CN114149508B - Fusion protein combined with CD40L and application thereof - Google Patents

Fusion protein combined with CD40L and application thereof Download PDF

Info

Publication number
CN114149508B
CN114149508B CN202111413828.6A CN202111413828A CN114149508B CN 114149508 B CN114149508 B CN 114149508B CN 202111413828 A CN202111413828 A CN 202111413828A CN 114149508 B CN114149508 B CN 114149508B
Authority
CN
China
Prior art keywords
ser
leu
val
lys
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111413828.6A
Other languages
Chinese (zh)
Other versions
CN114149508A (en
Inventor
张鹏
郭树华
马琳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Pharmaceutical Technology Co ltd
Original Assignee
Suzhou Pharmaceutical Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Pharmaceutical Technology Co ltd filed Critical Suzhou Pharmaceutical Technology Co ltd
Priority to CN202111413828.6A priority Critical patent/CN114149508B/en
Publication of CN114149508A publication Critical patent/CN114149508A/en
Application granted granted Critical
Publication of CN114149508B publication Critical patent/CN114149508B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a fusion protein combined with CD40L and application thereof, belonging to the field of biological medicine. The fusion protein combined with CD40L does not contain a human antibody Fc structural domain, and researches show that the fusion protein can combine with CD40L and does not cause activation of any blood platelet, so that the safety of the fusion protein is greatly improved, the treatment side effect is reduced, and a new thought and method are provided for the treatment of immune system diseases. In addition, the fusion protein shows extremely high expression in the construction and screening processes of stable transgenic cell lines, and is beneficial to large-scale development and production.

Description

Fusion protein combined with CD40L and application thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a fusion protein combined with CD40L and application thereof.
Background
CD40L (SEQ ID NO: 24), also known as CD154, belongs to a member of the Tumor Necrosis Factor (TNF) family. CD40L is a type II transmembrane glycoprotein expressed primarily in mature activated CD 4-positive T lymphocytes and platelets, partially in natural killer cells, dendritic cells, monocytes macrophages, B lymphocytes, and endothelial cells, epithelial cells and vascular smooth muscle cells of nonhematopoietic origin, and The like (D' Orlando O, et al, "Outside inside signalling in CD-mediated B cell activation" ("J Biol Regul Homeost Agents.2007;21 (3-4): 49-62.; antoniades C, et al," The CD40/CD40 ligand systems: linking inflammation with atherothrombosis., "Journal of The American College of Cardiology,2009,54 (8): 669-677.). CD40L is typically present in both the form of a membrane-bound CD40 ligand (mCD 40L) and a soluble CD40L (sCD 40L). Both mCD40L and sCD40L bind to the corresponding receptor CD40 (SEQ ID NO: 25) in trimeric form and play an important role in initiating immune and modulating inflammatory responses.
Activation of CD40 binding to CD40L activates downstream signaling pathways including nuclear transcription factor NF-. Kappa.B, promotes activation of T cells and B cells, and increases expression of inflammatory factors such as IL-1, IL-6, IL-8, IL-12, IFN-. Gamma., TNF-. Alpha., monocyte chemotactic protein (MCP-1) and macrophage inflammatory protein-1 (MIP-1) on monocytes, macrophages, fibroblasts, smooth muscle cells and endothelial cells. CD40/CD40L plays an important role in the development of autoimmune diseases and in the regulation of inflammatory responses through this series of biological effects (Karnel, jodi L., et al, "Targeting the CD40-CD40L pathway in autoimmune diseases: humoral immunity and bed." Advanced Drug Delivery Reviews 141 (2018)). Prolonged overactivation of the immune system can induce autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, etc., while the development of immune antagonistic drugs against the CD40/CD40L pathway has the potential to treat a variety of autoimmune diseases.
Antibodies developed against CD40L protein have been reported in WO2002018445, CN1247472A, CN108135969A, US20180193454A1, CN106687133A and other patents, and the half-life of the antibodies in vivo is prolonged by containing a human antibody Fc domain or by PEGylation. However, anti-CD 40L antibodies containing human antibody Fc have developed severe thromboembolism in clinical trials, leading to failure in drug development, because binding of the antibody Fc fragment to its receptor fcγriia leads to activation and aggregation of platelets, which in turn, leads to thromboembolism. On the other hand, it is considered that this risk is eliminated by means of pegylation, but the immunogenicity of the protein subjected to pegylation is increased, and the pegylation also affects the functional domain of the protein to reduce the activity, and moreover, the presence of the conjugation site in the pegylation is indeterminate to increase the difficulty of process quality control.
There is currently no therapeutic antibody drug against CD40L on the market, and there is a need to develop fusion protein molecules that bind CD40L for the treatment of immune system diseases, while further reducing its side effects.
Disclosure of Invention
In view of the above shortcomings, the present invention provides a fusion protein that binds CD40L and uses thereof. The fusion protein combined with CD40L does not contain a human antibody Fc structural domain, and researches show that the fusion protein can combine with CD40L and does not cause activation of any blood platelet, so that the safety of the fusion protein is greatly improved, the treatment side effect is reduced, and a new thought and method are provided for the treatment of immune system diseases. In addition, the fusion protein shows extremely high expression in the construction and screening processes of stable transgenic cell lines, and is beneficial to large-scale development and production.
In order to achieve the above object, the present invention has the following technical scheme:
in one aspect, the invention provides a fusion protein comprising an antibody fragment that binds human CD40L protein and human serum albumin or an antibody fragment that binds human serum albumin.
Specifically, the antibody fragment binding to human CD40L protein comprises:
(1) Light chain complementarity determining region 1LCDR1 of the amino acid sequence shown in SEQ ID NO. 1 or a variant thereof;
(2) Light chain complementarity determining region 2LCDR2 of the amino acid sequence shown in SEQ ID NO. 2 or a variant thereof;
(3) Light chain complementarity determining region 3LCDR3 of the amino acid sequence shown in SEQ ID NO. 3 or a variant thereof;
(4) Heavy chain complementarity determining region 1HCDR1 of the amino acid sequence shown in SEQ ID NO. 4 or a variant sequence thereof;
(5) Heavy chain complementarity determining region 2HCDR2 of the amino acid sequence shown in SEQ ID NO. 5 or a variant sequence thereof;
(6) Heavy chain complementarity determining region 3HCDR3 of the amino acid sequence shown in SEQ ID NO. 6 or a variant sequence thereof;
preferably, the variant sequence is a CDR sequence having one or several amino acid substitutions, deletions or additions compared to the CDR from which it is derived; the substitutions are conservative substitutions.
Further specifically, the antibody fragment binding to human CD40L protein comprises:
(1) A light chain variable region VL of an amino acid sequence shown in SEQ ID NO. 7; and/or a heavy chain variable region VH of the amino acid sequence shown in SEQ ID NO. 8;
or (2), a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VL in (1); and/or a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the VH of (1);
Or (3) a VL having one or more amino acid substitutions, deletions or additions or any combination thereof as compared to the VL in (1); and/or VH having one or several amino acid substitutions, deletions or additions or any combination thereof as compared to VH in (1); the substitutions are conservative substitutions.
Further specifically, the antibody fragment binding to human CD40L protein comprises:
(1) A light chain LC comprising the amino acid sequence shown in SEQ ID NO. 9; and/or, the heavy chain HC of the amino acid sequence shown in SEQ ID NO. 10;
or (2), a heavy chain and a light chain, wherein the heavy chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the heavy chain and the light chain in (1); and/or, the light chain has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
Further specifically, the antibody fragment that binds to human CD40L protein is in the form of a Fab or scFv.
Specifically, the human serum albumin comprises an amino acid sequence shown as SEQ ID NO. 11.
Specifically, the antibody fragment combined with the human serum albumin is a single domain antibody, and the single domain antibody comprises an amino acid sequence shown as SEQ ID NO. 12.
Specifically, the human serum albumin or the antibody fragment binding to the human serum albumin is connected to the N end or the C end of the light chain or the heavy chain of the antibody fragment binding to the human CD40L protein through a flexible connecting peptide.
More specifically, the flexible connecting peptide is any one of the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 14 or SEQ ID NO. 15, preferably the amino acid sequence shown in SEQ ID NO. 15.
Specifically, the fusion protein comprises:
(1) LC as shown in SEQ ID No. 16, and/or HC as shown in SEQ ID No. 10; or alternatively, the first and second heat exchangers may be,
(2) LC as shown in SEQ ID No. 17, and/or HC as shown in SEQ ID No. 10; or alternatively, the first and second heat exchangers may be,
(3) LC as shown in SEQ ID NO. 18, and/or HC as shown in SEQ ID NO. 10; or alternatively, the first and second heat exchangers may be,
(4) LC as shown in SEQ ID NO. 19, and/or HC as shown in SEQ ID NO. 10; or alternatively, the first and second heat exchangers may be,
(5) LC as shown in SEQ ID NO. 9, and/or HC as shown in SEQ ID NO. 20; or alternatively, the first and second heat exchangers may be,
(6) LC as shown in SEQ ID NO. 9, and/or HC as shown in SEQ ID NO. 21; or alternatively, the first and second heat exchangers may be,
(7) LC as shown in SEQ ID No. 9, and/or HC as shown in SEQ ID No. 22; or alternatively, the first and second heat exchangers may be,
(8) LC as shown in SEQ ID NO. 9, and/or HC as shown in SEQ ID NO. 23.
In another aspect, the invention provides a series of nucleic acid molecules encoding said fusion proteins.
In particular, the nucleic acid molecules comprise one or more codon optimized nucleic acid molecules.
In yet another aspect, the invention provides a series of vectors comprising one or more nucleic acid molecules described herein.
In particular, the vectors include, but are not limited to, plasmids, viruses, phages.
In yet another aspect, the invention provides a series of host cells comprising the nucleic acid molecule described above or the vector described above.
In particular, the host cells include, but are not limited to, microorganisms, plants or animal cells, into which the vectors of the present invention can be introduced by methods known to those skilled in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like.
In yet another aspect, the invention provides a pharmaceutical composition comprising the fusion protein, nucleic acid molecule, vector and/or host cell described above.
Specifically, the pharmaceutical composition further comprises an optional pharmaceutically acceptable carrier.
Further specifically, the pharmaceutically acceptable carrier includes, but is not limited to: diluents, excipients, fillers, wetting agents, disintegrants, flavoring agents and binders.
In particular, the pharmaceutical composition further comprises a combination therapeutic agent including, but not limited to, chemotherapeutic agents, radiation therapeutic agents, immunosuppressants, cytotoxic drugs.
In yet another aspect, the present invention provides a method of producing the above fusion protein, said method comprising culturing the above host cell under conditions such that the fusion protein is expressed.
In yet another aspect, the invention provides the use of the fusion protein, nucleic acid molecule, vector, host cell, and/or pharmaceutical composition in the manufacture of a medicament, kit, and/or medical device for the treatment of an autoimmune disease, an anti-organ transplant rejection, or a desensitization treatment prior to organ transplantation.
In particular, the autoimmune diseases include, but are not limited to: rheumatoid arthritis, behcet's disease, multiple sclerosis (including relapsing remitting, secondary progressive and primary progressive), systemic sclerosis, optic neuritis, asthma, sjogren's Syndrome, autoimmune hemolytic anemia, psoriasis, vitiligo, dry eye, uveitis, sympathoophthalmitis, vogt-Koyanagi-Harada Syndrome, conjunctival pemphigoid, cicatricial pemphigoid, stevens-Johnson Syndrome, behcet's disease, granulomatous vasculitis or Wegener's granulomatosis, alopecia areata, age-related macular degeneration (AMD), oophoritis, systemic lupus erythematosus, lupus nephritis, drug-induced autoimmune diseases or drug-induced lupus thrombocytopenic purpura, myasthenia gravis, thyroiditis, allergic rhinitis, nasal polyp, hemolytic anemia, inflammatory bowel disease, crohn's disease, ulcerative colitis, celiac disease, igG 4-related diseases, X-linked high IgM Syndrome, mixed connective tissue disease, chronic lymphothyroiditis, hyperthyroidism, thyroid-related eye disease, graft-versus-host disease, type I diabetes, vasculitis, compulsive spondylitis, atopic dermatitis, eczema, primary biliary cirrhosis, polymyositis/dermatomyositis, myocarditis, autoimmune encephalitis, neuromyelitis optica lineage diseases and atherosclerosis, insulin resistance.
Specifically, the organ transplantation includes organ transplantation such as kidney, heart, lung, liver, pancreas, etc., and joint transplantation thereof, and transplantation of tissue or cells such as bone marrow, cornea, skin, small intestine, heart valve, blood vessel, trachea, cartilage tissue, tendon, bone, etc.
In yet another aspect, the present invention provides a method of treating an autoimmune disease by administering to a subject in need thereof an effective amount of the fusion protein, and/or pharmaceutical composition described above.
In yet another aspect, the present invention provides a drug delivery device comprising: (1) An infusion module for administering the pharmaceutical composition to a subject in need thereof, and (2) optionally a efficacy monitoring module.
Compared with the prior art, the invention has the following positive and beneficial effects:
(1) The present invention provides a fusion protein that is capable of partially or completely blocking the binding of human CD40 to human CD40L and further partially or completely blocking the biological effects of human CD40L, including, for example, promoting proliferation, differentiation of T cells and production of cytokines, promoting proliferation, differentiation of B cells and secretion of antibodies, promoting maturation and antigen presentation of APC cells, and production of cytokines such as IL-1, IL-6, IL-8, IL-12 and TNF- α.
(2) The fusion protein does not contain a human antibody Fc structural domain, does not cause activation of any blood platelet while maintaining the biological activity or blocking the activity, greatly improves the safety of the fusion protein while ensuring the biological activity, reduces the treatment side effect, and provides a new thought and method for treating immune system diseases.
(3) The fusion protein of the invention shows extremely high expression in the construction and screening process of stable transgenic cell lines, is beneficial to large-scale development and production, and reduces production cost.
Drawings
FIG. 1 is a graph showing the results of detecting the purity of fusion protein 1.
FIG. 2 is a graph showing the results of detecting the purity of fusion protein 2.
FIG. 3 is a graph showing the results of detecting the purity of fusion protein 3.
FIG. 4 is a graph showing the results of detecting the purity of fusion protein 4.
FIG. 5 is a graph showing the results of blocking the binding of CD40 to CD40L by the fusion protein.
FIG. 6 is a graph showing the results of the platelet activation test by the positive control antibody.
FIG. 7 is a graph showing the results of detection of platelet activation by fusion protein 1.
FIG. 8 is a graph showing the results of platelet activation detection by a negative control antibody.
FIG. 9 is a graph showing the results of platelet activation assay by the negative control.
FIG. 10 is a graph showing the results of the platelet activation assay by Letolizumab.
FIG. 11 is a graph showing scoring results of the mouse EAE model.
FIG. 12 is a graph showing the results of body weight change in the EAE model of mice.
FIG. 13 is an exemplary graph of the results of LFB staining of mice in the EAE group administered with fusion protein 1.
FIG. 14 is an exemplary graph of spinal cord LFB staining results from PBS-treated EAE mice.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Definition of terms
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
The three-letter and one-letter codes for amino acids used in the present invention are as described in J.biol. Chem,243, p3558 (1968, IUPAC-IUB Commission).
The antibody disclosed by the invention refers to an immunoglobulin, and is a tetrapeptide chain structure formed by linking two identical heavy chains (H) and two identical light chains (L) through inter-chain disulfide bonds (S-S). The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or isotypes, i.e., igM, igD, igG, igA, igE, with their respective heavy chains being μ, δ, γ, α, ε chains, respectively. The Ig of the same class can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, for example, igG can be classified into IgG1, igG2, igG3 and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each of the five classes of Ig may have either a kappa chain or a lambda chain.
The sequences of the heavy and light chains of antibodies, near the N-terminus, vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each light chain variable region (VL or LCVR) and heavy chain variable region (VH or HCVR) consists of 3 CDR regions and 4 FR regions, arranged in order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR3 and the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.
The term "complementarity determining region CDR" refers to: the antibody variable domains are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or regions containing antigen-contacting residues ("antigen-contacting points"). CDRs are mainly responsible for binding to the epitope. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3. CDRs may be determined according to various numbering systems known in the art, for example Kabat, chothia or AbM or IMGT numbering systems. The CDR partitioning of the present invention is based on the definition of CDRs by Kabat et al (Kabat et al Sequences of Proteins of Immunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, national Institutes of Health (1987)).
The term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
The terms "specific binding", "selective binding" refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the dissociation equilibrium constant (KD) of the interaction. In this context, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is the ratio of the dissociation rate constant and the binding rate constant (kdis and kon, respectively), which is used to describe the binding affinity between an antibody and an antigen. The smaller the dissociation equilibrium constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. Typically, the antibody is present at about less than 10 -8 M, e.g. less than about 10 -9 M、10 -10 M、10 -11 Affinity (KD) binding of M or less.
The term "flexible connecting peptide" refers to: a linker peptide consisting of amino acids, such as glycine and/or serine residues, used alone or in combination, to link the individual variable domains in the antibody. The flexible linkages that can be used have many different forms of sequence, such as GGGGSGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSAGSAAGSGEF, EPSGPISTINSPPSKESHKSP, GTVAAPSVFIFPPSD, GGGGIAPSMVGGGGS, GGGGKVEGAGGGGGS, GGGGSMKSHDGGGGS, GGGGNLITIVGGGGS, GGGGVVPSLPGGGGS, GGEKSIPGGGGS, RPLSYRPPFPFGFPSVRP, YPRSIYIRRRHPSPSLTT, TPSHLSHILPSFGLPTFN, RPVSPFTFPRLSNSWLPA, SPAAHFPRSIPRPGPIRT, APGPSAPSHRSLPSRAFG, PRNSIHFLHPLLVAPLGA, MPSLSGVLQVRYLSPPDL, SPQYPSPLTLTLPPHPSL, NPSLNPPSYLHRAPSRIS, LPWRTSLLPSLPLRRRP, PPLFAKGPVGLLSRSFPP, VPPAPVVSLRSAHARPPY, PNVAHVLPLLTVPWDNLR, etc.
The term "single domain antibody" refers to: the minimum antigen binding unit of the antibody molecule is a novel antibody which only retains the heavy chain variable region and has antigen binding activity, and is cloned from camelid and cartilaginous fish serum, and the single domain antibody in the invention comprises an unmodified heavy chain variable region sequence or a humanized modified heavy chain variable region sequence from nature.
The term "Fab" fragment includes both the heavy chain variable domain and the light chain variable domain, and also includes the constant domain of the light chain as well as the first constant domain of the heavy chain (CH 1). In some embodiments, the fusion protein may be a fusion of Fab and HSA or anti-HSA single domain antibodies, or a fusion of scFv and HSA or anti-HSA single domain antibodies, and the manner of attachment of the fusion protein may begin at the amino or carboxy terminus of the CD40L antibody fragment.
The term "mutant" refers to: novel molecules formed by gene level or amino acid level insertion, deletion or substitution processes that differ from the original polypeptide or protein sequence and have amino acid sequences that are substantially homologous to the original molecule, which may result in the novel molecule having increased, similar or decreased functions or properties as the original molecule due to one or more different amino acid deletions, insertions, and or substitutions. "mutant" in the present invention refers to a molecule that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a prosequence at the amino acid level. A variant nucleic acid sequence may be a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference nucleotide sequence. Identity of two amino acid sequences or two nucleic acid sequences can be determined more easily by visual inspection and/or mathematical calculation, or by comparing sequence information using known computer programs for sequence comparison (e.g., clustal package version 1.83). A variant may comprise a sequence of amino acids with at least one conservative substitution, which means that a given amino acid residue is substituted by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as Ile, val, leu or Ala for each other, or the substitution of one polar residue for another, such as between Lys and Arg; substitution between Glu and Asp; or substitution between Gln and Asn. Other such conservative substitutions, for example substitutions of the entire region with similar hydrophobic characteristics, are well known (Kyte, jack & Doolittle, russell. (1982), "A Simple Method for Displaying the Hydropathic Character of a protein.," J.mol. Biol.. 157.105-132.). For example, a "conservative amino acid substitution" may involve substitution of a natural amino acid residue with a non-natural residue such that there is little effect on the polarity or charge of the amino acid residue at that position. Alternatively, substitutions of one or more amino acids present in the original polypeptide are not conservative, which may result in a variant having altered properties as compared to the reference antibody. The desired amino acid substitutions (whether conservative or non-conservative) may be determined by one of ordinary skill in the art when such substitutions are desired. The term "variant" also includes peptides or polypeptides that are substantially homologous to a reference peptide sequence, but which have an amino acid sequence that differs from the reference sequence in that one or more amino acids have been chemically modified or substituted with an amino acid analog.
The term "single chain antibody (scFv)" is an antibody in which the VL and VH regions are joined by a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, wherein the linker is long enough to allow the protein chain to fold upon itself, thereby forming a monovalent antigen binding site (see, e.g., bird et al, 1988,Science 242:423-26 and Huston et al, 1988,Proc.Natl.Acad.Sci.USA 85:5879-83).
The terms "N-terminus" and "C-terminus" refer to the free amino terminus (-NH) of the amino acid sequence of a polypeptide or polypeptide fragment, and of a protein or protein fragment 2 N-terminal) and carboxy-terminal (-COOH, C-terminal).
The term "identity" generally refers to the percentage of amino acid residues or nucleotides in a query sequence that are identical to residues in a second reference polypeptide sequence or portion thereof after aligning the sequences, introducing GAPS (GAPS) as necessary to achieve the maximum percent sequence identity, and not taking into account any conservative substitutions as part of the sequence identity. The alignment used to determine the percent amino acid/nucleotide sequence identity can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR). One skilled in the art can determine suitable parameters for measuring the alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences compared. The percent identity may be determined over the length of the entire defined polypeptide/polynucleotide sequence, or may be determined over a shorter length, such as over the length of a fragment obtained from a larger defined polypeptide/polynucleotide sequence. It is understood that any fragment length supported by the sequences shown herein can be used as a length in a table, drawing or sequence listing as a percentage of measurable identity. Sequences having "% identity" retain important biological activity, such as antibody binding specificity, of the sequence to which they are compared or from which they are derived. Sequences having one or several amino acid substitutions, deletions or additions, or any combination thereof, retain important biological activity, such as antibody binding specificity, of the sequences to which they are compared or from which they are derived. Nucleotide sequences having "% identity" or nucleotide sequences differing by no more than 3, 6, 15, 30 or 45 nucleotides are capable of performing a function similar to the nucleotide sequence from which they are compared or derived, e.g., the expressed protein is capable of specifically binding to the same antigen or molecule.
The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus (e.g., AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9, etc.), herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.
The term "human serum albumin" refers to: human Serum Albumin (HSA), a plasma protein capable of long-term retention in plasma, has an in vivo half-life of up to 19 days. HSA has a relative molecular mass of about 66000 daltons and consists of 3 homologous domains, and is one of the major components of human plasma (content up to 40 g/L). Because of its good water solubility, it is non-immunogenic and itself acts as a carrier for a variety of biomolecules in vivo.
The term "host cell" refers to: cells useful for expressing nucleic acids, e.g., the nucleic acids of the invention. The host cell may be a prokaryote, e.g., escherichia coli (e.coli), or it may be a eukaryote, e.g., a single cell eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), an animal cell (e.g., a human cell, monkey cell, hamster cell, rat cell, mouse cell, or insect cell), or a hybridoma. Examples of host cells include COS-7 cell lines of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al, 1981, cell 23:175), 3T3 cells (ATCC CCL 163), chinese Hamster Ovary (CHO) cells or derivatives thereof such as Veggie CHO and related cell lines grown on serum-free medium (see Rasmussen et al, 1998,Cytotechnology 28:31) or CHO lines DX-B11 (see Urlaub et al, 1980,Proc.Natl.Acad.Sci.USA 77:4216-20) (said cell lines defective in DHFR), heLa cells, BHK (ATCC CRL 10) cell lines, CV1/EBNA cell lines derived from African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al, 1991,EMBO J.10:2821), human embryonic kidney cells such as 293, human epidermal A431 cells, human Colo20 cells, other transformed primate cell lines, normal diploid cells, primate tissue derived from in vitro cultures of primate explants, HL-60, U937 or Jurt cells. Generally, a host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which nucleic acid can therefore be expressed in the host cell.
The term "recombinant host cell" may be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. If the regulatory sequences are not introduced into the host cell so that they are operably linked to the nucleic acid, the host cell may also be a cell comprising the nucleic acid but not expressing it at the desired level. It is to be understood that the term host cell refers not only to a particular cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, for example, mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
The term "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, which are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier may be suitable for intravenous, intraperitoneal, intramuscular or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional medium or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds may also be incorporated into the compositions. The carrier or excipient can be used for treating diseases simultaneously or sequentially with other medicaments or active substances, wherein the medicaments or active substances can be selected from cortisone and prednisone, methylprednisolone, methotrexate, cyclophosphamide, leflunomide, mycophenolate mofetil, cyclosporin A, tacrolimus, glatiramer, azathioprine, fingolimod, teriflunomide, laquinimod, dimethyl fumarate, anthraquinone medicaments, selective sphingosine 1-phosphate (S1P) receptor modulators, potassium channel blocker, elkuizumab, natalizumab, interferon, anti-CD 19 monoclonal antibody, anti-CD 20 monoclonal antibody, B cell depleting agent, anti-IL-4R monoclonal antibody, anti-IL-33 monoclonal antibody, anti-IL-12 monoclonal antibody, anti-IL-23 monoclonal antibody, anti-IL-17 monoclonal antibody, anti-IL-6R monoclonal antibody, anti-TNF monoclonal antibody and the like.
The term "pharmaceutical composition" generally refers to a formulation that exists in a form that allows for the biological activity of the active ingredient to be effective and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the composition is to be administered. The composition is sterile.
The term "subject" refers to a mammal, such as a primate mammal, e.g., a non-human primate mammal or a human. In certain embodiments, the subject (e.g., human) has, or is at risk of having, an autoimmune disease.
The term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of a disease; a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.
The term "autoimmune disease" refers to a disease caused by the damage of autoantibodies or/and sensitized lymphocytes to the tissues of the organs of the body, which is caused by the reduced or destroyed immune tolerance of the immune system to the self-components for some reason, and is manifested as dysfunction of the corresponding tissues and organs. Autoimmune diseases may be selected from the following: allergic reactions, ankylosing spondylitis, asthma, psoriasis, sjogren's syndrome, atherosclerosis, autoimmune hepatitis, autoimmune parotitis, colitis, igG 4-related diseases, behcet's disease, immune vasculitis, immune myocarditis, graft-related diseases, crohn's disease, diabetes (including type 1 and/or type 2 diabetes), glomerulonephritis, graves ' disease, guillain-barre syndrome, hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura (also known as primary immune thrombocytopenia), inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus (e.g., pemphigus vulgaris and leaf-shaped pemphigus), rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (systemic sclerosis), spondyloarthropathies, thyroiditis, transplant rejection. Autoimmune mediated disorders include, but are not limited to, conditions in which the affected tissue is the primary target, and in some cases the secondary target. Such conditions include, but are not limited to, AIDS, atopic allergies, bronchial asthma, eczema, tissue and organ transplantation (e.g., solid organ transplantation), chronic fatigue syndrome, alzheimer's disease, parkinson's disease, myocardial infarction, stroke, epilepsy, allergic reactions. In a specific embodiment, the autoimmune disease model is experimental allergic encephalomyelitis in mice, corresponding to multiple sclerosis in humans.
In the present invention, unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.
Exemplary techniques for use in connection with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification techniques are known in the art. Many such techniques and procedures are described, for example, in Sambrook et al, molecular μlar Cloning: a Laboratory Manual (version 2, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (1989)), among many others. In addition, exemplary techniques for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment are also known in the art.
In this application, the use of "or" means "and/or" unless stated otherwise. In the case of multiple dependent claims, the use of "or" in the alternative only refers to more than one of the foregoing independent or dependent claims.
As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the range and to include fractions thereof (such as tenths and hundredths of integers) as appropriate, unless otherwise indicated.
Units, prefixes, and symbols are all expressed in their international system of units (SI) accepted form. Numerical ranges include numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure which can be had by reference to the specification as a whole.
Abbreviations
CDR: complementarity determining regions in immunoglobulin variable regions.
VH: antibody heavy chain variable regions.
VL: antibody light chain variable regions.
HC: antibody heavy chain.
LC (liquid crystal): an antibody light chain.
IgG: immunoglobulin G.
AbM: abM CDR definition was derived from Martin's related studies (Martin ACR, cheethane JC, rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), which integrates part of the definitions of both Kabat and Chothia.
Kabat: the immunoglobulin alignment and numbering system as proposed by Elvin a.kabat (see, e.g., kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md, 1991).
Chothia: the immunoglobulin numbering system proposed by Chothia et al is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, e.g., chothia & Lesk (1987) J. Mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883).
IMGT: based on the international immunogenetic information system initiated by Lefranc et al (The international ImMunoGeneTics information system
Figure BDA0003375174570000141
(IMGT)) can be found in Lefranc et al, dev. Comparat. Immunol.27:55-77,2003.
mAb: a monoclonal antibody.
EC50: resulting in a concentration of 50% efficacy or binding.
IC50: resulting in a 50% inhibitory concentration.
ELISA: enzyme-linked immunosorbent assay.
And (2) PCR: polymerase chain reaction.
HRP: horseradish peroxidase.
hFc: human IgG antibody Fc fragment.
KD: dissociation equilibrium constant.
HCDR1: complementarity determining region 1 in the immunoglobulin heavy chain variable region.
HCDR2: complementarity determining region 2 in the immunoglobulin heavy chain variable region.
HCDR3: complementarity determining region 3 in the immunoglobulin heavy chain variable region.
LCDR1: complementarity determining region 1 in the immunoglobulin light chain variable region.
LCDR2: complementarity determining region 2 in the immunoglobulin light chain variable region.
LCDR3: complementarity determining region 3 in the immunoglobulin light chain variable region.
EXAMPLE 1 expression and purification of fusion proteins
In certain embodiments, the antigen binding proteins provided herein include heavy chain HC and light chain LC as follows:
(1) Fusion protein 1, HC as shown in SEQ ID NO. 21, and/or LC as shown in SEQ ID NO. 9; or alternatively, the first and second heat exchangers may be,
(2) Fusion protein 2, HC as shown in SEQ ID NO. 31, and/or LC as shown in SEQ ID NO. 9; or alternatively, the first and second heat exchangers may be,
(3) Fusion protein 3, HC as shown in SEQ ID NO. 23, and/or LC as shown in SEQ ID NO. 9;
(4) Fusion protein 4, HC as shown in SEQ ID NO. 32, and/or LC as shown in SEQ ID NO. 9.
The light chain sequence and the heavy chain fragment sequence of the fusion protein are respectively subjected to amino acid codon optimization according to a human host cell expression system, then a signal peptide nucleotide sequence for protein expression is added, gene synthesis is carried out, then the synthesized target gene is cloned to a vector pTT5 (ampicillin resistance) through 5'EcoRI and 3' HindIII, and after construction, the light chain vector and the heavy chain vector are respectively subjected to combined expression. Selecting clones for sequencing, selecting thalli with correct sequencing for seed preservation, and performing expansion culture of thalli, wherein the expanded thalli are used for extracting plasmids.
Cell transfection and protein isolation and purification were performed on the extracted plasmids as follows:
1. determination of cell density, viability should be greater than95%, HEK293 cells (purchased from ATCC under the trade designation CRL-1573.3) were density-adjusted to 3X 10 with (pre-warmed) HEK293 medium 6 The cells/mL were gently shaken and the cells were aliquoted (the transfection amount was 90% of the transfection system), taking care that the cell volume in the shake flask was not more than 1/3 of the shake flask format, and put into a shaker for use.
2. Calculating the volume of the transfection buffer opti-MEM according to the volume of transfected cells, wherein the volume is 1/10 of that of the transfection system; calculating the amount of the transfection reagent PEI, wherein the proportion of the amount of the transfection reagent PEI is 3 mu L/mL of transfected cells; the total amount of transfected DNA was calculated at a ratio of 1. Mu.g/mL transfected cells.
The specific transfection procedure was as follows:
taking 1 50mL centrifuge tube, adding MEM with 10% of transfection system, adding plasmid, mixing, filtering, standing for 5min, adding PEI into DNA suspension, mixing gently (mixing gently for 2-3 times), and standing for 15-20min. Then the compound is gently added into the packaged cells, and the shake flask is gently shaken while being added; the transfected cells were placed in a shaking table at 37℃for cultivation. The fusion protein in the supernatant expressed by HEK293 cells is separated and purified by a conventional affinity purification method, and the brief steps are that a filling column is balanced by 5-10 times of volume of binding buffer, a supernatant sample is loaded, and the binding buffer is used for eluting the hybrid protein and the protein. After purification, the characterization result shows that the fusion protein 1 has a monomer purity of 97.8%, and the fusion proteins 2, 3 and 4 have lower purities after protein purification, namely 67.4%, 77.3% and 87.5%, respectively, and the detection results are shown in figures 1-4.
Next, further evaluation studies were performed on the fusion protein 1.
Example 2 detection of binding of fusion proteins to human CD40L and HSA antigens
The affinity of the purified fusion protein 1 of example 1 with CD40L protein (manufacturer: beijing Baiposis Biotechnology Co., ltd., cat# CDL-H5248) and HSA (manufacturer: beijing Baiposis Biotechnology Co., ltd., cat# HSA-H5220) was measured using a Gator non-labeled bioanalyzer. CD40L and HSA are captured by a biosensor with an anti-His tag (the loading concentration is 10 mu g/mL), and then the captured sample and the fusion protein are subjected to dynamic detection of binding and dissociation. Kinetic use 1:1 fitting analysis was performed in conjunction with the model. The method comprises the following brief action steps: protein loading was 200s, binding 180s, dissociation 300s, regeneration 30s. The affinities determined are shown in table 1 below.
TABLE 1
Figure BDA0003375174570000161
Figure BDA0003375174570000171
As shown in the above table, fusion protein 1 binds with high affinity to CD40L and HSA, also indicating that the expressed fusion protein has the correct folded conformation and that the functional domains that bind CD40L and to HSA do not interact. Meanwhile, due to the normal binding with HSA, the fusion protein 1 is ensured to have a good half-life in vivo (Low S, et al, "VHH antibody targeting the chemokine receptor CX3CR1 inhibits progression of atherocross." MAbs.2020 Jan-Dec;12 (1): 1709322.).
Example 3 determination of the blocking of CD40 binding to CD40L by the fusion protein
CD40 protein (manufacturer: beijing Bai Sai Biotechnology Co., ltd., product No. CD 0-H5228) was coated at 4℃in ELISA plates overnight, and after the completion of the blocking, the fusion protein 1 obtained in example 1 was subjected to gradient dilution at an initial concentration of 50. Mu.g/mL and then mixed with biotinylated CD40L protein (manufacturer: beijing Bai Sai Biotechnology Co., ltd., product No. CDL-H82F 1). And adding 100 mu L of mixed solution into each hole of the ELISA plate, incubating at 37 ℃, adding the ELISA secondary antibody after 6 times of plate washing, incubating at 37 ℃, and performing color development and result reading after 6 times of plate washing. The results of the measurement are shown in FIG. 5, and the IC50 value thereof was 0.1182. Mu.g/mL.
The results are shown in FIG. 5, where fusion protein 1 can form an effective binding block for CD40 and CD40L at lower concentrations, further indicating that the fusion protein has the ability to block CD40 and CD40L signaling.
Example 4 detection of the activated platelet Effect of fusion proteins
Reference example 1 was made to positive control antibodies with platelet activating function (see patent WO2001030386, wherein heavy chain sequence SEQ ID NO:26, light chain sequence SEQ ID NO: 27) and negative control antibodies without platelet activating function (see patent US20180193454, wherein heavy chain sequence SEQ ID NO:28, light chain sequence SEQ ID NO: 29) as well as to antibodies Letolizumab engineered by Fc segment (see patent CN106132429A, sequence SEQ ID NO: 30) for gene synthesis and expression purification of antibodies. The evaluation of the extent of platelet activation was performed by the different antibodies inducing the platelet cell surface marker PAC-1.
Specifically, whole blood was drawn from healthy volunteers into 3.2% sodium citrate tubes, and the first 2mL was discarded. Platelet rich plasma was prepared by centrifugation at 120g for 15 min and platelet counts were normalized to 1 x 10 with phosphate buffered saline 5 Individual cells/mL. Immunocomplexes of recombinant human CD40L (manufacturer: beijing Baiposis Biotechnology Co., ltd., cat# CDL-H5248) with test positive control antibody, negative control antibody, antibody Letolizumab and fusion protein 1 were prepared by pre-incubation at room temperature for 15 minutes at a 3:1 CD40L to antibody molar ratio. The immunocomplex mixture was diluted to a final concentration of 5 μg/mL CD40L in normalized PBS/platelet solution and then incubated for 30 minutes at 37 ℃. The negative controls were untreated platelets alone and CD40L. After incubation for 30 minutes, anti-human PAC-1-FITC (manufacturer: semerle Feishmanic technologies (China) Co., ltd., cat# MA 5-28564) conjugated antibodies were added to all samples and incubated for 15 minutes. Sample 1:1 was diluted into 2% paraformaldehyde in PBS buffer, fixed on ice for 30 min, and centrifuged at 100g for 5 min to pellet the cells. Cells were resuspended in PBS. Fluorescence activated cell detection was performed on a flow cytometer.
The results of the assay are shown in FIGS. 6-10, where the positive control antibody has a significant platelet activating effect (21.9% activated red blood cells), and neither the fusion protein 1 of the invention nor the negative control antibody nor the negative control has significant platelet activation, but the Fc engineered Letolizumab antibody still shows the ability to activate platelets (10.6% activated red blood cells, indicating that thromboembolism can still result). The results of this example demonstrate that the fusion protein 1 of the present invention is a safer molecular form, can be used without causing platelet activation, aggregation, and can avoid the risk of thromboembolism in subjects, which provides safety guarantee for the development of post-drug molecules.
Example 5 test of the efficacy of fusion protein 1 in the treatment of Experimental allergic encephalomyelitis in mice
The experimental allergic encephalomyelitis model (experimental autoimmune encephalomyelitis, EAE) was constructed by subcutaneously injecting 18 mice with MOG35-55/CFA emulsifier (MOG 35-55 from Tocris Bioscience, cat No. 2568; CFA from Merck KgaA, cat No. F5881, the administration of the emulsifier being Day 0) and PTX (from APExBIO Technology, cat No. B7273) at Day0 and Day2 tail veins.
Mice were divided into 2 groups by the random block method, and the group information is shown in Table 2 below, PBS and fusion protein were injected intravenously at the tails of day1, day4, day8, day11, day14, day18, day21 and day24, respectively, at 30mpk, after which the mice were weighed according to the experimental plan and scored by observing whether they developed the corresponding clinical symptoms (e.g., tail weakness, impaired eversion and hind limb paralysis). A portion of the mice were selected from Group1, PBS, i.v., Q3D 8times and Group2, fusion proteins 30mpk, i.v., Q3D 8times, euthanized and spinal cord from the mice, and histopathological examination was performed after tissue fixation staining.
TABLE 2 experimental grouping information
Group of Dosage (mg/k)g) Quantity of Administration mode
Group1,PBS,i.v.,Q3D*8times / 9 Tail vein injection
Group2, fusion protein 30mpk, i.v., Q3D 8times 30 9 Tail vein injection
Clinical scoring system:
0 point: the mice are healthy;
1, the method comprises the following steps: tail weakness in mice;
2, the method comprises the following steps: mice are impaired in eversion and reflection;
3, the method comprises the following steps: paralysis of one hind limb of the mouse;
4, the following steps: paralysis of two hind limbs of the mice;
5, the method comprises the following steps: weight loss of mice was 20% or more;
and 6, dividing: mice die or are dying.
The experimental results are shown in fig. 11-12, where the EAE model score of mice in the group treated with fusion protein 1 showed significant statistical differences in the model onset process compared to the control group, and 5 mice showed a score of 0 (clinical cure), which shows excellent efficacy of the fusion protein. Meanwhile, the statistical difference of the growth of the weight of the mice appears along with the experimental study, which indicates that the fusion protein 1 has no toxicity and can keep the healthy growth of the mice.
The results of LFB (Luxol Fast Blue) staining of the spinal cord of mice showed that severe demyelination occurred at the edges of the spinal cord of severely ill mice, whereas the spinal cord of mice receiving the fusion protein treatment was relatively intact and no significant demyelination lesions were seen. This demonstrates that the fusion protein can be used to effectively treat injuries to spinal cord tissue and nervous system in mice.
EXAMPLE 6 screening of CHO stable cell lines
In order to obtain a cell line suitable for process development, the fusion protein 1 of the present invention was subjected to stable cell line construction, briefly, the amino acid of the fusion protein 1 was synthesized and constructed into eukaryotic expression vectors after codon optimization according to eukaryotic expression hosts, the expression vectors were transfected into CHO cells by electrotransfection (supplier: european certified cell culture collection center, cat# 85051005), subcloned dilution plating was performed when cell viability was restored to 90% or more, and 96-well cell culture and screening were performed at a density of 0.5 cells/well. Selecting clone with higher expression quantity for feed supplement fed-batch culture, harvesting culture supernatant for detecting the expression quantity when the 14 th day of fed-batch culture or the cell activity rate is less than 80%, wherein the expression quantity of the fusion protein 1 is 10.30g/L, and selecting the cell strain for large-scale culture. The cell strains obtained by constructing other fusion proteins by adopting the same method have lower expression levels.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
SEQUENCE LISTING
<110> Pratensine medical science and technology Co., ltd
<120> a fusion protein binding to CD40L and use thereof
<130> 20211117
<160> 32
<170> PatentIn version 3.5
<210> 1
<211> 15
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 1
Arg Ala Ser Gln Arg Val Ser Ser Ser Thr Tyr Ser Tyr Met His
1 5 10 15
<210> 2
<211> 7
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 2
Tyr Ala Ser Asn Leu Glu Ser
1 5
<210> 3
<211> 9
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 3
Gln His Ser Trp Glu Ile Pro Pro Thr
1 5
<210> 4
<211> 5
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 4
Ser Tyr Tyr Met Tyr
1 5
<210> 5
<211> 17
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 5
Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe Lys
1 5 10 15
Ser
<210> 6
<211> 9
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 6
Ser Asp Gly Arg Asn Asp Met Asp Ser
1 5
<210> 7
<211> 111
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 7
Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser
20 25 30
Thr Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Trp
85 90 95
Glu Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 8
<211> 118
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 8
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr
20 25 30
Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Ser Asp Gly Arg Asn Asp Met Asp Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 9
<211> 218
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 9
Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser
20 25 30
Thr Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Trp
85 90 95
Glu Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 10
<211> 224
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 10
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr
20 25 30
Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Ser Asp Gly Arg Asn Asp Met Asp Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
<210> 11
<211> 585
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 11
Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu
1 5 10 15
Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
20 25 30
Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 45
Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
50 55 60
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu
65 70 75 80
Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
100 105 110
Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125
Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
130 135 140
Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg
145 150 155 160
Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175
Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190
Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205
Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220
Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
225 230 235 240
Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
245 250 255
Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
260 265 270
Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285
Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300
Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala
305 310 315 320
Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335
Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr
340 345 350
Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu
355 360 365
Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
370 375 380
Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu
385 390 395 400
Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415
Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys
435 440 445
Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460
Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser
465 470 475 480
Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495
Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510
Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala
515 520 525
Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540
Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys
545 550 555 560
Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575
Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 585
<210> 12
<211> 115
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 12
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 13
<211> 18
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 13
Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser
1 5 10 15
Leu Asp
<210> 14
<211> 14
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 14
Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr
1 5 10
<210> 15
<211> 9
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 15
Gly Gly Gly Gly Ser Gly Gly Gly Ser
1 5
<210> 16
<211> 812
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 16
Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser
20 25 30
Thr Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Trp
85 90 95
Glu Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Ser Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp
225 230 235 240
Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln
245 250 255
Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu
260 265 270
Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn
275 280 285
Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val
290 295 300
Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys
305 310 315 320
Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn
325 330 335
Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
340 345 350
Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu
355 360 365
Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe
370 375 380
Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp
385 390 395 400
Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly
405 410 415
Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys
420 425 430
Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln
435 440 445
Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp
450 455 460
Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys
465 470 475 480
Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp
485 490 495
Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu
500 505 510
Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp
515 520 525
Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys
530 535 540
Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu
545 550 555 560
Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu
565 570 575
Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp
580 585 590
Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val
595 600 605
Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln
610 615 620
Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys
625 630 635 640
Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn
645 650 655
Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg
660 665 670
Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys
675 680 685
Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys
690 695 700
Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val
705 710 715 720
Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe
725 730 735
His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys
740 745 750
Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys
755 760 765
Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys
770 775 780
Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys
785 790 795 800
Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
805 810
<210> 17
<211> 342
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 17
Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser
20 25 30
Thr Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Trp
85 90 95
Glu Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
225 230 235 240
Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
245 250 255
Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
260 265 270
Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala
275 280 285
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr
290 295 300
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val
305 310 315 320
Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr
325 330 335
Leu Val Thr Val Ser Ser
340
<210> 18
<211> 812
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 18
Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu
1 5 10 15
Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
20 25 30
Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 45
Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
50 55 60
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu
65 70 75 80
Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
100 105 110
Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125
Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
130 135 140
Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg
145 150 155 160
Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175
Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190
Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205
Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220
Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
225 230 235 240
Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
245 250 255
Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
260 265 270
Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285
Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300
Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala
305 310 315 320
Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335
Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr
340 345 350
Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu
355 360 365
Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
370 375 380
Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu
385 390 395 400
Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415
Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys
435 440 445
Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460
Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser
465 470 475 480
Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495
Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510
Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala
515 520 525
Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540
Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys
545 550 555 560
Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575
Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Gly Gly Ser Gly Gly
580 585 590
Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser
595 600 605
Pro Gly Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Arg Val Ser
610 615 620
Ser Ser Thr Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln
625 630 635 640
Pro Pro Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val
645 650 655
Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
660 665 670
Ile Ser Ser Val Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His
675 680 685
Ser Trp Glu Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
690 695 700
Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
705 710 715 720
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
725 730 735
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
740 745 750
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
755 760 765
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
770 775 780
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
785 790 795 800
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
805 810
<210> 19
<211> 342
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 19
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Asp Ile Val Leu
115 120 125
Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr
130 135 140
Ile Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser Thr Tyr Ser Tyr
145 150 155 160
Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
165 170 175
Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly
180 185 190
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Glu Pro
195 200 205
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Trp Glu Ile Pro Pro
210 215 220
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
225 230 235 240
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
245 250 255
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
260 265 270
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
275 280 285
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
290 295 300
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
305 310 315 320
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
325 330 335
Phe Asn Arg Gly Glu Cys
340
<210> 20
<211> 818
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 20
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr
20 25 30
Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Ser Asp Gly Arg Asn Asp Met Asp Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Ser Asp Ala His Lys Ser Glu Val
225 230 235 240
Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val
245 250 255
Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His
260 265 270
Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala
275 280 285
Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly
290 295 300
Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met
305 310 315 320
Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu
325 330 335
Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu
340 345 350
Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu
355 360 365
Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala
370 375 380
Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu
385 390 395 400
Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp
405 410 415
Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys
420 425 430
Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala
435 440 445
Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val
450 455 460
Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His
465 470 475 480
Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr
485 490 495
Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys
500 505 510
Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn
515 520 525
Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu
530 535 540
Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu
545 550 555 560
Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val
565 570 575
Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys
580 585 590
Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp
595 600 605
Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn
610 615 620
Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu
625 630 635 640
Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu
645 650 655
Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys
660 665 670
His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val
675 680 685
Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp
690 695 700
Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys
705 710 715 720
Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn
725 730 735
Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys
740 745 750
Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His
755 760 765
Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe
770 775 780
Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys
785 790 795 800
Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu
805 810 815
Gly Leu
<210> 21
<211> 348
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 21
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr
20 25 30
Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Ser Asp Gly Arg Asn Asp Met Asp Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser
225 230 235 240
Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala
245 250 255
Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln
260 265 270
Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly
275 280 285
Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
290 295 300
Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
305 310 315 320
Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser
325 330 335
Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser
340 345
<210> 22
<211> 818
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 22
Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu
1 5 10 15
Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
20 25 30
Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 45
Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
50 55 60
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu
65 70 75 80
Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95
Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
100 105 110
Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125
Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
130 135 140
Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg
145 150 155 160
Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175
Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190
Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205
Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220
Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
225 230 235 240
Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
245 250 255
Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
260 265 270
Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285
Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300
Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala
305 310 315 320
Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335
Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr
340 345 350
Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu
355 360 365
Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
370 375 380
Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu
385 390 395 400
Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415
Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys
435 440 445
Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460
Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser
465 470 475 480
Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495
Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510
Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala
515 520 525
Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540
Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys
545 550 555 560
Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575
Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Gly Gly Ser Gly Gly
580 585 590
Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro
595 600 605
Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr
610 615 620
Ser Tyr Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu
625 630 635 640
Trp Ile Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu
645 650 655
Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr
660 665 670
Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
675 680 685
Tyr Cys Thr Arg Ser Asp Gly Arg Asn Asp Met Asp Ser Trp Gly Gln
690 695 700
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
705 710 715 720
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
725 730 735
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
740 745 750
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
755 760 765
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
770 775 780
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
785 790 795 800
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
805 810 815
Lys Thr
<210> 23
<211> 348
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 23
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30
Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu
115 120 125
Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys Leu
130 135 140
Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr Tyr Met Tyr Trp
145 150 155 160
Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile Asn
165 170 175
Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe Lys Ser Lys Ala
180 185 190
Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser
195 200 205
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Ser Asp
210 215 220
Gly Arg Asn Asp Met Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val
225 230 235 240
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
245 250 255
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
260 265 270
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
275 280 285
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
290 295 300
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
305 310 315 320
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
325 330 335
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
340 345
<210> 24
<211> 261
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 24
Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly
1 5 10 15
Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu
20 25 30
Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg
35 40 45
Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val
50 55 60
Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser
65 70 75 80
Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys
85 90 95
Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu
100 105 110
Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser
115 120 125
Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
130 135 140
Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
145 150 155 160
Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
165 170 175
Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
180 185 190
Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
195 200 205
Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
210 215 220
Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
225 230 235 240
Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
245 250 255
Gly Leu Leu Lys Leu
260
<210> 25
<211> 277
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 25
Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr
1 5 10 15
Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu
20 25 30
Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val
35 40 45
Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu
50 55 60
Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His
65 70 75 80
Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr
85 90 95
Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr
100 105 110
Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly
115 120 125
Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu
130 135 140
Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys
145 150 155 160
Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln
165 170 175
Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu
180 185 190
Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile
195 200 205
Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn
210 215 220
Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp
225 230 235 240
Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His
245 250 255
Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser
260 265 270
Val Gln Glu Arg Gln
275
<210> 26
<211> 448
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 26
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr
20 25 30
Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Ser Asp Gly Arg Asn Asp Met Asp Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 27
<211> 218
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 27
Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser
20 25 30
Thr Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Trp
85 90 95
Glu Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 28
<211> 450
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 28
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Asn Gly
20 25 30
Phe Trp Ile Trp Ile Arg Lys Pro Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Gly Val Tyr Tyr Cys Ala
85 90 95
Tyr Arg Ser Tyr Gly Arg Thr Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Arg
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 29
<211> 214
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 29
Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ser Asn Leu Gly His Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asp Asp Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 30
<211> 353
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 30
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Trp Glu
20 25 30
Leu Met Gly Trp Ala Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Glu Gly Pro Gly Asp Val Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Lys Val Gly Lys Asp Ala Lys Ser Asp Tyr Arg Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Glu Pro Lys Ser Ser Asp Lys
115 120 125
Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser
130 135 140
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
145 150 155 160
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
165 170 175
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
180 185 190
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
195 200 205
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
210 215 220
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
225 230 235 240
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
245 250 255
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
260 265 270
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
275 280 285
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
290 295 300
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
305 310 315 320
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
325 330 335
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
340 345 350
Lys
<210> 31
<211> 357
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 31
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr
20 25 30
Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Ser Asp Gly Arg Asn Asp Met Asp Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser
225 230 235 240
Leu Asp Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
245 250 255
Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
260 265 270
Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
275 280 285
Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp
290 295 300
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr
305 310 315 320
Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr
325 330 335
Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu
340 345 350
Val Thr Val Ser Ser
355
<210> 32
<211> 349
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 32
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr
20 25 30
Tyr Met Tyr Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Asp Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Ser Asp Gly Arg Asn Asp Met Asp Ser Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu
225 230 235 240
Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys
245 250 255
Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg
260 265 270
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser
275 280 285
Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
290 295 300
Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu
305 310 315 320
Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu
325 330 335
Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser
340 345

Claims (6)

1. A fusion protein, said fusion protein comprising: LC as shown in seq id No. 9, and HC as shown in seq id No. 21.
2. A nucleic acid molecule encoding the fusion protein of claim 1.
3. A vector comprising the nucleic acid molecule of claim 2.
4. A host cell comprising the nucleic acid molecule of claim 2 or the vector of claim 3.
5. A pharmaceutical composition comprising the fusion protein of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3 and/or the host cell of claim 4.
6. Use of the fusion protein of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, the host cell of claim 4, and/or the pharmaceutical composition of claim 5 for the preparation of a medicament, kit and/or medical device for the treatment of autoimmune diseases, anti-organ transplant rejection or pre-organ transplant desensitization therapy.
CN202111413828.6A 2021-11-25 2021-11-25 Fusion protein combined with CD40L and application thereof Active CN114149508B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111413828.6A CN114149508B (en) 2021-11-25 2021-11-25 Fusion protein combined with CD40L and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111413828.6A CN114149508B (en) 2021-11-25 2021-11-25 Fusion protein combined with CD40L and application thereof

Publications (2)

Publication Number Publication Date
CN114149508A CN114149508A (en) 2022-03-08
CN114149508B true CN114149508B (en) 2023-04-28

Family

ID=80457577

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111413828.6A Active CN114149508B (en) 2021-11-25 2021-11-25 Fusion protein combined with CD40L and application thereof

Country Status (1)

Country Link
CN (1) CN114149508B (en)

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ZA200702427B (en) * 2004-09-17 2008-12-31 Domantis Ltd Compositions monovalent for CD40L binding and methods of use
CN101557817A (en) * 2006-09-14 2009-10-14 人类基因科学公司 Albumin fusion proteins
TWI417299B (en) * 2007-03-22 2013-12-01 Biogen Idec Inc Binding proteins, including antibodies, antibody derivatives and antibody fragments, that specifically bind cd154 and uses thereof
CN101255197B (en) * 2008-03-28 2012-01-25 浙江海正药业股份有限公司 Fusion protein for serum albumin and interleukin 1 receptor antagonist and uses thereof
US9475879B2 (en) * 2011-04-21 2016-10-25 Bristol-Myers Squibb Company Antibody polypeptides that antagonize CD40
PE20141478A1 (en) * 2011-10-13 2014-10-23 Bristol Myers Squibb Co POLYPEPTIDES OF ANTIBODIES THAT ANTAGONIZE CD40L
MA41459A (en) * 2015-02-03 2017-12-12 Als Therapy Development Inst ANTI-CD40L ANTIBODIES AND METHODS FOR TREATING CD40L ILLNESSES OR DISORDERS
EP3380517B1 (en) * 2015-11-27 2021-08-04 Ablynx NV Polypeptides inhibiting cd40l
GB201521383D0 (en) * 2015-12-03 2016-01-20 Ucb Biopharma Sprl And Ucb Celltech Method
EP3856770A4 (en) * 2018-09-26 2022-07-27 Viela Bio, Inc. Cd40l antagonist and uses thereof
CN110950967B (en) * 2019-12-13 2022-05-13 山东民康生物科技有限公司 Anti-human serum albumin nano antibody and IL-2 fusion protein and preparation method thereof
KR20210095781A (en) * 2020-01-24 2021-08-03 주식회사 에이프릴바이오 A multi-specific antibody comprising a fusion construct consisting of a Fab and a bioactive effector moiety
CN112724259B (en) * 2020-11-16 2022-12-20 天津林达生物科技有限公司 Fusion protein of human serum albumin and interleukin 2 and application thereof

Also Published As

Publication number Publication date
CN114149508A (en) 2022-03-08

Similar Documents

Publication Publication Date Title
AU2009296937B2 (en) Anti-CD147 antibodies, methods, and uses
JP4294082B2 (en) Agonist antibody to human thrombopoietin receptor
JP5435986B2 (en) Human anti-IL-1R1 monoclonal antibody for treatment
JP2021527110A (en) Antibodies capable of blocking the interaction between CD47 and SIRPa and their applications
WO2019228266A1 (en) Anti-interleukin-17a antibody, pharmaceutical composition thereof and use thereof
CN108690138A (en) It is a kind of can be with people CD19 or CD20 and people the CD3 bispecific antibody combined and its application
CN110092837A (en) UTI fusion protein
CN114149508B (en) Fusion protein combined with CD40L and application thereof
US11479603B2 (en) Polypeptide and antibody bound to polypeptide
EP3808847A1 (en) Apj antibody, fusion protein thereof with elabela, and pharmaceutical compositions and use thereof
EP3777889A1 (en) Use of probdnf regulator in b cell-related diseases
CN114874330B (en) Neutralizing monoclonal antibodies targeting single chain antibodies
US20220356259A1 (en) Isolated antigen-binding protein and application thereof
US20230391879A1 (en) Caninized antibodies to canine interleukin-31 receptor alpha
WO2023165514A1 (en) Antigen-binding molecule specifically binding to flt3 and cd3 and pharmaceutical use thereof
WO2023169524A1 (en) Interleukin 2 mutant and complex containing same
WO2023046097A1 (en) Anti-tigit humanized antibody or antigen-binding fragment thereof and application thereof
WO2021093753A1 (en) Molecule capable of binding to human 4-1bb, and application of molecule
WO2023111148A1 (en) Caninized antibodies to canine interleukin-31 receptor alpha 1
CN115215936A (en) CSF1R antigen binding proteins
CN116133684A (en) Anti-human NR1 antibody derivatives
TW202246333A (en) An antigen-binding molecule that specifically binds bcma and cd3 and the pharmaceutical use thereof
KR20220038775A (en) Anti-human P40 protein domain antibodies and uses thereof
JP2024505563A (en) GITR antagonists and methods of use thereof
CN117062837A (en) Antigen binding molecule capable of specifically binding RANKL and NGF and medical application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant