CN114380917B

CN114380917B - Bispecific single domain antibodies against IL-17A and TNF α and uses thereof

Info

Publication number: CN114380917B
Application number: CN202210298224.XA
Authority: CN
Inventors: 苏志鹏; 张云; 孟巾果; 王乐飞; 郑民强; 谢维
Original assignee: Nanjing Rongjiekang Biotechnology Co ltd
Current assignee: Nanjing Rongjiekang Biotechnology Co ltd
Priority date: 2022-03-25
Filing date: 2022-03-25
Publication date: 2022-06-14
Anticipated expiration: 2042-03-25
Also published as: CN114380917A

Abstract

The invention belongs to the field of immunology and relates to a bispecific single domain antibody aiming at IL-17A and TNF alpha and application thereof. The bispecific single domain antibody comprises (a) a first antigen-binding moiety for specifically binding IL-17A; and (b) a second antigen-binding moiety for specifically binding TNF α; the first antigen-binding moiety and the second antigen-binding moiety are fused to each other. The beneficial effects of the invention are: the invention screens out the bispecific single-domain antibody specifically aiming at IL-17A and TNF alpha by using a biological genetic engineering technology, has better antibody affinity, can block specific cells from releasing cytokines, has good binding activity and excellent stability, and has obvious treatment effect aiming at autoimmune diseases.

Description

Bispecific single domain antibodies against IL-17A and TNF α and uses thereof

Technical Field

The present invention relates to a bispecific antibody capable of specifically binding to IL-17A and TNF α, respectively (hereinafter, abbreviated as "IL-17A/TNF α bispecific antibody"), and a pharmaceutical composition containing the bispecific antibody as an active ingredient, and a pharmaceutical therapeutic use thereof.

Background

Psoriasis is a relatively 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 with joint swelling and pain, stiffness and dyskinesia, part of the psoriatic arthritis can affect the spine, severe patients can cause disability, and the physical and psychological health of the patients is seriously affected. Psoriasis is classified according to its clinical features, mainly in the following categories: the psoriasis is of the vulgaris type, of the articular type, of the pustular type and of the erythrodermic type, more than 90% of the psoriasis belonging to the vulgaris type, the other types of this disease being caused by the sudden withdrawal of the patient during the treatment period by external irritant drugs, by overuse of glucocorticoids and by immunosuppressive agents.

The pathogenesis of psoriasis is unclear. At present, the treatment scheme about PsA mainly comprises nonsteroidal 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 state of illness. 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 of the psoriasis. 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 quality of life and even 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 lymphocytes are activated into memory-effect T lymphocytes, the memory-effect T lymphocytes enter circulation and migrate to the skin, gather at the lesion part, secrete various cytokines to play various biological functions and cause the disease.

TNF-alpha (as well as TNF alpha) is a cytokine with a wide range of biological activities, and the activity of TNF-alpha accounts for 70-95% of the total activity of the TNF family possessing TNF-alpha, TNF-beta, TNF-gamma, and under abnormal conditions, especially elevated levels, can lead to immunopathological responses such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, Crohn's disease, and the like. Researches find that the TNF-alpha level in skin lesions, serum, articular cavity synovium or cell culture supernatant of patients with psoriasis is obviously increased compared with that in a normal control group, the TNF-alpha level in the skin lesions, the serum, the articular cavity synovium or the cell culture supernatant of the patients with psoriasis is reduced in different degrees after treatment, and the disease severity of the patients with psoriasis is positively correlated with the TNF-alpha level, which indicates that the TNF-alpha has an important role in the pathogenesis of psoriasis.

TNF-alpha is mainly secreted by macrophages, other types of cells such as lymphocytes, smooth muscle cells, fibroblasts and the like can also generate and release TNF-alpha under certain conditions, and after a switch of an immunopathological mechanism of psoriasis is opened, cells Th1 and Th2 in T lymphocytes can generate TNF-alpha so as to play a pathogenic biological effect. TNF-alpha is known to have the effects of killing or inhibiting tumor cells, resisting infection, and also to participate in inflammatory reactions, promoting cell proliferation and differentiation, both of which play important roles in the pathogenesis of psoriasis.

IL-17A is the first member of the IL-17 cytokine family to be found and is secreted mainly by Th17 cells. In 1986, Mosmann, coffman and related panelists discovered two subsets of helper T cells-Th 1 and Th2(Mosmann et al, 1986). In addition to Th17 cells, other T cells (including CD8+ T cells, γ δ 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 (Iwakuraatal, 2011 a).

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. In the field of autoimmunity, TNF α antagonists and Interleukin (IL) class drugs are currently the focus of research and development, with the IL-12 family and IL-17 being of greatest interest. As for IL inhibitors for psoriasis which have been marketed globally by 7 months of 2018, secukinumab (Cosentyx), usteklizumab, eculizumab, broludamumab, gusucumab, tiltrakizumab; there are Risankizumab, Bimekizumab, Mirikizumab, CJM112, COVA322, ABT-122, ALX-0761, CNTO-6785, NI-1401 and the like, which are still in the clinical trial stage. No varieties are approved in the market at home, the IL-17A inhibitor SHR-1314 developed by Jiangsu Henrui medicine corporation is currently in the clinical test stage, and the secukinumab of Beijing Nowa pharmacy Co., Ltd and the ektexus mab of Li Laisu State pharmacy Co., Ltd are also in the clinical test stage.

Although the configuration and variety of bispecific antibodies are increasing, there are many corresponding problems with existing bispecific antibodies. First, in terms of configuration, many configurations of the existing bispecific antibody are greatly different from those of a naturally-occurring antibody, so that the antibody is poor in stability and specificity, easily generates homodimers, has no good pharmacokinetic characteristic, and even generates immunogenicity. Therefore, it is one of the technical problems to be solved in the art to provide a bispecific antibody against TNF α and IL-17A with high stability and good effect.

Disclosure of Invention

The invention of this patent aims to provide a bispecific single domain antibody capable of specifically binding to IL-17A and TNF alpha, respectively, and uses thereof.

A first aspect of the invention provides a bispecific single domain antibody directed to IL-17A and TNF α, comprising (a) a first antigen-binding moiety for specifically binding IL-17A; and (b) a second antigen-binding moiety for specifically binding TNF α; the first antigen-binding moiety and the second antigen-binding moiety are fused to each other;

the bispecific single domain antibody has less than 2 x 10 with respect to IL-17A^-12M, the bispecific single domain antibody having less than 3 x 10 for TNF alpha ^-10M, said dissociation constant being represented by BLI: (KD)Biofilm interferometry).

In some preferred embodiments, the bispecific single domain antibody has less than 1.9 x 10 with respect to IL-17A protein^-12M、1.8*10^-12M、1.7*10^-12M、1.6*10^-12M、1.5*10^-12M、1.4*10^-12M、1.3*10^-12M、1.2*10^-12M、1.1*10^-12M、1.0*10^-12Dissociation constant (KD) of M.

In further preferred embodiments, the bispecific single domain antibody has less than 2.4 x 10 with respect to the TNF α protein^-10M、2.3*10^-10M、2.2*10^-10M、2.1*10^-10M、2.0*10^-10M、1.9*10^-10M、1.8*10^-10M、1.7*10^-10M、1.6*10^-10M、1.5*10^-10M、1.4*10^-10M、1.3*10^-10M、1.2*10^-10Dissociation constant (KD) of M.

In another preferred embodiment, the bispecific single domain antibody has (1.2-2.4) × 10 for TNF α protein^-10Dissociation constant (KD) of M.

In another preferred embodiment, the bispecific single domain antibody has 1 x 10 with respect to the IL-17A protein^- ¹²M dissociation constant (KD), 1.2 x 10 for TNF α protein^-10Dissociation constant (KD) of M.

In another preferred embodiment, the bispecific single domain antibody has 1 x 10 with respect to the IL-17A protein^- ¹²M has a dissociation constant (KD) of 2.4 x 10 for TNF α protein^-10Dissociation constant (KD) of M.

The first antigen-binding portion and the second antigen-binding portion described above each comprise an amino acid sequence consisting of 3 CDR regions (CDR1 to CDR3) and 4 hinge regions (FR1 to FR 4): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I); it is worth noting that the amino acid sequences of CDR1, CDR2 and CDR3 can be readily combined with each other to conform to formula (I). The skilled person knows how to determine without any difficulty how these sequences can be combined to obtain a single domain antibody of the invention having the desired dissociation constant (KD).

For ease of distinction, write: the 3 CDR regions of the first antigen-binding portion are CDR1, CDR2, CDR3 in that order; the 3 CDR regions of the second antigen-binding portion are CDR1, CDR2, CDR3 in that order. The first antigen-binding portion is a single domain antibody directed against IL-17A. The second antigen-binding portion is a single domain antibody directed to TNF α.

Preferably, the bispecific single domain antibody comprises (a) a first antigen-binding moiety for specifically binding IL-17A; and (b) a second antigen-binding moiety for specifically binding TNF α; the first antigen-binding moiety and the second antigen-binding moiety are fused to each other;

the first antigen-binding portion is a heavy chain comprising CDR1, CDR2 and CDR 3:

the amino acid sequence of CDR1 is shown in SEQ ID NO. 9 or SEQ ID NO. 10, or the amino acid sequence of HCDR1 is a variant sequence of SEQ ID NO. 9 or SEQ ID NO. 10, the variant sequence has at most 5 amino acid mutations relative to SEQ ID NO. 9 or SEQ ID NO. 10;

the amino acid sequence of CDR2 is shown as SEQ ID NO. 12 or SEQ ID NO. 13, or the amino acid sequence of HCDR2 is the variant sequence of SEQ ID NO. 12 or SEQ ID NO. 13, the variant sequence has at most 5 amino acid mutations relative to SEQ ID NO. 12 or SEQ ID NO. 13;

The amino acid sequence of CDR3 is shown in SEQ ID NO. 15 or SEQ ID NO. 16, or the amino acid sequence of HCDR3 is the variant sequence of SEQ ID NO. 15 or SEQ ID NO. 16, the variant sequence has at most 5 amino acid mutations relative to SEQ ID NO. 15 or SEQ ID NO. 16;

the second antigen-binding moiety is a heavy chain comprising CDR1, CDR2 and CDR 3:

the amino acid sequence of CDR1 is shown in SEQ ID NO. 11, or the amino acid sequence of CDR1 is a variant sequence of SEQ ID NO. 11, the variant sequence has at most 5 amino acid mutations relative to SEQ ID NO. 11;

the amino acid sequence of CDR2 is shown in SEQ ID NO. 14, or the amino acid sequence of CDR2 is a variant sequence of SEQ ID NO. 14, and the variant sequence has at most 5 amino acid mutations relative to SEQ ID NO. 14;

the amino acid sequence of CDR3 is shown in SEQ ID NO. 17, or the amino acid sequence of CDR3 is a variant sequence of SEQ ID NO. 17, and the variant sequence has at most 5 amino acid mutations relative to SEQ ID NO. 17.

In another preferred embodiment, the first and second antigen-binding portions further comprise a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR 4;

(ii) a first antigen-binding portion whose FR1 sequence is as set forth in SEQ ID No. 18 or has at least 80% homology with the amino acid sequence set forth in SEQ ID No. 18; the FR2 sequence is shown as SEQ ID NO. 20 or SEQ ID NO. 21 or has at least 80 percent of homology with the amino acid sequence shown as SEQ ID NO. 20 or SEQ ID NO. 21; the FR3 sequence is shown as SEQ ID NO. 23 or SEQ ID NO. 24 or has at least 80 percent of homology with the amino acid sequence shown as SEQ ID NO. 23 or SEQ ID NO. 24; the FR4 sequence is shown as SEQ ID NO. 26 or has at least 80 percent of homology with the amino acid sequence shown as SEQ ID NO. 26;

(ii) the FR1 sequence for the second antigen-binding portion is as shown in SEQ ID NO. 19 or has at least 80% homology with the amino acid sequence shown in SEQ ID NO. 19; the FR2 sequence is shown as SEQ ID NO. 22 or has at least 80 percent of homology with the amino acid sequence shown as SEQ ID NO. 22; the FR3 sequence is shown as SEQ ID NO. 25 or has at least 80 percent of homology with the amino acid sequence shown as SEQ ID NO. 25; the FR4 sequence is shown as SEQ ID NO. 27 or has at least 80 percent homology with the amino acid sequence shown as SEQ ID NO. 27.

It is worth mentioning that the amino acid sequences of FR1, FR2, FR3 and FR4 can be easily combined with each other to conform to formula (I). The skilled person knows how to determine without any difficulty how to what extent these sequences can be combined in order to obtain a bispecific single domain antibody of the invention with a desired dissociation constant (KD).

Preferably, the first antigen-binding portion and the second antigen-binding portion are both humanized single domain antibodies.

The first antigen-binding moiety and the second antigen-binding moiety are fused to each other via an Fc-fragment of human IgG, the first antigen-binding moiety being coupled to the amino terminus of the Fc-fragment of human IgG, and the second antigen-binding moiety being coupled to the carboxy terminus of the Fc-fragment of human IgG. The Fc fragment of human IgG can be human IgG1 Fc fragment, human IgG4 Fc fragment, or Fc fragment of other human IgG. More preferably, bispecific single domain antibodies against IL-17A and TNF α can be constructed in the following manner:

1) The amino acid sequence of CDR1 of the first antigen binding portion is set forth as SEQ ID NO. 9, the amino acid sequence of CDR2 is set forth as SEQ ID NO. 12, and the amino acid sequence of CDR3 is set forth as SEQ ID NO. 15; the FR1 sequence of the first antigen binding portion is shown as SEQ ID NO. 18, the FR2 sequence is shown as SEQ ID NO. 20, the FR3 sequence is shown as SEQ ID NO. 23, and the FR4 sequence is shown as SEQ ID NO. 26;

the amino acid sequence of CDR1 of the second antigen binding portion is shown as SEQ ID NO. 11, the amino acid sequence of CDR2 is shown as SEQ ID NO. 14, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 17; the FR1 sequence of the second antigen-binding portion is shown as SEQ ID NO. 19, the FR2 sequence is shown as SEQ ID NO. 22, the FR3 sequence is shown as SEQ ID NO. 25, and the FR4 sequence is shown as SEQ ID NO. 27;

the linkage between the first antigen-binding moiety and the second antigen-binding moiety is accomplished via the Fc-fragment of human IgG1 or the Fc-fragment of human IgG 4.

2) The amino acid sequence of CDR1 of the first antigen binding portion is shown as SEQ ID NO. 10, the amino acid sequence of CDR2 is shown as SEQ ID NO. 13, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 16; the FR1 sequence of the first antigen binding portion is shown as SEQ ID NO. 18, the FR2 sequence is shown as SEQ ID NO. 21, the FR3 sequence is shown as SEQ ID NO. 24, and the FR4 sequence is shown as SEQ ID NO. 26;

the linkage between the first antigen-binding moiety and the second antigen-binding moiety is accomplished through the Fc segment of human IgG1 (i.e., hFC 1) or human IgG4 (i.e., hFC 4).

All of 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".

The "bispecific single domain antibody against IL-17A and TNF α" of the present invention includes not only the complete bispecific single domain antibody, but also fragments, derivatives and analogs of said 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 (i ii) a polypeptide formed by fusing the mature polypeptide to another compound (such as a compound that increases the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to the sequence of the polypeptide (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 well within the skill of those in the art in light of the teachings herein.

In a third aspect, the invention provides the amino acid sequence of a bispecific single domain antibody against IL-17A and TNF α, said bispecific single domain antibody having the amino acid sequence as shown in SEQ ID No.1-4, respectively, or said bispecific single domain antibody having at least 80% sequence homology with the amino acid sequence of SEQ ID No. 1-4.

In some embodiments, the bispecific single domain antibody against IL-17A and TNF α binds to a polypeptide selected from the group consisting of SEQ ID NOs: 1-4 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 and TNF α proteins.

In other preferred embodiments, the bispecific single domain antibody against IL-17A and TNF α binds to a polypeptide selected from SEQ ID NOs: 1-4 have at least 95% sequence homology and are capable of specifically binding IL-17A and TNF α proteins.

In a fourth aspect, the invention provides an Fc fusion antibody or a humanized antibody of the bispecific single domain antibody against IL-17A and TNF α.

A fifth aspect of the present invention provides a nucleotide molecule encoding the aforementioned bispecific single domain antibody against IL-17A and TNF α or the aforementioned Fc fusion antibody or the aforementioned humanized antibody, the nucleotide sequences of which are set forth in SEQ ID NOs: 5-8, or a variant of SEQ ID NO: 5-8 have at least 80% sequence homology.

In one embodiment, the nucleic acid molecule encoding said bispecific single domain antibody against IL-17A and TNF α is identical to a sequence selected from SEQ ID NOs: 5-8 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology, and encodes single domain antibodies to IL-17A and TNF α that are capable of specifically binding to IL-17A and TNF α proteins.

A sixth aspect of the present invention provides an expression vector comprising a nucleotide molecule encoding a bispecific single domain antibody against IL-17A and TNF α, or an Fc fusion antibody or a humanized antibody thereof, the nucleotide sequences of which are set forth in SEQ ID NO: 5-8.

In a preferred embodiment, the expression vector used is RJK-V4-3 (the nucleotide molecules encoding the bispecific single domain antibody against IL-17A and TNF α are integrated into RJK-V4-3 by genetic engineering means), and other general expression vectors may be selected as desired.

In a seventh aspect the invention provides a host cell capable of expressing the bispecific single domain antibody of IL-17A and TNF α as described above, or which comprises the expression vector as described above. 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 and 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 combinations thereof.

In another preferred embodiment, the eukaryotic cell is selected from the group consisting of: pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma or combinations 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 a combination thereof.

In another preferred embodiment, the host cell is preferably a mammalian cell, more preferably a HEK293 cell, CHO cell, BHK cell, NSO cell or COS cell.

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.

An eighth aspect of the invention provides a recombinant protein comprising the aforementioned bispecific single domain antibody against IL-17A and TNF α.

A ninth aspect of the invention provides a pharmaceutical composition comprising the aforementioned bispecific single domain antibody against IL-17A and TNF α 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: intravenously, transdermally (directly applying or applying plaster to affected part).

The pharmaceutical compositions of the invention are useful for binding IL-17A/TNF α protein molecules directly and thus for the treatment of disorders associated with aberrant expression of IL-17A and/or TNF α such as psoriasis. In addition, other therapeutic agents may also be used simultaneously. 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 bispecific single domain antibody (or conjugate thereof) of the present invention as described above 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. In addition, the pharmaceutical compositions of the present invention may also be used with other therapeutic agents.

A tenth aspect of the present invention provides an agent for treating a disorder associated with abnormal expression of TNF α and/or IL-17A or an autoimmune disease, which comprises the aforementioned IL-17A and TNF α bispecific single domain antibody as an active ingredient. For example, autoimmune diseases (including but not limited to psoriasis, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, crohn's disease). Preferably, the disorder associated with aberrant expression of TNF α and/or IL-17A may be psoriasis.

An eleventh aspect of the invention provides a kit for detecting TNF α and/or IL-17A levels comprising the aforementioned bispecific single domain antibody directed against IL-17A and TNF α. In a preferred embodiment of the invention, the kit further comprises a container, instructions for use, buffers and the like.

In a preferred embodiment, the kit comprises an antibody recognizing IL-17A and/or TNF α protein, a lysis medium for solubilizing the sample, general reagents and buffers required for the assay, 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 secondary 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 is an antibody to the aforementioned bispecific single domain antibody (i.e. as an anti-antibody), which may be a single domain antibody, a monoclonal antibody, a polyclonal antibody or any other form of antibody.

In a twelfth aspect of the invention, there is provided a method of producing a bispecific antibody against IL-17A and TNF α comprising the steps of:

(a) culturing the host cell of the seventh aspect of the invention under conditions suitable for the production of the bispecific single domain antibody, thereby

Obtaining a culture comprising said bispecific single domain of IL-17A and TNF α; and

(b) isolating or recovering said bispecific single domain antibody to IL-17A and TNF α from said culture; and

(c) optionally, purifying and/or modifying the bispecific single domain antibody of IL-17A and TNF α obtained in step (b).

A thirteenth aspect of the invention provides the use of a bispecific single domain antibody against IL-17A and TNF α as described above in the manufacture of a medicament for inhibiting the expression of the IL-17A or TNF α gene or an anti-psoriatic medicament.

A fourteenth aspect of the invention provides the use of a bispecific single domain antibody against IL-17A and TNF α as described above in a medicament for blocking the binding of IL-17A to IL-17R.

A fifteenth aspect of the invention provides the use of the aforementioned bispecific single domain antibody against IL-17A and TNF α or the aforementioned pharmaceutical composition in the manufacture of a medicament for the treatment of a disease.

In a preferred embodiment, the disease is a disorder associated with aberrant expression of IL-17A and/or TNF α.

In a preferred embodiment, the disease is an autoimmune disease.

In a preferred embodiment, the disease is psoriasis or arthritis.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention of the directed against IL-17A and TNF alpha bispecific single domain antibodies, with regard to IL-17A less than 2 x 10^-12M, the bispecific single domain antibody having less than 3 x 10 for TNF alpha^-10The dissociation constant (KD) of M, represents its strong affinity. And the single-domain antibody can be simultaneously and specifically combined with IL-17A and TNF alpha protein with correct spatial structures.

(2) The bispecific single domain antibody aiming at IL-17A and TNF alpha can block the interaction of the combination action of IL-17A and IL-17R, and has higher blocking activity than a secukinumab (positive drug), so the bispecific single domain antibody has great application prospect in preparing drugs for treating autoimmune diseases, especially in preparing drugs for treating psoriasis.

(3) The bispecific single-domain antibody aiming at IL-17A and TNF alpha has great advantages in stability. The bispecific single domain antibodies of the invention exhibit excellent stability performance in both accelerated stability experiments and in low pH challenges.

(4) The bispecific single domain antibody aiming at IL-17A and TNF alpha not only has good druggability, but also shows remarkable effect in drug effect experiments of mouse psoriasis-like diseases induced by IMQ, wherein 2G3-hFC1-1B2 (test article 1) and 1A10-hFC1-1B2 (test article 2) can inhibit the reduction of mouse weight; the occurrence degree of the skin rash and desquamation of the mouse can be obviously weakened; can obviously alleviate pathological changes caused by IMQ.

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 screen in example 3;

FIG. 2 is a library enrichment scenario for the targeted TNF α screen of example 3;

FIG. 3 is a graph showing the results of an assay in which the antibody of example 12 blocks the binding of IL-17A to IL-17R;

FIG. 4 is a graph of the results of the experiment in which the antibody samples Tab, hIgG, 1A10-hFC1-1B2, 2G3-hFC1-1B2 induced the Hela cells to release IL-6 in example 13;

FIG. 5 is a graph showing the results of the experiment that the antibody samples 1A10-hFC4-1B2, 2G3-hFC4-1B2 in example 13 induce Hela cells to release IL-6;

FIG. 6 is a graph of the results of experiments in which antibody samples 1A10-hFC1-1B2, 2G3-hFC1-1B2 induced Hs27 cells to release CXCL1 in example 14;

FIG. 7 is a graph showing the results of L929 apoptosis induction by antibody samples 1A10-hFC1-1B2, 2G3-hFC1-1B2, 1A10-hFC4-1B2, and 2G3-hFC4-1B2 in example 15;

FIG. 8 is a graph showing the results of the experiment in which the antibody samples Tab, hIgG, induced apoptosis in L929 of example 15;

FIG. 9 is a weight change profile of mice during the test in example 21;

FIG. 10 shows the results of pathological examination of the animal in example 21;

FIG. 11 is the change in clinical scores of animals during the test in example 21.

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.

Single domain antibodies (sdabs, also referred to as nanobodies or VHHs by the developer Ablynx) 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 the immunoglobulin chain, 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 the exact same sequence have 100% homology.

In the present invention, a single domain antibody against IL-17A and TNF α can be obtained by using a sequence having high homology with the CDR1-3 sequence disclosed in the present invention. In some embodiments, the polypeptide of SEQ ID NO: 1-4, or sequences "at least 85% homologous", "at least 90% homologous", "at least 95% homologous", or "at least 98% homologous".

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 objects 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-terminal and/or N-terminal. 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 by 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 substituted with Ser. In addition, the technicians in this field knows, single domain antibody creativity is in the CDR1-3 region, and the framework region sequence FR1-4 is not invariable, FR1-4 sequence can adopt the present invention discloses the sequence of the conservative sequence variants.

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 heavy chain variable region of a target antibody (e.g., an animal antibody) with the constant region of a human antibody, or 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.

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 embodiments:

while embodiments of the invention have been disclosed above, it is not intended to be limited to the details shown in the description and the examples, which are set forth, but are fully applicable to various fields of endeavor as are suited to the particular use contemplated, and further modifications will readily occur to those skilled in the art, since the invention is not limited to the details shown and described without departing from the general concept as defined by the appended claims and their equivalents. The methods and reagents not described in the present examples are those known to those skilled in the art.

Example 1: preparation of human IL-17A and TNF alpha recombinant extracellular domain proteins:

the human recombinant extracellular domain protein used in this patent is obtained by expression and purification of the company itself, and the preparation process of the human IL-17A, TNF alpha recombinant extracellular domain protein is as follows.

The preparation process of the human IL-17A recombinant extracellular domain protein comprises the following steps:

(1) the coding sequence for IL-17A was retrieved at NCBI and included as NM-002190.2, and the amino acid sequence accession number for this sequence was NP-002181.1 and Uniprot ID was Q16552.

(2) The amino acid sequence corresponding to NP-002181.1 was analyzed for the transmembrane region and extracellular region of the protein by TMHMM and SMART websites, respectively.

(3) IL-17A is a secreted protein, and the 1-23 positions are signal peptides of the protein.

(4) Cloning the nucleotide sequence of the amino acid at the position 24-155 of the coding IL-17A protein into a vector pcDNA3.4 by using a gene synthesis mode;

(5) carrying out Sanger sequencing on the constructed vector, comparing with an original sequence, carrying out batch extraction on the recombinant plasmid after confirming no errors, removing endotoxin, transfecting and suspending 293F to carry out expression and purification on target protein, wherein the purity of the purified protein is up to 90%, and the requirement of animal immunity is met.

The preparation process of the human TNF alpha recombinant extracellular domain protein comprises the following steps:

(1) the coding sequence for TNF α was retrieved at NCBI and included under accession number NM _000594.3, and the sequence encoded the resulting amino acid sequence accession number NP _000585.2 and Uniprot ID P01375.

(2) The amino acid sequence corresponding to NP-000585.2 was analyzed for the transmembrane region and extracellular end of the protein by TMHMM and SMART websites, respectively.

(3) The extracellular end of the TNF alpha protein is 57-233 amino acids.

(4) The nucleotide sequence of 57-233 amino acids of TNF alpha protein is cloned into 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 the libraries against single domain antibodies against the IL-17A, TNF alpha protein, respectively:

1mg of the human recombinant IL-17A protein and the TNF alpha protein purified and obtained in example 1 are respectively mixed with Freund's complete adjuvant with the same volume, and are respectively used for immunizing Alexandrine camel of inner Mongolia once a week for 7 times in a continuous way, except for the first immunization, 1mg of the recombinant protein and Freund's incomplete adjuvant with the same volume are respectively used for animal immunization in the other six times, and the immunization process is to intensively stimulate the camel to generate antibodies aiming at the recombinant 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 connecting the enzyme-digested fragment with the vector. The connected fragments are electrically transformed into competent cells TG1, phage display libraries of IL-17A and TNF alpha protein are respectively constructed and the library capacity is determined, the size of the library capacity is about 1 × 10⁹Meanwhile, the correct insertion rate of the detection library in the target fragment is identified through colony PCR. Calculated, the correct insertion rate for the constructed IL-17A library was 95%. The correct insertion rate was 97% for the constructed library of TNF α.

Example 3: single domain antibody screening against IL-17A, TNF α protein:

single domain antibody screening of IL-17A protein:

mu.L of recombinant TG1 cells from IL-17A of example 2 were cultured in 2 XTY medium during which TG1 cells were infected by addition of 40. mu.L of helper phage VCSM13 and cultured overnight to amplify phages, which were precipitated with PEG/NaCl the next day and collected by centrifugation.

NaHCO diluted at 100 mM pH8.3₃500 mu g of IL-17A protein in the kit 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 blocking was complete, 100. mu.l of the amplified phage library (approximately 2X 10)¹¹Individual phage particles) at room temperature for 1 h; 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 the logarithmic growth phase, cultured at 37 ℃ for 1h, phage was generated and collected for the next round of screening, the same screening procedure was repeated for 1 round, and enrichment was obtained step by step, with the enrichment profile shown in FIG. 1.

Single domain antibody screening of TNF α proteins:

200 μ L of recombinant TG1 cells of TNF α from example 2 were cultured in 2 XTY medium, while adding 40 μ L of helper phage VCSM13 to infect TG1 cells and cultured overnight to amplify phages, which were precipitated with PEG/NaCl the next day and collected by centrifugation.

NaHCO diluted at 100 mM pH8.3₃500 mu g of TNF alpha protein in the kit 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 blocking was complete, 100. mu.l of the amplified phage library (approximately 2X 10)¹¹Individual phage particles) at room temperature for 1 h; after 1 hour of action, the unbound phage were washed off 15 times with PBS + 0.05% Tween-20.

The phage specifically bound to TNF alpha 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 process was repeated for 1 round to gradually enrich. The enrichment profile is shown in FIG. 2.

Example 4: specific positive clones were screened against IL-17A, TNF α using phage enzyme-linked immunosorbent assay (ELISA):

The screening of single-domain antibodies against IL-17A and TNF α proteins, respectively, was carried out according to the screening method of example 3, and the phage enrichment factor for IL-17A and TNF α was 10 or more, and after the screening was completed, the following operations were carried out for positive clones of IL-17A, TNF α, respectively:

384 single colonies from the positive clones obtained by screening were inoculated into 96-well plates of 2 XTY medium containing 100. mu.g/mL ampicillin, respectively, a blank was set, and after incubation at 37 ℃ until logarithmic phase, IPTG was added to 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 recombinant protein was released to 100 mM NaHCO pH8.3₃And 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, and 100. mu.l of Mouse Anti-HA tag Antibody (HRP) (Mouse Anti-HA horseradish peroxidase label) diluted 1:2000 was addedAntibody, Thermo Fisher), 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 bacteria of the positive cloning wells were shaken in LB medium containing 100. mu.g/mL ampicillin to extract plasmids and sequenced.

The gene sequences of the individual 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, while strains having different sequences were regarded as different clones, and finally a single domain antibody specific to IL-17A protein (non-humanized 1a10 single domain antibody, non-humanized 2G3 single domain antibody) and a single domain antibody specific to TNF α (non-humanized 1B2 single domain antibody) were obtained. The amino acid sequence of the non-humanized 1A10 single-domain antibody is shown as SEQ ID NO.28, and the amino acid sequence of the non-humanized 2G3 single-domain antibody is shown as SEQ ID NO. 29; the amino acid sequence of the non-humanized 1B2 single-domain antibody is shown in SEQ ID NO. 30. Hereinafter, a humanized antibody humanized on the basis of the non-humanized 1a10 single domain antibody is designated as a humanized antibody 1a 10. A humanized antibody obtained by humanization on the basis of a non-humanized 2G3 single domain antibody was designated as a humanized antibody 2G 3. A humanized antibody obtained by humanization on the basis of the non-humanized 1B2 single domain antibody was designated as a humanized antibody 1B 2.

The amino acid sequence of the single domain antibody has a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and constitutes the entire VHH.

Example 5: humanization of Single Domain antibodies against IL-17A and TNF α, respectively

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 single-domain antibody sequences of the company to obtain amino terminal proportion data of each site of a framework region;

(2) comprehensive weighted 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 weighted analysis is carried out according to the weight of 90 percent of human source camel source 10 percent by combining the analysis results of the amino acid proportion in the two species, the weighted proportion of the amino acid at each site is counted, and the sequences are ordered from high to low; according to the final weighting result, only reserving the amino acid types accounting for more than 10% of the single site of the framework region, and calculating the final weight of the amino acids accounting for more than 10% of the single site of the framework region according to the standard that the reserved ratio is integrated into 1 to be used as the design basis of the 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 the high concentration site, otherwise, the site is considered to belong to the 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 were constructed from antibody strains, non-humanized 1a10, 2G3, and 1B2, and panning was performed using the corresponding antigens against the constructed libraries, to finally obtain antibody sequences with high affinity and high degree of humanization, and the CDR sequences of the obtained humanized antibodies were as shown in table 1. The humanized antibody sequence FR1-FR4 is shown in Table 2, and the humanized antibody amino acid sequence is shown in Table 3.

TABLE 1 humanized antibody CDR1-CDR3 sequences

TABLE 2 humanized antibody sequences FR1-FR4 sequences

Portions of the sequences of humanized antibodies of tables 31A 10, 2G3, 1B2

TABLE 4 SEQ ID NO.1-4 sequences

SEQ ID NO.1 of Table 4 consists of three parts in sequence: namely humanized 1A10 part-hFc 1 part-humanized 1B2 part, wherein the sequences of the parts specifically constitute the following:

humanized 1a10 part: QVQLVESGGGLVKPGGSLRLSCAASEYAFRLNRMGWVRQAPGKEREGVAAIGTRGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAGLGGDTRTPIYDISYWGQGTLVTVSS (humanized 1A10 part: 1-124 of the sequence located in SEQ ID NO. 1)

Part of hFc 1: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQESLSLSPGKEPKSCDKTHTCPPCP (hFc 1 part: 125-371 located in SEQ ID NO. 1)

Humanized 1B2 part:

QVQLVESGGGSVQAGGSLRLSCAASGYTYYSDACMAWFRQAPGKEREGVAVIDRDHITQHADSVKGRFTISKDNAKNTLTLQMNSLEPEDTAMYYCAAGHPPSPRFGCGLGYQYYNYWGQGTQVTVSS (part of humanised 1B 2: 372-499 located in the sequence of SEQ ID NO. 1)

The sequence SEQ ID NO.2 in Table 4 consists of three parts in sequence: namely humanized 2G3 part-hFc 1 part-humanized 1B2 part, wherein the sequences of the parts specifically constitute the following:

Humanized 2G3 part:

QVQLVESGGGLVKPGGSLRLSCAASGYTYSSYCMGWFRQAPGKGREGVATIDNRGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCATGGGGYCSARLGEADFEFWGQGTLVTVSS (humanised part 2G 3: located in SEQ ID NO.2 sequence 1-125)

The hFc1 part is located at 126-372 of the sequence SEQ ID NO.2 and the humanized 1B2 part is located at 373-500 of the sequence SEQ ID NO. 2.

The sequence SEQ ID NO.3 in Table 4 consists of three parts in sequence: namely humanized 1A10 part (position 1-124 of the sequence) -hFc4 part (position 125-364 of the sequence) -humanized 1B2 part (position 365-492) differs from SEQ ID NO.1 in that the hFc1 sequence part is replaced by the hFc4 sequence, and the hFc4 sequence is as follows: ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQESLSLSLGESKYGPPCPPCP;

the sequence SEQ ID NO.4 in Table 4 consists of three parts in sequence: namely a humanized 2G3 part (positions 1-125 of the sequence) -a hFc4 part (position 365 of the sequence) -a humanized 1B2 part (366-493).

Example 6: construction of Fc fusion antibody eukaryotic expression vector of bispecific single domain antibody for IL-17A and TNF alpha protein

(1) The nucleotide sequence of the humanized single-domain antibody against IL-17A or TNF alpha obtained in example 5 was synthesized into a carrier containing human IgG fragments designed and modified by the same company, and then a series of following steps were performed to finally express humanized bispecific antibody with different types of combinations, wherein the carrier types in this example are two types: vector 1 (hFC 4 vector) and vector 2 (hFC 1 vector), the methods of engineering vector 1 and vector 2 were as described in example 10;

(2) transforming the recombinant eukaryotic expression vector constructed by the company into DH5 alpha escherichia coli, culturing, carrying out plasmid extraction, and removing endotoxin;

(3) carrying out sequencing identification on the extracted plasmid;

(4) after identifying no errors, the anti-IL-17A single domain antibody sequence is subcloned into a eukaryotic expression vector containing the anti-TNF alpha single domain antibody sequence: the specific operation is that restriction endonucleases Xba I and BamH I are used to cut the anti-IL-17A single domain antibody sequence from the eukaryotic expression vector where the antibody sequence is positioned, and the anti-IL-17A single domain antibody sequence is connected with the eukaryotic expression vector containing the anti-TNF alpha single domain antibody sequence with the same restriction endonucleases cohesive end, transformed, sequenced and identified; carrying out plasmid extraction on the correctly sequenced clone to remove endotoxin; carrying out sequencing identification on the extracted plasmid; and preparing the recombinant vector without errors for subsequent eukaryotic cell transfection expression.

In the subsequent eukaryotic cell transfection expression, the obtained antibodies comprise four antibodies which are numbered in sequence: 1A10-hFC1-1B2, 2G3-hFC1-1B2, 1A10-hFC4-1B2, and 2G3-hFC4-1B 2.

1A10-hFC1-1B2 are numbered as follows, humanized antibody 1A10 is linked to the amino terminus of hFC1, and humanized antibody 1B2 is linked to the carboxy terminus of hFC 1; the meaning of 2G3-hFC1-1B2 is: the humanized antibody 2G3 is connected with the amino terminal of hFC1, and the humanized antibody 1B2 is connected with the carboxyl terminal of hFC 1; the meaning of 1A10-hFC4-1B2 is: the humanized antibody 1A10 is connected with the amino terminal of hFC4, and the humanized antibody 1B2 is connected with the carboxyl terminal of hFC 4; the meaning of 2G3-hFC4-1B2 is: humanized antibody 2G3 was linked to the amino terminus of hFC4 and humanized antibody 1B2 was linked to the carboxy terminus of hFC 4. The amino acid sequence of the hFC1 is shown as the amino acid at the 125-371 position in the SEQ ID NO.1 or the amino acid at the 126-372 position in the SEQ ID NO. 2; the amino acid sequence of the hFC4 is shown as the amino acid at the 125-364 th position in SEQ ID NO.3 or the amino acid at the 126-365 th position in SEQ ID NO. 4.

1A10-hFC1-1B2 is all named as 1A10-V14 FT-hFC1-1B 2-VF;

2G3-hFC1-1B2 is fully called 2G 3-V34R-hFC 1-1B 2-VF;

1A10-hFC4-1B2 is all named as 1A10-V14 FT-hFC 4-1B 2-VF;

The 2G3-hFC4-1B2 is totally called 2G 3-V34R-hFC 4-1B 2-VF.

Example 7: bispecific single domain antibodies against IL-17A and TNF α proteins expressed in suspension ExpicCHO-S cells

(1) 3 days before transfection at 2.5X 10⁵(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; the cell concentration is about 4X 10⁶ -6×10⁶Viable cells/mL;

(2) one day prior to transfection, ExpicHO-S-chamber cells were diluted to a concentration of 3.5X 10⁶Viable 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 10⁶-10×10⁶Viable cells/mL;

(4) cells were diluted to 6X 10 with fresh ExpiCHO ™ expression medium preheated to 37 ℃⁶Viable 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 an Expifactamine CHO/plasmid DNA (plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the bispecific single domain antibody against IL-17A and TNF alpha protein prepared in step (4) of example 6) complex for 1-5 minutes at room temperature, then gently adding into the prepared cell suspension, and gently swirling the flask during the addition;

(8) cells were incubated at 37 ℃ with 8% CO₂Carrying out shake culture in humidified air;

(9) on day 1 post-transfection (18-22 h later), 600ul Expifeacamine ™ CHO Enhancer and 24mL ExpicHO feed were added.

(10) Supernatants were collected approximately 8 days after transfection (cell viability below 70%).

Example 8: expression of bispecific single domain antibodies against IL-17A and TNF α proteins 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 10⁵The 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 10⁶-3×10⁶Viable 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 ⁶-3×10⁶Viable cells/mL.

(3) Cells were diluted to 1X 10 with preheated OPM-293 CD05 Medium⁶Viable 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 Opti-MEM medium, and swirling or beating to mix evenly; plasmid DNA (plasmid DNA is the Fc fusion antibody eukaryotic expression vector for the bispecific single domain antibody against IL-17A and TNF α protein prepared in step (4) of example 6) was diluted with 4mL of Opt-MEM medium, vortexed, mixed, and filtered through a 0.22um filter. 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 ℃ with 5% CO₂And shake culture at 120 rpm.

(7) 5mL OPM-CHO PFF05 feed was added at 24h, 72h post transfection.

(8) Supernatants were collected approximately 7 days after transfection (cell viability below 70%).

Example 9: purification of bispecific single domain antibodies against IL-17A and TNF alpha proteins

(1) Filtering the protein expression supernatant obtained in example 7 or 8 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 using 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 bound to the packing material;

(4) washing the impurity protein bound on the column by low-salt and high-salt buffer solutions;

(5) eluting the target protein bound on the column by using a low pH buffer solution;

(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 10: construction of bispecific single domain antibody eukaryotic expression vector for resisting IL-17A/TNF alpha protein

The universal targeting vectors for single domain antibodies mentioned in the previous examples include two, vector 1 and vector 2.

The vector 1 is obtained by fusing Fc segment (namely hFC 4) in heavy chain coding sequence of human IgG4 on the basis of Invitrogen commercial vector pCDNA3.4 (vector data link: https:// Assets. thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_4_ topo _ ta _ cloning _ kit _ man. pdf), namely the vector comprises Hinge region (Hinge) CH2 and CH3 regions of IgG4 heavy chain. The vector 2 is obtained by fusing Fc segment (namely hFC 1) in heavy chain coding sequence of human IgG1 on the basis of Invitrogen commercial vector pCDNA3.4 (vector data link: https:// Assets. thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_4_ topo _ ta _ cloning _ kit _ man. pdf), namely the vector comprises Hinge region (Hinge) CH2 and CH3 regions of IgG1 heavy chain.

The specific modification scheme of the vector 1 is as follows:

(1) selecting restriction sites XbaI and AgeI on pcDNA3.4;

(2) introducing a Multiple Cloning Site (MCS) and a 6 XHis tag at the 5 'end and the 3' end of the Fc fragment coding sequence respectively by means of overlapping PCR;

(3) amplifying the Fc fragment (hFC 4) by means of PCR using a pair of primers with XbaI and AgeI cleavage sites respectively;

(4) using restriction enzymes XbaI and AgeI to respectively enzyme-cut the pcDNA3.4 and the recombinant DNA fragment obtained in the step (3);

(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 1.

The specific modification scheme of the vector 2 is as follows:

(1) selecting restriction sites XbaI and AgeI on pcDNA3.4;

(3) amplifying the Fc fragment (hFC 1) by means of PCR using a pair of primers with XbaI and AgeI cleavage sites respectively;

(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 2.

Example 11: expression and purification of Tool antibodies targeting human IL-17A (Tool antibodies, Tab) and human TNF alpha protein (Tool antibodies, Tab)

The tool antibody Tab (Secukinumab) of IL-17A is secukinumab, with the sequence from IMGT. The tool antibody Tab for TNF α is adalimumab (humira), i.e., adalimumab, sequence from IMGT.

Will search toThe above sequences were separately 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 ₂Culturing at 130rpm for about 7-8 days; collecting the expression supernatant when the cell viability is reduced to 50%, 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 IL-17A tool antibody and the TNF alpha tool antibody obtained by the method have the concentration of not less than 2 mg/ml and the purity of more than 95 percent.

Example 12: assay for bispecific single domain antibodies directed against IL-17A and TNF α proteins to block the binding of IL-17A to IL-17R

(1) Coating 50. mu.L of 1. mu.g/mL IL-17RA & IL-17RC (Acro, Cat # ILC-H5257, Lot # G173a-2085F 1-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)) and the VHH-hFc diluted in a gradient were mixed to obtain a mixture (the concentration of Biotin-IL-17A in the mixture was 3.8 ng/ml). Here, VHH-hFc means that the bispecific single domain antibody prepared in example 8 (expressed in 293F cells, fused with Fc) was purified in example 9. Furthermore, hIgG is also provided; wherein hIgG refers to isotype control, immunoglobulin molecules that do not bind to any target, 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; 50 μ L of Streptavidin-HRP was added and incubated at 37 ℃ for 30 min.

(6) Washing the plate (several times of washing); 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 keep the microplate reader reading.

(8) The curve was plotted and EC50 was calculated and the results are shown in fig. 3. The abscissa in FIG. 3 represents the specific concentration of VHH-hFc in a gradient dilution. Wherein 1A10-hFC1-1B2, 2G3-hFC1-1B2, 1A10-hFC4-1B2 and 2G3-hFC4-1B2 sequentially correspond to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.

Example 13: the bispecific single-domain antibody aiming at the IL-17A and the TNF alpha protein neutralizes the human IL-17A to induce the IL-6 release experiment of Hela cells. The method is operated according to a conventional method in the field, and comprises the following specific steps:

(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 antibody herein was the bispecific single domain antibody prepared in example 8 (expressed in 293F cells) purified in example 9; besides, hIgG and Tab controls are also set respectively; tab herein, made in example 11, is a tool antibody targeting IL-17A;

(3) After incubation in a cell incubator for 24h at 37 ℃, detecting the IL-6 concentration in the supernatant by using a human IL-6 ELISA kit;

(4) according to the detection result, EC50 for neutralizing IL-17A by the antibody to induce the Hela cells to release IL-6 is calculated, and the result is shown in figure 4 and figure 5.

Wherein the abscissa represents the specific concentration of the antibody diluted in the aforementioned gradient; as can be seen from FIGS. 4-5, bispecific single domain antibodies against IL-17A and TNF α proteins were able to efficiently neutralize IL-17A and induce the release of IL-6 by HeLa cells.

Example 14: the experiments for neutralizing the human IL-17A induced release of CXCL1 from Hs27 cells by bispecific single domain antibodies against IL-17A and TNF α were performed according to the routine procedures in the art, and the specific steps are as follows:

(1) 10000 and 50 mul of Hs27 cells which are recovered and passaged for 3 times or more are paved into a 96-well plate;

(2) gradient diluted antibodies were mixed with equal volumes of 4 x 47.7 ng/ml human IL-17A and this mixture was added to cell culture wells at 50 μ l per well; the antibody is the bispecific single domain antibody prepared in example 8 (expressed in 293F cells) purified in example 9; besides, hIgG and Tab controls are also set respectively; tab herein, made in example 11, is a tool antibody targeting IL-17A;

(3) After incubation in a cell culture box at 37 ℃ for 17h, detecting the concentration of CXCL1 in the supernatant by using a human CXCL1 HTRF kit;

(4) according to the detection result, the EC50 of the antibody neutralizing IL-17A to induce the release of CXCL1 from Hs27 cells is calculated, and the result is shown in FIG. 6.

Wherein the abscissa represents the specific concentration of the antibody diluted in the aforementioned gradient; the ordinate represents the concentration of CXCL1 in the supernatant (in pg/ml); as can be seen from FIG. 6, bispecific single domain antibodies directed against IL-17A and TNF α were effective in neutralizing human IL-17A and inducing the release of CXCL1 from Hs27 cells.

Example 15: bispecific single domain antibody neutralization of human TNF α against IL-17A and TNF α induced apoptosis of L929 experiments, this example was performed according to routine methods in the art, with the following specific steps:

(1) laying the recovered L929 cells which are passaged for 3 times or more into a 96-well plate according to 10000 cells per well;

(2) gradient diluted antibodies were mixed with equal volumes of 4 x 11.82 ng/mL TNF α solution and added to the wells; the antibody herein was the bispecific single domain antibody prepared in example 8 (expressed in 293F cells) purified in example 9; besides, hIgG and Tab controls are also respectively arranged; tab, made in example 11, is a tool antibody targeting TNF α;

(3) After incubation in a Cell incubator for 24h at 37 ℃, Cell Titer Glo is used for detecting Cell viability, and a Luminescence value is read;

(4) the EC50 concentration for neutralizing TNF α induced apoptosis of L929 cells by the antibody was calculated based on the assay results, which are shown in fig. 7 and fig. 8.

Wherein the abscissa represents the specific concentration of the antibody diluted in the aforementioned gradient; from FIGS. 7 and 8, it can be seen that bispecific single domain antibodies against IL-17A and TNF α were effective in inducing apoptosis of L929 cells.

Example 16: affinity kinetic assay of bispecific single domain antibodies against IL-17A/TNF α, comprising the following steps:

(1) preparing an SD buffer solution: an appropriate amount of tween 20 was dissolved in 1 × PBS (ph7.4) so that tween 20 was 0.02% by mass.

(2) Preparing an antibody working solution: the antibody (bispecific single domain antibody prepared in example 8 (expressed in 293F cells, fused with Fc) purified in example 9) was prepared in SD buffer at 10. mu.g/mL.

(3) Preparing an antigen working solution: antigen (human TNF alpha or IL-17A) in SD buffer solution preparation 200 nM, and then according to 2 times of gradient dilution, set up a total of 5 concentration gradient, in addition to set up a zero concentration.

(4) Glycine stock (0.1M, ph 2.0) preparation: after 7.5 g of glycine solid is dissolved in a proper amount of deionized water, the pH value is adjusted to 2.0 by hydrochloric acid, and then the volume is adjusted to 100 mL by deionized water.

(5) Preparing a glycine working solution (0.01M, pH2.0): taking a proper amount of glycine stock solution, diluting the glycine stock solution by 10 times by using deionized water, and uniformly mixing.

(6) The system comprises: starting Octet 96 and Data Acquiston software in a computer matched with the Octet 96, cleaning the bottom surface and the side surface of the acquisition probe by taking a proper amount of 75% ethanol through a piece of lens wiping paper, and preheating the instrument for more than 15 min.

(7) Sensor pre-wetting: the Sensor is soaked in SD buffer solution for more than 10 minutes before the experiment is started for standby.

(8) Arranging samples to be tested: the materials are arranged in a 96-hole blackboard according to the following table, and 200 mu L/hole can be adjusted according to actual conditions.

(9) And (3) analysis program: the time can be adjusted to the specific experimental conditions, as shown in table 5 below.

TABLE 5 analytical procedures

(10) And (5) closing the instrument and the computer after the analysis is finished.

(11) And (3) data analysis: KD, Kon, Koff, X was calculated in Data Analysis software²，Full R²And the correlation constant is equal. The results are shown in tables 6 and 7.

TABLE 6 affinity data for bispecific antibodies against IL-17A

Table 7 affinity data for bispecific antibodies against TNFa

Example 17: thermostability assay (DSF) of bispecific Single Domain antibodies directed against IL-17A and TNFa proteins

Measurement of Tm value: diluting the sample concentration to 0.2mg/ml with 1 XPBS, adding sypro Orange dye to the diluted sample, the dye final concentration is 5 ×, detecting on a machine, the instrument model: q3, brand: thermo Scientific, results are shown in table 8.

TABLE 8 thermal stability test results

Example 18: development of bispecific single domain antibody purification process method for anti-IL-17A/TNFa protein

Aiming at the antibody, a purification process method suitable for the antibody needs to be developed, so that the purity of the antibody product is improved, and the requirements of subsequent experiments are met. The specific procedures are shown in tables 9-10 below, and the purification results are shown in Table 11. Bispecific single domain antibodies to eukaryotic expressed IL-17A and TNF α proteins were subjected to affinity chromatography followed by ion exchange chromatography.

TABLE 9 affinity chromatography

TABLE 10 ion exchange chromatography

TABLE 11 purity analysis by SEC of samples after two chromatography steps

Example 19: preliminary characterization analysis of bispecific single domain antibodies against IL-17A and TNFa proteins

The preliminary characterization test results and the molecular weight results are shown in tables 12 and 13, respectively. Wherein, the sample A is 1A10-hFC1-1B2, and the sample B is 2G3-hFC1-1B 2.

TABLE 12 preliminary characterization test results

TABLE 13 complete molecular weights

Example 20: bispecific single domain antibody stability experiments against IL-17A and TNFa proteins

Stability of acceleration

The chromatographically purified samples obtained according to example 18 at a concentration of 10mg/mL were tested for the corresponding test items according to the protocol and the corresponding time points in the table below. Accelerated stability sample processing conditions and test items are shown in table 14.

TABLE 14 accelerated stability conditions and test items

Accelerated stability at 40 deg.C

The experimental method comprises the following steps: placing the candidate molecules in a constant temperature and humidity box at 40 ℃ in a dark place, standing for 0, 7 and 14 days, and sampling to be detected;

a buffer system: a patent pharmaceutical platform prescription;

and (4) judging the standard: whether the variation of the physicochemical properties under high temperature conditions is within an acceptable range: the change of the main peak of icIEF is less than 15%; the SEC main peak change is less than 10 percent, and the CE main peak change is less than 5 percent; the results of the accelerated stability test at 40 ℃ are shown in Table 15.

TABLE 1540 ℃ accelerated stability test results

Accelerated stability at 25 deg.C

The experimental method comprises the following steps: placing candidate molecules in a constant temperature and humidity box at 25 ℃ in a dark place, standing for 0, 7 and 14 days, and then sampling to be detected;

a buffer system: a patent pharmaceutical platform prescription A;

and (4) judging the standard: whether the variation of the physicochemical properties under high temperature conditions is within an acceptable range: the change of the main peak of icIEF is less than 15%; the SEC main peak change is less than 10 percent, and the CE main peak change is less than 5 percent; the results of the 25 ℃ accelerated stability test are shown in Table 16.

TABLE 1625 deg.C accelerated stability test results

Stability to oxidation acceleration

The experimental method comprises the following steps: placing the candidate molecule in a light-proof buffer solution containing 0.1% tBHP, standing for 3 days at 25 ℃, and sampling for detection after 7 days;

a buffer system: a patent pharmaceutical platform prescription;

And (4) judging the standard: whether the variation of the physicochemical properties under oxidizing conditions is within an acceptable range: the change of the main peak of icIEF is less than 15%; SEC major peak variation less than 10%; CE main peak variation less than 5%; the results of the oxidation acceleration stability test are shown in table 17.

TABLE 17 Oxidation acceleration stability test results

Freeze thaw stability

The experimental method comprises the following steps: storing the candidate molecules in a refrigerator at-80 deg.C, freezing for 24 hr, thawing at 25 deg.C, mixing, freezing again, and repeating for 2 times and 4 times;

a buffer system: a patent pharmaceutical platform prescription A;

and (4) judging the standard: whether the variation of the physicochemical properties under freeze-thaw conditions is within an acceptable range: SEC major peak variation less than 10%; CE major peak variation less than 5%; the results of the freeze-thaw stability experiments are shown in table 18.

TABLE 18 Freeze thaw stability test results

Low pH challenge

The experimental method comprises the following steps: placing the candidate molecules in a buffer solution with pH of 3.0 in a dark place, standing at room temperature for 2 to 4 hours, and sampling to be detected;

a buffer system: 100 mM acetate buffer, pH 3.0;

and (4) judging the standard: whether the physicochemical properties under low pH conditions vary within acceptable ranges: SEC major peak variation less than 10%; CE major peak variation less than 5%; the results of the low pH challenge experiments are shown in table 19.

TABLE 19 Low pH challenge test results

Example 21: detection of preliminary drug efficacy of antibody in humanized mouse

A psoriasis model is established by using a humanized mouse and is used for testing the drug effect of the test article 1 and the test article 2. The 18 mice were randomly divided into 3 groups according to body weight, and each group had 6 mice, which were labeled G1-G3. Starting from Day0, each group of mice was evenly coated with IMQ cream on the bare skin of the back 1 time a Day for 5 consecutive days to construct a psoriasis-like mouse model. IgG Isotype, 2G3-hFC1-1B2 (test article 1) and 1A10-hFC1-1B2 (test article 2) were subcutaneously injected into mice in groups G1-G3, respectively. Throughout the in vivo experiment, the skin rash and desquamation conditions of the mice model area were scored daily. And (5) after the in vivo experiment is finished, collecting the skin tissue on the back of the mouse for pathological examination.

FIG. 9 is a graph of the change in body weight of mice during the experiment, and FIG. 10 is the result of pathological examination of animals; figure 11 is the change in animal clinical score during the trial. Wherein the clinical score is based on the clinical scoring criteria PASI.

In FIG. 9, A shows specific body weights of mice of groups G1-G3 during the experiment, B shows the percentage of body weights of mice of groups G1-G3 during the experiment, C shows a histogram of body weights of mice of each group at D0, D shows a histogram of body weights of mice of each group at D5, and E shows the change in body weights of mice of each group during the experiment;

In fig. 10, a shows the pathology score of the mice during the experiment, B shows the epidermal thickness of the skin tissue of the back of the mice;

in fig. 11, a shows the score of erythema on the skin of the mouse model region, B shows the score of desquamation of the skin of the mouse model region, and C shows the total clinical score of the skin of the mouse model region;

the results were as follows: the body weights of the mice in each group during the experiment show a trend of descending and then ascending, and the body weight recovery speed of the mice given with 2G3-hFC1-1B2 (test article 1) and 1A10-hFC1-1B2 (test article 2) intervention is obviously faster than that of the negative control mice. Compared with the G1 group mice, the red rash occurrence degree and the desquamation degree of the G2 and G3 group mice are also obviously reduced (P < 0.01), and the reduction of the clinical total score is also obviously different (P < 0.001). Similarly, the pathological changes in groups G2 and G3 were significantly reduced compared to the extent of the psoriasis-associated pathological changes in the group G1 mice.

In the IMQ-induced drug effect experiment of psoriasis-like diseases of mice, 2G3-hFC1-1B2 (test article 1) and 1A10-hFC1-1B2 (test article 2) can inhibit the weight reduction of the mice; the occurrence degree of the skin rash and desquamation of the mouse can be obviously weakened; can obviously alleviate pathological changes caused by IMQ.

While embodiments of the invention have been disclosed above, it is not intended to be limited to the details shown in the description and the examples, which are set forth, but are fully applicable to various fields of endeavor as are suited to the particular use contemplated, and further modifications will readily occur to those skilled in the art, since the invention is not limited to the details shown and described without departing from the general concept as defined by the appended claims and their equivalents.

Sequence listing

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Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

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Pro Cys Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr

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Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

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Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

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Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

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Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

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Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

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Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

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Ala Leu His Ser His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Leu Gly

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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

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Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

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Thr Gly Gly Gly Gly Tyr Cys Ser Ala Arg Leu Gly Glu Ala Asp Phe

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Glu Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys

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Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

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Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

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Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

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Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His

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Glu Ala Leu His Ser His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Leu

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Gly Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gln Val Gln

355 360 365

Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg

370 375 380

Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Tyr Ser Asp Ala Cys Met

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Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Val

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Ile Asp Arg Asp His Ile Thr Gln His Ala Asp Ser Val Lys Gly Arg

420 425 430

Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Thr Leu Gln Met

435 440 445

Asn Ser Leu Glu Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly

450 455 460

His Pro Pro Ser Pro Arg Phe Gly Cys Gly Leu Gly Tyr Gln Tyr Tyr

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Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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<213> Artificial Sequence (Artificial Sequence)

<400> 5

caggtgcagc tggtggagag cggcggcggc ctggtgaagc ccggcggcag cctgaggctg 60

agctgcgccg ccagcgagta cgccttcagg ctgaacagga tgggctgggt gaggcaggcc 120

cccggcaagg agagggaggg cgtggccgcc atcggcacca ggggcggcag cacctactac 180

gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa caccctgtac 240

ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc cgccggcctg 300

ggcggcgaca ccaggacccc catctacgac atcagctact ggggccaggg caccctggtg 360

accgtgagca gcgagcccaa gagctgcgac aagacccaca cctgcccccc ctgccccgcc 420

cccgagctgc tgggcggccc cagcgtgttc ctgttccccc ccaagcccaa ggacaccctg 480

atgatcagca ggacccccga ggtgacctgc gtggtggtgg acgtgagcca cgaggacccc 540

gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagccc 600

agggaggagc agtacaacag cacctacagg gtggtgagcg tgctgaccgt gctgcaccag 660

gactggctga acggcaagga gtacaagtgc aaggtgagca acaaggccct gcccgccccc 720

atcgagaaga ccatcagcaa ggccaagggc cagcccaggg agccccaggt gtacaccctg 780

ccccccagca gggacgagct gaccaagaac caggtgagcc tgacctgcct ggtgaagggc 840

ttctacccca gcgacatcgc cgtggagtgg gagagcaacg gccagcccga gaacaactac 900

aagaccaccc cccccgtgct ggacagcgac ggcagcttct tcctgtacag caagctgacc 960

gtggacaaga gcaggtggca gcagggcaac gtgttcagct gcagcgtgct gcacgaggcc 1020

ctgcacagcc actacaccca ggagagcctg agcctgagcc ccggcaagga gcccaagagc 1080

tgcgacaaga cccacacctg ccccccctgc ccccaggtgc agctggtgga gagcggcggc 1140

ggcagcgtgc aggccggcgg cagcctgagg ctgagctgcg ccgccagcgg ctacacctac 1200

tacagcgacg cctgcatggc ctggttcagg caggcccccg gcaaggagag ggagggcgtg 1260

gccgtgatcg acagggacca catcacccag cacgccgaca gcgtgaaggg caggttcacc 1320

atcagcaagg acaacgccaa gaacaccctg accctgcaga tgaacagcct ggagcccgag 1380

gacaccgcca tgtactactg cgccgccggc caccccccca gccccaggtt cggctgcggc 1440

ctgggctacc agtactacaa ctactggggc cagggcaccc aggtgaccgt gagcagc 1497

<210> 6

<211> 1500

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

caggtgcagc tggtggagag cggcggcggc ctggtgaagc ccggcggcag cctgaggctg 60

agctgcgccg ccagcggcta cacctacagc agctactgca tgggctggtt caggcaggcc 120

cccggcaagg gcagggaggg cgtggccacc atcgacaaca ggggcagcac cagctacgcc 180

gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cctgtacctg 240

cagatgaaca gcctgaggcc cgaggacacc gccgtgtact actgcgccac cggcggcggc 300

ggctactgca gcgccaggct gggcgaggcc gacttcgagt tctggggcca gggcaccctg 360

gtgaccgtga gcagcgagcc caagagctgc gacaagaccc acacctgccc cccctgcccc 420

gcccccgagc tgctgggcgg ccccagcgtg ttcctgttcc cccccaagcc caaggacacc 480

ctgatgatca gcaggacccc cgaggtgacc tgcgtggtgg tggacgtgag ccacgaggac 540

cccgaggtga agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag 600

cccagggagg agcagtacaa cagcacctac agggtggtga gcgtgctgac cgtgctgcac 660

caggactggc tgaacggcaa ggagtacaag tgcaaggtga gcaacaaggc cctgcccgcc 720

cccatcgaga agaccatcag caaggccaag ggccagccca gggagcccca ggtgtacacc 780

ctgcccccca gcagggacga gctgaccaag aaccaggtga gcctgacctg cctggtgaag 840

ggcttctacc ccagcgacat cgccgtggag tgggagagca acggccagcc cgagaacaac 900

tacaagacca ccccccccgt gctggacagc gacggcagct tcttcctgta cagcaagctg 960

accgtggaca agagcaggtg gcagcagggc aacgtgttca gctgcagcgt gctgcacgag 1020

gccctgcaca gccactacac ccaggagagc ctgagcctga gccccggcaa ggagcccaag 1080

agctgcgaca agacccacac ctgccccccc tgcccccagg tgcagctggt ggagagcggc 1140

ggcggcagcg tgcaggccgg cggcagcctg aggctgagct gcgccgccag cggctacacc 1200

tactacagcg acgcctgcat ggcctggttc aggcaggccc ccggcaagga gagggagggc 1260

gtggccgtga tcgacaggga ccacatcacc cagcacgccg acagcgtgaa gggcaggttc 1320

accatcagca aggacaacgc caagaacacc ctgaccctgc agatgaacag cctggagccc 1380

gaggacaccg ccatgtacta ctgcgccgcc ggccaccccc ccagccccag gttcggctgc 1440

ggcctgggct accagtacta caactactgg ggccagggca cccaggtgac cgtgagcagc 1500

<210> 7

<211> 1476

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

caggtgcagc tggtggagag cggcggcggc ctggtgaagc ccggcggcag cctgaggctg 60

agctgcgccg ccagcgagta cgccttcagg ctgaacagga tgggctgggt gaggcaggcc 120

cccggcaagg agagggaggg cgtggccgcc atcggcacca ggggcggcag cacctactac 180

gccgacagcg tgaagggcag gttcaccatc agcagggaca acgccaagaa caccctgtac 240

ctgcagatga acagcctgag ggccgaggac accgccgtgt actactgcgc cgccggcctg 300

ggcggcgaca ccaggacccc catctacgac atcagctact ggggccaggg caccctggtg 360

accgtgagca gcgagagcaa gtacggcccc ccctgccccc cctgccccgc ccccgagttc 420

ctgggcggcc ccagcgtgtt cctgttcccc cccaagccca aggacaccct gatgatcagc 480

aggacccccg aggtgacctg cgtggtggtg gacgtgagcc aggaggaccc cgaggtgcag 540

ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc cagggaggag 600

cagttcaaca gcacctacag ggtggtgagc gtgctgaccg tgctgcacca ggactggctg 660

aacggcaagg agtacaagtg caaggtgagc aacaagggcc tgcccagcag catcgagaag 720

accatcagca aggccaaggg ccagcccagg gagccccagg tgtacaccct gccccccagc 780

caggaggaga tgaccaagaa ccaggtgagc ctgacctgcc tggtgaaggg cttctacccc 840

agcgacatcg ccgtggagtg ggagagcaac ggccagcccg agaacaacta caagaccacc 900

ccccccgtgc tggacagcga cggcagcttc ttcctgtaca gcaggctgac cgtggacaag 960

agcaggtggc aggagggcaa cgtgttcagc tgcagcgtgc tgcacgaggc cctgcacagc 1020

cactacaccc aggagagcct gagcctgagc ctgggcgaga gcaagtacgg ccccccctgc 1080

cccccctgcc cccaggtgca gctggtggag agcggcggcg gcagcgtgca ggccggcggc 1140

agcctgaggc tgagctgcgc cgccagcggc tacacctact acagcgacgc ctgcatggcc 1200

tggttcaggc aggcccccgg caaggagagg gagggcgtgg ccgtgatcga cagggaccac 1260

atcacccagc acgccgacag cgtgaagggc aggttcacca tcagcaagga caacgccaag 1320

aacaccctga ccctgcagat gaacagcctg gagcccgagg acaccgccat gtactactgc 1380

gccgccggcc acccccccag ccccaggttc ggctgcggcc tgggctacca gtactacaac 1440

tactggggcc agggcaccca ggtgaccgtg agcagc 1476

<210> 8

<211> 1479

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caggtgcagc tggtggagag cggcggcggc ctggtgaagc ccggcggcag cctgaggctg 60

agctgcgccg ccagcggcta cacctacagc agctactgca tgggctggtt caggcaggcc 120

cccggcaagg gcagggaggg cgtggccacc atcgacaaca ggggcagcac cagctacgcc 180

gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cctgtacctg 240

cagatgaaca gcctgaggcc cgaggacacc gccgtgtact actgcgccac cggcggcggc 300

ggctactgca gcgccaggct gggcgaggcc gacttcgagt tctggggcca gggcaccctg 360

gtgaccgtga gcagcgagag caagtacggc cccccctgcc ccccctgccc cgcccccgag 420

ttcctgggcg gccccagcgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc 480

agcaggaccc ccgaggtgac ctgcgtggtg gtggacgtga gccaggagga ccccgaggtg 540

cagttcaact ggtacgtgga cggcgtggag gtgcacaacg ccaagaccaa gcccagggag 600

gagcagttca acagcaccta cagggtggtg agcgtgctga ccgtgctgca ccaggactgg 660

ctgaacggca aggagtacaa gtgcaaggtg agcaacaagg gcctgcccag cagcatcgag 720

aagaccatca gcaaggccaa gggccagccc agggagcccc aggtgtacac cctgcccccc 780

agccaggagg agatgaccaa gaaccaggtg agcctgacct gcctggtgaa gggcttctac 840

cccagcgaca tcgccgtgga gtgggagagc aacggccagc ccgagaacaa ctacaagacc 900

accccccccg tgctggacag cgacggcagc ttcttcctgt acagcaggct gaccgtggac 960

aagagcaggt ggcaggaggg caacgtgttc agctgcagcg tgctgcacga ggccctgcac 1020

agccactaca cccaggagag cctgagcctg agcctgggcg agagcaagta cggccccccc 1080

tgccccccct gcccccaggt gcagctggtg gagagcggcg gcggcagcgt gcaggccggc 1140

ggcagcctga ggctgagctg cgccgccagc ggctacacct actacagcga cgcctgcatg 1200

gcctggttca ggcaggcccc cggcaaggag agggagggcg tggccgtgat cgacagggac 1260

cacatcaccc agcacgccga cagcgtgaag ggcaggttca ccatcagcaa ggacaacgcc 1320

aagaacaccc tgaccctgca gatgaacagc ctggagcccg aggacaccgc catgtactac 1380

tgcgccgccg gccacccccc cagccccagg ttcggctgcg gcctgggcta ccagtactac 1440

aactactggg gccagggcac ccaggtgacc gtgagcagc 1479

<210> 9

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Glu Tyr Ala Phe Arg Leu Asn Arg

1 5

<210> 10

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gly Tyr Thr Tyr Ser Ser Tyr Cys

1 5

<210> 11

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gly Tyr Thr Tyr Tyr Ser Asp Ala Cys

1 5

<210> 12

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Ile Gly Thr Arg Gly Gly Ser Thr

1 5

<210> 13

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Ile Asp Asn Arg Gly Ser Thr

1 5

<210> 14

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Ile Asp Arg Asp His Ile Thr

1 5

<210> 15

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ala Ala Gly Leu Gly Gly Asp Thr Arg Thr Pro Ile Tyr Asp Ile Ser

1 5 10 15

Tyr

<210> 16

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Ala Thr Gly Gly Gly Gly Tyr Cys Ser Ala Arg Leu Gly Glu Ala Asp

1 5 10 15

Phe Glu Phe

<210> 17

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Ala Ala Gly His Pro Pro Ser Pro Arg Phe Gly Cys Gly Leu Gly Tyr

1 5 10 15

Gln Tyr Tyr Asn Tyr

20

<210> 18

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 19

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 20

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Met Gly Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala

1 5 10 15

Ala

<210> 21

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val Ala

1 5 10 15

Thr

<210> 22

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala

1 5 10 15

Val

<210> 23

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 24

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 25

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Gln His Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn

1 5 10 15

Ala Lys Asn Thr Leu Thr Leu Gln Met Asn Ser Leu Glu Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 26

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 27

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 28

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

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> 29

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

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

85 90 95

Tyr Cys Ser Ala Arg Leu Gly Glu Ala Asp Phe Glu Phe Trp Gly Gln

100 105 110

Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 30

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Glu Val Ser Gly Tyr Thr Tyr Tyr Ser Asp Ala Cys Met Ala Trp

20 25 30

Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Val Ile Asp

35 40 45

Arg Asp His Ile Thr Gln His Ala Asp Ser Val Lys Gly Arg Phe Thr

50 55 60

Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Thr Leu Gln Met Asn Ser

65 70 75 80

Leu Glu Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly His Pro

85 90 95

Pro Ser Pro Arg Phe Gly Cys Gly Leu Gly Tyr Gln Tyr Tyr Asn Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

Claims

1. A bispecific single domain antibody directed to IL-17A and TNF α, characterized in that said bispecific single domain antibody comprises (a) a first antigen-binding moiety for specifically binding IL-17A; and (b) a second antigen-binding moiety for specifically binding TNF α; the first antigen-binding moiety and the second antigen-binding moiety are fused to each other; the first antigen-binding portion is a heavy chain comprising CDR1, CDR2, and CDR 3; the second antigen-binding portion is a heavy chain comprising CDR1, CDR2, and CDR 3;

The bispecific single-domain antibody aiming at the IL-17A and the TNF alpha is 1) or 2) as follows:

1) the amino acid sequence of CDR1 of the first antigen binding portion is shown as SEQ ID NO. 9, the amino acid sequence of CDR2 is shown as SEQ ID NO. 12, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 15;

the amino acid sequence of CDR1 of the second antigen binding portion is shown as SEQ ID NO. 11, the amino acid sequence of CDR2 is shown as SEQ ID NO. 14, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 17;

2) the amino acid sequence of CDR1 of the first antigen binding portion is shown as SEQ ID NO. 10, the amino acid sequence of CDR2 is shown as SEQ ID NO. 13, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 16;

the amino acid sequence of CDR1 of the second antigen binding portion is shown as SEQ ID NO. 11, the amino acid sequence of CDR2 is shown as SEQ ID NO. 14, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 17.

2. The bispecific single domain antibody against IL-17A and TNF α of claim 1, wherein said first and second antigen-binding moieties further comprise a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR 4;

when the bispecific single domain antibody aiming at IL-17A and TNF alpha is shown in the 1), the FR1 sequence of the first antigen binding part is shown as SEQ ID NO. 18, the FR2 sequence is shown as SEQ ID NO. 20, the FR3 sequence is shown as SEQ ID NO. 23, and the FR4 sequence is shown as SEQ ID NO. 26; the FR1 sequence of the second antigen-binding portion is shown as SEQ ID NO. 19, the FR2 sequence is shown as SEQ ID NO. 22, the FR3 sequence is shown as SEQ ID NO. 25, and the FR4 sequence is shown as SEQ ID NO. 27;

When the bispecific single domain antibody aiming at IL-17A and TNF alpha is shown in the 2), the FR1 sequence of the first antigen binding part is shown as SEQ ID NO. 18, the FR2 sequence is shown as SEQ ID NO. 21, the FR3 sequence is shown as SEQ ID NO. 24, and the FR4 sequence is shown as SEQ ID NO. 26; the FR1 sequence of the second antigen-binding portion is shown as SEQ ID NO. 19, the FR2 sequence is shown as SEQ ID NO. 22, the FR3 sequence is shown as SEQ ID NO. 25, and the FR4 sequence is shown as SEQ ID NO. 27.

3. The bispecific single domain antibody according to claim 1, wherein said first antigen-binding moiety and said second antigen-binding moiety are both humanized single domain antibodies.

4. The bispecific single domain antibody according to claim 1, wherein said first and second antigen-binding moieties are fused to each other via the Fc-domain of human IgG, the first antigen-binding moiety being coupled to the amino-terminus of the Fc-domain of human IgG, and the second antigen-binding moiety being coupled to the carboxy-terminus of the Fc-domain of human IgG.

5. The bispecific single-domain antibody aiming at IL-17A and TNF alpha is characterized in that the amino acid sequences of the bispecific single-domain antibody are respectively shown in SEQ ID NO. 1-4.

6. A nucleotide molecule encoding the bispecific single domain antibody against IL-17A and TNF α of any one of claims 1 to 4, wherein when said bispecific single domain antibody against IL-17A and TNF α is said 1), the nucleotide sequence of said bispecific single domain antibody is as set forth in SEQ ID NO: 5 or SEQ ID NO: 7 is shown in the specification; when the bispecific single domain antibody aiming at IL-17A and TNF alpha is shown in the 2), the nucleotide sequence of the bispecific single domain antibody is shown in SEQ ID NO: 6 or SEQ ID NO: shown in fig. 8.

7. An expression vector comprising a nucleotide molecule encoding the bispecific single domain antibody against IL-17A and TNF α of any one of claims 1 to 4 or the nucleotide molecule of claim 6.

8. A host cell expressing a bispecific single domain antibody against IL-17A and TNF α according to any one of claims 1 to 4, or comprising an expression vector according to claim 7.

9. The host cell of claim 8, wherein the host cell comprises prokaryotic cells and eukaryotic cells.

10. A recombinant protein comprising the bispecific single domain antibody against IL-17A and TNF α of any one of claims 1 to 4.

11. A pharmaceutical composition comprising a bispecific single domain antibody against IL-17A and TNF α selected from any one of claims 1-4 and a pharmaceutically acceptable carrier.

12. Agent for the treatment of psoriasis, characterized in that it has as active ingredient a bispecific single domain antibody against IL-17A and TNF α according to any one of claims 1 to 4.

13. A kit for detecting TNF α and/or IL-17A levels comprising a bispecific single domain antibody against IL-17A and TNF α of any of claims 1 to 4.

14. Use of a bispecific single domain antibody against IL-17A and TNF α according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 11, for the preparation of a medicament for the treatment of a disease, characterized in that said disease is psoriasis.