CN114920838B - 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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CN114920838B
CN114920838B CN202210489565.5A CN202210489565A CN114920838B CN 114920838 B CN114920838 B CN 114920838B CN 202210489565 A CN202210489565 A CN 202210489565A CN 114920838 B CN114920838 B CN 114920838B
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CN114920838A (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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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 a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3. 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 uses biological gene engineering technology to screen out the single domain antibody specific to IL-17A, the antibody has better affinity, and can block the specific cell to release the cell factor, and has good binding activity through prokaryotic expression and eukaryotic expression, and has certain drug property.

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"), a pharmaceutical composition containing the single domain antibody as an active ingredient, and a pharmaceutical therapeutic use thereof.
Background
Psoriasis is a relatively common chronic inflammatory skin disease commonly known as psoriasis. The disease is easy to recur or aggravate in winter, the global prevalence rate is about 2% -3% in spring and autumn, 1/3 of psoriasis patients have psoriatic arthritis (PsA), joint swelling and pain, stiffness and dyskinesia are often accompanied, the spine can be partially affected, serious people can cause disability, and serious influence is generated on physical and mental health of the patients. Psoriasis is classified according to its clinical characteristics, mainly in the following categories: psoriasis, of the type of the ordinary, articular, pustular and erythroderma, is more than 90% of the other types of the disease, which are caused by the use of topical irritant drugs, excessive use of glucocorticoids and sudden withdrawal of drugs during immunosuppression in patients during treatment.
The pathogenesis of psoriasis is not known. The current treatment schemes for PsA mainly comprise nonsteroidal anti-inflammatory drugs, glucocorticoids, antirheumatic drugs, azathioprine, tretinoin and the like, and methods of physical treatment, traditional Chinese medicine treatment and the like for relieving symptoms and controlling illness states. Traditional therapies have been increasingly prone to biological agents in clinic due to poor efficacy and adverse effects such as increased blood pressure, increased blood glucose, osteoporosis, peptic ulcers, skin atrophy, etc. caused by prolonged hormone use, tumor necrosis factor inhibitors are often selected as the primary biological therapy for PsA patients, and anti-IL-17 type biological agents.
Psoriasis has keratinocyte hyperproliferation, inflammatory cell infiltration, and neovascularization as three elements of its histopathological changes. Patients have a variety of immune cells, immune molecules, intracellular signaling systems and other dysfunctions, and have a certain incubation period for onset, and administration of antimalarial, antipsychotic lithium preparations, antihypertensive beta blockers and angiotensin converting enzyme inhibitors during this period induce the onset of the disease. Severely affects the quality of life and even physical and mental health of the patient.
The pathogenesis of psoriasis is not known, and it is currently considered an autoimmune disorder in a polygenic genetic background. The pathogenesis of the tumor is related to T lymphocytes, mainly CD4+Th1 lymphocyte mediated immunity, and the pathogenic process comprises the steps of activating the initial T lymphocytes into memory-effect T lymphocytes, allowing the memory-effect T lymphocytes to enter circulation and migrate to the skin, gathering at a lesion site, secreting multiple cytokines and exerting multiple biological functions to cause the tumor.
IL-17A is the earliest discovered member of the IL-17 cytokine family, and is secreted primarily by Th17 cells. In 1986, mosmann, coffman and related panelists found two subpopulations of helper T cells-Th 1 and Th2 (mosmannet, 1986). In addition to Th17 cells, other T cells (including cd8+ T cells, γδ 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 processes of many autoimmune diseases (such as rheumatoid arthritis and encephalomyelitis), and plays an important role in protecting against bacterial and fungal infection.
Psoriasis is an autoimmune skin disease with hyperproliferative epidermal cells, and studies have shown that IL-17A deficient mice have reduced epidermal hyperplasia (rizzo et al, 2011); clinical data also show an important pathological role of Th17 cells and IL-17A in psoriasis (huebert et al, 2010; wilson et al, 2007). Based on the above research results on IL-17A in autoimmune diseases, treatment of autoimmune diseases by inhibiting differentiation of Th17 cells and blocking IL-17A signaling pathway has become a current hotspot.
Psoriasis causes are refractory and are listed as an important research topic in the field of dermatology in the world, and are one of important diseases prevention and treatment in dermatology in the world. With the marketing of a range of new drugs, psoriasis patients have more and better medication options. TNFa antagonists and Interleukins (IL) class of drugs are currently being developed as hot spots in the autoimmune field, with the IL-12 family and IL-17 being of greatest interest.
Currently, there is still a lack of a strong affinity, pharmaceutically valuable single domain antibody product against IL-17A in the prior art.
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, the single domain antibody is composed of a heavy chain, the heavy chain comprises a heavy chain CDR1 shown in any one of SEQ ID NO: 19-25, a heavy chain CDR2 shown in any one of SEQ ID NO: 26-32 and a heavy chain CDR3 shown in any one of SEQ ID NO: 33-41. Anti IL-17A single domain antibodies, i.e., directed against IL-17A single domain antibodies.
Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1) - (9):
(1) CDR1 shown in SEQ ID NO. 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) - (9) correspond in sequence to SEQ ID NO. 1-9.
All of the above sequences may be replaced by sequences having "at least 80% homology" to the sequence or sequences with 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 the amino acid residues in any one or more of the CDRs of heavy chain CDR1, CDR2 and CDR3 may be substituted with their conserved amino acids, respectively. In particular, in the heavy chain CDR1, 1 to 5 amino acid residues may be replaced by their conserved amino acids; in the heavy chain CDR2, 1 to 5 amino acid residues may be replaced by their conserved amino acids; 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 identical positions 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 with identical sequences have 100% homology.
In some embodiments, sequences that replace only one or a few amino acids, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, as compared to the preceding sequences, may also achieve the object. 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 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. In fact, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution would preferably be conservative amino acid substitutions, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combinations in a single domain antibody. The conserved amino acid, which may be generally described as an amino acid substitution of an amino acid residue 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, e.g., conservative amino acid substitutions are those in which one or a few amino acids in the following groups (a) - (d) are substituted for another or a few amino acids in the same group: (a) a polar negatively charged residue and an uncharged amide thereof: asp, asn, glu, gln; (b) a polar positively charged residue: his, arg, lys; (c) aromatic residues: phe, trp, tyr; (d) aliphatic nonpolar or low polar residues: ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are as follows: asp is substituted with Glu; asn is substituted with Gln or His; glu is substituted with Asp; gln is substituted with Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys is substituted by Arg, gln; phe is replaced by Met, leu, tyr; trp is substituted with Tyr; tyr is substituted with Phe, trp; substitution of Ala with Gly or Ser; ser is substituted by Thr; thr is replaced by Ser; substitution of Gly with Ala or Pro; met is substituted with Leu, tyr or Ile; leu is substituted with Ile or Val; lie is substituted with Leu or Val; val is substituted with Ile or Leu; cys is replaced by Ser. In addition, those skilled in the art will recognize that the creativity of single domain antibodies is represented in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable, and that the sequences of FR1-4 may take the form of conservative sequence variants of the sequences disclosed herein.
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 analogues of the anti-IL-17A single domain antibody. As used herein, the terms "fragment," "derivative," and "analog" are synonymous and refer to a polypeptide that retains substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as 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 within the purview of one skilled in the art and would be well known in light of the teachings herein.
In a preferred embodiment, the antibody sequence further comprises a framework region FR; the framework regions FR include the amino acid sequences of FR1, FR2, FR3 and FR 4; the amino acid sequences of the framework regions FR are respectively:
42-44, said FR1 variant comprising up to 5 amino acid substitutions in said FR 1;
45-52, said FR2 variant comprising up to 5 amino acid substitutions in said FR 2;
53-59, or a variant of FR3 as set forth in any one of SEQ ID nos. 53-59, said variant of FR3 comprising up to 5 amino acid substitutions in said FR 3;
60-62, or a variant of 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.
In a second aspect of the invention, there is provided an 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 sequences 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 hybridizes to a polypeptide selected from the group consisting of SEQ ID NOs: 1-9 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and is capable of specifically binding to IL-17A protein.
In a third aspect of the invention, there is provided a bispecific antibody comprising a first antigen binding moiety and a second antigen binding moiety, the first antigen binding moiety being a single domain antibody, the amino acid sequences of which are shown in SEQ ID NO.1-9 or have at least 80% homology with SEQ ID NO.1-9, respectively. The second antigen binding portion is another antibody, for example, an antibody against TNFα, IL-6R, IL-6, IL-23R (which may be a monoclonal antibody, a polyclonal antibody, a single domain antibody, or any other form of antibody); 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.
In a fourth aspect, the invention provides the use of any one of the foregoing anti-IL-17A single domain antibodies in the preparation of bispecific antibodies.
A fifth aspect of the invention provides an Fc fusion antibody or a humanized antibody of any of the foregoing anti-IL-17A single domain antibodies.
In a sixth aspect, the present invention provides a nucleotide molecule encoding the aforementioned anti-IL-17A single domain antibody or the aforementioned Fc fusion antibody or the aforementioned humanized antibody, which has the nucleotide sequence shown in SEQ ID NO:10-18, or with SEQ ID NO:10-18 has at least 95% sequence homology.
In one embodiment, the nucleic acid molecule encoding the anti-IL-17A single domain antibody hybridizes to a nucleic acid molecule selected from the group consisting of SEQ ID NO:10-18, and encoding an anti-IL-17A single domain antibody capable of specifically binding to an IL-17A protein.
In a seventh aspect, the invention provides an expression vector comprising a nucleotide molecule encoding a single domain antibody or an Fc fusion antibody or a humanized antibody against IL-17A, having the nucleotide sequence set forth in SEQ ID NO: 10-18.
In a preferred embodiment, the expression vector used is RJK-V4-3 (the nucleotide molecules encoding the anti-IL-17A single domain antibody or its Fc fusion antibody or humanized antibody are integrated into RJK-V4-3 by genetic engineering means), and other universal expression vectors may be selected as desired.
An eighth aspect of the invention provides a host cell capable of expressing the aforementioned anti-IL-17A single domain antibody, fc fusion antibody or humanized antibody, or comprising the aforementioned expression vector. 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: coli, bacillus subtilis, lactobacillus, streptomyces, proteus mirabilis, or combinations 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 myxoplasma gondii, 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 ExpiCHO-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 anti IL-17A single domain antibody. The recombinant protein can be a single-domain antibody shown in SEQ ID NO.1-9, a single-domain antibody with at least 80% homology with SEQ ID NO.1-9, a multi-epitope antibody, a multi-specific antibody and a multivalent antibody; for example, the multi-epitope antibody may consist of more than one of the sequences set forth in SEQ ID NOS.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, the bispecific antibodies described above, as well as trispecific antibodies; furthermore, the recombinant proteins may be fragments, derivatives and analogues of the aforementioned antibodies.
In a tenth aspect, 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 nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally determined by the isoelectric point of the antibody (the pH of the aqueous carrier medium is required to deviate from and from 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 and transdermal (directly applied or plastered on affected part).
The pharmaceutical compositions of the invention can be used directly to bind IL-17A protein molecules and thus can be used to treat psoriasis. In addition, other psoriasis treatment agents may be used in combination.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the foregoing single domain antibodies, together with 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 formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions.
An eleventh aspect of the present invention is to provide an agent for treating psoriasis, which comprises the aforementioned anti-IL-17A single domain antibody as an active ingredient.
In a twelfth aspect, the invention provides a kit for detecting IL-17A levels, which contains the aforementioned anti-IL-17A single domain antibody. In a preferred embodiment of the invention, the kit further comprises a container, instructions for use, buffers, etc.
In a preferred embodiment, the kit comprises antibodies recognizing the IL-17A protein, a lysis medium for lysing the sample, universal reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit may be an in vitro diagnostic device.
In a preferred embodiment, the kit further comprises a second antibody and an enzyme or fluorescent or radiolabel for detection, and a buffer.
In a preferred embodiment, the second antibody of the kit may be an antibody (as an anti-antibody) to the aforementioned single domain antibody of IL-17A, 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 a single domain antibody, 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 of IL-17A obtained in step (b).
In a fourteenth aspect, the present invention provides the use of a single domain antibody or pharmaceutical composition against the aforementioned anti-IL-17A for the preparation of a medicament for inhibiting the expression of the IL-17A gene or an anti-psoriasis medicament.
In a fifteenth aspect, the present invention provides the use of a single domain antibody against IL-17A as described above or a pharmaceutical composition as described above 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 against the aforementioned anti-IL-17A for the preparation 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 IL-17A and IL-17R binding site, has higher blocking activity than the prior medicine, and has great application prospect in preparing the medicine for treating autoimmune diseases.
(3) The single domain antibody obtained by the invention has flexible expression system selection, can be expressed in a prokaryotic system or a eukaryotic system of yeast cells or mammalian cells, has low expression cost in the prokaryotic expression system, and can reduce the post production cost.
(4) The single-domain antibody obtained by the invention has simple reconstruction of the multi-combination form of the antibody, can obtain multivalent and multi-specific antibodies through simple serial connection in a genetic engineering mode, has low immune heterogeneity and can not generate stronger immune response under the condition of not undergoing humanized reconstruction.
(5) The invention provides single domain antibodies with a broader range of affinities, ranging from nM to pM, which provide multiple options for later use of the antibodies without affinity maturation.
Drawings
In order to more clearly illustrate the technical solution of the present application, the drawings that are needed in the embodiments will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 is a library enrichment profile of the IL-17A-targeting antibody screen of example 3;
FIG. 2 is a graph of the antibody antigen binding response curve assay (a portion of clones) in example 13;
FIG. 3 is a graph showing the quantitative response curves of the binding of the antibody antigen in example 13 (another clone, tab1, hIgG);
FIG. 4 is a graph of an assay (a portion of clones) of example 14 in which antibodies block IL-17A binding to IL-17R;
FIG. 5 is a graph of the antibody of example 14 blocking IL-17A binding to IL-17R (other clone, hIgG);
FIG. 6 is a graph of the results of an experiment (a portion of clones, tab, hIgG) for neutralizing human IL-17A (eukaryotic sample) induced by Hela cells to release IL-6 in the antibody of example 15;
FIG. 7 is a graph (another clone) showing the experimental results of IL-6 release by Hela cells induced by the neutralization of human IL-17A (eukaryotic sample) by the antibodies of example 15;
FIG. 8 is a graph (Tab, hIgG, 2G 3) of the experimental results of IL-6 release by HeLa cells induced by the neutralizing human IL-17A (humanized sample) of the antibody of example 16;
FIG. 9 is a graph (2G 3, 1A 10) showing the results of an experiment in which the neutralizing human IL-17A (humanized sample) of the antibody of example 16 induced Hela cells to release IL-6;
FIG. 10 is a graph (1A 10) showing the results of an experiment in which neutralizing human IL-17A (humanized sample) induced Hela cells to release IL-6 by the antibody of example 16.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
As used herein, a "single domain antibody" (sdAb, also called nanobody or VHH by the developer Ablynx) is well known to those skilled in the art. A single domain antibody is an antibody whose complementarity determining region is part of a single domain polypeptide. Thus, a single domain antibody comprises a single complementarity determining region (single CDR1, single CDR2, and single CDR 3). Examples of single domain antibodies are heavy chain-only antibodies (which naturally do not comprise light chains), single domain antibodies derived from conventional antibodies, and engineered antibodies.
The single domain antibodies may be derived from any species including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules may be derived from antibodies provided by camelidae species (e.g. camels, dromedaries, llamas and dromedaries). Like whole antibodies, single domain antibodies are capable of selectively binding to a particular antigen. A single domain antibody may contain only the variable domains of an immunoglobulin chain, which domains have CDR1, CDR2 and CDR3, as well as 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 identical positions 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 with identical sequences have 100% homology.
As used herein, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc segment of an antibody of interest to a functional protein molecule having biological activity using genetic engineering techniques.
The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (e.g., an animal antibody) to the constant region of a human antibody, or by grafting the complementarity determining regions (CDR 1 to CDR 3 sequences) of a target antibody into the variable region of a human antibody, or by subjecting a target antibody to amino acid mutation according to the characteristics of the framework regions (FR 1 to FR 4) of a human antibody. Humanized antibodies can be synthesized or site-directed mutagenesis.
In the present invention, a single domain antibody against IL-17A can be obtained from a sequence having high sequence homology with CDR1-3 disclosed in the present invention. In some embodiments, sequences having "at least 80% homology" or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" to the sequences in SEQ ID No.1-9 may achieve the object of the invention.
In some embodiments, the polypeptide that hybridizes to SEQ ID NO:1-9, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, may also achieve the object of the invention. In fact, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combinations in a single domain antibody, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution will preferably be conservative amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which substitution 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, e.g., conservative amino acid substitutions are those in which one or a few amino acids in the following groups (a) - (d) are substituted for another or a few amino acids in the same group: (a) a polar negatively charged residue and an uncharged amide thereof: asp, asn, glu, gln; (b) a polar positively charged residue: his, arg, lys; (c) aromatic residues: phe, trp, tyr; (d) aliphatic nonpolar or low polar residues: ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are as follows: asp is substituted with Glu; asn is substituted with Gln or His; glu is substituted with Asp; gln is substituted with Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys is substituted by Arg, gln; phe is replaced by Met, leu, tyr; trp is substituted with Tyr; tyr is substituted with Phe, trp; substitution of Ala with Gly or Ser; ser is substituted by Thr; thr is replaced by Ser; substitution of Gly with Ala or Pro; met is substituted with Leu, tyr or Ile; leu is substituted with Ile or Val; lie is substituted with Leu or Val; val is substituted with Ile or Leu; cys is replaced by Ser. In addition, those skilled in the art will recognize that the creativity of single domain antibodies is represented in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable, and that the sequences of FR1-4 may take the form of conservative sequence variants of the sequences disclosed herein.
Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.
The preparation method comprises the steps of preparing target protein and a truncated form of the target protein through a genetic engineering technology, immunizing an inner Mongolian alashan alpaca with the obtained antigen protein, obtaining peripheral blood lymphocytes or spleen cells of the alpaca after multiple immunization, recombining a camel source antibody variable region coding sequence into a phage display carrier through a genetic engineering mode, screening out a specific antibody aiming at the antigen protein through the phage display technology, and further detecting the binding capacity of the specific antibody and the antigen and application of the specific antibody in treatment of autoimmune diseases.
The above technical solutions will now be described in detail by way of specific embodiments:
example 1: preparation of human IL-17A recombinant extracellular domain protein:
the human recombinant extracellular domain protein used in the patent is obtained by self-expression and purification of a company, and the design scheme of the human IL-17A recombinant extracellular domain protein expression vector is as follows:
(1) The coding sequence for IL-17A, which was identified as NM-002190.2, was retrieved from NCBI and encoded to give an amino acid sequence with accession number NP-002181.1,Uniprot ID as Q16552.
(2) The amino acid sequences corresponding to NP 002181.1 were analyzed for the transmembrane region and extracellular end of the protein via TMHMM and SMART websites, respectively.
(3) The analysis result shows that IL-17A is a secretion type protein, and the 1-23 positions are signal peptides of the protein.
(4) The nucleotide sequence encoding amino acids 24-155 of the IL-17A protein was cloned into the vector pcDNA3.4 by means of gene synthesis.
(5) And (3) carrying out Sanger sequencing on the constructed vector, comparing the original sequences, carrying out batch extraction on the recombinant plasmid after confirming no errors, removing endotoxin, carrying out expression and purification of target protein by transfecting suspension 293F, and ensuring that the purity of the purified protein is up to 90%, thereby meeting the requirements of animal immunization.
Example 2: construction of a single domain antibody library against IL-17A protein:
1mg of the recombinant human IL-17A protein obtained by purification in example 1 was mixed with an equal volume of Freund's complete adjuvant, and an inner Mongolian Alexander bactrian camel was immunized once a week for a total of 7 continuous immunizations, and the remaining six immunizations were animal immunizations with 1mg of IL-17A protein mixed with Freund's incomplete adjuvant in equal volumes except for the first immunization, in order to intensively stimulate the camel to produce antibodies against IL-17A protein.
After the 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 a nested PCR reaction using the cDNA as a template.
Then, the pMECS vector and the VHH fragment were digested separately using restriction enzymes, and the digested fragments and vector were ligated. Electrotransformation of the ligated fragments into competent cells TG1, construction of a phage display library of IL-17A protein and measurement of the library capacity, which was approximately 1X 10 9 At the same time, the correct insertion rate of the library at the fragment of interest was detected by colony PCR identification.
The results showed that after PCR amplification of 40 randomly selected colonies from the library, 38 clones amplified bands of predicted size and 2 clones amplified bands incorrectly, so the correct insertion rate was 38.times.40.times.100%. Apprxeq.95%.
Example 3: screening of single domain antibodies against IL-17A protein:
200. Mu.L of the recombinant TG1 cells of example 2 were cultured in 2 XTY medium, during which 40. Mu.L of helper phage VCSM13 was added to infect TG1 cells, and cultured overnight to amplify phage, the phage was precipitated the next day with PEG/NaCl, and the amplified phage was collected by centrifugation.
NaHCO diluted at 100 mM pH8.3 3 500 mug of IL-17A protein coupled to an ELISA plate, and left overnight at 4 ℃ while setting up a negative control well (medium control); the next day 200 μl of 3% skim milk was added and blocked at room temperature for 2h; after blocking was completed, 100. Mu.l of amplified phage library (approximately 2X 10 11 Individual phage particles), 1h at room temperature; after 1 hour of action, washing with PBS+0.05% Tween-20 for 15 times,to wash away unbound phage.
The phage specifically binding to IL-17A protein was dissociated with trypsin at a final concentration of 25mg/mL, and E.coli TG1 cells in the logarithmic growth phase were infected, cultured at 37℃for 1 hour, phage were generated and collected for the next round of screening, and the same screening process was repeated for 1 round, and enrichment was gradually obtained.
When the enrichment multiple reaches more than 10 times, the enrichment effect is shown in figure 1.
In fig. 1, P/n=number of monoclonal bacteria grown after infection of TG1 bacteria by phage with positive Kong Xi removal from biopanning/number of monoclonal bacteria grown after infection of TG1 bacteria by phage with positive Kong Xi removal, which parameter increases gradually after enrichment occurs; I/E = total phage added to positive wells per round of biopanning/total phage removed from positive Kong Xi per round of biopanning, which parameter gradually approaches 1 after enrichment has occurred.
Example 4: screening of specific positive clones for IL-17A by phage enzyme-linked immunosorbent assay (ELISA):
screening was performed according to the screening method described in example 3 above for 3 rounds of screening against a single domain antibody against IL-17A protein, the phage enrichment factor against IL-17A protein was 10 or more, 384 single colonies were selected from positive clones obtained by screening after the end of screening, inoculated into 96-well plates containing 2 XSTY medium of 100. Mu.g/mL ampicillin, respectively, and a blank was set, and after culturing at 37℃to the logarithmic phase, IPTG was added at a final concentration of 1 mM, and culturing was carried out at 28℃overnight.
Obtaining a crude extract antibody by using a permeation swelling method; the IL-17A recombinant protein was released to 100 mM NaHCO pH8.3, respectively 3 100. Mu.g of protein was coated in an ELISA plate (ELISA plate) at 4℃overnight. Transferring 100 mu L of the obtained crude antibody extract to an ELISA plate added with antigen, and incubating for 1h at room temperature; washing unbound Antibody with PBST, adding 100 μl of Mouse Anti-HA tag Anti-body (HRP) (Mouse Anti-HA horseradish peroxidase labeled Antibody, thermo Fisher) diluted 1:2000, and incubating for 1h at room temperature; washing unbound antibody with PBST, adding horse radish peroxidase chromogenic solution, reacting at 37deg.C for 15min, and adding stop reagent The absorption value of the liquid is read at a wavelength of 450nm on an enzyme-labeled instrument.
When the OD value of the sample hole is more than 5 times that of the control hole, judging that the sample hole is a positive cloning hole; the positive clone well was transferred to LB medium containing 100. Mu.g/mL ampicillin to extract plasmids and sequenced.
Gene sequences of the respective clones were analyzed according to sequence alignment software VectorNTI, strains with the same CDR1, CDR2 and CDR3 sequences were regarded as the same clone strain, and strains with different sequences were regarded as different clone strains, and finally single domain antibodies specific to IL-17A protein (SEQ ID NOS.1-9 and other single domain antibodies not showing sequences, including 1C9, 1D1, etc. in the drawings) were obtained.
The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure, which forms the whole VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally the single-domain antibody protein is obtained.
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 shown as SEQ ID NO.1-9 respectively, and the DNA sequences for encoding the 9 single-domain antibodies are shown as SEQ ID NO.10-18 respectively.
TABLE 1 9 CDR1 sequences of single domain antibodies
TABLE 2 9 CDR2 sequences of single domain antibodies
TABLE 3 9 CDR3 sequences of single domain antibodies
TABLE 4 9 FR1 sequences of single domain antibodies
TABLE 5 9 FR2 sequences of single domain antibodies
TABLE 6 9 FR3 sequences of single domain antibodies
TABLE 7 9 FR4 sequences of single domain antibodies
The amino acid sequences SEQ ID NO.1-9 of the single domain antibodies correspond to the single domain antibodies 1A10,1D7,1E6,2E2,2G3,3D1,3E1,3H2,4A12 one by one in sequence. The nucleotide sequences SEQ ID NO.10-18 of the single domain antibodies also correspond one-to-one with the single domain antibodies 1A10,1D7,1E6,2E2,2G3,3D1,3E1,3H2,4A12 in sequence.
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 by sequencing (pMECS-VHH) in example 4 were electrotransformed into E.coli HB2151 and plated onto LB+amp+glucose-containing culture plates, which were incubated overnight at 37 ℃; individual colonies were selected and inoculated in 5mL of LB medium containing ampicillin, and shake-cultured overnight at 37 ℃.
Inoculating 1mL of overnight culture strain into 330mLTB culture solution, shake culturing at 37deg.C until OD600nm reaches 0.6-0.9, adding 1M IPTG, shake culturing at 28deg.C overnight; centrifuging, collecting escherichia coli, and obtaining an antibody crude extract by using a permeation swelling method;
Purifying the antibody by nickel column affinity chromatography, wherein the purified part of single domain antibody comprises VHH 1-9.
The single domain antibodies of VHH1-9 correspond in sequence to the amino acid sequences SEQ ID NO.1-9, i.e.VHH 1-9 correspond respectively: 1A10,1D7,1E6,2E2,2G3,3D1,3E1,3H2,4A12.
Example 6: humanization of Single-Domain antibodies against IL-17A
The humanization method is completed by adopting a method of carrying out high-throughput screening on the mutation library of the antibody framework region constructed based on the analysis result of big data. The method comprises the following detailed steps:
(1) Sequence analysis of human/camel antibody data: performing amino acid preference analysis on 13873 Nb (Human) sequences downloaded in batches from NCBI websites, and simultaneously performing amino acid preference analysis on 2000 nanometer 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 sources: the source/camel source antibody sequences are uniformly numbered according to the IMGT numbering rule and are in one-to-one correspondence, the analysis results of the amino acid proportion in the two species are combined, weighting analysis is carried out according to the weight of 10% of the humanized 90% camel source, the proportion of the amino acid of each site after weighting is counted, and the sequence is ordered from high to low; according to the final weighting result, only preserving the amino acid types with the proportion of more than 10% at a single site of the framework region, and calculating the final weight of the amino acid with the proportion of more than 10% according to the standard that the proportion is integrated to be 1 after preservation, so as to be used as the design basis of a subsequent amino acid custom library;
(3) Scheme design of amino acid custom library: the method comprises the steps of providing an independent site to be mutated, providing n as the number of amino acids of which the number is more than 10%, providing V as the ratio of the highest value to the lowest value of the proportion of the amino acids of which the number is more than 10%, 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 locus is considered to be a 'high concentration locus', otherwise, the locus is considered to be a 'medium-low concentration locus'. According to the method, the customized amino acid library is divided into two libraries with high concentration, medium concentration and low concentration, and the construction of the amino acid customized library is respectively carried out, wherein the final weight in the step (2) is the reference basis of the types and the proportions of the site amino acids in the library.
(4) High throughput screening of amino acid custom libraries:
and respectively constructing humanized antibody libraries for antibody strains, 1A10,1D7,1E6,2E2,2G3,3D1,3E1,3H2 and 4A12, respectively panning corresponding antigens aiming at the constructed libraries, and finally obtaining the antibody sequences with higher affinity and higher humanization degree.
For example, the following humanized antibodies can be obtained: 8 humanized antibodies (2G 3-V17, 2G3-V18, 2G3-V24, 2G3-V25, 2G3-V46, 2G3-V50, 2G3-V31, 2G 3-V34) obtained by humanizing the 2G3 single domain antibody, 8 humanized antibodies (1A 10-V1, 1A10-V4, 1A10-V9, 1A10-V10, 1A10-V5, 1A10-V6, 1A10-V15 or 1A 10-V14) obtained by humanizing the 1A10 single domain antibody, and the 8 humanized antibodies (1A 10-V1, 1A10-V9, 1A10-V15 or 1A 10-V14) obtained by humanizing the 8 1A10 single domain antibody are all not identical in sequence.
Example 7: construction of Fc fusion antibody eukaryotic expression vector of anti-IL-17A single domain antibody
(1) Subcloning the target sequence obtained in example 4 into a eukaryotic expression vector: the antibodies selected in example 4 were subjected to Sanger sequencing to obtain their nucleotide sequences;
(2) Synthesizing the nucleotide sequence (SEQ ID NO: 10-18) into a vector RJK-V4-3 designed and modified by the company in a sequence synthesis mode to obtain a recombinant eukaryotic expression vector, wherein the modification method of the vector is as described in example 11;
(3) Converting the recombinant eukaryotic expression vector constructed in the step (2) into DH5 alpha escherichia coli, culturing to extract plasmids, and removing endotoxin;
(4) Sequencing and identifying the extracted plasmid;
(5) The recombinant vector after confirmation was prepared for subsequent eukaryotic cell transfection and expression, and the antibody was purified by the method of example 10 after the VHH Fc protein was expressed by the method of example 8 or 9.
Example 8: single domain antibodies against IL-17A protein are expressed in suspension ExpiCHO-S cells
(1) 3 days before transfection at 2.5X10 5 The cells were passaged/mL and the ExpiCHO-S ™ cells were expanded, and the calculated desired cell volume was transferred to 500mL shake flasks with fresh pre-warmed 120mL (final volume) of ExpiCHO ™ expression medium; to achieve a cell concentration of about 4X 10 6 -6×10 6 Living cells/mL;
(2) The day before transfection, the ExpiCHO-S ™ cells were diluted to a concentration of 3.5X10 6 Living cells/mL, allowing the cells to incubate overnight;
(3) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 7X 10 before transfection 6 -10×10 6 Living cells/mL;
(4) Cells were diluted to 6×10 with fresh expiocho ™ expression medium pre-warmed to 37 ℃ 6 Each living cell/mL. The calculated desired cell volume was transferred to a 500mL shake flask containing fresh pre-warmed 100mL (final volume) of expcho ™ expression medium;
(5) Gently mixing the epifectamine ™ CHO reagent upside down, diluting the epifectamine ™ CHO reagent with 3.7mL of OptiPRO ™ medium, and stirring or mixing;
(6) Diluting plasmid DNA with refrigerated 4mL of OptiPRO ™ culture medium, and mixing;
(7) Incubating ExpiFectamine 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 to the prepared cell suspension, and gently agitating the shake flask during the addition;
(8) Cells were incubated at 37℃with 8% CO 2 Shake culturing in humidified air;
(9) 600ul of ExpiFectamine ™ CHO Enhancer and 24mL of ExpiCHO feed were added on day 1 (18-22 hours post transfection).
(10) Supernatants were collected about 8 days after transfection (cell viability below 70%).
Example 9: expression of anti-IL-17A protein single domain antibodies in suspension 293F cells
Recombinant single domain antibody expression experimental procedure (500 mL shake flask for example):
(1) 3 days before transfection at 2.5X10 5 The cells were passaged/mL and expanded 293F cells, and the calculated desired cell volume was transferred to a 500mL shake flask containing fresh pre-warmed 120mL (final volume) OPM-293 CD05 Medium. To achieve a cell concentration of about 2X 10 6 -3×10 6 Living cells/mL.
(2) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 2X 10 before transfection 6 -3×10 6 Living cells/mL.
(3) Dilution of cells to 1X 10 with pre-warmed OPM-293 CD05 Medium 6 Each living cell/mL. The calculated cell volume required was 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 culture medium, and stirring or blowing to mix uniformly; plasmid DNA (plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-IL-17A single domain antibody prepared in example 7) was diluted with 4mL of Opt-MEM medium, mixed by vortexing, and filtered with a 0.22um filter head. Incubate at room temperature for 5min.
(5) Diluted PEI reagent was added to the diluted DNA and mixed upside down. PEI/plasmid DNA complexes were incubated for 15-20 minutes at room temperature and then gently added to the prepared cell suspension, during which time the shake flask was gently swirled.
(6) Cells were incubated at 37℃with 5% CO 2 Shake culturing at 120 rpm.
(7) 5mL OPM-CHO PFF05 feed was added 24h, 72h post transfection.
(8) Supernatants were collected about 7 days after transfection (cell viability below 70%).
Example 10: purification of anti-IL-17A protein single domain antibodies
(1) The protein expression supernatant obtained in example 8 or 9 was filtered with a disposable filter head of 0.45 μm to remove insoluble impurities;
(2) Purifying the filtrate by using a Protein purifier to perform affinity chromatography, and purifying by using agarose filler coupled with Protein A by utilizing the binding capacity of human 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 combined with the packing;
(4) Washing the column-bound impurity proteins with a low-salt and high-salt buffer;
(5) The target protein combined on the column is subjected to a system by using a low pH buffer solution;
(6) Rapidly adding the eluent into Tris-HCl solution with pH of 9.0 for neutralization;
(7) And (3) dialyzing the neutralized protein solution, performing SDS-PAGE analysis to determine that the protein purity is above 95%, and preserving the protein at a low temperature for later use after the concentration is above 0.5 mg/mL.
Example 11: construction of Single-Domain antibody eukaryotic expression vector RJK-V4-3
The mentioned nanobody universal targeting vector RJK-V4-3 is modified by the company after fusion of Fc region in heavy chain coding sequence of human IgG4 based on the invitrogen commercial vector pCDNA3.4 (vector data link: https:// assems. Thermo-former. Com/TFS-assems/LSG/manals/pcdna3_4_topo_ta_cloning_kit_man. Pdf), i.e. the vector comprises Hinge region (Hinge) CH2 and CH3 region of IgG4 heavy chain. The concrete improvement scheme is as follows:
(1) Selecting restriction enzyme cutting sites XbaI and AgeI on pcDNA3.4;
(2) Introducing multiple cloning sites (MCS, multiple Cloning Site) and a 6 XHis tag at the 5 'end and the 3' end of the coding sequence of the Fc fragment respectively by means of overlapping PCR;
(3) Amplifying the fragments by PCR using a pair of primers with XbaI and AgeI cleavage sites, respectively;
(4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction enzymes XbaI and AgeI, respectively;
(5) And (3) connecting the digested vector and the inserted fragment under the action of T4 ligase, then converting the connection product into escherichia coli, amplifying, and checking by sequencing to obtain the recombinant plasmid.
Example 12: expression and purification of Tool antibodies (Tab 1) targeting human IL-17A
Tab1 is herein referred to as Tab. Tab1 (Secukinumab) is Stuzumab, the sequence is from IMGT.
The searched sequences were commissioned for mammalian cell expression system codon optimization by general biosystems (Anhui) Inc., and cloned into pcDNA3.1 vector. After resistance selection, plasmid positive bacteria were selected for amplification and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, cat# 740412.50). According to each 100mL cell added with 100 μg plasmid (40 μg heavy chain +60 μg light chain), using PEI in 293F cells (medium: freeStyle 293 Expression medium, thermo, cat #12338026 + F-68, thermo, cat # 24040032) transient expression; after 6-24 h of transfection 5% volume of 10% Peptone (Sigma, cat#P0521-100G), 8% CO were added 2 Culturing at 130rpm for about 7-8 days; when the cell viability was reduced to 50%, the expression supernatant was collected and purified using a gravity column of ProteinA (GE, cat#17-5438-02); after PBS dialysis, concentration was determined using Nanodrop, SEC to identify purity, and indirect ELISA to verify binding capacity;
Tab obtained by the method has the concentration of not less than 2 mg/ml and the purity of more than 95 percent.
Example 13: antigen binding quantitative profile assay for antibodies
This example was performed using standard enzyme-linked immunosorbent assay (ELISA) protocols.
(1) 50. Mu.L of IL-17A & F (Acro, cat#ILF-H52WF, lot#G41-88ZF 1-RD) at 1. Mu.g/mL was coated overnight at 4 ℃.
(2) Washing the plate; 200. Mu.L of 5% milk was added and blocked at 37℃for 2h.
(3) VHH-hFc was diluted to 2ug/mL and then the antibody was diluted 5-fold gradient for a total of 8 concentration gradients. VHH-hFc here refers to the Fc fusion antibody (expressed in 293F cells) of the single domain antibody against IL-17A protein prepared in example 9, purified in example 10. In addition, hIgG and Tab controls are also respectively arranged; tab1 was prepared from example 12;
(4) Washing the plate; add 50. Mu.L of single domain antibody diluted in step (3), double wells and incubate at 37℃for 1h.
(5) Washing the plate; 50. Mu.L of HRP-coat anti-hIgG secondary antibody was added and incubated at 37℃for 30min.
(6) Washing the plate (washing several times); 50. Mu.L of TMB which had previously recovered the room temperature was added thereto, and the reaction was continued at the normal temperature in the dark for 15 minutes.
(7) Add 50. Mu.L of stop solution (1N HCl) and store the microplate reader reading.
(8) Drawing a curve, calculating EC50, as shown in fig. 2-3, wherein igg designates a type control, immunoglobulin molecules that do not bind to any target, commercially available; it can be seen that both the single domain antibodies 1A10,1D7,1E6,2E2,2G3,3D1,3E1,3H2, 4A12 are excellent in IL-17A protein binding potency and specificity.
In FIG. 2, 2A shows the binding of the single domain antibodies 1A10, 1C9, 1D1, 1D4, 1D7, 1E2 to IL-17A protein, and 2B shows the binding of the single domain antibodies 1E6, 1H2, 2D8, 2E2, 2F5, 2G3 to IL-17A protein. In FIG. 3, 3A shows the binding force of the single domain antibodies 3A6, 3A8, 3B11, 3D1, 3E2 to IL-17A protein, 3B shows the binding force of the single domain antibodies 3F8, 3H2, 4A1, 4A12, 4F4, 1H3 to IL-17A protein, and 3C shows the binding force of the single domain antibodies 3B10, 4A1, 4G4, tab1, hIgG to IL-17A protein.
Example 14: determination of antibodies blocking IL-17A binding to IL-17R
This example was performed using standard enzyme-linked immunosorbent assay (ELISA) protocols.
(1) 50. Mu.L of 1. Mu.g/mL IL-17RA & IL-17RC (Acro, cat#ILC-H5257, lot#G173a-2085F 1-TE) was coated overnight at 4 ℃.
(2) Washing the plate; 200. Mu.L of 5% milk was added and blocked at 37℃for 2h.
(3) Biotin-IL-17A (Acro, cat#ILA-H82Q1, lot#CBV296P1-97RF 1-QJ)) was mixed with the gradient diluted VHH-hFc to obtain a mixed solution (the concentration of Biotin-IL-17A in the mixed solution was 3.8 ng/ml). VHH-hFc here refers to the Fc fusion antibody (expressed in 293F cells) of the single domain antibody against IL-17A protein prepared in example 9, purified in example 10. In addition, an hIgG control was also set; hIgG indicates a isotype control, immunoglobulin molecules that do not bind to any target, are commercially available;
(4) Washing the plate; mu.L of the above mixture was added, and the wells were double-plated and incubated at 37℃for 1 hour.
(5) Washing the plate; 50. Mu.L of strepavidin-HRP was added and incubated at 37℃for 30min.
(6) Washing the plate (washing several times); 50. Mu.L of TMB which had previously recovered the room temperature was added thereto, and the reaction was continued at the normal temperature in the dark for 15 minutes.
(7) Add 50. Mu.L of stop solution (1N HCl) and store the microplate reader reading.
(8) EC50 was calculated by plotting a curve. The results are shown in FIGS. 4-5, and the abscissa in FIGS. 4-5 represents the specific concentration of VHH-hFc in gradient dilutions. As can be seen, both single domain antibodies 1A10,1D7,1E6,2E2,2G3,3D1,3E1,3H2,4A12 effectively blocked IL-17A binding to IL-17R.
In fig. 4, 4A shows measurement results of 1a10, 1C9, 1D7,1E 2, 1D1, 1D4, 4B shows measurement results of single domain antibodies 3A6, 3A8, 3E1, 3E2, 3B11, 3D1, and 4C shows measurement results of 3F8, 3H2, 4F4, 1H3, 4A1, 4A 12. In fig. 5, 5A shows the measurement results of 3B10, 4A1, hIgG, and 4G4, and 5B shows the measurement results of the single domain antibodies 1E6, 1H2, 2F5, 2G3, 2D8, and 2E 2.
Example 15: antibody (eukaryotic sample) neutralization of human IL-17A-induced Hela cells to release IL-6 experiments
The following operations are carried out according to methods common to those skilled in the art:
(1) Spreading 10000 Hela cells, 50 μl/well into 96-well plate after resuscitating for 3 times or more;
(2) The antibody was mixed in gradient dilution with equal volumes of 4 x 55ng/ml human IL-17A (Novo, cat#C774, lot#0331872-4147) and the mixture was added to the cell culture wells at 50 μl per well; the antibody herein was obtained by purifying the Fc fusion antibody (expressed in 293F cells) of the single domain antibody against IL-17A protein obtained in example 9, as described in example 10; in addition, hIgG and Tab controls are also respectively arranged; hIgG indicates a isotype control, immunoglobulin molecules that do not bind to any target, are commercially available; tab was prepared from example 12;
(3) After incubation of the cell incubator at 37℃for 24h, the supernatant was assayed for IL-6 concentration using a human IL-6 ELISA kit;
the EC50 of the neutralizing IL-17A-induced Hela cells of the antibodies to release IL-6 was calculated based on the results of the assays, as shown in FIGS. 6-7, wherein the abscissa represents the molar concentration of the aforementioned gradient diluted antibodies; the ordinate indicates the IL-6 concentration (in pg/ml) in the supernatant; from FIGS. 6-7, it can be seen that eukaryotic antibodies were effective in neutralizing IL-17A and inducing IL-6 release from Hela cells.
In fig. 6, 6A shows experimental results of Tab and hIgG, 6B shows experimental results of 2G3, 3D1, 1D4, and 2E2, and 6C shows experimental results of 1a10, 1D1, 3E1, and 3H 2. In fig. 7, 7A shows experimental results of 4a12, 4F4, 7B shows experimental results of 1C9, 1D7, 1E2, 1E6, 7C shows experimental results of 3E2, 3F8, 2F5, 3A6, and 7D shows experimental results of 4A1, 1H 3.
Example 16: antibody (humanized sample) neutralization of human IL-17A Induction of Hela cells to release IL-6 experiments
The following operations are carried out according to methods common to those skilled in the art:
(1) Spreading 10000 Hela cells, 50 μl/well into 96-well plate after resuscitating for 3 times or more;
(2) Mixing the gradient diluted antibody with equal volume of 4 x 55ng/ml human IL-17A, adding 50 μl per well to the cell culture 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 Fc fusion antibody eukaryotic expression vectors of humanized single domain antibodies against IL-17A by example 7 (the nucleotide sequences of the humanized antibodies were constructed into vectors RJK-V4-3), and then by expression of example 9 and purification of example 10. In addition, hIgG and Tab controls are also respectively arranged; hIgG indicates a isotype control, immunoglobulin molecules that do not bind to any target, are commercially available; tab was prepared from example 12;
(3) After incubation of the cell incubator at 37℃for 24h, the supernatant was assayed for IL-6 concentration using a human IL-6 ELISA kit;
The EC50 of the neutralizing IL-17A-induced Hela cells of the antibodies to release IL-6 was calculated based on the results of the assays, as shown in FIGS. 8-10, wherein the abscissa represents the specific concentration of the aforementioned gradient diluted antibodies; the ordinate indicates the IL-6 concentration (in pg/ml) in the supernatant; from FIGS. 8-10, it can be seen that humanized antibodies efficiently neutralize IL-17A and induce the release of IL-6 by HeLa cells, and have a technical effect superior to that of antibodies (eukaryotic samples).
In FIG. 8, 8A shows the experimental results of Tab and hIgG, 8B shows the experimental results of 2G3-V17 and 2G3-V18, and 8C shows the experimental results of 2G3-V24 and 2G 3-V25. 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 experimental results of 1A10-V9, 1A10-V10, 10B shows the experimental results of 1A10-V5, 1A10-V6, and 10C shows the experimental results of 1A10-V15, 1A 10-V14.
Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.
Sequence listing
<110> Nanjing Rongjiekang biotechnology Co., ltd
<120> an anti-IL-17A single domain antibody and uses thereof
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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
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Cys Ala Ala Ser Glu Tyr Ala Trp Arg Leu Asn Arg Met Gly Trp Val
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Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Val Ser Ile Gly Thr
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Arg Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
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Ile Ala Gln Asp Asn Ala Lys Asn Thr Met Tyr Leu Gln Met Asn Ser
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Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Leu Gly
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Gly Asp Ser Arg Thr Pro Ile Tyr Asp Ile Ser Tyr Trp Gly Gln Gly
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Thr Gln Val Thr Val Ser Ser
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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
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Cys Ala Val Ser Gly Tyr Phe Tyr Ser Ser Ala Cys Met Gly Trp Phe
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Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ser Ile Tyr Ile
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Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
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Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
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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
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Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
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Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr Cys Met Gly Trp Phe
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Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Thr Ile Asp Asn
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His Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
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Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
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Lys Pro Glu Asp Ala Ala Met Tyr Tyr Cys Ala Thr Gly Gly Gly Gly
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Gly Thr Gln Val Thr Val Ser Ser
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<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
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Cys Ala Ala Ser Gly Tyr Thr Tyr Arg Ser Tyr Phe Met Ala Trp Phe
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Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Val Ile Asp Ser
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Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Ser Arg Phe Thr Val
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Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
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Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Ala Thr Glu
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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
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Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr Cys Met Gly Trp Phe
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Arg Gln Thr Pro Glu Lys Glu Arg Glu Gly Val Ala Thr Ile Asp Asn
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His Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
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Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
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Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Thr
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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
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Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Gly Gly
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Thr Gln Val Thr Val Ser Ser
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<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
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Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Ser
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Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
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Ser Glu Asp Asn Ala Lys Thr Thr Leu Phe Leu Gln Met Asn Ser Leu
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Gly Arg Gly Thr Gln Val Thr Val Ser Ser
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<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 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
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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 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
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<213> Artificial sequence (Artificial Sequence)
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gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
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gagggcgtgg tgagcatcgg caccaggggc ggcagcacct actacgccga cagcgtgaag 180
ggcaggttca ccatcgccca ggacaacgcc aagaacacca tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg gcctgggcgg cgacagcagg 300
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<210> 11
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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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
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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 (12)

1. A single domain antibody against IL-17A, characterized in that: the single domain antibody is composed of a heavy chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3;
the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are as follows:
CDR1 shown in SEQ ID NO. 22, CDR2 shown in SEQ ID NO. 32, and CDR3 shown in SEQ ID NO. 39.
2. The anti-IL-17A single domain antibody of claim 1, wherein: the single domain antibody also comprises a framework region FR; the framework regions FR include the amino acid sequences of FR1, FR2, FR3 and FR 4; the amino acid sequences of the framework regions FR are respectively:
42, said FR1 variant comprising up to 5 amino acid substitutions in said FR 1;
46. FR2 or a variant of FR2 shown in SEQ ID NO. 46, said variant of FR2 comprising at most 5 amino acid substitutions in said FR 2;
56, said FR3 variant comprising up to 5 amino acid substitutions in said FR 3;
the FR4 shown in SEQ ID NO. 60 or a variant of FR4, said variant of FR4 comprising at most 5 amino acid substitutions in said FR 4.
3. A single domain antibody against IL-17A, characterized in that: the amino acid sequence of the single domain antibody is shown as SEQ ID NO. 9.
4. The Fc fusion protein or humanized antibody of the single domain antibody of anti-IL-17A of any one of claims 1 to 3.
5. A nucleotide molecule encoding the anti-IL-17A single domain antibody of any one of claims 1 to 3, characterized in that: the nucleotide sequence is shown as SEQ ID NO: shown at 18.
6. 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 4 or the nucleotide molecule of claim 5.
7. A host cell, characterized in that: which can express 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 4, or which comprises the expression vector of claim 6.
8. The host cell of claim 7, wherein: the host cell is a eukaryotic cell or a prokaryotic cell.
9. A pharmaceutical composition characterized by: the pharmaceutical composition comprising a single domain antibody selected from the group consisting of anti-IL-17A of any one of claims 1 to 3, and a pharmaceutically acceptable carrier.
10. A medicament for treating psoriasis, characterized in that: comprising as active ingredient the anti-IL-17A single domain antibody of any one of claims 1 to 3.
11. A kit for detecting IL-17A levels, characterized in that: the kit contains the anti-IL-17A single domain antibody of any one of claims 1 to 3.
12. Use of the anti-IL-17A single domain antibody of any one of claims 1 to 3 or the pharmaceutical composition of claim 9 in the manufacture of a medicament for the treatment of a disease, characterized in that: the disease is psoriasis.
CN202210489565.5A 2022-03-25 2022-03-25 anti-IL-17A single domain antibody and application thereof Active CN114920838B (en)

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