are obtained by commercial purchase; tab was obtained from example 12;
(3) after incubation for 24h at 37 ℃ in a cell culture box, detecting the concentration of IL-6 in the supernatant by using a human IL-6 ELISA kit;
EC50 for neutralizing IL-17A to induce IL-6 release from Hela cells was calculated based on the results of the measurement, and the results are shown in FIGS. 6 to 7, in which the abscissa indicates the molar concentration of the antibody at the aforementioned dilution gradient; the ordinate represents the IL-6 concentration in the supernatant (in pg/ml); as can be seen from FIGS. 6-7, eukaryotic antibodies were effective in neutralizing IL-17A and inducing the release of IL-6 from Hela cells.
In fig. 6, 6A shows the experimental results of Tab and hIgG, 6B shows the experimental results of 2G3, 3D1, 1D4 and 2E2, and 6C shows the experimental results of 1a10, 1D1, 3E1 and 3H 2. In fig. 7, 7A shows the experimental results of 4a12, 4F4, 7B shows the experimental results of 1C9, 1D7, 1E2, 1E6, 7C shows the experimental results of 3E2, 3F8, 2F5, 3a6, and 7D shows the experimental results of 4a1, 1H 3.
Example 16: antibody (humanized sample) neutralization human IL-17A induced Hela cell to release IL-6 experiment
The following operations are carried out according to methods common to the person skilled in the art:
(1) spreading the Hela cells which are recovered and passaged for 3 times or more into a 96-well plate according to 10000 cells and 50 mul per well;
(2) gradient diluted antibodies were mixed with equal volumes of 4 x 55ng/ml human IL-17A and this mixture was added to cell culture wells at 50 μ l per well; the antibodies herein are: humanized antibodies 2G3-V17, 2G3-V18, 2G3-V24, 2G3-V25, 2G3-V46, 2G3-V50, 2G3-V31, 2G3-V34, 1A10-V1, 1A10-V4, 1A10-V9, 1A10-V10, 1A10-V5, 1A10-V6, 1A10-V15 or 1A10-V14 were obtained by constructing an Fc fusion antibody eukaryotic expression vector of a humanized single domain antibody against IL-17A (constructing the nucleotide sequence of the humanized antibody into the vector RJK-V4-3), expressing it by example 9 and purifying it by example 10. Besides, hIgG and Tab controls are also set respectively; hIgG refers to isotype control, an immunoglobulin molecule that does not bind to any target, and is purchased commercially; tab was obtained from example 12;
(3) after incubation for 24h at 37 ℃ in a cell culture box, detecting the concentration of IL-6 in the supernatant by using a human IL-6 ELISA kit;
calculating EC50 for neutralizing IL-17A to induce the Hela cells to release IL-6 according to the detection results, which are shown in FIGS. 8-10, wherein the abscissa represents the specific concentration of the antibody at the aforementioned gradient dilution; the ordinate represents the IL-6 concentration in the supernatant (in pg/ml); from FIGS. 8-10, it can be seen that the humanized antibody was able to neutralize IL-17A and induce the release of IL-6 from Hela cells, and the technical effect thereof was superior to that of the antibody (eukaryotic sample).
In FIG. 8, 8A shows the results of Tab and hIgG experiments, 8B shows the results of 2G3-V17 and 2G3-V18 experiments, and 8C shows the results of 2G3-V24 and 2G3-V25 experiments. In FIG. 9, 9A shows the experimental results of 2G3-V46, 2G3-V50, 9B shows the experimental results of 2G3-V31, 2G3-V34, and 9C shows the experimental results of 1A10-V1, 1A 10-V4. In FIG. 10, 10A shows the results of experiments with 1A10-V9, 1A10-V10, 10B shows the results of experiments with 1A10-V5, 1A10-V6, and 10C shows the results of experiments with 1A10-V15, 1A 10-V14.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Sequence listing
<110> Nanjing Congjiekang Biotech Co., Ltd
<120> anti-IL-17A single domain antibody and use thereof
<160> 62
<170> SIPOSequenceListing 1.0
<210> 1
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Glu Tyr Ala Trp Arg Leu Asn Arg Met Gly Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Val Ser Ile Gly Thr
35 40 45
Arg Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ala Gln Asp Asn Ala Lys Asn Thr Met Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Leu Gly
85 90 95
Gly Asp Ser Arg Thr Pro Ile Tyr Asp Ile Ser Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 2
<211> 122
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Val Ser Gly Tyr Phe Tyr Ser Ser Ala Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile Tyr Ile
35 40 45
Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Gly Gly
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Gly Tyr Cys Tyr Ile Gly Glu Ser Ser Ser Tyr Glu Tyr Asn Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 3
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Thr Ile Asp Asn
35 40 45
His Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Thr Gly Gly Gly Gly
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Tyr Cys Ser Ala Arg Leu Gly Glu Gly Asp Phe Glu Phe Trp Gly Gln
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Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 4
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Thr
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Arg Ser Tyr Phe Met Ala Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Val Ile Asp Ser
35 40 45
Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Ser Arg Phe Thr Val
50 55 60
Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Ala Thr Glu
85 90 95
Gly Ser Leu Ser Trp Tyr Glu Pro His Thr Pro Leu Thr Pro His Glu
100 105 110
Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr Cys Met Gly Trp Phe
20 25 30
Arg Gln Thr Pro Glu Lys Glu Arg Glu Gly Val Ala Thr Ile Asp Asn
35 40 45
His Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Thr Gly Gly Gly Gly
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Tyr Cys Ser Ala Arg Leu Gly Glu Ala Asp Phe Glu Phe Trp Gly Gln
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Gly Thr Gln Val Thr Val Ser Ser
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<210> 6
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Trp Thr Phe Ser Thr Leu Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Thr
35 40 45
Gly Gly Gly Ser Pro Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Gly Gly
85 90 95
Gly Tyr Cys Pro Leu Ile Asp Tyr Glu Tyr Asn Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 7
<211> 122
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr Cys Met Gly Trp Phe
20 25 30
Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Ser
35 40 45
Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Glu Asp Asn Ala Lys Thr Thr Leu Phe Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ile Gly Gly Gly Gly
85 90 95
Tyr Cys Thr Pro Ala Thr Leu Val Ser Asp Leu Asp Phe Gly Asn Trp
100 105 110
Gly Arg Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 8
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Asn Ala Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Tyr Ile Tyr Thr
35 40 45
Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Gly Gly
85 90 95
Gly Tyr Cys Arg Ala Pro Pro Glu Glu Tyr Val Phe Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 9
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Arg Ser Tyr Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Met Asn Ser
35 40 45
Asp Gly Thr Thr Thr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Lys Asp Ser Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ile Leu Asp Gly Tyr
85 90 95
Cys Tyr Arg Pro Ile Arg Thr Gly Tyr His Asp Arg Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 10
<211> 357
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
gagtacgcct ggaggctgaa caggatgggc tgggtgaggc aggcccccgg caaggagagg 120
gagggcgtgg tgagcatcgg caccaggggc ggcagcacct actacgccga cagcgtgaag 180
ggcaggttca ccatcgccca ggacaacgcc aagaacacca tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg gcctgggcgg cgacagcagg 300
acccccatct acgacatcag ctactggggc cagggcaccc aggtgaccgt gagcagc 357
<210> 11
<211> 366
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgtgagc 60
ggctacttct acagcagcgc ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccagcatcta catcggcagc ggcagcacct actacgccga cagcgtgaag 180
ggcaggttca ccatcagcca ggacaacgcc aagaacaccg tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg gcggcggcgg ctactgctac 300
atcggcgaga gcagcagcta cgagtacaac tactggggcc agggcaccca ggtgaccgtg 360
agcagc 366
<210> 12
<211> 360
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct acagcagcta ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccaccatcga caaccacggc agcaccagct acgccgacag cgtgaagggc 180
aggttcacca tcagcaagga caacgccaag aacaccctgt acctgcagat gaacagcctg 240
aagcccgagg acgccgccat gtactactgc gccaccggcg gcggcggcta ctgcagcgcc 300
aggctgggcg agggcgactt cgagttctgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 13
<211> 378
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgacctg cgccgccagc 60
ggctacacct acaggagcta cttcatggcc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccgtgatcga cagcgacggc agcaccagct acgccgacag cgtgaagagc 180
aggttcaccg tgagcaagga caacgccaag aacaccctgt acctgcagat gaacagcctg 240
aagcccgagg acaccgccat gtactactgc gccgccaggg ccaccgaggg cagcctgagc 300
tggtacgagc cccacacccc cctgaccccc cacgagtaca actactgggg ccagggcacc 360
caggtgaccg tgagcagc 378
<210> 14
<211> 360
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct acagcagcta ctgcatgggc tggttcaggc agacccccga gaaggagagg 120
gagggcgtgg ccaccatcga caaccacggc agcaccagct acgccgacag cgtgaagggc 180
aggttcacca tcagcaagga caacgccaag aacaccctgt acctgcagat gaacagcctg 240
aagcccgagg acgccgccat gtactactgc gccaccggcg gcggcggcta ctgcagcgcc 300
aggctgggcg aggccgactt cgagttctgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 15
<211> 357
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctggacct tcagcaccct gtgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccgccatcta caccggcggc ggcagcccct actacgccga cagcgtgaag 180
ggcaggttca ccatcagcca ggacaacgcc aagaacaccc tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg gcggcggcgg ctactgcccc 300
ctgatcgact acgagtacaa ctactggggc cagggcaccc aggtgaccgt gagcagc 357
<210> 16
<211> 366
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct acagcagcta ctgcatgggc tggttcaggc aggtgcccgg caaggagagg 120
gagggcgtgg ccgccatcga cagcgacggc agcaccagct acgccgacag cgtgaagggc 180
aggttcacca tcagcgagga caacgccaag accaccctgt tcctgcagat gaacagcctg 240
aagcccgagg acaccgccat gtactactgc gccatcggcg gcggcggcta ctgcaccccc 300
gccaccctgg tgagcgacct ggacttcggc aactggggca ggggcaccca ggtgaccgtg 360
agcagc 366
<210> 17
<211> 357
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct acagcaacgc ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg cctacatcta caccggcggc ggcagcacct actacgccga cagcgtgaag 180
ggcaggttca ccatcagcca ggacaacgcc aagaacaccg tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg gcggcggcgg ctactgcagg 300
gccccccccg aggagtacgt gttctggggc cagggcaccc aggtgaccgt gagcagc 357
<210> 18
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct acaggagcta ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccgccatgaa cagcgacggc accaccacct acaccgacag cgtgaagggc 180
aggttcacca tcagcaagga cagcgccaag aacaccctgt acctgcagat gaacagcctg 240
aagcccgagg acaccgccat gtactactgc gccatcctgg acggctactg ctacaggccc 300
atcaggaccg gctaccacga caggggccag ggcacccagg tgaccgtgag cagc 354
<210> 19
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Glu Tyr Ala Trp Arg Leu Asn Arg
1 5
<210> 20
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Gly Trp Thr Phe Ser Thr Leu Cys
1 5
<210> 21
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Gly Tyr Phe Tyr Ser Ser Ala Cys
1 5
<210> 22
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Gly Tyr Thr Tyr Arg Ser Tyr Cys
1 5
<210> 23
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Gly Tyr Thr Tyr Arg Ser Tyr Phe
1 5
<210> 24
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Gly Tyr Thr Tyr Ser Asn Ala Cys
1 5
<210> 25
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Gly Tyr Thr Tyr Ser Ser Tyr Cys
1 5
<210> 26
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Ile Asp Asn His Gly Ser Thr
1 5
<210> 27
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Ile Asp Ser Asp Gly Ser Thr
1 5
<210> 28
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Ile Gly Thr Arg Gly Gly Ser Thr
1 5
<210> 29
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Ile Tyr Ile Gly Ser Gly Ser Thr
1 5
<210> 30
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Ile Tyr Thr Gly Gly Gly Ser Pro
1 5
<210> 31
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Ile Tyr Thr Gly Gly Gly Ser Thr
1 5
<210> 32
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Met Asn Ser Asp Gly Thr Thr
1 5
<210> 33
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Ala Ala Gly Gly Gly Gly Tyr Cys Pro Leu Ile Asp Tyr Glu Tyr Asn
1 5 10 15
Tyr
<210> 34
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Ala Ala Gly Gly Gly Gly Tyr Cys Arg Ala Pro Pro Glu Glu Tyr Val
1 5 10 15
Phe
<210> 35
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Ala Ala Gly Gly Gly Gly Tyr Cys Tyr Ile Gly Glu Ser Ser Ser Tyr
1 5 10 15
Glu Tyr Asn Tyr
20
<210> 36
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Ala Ala Gly Leu Gly Gly Asp Ser Arg Thr Pro Ile Tyr Asp Ile Ser
1 5 10 15
Tyr
<210> 37
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Ala Ala Arg Ala Thr Glu Gly Ser Leu Ser Trp Tyr Glu Pro His Thr
1 5 10 15
Pro Leu Thr Pro His Glu Tyr Asn Tyr
20 25
<210> 38
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Ala Ile Gly Gly Gly Gly Tyr Cys Thr Pro Ala Thr Leu Val Ser Asp
1 5 10 15
Leu Asp Phe Gly Asn
20
<210> 39
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Ala Ile Leu Asp Gly Tyr Cys Tyr Arg Pro Ile Arg Thr Gly Tyr His
1 5 10 15
Asp
<210> 40
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Ala Thr Gly Gly Gly Gly Tyr Cys Ser Ala Arg Leu Gly Glu Ala Asp
1 5 10 15
Phe Glu Phe
<210> 41
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Ala Thr Gly Gly Gly Gly Tyr Cys Ser Ala Arg Leu Gly Glu Gly Asp
1 5 10 15
Phe Glu Phe
<210> 42
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser
20
<210> 43
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Val Ser
20
<210> 44
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Thr
1 5 10 15
Cys Ala Ala Ser
20
<210> 45
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Val
<210> 46
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ala
<210> 47
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ser
<210> 48
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Thr
<210> 49
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Tyr
<210> 50
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
Met Gly Trp Phe Arg Gln Thr Pro Glu Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Thr
<210> 51
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ala
<210> 52
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
Met Gly Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Val
1 5 10 15
Ser
<210> 53
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 53
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Glu Asp Asn
1 5 10 15
Ala Lys Thr Thr Leu Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 54
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 54
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Ala Ala Met Tyr Tyr Cys
35
<210> 55
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 55
Ser Tyr Ala Asp Ser Val Lys Ser Arg Phe Thr Val Ser Lys Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 56
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 56
Thr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 57
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 57
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ala Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 58
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 58
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 59
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 59
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 60
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 60
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 61
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 61
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 62
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 62
Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser
1 5 10

Claims (15)

1. A single domain antibody against IL-17A, characterized by: the single domain antibody is composed of a heavy chain, wherein the heavy chain comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR 3;
the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are one of the following (2) - (4) or (6) - (9):
(2) CDR1 shown in SEQ ID NO. 21, CDR2 shown in SEQ ID NO. 29, CDR3 shown in SEQ ID NO. 35;
(3) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 26, CDR3 shown in SEQ ID NO. 41;
(4) CDR1 shown in SEQ ID NO. 23, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 37;
(6) CDR1 shown in SEQ ID NO. 20, CDR2 shown in SEQ ID NO. 30, CDR3 shown in SEQ ID NO. 33;
(7) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 27, CDR3 shown in SEQ ID NO. 38;
(8) CDR1 shown in SEQ ID NO. 24, CDR2 shown in SEQ ID NO. 31, CDR3 shown in SEQ ID NO. 34;
(9) CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 32, and CDR3 shown in SEQ ID NO. 39.
2. An anti-IL-17A single domain antibody according to claim 1, characterized in that: the single domain antibody further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR 4; the amino acid sequences of the FR regions of the framework regions are:
a variant of FR1 or FR1 as set forth in any one of SEQ ID NOs 42-44, said variant of FR1 comprising a substitution of up to 5 amino acids in said FR 1;
a variant of FR2 or FR2 represented by any one of SEQ ID NOS.45-52, said variant of FR2 comprising a substitution of up to 5 amino acids in said FR 2;
a variant of FR3 or FR3 represented by any one of SEQ ID NOS: 53-59, said variant of FR3 comprising a substitution of up to 5 amino acids in said FR 3;
a variant of FR4 or FR4 as set forth in any one of SEQ ID NOs 60-62, said variant of FR4 comprising up to 5 amino acid substitutions in said FR 4.
3. A single domain antibody against IL-17A, characterized by: the amino acid sequences of the single-domain antibodies are respectively shown in any one of SEQ ID NO.2-4 or SEQ ID NO. 6-9.
4. A bispecific antibody characterized in that: the bispecific antibody comprises a first antigen-binding moiety which is a single domain anti-IL-17A antibody of any one of claims 1 to 3 and a second antigen-binding moiety.
5. Use of a single domain antibody against IL-17A according to any one of claims 1 to 3 for the preparation of a bispecific antibody.
6. An Fc fusion antibody or a humanized antibody of a single domain antibody against IL-17A according to any one of claims 1 to 3.
7. A recombinant protein comprising an anti-IL-17A single domain antibody according to any one of claims 1 to 3.
8. A nucleotide molecule encoding the anti-IL-17A single domain antibody of any one of claims 1 to 3, characterized in that: the nucleotide sequences are respectively shown as SEQ ID NO: 11-13 or SEQ ID NO: 15-18, respectively.
9. An expression vector, characterized in that: comprising a nucleotide molecule encoding the anti-IL-17A single domain antibody of any one of claims 1 to 3 or the Fc fusion antibody or humanized antibody of claim 6 or the nucleotide molecule of claim 8.
10. A host cell, characterized in that: which can express a single domain anti-IL-17A antibody according to any one of claims 1 to 3 or an Fc fusion antibody or humanized antibody according to claim 6, or which comprises an expression vector according to claim 9.
11. The host cell of claim 10, wherein: the host cell is a eukaryotic cell or a prokaryotic cell.
12. A pharmaceutical composition characterized by: the pharmaceutical composition comprises a single domain antibody against IL-17A selected from any one of claims 1 to 3, and a pharmaceutically acceptable carrier.
13. A medicament for treating psoriasis characterized by: which comprises as an active ingredient an anti-IL-17A single domain antibody according to any one of claims 1 to 3.
14. A kit for detecting IL-17A levels, characterized in that: the kit contains a single domain antibody against IL-17A according to any one of claims 1 to 3.
15. Use of an anti-IL-17A single domain antibody according to any one of claims 1 to 3 or a pharmaceutical composition according to claim 12 for the preparation of a medicament for the treatment of a disease characterized in that: the disease is psoriasis.
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