CN114075279B - pH-independent long-acting type antiserum albumin nano antibody and application thereof - Google Patents

pH-independent long-acting type antiserum albumin nano antibody and application thereof Download PDF

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CN114075279B
CN114075279B CN202010820234.6A CN202010820234A CN114075279B CN 114075279 B CN114075279 B CN 114075279B CN 202010820234 A CN202010820234 A CN 202010820234A CN 114075279 B CN114075279 B CN 114075279B
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nanobody
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CN114075279A (en
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万亚坤
朱敏
盖军伟
李光辉
沈晓宁
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Shanghai Novamab Biopharmaceuticals Co Ltd
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    • 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
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    • 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
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
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    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • 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
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    • 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]
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
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    • 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®
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

The invention provides a pH-independent long-acting type anti-serum albumin nano antibody and application thereof. Specifically, the invention provides amino acid sequences of VHH chain framework region FR and complementarity determining region CDR of a pH independent long acting anti-serum albumin nanobody. The invention also provides a nucleotide sequence for encoding the nano antibody. The antiserum albumin nano antibody provided by the invention can be combined with serum albumin of human, mouse, rat and cynomolgus monkey under different pH conditions; and the half life of the protein medicine can be obviously prolonged, and a research and development basis is provided for the development of long-acting protein medicines.

Description

pH-independent long-acting type antiserum albumin nano antibody and application thereof
Technical Field
The invention relates to the technical field of biomedicine or biopharmaceuticals, in particular to a pH-independent long-acting type anti-serum albumin nano antibody and application thereof.
Background
As an important parameter of pharmacokinetics, the half-life of an antibody comprehensively represents the process of absorption and distribution of the antibody in vivo. The half-life period can be improved effectively, the drug effect of the antibody can be improved, the dosage and the frequency of administration can be reduced, the possible side effect can be reduced, and the treatment burden of patients can be reduced. How to change the half-life of antibody drugs has been an important research direction in the field of antibody engineering. Various factors affecting the drug half-life of antibodies, including: molecular weight, fcRn binding, isoelectric point, glycosylation, target-mediated clearance, anti-drug antibodies, and the like. Current strategies for achieving half-life extension of antibody drugs include glycosylation engineering, pegylation, albumin fusion, transferrin fusion, fc fusion, inert protein fusion, negative electric protein fusion, and the like.
Human serum albumin is the most abundant soluble protein in human plasma, and is also a carrier for many endogenous factors and exogenous drugs. Human serum albumin consists of 585 amino acids and has a relative molecular mass of about 66.5kDa. Because the human serum albumin is not easy to penetrate kidney glomeruli under normal conditions, the half-life period of the human serum albumin in blood plasma is long (can reach 14-20 days and is about 19 days on average), and the human serum albumin has no enzymatic and immunological activities, is safe and nontoxic, has good biocompatibility and extremely wide in-vivo distribution, and is an ideal drug carrier. Various long-acting techniques of human serum albumin-based drugs have been widely used and developed, and currently mainly include the construction of human serum albumin fusion proteins, the coupling with human serum albumin through covalent chemical bonds, the reversible binding with human serum albumin through non-covalent bonds, and the like.
Nanobody (Nb), a heavy chain single domain antibody VHH (variable domain of heavychain of heavy-chain antibody) -heavy chain antibodies (hcabs) in which naturally deleted light chains are present in the camelid. The single domain antibody consisting of only one heavy chain variable region obtained by cloning the variable region thereof is the smallest unit of the stable, bindable antigen having a complete function that is currently available. The nano antibody has the characteristics of high stability, good water solubility, simple humanization, high targeting property, strong penetrability and the like, and plays an unexpectedly large function in immune experiments, diagnosis and treatment. Ablynx corporation, the army corporation in the world nanobody field, is the earliest enterprise to use nanobodies for therapeutic antibody development, which already has more than 45 proprietary and cooperative nanobodies, and among them, there are various products that are fusion proteins constructed with anti-human serum albumin as a long-acting factor, such as products targeting IL-6R, TNF-alpha, RANKL or IL-17A/IL17F, and the like.
Although there are several patent documents in the prior art reporting nanobodies against human serum albumin, there are some disadvantages in application of the existing nanobodies, and there is a need in the art to develop new nanobodies with better functions.
Disclosure of Invention
The invention aims to provide a pH-independent long-acting type anti-serum albumin nano antibody and application thereof.
Specifically, the invention provides 4 nano antibodies which specifically bind to serum albumin, and simultaneously provides a coding sequence, a preparation method and application of the nano antibodies.
In a first aspect of the invention there is provided an anti-serum albumin nanobody which is capable of specifically binding to serum albumin and the complementarity determining region CDRs of the VHH chain of the nanobody are one or more selected from the group consisting of:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3;
(2) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 12;
(3) CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20, and CDR3 shown in SEQ ID NO. 21; and
(4) CDR1 shown in SEQ ID NO. 28, CDR2 shown in SEQ ID NO. 29, and CDR3 shown in SEQ ID NO. 30.
In another preferred embodiment, the anti-serum albumin nanobody is a pH-independent long-acting anti-serum albumin nanobody.
In another preferred embodiment, the anti-serum albumin nanobody comprises an immunoglobulin single variable domain that specifically binds serum albumin.
In another preferred embodiment, the anti-serum albumin nanobody comprises two or more immunoglobulin single variable domains that specifically bind serum albumin.
In another preferred embodiment, the immunoglobulin single variable domain comprises a CDR1, CDR2, and CDR3 selected from the group consisting of:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3;
(2) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 12;
(3) CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20, and CDR3 shown in SEQ ID NO. 21; and
(4) CDR1 shown in SEQ ID NO. 28, CDR2 shown in SEQ ID NO. 29, and CDR3 shown in SEQ ID NO. 30.
In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and which retains the ability to specifically bind serum albumin in a pH independent manner.
In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
In another preferred embodiment, the serum albumin is human or non-human mammalian serum albumin.
In another preferred embodiment, the serum albumin is human, mouse, rat or cynomolgus serum albumin.
In another preferred embodiment, the anti-serum albumin nanobody has a pH-independent property.
In another preferred embodiment, the anti-serum albumin nanobody is capable of binding serum albumin at different pH conditions.
In another preferred embodiment, the anti-serum albumin nanobody is capable of binding serum albumin at a pH of 3.0-10.0, preferably pH4.0-9.0, more preferably pH 5.0-8.0.
In another preferred embodiment, the monoclonal antibody has a binding activity to serum albumin of A1 at about pH 7.4 or more (preferably 7.8 or more, more preferably 8.0 or more; and pH 8.5 or less); the monoclonal antibody has a binding activity to serum albumin of A2 at a pH of about 5.0 or less (preferably 4.8 or less, more preferably 4.5 or less; and a pH of 4.0 or more); then 0.5.ltoreq.A1/A2.ltoreq.2, preferably 0.7.ltoreq.A1/A2.ltoreq.1.5, more preferably 0.8.ltoreq.A1/A2.ltoreq.1.2.
In another preferred embodiment, the framework region of the nanobody comprises FRl, FR2, FR3 and FR4 selected from the group consisting of:
(1) FR1 shown in SEQ ID NO. 4, FR2 shown in SEQ ID NO. 5, FR3 shown in SEQ ID NO. 6 and FR4 shown in SEQ ID NO. 7;
(2) FR1 shown in SEQ ID NO. 13, FR2 shown in SEQ ID NO. 14, FR3 shown in SEQ ID NO. 15 and FR4 shown in SEQ ID NO. 16;
(3) FR1 shown in SEQ ID NO. 22, FR2 shown in SEQ ID NO. 23, FR3 shown in SEQ ID NO. 24 and FR4 shown in SEQ ID NO. 25;
(4) FR1 shown in SEQ ID NO. 31, FR2 shown in SEQ ID NO. 32, FR3 shown in SEQ ID NO. 33 and FR4 shown in SEQ ID NO. 34;
(5) FR1 shown in SEQ ID NO. 37, FR2 shown in SEQ ID NO. 38, FR3 shown in SEQ ID NO. 39 and FR4 shown in SEQ ID NO. 40;
(6) FR1 shown in SEQ ID NO. 43, FR2 shown in SEQ ID NO. 44, FR3 shown in SEQ ID NO. 45 and FR4 shown in SEQ ID NO. 46;
(7) FR1 shown in SEQ ID NO. 49, FR2 shown in SEQ ID NO. 50, FR3 shown in SEQ ID NO. 51 and FR4 shown in SEQ ID NO. 52; and
(8) FR1 shown in SEQ ID NO. 55, FR2 shown in SEQ ID NO. 56, FR3 shown in SEQ ID NO. 57 and FR4 shown in SEQ ID NO. 58.
In another preferred embodiment, the amino acid sequence of the VHH chain in the antibody is selected from the group consisting of: SEQ ID NO. 8, SEQ ID NO. 17, SEQ ID NO. 26, SEQ ID NO. 35, SEQ ID NO. 41, SEQ ID NO. 47, SEQ ID NO. 53, SEQ ID NO. 59, or a combination thereof.
In another preferred embodiment, the amino acid sequence of the VHH chain in the antibody is selected from the group consisting of: SEQ ID NO. 41, SEQ ID NO. 47, SEQ ID NO. 53, SEQ ID NO. 59, or a combination thereof.
In another preferred embodiment, the nanobody comprises a humanized antibody, a camelid antibody, or a chimeric antibody.
In another preferred embodiment, the serum albumin-specific nanobody of the invention also encompasses an anti-serum albumin antibody molecule capable of binding to human serum albumin with a VHH consisting of the amino acid sequence of any one of SEQ ID NO:8, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53 or SEQ ID NO: 59.
In a second aspect of the invention there is provided an anti-serum albumin antibody comprising one or more VHH chains of a nanobody according to the first aspect of the invention.
In another preferred example, the anti-serum albumin antibody may be a monomer, a bivalent antibody, and/or a multivalent antibody.
In a third aspect of the invention, there is provided an isolated polynucleotide encoding an antisera albumin nanobody according to the first aspect of the invention or an antisera albumin antibody according to the second aspect of the invention.
In another preferred embodiment, the polynucleotides are in a combinatorial form.
In another preferred embodiment, the sequence of the polynucleotide is selected from the group consisting of: SEQ ID NO. 9, SEQ ID NO. 18, SEQ ID NO. 27, SEQ ID NO. 36, SEQ ID NO. 42, SEQ ID NO. 48, SEQ ID NO. 54, SEQ ID NO. 60, or a combination thereof.
In another preferred embodiment, the invention relates to a nucleic acid molecule encoding an anti-serum albumin nanobody of the invention. The nucleic acid of the invention may be RNA, DNA or cDNA.
In a fourth aspect of the invention there is provided an expression vector expressing a polynucleotide of the third aspect of the invention.
In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.
In another preferred embodiment, the viral vector comprises a lentiviral vector, an adenoviral vector, an AAV viral vector, a retroviral vector, or a combination thereof.
In a fifth aspect of the invention there is provided a host cell comprising an expression vector according to the fourth aspect of the invention, or having integrated into its genome a polynucleotide according to the third aspect of the invention.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of: coli, yeast cells, mammalian cells.
In a sixth aspect of the present invention, there is provided a method of producing an anti-serum albumin nanobody comprising the steps of:
(a) Culturing the host cell of the fifth aspect of the invention under conditions suitable for nanobody production, thereby obtaining a culture comprising said anti-serum albumin nanobody; and
(b) Isolating or recovering said anti-serum albumin nanobodies from said culture; optionally, a plurality of metal sheets
(c) Purifying and/or modifying the antisera albumin nanobody obtained in step (b).
In another preferred embodiment, the anti-serum albumin nanobody has an amino acid sequence as shown in SEQ ID NO. 8, SEQ ID NO. 17, SEQ ID NO. 26, SEQ ID NO. 35, SEQ ID NO. 41, SEQ ID NO. 47, SEQ ID NO. 53 or SEQ ID NO. 59.
In a seventh aspect of the invention, there is provided an immunoconjugate comprising:
(a) An anti-serum albumin nanobody according to the first aspect of the invention, or an anti-serum albumin antibody according to the second aspect of the invention; and
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, cytokine, radionuclide, enzyme, gold nanoparticle/nanorod, nanomagnetic particle, viral coat protein, or VLP, or a combination thereof.
In another preferred embodiment, components (a) and (b) are operably linked.
In another preferred embodiment, the coupling moiety is a chemical label or a biomarker.
In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and/or a chemical drug.
In another preferred embodiment, the biomarker is biotin, avidin, or an enzyme label.
In another preferred embodiment, the radionuclide comprises:
(i) A diagnostic isotope selected from the group consisting of: tc-99m, ga-68, F-18, I-123, I-125, I-131, in-111, ga-67, cu-64, zr-89, C-11, lu-177, re-188, or a combination thereof; and/or
(ii) A therapeutic isotope selected from the group consisting of: lu-177, Y-90, ac-225, as-211, bi-212, bi-213, cs-137, cr-51, co-60, dy-165, er-169, fm-255, au-198, ho-166, I-125, I-131, ir-192, fe-59, pb-212, mo-99, pd-103, P-32, K-42, re-186, re-188, sm-153, ra223, ru-106, na24, sr89, tb-149, th-227, xe-133, yb-169, yb-177, or combinations thereof.
In another preferred embodiment, the coupling moiety is a detectable label.
In another preferred embodiment, the coupling moiety is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)) or any form of nanoparticle.
In an eighth aspect of the invention, there is provided a conjugate comprising:
(a) An anti-serum albumin nanobody according to the first aspect of the invention, or an anti-serum albumin antibody according to the second aspect of the invention; and operatively connected to
(b) A modification marker selected from the group consisting of: chemical markers and biological markers.
In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and/or a chemical drug.
In another preferred embodiment, the biomarker is biotin, avidin, or an enzyme label.
In a ninth aspect of the invention, there is provided a multispecific antibody comprising: the anti-serum albumin nanobody according to the first aspect of the invention or the anti-serum albumin antibody according to the second aspect of the invention.
In another preferred embodiment, the multispecific antibody further comprises a second antigen-binding region that targets a target selected from the group consisting of: IL-4R, IL-4Rα, TNF- α, VEGF, PD-1, PD-L1, 4-1BB, CD47, TIM3, CTLA4, IL-17A, CD, CD22, CD38, IL-5, TSLP, BCMA, GLP-1, trop2, TIGIT, or combinations thereof.
In another preferred embodiment, the second antigen binding region is a nanobody.
In another preferred embodiment, the multispecific antibody comprises one or more second antigen-binding regions.
In another preferred embodiment, the multispecific antibody further comprises an Fc fragment of an antibody.
In another preferred embodiment, the multispecific antibody is a bispecific antibody that targets serum albumin and IL 4R.
In another preferred embodiment, the multispecific antibody comprises one anti-serum albumin nanobody and two anti-IL 4R nanobodies.
In another preferred embodiment, the multispecific antibody is a trivalent antibody.
In another preferred embodiment, the bispecific antibody has the structure of formula I:
I-I-B formula I
Wherein, the liquid crystal display device comprises a liquid crystal display device,
"-" is a peptide bond;
i is an anti-IL 4R nanobody;
b is an antiserum albumin nanobody according to the first aspect of the invention.
In a tenth aspect of the present invention, there is provided a fusion protein comprising:
(i) An anti-serum albumin nanobody according to the first aspect of the invention, or an anti-serum albumin antibody according to the second aspect of the invention;
(ii) Optionally a polypeptide molecule or fragment having therapeutic functions.
In another preferred embodiment, the polypeptide molecule or fragment having therapeutic function includes, but is not limited to: a polypeptide molecule or fragment that targets IL-4R, IL-4 ra, TNF-a, VEGF, PD-1, PD-L1, 4-1BB, CD47, TIM3, CTLA4, IL-17A, CD19, CD22, CD38, IL-5, TSLP, BCMA, GLP-1, trop2, or TIGIT.
In another preferred embodiment, the polypeptide molecule or fragment having therapeutic function includes, but is not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analog, albumin, antibody fragment, cytokine.
In another preferred embodiment, the polypeptide molecule or fragment having therapeutic function comprises a single chain antibody (scFv), a diabody, a monoclonal antibody, or a chimeric antibody.
In another preferred embodiment, the fusion protein further comprises a tag sequence that facilitates expression and/or purification.
In another preferred embodiment, the tag sequence is selected from the group consisting of: 6His tag, GGGS sequence, FLAG tag.
In another preferred embodiment, the fusion protein comprises a bispecific antibody or a chimeric antibody.
In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) An anti-serum albumin nanobody according to the first aspect of the invention, an anti-serum albumin antibody according to the second aspect of the invention, an immunoconjugate according to the seventh aspect of the invention, a conjugate according to the eighth aspect of the invention, a multispecific antibody according to the ninth aspect of the invention or a fusion protein according to the tenth aspect of the invention;
(ii) A pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition further comprises other biologically active substances, such as a medicament for treating tumors.
In a twelfth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: the antisera albumin nanobody according to the first aspect of the invention, the antisera albumin antibody according to the second aspect of the invention, the immunoconjugate according to the seventh aspect of the invention, the conjugate according to the eighth aspect of the invention, the multispecific antibody according to the ninth aspect of the invention, the fusion protein according to the tenth aspect of the invention, or a combination thereof, is used for the preparation of a long-acting protein medicament.
In a thirteenth aspect of the invention there is provided the use of an anti-serum albumin nanobody according to the first aspect of the invention, an anti-serum albumin antibody according to the second aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention: (a) Reagents, assay plates or kits for preparing a reagent for detecting serum albumin; (b) For the preparation of a medicament (long acting protein drug) that binds serum albumin.
In another preferred embodiment, the reagent is a diagnostic reagent.
In another preferred embodiment, the reagent is used to detect serum albumin or a fragment thereof in a sample.
In another preferred embodiment, the agent comprises an immunoconjugate according to the seventh aspect of the invention, a conjugate according to the eighth aspect of the invention, a multispecific antibody according to the ninth aspect of the invention, or a fusion protein according to the tenth aspect of the invention.
In a fourteenth aspect of the present invention there is provided an immunoadsorbent material for purifying serum albumin, wherein the immunoadsorbent material comprises an anti-serum albumin nanobody as described in the first or second aspect, a VHH chain of an anti-serum albumin nanobody as described in the first or second aspect of the invention.
In another preferred embodiment, the immunoadsorbent material further comprises a carrier.
In another preferred embodiment, the carrier includes, but is not limited to: magnetic beads, agarose gel, silica gel microspheres, and porous materials.
In a fifteenth aspect of the present invention, there is provided a method of detecting serum albumin or a fragment thereof in a sample in vitro, the method comprising the steps of:
(1) Contacting the sample in vitro with an anti-serum albumin nanobody according to the first aspect of the invention, an anti-serum albumin antibody according to the second aspect of the invention, or an immunoconjugate according to the seventh aspect of the invention;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of serum albumin or a fragment thereof in the sample.
In another preferred embodiment, the detection comprises diagnostic or non-diagnostic.
In a sixteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:
(a) Culturing a host cell according to the fifth aspect of the invention under conditions suitable for expression;
(b) Isolating from the culture a recombinant polypeptide comprising an anti-serum albumin nanobody according to the first aspect of the invention, an anti-serum albumin antibody according to the second aspect of the invention, a multispecific antibody according to the ninth aspect of the invention, and a fusion protein according to the tenth aspect of the invention.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1A shows the binding activity of albumin nanobody Nb1-15 to human, mouse, rat, cynomolgus serum albumin at different pH conditions.
FIG. 1B shows the binding activity of albumin nanobody Nb1-60 to human, mouse, rat, cynomolgus serum albumin at different pH conditions.
FIG. 1C shows the binding activity of albumin nanobody Nb1-83 to human, mouse, rat, cynomolgus serum albumin at different pH conditions.
FIG. 1D shows the binding activity of albumin nanobody Nb3-24 to human, mouse, rat, cynomolgus serum albumin at different pH conditions.
FIG. 2A shows the binding activity of IL4R nanobody fusion humanized albumin nanobody HuNb1-15 to human, mouse, rat, cynomolgus monkey serum albumin under different pH conditions.
FIG. 2B shows the binding activity of IL4R nanobody to human, mouse, rat, cynomolgus serum albumin at different pH conditions after fusion of humanized albumin nanobody HuNb 1-60.
FIG. 2C shows the binding activity of IL4R nanobody to human, mouse, rat, cynomolgus serum albumin at different pH conditions after fusion of humanized albumin nanobody HuNb 1-83.
FIG. 2D shows the binding activity of IL4R nanobody to human, mouse, rat, cynomolgus serum albumin at different pH conditions after fusion of humanized albumin nanobody HuNb 3-24.
FIG. 3 shows the half-life detection results of IL4R-HSA trivalent antibodies in mice.
FIG. 4 shows the half-life detection results of IL4R-HSA trivalent antibodies in rats.
FIG. 5 shows the half-life detection results of IL4R-HSA trivalent antibodies in cynomolgus monkeys.
Detailed Description
Through extensive and intensive research, the inventor successfully obtains a group of antiserum albumin nanobodies through a large number of screening, and experimental results show that the 4 antiserum albumin nanobodies obtained by the invention can be effectively combined with serum albumin. The present invention has been completed on the basis of this finding.
Specifically, the invention utilizes human serum albumin extracellular antigen protein to immunize camels, and obtains a high-quality immune nanobody gene library. And coupling serum albumin molecules on an ELISA plate, displaying the correct space structure of serum albumin, and screening an immune nanobody gene library (camel heavy chain antibody phage display gene library) by using phage display technology by using the antigen in the form, so as to obtain serum albumin specific nanobody genes.
As used herein, the terms "nanobody of the invention", "nanobody of the antisera albumin of the invention", are used interchangeably and refer to nanobodies that specifically recognize and bind to serum albumin (including human serum albumin). Particularly preferred are nanobodies of the amino acid sequence of the VHH chain as shown in SEQ ID NO. 41, SEQ ID NO. 47, SEQ ID NO. 53 or SEQ ID NO. 59.
As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
As used herein, the terms "single domain antibody (single domain antibody, sdAb, or VHH)", "nanobody" (nanobody) have the same meaning, referring to the variable region of a cloned antibody heavy chain, to construct a nanobody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Typically, the naturally deleted light and heavy chain constant region 1 (CH 1) antibodies are obtained first, and then the variable region of the heavy chain of the antibody is cloned to construct nanobodies (VHHs) consisting of only one heavy chain variable region.
Nanobody/single domain antibody (Nanobody) is a novel small molecule antibody fragment obtained by cloning a heavy chain variable region (VHH) of a camelid natural heavy chain antibody. Nanobody (Nb) has excellent biological characteristics, has a molecular weight of 12-15kDa, is one tenth of that of an intact antibody, has good tissue penetrability, high specificity and good water solubility. Because of the special structural property, the antibody has the advantages of the traditional antibody and the small molecular medicine, almost perfectly overcomes the defects of long development period, lower stability, harsh preservation condition and the like of the traditional antibody, gradually becomes an emerging force in the treatment of the new generation antibody, and shows wide application prospect in immunodiagnosis and treatment.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
Immunoconjugates and fusion expression products include, as known to those of skill in the art: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules in combination with antibodies or fragments thereof of the present invention. The invention also includes cell surface markers or antigens that bind to the anti-serum albumin antibodies or fragments thereof.
As used herein, the terms "heavy chain variable region" and "V H "interchangeably used.
As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".
In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.
In a preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably to refer to a polypeptide that specifically binds serum albumin, such as a protein or polypeptide having a heavy chain variable region. They may or may not contain an initiating methionine.
The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.
In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the variable region of the heavy chain, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of which 4 FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.
The variable regions of the heavy chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to antigens. Thus, the invention includes those molecules having antibody heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.
The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.
The antibody of the present invention refers to a polypeptide having serum albumin binding activity, comprising the above-mentioned CDR regions. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
The invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the nanobodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively mutated polypeptides are preferably produced by amino acid substitution according to Table 1.
TABLE 1
Initial residues Representative substitution Preferred substitution
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.
The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.
At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl 2 The process is carried out using procedures well known in the art. Another approach is to use MgCl 2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.
Therapeutic agents that may be conjugated or coupled to an antibody of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. drug-activated enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (e.g., cisplatin) or any form of nanoparticle, etc.
Pharmaceutical composition
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier or excipient, and optionally other biologically active substances. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intraperitoneal, intravenous, or topical administration.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Detection method
The invention also relates to a method for detecting serum albumin. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving a sample in a medium; detecting the level of serum albumin in the solubilized sample.
In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.
Kit for detecting a substance in a sample
The invention also provides a kit comprising an antibody (or fragment thereof) or assay plate of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, and the like.
The invention also provides a detection kit for detecting serum albumin level, which comprises an antibody for recognizing serum albumin, a lysis medium for dissolving a sample, and a general reagent and a buffer solution required for detection, such as various buffers, detection marks, detection substrates and the like. The detection kit may be an in vitro diagnostic device.
Application of
As described above, the nano antibody of the invention has wide biological application value and clinical application value, and the application relates to the application of the antiserum albumin nano antibody in preparing long-acting protein medicines, thus providing a research and development basis for the development of the long-acting protein medicines.
The main advantages of the invention include:
(a) The nano antibody can be combined with serum albumin of human, mouse, rat and cynomolgus monkey under different pH conditions.
(b) The nano antibody can obviously prolong the half life of the protein drug, and provides a research and development basis for the development of long-acting protein drugs.
(c) The nano antibody is suitable for prokaryotic expression and eukaryotic expression, has the advantages of extremely high solubility, difficult aggregation, high temperature resistance, strong acid, strong alkali and other denaturation conditions, and is suitable for laboratory and industrial development.
(d) The protein medicine prepared by the nano antibody of the invention can not influence the targeting property and activity of the medicine.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Example 1: screening and expression of antiserum albumin nano antibody
Immunizing 1 Sinkiang Bactrian camel with human serum albumin, separating total RNA from peripheral blood of the camel after 7 times of immunization, performing reverse transcription and PCR amplification to obtain VHH gene, cloning the VHH gene onto a phage vector pMECS, and transforming the phage gene into TG1 host cells to construct a phage display library. Then, the phage display technology is used for library screening, and phage enrichment containing antibody genes is finally obtained by 30 times through 4 rounds of screening processes of adsorption-washing-enrichment. 400 phage clones enriched in the above were randomly selected for PE-ELISA identification, all obtained positive clones were sequenced and identified, all antibodies with different sequences were used as candidate objects, and various antibodies were purified for subsequent identification by E.coli expression.
Example 2: binding of anti-serum albumin nanobodies to albumin at different pH conditions
Diluting serum of mice, rats and cynomolgus monkey and human serum albumin with pH8.2 NaHCO3 to 10ug/mL, coating the solution on an enzyme-labeled plate, and standing at 4 ℃ overnight; PBST washes the ELISA plate, and 300uL of 1% skimmed milk is added into each hole to be sealed for 2 hours at room temperature; PBST washes the ELISA plate, adds 100uL of antibody to be tested diluted in gradient (each antibody is diluted with PBS solution with pH5.0 and pH7.4 respectively, 3-fold gradient dilution is started from 300 ug/mL), incubate for 1 hour at 37 ℃; the ELISA plate was washed with PBST, 100uL of diluted Goat pAb to Nanobody (HRP) (diluted 1:1000 in PBS) was added and incubated at 37℃for 1 hour; the enzyme-labeled plate is washed by PBST, 100uL of TMB color development liquid is added, the reaction is carried out for 5 minutes at room temperature in a dark place, 50uL of 2M sulfuric acid is added to stop the color development, and the absorption value is read at the wavelength of 450nm of the enzyme-labeled instrument. The results are shown in FIGS. 1A-1D: the four anti-serum albumin nano antibodies can be combined with serum albumin of human, mice, rats and cynomolgus monkeys simultaneously, and can be combined with serum albumin of various species under different pH conditions (pH 5.0 and pH 7.4). The numerical statistics are shown in Table 2.
TABLE 2 binding of anti-serum Albumin nanobodies to Albumin of different species
Figure BDA0002634182330000171
Example 3: humanization of anti-serum albumin nanobodies
The amino acid sequences of the 4 nano antibodies are placed in a structural database to search for homologous structures, and the antibody sequences are shown in Table 3.
TABLE 3 sequence numbering of anti-serum albumin nanobodies
Antibody numbering Nb1-15 Nb1-60 Nb1-83 Nb3-24
CDR1 SEQ ID NO.1 SEQ ID NO.10 SEQ ID NO.19 SEQ ID NO.28
CDR2 SEQ ID NO.2 SEQ ID NO.11 SEQ ID NO.20 SEQ ID NO.29
CDR3 SEQ ID NO.3 SEQ ID NO.12 SEQ ID NO.21 SEQ ID NO.30
FR1 SEQ ID NO.4 SEQ ID NO.13 SEQ ID NO.22 SEQ ID NO.31
FR2 SEQ ID NO.5 SEQ ID NO.14 SEQ ID NO.23 SEQ ID NO.32
FR3 SEQ ID NO.6 SEQ ID NO.15 SEQ ID NO.24 SEQ ID NO.33
FR4 SEQ ID NO.7 SEQ ID NO.16 SEQ ID NO.25 SEQ ID NO.34
Full-length amino acid sequence SEQ ID NO.8 SEQ ID NO.17 SEQ ID NO.26 SEQ ID NO.35
Full-length base sequence SEQ ID NO.9 SEQ ID NO.18 SEQ ID NO.27 SEQ ID NO.36
Comparing the structures with high sequence isogeny, finally selecting proteins including 3dwt according to the resolution of the crystal structure and the constructed evolutionary tree, carrying out multi-template homologous modeling of the target nano antibody sequence, and selecting the structure with the lowest molpdf according to the high-low ranking of the scoring function; and then calculating solvent accessibility of the residues by using the ProtSA server on the modeled optimal structure, and performing sequence alignment on the modeled optimal structure and DP-47 to replace the corresponding residues exposed to the solvent. Finally, a humanized anti-serum albumin nanobody is determined, and the humanized antibody sequence corresponds to the following table 4:
TABLE 4 humanized anti-serum Albumin nanobody sequence numbering
Antibody numbering HuNb1-15 HuNb1-60 HuNb1-83 HuNb3-24
CDR1 SEQ ID NO.1 SEQ ID NO.10 SEQ ID NO.19 SEQ ID NO.28
CDR2 SEQ ID NO.2 SEQ ID NO.11 SEQ ID NO.20 SEQ ID NO.29
CDR3 SEQ ID NO.3 SEQ ID NO.12 SEQ ID NO.21 SEQ ID NO.30
FR1 SEQ ID NO.37 SEQ ID NO.43 SEQ ID NO.49 SEQ ID NO.55
FR2 SEQ ID NO.38 SEQ ID NO.44 SEQ ID NO.50 SEQ ID NO.56
FR3 SEQ ID NO.39 SEQ ID NO.45 SEQ ID NO.51 SEQ ID NO.57
FR4 SEQ ID NO.40 SEQ ID NO.46 SEQ ID NO.52 SEQ ID NO.58
Full-length amino acid sequence SEQ ID NO.41 SEQ ID NO.47 SEQ ID NO.53 SEQ ID NO.59
Full-length base sequence SEQ ID NO.42 SEQ ID NO.48 SEQ ID NO.54 SEQ ID NO.60
Example 4: construction and expression of HSA-IL4R trivalent nanobody
The IL4R nanobody is selected as a representative to construct a long-acting protein drug with the humanized albumin nanobody so as to verify the function of the humanized albumin nanobody for prolonging the in vivo half-life of the protein. The 4 candidate humanized albumin nanobodies are respectively connected in series with IL4R nanobodies (the sequences of the humanized albumin nanobodies are derived from patent CN 2019110547879) to form trivalent nanobodies, and the structural sequences of the trivalent nanobodies are shown in the following table 5:
TABLE 5 multivalent antibody structures and sequences
Antibody numbering Structure of the Amino acid sequence Base sequence
MY8154 IL4R Nb103-Nb103-HSA HuNb1-15 SEQ ID NO.61 SEQ ID NO.62
MY8267 IL4R Nb103-Nb103-HSA HuNb1-60 SEQ ID NO.63 SEQ ID NO.64
MY8162 IL4R Nb103-Nb103-HSA HuNb1-83 SEQ ID NO.65 SEQ ID NO.66
MY8268 IL4R Nb103-Nb103-HSA HuNb3-24 SEQ ID NO.67 SEQ ID NO.68
MY8171 IL4R Nb103-Nb103 SEQ ID NO.69 SEQ ID NO.70
The multivalent antibody was expressed using pichia pastoris. Briefly, the expression method is as follows: (1) Constructing the bases of the nanometer antibody sequences shown in SEQ ID NO.62, SEQ ID NO.64, SEQ ID NO.66, SEQ ID NO.68 and SEQ ID NO.70 into a pPICZaA carrier; (2) Electrotransformation into X-33 competent cells after linearization with SacI restriction enzyme; (3) Electroblotted samples were each plated on YPD plate medium containing bleomycin resistance at different concentrations and incubated in a 30℃incubator for 3 days, see for specific embodiments pPICZaA vector instructions from Invitrogen; (4) After the monoclonal grows on the plate culture medium, selecting the monoclonal and placing the monoclonal in a BMGY culture medium, when the OD value of the BMGY culture medium reaches about 20, collecting thalli, replacing the thalli into the BMMY culture medium, and culturing at 28 ℃ and 250 rpm; (5) after every 24 hours 1% methanol was added to the final volume; continuously inducing for 5 days, and ending the culture; (6) The obtained supernatant was purified by cation chromatography to obtain the objective antibody.
The above expressed purified antibodies were subjected to different pH conditions to detect their binding to albumin of various genus. The detection method is the same as that of example 2, and the results are shown in fig. 2A to 2D: the humanized trivalent antibody fused with the human IL4R nanobody can still be combined with serum albumin of different species, and the combination activity of the humanized trivalent antibody is not changed depending on pH change. The numerical statistics are shown in Table 6.
TABLE 6 binding of humanized trivalent antibodies to serum Albumin of different species
Figure BDA0002634182330000191
Figure BDA0002634182330000201
Example 5: half-life of antiserum albumin nanobody in mouse
The multivalent antibody expressed by 100ug yeast cells is respectively injected into mice intravenously, and after administration, orbital blood is taken for 5min, 3h, 8h, 24h, 48h, 96h and 144h respectively, and plasma is separated after centrifugation. Wherein the blood collection time points after administration of the control antibody MY8171 are 5min, 15min, 30min, 1.5h and 3h. PK analysis was then performed using ELISA detection. Coating enzyme-labeled version with 1ug/mL hIL4R protein at 100 uL/well overnight; PBST plates were washed 5 times, and 300uL of 0.3% Casein was added and blocked at room temperature for 2 hours; PBST plates were washed 5 times and diluted serum samples were added to the corresponding wells and incubated for 1 hour at 37 ℃; PBST plates were washed 5 times and incubated at 37℃for 1 hour with the addition of coat 1 pAb to Nanobody-HRP (1:1000 dilution) at 100 uL/well; PBST plates were washed 5 times, added with TMB developing solution, left to stand at room temperature for developing for 5 minutes, and then added with 2M sulfuric acid to terminate the reaction, and the absorption value was read at a wavelength of 450nm by an ELISA reader. Converting the detection result according to a standard curve to obtain a time-drug concentration relation diagram, wherein the result is shown in fig. 3: the half-life of the divalent antibody MY8171 without albumin nanobody in the mouse is only about 1.86 hours, while the half-life of the trivalent antibody with antiserum albumin nanobody in the mouse is obviously prolonged (as shown in table 7), and the half-life of the trivalent antibody with antiserum albumin nanobody in the mouse can be prolonged by about 22 times at most.
TABLE 7 half-life of different antisera Albumin nanobodies in mice
Antibody numbering MY8171 MY8154 MY8267 MY8162 MY8268
T 1/2 (hours) 1.86 16.50 30.35 8.98 40.99
Example 6: half-life of antiserum albumin nanobody in rat body
The multivalent antibodies expressed by 400ug yeast cells are respectively injected into rats intravenously, and the orbital blood is respectively taken for 5min, 3h, 8h, 24h, 48h, 96h and 144h after administration, and the plasma is separated after centrifugation. Wherein the blood collection time points after administration of the control antibody MY8171 are 5min, 15min, 30min, 1.5h and 3h. PK analysis was then performed using ELISA detection. The detection method is the same as in example 5. Converting the detection result according to a standard curve to obtain a time-drug concentration relation diagram, wherein the result is shown in fig. 4: the half-life of the divalent antibody MY8171 without albumin nanobody in the rat is only about 0.82 hours, while the half-life of the trivalent antibody with antiserum albumin nanobody in the mouse is obviously prolonged (as shown in table 8), and the half-life of the trivalent antibody with antiserum albumin nanobody in the mouse can be prolonged by about 63 times at most.
TABLE 8 half-life of different antisera Albumin nanobodies in rats
Antibody numbering MY8171 MY8154 MY8267 MY8268
T 1/2 (hours) 0.82 41.63 51.96 51.42
Example 7: half-life of antiserum albumin nano-antibody in cynomolgus monkey body
Two antibodies of MY8267 and MY8268 were respectively injected intravenously into the left anterior limb of a cynomolgus monkey at a dose of 2mg/kg, blood was taken from the right anterior limb 5min, 20min, 1h, 2h, 4h, 8h, 16h, 1day, 2day, 4day, 6day, 8day, 11day, 14day, 17day, 20day, 23day, 26day, 29day, 32day, 35day, respectively, and plasma was separated after centrifugation. PK analysis was then performed using ELISA detection. The detection method is the same as in example 5. Converting the detection result according to a standard curve to obtain a time-drug concentration relation diagram, wherein the result is shown in fig. 5: the half-life of the trivalent antibody with the anti-serum albumin nanobody in the cynomolgus monkey body can reach about 9-15 days (table 9).
TABLE 9 half-life of different antisera Albumin nanobodies in cynomolgus monkey
Antibody numbering MY8267 MY8268
T 1/2 (days) 9.53 14.21
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
<110> Shanghai Lo Start biomedical technology Co., ltd
<120> pH-independent long-acting type anti-serum albumin nanobody and application thereof
<130> P2020-1480
<160> 70
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 1
Gly Tyr Thr Tyr Thr Thr Ala Cys Met Ala
1 5 10
<210> 2
<211> 8
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 2
Phe Tyr Thr His Ser Gly Asn Ala
1 5
<210> 3
<211> 17
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 3
Ala Ala Ala Asn Tyr Gly Trp Gly Cys Ser Arg Trp Ser Asp Tyr Asn
1 5 10 15
Tyr
<210> 4
<211> 25
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 4
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser
20 25
<210> 5
<211> 15
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 5
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val Ala Arg
1 5 10 15
<210> 6
<211> 38
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 6
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Lys Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 7
<211> 11
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 7
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 8
<211> 124
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 8
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Thr Tyr Thr Thr Ala
20 25 30
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val
35 40 45
Ala Arg Phe Tyr Thr His Ser Gly Asn Ala Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Lys Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Tyr Gly Trp Gly Cys Ser Arg Trp Ser Asp Tyr Asn
100 105 110
Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 9
<211> 372
<212> DNA
<213> Bactrian camel (Camelus bactrianus)
<400> 9
caggtgcagc tgcaggaatc tggtggtggt tctgttcagg cgggtggttc tctgcgtctg 60
tcttgcgcgg tttctggtta cacctacacc accgcgtgca tggcgtggtt ccgtcaggcg 120
ccgggtaaag aacgtgaacg tgttgcgcgt ttctacaccc actctggtaa cgcgtactac 180
gcggactctg ttaaaggtcg tttcaccatc tctcaggaca acgcgaaaaa aaccgtttac 240
ctgcagatga actctctgaa accggaagac accgcgatgt actactgcgc ggcggcgaac 300
tacggttggg gttgctctcg ttggtctgac tacaactacc gtggtcaggg tacccaggtt 360
accgtttctt ct 372
<210> 10
<211> 10
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 10
Gly Phe Arg Phe Ser Ser Tyr Trp Met Tyr
1 5 10
<210> 11
<211> 8
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 11
Ile Asn Ser Ser Gly Gly Tyr Thr
1 5
<210> 12
<211> 19
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 12
Ala Thr Asp Ser Gly Asp Gly Lys Arg Tyr Trp Ser Gly Glu Tyr Phe
1 5 10 15
Tyr Arg Ser
<210> 13
<211> 25
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 13
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 14
<211> 15
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 14
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala
1 5 10 15
<210> 15
<211> 38
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 15
Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
20 25 30
Thr Ala Val Tyr Phe Cys
35
<210> 16
<211> 11
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 16
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 17
<211> 126
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 17
Gln Val Gln Leu Gln 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 Arg Phe Ser Ser Tyr
20 25 30
Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Asn Ser Ser Gly Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Thr Asp Ser Gly Asp Gly Lys Arg Tyr Trp Ser Gly Glu Tyr Phe
100 105 110
Tyr Arg Ser Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 18
<211> 378
<212> DNA
<213> Bactrian camel (Camelus bactrianus)
<400> 18
caggtgcagc tgcaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatt ccgcttcagt agctactgga tgtactgggt ccgccaggct 120
ccagggaagg ggctcgagtg ggtctcagct attaatagta gtggtggtta cacacgctat 180
gcagactccg tgaagggccg attcaccatc tccagagaca atgccaagaa cacgctgttt 240
ctgcaaatga acagcctgaa aactgaggac actgccgtgt atttctgcgc cacagattcg 300
ggtgacggaa aacggtattg gagtggtgaa tacttctacc gctctagggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 19
<211> 10
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 19
Gly Thr Arg Tyr Ser Ala Pro Cys Met Gly
1 5 10
<210> 20
<211> 7
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 20
Ile Ala Gly Asp Gly Thr Thr
1 5
<210> 21
<211> 18
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 21
Ala Ala Asp Pro Phe Thr Trp Cys Ser Arg Arg Arg Pro Tyr Gln Tyr
1 5 10 15
Ala His
<210> 22
<211> 25
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 22
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Thr
20 25
<210> 23
<211> 15
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 23
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala
1 5 10 15
<210> 24
<211> 38
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 24
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Ala
1 5 10 15
Ala Glu Asn Thr Leu Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 25
<211> 11
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 25
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 26
<211> 124
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 26
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Thr Gly Thr Arg Tyr Ser Ala Pro
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Ala Gly Asp Gly Thr Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Ala Ala Glu Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Asp Pro Phe Thr Trp Cys Ser Arg Arg Arg Pro Tyr Gln Tyr Ala
100 105 110
His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 27
<211> 372
<212> DNA
<213> Bactrian camel (Camelus bactrianus)
<400> 27
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tttgagactc 60
tcctgtgcag cgactggaac aagatatagt gcgccttgta tgggctggtt ccgccaggct 120
ccaggaaagg agcgcgaggg ggtcgcagcg attgcaggtg acggtaccac aagctacgca 180
gactccgtga agggccgatt caccatctcc aaagacgccg ccgagaacac tctgtatctg 240
caaatgacta gtctgaaacc tgaggacact gccatgtact actgtgcggc agacccgttt 300
acttggtgta gcaggcgccg gccttatcag tatgcgcact ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210> 28
<211> 10
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 28
Gly Phe Thr Phe Ser Arg Tyr Trp Met Phe
1 5 10
<210> 29
<211> 8
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 29
Ile Asn Ser Gly Gly Thr Tyr Thr
1 5
<210> 30
<211> 19
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 30
Ala Thr Asn Ser Gly Asp Gly Lys Arg Tyr Cys Ser Gly Gly Tyr Cys
1 5 10 15
Tyr Arg Ser
<210> 31
<211> 25
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 31
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 32
<211> 15
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 32
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ser Asp
1 5 10 15
<210> 33
<211> 38
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 33
Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 34
<211> 11
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 34
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 35
<211> 126
<212> PRT
<213> Bactrian camel (Camelus bactrianus)
<400> 35
Gln Val Gln Leu Gln 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 Ser Arg Tyr
20 25 30
Trp Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Ser Asp Ile Asn Ser Gly Gly Thr Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Ser Gly Asp Gly Lys Arg Tyr Cys Ser Gly Gly Tyr Cys
100 105 110
Tyr Arg Ser Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 36
<211> 378
<212> DNA
<213> Bactrian camel (Camelus bactrianus)
<400> 36
caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctgggtt caccttcagt aggtactgga tgttctgggt ccgccaggct 120
ccagggaagg ggctcgaatg gatctcagat attaatagtg gtggaactta cacacgctat 180
gcagactccg tgaagggccg gttcaccgtc tccagagaca atgccaagaa cacgctgtat 240
ctgcagatga acagcctgaa aactgaggac actgccgtgt attactgcgc cacaaattcg 300
ggtgacggaa aacggtattg cagtggtggt tactgctacc gctctagggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 37
<211> 25
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 37
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser
20 25
<210> 38
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 38
Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Val Ala Arg
1 5 10 15
<210> 39
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 39
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Lys Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 40
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 40
Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 41
<211> 132
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 41
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Thr Tyr Thr Thr Ala
20 25 30
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Val
35 40 45
Ala Arg Phe Tyr Thr His Ser Gly Asn Ala Phe Tyr Thr His Ser Gly
50 55 60
Asn Ala Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln
65 70 75 80
Asp Asn Ala Lys Lys Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
85 90 95
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala Asn Tyr Gly Trp Gly
100 105 110
Cys Ser Arg Trp Ser Asp Tyr Asn Tyr Arg Gly Gln Gly Thr Leu Val
115 120 125
Thr Val Ser Ser
130
<210> 42
<211> 396
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
gaggttcaat tgcaagaatc tggtggtggt ttggttcaac caggtggttc tttgagattg 60
tcttgtgctg tttctggtta cacttacact actgcttgta tggcttggtt cagacaagct 120
ccaggtaagg gtttggaaag agttgctaga ttctacactc actctggtaa cgctttctac 180
actcactctg gtaacgctta ctacgctgac tctgttaagg gtagattcac tatttctcaa 240
gacaacgcta agaagactgt ttacttgcaa atgaactctt tgaagccaga agacactgct 300
gtttactact gtgctgctgc taactacggt tggggttgtt ctagatggtc tgactacaac 360
tacagaggtc aaggtacttt ggttactgtt tcttct 396
<210> 43
<211> 25
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 43
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 44
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 44
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala
1 5 10 15
<210> 45
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 45
Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 46
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 46
Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 47
<211> 126
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 47
Glu Val Gln Leu Gln 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 Arg Phe Ser Ser Tyr
20 25 30
Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Asn Ser Ser Gly Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 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
Ala Thr Asp Ser Gly Asp Gly Lys Arg Tyr Trp Ser Gly Glu Tyr Phe
100 105 110
Tyr Arg Ser Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 48
<211> 378
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
gaggttcaat tgcaagaatc tggtggtggt ttggttcaac caggtggttc tttgagattg 60
tcttgtgctg cttctggttt tagattttct tcttactgga tgtactgggt tagacaagct 120
ccaggtaaag gattggaatg ggtttctgct attaactctt ctggtggtta cactagatac 180
gctgattctg ttaagggtag attcactata tccagagata acgctaagaa cactttgtac 240
ttgcagatga actctttgag agctgaagat actgctgttt actactgtgc taccgattct 300
ggagatggaa agagatactg gtccggtgag tatttctaca gatccagagg tcaaggtacc 360
ttggttaccg tttcttct 378
<210> 49
<211> 25
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 49
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Thr
20 25
<210> 50
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 50
Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Ala
1 5 10 15
<210> 51
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 51
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 52
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 52
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 53
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 53
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Thr Gly Thr Arg Tyr Ser Ala Pro
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Ala Gly Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Asp Pro Phe Thr Trp Cys Ser Arg Arg Arg Pro Tyr Gln Tyr Ala
100 105 110
His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 54
<211> 372
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
gaggttcaat tgcaagaatc tggtggtggt ttggttcaac caggtggttc tttgagattg 60
tcttgtgctg ctactggtac tagatactct gctccatgta tgggttggtt tagacaagct 120
ccaggaaagg gtttggaagg tgttgctgct attgctggtg acggtactac ttactacgct 180
gattctgtta agggtagatt cactatttcc agagataact ctaagaacac tttgtacttg 240
caaatgaact ctttgaaaac tgaagatact gctatgtact actgtgctgc tgatccattc 300
acttggtgtt ccagaagaag gccataccaa tacgctcatt ggggtcaagg tactttggtt 360
actgtttctt ct 372
<210> 55
<211> 25
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 55
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 56
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 56
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ser Asp
1 5 10 15
<210> 57
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 57
Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 58
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 58
Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 59
<211> 126
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 59
Glu Val Gln Leu Gln 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 Ser Arg Tyr
20 25 30
Trp Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Ser Asp Ile Asn Ser Gly Gly Thr Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 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
Ala Thr Asn Ser Gly Asp Gly Lys Arg Tyr Cys Ser Gly Gly Tyr Cys
100 105 110
Tyr Arg Ser Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 60
<211> 378
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
gaggttcaat tgcaagaatc tggtggtggt ttggttcaac caggtggttc tttgagattg 60
tcttgtgctg cttctggttt tactttttcc agatactgga tgttttgggt tagacaagct 120
ccaggtaaag gtttggaatg gatttctgat attaattctg gtggtactta cactagatac 180
gctgattctg ttaagggtag attcactatt tcaagagata acgctaagaa cactttgtac 240
ttgcaaatga actctttgag agctgaagat actgctgttt actactgtgc tactaactcc 300
ggtgacggta aaagatactg ttctggtgga tactgttaca gatctcgtgg tcaaggtact 360
ttggttactg tttcctct 378
<210> 61
<211> 388
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 61
Glu Val Gln Leu Gln 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 Ser Thr Ser Tyr Arg Tyr
20 25 30
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val
35 40 45
Ala Ser Ile Arg Pro Arg Ser Gly Arg Ala 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 Met Tyr Tyr Cys
85 90 95
Ala Ala Ser Asp Asn Asp Gly Asn Cys Gln Asp Tyr Trp Gly Lys Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser
115 120 125
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Tyr Arg Tyr
145 150 155 160
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val
165 170 175
Ala Ser Ile Arg Pro Arg Ser Gly Arg Ala Tyr Tyr Ala Asp Ser Val
180 185 190
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys
210 215 220
Ala Ala Ser Asp Asn Asp Gly Asn Cys Gln Asp Tyr Trp Gly Lys Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser
245 250 255
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
260 265 270
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Thr Tyr Thr Thr Ala
275 280 285
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Val
290 295 300
Ala Arg Phe Tyr Thr His Ser Gly Asn Ala Phe Tyr Thr His Ser Gly
305 310 315 320
Asn Ala Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln
325 330 335
Asp Asn Ala Lys Lys Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
340 345 350
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala Asn Tyr Gly Trp Gly
355 360 365
Cys Ser Arg Trp Ser Asp Tyr Asn Tyr Arg Gly Gln Gly Thr Leu Val
370 375 380
Thr Val Ser Ser
385
<210> 62
<211> 1164
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
gaagttcaat tgcaagaatc cggtggtggt ttggttcaac caggtggttc tttgagattg 60
tcttgtgctg cttccggttc cacttcttac agatactgta tggcttggtt tagacaagcc 120
cctggtaaag gattggaagc tgttgcctct attagaccta gatccggaag agcttactat 180
gctgattctg ttaagggtag attcaccatt tctcgtgata actccaagaa cactttgtac 240
ttgcaaatga actctttgag agccgaagat actgctatgt actactgtgc tgcttctgat 300
aatgatggta actgtcaaga ttattgggga aagggaacac ttgttactgt ttcttctggt 360
ggaggtggtt ctggtggtgg ttctgaagtt caattgcaag agtctggtgg tggattggtt 420
caacctggtg gttccttgag gttgtcttgt gctgcttctg gttctacttc ttacagatat 480
tgtatggcct ggtttagaca agctccaggt aaaggacttg aagctgttgc ttctattaga 540
ccaagatctg gtagagctta ctacgctgat tcagttaaag gtagattcac tatttccaga 600
gataactcta agaacacttt gtacctgcaa atgaattctc tgagagctga agatactgcc 660
atgtactatt gtgcagcttc tgataacgat ggtaattgtc aagattactg gggaaagggt 720
actttggtta ctgtttcctc cggtggtggt ggatctggag gtggttctga ggttcaattg 780
caagaatctg gtggtggttt ggttcaacca ggtggttctt tgagattgtc ttgtgctgtt 840
tctggttaca cttacactac tgcttgtatg gcttggttca gacaagctcc aggtaagggt 900
ttggaaagag ttgctagatt ctacactcac tctggtaacg ctttctacac tcactctggt 960
aacgcttact acgctgactc tgttaagggt agattcacta tttctcaaga caacgctaag 1020
aagactgttt acttgcaaat gaactctttg aagccagaag acactgctgt ttactactgt 1080
gctgctgcta actacggttg gggttgttct agatggtctg actacaacta cagaggtcaa 1140
ggtactttgg ttactgtttc ttcc 1164
<210> 63
<211> 382
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 63
Glu Val Gln Leu Gln 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 Ser Thr Ser Tyr Arg Tyr
20 25 30
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val
35 40 45
Ala Ser Ile Arg Pro Arg Ser Gly Arg Ala 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 Met Tyr Tyr Cys
85 90 95
Ala Ala Ser Asp Asn Asp Gly Asn Cys Gln Asp Tyr Trp Gly Lys Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser
115 120 125
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Tyr Arg Tyr
145 150 155 160
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val
165 170 175
Ala Ser Ile Arg Pro Arg Ser Gly Arg Ala Tyr Tyr Ala Asp Ser Val
180 185 190
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys
210 215 220
Ala Ala Ser Asp Asn Asp Gly Asn Cys Gln Asp Tyr Trp Gly Lys Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser
245 250 255
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
260 265 270
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Arg Phe Ser Ser Tyr
275 280 285
Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
290 295 300
Ser Ala Ile Asn Ser Ser Gly Gly Tyr Thr Arg Tyr Ala Asp Ser Val
305 310 315 320
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
325 330 335
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
340 345 350
Ala Thr Asp Ser Gly Asp Gly Lys Arg Tyr Trp Ser Gly Glu Tyr Phe
355 360 365
Tyr Arg Ser Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
370 375 380
<210> 64
<211> 1146
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
gaagttcaat tgcaagaatc cggtggtggt ttggttcaac caggtggttc tttgagattg 60
tcttgtgctg cttccggttc cacttcttac agatactgta tggcttggtt tagacaagcc 120
cctggtaaag gattggaagc tgttgcctct attagaccta gatccggaag agcttactat 180
gctgattctg ttaagggtag attcaccatt tctcgtgata actccaagaa cactttgtac 240
ttgcaaatga actctttgag agccgaagat actgctatgt actactgtgc tgcttctgat 300
aatgatggta actgtcaaga ttattgggga aagggaacac ttgttactgt ttcttctggt 360
ggaggtggtt ctggtggtgg ttctgaagtt caattgcaag agtctggtgg tggattggtt 420
caacctggtg gttccttgag gttgtcttgt gctgcttctg gttctacttc ttacagatat 480
tgtatggcct ggtttagaca agctccaggt aaaggacttg aagctgttgc ttctattaga 540
ccaagatctg gtagagctta ctacgctgat tcagttaaag gtagattcac tatttccaga 600
gataactcta agaacacttt gtacctgcaa atgaattctc tgagagctga agatactgcc 660
atgtactatt gtgcagcttc tgataacgat ggtaattgtc aagattactg gggaaagggt 720
actttggtta ctgtttcctc cggtggtggt ggatctggag gtggttctga agttcaattg 780
caagaatctg gtggtggttt ggttcaacca ggtggttctt tgagattgtc ttgtgctgct 840
tctggtttta gattttcttc ttactggatg tactgggtta gacaagctcc aggtaaagga 900
ttggaatggg tttctgctat taactcttct ggtggttaca ctagatacgc tgattctgtt 960
aagggtagat tcactatatc cagagataac gctaagaaca ctttgtactt gcagatgaac 1020
tctttgagag ctgaagatac tgctgtttac tactgtgcta ccgattctgg agatggaaag 1080
agatactggt ccggtgagta tttctacaga tccagaggtc aaggtacctt ggttaccgtt 1140
tcttcc 1146
<210> 65
<211> 380
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 65
Glu Val Gln Leu Gln 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 Ser Thr Ser Tyr Arg Tyr
20 25 30
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val
35 40 45
Ala Ser Ile Arg Pro Arg Ser Gly Arg Ala 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 Met Tyr Tyr Cys
85 90 95
Ala Ala Ser Asp Asn Asp Gly Asn Cys Gln Asp Tyr Trp Gly Lys Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser
115 120 125
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Tyr Arg Tyr
145 150 155 160
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val
165 170 175
Ala Ser Ile Arg Pro Arg Ser Gly Arg Ala Tyr Tyr Ala Asp Ser Val
180 185 190
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys
210 215 220
Ala Ala Ser Asp Asn Asp Gly Asn Cys Gln Asp Tyr Trp Gly Lys Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser
245 250 255
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
260 265 270
Ser Leu Arg Leu Ser Cys Ala Ala Thr Gly Thr Arg Tyr Ser Ala Pro
275 280 285
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
290 295 300
Ala Ala Ile Ala Gly Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys
305 310 315 320
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
325 330 335
Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr Tyr Cys Ala
340 345 350
Ala Asp Pro Phe Thr Trp Cys Ser Arg Arg Arg Pro Tyr Gln Tyr Ala
355 360 365
His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
370 375 380
<210> 66
<211> 1140
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
gaagttcaat tgcaagaatc cggtggtggt ttggttcaac caggtggttc tttgagattg 60
tcttgtgctg cttccggttc cacttcttac agatactgta tggcttggtt tagacaagcc 120
cctggtaaag gattggaagc tgttgcctct attagaccta gatccggaag agcttactat 180
gctgattctg ttaagggtag attcaccatt tctcgtgata actccaagaa cactttgtac 240
ttgcaaatga actctttgag agccgaagat actgctatgt actactgtgc tgcttctgat 300
aatgatggta actgtcaaga ttattgggga aagggaacac ttgttactgt ttcttctggt 360
ggaggtggtt ctggtggtgg ttctgaagtt caattgcaag agtctggtgg tggattggtt 420
caacctggtg gttccttgag gttgtcttgt gctgcttctg gttctacttc ttacagatat 480
tgtatggcct ggtttagaca agctccaggt aaaggacttg aagctgttgc ttctattaga 540
ccaagatctg gtagagctta ctacgctgat tcagttaaag gtagattcac tatttccaga 600
gataactcta agaacacttt gtacctgcaa atgaattctc tgagagctga agatactgcc 660
atgtactatt gtgcagcttc tgataacgat ggtaattgtc aagattactg gggaaagggt 720
actttggtta ctgtttcctc cggtggtggt ggatctggag gtggttctga ggttcaattg 780
caagaatctg gtggtggttt ggttcaacca ggtggttctt tgagattgtc ttgtgctgct 840
actggtacta gatactctgc tccatgtatg ggttggttta gacaagctcc aggaaagggt 900
ttggaaggtg ttgctgctat tgctggtgac ggtactactt actacgctga ttctgttaag 960
ggtagattca ctatttccag agataactct aagaacactt tgtacttgca aatgaactct 1020
ttgaaaactg aagatactgc tatgtactac tgtgctgctg atccattcac ttggtgttcc 1080
agaagaaggc cataccaata cgctcattgg ggtcaaggta ctttggttac tgtttcttcc 1140
<210> 67
<211> 382
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 67
Glu Val Gln Leu Gln 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 Ser Thr Ser Tyr Arg Tyr
20 25 30
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val
35 40 45
Ala Ser Ile Arg Pro Arg Ser Gly Arg Ala 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 Met Tyr Tyr Cys
85 90 95
Ala Ala Ser Asp Asn Asp Gly Asn Cys Gln Asp Tyr Trp Gly Lys Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser
115 120 125
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Tyr Arg Tyr
145 150 155 160
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val
165 170 175
Ala Ser Ile Arg Pro Arg Ser Gly Arg Ala Tyr Tyr Ala Asp Ser Val
180 185 190
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys
210 215 220
Ala Ala Ser Asp Asn Asp Gly Asn Cys Gln Asp Tyr Trp Gly Lys Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser
245 250 255
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
260 265 270
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
275 280 285
Trp Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
290 295 300
Ser Asp Ile Asn Ser Gly Gly Thr Tyr Thr Arg Tyr Ala Asp Ser Val
305 310 315 320
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
325 330 335
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
340 345 350
Ala Thr Asn Ser Gly Asp Gly Lys Arg Tyr Cys Ser Gly Gly Tyr Cys
355 360 365
Tyr Arg Ser Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser
370 375 380
<210> 68
<211> 1146
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 68
gaagttcaat tgcaagaatc cggtggtggt ttggttcaac caggtggttc tttgagattg 60
tcttgtgctg cttccggttc cacttcttac agatactgta tggcttggtt tagacaagcc 120
cctggtaaag gattggaagc tgttgcctct attagaccta gatccggaag agcttactat 180
gctgattctg ttaagggtag attcaccatt tctcgtgata actccaagaa cactttgtac 240
ttgcaaatga actctttgag agccgaagat actgctatgt actactgtgc tgcttctgat 300
aatgatggta actgtcaaga ttattgggga aagggaacac ttgttactgt ttcttctggt 360
ggaggtggtt ctggtggtgg ttctgaagtt caattgcaag agtctggtgg tggattggtt 420
caacctggtg gttccttgag gttgtcttgt gctgcttctg gttctacttc ttacagatat 480
tgtatggcct ggtttagaca agctccaggt aaaggacttg aagctgttgc ttctattaga 540
ccaagatctg gtagagctta ctacgctgat tcagttaaag gtagattcac tatttccaga 600
gataactcta agaacacttt gtacctgcaa atgaattctc tgagagctga agatactgcc 660
atgtactatt gtgcagcttc tgataacgat ggtaattgtc aagattactg gggaaagggt 720
actttggtta ctgtttcctc cggtggtggt ggatctggag gtggttctga ggttcaattg 780
caagaatctg gtggtggttt ggttcaacca ggtggttctt tgagattgtc ttgtgctgct 840
tctggtttta ctttttccag atactggatg ttttgggtta gacaagctcc aggtaaaggt 900
ttggaatgga tttctgatat taattctggt ggtacttaca ctagatacgc tgattctgtt 960
aagggtagat tcactatttc aagagataac gctaagaaca ctttgtactt gcaaatgaac 1020
tctttgagag ctgaagatac tgctgtttac tactgtgcta ctaactccgg tgacggtaaa 1080
agatactgtt ctggtggata ctgttacaga tctcgtggtc aaggtacttt ggttactgtt 1140
tcctct 1146
<210> 69
<211> 247
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 69
Glu Val Gln Leu Gln 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 Ser Thr Ser Tyr Arg Tyr
20 25 30
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val
35 40 45
Ala Ser Ile Arg Pro Arg Ser Gly Arg Ala 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 Met Tyr Tyr Cys
85 90 95
Ala Ala Ser Asp Asn Asp Gly Asn Cys Gln Asp Tyr Trp Gly Lys Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser
115 120 125
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Tyr Arg Tyr
145 150 155 160
Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val
165 170 175
Ala Ser Ile Arg Pro Arg Ser Gly Arg Ala Tyr Tyr Ala Asp Ser Val
180 185 190
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
195 200 205
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys
210 215 220
Ala Ala Ser Asp Asn Asp Gly Asn Cys Gln Asp Tyr Trp Gly Lys Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser
245
<210> 70
<211> 741
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 70
gaagttcaat tgcaagaatc cggtggtggt ttggttcaac caggtggttc tttgagattg 60
tcttgtgctg cttccggttc cacttcttac agatactgta tggcttggtt tagacaagcc 120
cctggtaaag gattggaagc tgttgcctct attagaccta gatccggaag agcttactat 180
gctgattctg ttaagggtag attcaccatt tctcgtgata actccaagaa cactttgtac 240
ttgcaaatga actctttgag agccgaagat actgctatgt actactgtgc tgcttctgat 300
aatgatggta actgtcaaga ttattgggga aagggaacac ttgttactgt ttcttctggt 360
ggaggtggtt ctggtggtgg ttctgaagtt caattgcaag agtctggtgg tggattggtt 420
caacctggtg gttccttgag gttgtcttgt gctgcttctg gttctacttc ttacagatat 480
tgtatggcct ggtttagaca agctccaggt aaaggacttg aagctgttgc ttctattaga 540
ccaagatctg gtagagctta ctacgctgat tcagttaaag gtagattcac tatttccaga 600
gataactcta agaacacttt gtacctgcaa atgaattctc tgagagctga agatactgcc 660
atgtactatt gtgcagcttc tgataacgat ggtaattgtc aagattactg gggaaagggt 720
actttggtta ctgtttcctc c 741

Claims (19)

1. An anti-serum albumin nanobody, wherein said nanobody is capable of specifically binding serum albumin and the VHH chain of said nanobody has complementarity determining region CDRs as follows:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3.
2. The nanobody of claim 1, wherein the VHH chain of the nanobody further comprises a framework region FR selected from the group consisting of:
(1) FR1 shown in SEQ ID NO. 4, FR2 shown in SEQ ID NO. 5, FR3 shown in SEQ ID NO. 6 and FR4 shown in SEQ ID NO. 7; and
(2) FR1 shown in SEQ ID NO. 37, FR2 shown in SEQ ID NO. 38, FR3 shown in SEQ ID NO. 39 and FR4 shown in SEQ ID NO. 40.
3. The nanobody of claim 1, wherein the amino acid sequence of the VHH chain of the nanobody is selected from the group consisting of: SEQ ID NO. 8 and SEQ ID NO. 41.
4. An anti-serum albumin antibody comprising the VHH chain of the nanobody of claim 1.
5. A polynucleotide encoding a protein selected from the group consisting of: the nanobody of claim 1 or the antibody of claim 4.
6. The polynucleotide of claim 5, wherein the nucleotide sequence of said polynucleotide is selected from the group consisting of: SEQ ID NO. 9 and SEQ ID NO. 42.
7. An expression vector comprising the polynucleotide of claim 5.
8. A host cell comprising the expression vector of claim 7, or having integrated into its genome the polynucleotide of claim 5.
9. A method of producing an anti-serum albumin nanobody comprising the steps of:
(a) Culturing the host cell of claim 8 under conditions suitable for nanobody production, thereby obtaining a culture comprising anti-serum albumin nanobody;
(b) Isolating and/or recovering said anti-serum albumin nanobodies from said culture; and
(c) Optionally purifying and/or modifying the antisera albumin nanobody obtained in step (b).
10. A fusion protein, said fusion protein comprising:
(i) The nanobody of claim 1;
(ii) Optionally a polypeptide molecule or fragment having therapeutic functions.
11. An immunoconjugate, the immunoconjugate comprising:
(a) The antisera albumin nanobody of claim 1, or the antisera albumin antibody of claim 4; and
(b) A coupling moiety selected from the group consisting of: a detectable label, a drug, a cytokine, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein, or VLP, or a combination thereof.
12. The immunoconjugate of claim 11, wherein the conjugated moiety is a radionuclide or an enzyme.
13. A conjugate, the conjugate comprising:
(a) The antisera albumin nanobody of claim 1, or the antisera albumin antibody of claim 4; and operatively connected to
(b) A modification marker selected from the group consisting of: chemical markers and biological markers.
14. A multispecific antibody, the multispecific antibody comprising: the antisera albumin nanobody of claim 1, or the antisera albumin antibody of claim 4.
15. A pharmaceutical composition, said pharmaceutical composition comprising:
(i) The antisera albumin nanobody of claim 1, the antisera albumin antibody of claim 4, the immunoconjugate of claim 11, the conjugate of claim 13, the multispecific antibody of claim 14, or the fusion protein of claim 10;
(ii) A pharmaceutically acceptable carrier.
16. Use of the antisera albumin nanobody of claim 1, the antisera albumin antibody of claim 4, or the immunoconjugate of claim 11: (a) Reagents, assay plates or kits for preparing a reagent for detecting serum albumin; (b) for the preparation of a medicament for binding serum albumin.
17. An immunoadsorbed material for purifying serum albumin, wherein the immunoadsorbed material comprises the anti-serum albumin nanobody of claim 1 or the anti-serum albumin antibody of claim 4 or the VHH chain of the anti-serum albumin nanobody of claim 1.
18. A method for the in vitro non-diagnostic detection of serum albumin or fragments thereof in a sample, said method comprising the steps of:
(1) Contacting the sample with the anti-serum albumin nanobody of claim 1, the anti-serum albumin antibody of claim 4, or the immunoconjugate of claim 11 in vitro;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of serum albumin or a fragment thereof in the sample.
19. A method of producing a recombinant polypeptide, the method comprising:
(a) Culturing the host cell of claim 8 under conditions suitable for expression;
(b) Isolating from the culture a recombinant polypeptide comprising the anti-serum albumin nanobody of claim 1, the anti-serum albumin antibody of claim 4, the multispecific antibody of claim 14, the fusion protein of claim 10.
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