CN115433274A - Nano antibody of targeted Human Serum Albumin (HSA) and application thereof - Google Patents

Nano antibody of targeted Human Serum Albumin (HSA) and application thereof Download PDF

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CN115433274A
CN115433274A CN202210624226.3A CN202210624226A CN115433274A CN 115433274 A CN115433274 A CN 115433274A CN 202210624226 A CN202210624226 A CN 202210624226A CN 115433274 A CN115433274 A CN 115433274A
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antibody
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王荣娟
曾大地
刘大涛
张畅
杨莹莹
王双
张锦超
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Beijing Konuoxincheng Technology Co ltd
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Abstract

The invention discloses a nano antibody of targeted Human Serum Albumin (HSA), a derivative thereof and application of the nano antibody in preparing a medicament. Constructing a phage surface display VHH antibody library by using human HSA immune camels of the same group, coating an enzyme-linked plate with human serum albumin and cynomolgus monkey serum albumin, and obtaining VHH antibody clone with the binding capacity to HSA and cyno SA by a solid-phase screening method. Preparing a corresponding chimeric antibody to detect the antigen affinity, and selecting two VHH antibodies 2H4 and 2MG6 for humanized transformation and sequence optimization. The in vivo activity of the humanized VHH antibody hz2MG6 in mice is determined, and the result shows that the half-life of the drug can be effectively prolonged by combining the drug with the anti-HSA single domain antibody.

Description

Nano antibody of targeting Human Serum Albumin (HSA) and application thereof
PRIORITY INFORMATION
The present application claims priority and benefit of a patent application having patent application number 202110616699.4, filed on 3/6/2021 with the intellectual property office of china, and is incorporated herein by reference in its entirety.
Technical Field
The invention belongs to the field of antibody engineering, and particularly relates to a therapeutic single-domain antibody for diagnosing or treating tumors, in particular to a nano antibody targeting Human Serum Albumin (HSA), a derivative protein thereof and application thereof in preparing a medicament, particularly application in loading the medicament on albumin and prolonging half-life of the medicament.
Background
Single domain antibodies (sdabs) are a special class of antibodies that comprise only one heavy chain of an antibody. Similar to conventional diabodies, it can selectively bind to a specific antigen. Single domain antibodies were first found in camelids and later in the nurse shark class chondroiidae. Single domain antibodies the single heavy chain antibody variable region (VHH) is a single functional domain that binds antigen intact, only 12-15kDa. The VHH has the advantages of simple structure, high specificity, high affinity, low immunogenicity, good permeability when being combined with antigen, capability of contacting hidden targets which cannot be contacted by conventional antibodies when tumor therapy is carried out, and the like. Furthermore, since the single-domain antibody has only one chain, the problem of mismatch in the fusion of the diabody does not occur. Based on these advantages, the use of single domain antibodies as antigen binding sequences for bispecific antibodies has great advantages, and is becoming a focus of development (Serge Muydermans (2013), annu. Rev. Biochem.82: 775-797).
Human Serum Albumin (HSA) is the most abundant protein in Human plasma, approximately half of the serum protein, produced in the liver, soluble in water and present in monomeric form. Serum albumin consists of 585 amino acids, contains 1 free Cys and 17 disulfide bonds, and has a molecular weight of about 66.5KD. Unlike other protein polypeptides, HSA specifically binds to the neonatal receptor (FcRn) in the acidic intracellular environment after entry into the cell, thereby protecting it from lysosomal degradation and return to the cell surface with FcRn, and thus its serum half-life can reach about 21 days. HSA is an ideal drug carrier (Elsadek, B., & Kratz, F. (2012). Impact of albumin on drug delivery-new applications on the horizontal.J. Control Release,157 (1), 4-28), and fusion expression of a target protein and HSA or binding of the target protein and HSA in a non-covalent linkage form can effectively prolong the half-life of the target protein.
Disclosure of Invention
In the prior art, an ScFv, fab or whole IgG anti-HSA antibody molecule has a complex structure and larger molecules, and although an active molecule can be connected to HSA, the function and the method of the active molecule are affected, and the loading efficiency is lower; the nano antibody has small molecules and is easy to operate, but the humanized degree is low, the affinity is not high, and the half-life prolonging property needs to be further improved.
In view of the defects of the prior art, the invention provides a single-domain antibody targeting Human Serum Albumin (HSA), a derivative thereof and application of the single-domain antibody in preparation of a medicament.
Single variable domain antibodies are currently the smallest antibody molecule with a molecular weight of 1/10 that of the common antibody, originally found in camel blood by the Belgian scientist Hamers, R, and are a class of great interest in engineered antibody products. The single variable domain antibody not only has the antigen reactivity of the monoclonal antibody, but also has some unique functional characteristics, such as small molecular mass, strong stability, good solubility, easy expression, strong targeting property, simple humanization and the like, and is particularly suitable for developing bi-or multi-specific therapeutic antibodies and developing Car-T/M/NK and other therapies. Development of single variable domain antibodies and/or single variable domain antibody-based bi/multispecific antibodies has become a research and development hotspot.
Studies have shown that HSA binding proteins or peptides also have a longer half-life in vivo, and thus anti-HSA antibodies can be used to extend the half-life of protein or polypeptide drugs. The obtained anti-HSA antibody can be obtained by preparing hybridoma or immune library through an antigen immune mouse for screening and performing humanized transformation; human anti-HSA antibodies can also be directly screened by antibody library technology; the antibody obtained by the above technology can be ScFv, fab or whole antibody IgG consisting of VH and VL. The anti-HSA nano antibody is obtained by immunizing alpaca/camel and preparing immune repertoire or screening by HSA antigen through natural nano antibody repertoire. The antibody Ozoraluzumab (ATN-103) was developed as a trispecific antibody by the company Ablynx using a nanobody of HSA. Ozoralizumab contains 3 nanobody domains, 2 of which target TNF α, and 1 of which binds to HSA to extend the half-life of the drug (Fleischmann 1, r.m. (2019). A clinical three-phase study (NCT 04077567) is currently underway.
The inventor constructs a phage surface display VHH antibody library by using human HSA immune camels of the same group, adopts human serum albumin and Cynomolgus monkey serum albumin to coat an enzyme-linked plate, and obtains VHH antibody clone with binding capacity to HSA and Cynomolgus monkey serum albumin (cyno SA) by a solid phase screening method. Preparing corresponding chimeric antibody to detect antigen affinity, and selecting two VHH antibodies 2H4 and 2MG6 for humanization and sequence optimization. The in vivo activity assay of mice is carried out by using humanized VHH antibody hz2MG6 with cross reaction to Mouse Serum Albumin (MSA), and the result shows that the half-life of the drug can be effectively prolonged by combining the drug with the anti-HSA single domain antibody.
In particular, in one aspect, the invention provides a molecule having albumin binding function. According to an embodiment of the invention, the molecule with albumin binding function comprises at least one albumin binding domain, said albumin binding domain comprising Complementarity Determining Regions (CDRs) 1,2 and 3, said CDRs1, 2 and 3 having the amino acid sequence as shown in CDRs1, 2 and 3 of a selected VHH or a mutant thereof, wherein said mutant is obtained by performing one or several amino acid mutations on the basis of CDRs1, 2 and 3 of said selected VHH, preferably said one or several amino acid mutations are substitution mutations of one, two, three, four or five amino acids,
wherein the amino acid sequence of CDRs1, 2 and 3 of the selected VHH is one of:
(1) The amino acid sequences of CDRs1, 2 and 3 of the selected VHH are shown in SEQ ID No.43, 44 and 45 respectively;
(2) The amino acid sequences of CDRs1, 2 and 3 of the selected VHH are shown in SEQ ID No.46, 47 and 48 respectively;
(3) The amino acid sequences of CDRs1, 2 and 3 of the selected VHH are set forth in SEQ ID nos. 43, 49 and 50, respectively;
(4) The amino acid sequences of the CDRs1, 2 and 3 of the selected VHH are shown in SEQ ID nos. 51, 52 and 53, respectively.
According to an embodiment of the invention the amino acid sequences of CDRs1, 2 and 3 of said mutant of said selected VHH are one of the following:
(1) The amino acid sequence of CDR1 of the mutant of the selected VHH is shown in SEQ ID NO.51, and the amino acid sequence of CDR2 is shown in GIYTVGGSTFYTDX 10 VKG shows that the amino acid sequence of CDR3 is GX 1 TDTAX 2 LX 3 X 4 PX 5 SFGY is shown;
(2) The amino acid sequence of CDR1 and the amino acid sequence of CDR2 of said mutant of said selected VHH are shown in SEQ ID NO.71 and AIYTX, respectively 6 SGLSGTTYYADX 7 VKG shows that the amino acid sequence of CDR3 is DWKYX 8 STRCGLEVEYDX 9 As shown in the drawings, the first and second,
wherein,
X 1 is R, E or A;
X 2 is K, E or A;
X 3 is R, E or A;
X 4 is D or E;
X 5 is R, E or A;
X 6 is D or S;
X 7 is S or T;
X 8 is D or E;
X 9 is S or A;
X 10 is S or T.
According to an embodiment of the invention, said selected VHH or said mutant thereof comprises a heavy chain variable region, wherein,
(1) The heavy chain variable region comprises the sequence set forth in SEQ ID NO 1 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;
(2) The heavy chain variable region comprises the sequence set forth in SEQ ID No. 3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(3) The heavy chain variable region comprises the sequence set forth in SEQ ID NO.5 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(4) The heavy chain variable region comprises the sequence set forth in SEQ ID NO.7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
According to an embodiment of the invention, the amino acid sequence of the heavy chain variable region of the mutant is mutated in any combination selected from the group consisting of the following on the basis of the VHH shown in SEQ ID NO 5:
V27F+S28T，
D54S+S66T，
D54E+S66A。
according to an embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the mutant is shown as SEQ ID NO 36, 38, 39 or 40.
According to an embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the mutant is mutated on the basis of the VHH shown in SEQ ID No.7 in any combination selected from:
A14P，
A14P+S27F，
A14P+S26T，
A14P+S27F+S62T，
A14P+S27F+S62T+R99E，
A14P+S27F+S62T+R99A，
A14P+S27F+S62T+K104E，
A14P+S27F+S62T+K104A，
A14P+S27F+S62T+R106E，
A14P+S27F+S62T+R106A，
A14P+S27F+S62T+R109E，
A14P+S27F+S62T+R109A，
A14P+S27F+S62T+R106A+R99E，
A14P+S27F+S62T+R106A+R99A，
A14P+S27F+S62T+K104E+R99E，
A14P+S27F+S62T+K104E+R99A，
A14P+S27F+S62T+D107E，
A14P+S27F+S62T+R109E+R99E，
A14P+S27F+S62T+R109E+K104A，
A14P+S27F+S62T+R109E+R106A，
A14P+S27F+S62T+R109E+R99A。
according to an embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the mutant is as shown in SEQ ID NOs 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35.
According to embodiments of the invention, the albumin binding domain is capable of specifically binding to Human Serum Albumin (HSA) and cynomolgus monkey serum albumin (cyno SA) without specific binding to Bovine Serum Albumin (BSA); and the albumin binding structural domain is a nano antibody or a functional derivative thereof obtained by screening a nano antibody library by taking albumin as bait protein.
According to embodiments of the invention, the albumin binding domain is also capable of specifically binding Murine Serum Albumin (MSA).
According to an embodiment of the invention, the molecule having albumin binding function comprises two or more albumin binding domains, said two or more albumin binding domains having the same or different amino acid sequences.
According to an embodiment of the present invention, the molecule having albumin binding function further comprises at least one bioactive domain or functional fragment, wherein the bioactive domain or functional fragment comprises a carrier protein, an active peptide, a tag peptide, a chaperone protein, or the like, which is linked to the albumin binding domain by a covalent bond, a non-covalent bond, or a linker;
wherein preferably, the carrier protein comprises BSA, OVA, HSA, KLH, fc, etc.; the active peptides include linear peptides or cyclic peptides, having enzymatic activity, cytotoxic activity, antigen-binding activity, etc.; the tag peptide comprises a purification tag peptide, a tracer tag peptide and the like; the chaperone protein includes HSP60, HSP70, HSP90 and the like.
According to an embodiment of the invention, the biologically active domain or functional fragment comprises a polypeptide, a synthetic small molecule, a naturally active molecule, and the molecule with albumin binding function is present in the form of a monomer, a homomultimer, a heteromultimer.
According to an embodiment of the present invention, the molecule having an albumin binding function is a nanobody, a heavy chain antibody, a VHH-Fc fusion protein, a monovalent antibody, a bivalent antibody, a multivalent antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, an immunoconjugate.
Further, the present invention provides a molecule having an albumin binding function. According to an embodiment of the invention, the molecule comprises at least one albumin binding domain capable of specifically binding to Human Serum Albumin (HSA) and cynomolgus monkey serum albumin (cyno SA) without specific binding to Bovine Serum Albumin (BSA); the albumin binding structural domain is a nano antibody or a functional derivative thereof obtained by screening a nano antibody library by taking albumin as bait protein.
Further, according to embodiments of the present invention, the albumin binding domain is also capable of specifically binding Murine Serum Albumin (MSA).
Further, according to an embodiment of the present invention, the albumin binding domain comprises:
(1) CDRs region, it has CDRs region sequence of VHH shown in any one of SEQ ID NO 1, 3, 5, 7;
or the like, or a combination thereof,
(2) CDRs which are obtained by mutating one or more amino acids based on the CDRs of the VHH shown in any one of SEQ ID NO 1, 3, 5 and 7, preferably the one or more mutations are substitution mutations of one, two, three, four or five amino acids.
Still further according to an embodiment of the invention, the site where one or several amino acid mutations are made on the basis of the CDRs region of the VHH represented by any one of SEQ ID NOs 1, 3, 5, 7 is selected from the group consisting of: amino acids 14, 27, 28, 54, 62, 66, 99, 104, 106, 107, 109.
Further, according to an embodiment of the invention, the albumin binding domain comprises:
(1) A VHH selected from any one of SEQ ID NOs 1, 3, 5, 7;
(2) A humanized VHH obtained by transplanting CDRs on the basis of the VHH shown in any one of SEQ ID NOs 1, 3, 5 and 7;
or the like, or a combination thereof,
(3) 1, 3, 5, 7, and then obtaining VHH through sequence optimization.
Still further in accordance with an embodiment of the present invention, the albumin binding domain comprises an amino acid sequence selected from any one of SEQ ID NOs 1, 3, 5, 7, 13, 15-36, 38-40.
Further, according to an embodiment of the present invention, the molecule having an albumin binding function comprises two or more albumin binding domains having the same or different amino acid sequences.
Further, according to embodiments of the present invention, the molecule having albumin binding function further comprises at least one bioactive domain or functional fragment, wherein the bioactive domain or functional fragment comprises a carrier protein, an active peptide, a tag peptide, a chaperone protein, or the like, which is linked to the albumin binding domain by a covalent bond, a non-covalent bond, or a linker;
wherein preferably, the carrier protein comprises BSA, OVA, HSA, KLH, fc, etc.; the active peptides include linear peptides or cyclic peptides, having enzymatic activity, cytotoxic activity, antigen-binding activity, etc.; the tag peptide comprises a purification tag peptide, a tracing tag peptide and the like; the chaperone protein includes HSP60, HSP70, HSP90 and the like.
Further, according to embodiments of the present invention, the functional domain or functional fragment having biological activity includes polypeptides, synthetic small molecules, naturally active molecules, and the molecules having albumin binding function exist in the form of monomers, homomultimers, heteromultimers.
Further, according to an embodiment of the present invention, the molecule having an albumin binding function is a nanobody, a heavy chain antibody, a VHH-Fc fusion protein, a monovalent antibody, a bivalent antibody, a multivalent antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, an immunoconjugate.
In a third aspect, the present invention provides a method of producing an albumin binding domain. According to an embodiment of the invention, the method comprises:
(1) Immunizing an animal capable of producing a native heavy chain antibody with HSA;
(2) Separating PBMC amplified heavy chain variable region encoding nucleic acid, and constructing a phage heavy chain antibody library;
(3) Performing affinity panning by using an HSA coated solid phase carrier to remove unbound phage;
(4) Eluting and amplifying the combined phage from the solid phase;
(5) Repeating steps (3) - (4) two to four times;
(6) Performing phagemid rescue on the specifically bound phage, and performing phage ELISA identification;
(7) Phage clones positive for phage ELISA identification were sequenced to obtain nucleic acids encoding the albumin binding domain.
Further, in accordance with an embodiment of the present invention, the method further comprises:
(8) Performing humanized transformation on the albumin binding structural domain;
(9) Sequence optimization of the humanised engineered albumin binding domain.
In a fourth aspect, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises any one of the molecules with albumin binding function described above, and optionally pharmaceutically acceptable excipients.
In a fifth aspect, the present invention provides a polynucleotide. According to an embodiment of the invention, the molecule having an albumin binding function or a protein fraction thereof according to any of the preceding claims is encoded.
In a sixth aspect, the present invention provides a nucleic acid construct. According to an embodiment of the invention, the construct comprises the aforementioned polynucleotide.
In a seventh aspect, the present invention provides a host cell. According to an embodiment of the invention, the host cell comprises the aforementioned polynucleotide or the aforementioned nucleic acid construct.
In an eighth aspect, the present invention provides a method for preparing a molecule having albumin binding function. According to an embodiment of the invention, the method comprises:
(1) Culturing the host cell of claim 18 under conditions suitable for expression of the recombinant foreign protein,
(2) Optionally, the molecule having albumin binding function is isolated and purified from the cell culture.
In a ninth aspect, the present invention provides an albumin binding domain of any one of the molecules with albumin binding function.
In a tenth aspect, the present invention provides the use of the aforementioned albumin binding domain in the manufacture of a medicament and/or for extending the half-life of a medicament.
Further, according to an embodiment of the present invention, the use comprises binding an active molecule having a prophylactic or therapeutic effect to the albumin binding domain via a covalent bond, a non-covalent bond, or a linker molecule.
For a better understanding of the present invention, certain terms are first defined. Other definitions are listed throughout the detailed description section.
Unless otherwise indicated, the term "immunoglobulin sequence" is used as a generic term, whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody, including full-size antibodies, individual chains thereof, and all portions, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or VH/VL domains, respectively). Furthermore, the term "sequence" as used herein (e.g. in terms such as "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence" or "protein sequence") should generally be understood to include both related amino acid sequences as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more restrictive interpretation.
An immunoglobulin single variable domain may be used as a "binding unit", "binding domain" or "building block" (these terms are used interchangeably) for the preparation of a polypeptide containing one or more further immunoglobulin single variable domains that may serve as a binding unit (i.e., for the same or different epitopes of the same target and/or for one or more different targets).
The term "immunoglobulin single variable domain" ("ISVD"), used interchangeably with "single variable domain" ("SVD"), defines a molecule in which an antigen binding site is present on and formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins or fragments thereof in which two immunoglobulin domain variable domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the Complementarity Determining Regions (CDRs) of both VH and VL would favor the antigen binding site, i.e. a total of 6 CDRs would be involved in the formation of the antigen binding site. In contrast, the binding site of an immunoglobulin single variable domain is formed by a single VH or VL domain. Thus, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.
The terms "immunoglobulin single variable domain" and "single variable domain" therefore do not include conventional immunoglobulins or fragments thereof that require the interaction of at least two variable domains to form an antigen binding site. However, these terms include fragments of conventional immunoglobulins in which the antigen binding site is formed by a single variable domain.
Typically, a single variable domain will be an amino acid sequence consisting essentially of 4 framework regions (FR 1 to FR4, respectively) and 3 complementarity determining regions (CDR 1 to CDR3, respectively). Such single variable domains and fragments are most preferred such that they comprise an immunoglobulin fold or are capable of forming an immunoglobulin fold under suitable conditions. Thus, a single variable domain may, for example, comprise a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e. a functional antigen binding unit consisting essentially of a single variable domain, such that the single antigen binding unit does not need to interact with another variable domain to form a functional antigen binding unit, as is the case, for example, for variable domains present in, for example, conventional antibodies and scFv fragments that need to interact with another variable domain, for example, by VH/VL interactions, to form a functional antigen binding domain).
In one embodiment of the invention, the immunoglobulin single variable domain is a light chain variable domain sequence (e.g., a VL sequence) or a heavy chain variable domain sequence (e.g., a VH sequence); more specifically, the immunoglobulin single variable domain may be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody.
For example, a single variable domain or immunoglobulin single variable domain (or amino acids suitable for use as an immunoglobulin single variable domain) may be a (single) domain antibody (or amino acids suitable for use as a (single) domain antibody), "dAb" or dAb (or amino acids suitable for use as a dAb) or nanobody (as defined herein and including but not limited to VHH); other single variable domains, or any suitable fragment of any of them.
For a general description of (single) domain antibodies, reference is also made to the prior art cited herein and to EP0368684. For the term "dAb", reference is made, for example, to Ward et al, 1989 (Nature 341: 544-546), to Holt et al, 2003 (Trends Biotechnol.21: 484-490); and reference is made to other published patent applications such as WO 04/068820, WO 06/030220, WO06/003388, WO 06/059108, WO 07/049017, WO 07/085815 and Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, the single variable domains may be derived from certain species of sharks (e.g. the so-called "IgNAR domains", see e.g. WO 05/18629).
In particular, the immunoglobulin single variable domain may be
Figure BDA0003676091210000081
(as defined herein) or a suitable fragment thereof. [ Note:
Figure BDA0003676091210000082
is a registered trademark of Ebolenx N.V. Inc]For a general description of nanobodies reference is made to the further description below, as well as to the prior art cited herein, as described, for example, in WO 08/020079 (page 16).
For further description of VHH and nanobodies, reference is made to the review article of muydermans 2001 (review in Molecular Biotechnology) 74, 277-302, and to the following patent applications mentioned as general background: WO 94/04678, WO 95/04079 and WO 96/34103 to VrijeUniversiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 from Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO03/055527 to Vlaams institute voor Biotechnology (VIB); algonomics N.V. and WO 03/050531 of Ebolks GmbH; WO 01/90190 of the National Research Council of Canada (National Research Council of Canada); WO 03/025020 of the Institute of Antibodies (Institute of Antibodies); and WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO06/40153, WO 06/079372, WO 06/122786, WO 06/122787, and WO 06/122825 of Ebol Keys GmbH and additional published patent applications of Ebol Keys GmbH. Reference is also made to the further prior art mentioned in these applications and in particular to the list of references mentioned at pages 41-43 of international application WO 06/040153, which list and reference are incorporated herein by reference. As described in these references, nanobodies (especially VHH sequences and partially humanized nanobodies) may be characterized, inter alia, by the presence of one or more "marker residues" in one or more framework sequences. Further descriptions of nanobodies may be found, for example, in WO 08/101985 and WO 08/142164, including humanization and/or camelization of nanobodies, as well as other modifications, moieties or fragments, derivatives or "nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences) and various modifications that increase the half-life of nanobodies and their preparation.
Thus, in the sense of the present invention, the term "immunoglobulin single variable domain" or "single variable domain" includes polypeptides derived from heavy chain antibodies of non-human origin, preferably camelids. As previously described, they may be humanized. Furthermore, the term includes polypeptides which have been "camelized" from non-camelid sources such as mice or humans, for example as described in Davies and Riechmann 1994 (FEBS 339.
The term "immunoglobulin single variable domain" encompasses immunoglobulin sequences of different origin, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. It also includes fully human, humanized or chimeric immunoglobulin sequences. For example, it includes camelid and humanized camelid immunoglobulin sequences, or camelized immunoglobulin single variable domains, such as the camelized dAbs described by Ward et al, 1989 (see, e.g., WO 94/04678 and Davies and Riechmann 1994, 1995 and 1996) and camelized VH.
Likewise, such immunoglobulin single variable domains may be derived in any suitable manner from any suitable source and may for example be naturally occurring VHH sequences (i.e. from a suitable camelid species) or synthetic or semi-synthetic amino acid sequences including but not limited to partially or fully "humanized" VHHs, "camelized" immunoglobulin sequences (and in particular camelized VH), as well as nanobodies and/or VHHs obtained by: such as affinity maturation (e.g., starting from synthetic, random or naturally occurring immunoglobulin sequences such as VHH sequences), CDR grafting, veneering (veneering), combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.
The amino acid sequence and structure of an immunoglobulin single variable domain may be considered-without limitation-to consist of four framework regions or "FRs", which are referred to in the art and herein as "framework region 1" or "FR1", respectively; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4"; the framework regions are separated by three complementarity determining regions or "CDRs," which are referred to in the art as "complementarity determining region 1" or "CDR1," respectively; "complementarity determining region 2" or "CDR2"; and "complementarity determining region 3" or "CDR3".
The total number of amino acid residues in an immunoglobulin single variable domain may be in the range of 110-120, preferably 112-115, and most preferably 113.
As further described in WO 08/020079 (incorporated herein by reference) in paragraphs 58 and 59 q), the amino acid residues of the immunoglobulin single variable domain are numbered according to the general numbering ("Kabat numbering") given by Kabat et al for VH domains ("Sequence of proteins of immunological interest", american public health services (NIH Bethesda, MD, publication No. 91), as described in Riechmann and mumdermans 2000 (j.immunol.methods 240-185-195; see e.g. figure 2 of the publication) applied to camelid VHH domains and, correspondingly, FR1 of the immunoglobulin single variable domain comprises amino acid residues in positions 1-30, CDR1 of the immunoglobulin single variable domain comprises amino acid residues in positions 31-35, FR2 of the immunoglobulin single variable domain comprises amino acids in positions 36-49, CDR2 of the immunoglobulin single variable domain comprises amino acid residues in positions 50-65, FR3 of the immunoglobulin single variable domain comprises amino acid residues in positions 66-94, CDR3 of the immunoglobulin single variable domain comprises amino acid residues in positions 95-102 and FR4 of the immunoglobulin single variable domain comprises amino acid residues in positions 103-113.
Based on the examples of immunoglobulin single variable domain sequences given herein and in WO 08/020079, in WO 06/040153 and in further references cited therein concerning immunoglobulin single variable domains, it will be clear that the exact number of amino acid residues will also depend on the length of the particular CDRs present in the immunoglobulin single variable domain. With respect to CDRs, as is well known in the art, there are a number of conventions that define and describe the CDRs of a VH or VHH fragment, such as the Kabat definition (which is based on sequence variability and is most commonly used) and Chothia definition (which is based on the position of structural loop regions). For example, reference website http:// www. Bio in. For the purposes of the present description and claims, even though one may also refer to CDRs according to Kabat, it is most preferred to define the CDRs based on the Abm definition (which is based on the Abm antibody modeling software of oxford molecular), as this is considered the best compromise between Kabat and Chothia definitions. Refer again to the website http:// www. Bio in. Org. Uk/abs /).
In one embodiment, FR4 comprises the C-terminal amino acid sequence VTVSS, i.e., each of positions 109, 110, 111, 112, and 113. The invention also includes ISVD that terminates at bit 109, 110, 111, or 112. In one aspect of the invention, FR4 terminates with the C-terminal amino acid sequence VTVS (positions 109-112), FR4 terminates with the C-terminal amino acid sequence VTV (positions 109-111), FR4 terminates with the C-terminal amino acid sequence VT (positions 109-110), or FR4 terminates with the C-terminal amino acid sequence V (position 109). The C-terminal extension may be present C-terminally of the last (most C-terminally located) amino acid residue of FR4 of the last ISVD, e.g. V109, T110, V111, S112 or S113, wherein the cysteine moiety of the invention is preferably present or located C-terminally of the C-terminal extension. In one embodiment, FR4 comprises the C-terminal amino acid sequence VTVSS and the C-terminal extension is a cysteine (e.g., the polypeptide of the invention terminates with VTVSSC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VTVS and the C-terminal extension is a cysteine (e.g., the polypeptide of the invention terminates with a VTVSC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VTV and the C-terminal extension is a cysteine (e.g., the polypeptide of the invention terminates in a VTVC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VT and the C-terminal extension is a cysteine (e.g., the polypeptide of the invention terminates with a VTC). In one embodiment, FR4 comprises the C-terminal amino acid V and the C-terminal extension is cysteine (e.g., the polypeptide of the invention terminates with VC).
In one embodiment, the invention relates to a dimer as described herein, wherein said ISVD is selected from the group consisting of: single domain antibodies, amino acid sequences suitable for use as single domain antibodies, amino acid sequences suitable for use as domain antibodies, dabs, amino acid sequences suitable for use as dabs, nanobodies, VHHs, humanized VHHs, and camelized VHs. Preferably, the ISVD comprises between 100 and 140 amino acids, such as between 110-130 amino acids.
The term "phage display library" refers to a "library" of phage on the surface of which exogenous peptides or proteins are expressed. The foreign peptide or polypeptide is displayed on the outer surface of the phage capsid. The foreign peptide can be displayed as a recombinant fusion protein incorporated as part of the phage coat protein; a recombinant fusion protein that is not normally a phage coat protein, but is capable of becoming incorporated into the outer surface of the capsid; or a protein or peptide that becomes covalently or non-covalently linked to the protein. This is achieved by inserting an exogenous nucleic acid sequence into a nucleic acid that can be packaged into a phage particle. The exogenous nucleic acid sequence can, for example, be inserted into the coding sequence of a bacteriophage coat protein gene. If the foreign sequence is cloned in-frame, the protein it encodes will be expressed as part of the coat protein. Thus, a library of nucleic acid sequences, such as a library of antibody lineages made from gene segments encoding the entire B cell lineage of one or more individuals, can be so inserted into a phage to generate a "phage library. When those peptides and proteins representing the peptides and proteins encoded by the nucleic acid library are displayed by phage, a "peptide display library" is generated. Although a variety of bacteriophages are used in the library construction, filamentous bacteriophages are commonly used (Dunn (1996) curr. Opin. Biotechnol.7: 547-553). See, e.g., the description of phage display libraries below.
The term "chimeric antibody" refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to those of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in the antibody derived from another species (typically human) to avoid eliciting an immune response in that species.
The term "chimeric antibody" refers to a portion of the heavy and/or light chain that is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; morrison SL et al, proc.natl.acad.sci.usa,81 6851-6855,1984). For example, the term "chimeric antibody" can include an antibody (e.g., a human murine chimeric antibody) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human antibody).
The term "multispecific antibody", an antibody of the invention may be monospecific, bispecific or multispecific. Multispecific antibodies may be specific for different epitopes of a polypeptide of interest or may contain antigen binding domains specific for more than one polypeptide of interest. See, e.g., tutt et al, 1991, "journal of immunology" 147:60-69; kufer et al, 2004, "trends in Biotechnol. (trends Biotechnol.) 22:238-244. The antibody of the invention may be linked to or co-expressed with another functional molecule (e.g. another peptide or protein). For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second binding specificity. For example, the invention includes bispecific antibodies in which one arm of the immunoglobulin is specific for the N-terminal region of hemolysin a or a fragment thereof, and the other arm of the immunoglobulin is specific for the C-terminal region of hemolysin a or a second therapeutic agent target, or is bound to a therapeutic agent moiety. An exemplary bispecific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces the binding of the bispecific antibody to protein a as compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds protein a and the second Ig CH3 domain contains a mutation that reduces or eliminates protein a binding, such as an H95R modification (numbering according to IMGT exons; H435R according to EU numbering). The second CH3 may further comprise a Y96F modification (according to IMGT; according to EU Y436F). Other modifications that may be found within the second CH3 include: in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; according to EU D356E, L358M, N384S, K392N, V397M and V422I); in the case of IgG2 antibodies, N44S, K52N and V82I (IMGT; N384S, K392N and V422I according to EU); and in the case of IgG4 antibodies Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to IMGT; according to EU Q355R, N384S, K392N, V397M, R409K, E419Q and V422I). Variations on the bispecific antibody formats described above are encompassed within the scope of the invention. Other exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, for example, scFv-or bifunctional-based bispecific formats, igG-scFv fusions, dual Variable Domain (DVD) -Ig, quadromas (quadromas), knob-in-hole structures, common light chains (e.g., common light chains with knob-in-hole structures, etc.), crossmabs, crossfabs, (SEED) bodies, leucine zippers, duobibdoy, igG1/IgG2, dual Action Fab (DAF) -IgG, and Mab2 bispecific formats (for a review of the foregoing formats see, e.g., klein et al, 2012, mabs 4. Bispecific antibodies can also be constructed using peptide/nucleic acid binding, for example where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes of defined composition, valency, and geometry. (see, e.g., kazane et al, journal of the american society of chemistry (j.am. Chem. Soc.) -in electronic publication: 12/4/2012).
The term "single domain antibody" is obtained by genetic engineering methods and has mainly 3 classes, the first of which is the heavy chain variable domain obtained from camelid HCAb, as a single folded unit, which retains full antigen binding activity and is the smallest fragment of the natural antibody. The second type is a heavy chain variable region obtained from IgNAR of cartilaginous fish such as shark, and is denoted by VNAR. The third type is a heavy chain or light chain variable region obtained from a monoclonal antibody of human or murine origin, which retains antigen binding activity but has greatly reduced affinity and solubility.
The term "Fc region" or "Fc" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the hinge region, a CH2 domain, and a CH3 domain, which mediates binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component of the classical complement system (e.g., C1 q), including native sequence Fc regions and variant Fc regions. Typically, the human IgG heavy chain Fc region is the region from the amino acid residue at position Cys226 or Pro230 to the carboxy terminus thereof, although the boundaries may vary. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region may or may not be present. Fc may also refer to this region of isolation, or in the case of a protein polypeptide comprising Fc, such as "binding protein comprising an Fc region," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin). The native sequence Fc region in the antibodies of the invention includes human IgG1, igG2 (IgG 2A, igG 2B), igG3 and IgG4. In IgG, igA, and IgD antibody isotypes, the Fc region comprises the CH2 and CH3 constant domains of each of the two heavy chains of an antibody; the IgM and IgEFc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
The term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen against which it is directed. The term "immunological binding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific. The strength or affinity of an immunological binding interaction may be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. The immunological binding properties between two molecules can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "association rate constant" (Ka or Kon) and the "dissociation rate constant" (Kd or Koff) referring to a particular antibody-antigen interaction can be calculated from the concentration and the actual rate of association and dissociation, see Malmqvist M,1993, nature, 361. The ratio of Kd/Ka is equal to the dissociation constant KD, see Davies DR et al, 1990, annual Rev Biochem, 59. The KD, ka and KD values can be measured by any effective method. In a preferred embodiment, the dissociation constant is measured by bioluminescence interferometry. In other preferred embodiments, the dissociation constant can be measured using surface plasmon resonance techniques (e.g., biacore) or KinExa.
The terms "antibody drug conjugate", "immunoconjugate", consist of an antibody, a linker, and a drug, the linker being a cleavable linker combination or a non-cleavable linker. Antibodies are globular proteins containing a series of amino acid sites that can be used to couple drug-linkers. Due to their tertiary and quaternary structure, only solvent accessible amino acids are available for coupling. In fact, high yields of coupling usually occur on the epsilon-amino group of a lysine residue or on the sulfhydryl group of a cysteine residue. The large number of lysine side chains on the surface of the antibody protein results in a large number of sites available for drug conjugation, resulting in the production of antibody drug conjugates as a mixture containing different numbers of drug conjugates (drug/antibody ratio, DAR) and conjugation sites. The coupling product provided by the invention is still a mixture, but has a narrow DAR distribution range compared with the antibody drug conjugate obtained by the conventional coupling method. The average DAR value is close to 4, and the average DAR value is close to the range of the optimal antibody drug conjugate (2-4). Furthermore, the conjugate product is rarely free of naked antibody (DAR = 0), and this component does not contribute to cytotoxic killing. Also, the coupling product does not contain a heavy coupling product (DAR = 8), which is cleared rapidly in vivo relative to the low DAR component. Therefore, the heterogeneity of the antibody drug conjugate product provided by the invention is greatly improved.
The antibody constituting the antibody-drug conjugate of the present invention preferably retains its antigen-binding ability in its original wild state. Thus, the antibodies of the invention are capable of, preferably specifically, binding to an antigen. In order to develop effective cellular level targets for cancer diagnosis and treatment, researchers have sought transmembrane or other tumor-associated polypeptides. These targets are capable of being specifically expressed on the surface of one or more cancer cells, while expressing little or no expression on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. The confirmation of such tumor-associated factors can greatly improve the specific targeting property of antibody-based cancer treatment.
The terms "conjugate," "linkage," "coupling" refer to the association of two or more molecules. The linkage may also be genetic (i.e., recombinant fusion). In a specific context, the term includes reference to linking a ligand (e.g., an antibody moiety) to an effector molecule. Such linking may be accomplished using a variety of art-recognized techniques, such as by chemical or recombinant means. By "chemical means" is meant a reaction between the antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form one molecule.
The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of a cell and/or causes destruction of a cell. The term is intended to include radioactive isotopes (e.g., I131, I125, Y90, re 186), chemotherapeutic agents, and toxins (such as enzymatically active toxins of bacterial, fungal, plant or animal origin), or fragments thereof.
The terms "vector", "nucleic acid construct" and "nucleic acid construct" refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and an episomal mammalian vector). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are usually present in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term "nucleic acid molecule" is intended to include both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.
The term "polypeptide" refers to a chain comprising at least two amino acid residues linked in series, with no upper limit on the length of the chain. One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation or disulfide bonds. A "protein" may comprise one or more polypeptides.
The term "host cell" refers to a cell in which a vector can be propagated and the DNA expressed, which cell may be a prokaryotic cell or a eukaryotic cell. The term also includes any progeny of the subject host cell. It is understood that not all progeny may be identical to a parent cell, since mutations may occur during replication and such progeny are included.
The invention achieves the following beneficial technical effects:
firstly, on the basis of a camel VHH phage surface display library technology, a humanized anti-HSA nano antibody is prepared and obtained, the humanized anti-HSA nano antibody can be combined with HSA and cyno SA, and the humanized anti-HSA nano antibody on the surface not only keeps the solubility of the camel VHH antibody, but also obviously reduces the heterologous immune reaction to a human body.
Secondly, the humanized VHH is subjected to sequence optimization, and the humanized nano antibody with high affinity and good linearity is obtained. The camel VHH is screened by adopting an immune phage library, the sequence optimization is carried out on the corresponding humanized antibody, and the product nano antibody is subjected to the processes of camel in-vivo affinity maturation, humanized in-vitro sequence optimization and the like, so that the proper affinity, structure and property stability of the product nano antibody are ensured. The result shows that the binding capacity of the antibody to HSA and cyno SA is equivalent to or better than that of a positive control, and the pharmacokinetics of the anti-CD 73mFab antibody in vivo can be remarkably improved and the half-life period of the anti-CD 73mFab antibody can be enhanced by fusing the antibody to the anti-CD 73mFab antibody.
Thirdly, the anti-HSA nanobody of the present invention has versatility, and due to its small molecular weight and clear sequence structure, drugs for parenteral administration can be linked to the anti-HSA nanobody of the present invention in the form of covalent bond (disulfide bond, peptide bond, amide bond, etc.), linker (with bifunctional group), or non-covalent bond (hydrogen bond, ionic bond, hydrophobic interaction, etc.), and further loaded onto HSA, having broad application scenarios.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1: ELISA detection of binding of phage supernatants to HSA and cynoSA antigens
The abscissa is clone number and the ordinate is OD value
FIG. 2 is a schematic diagram: ELISA detection of binding of anti-HSA chimeric antibody to HSA
FIG. 3: ELISA detection of binding of anti-HSA chimeric antibody to cyno SA
FIG. 4 is a schematic view of: ELISA detection of binding of anti-HSA chimeric antibody to MSA
FIG. 5: ELISA detection of binding of anti-HSA humanized antibody to HSA
FIG. 6: ELISA detection of binding of anti-HSA humanized antibody to cyno SA
FIG. 7 is a schematic view of: ELISA for detection of binding of anti-HSA humanized antibody to MSA
FIG. 8: change curve of metabolism blood concentration of tested recombinant protein with HSA binding structure domain in Balb/C mice
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
The present invention will now be described with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, huang Petang et al) or according to product instructions. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.
Example 1: construction of camel nano antibody immune phage library
The camel immune library is constructed by immunizing a camel with an HSA antigen, separating Peripheral Blood Mononuclear Cells (PBMC), extracting total RNA (ribonucleic acid) for reverse transcription, amplifying a variable region of a heavy chain of a nano antibody (VHH) by using a reverse transcription product as a template, connecting the variable region of the heavy chain of the nano antibody with a phage display carrier, and electrically transferring escherichia coli TG1 competent cells.
Specifically, camels were immunized twice weekly for 4 times. 0.8mg of HSA extracellular region recombinant protein (purchased from Hualan bioengineering Co., ltd., product batch No. 201405030) was injected each time, and Freund's complete/incomplete adjuvant (Sigma, F5881, F5506) was administered in a subcutaneous multi-point injection manner. 1mL of blood serum was collected 2 weeks after each immunization, and the titers of whole antibody (IgG) and heavy chain antibody (HcAb) in the serum were measured by ELISA using the immunogen as the antigen. When the serum titer meets the requirement of library construction, collecting 100mL camel peripheral blood, separating PBMC with a separation kit (Tianjin tertiary ocean, cat: TBD2011 CM), extracting the total RNA of the PBMC, inverting to obtain cDNA serving as subsequent amplificationA template for a VHH fragment. Searching camel source VHH antibody genes according to related literatures and databases, designing and synthesizing VHH antibody library construction primers, and amplifying antibody variable region gene sequences by PCR. The vector and amplified antibody fragment are then cleaved enzymatically using an endonuclease. The ligation product is constructed by adopting a T4 ligase ligation mode, and is transferred into TG1 strain by an electrotransfection technology. Finally construct a 1.8X 10 8 The camel antihuman HSA VHH antibody immune library is used for screening specific antihuman HSA nano antibodies. In order to detect the accuracy of the library, 50 clones were randomly selected for colony PCR, and the result showed that the insertion rate reached 100%.
Example 2: anti-HSA specific Nanobody screening
And screening the constructed camel immune library by a solid-phase screening method to obtain the specific phage display nano antibody.
(1) And presenting an original library. The camel immune library is transferred to a 2YT culture medium containing ampicillin and tetracycline, cultured to a logarithmic growth phase, added with M13 helper phage and then added with kanamycin, and presented overnight at a lower temperature. The culture supernatant is collected the next day, and the PEG precipitate is used for concentrating the phage to obtain a high-titer antibody library presentation product for subsequent screening.
(2) And (4) screening. Screening of specific antibodies was performed by solid phase method. Coating a specific antigen HSA on the surface of an immune tube, respectively sealing the immune tube and an antibody library by using a sealing agent, adding the antibody library into the immune tube for incubation, then repeatedly washing, finally eluting by using acid with the pH value of 2.2, mixing and incubating an eluate after the eluate is neutralized to neutrality with XL-Blue in a logarithmic growth phase for infection, further performing phage display, and recovering specific phage particles. Screening for 2-3 rounds, and identifying the monoclonal antibody.
(3) And (7) identifying. XL-Blue coated plates infected with recovered specific phage particles were used for monoclonal identification after colonies were grown. Single clones were picked and cultured until logarithmic growth phase was reached, infected with M13 helper phage, and then cultured overnight at 30 ℃ with kanamycin. The supernatants were collected the next day and ELISA reactions were performed using ELISA plates coated with HSA, cyno SA and BSA, respectively, as shown in FIG. 1. Phagemids (phage display vectors containing antibody genes) extracted from phage clones which are positive for HAS and cyno SA reactions and negative for BSA reactions are sequenced to determine the VHH antibody gene sequence. 4 strains of phage-displayed nanobodies which can be simultaneously combined with HSA and cynoSA recombinant proteins are obtained by screening: 2H1,2H2,2H4,2MG6, and the sequencing results are as follows:
the amino acid sequence of the variable region of the clone 2H1 is shown as SEQ ID NO. 1, and the nucleotide sequence of the variable region is shown as SEQ ID NO. 2;
the amino acid sequence of the variable region of the clone 2H2 is shown as SEQ ID NO. 3, and the nucleotide sequence of the variable region is shown as SEQ ID NO. 4;
the amino acid sequence of the variable region of the cloned 2H4 is shown as SEQ ID NO.5, and the nucleotide sequence of the variable region is shown as SEQ ID NO. 6;
the amino acid sequence of the variable region of the clone 2MG6 is shown as SEQ ID NO.7, and the nucleotide sequence of the variable region is shown as SEQ ID NO. 8.
Example 3: preparation of antihuman HSA nano antibody and contrast antibody
And (3) performing VHH gene total synthesis on the same-target-point control nano antibody ALB8, cloning the same-target-point control nano antibody ALB8 into a eukaryotic transient expression vector containing a human Fc (hFc) encoding gene, and performing recombinant expression in HEK293 cells. And (3) after 5-6 days of cell transfection, taking culture supernatant, and purifying the expression supernatant by using a ProA affinity chromatography column to obtain ALB8 recombinant protein. Wherein, the ALB8 amino acid sequence of the contrast antibody is derived from US 2014/0228546 A1, the amino acid sequence of the variable region is shown as SEQ ID NO. 9, and the nucleotide sequence of the variable region is shown as SEQ ID NO. 10. The amino acid sequence of hFc is shown in SEQ ID NO. 11, and the nucleotide sequence is shown in SEQ ID NO. 12.
The correctly sequenced 2H1,2H2,2H4,2MG6 VHH genes were cloned into eukaryotic expression vectors containing the human Fc (hFc) encoding gene. After obtaining expression plasmids with correct sequences, 293F cells are transfected for transient expression, 5-6 days after cell transfection, culture supernatants are taken and purified by a ProA affinity chromatography column to obtain chimeric antibodies ch2H1, ch2H2, ch2H4 and ch2MG6.
Example 4: chimeric antibody affinity assays
The antibody affinity was determined by capturing the Fc fragment of the antibody with an Ocet QKe system instrument from Fortebio using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe. For the measurement, the chimeric antibody of ch2H1, ch2H2, ch2H4, ch2MG6 and the control antibody ALB8 were diluted to 4. Mu.g/ml with PBS buffer, and passed over the surface of an AHC probe (Cat: 18-0015, PALL) for 120 seconds. HSA recombinant protein (purchased from Hualan bioengineering GmbH, product batch No. 201405030) (60 nM) was used as mobile phase with an association time of 300s and a dissociation time of 300s. After the experiment, blank control response values are deducted, and the software is used for 1:1Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated as shown in table 1.
TABLE 1 determination of the affinity of the chimeric antibodies to the HSA recombinant protein
Sample(s) KD(M)
Ch2H1 2.79E-08
Ch2H2 1.62E-08
Ch2H4 7.79E-09
Ch2MG6 5.79E-10
ALB8 6.76E-09
The results in table 1 indicate that all chimeric antibodies are capable of correct assembly, all with the ability to specifically bind to the HSA recombinant protein.
Example 5: species-specific assay for ELISA detection of anti-HSA chimeric antibodies
HSA recombinant protein (purchased from Hualan bioengineering GmbH, product batch number: 201405030), cynomolgus monkey SA recombinant protein (Cat: # CSA-C52H4, lot. #3445-9C5F1-QD, ACRO), mouse SA recombinant protein (Cat: # MSA-M52H8, lot. #3441a-9CBF1-QF, ACRO) were coated overnight at 4 ℃ with a coating concentration of 1 μ g/mL; after washing the plate 3 times with PBS, 5% BSA in PBS was added, blocked at 37 ℃ for 60min, and the plate was washed 3 times with PBST; adding ch2H1, ch2H2, ch2H4, ch2MG6 and control antibody ALB8 (initial concentration is 1 μ g/mL, 3-fold gradient is sequentially diluted for 12 concentrations), incubating for 60min at 37 ℃, and washing the plate for 4 times by PBST; HRP-anti-human Fc (Cat: 109-035-098, jackson Immuno Research) was added at a dilution of 1; adding a TMB substrate for color development, incubating at 37 ℃ for 10min, and adding 2M HCl to stop the reaction; and reading and recording the absorbance A450nm-630nm of the pore plate under the wavelength of 450nm by taking 630nm as a reference wavelength, and calculating the EC50 value. The results are shown in FIGS. 2-4.
FIGS. 2-3 show that ch2H1, ch2H2, ch2H4, ch2MG6 and the control antibody can specifically bind to human and cynomolgus monkey SA recombinant proteins, and the corresponding half effective binding concentration EC50 values are shown in Table 2. Table 2, fig. 4, shows that Ch2H1, ch2MG6 and control antibody ALB8 also bind to mouse SA recombinant protein.
TABLE 2 ELISA detection of EC50 of anti-HSA chimeric antibody binding to serum albumin of different species
Figure BDA0003676091210000171
Figure BDA0003676091210000181
Example 6: humanization and recombinant expression analysis of anti-HSA nanobody
6.1 humanization of camel-derived Nanobody 2MG6
1. CDR grafting
The VHH sequence of camelid antibody 2MG6 was analyzed in general to determine the antigen Complementarity Determining (CDR) regions to which the antibody binds to the antigen and the framework regions (framework) that support the conserved three-dimensional conformation of the antibody. Then, based on the results of homology alignment, the most similar human antibody template VH3 (3-07) was selected as the basic template, and CDR grafting was performed in combination with the full sequence blast results, thereby achieving humanization of 2MG6 variable region (VHH) in the Framework region. The humanized amino acid sequence of CDR Grafted (CDR Grafted) of the 2MG6 antibody is shown as SEQ ID NO. 13; the nucleotide sequence is shown as SEQ ID NO. 14.
2. Mutant design
The hz2MG6 sequence was designed by mutation, and the mutation sites are shown in Table 3.
Table 3 Hz2MG6 mutant sequence design information
Figure BDA0003676091210000182
Figure BDA0003676091210000191
Note: A14P indicates that the 14 th amino acid A of Hz2MG6 shown in SEQ ID NO. 13 is mutated to P, and so on.
3. Recombination expression and affinity detection of humanized antibody hz2MG6
The humanized design of the variable region sequence of the hz2MG6 antibody is completely synthesized and cloned to a eukaryotic expression vector containing a human Fc (hFc) coding gene. After obtaining an expression plasmid with a correct sequence, carrying out site-directed mutagenesis on the expression plasmid according to the mutagenesis design, transferring the expression plasmid into escherichia coli for amplification, and obtaining a hz2MG6 mutagenesis expression plasmid; transferring into HEK293 cells for recombinant expression, transfecting the cells for 5-6 days, taking culture supernatant, and purifying the expression supernatant by using a ProA affinity chromatography column to obtain the humanized antibody and mutant recombinant protein thereof.
The antibody affinity was determined by capturing the Fc fragment of the antibody with an Ocet QKe system instrument from Fortebio using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe. For the assay, the hz2MG6 antibody and the chimeric antibody were diluted to 4ug/mL with PBS buffer and passed over the surface of AHC probe (Cat: 18-0015, PALL) for 300S. HSA recombinant protein (purchased from Hualan bioengineering GmbH, product batch No. 201405030) was used as the mobile phase, and the concentration of the HSA recombinant protein was 60nM. The binding time was 300s and the dissociation time was 300s. After the experiment, blank control response values were deducted, and the software was run for 1:1Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated.
The affinity of hz2MG6 and its mutants to the HSA recombinant protein was determined by ForteBio and the KD results are shown in Table 4, and the affinity of humanized 2MG was 10 -8 M to 10 -10 M, mutants with different affinities can provide more choices for constituting fusion proteins with different recombinant proteins, thereby more specifically adjusting the metabolic characteristics of the fusion proteins.
TABLE 4 results of affinity assay of hz2MG6 mutants
Name (R) KD value (M) Name (R) KD value (M)
hz2MG6 1.72E-09 hz2MG6m11 7.36E-10
hz2MG6m1 1.56E-09 hz2MG6m12 6.83E-10
hz2MG6m2 2.41E-10 hz2MG6m13 5.13E-08
hz2MG6m3 1.64E-09 hz2MG6m14 2.54E-08
hz2MG6m4 2.49E-10 hz2MG6m15 6.63E-08
hz2MG6m5 4.51E-09 hz2MG6m16 3.61E-08
hz2MG6m6 2.91E-09 hz2MG6m17 4.40E-10
hz2MG6m7 5.12E-07 hz2MG6m18 5.72E-09
hz2MG6m8 5.76E-09 hz2MG6m19 9.85E-09
hz2MG6m9 4.71E-07 hz2MG6m20 7.10E-09
hz2MG6m10 5.63E-09 hz2MG6m21 3.42E-09
6.2 humanization of camel-derived Nanobody 2H4
1. CDR grafting
First, the VHH sequence of camelid antibody 2H4 was analyzed in general to determine the Complementarity Determining Region (CDR) of the antigen to which the antibody binds and the framework region (framework) supporting the conserved three-dimensional conformation of the antibody. Then, based on the results of homology alignment, the most similar human antibody template VH3 (3-07) is selected as a basic template, and CDR grafting is carried out by combining the results of full sequence blast, thereby realizing humanization of 2H4 variable regions (VHH) in the Framework region. Renamed hzH4; the humanized variable region amino acid sequence of the hzH4 antibody CDR Grafted (CDR Grafted) is shown as SEQ ID NO: 36; the nucleotide sequence of the humanized variable region is shown as SEQ ID NO. 37.
2. Mutant design
The hzH4 sequence was subjected to mutation design, and the mutation sites are shown in Table 5.
TABLE 5 hZH4 humanized mutant sequence design
Figure BDA0003676091210000201
3. Recombination expression and affinity detection of humanized antibody hzH4
The humanized hzH4 antibody variable region sequence is synthesized completely and cloned to eukaryotic expression vector containing human Fc (hFc) encoding gene. After obtaining an expression plasmid with a correct sequence, carrying out site-directed mutagenesis on the expression plasmid according to the mutagenesis design, transferring the expression plasmid into escherichia coli for amplification, and obtaining an hzH4 mutagenesis expression plasmid; transferring into HEK293 cells for recombinant expression, transfecting the cells for 5-6 days, taking culture supernatant, and purifying the expression supernatant by using a ProA affinity chromatography column to obtain the humanized antibody and mutant recombinant protein.
The antibody affinity was determined by capturing the Fc fragment of the antibody with an Ocet QKe system instrument from Fortebio using an anti-human antibody Fc fragment capture Antibody (AHC) biological probe. For the assay, hzH4 antibody and mutant were diluted to 4ug/mL with PBS buffer and passed over the surface of AHC probe (Cat: 18-0015, PALL) for 300S. HSA recombinant protein (purchased from Hualan bioengineering GmbH, product batch No. 201405030) was used as the mobile phase, and the concentration of the HSA recombinant protein was 60nM. The binding time was 300s and the dissociation time was 300s. After the experiment, blank control response values were deducted, and the software was run for 1:1Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated.
The affinity of hzH4 and its mutants to the recombinant HSA protein was determined by ForteBio, and the results are shown in Table 6, where the affinity of humanized H4 was 10 -8 M to 10 -9 M, mutants with different affinities can provide more choices for forming fusion proteins with different recombinant proteins, thereby more specifically adjusting the metabolic characteristics of the fusion proteins.
TABLE 6 result of affinity assay of HzH4 mutant
Figure BDA0003676091210000202
Figure BDA0003676091210000211
Example 7: ELISA for detecting species cross reaction of anti-HSA humanized antibody and serum albumin combination
HSA recombinant protein (purchased from Hualan bioengineering GmbH, product batch number: 201405030), cynomolgus monkey SA recombinant protein (Cat: # CSA-C52H4, lot. #3445-9C5F1-QD, ACRO), mouse SA recombinant protein (Cat: # MSA-M52H8, lot. #3441a-9CBF1-QF, ACRO) were coated overnight at 4 ℃ with a coating concentration of 1 μ g/mL; after washing the plate 3 times with PBS, 5% BSA in PBS was added, blocking was performed at 37 ℃ for 60min, and the plate was washed 3 times with PBST; adding hz2MG6, hZH4 and control antibody ALB8 (initial concentration is 1 μ g/mL, 3-fold gradient is sequentially diluted for 12 concentrations), incubating at 37 deg.C for 60min, and washing plate with PBST for 4 times; HRP-anti-human Fc (Cat: 109-035-098, jackson Immuno Research) was added at a dilution of 1; adding a TMB substrate for color development, incubating at 37 ℃ for 10min, and adding 2M HCl to stop the reaction; and reading and recording the absorbance A450nm-630nm of the pore plate under the wavelength of 450nm by taking 630nm as a reference wavelength.
The experimental results show that both the hz2MG6 and the control antibody can be specifically combined with human, cynomolgus monkey SA and mouse SA recombinant proteins, the results are shown in FIGS. 5-7, and the calculated EC50 values are shown in Table 7. The results in fig. 5-7 and table 7 show that hzH4 binds specifically to human and cynomolgus monkey SA, but has no binding activity to mouse SA recombinant protein.
TABLE 7 EC50 for ELISA detection of binding of anti-HSA humanized antibodies to serum albumin of different species
Figure BDA0003676091210000212
Example 8: HSA antibodies extend the half-life of antibody drugs in mice
anti-HSA nanobodies hz2MG6 and ALB8 which are crossed with mice are selected to respectively construct recombinant expression vectors of anti-CD 73 antibodies TJD5-mFab (patent number: CN 109476755A), TJD5-mFab-hz2MG6 (SEQ ID NO. 41) and TJD5-mFab-ALB8 (SEQ ID NO. 42), and the recombinant expression vectors are transiently expressed in HEK293 cells and purified to prepare recombinant protein samples. The pharmacokinetics detection of the 3 recombinant proteins in a Balb/C mouse proves that the anti-HSA nano antibody can prolong the half life of the recombinant proteins in an animal body.
Experiment design: female 6-week-old Balb/c mice (Witonglihua) were given 250 ug/mouse test sample via tail vein, 6 mice were divided into two subgroups, 3 mice/subgroup, blood was collected by cross-bleeding at 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 8h, 24h, 30h, 48h, 72h, and blood was collected by tail-breaking, and serum was collected and stored at-20 deg.C.
After the blood collection was completed, coating human CD73-His (SEQ ID NO: NP-002517.1) on a 96-well ELISA plate at 0.5ug/ml and 100 ul/well overnight at 4 ℃ and after washing the plate 3 times with PBS, adding 5% BSA PBS, blocking at 37 ℃ and washing the plate 3 times with PBST; adding a serum sample to be detected (10000, 20000-fold dilution), setting TJD5-Fab and a standard curve hole (the initial concentration is 0.05 mu g/mL, 2-fold serial dilution, 12 gradients), incubating for 60min at 37 ℃, washing the plate by PBST for 4 times; adding HRP-goat anti-mouse Fab (Cat: A2304-1ML, lot #019M4758V, sigma) diluted at 1; adding TMB substrate (Cat: ME142, beijing Taitianhe Biotech Co., ltd.) for color development, incubating at 37 deg.C for 10min, and adding 2M HCl to terminate the reaction; and reading and recording the absorbance A450nm-630nm of the pore plate under the wavelength of 450nm by taking 630nm as a reference wavelength. Taking the concentration of the standard antibody as an axis Y and the OD value as an axis X, drawing a time-antibody concentration curve chart, performing linear fitting, and obtaining the curve according to a formula T 1/2 = 0.693/k |, calculating the half-life T of the drug 1/2 . The respective protein metabolism profiles are shown in fig. 8, and the fitting half-life results are shown in table 8. The results show that under the current conditions, the TJD5-mFab fused with the HSA nanobody has higher blood concentration at the same time point compared with the TJD5-mFab not fused with the nanobody, and T is metabolized 1/2 The effect of prolonging the half-life of the molecule fused or coupled with the anti-HSA nano antibody is shown.
TABLE 8 half-life of the tested recombinant proteins in Balb/C mice
Group of Half-life (hrs)
TJD5-mFab(n＝3) T1/2＝1.5
TJD5-mFab-hz2MG6(n＝3) T1/2＝23.6
TJD5-mFab-ALB8(n＝3) T1/2＝15.8
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Sequence listing
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<120> nano antibody targeting Human Serum Albumin (HSA) and application thereof
<150> 202110616699.4
<151> 2021-06-03
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Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 10
<211> 345
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gaggtgcagc tggtggagtc tggaggtggc ctggtgcagc ctggcaactc cctgaggctg 60
tcctgcgctg cctctggctt caccttctcc tccttcggca tgtcctgggt gaggcaggct 120
cctggcaagg gcctggagtg ggtgtcctcc atctctggct ccggatctga caccctgtac 180
gccgactccg tgaagggacg gttcaccatc tccagagaca acgccaagac caccctgtac 240
ctgcagatga actccctgag acccgaggac acagccgtgt actactgcac catcggaggc 300
tccctgtcca ggtcctccca gggcaccctg gtgaccgtgt cctcc 345
<210> 11
<211> 233
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 11
Ala Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys
1 5 10 15
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
20 25 30
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
35 40 45
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
50 55 60
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
65 70 75 80
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
85 90 95
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
100 105 110
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
115 120 125
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
130 135 140
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
145 150 155 160
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
165 170 175
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
180 185 190
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
195 200 205
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
210 215 220
Gln Lys Ser Leu Ser Leu Ser Pro Gly
225 230
<210> 12
<211> 699
<212> DNA
<213> Intelligent (Homo sapiens)
<400> 12
gctagcgagc ccaaatctag cgacaaaact cacacatgcc caccgtgccc agcacctgaa 60
ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 240
gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300
ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgcctcca 420
tctcgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540
acgcctcccg tgctggactc cgacggctcc ttcttcctct atagcaagct caccgtggac 600
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 660
aaccactaca cgcagaagag cctctccctg tccccgggt 699
<210> 13
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Lys Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 14
<211> 375
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gaggtgcagc tggtggagtc tggaggaggc ctggtgcagg ctggaggctc cctgaggctg 60
tcctgcgctg cctccggctc cacctacccc acagcctaca tcgcctggtt cagacaggct 120
cctggcaagg agcgggaggg agtggccggc atctacaccg tgggaggctc caccttctac 180
accgactccg tgaagggacg gttcaccatc tccagagaca acgccaagaa ctccctgtac 240
ctgcagatga actccctgag agccgaggac acagccgtgt acttctgcgc tgctggcagg 300
accgacaccg ccaagctgcg ggaccctcgg tccttcggct actggggcca gggcaccctg 360
gtgaccgtgt cctcc 375
<210> 15
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Lys Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 16
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Lys Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 17
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Lys Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 18
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Lys Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 19
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Glu Thr Asp Thr Ala Lys Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 20
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Ala Thr Asp Thr Ala Lys Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 21
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Glu Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 22
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Ala Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 23
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Lys Leu Glu Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 24
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Lys Leu Ala Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 25
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Lys Leu Arg Asp Pro Glu Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 26
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Lys Leu Arg Asp Pro Ala Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 27
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Glu Thr Asp Thr Ala Lys Leu Ala Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 28
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Ala Thr Asp Thr Ala Lys Leu Ala Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 29
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Glu Thr Asp Thr Ala Ala Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 30
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Ala Thr Asp Thr Ala Ala Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 31
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Lys Leu Arg Glu Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 32
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Glu Thr Asp Thr Ala Lys Leu Arg Asp Pro Arg Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 33
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Ala Leu Arg Asp Pro Glu Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 34
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Arg Thr Asp Thr Ala Lys Leu Ala Asp Pro Glu Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 35
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Glu Val Gln Leu Val 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 Tyr Pro Thr Ala
20 25 30
Tyr Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Gly Ala Thr Asp Thr Ala Lys Leu Arg Asp Pro Glu Ser Phe
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 36
<211> 129
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Glu Val Gln Leu Val 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 Val Ser Tyr Arg Pro Asn
20 25 30
Cys Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Tyr Thr Asp Ser Gly Leu Ser Gly Thr Thr Tyr Tyr Ala
50 55 60
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
65 70 75 80
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Ala Asp Trp Lys Tyr Asp Ser Thr Arg Cys Gly Leu
100 105 110
Glu Val Glu Tyr Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 38
<211> 387
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gaggtgcagc tggtggagtc tggaggaggc ctggtgcagc ctggaggctc cctgaggctg 60
tcctgcgctg cctctggcgt gtcctacaga cccaactgcg tggcctggtt caggcaggct 120
cctggcaagg agcgggaggg agtggctgcc atctacaccg actctggcct gtccggcacc 180
acctactacg ccgactccgt gaagggacgg ttcaccatct ccagggacaa cgccaagaac 240
tccctgtacc tgcagatgaa ctccctgaga gccgaggaca ccgccgtgta ctactgcgct 300
gccgactgga agtacgactc cacccggtgc ggactggagg tggagtacga ctcctgggga 360
cagggcaccc tggtgaccgt gtcctcc 387
<210> 38
<211> 129
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Glu Val Gln Leu Val 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 Tyr Arg Pro Asn
20 25 30
Cys Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Tyr Thr Asp Ser Gly Leu Ser Gly Thr Thr Tyr Tyr Ala
50 55 60
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
65 70 75 80
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Ala Asp Trp Lys Tyr Asp Ser Thr Arg Cys Gly Leu
100 105 110
Glu Val Glu Tyr Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 39
<211> 129
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Glu Val Gln Leu Val 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 Val Ser Tyr Arg Pro Asn
20 25 30
Cys Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Tyr Thr Ser Ser Gly Leu Ser Gly Thr Thr Tyr Tyr Ala
50 55 60
Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
65 70 75 80
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Ala Asp Trp Lys Tyr Asp Ser Thr Arg Cys Gly Leu
100 105 110
Glu Val Glu Tyr Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 40
<211> 129
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Glu Val Gln Leu Val 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 Val Ser Tyr Arg Pro Asn
20 25 30
Cys Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Tyr Thr Asp Ser Gly Leu Ser Gly Thr Thr Tyr Tyr Ala
50 55 60
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
65 70 75 80
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Ala Ala Asp Trp Lys Tyr Glu Ser Thr Arg Cys Gly Leu
100 105 110
Glu Val Glu Tyr Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 41
<211> 358
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly
20 25 30
Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Asn Tyr Gly Gly Ser Asn Gly Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Tyr Asp Ala Tyr Tyr Glu Ala Leu Asp Asp Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser
180 185 190
Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
245 250 255
Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
260 265 270
Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
275 280 285
Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala
290 295 300
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr
305 310 315 320
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val
325 330 335
Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr
340 345 350
Leu Val Thr Val Ser Ser
355
<210> 42
<211> 358
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly
20 25 30
Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Asn Tyr Gly Gly Ser Asn Gly Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Tyr Asp Ala Tyr Tyr Glu Ala Leu Asp Asp Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser
180 185 190
Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Gly
210 215 220
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
245 250 255
Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
260 265 270
Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
275 280 285
Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala
290 295 300
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr
305 310 315 320
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val
325 330 335
Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr
340 345 350
Leu Val Thr Val Ser Ser
355
<210> 43
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Pro Asn Cys Val Ala
1 5
<210> 44
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Ala Ile Tyr Thr Asp Ser Gly Ile Ser Gly Thr Thr Tyr Tyr Ala Asp
1 5 10 15
Ser Val Lys Gly
20
<210> 45
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Asp Trp Lys Tyr Asp Gly Ser Arg Cys Gly Leu Glu Val Glu Tyr Asp
1 5 10 15
Ser
<210> 46
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Asn Tyr Gly Met Ser
1 5
<210> 47
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Gly Ile Tyr Ser Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 48
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Thr Tyr Asn Val Gln Phe Ala Ala Thr Ala Gly Ile Lys Leu Thr Asp
1 5 10 15
Phe Asp Tyr
<210> 49
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
Ala Ile Tyr Thr Asp Ser Gly Leu Ser Gly Thr Thr Tyr Tyr Ala Asp
1 5 10 15
Ser Val Lys Gly
20
<210> 50
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
Asp Trp Lys Tyr Asp Ser Thr Arg Cys Gly Leu Glu Val Glu Tyr Asp
1 5 10 15
Ser
<210> 51
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Thr Ala Tyr Ile Ala
1 5
<210> 52
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Ser Val Lys
1 5 10 15
Gly
<210> 53
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 53
Gly Arg Thr Asp Thr Ala Lys Leu Arg Asp Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 55
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 55
Gly Ile Tyr Thr Val Gly Gly Ser Thr Phe Tyr Thr Asp Thr Val Lys
1 5 10 15
Gly
<210> 56
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 56
Gly Glu Thr Asp Thr Ala Lys Leu Arg Asp Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 57
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 57
Gly Ala Thr Asp Thr Ala Lys Leu Arg Asp Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 58
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 58
Gly Arg Thr Asp Thr Ala Glu Leu Arg Asp Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 59
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 59
Gly Arg Thr Asp Thr Ala Ala Leu Arg Asp Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 60
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 60
Gly Arg Thr Asp Thr Ala Lys Leu Glu Asp Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 61
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 61
Gly Arg Thr Asp Thr Ala Lys Leu Ala Asp Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 62
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 62
Gly Arg Thr Asp Thr Ala Lys Leu Arg Asp Pro Glu Ser Phe Gly Tyr
1 5 10 15
<210> 63
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 63
Gly Arg Thr Asp Thr Ala Lys Leu Arg Asp Pro Ala Ser Phe Gly Tyr
1 5 10 15
<210> 64
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 64
Gly Glu Thr Asp Thr Ala Lys Leu Ala Asp Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 65
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 65
Gly Ala Thr Asp Thr Ala Lys Leu Ala Asp Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 66
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 66
Gly Glu Thr Asp Thr Ala Ala Leu Arg Asp Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 67
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 67
Gly Ala Thr Asp Thr Ala Ala Leu Arg Asp Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 67
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 67
Gly Arg Thr Asp Thr Ala Lys Leu Arg Glu Pro Arg Ser Phe Gly Tyr
1 5 10 15
<210> 68
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 68
Gly Arg Thr Asp Thr Ala Ala Leu Arg Asp Pro Glu Ser Phe Gly Tyr
1 5 10 15
<210> 69
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 69
Gly Arg Thr Asp Thr Ala Lys Leu Ala Asp Pro Glu Ser Phe Gly Tyr
1 5 10 15
<210> 70
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 70
Gly Ala Thr Asp Thr Ala Lys Leu Arg Asp Pro Glu Ser Phe Gly Tyr
1 5 10 15
<210> 71
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 71
Pro Asn Cys Val Ala
1 5
<210> 72
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 72
Ala Ile Tyr Thr Asp Ser Gly Leu Ser Gly Thr Thr Tyr Tyr Ala Asp
1 5 10 15
Ser Val Lys Gly
20
<210> 73
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 73
Asp Trp Lys Tyr Glu Ser Thr Arg Cys Gly Leu Glu Val Glu Tyr Asp
1 5 10 15
Ala
<210> 74
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 74
Asp Trp Lys Tyr Asp Ser Thr Arg Cys Gly Leu Glu Val Glu Tyr Asp
1 5 10 15
Ser
<210> 75
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 75
Ala Ile Tyr Thr Ser Ser Gly Leu Ser Gly Thr Thr Tyr Tyr Ala Asp
1 5 10 15
Thr Val Lys Gly
20

Claims (20)

1. A molecule having albumin binding function, comprising at least one albumin binding domain, said albumin binding domain comprising Complementarity Determining Regions (CDRs) 1,2 and 3, said CDRs1, 2 and 3 having amino acid sequences as set forth in CDRs1, 2 and 3 of a selected VHH or a mutant thereof, wherein said mutant is obtained by performing one or more amino acid mutations based on CDRs1, 2 and 3 of said selected VHH, preferably said one or more amino acid mutations are substitution mutations of one, two, three, four or five amino acids,
wherein the amino acid sequences of CDRs1, 2 and 3 of the selected VHH are one of:
(1) The amino acid sequences of CDRs1, 2 and 3 of the selected VHH are shown in SEQ ID No.43, 44 and 45 respectively;
(2) The amino acid sequences of CDRs1, 2 and 3 of the selected VHH are shown in SEQ ID NO.46, 47 and 48, respectively;
(3) The amino acid sequences of CDRs1, 2 and 3 of the selected VHH are set forth in SEQ ID nos. 43, 49 and 50, respectively;
(4) The amino acid sequences of the CDRs1, 2 and 3 of the selected VHH are shown in SEQ ID nos. 51, 52 and 53, respectively.
2. Molecule with albumin binding function according to claim 1, characterized in that the amino acid sequences of CDRs1, 2 and 3 of said mutant of said selected VHH are one of the following:
(1) The amino acid sequence of CDR1 and CDR2 of the selected VHH mutant is shown in SEQ ID NO.51, and GIYTVGGSTFYTDX 10 VKG shows that the amino acid sequence of CDR3 is GX 1 TDTAX 2 LX 3 X 4 PX 5 SFGY is shown;
(2) The amino acid sequence of CDR1 and the amino acid sequence of CDR2 of said mutant of said selected VHH are shown in SEQ ID NO.71 and AIYTX, respectively 6 SGLSGTTYYADX 7 VKG shows that the amino acid sequence of CDR3 is DWKYX 8 STRCGLEVEYDX 9 As shown in the drawings, the above-described,
wherein,
X 1 is R, E or A;
X 2 is K, E or A;
X 3 is R, E or A;
X 4 is D or E;
X 5 is R, E or A;
X 6 is D or S;
X 7 is S or T;
X 8 is D or E;
X 9 is S or A;
X 10 is S or T.
3. The molecule having albumin binding function of claim 1, wherein the selected VHH or the mutant thereof comprises a heavy chain variable region, wherein,
(1) The heavy chain variable region comprises the sequence set forth in SEQ ID NO 1 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(2) The heavy chain variable region comprises the sequence set forth in SEQ ID No. 3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(3) The heavy chain variable region comprises the sequence set forth in SEQ ID NO.5 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
(4) The heavy chain variable region comprises the sequence set forth in SEQ ID NO.7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
4. The albumin binding molecule of claim 3 wherein the amino acid sequence of the heavy chain variable region of said mutant is mutated in the VHH of SEQ ID No.5 in a combination selected from the group consisting of:
V27F+S28T，
D54S+S66T，
D54E+S66A。
5. the albumin binding molecule of claim 4 wherein the amino acid sequence of said heavy chain variable region of said mutant is as set forth in SEQ ID NO 36, 38, 39 or 40.
6. The albumin binding molecule of claim 3 wherein the amino acid sequence of the heavy chain variable region of said mutant is mutated in the VHH of SEQ ID No.7 in a combination selected from the group consisting of:
A14P，
A14P+S27F，
A14P+S26T，
A14P+S27F+S62T，
A14P+S27F+S62T+R99E，
A14P+S27F+S62T+R99A，
A14P+S27F+S62T+K104E，
A14P+S27F+S62T+K104A，
A14P+S27F+S62T+R106E，
A14P+S27F+S62T+R106A，
A14P+S27F+S62T+R109E，
A14P+S27F+S62T+R109A，
A14P+S27F+S62T+R106A+R99E，
A14P+S27F+S62T+R106A+R99A，
A14P+S27F+S62T+K104E+R99E，
A14P+S27F+S62T+K104E+R99A，
A14P+S27F+S62T+D107E，
A14P+S27F+S62T+R109E+R99E，
A14P+S27F+S62T+R109E+K104A，
A14P+S27F+S62T+R109E+R106A，
A14P+S27F+S62T+R109E+R99A。
7. the molecule having albumin binding function of claim 6, wherein the amino acid sequence of the heavy chain variable region of said mutant is as shown in SEQ ID NO 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35.
8. The molecule having albumin binding function of claim 1, wherein the albumin binding domain is capable of specifically binding to Human Serum Albumin (HSA) and cynomolgus monkey serum albumin (cyno SA) without specific binding to Bovine Serum Albumin (BSA); and the albumin binding structural domain is a nano antibody or a functional derivative thereof obtained by screening a nano antibody library by taking albumin as bait protein,
optionally, the albumin binding domain is also capable of specifically binding Murine Serum Albumin (MSA).
9. The molecule having albumin binding function according to any one of claims 1 to 9, comprising two or more albumin binding domains having the same or different amino acid sequences,
optionally, the albumin binding domain further comprises at least one biologically active domain or functional fragment comprising a carrier protein, active peptide, tagged peptide, or chaperone protein component, covalently, non-covalently, or linked to the albumin binding domain;
preferably, the carrier proteins include BSA, OVA, HSA, KLH, and Fc; the active peptide includes a linear peptide or a cyclic peptide and has an enzymatic activity, a cytotoxic activity and an antigen-binding activity; the tag peptide comprises a purification tag peptide and a tracing tag peptide; the chaperone proteins include HSP60, HSP70 and HSP90.
10. The molecule having albumin binding function of claim 11 wherein said biologically active domain or functional fragment comprises a polypeptide, a synthetic small molecule, a naturally active molecule, and wherein said molecule having albumin binding function is present in the form of a monomer, a homomultimer, or a heteromultimer,
optionally, the molecule with albumin binding function is a nanobody, a heavy chain antibody, a VHH-Fc fusion protein, a monovalent antibody, a bivalent antibody, a multivalent antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, an immunoconjugate.
11. A method of producing an albumin binding domain, comprising:
(1) Immunizing an animal capable of producing a native heavy chain antibody with HSA;
(2) Separating PBMC amplified heavy chain variable region encoding nucleic acid, and constructing a phage heavy chain antibody library;
(3) Performing affinity panning by using an HSA coated solid phase carrier to remove unbound phage;
(4) Eluting and amplifying the combined phage from the solid phase;
(5) Repeating steps (3) - (4) two to four times;
(6) Carrying out phagemid rescue on the specifically combined phage, and carrying out phage ELISA identification;
(7) Phage clones positive for phage ELISA identification were sequenced to obtain nucleic acids encoding the albumin binding domain.
12. The method of claim 11, further comprising:
(8) Performing humanized transformation on the albumin binding structural domain;
(9) Sequence optimization of the humanised engineered albumin binding domain.
13. A composition comprising the molecule having albumin binding function of any one of claims 1-10; and
optionally pharmaceutically acceptable excipients.
14. A polynucleotide encoding the molecule having albumin binding function according to any one of claims 1 to 10.
15. A nucleic acid construct comprising the polynucleotide of claim 14.
16. A host cell comprising the polynucleotide of claim 14 or the nucleic acid construct of claim 15.
17. A method for preparing a molecule having albumin binding activity, comprising:
(1) Culturing the host cell of claim 16 under conditions suitable for expression of the recombinant foreign protein,
(2) Optionally, the molecule having albumin binding function is isolated and purified from the cell culture.
18. Albumin binding domain in a molecule having albumin binding function according to any of claims 1-10.
19. Use of a molecule having albumin binding function according to any of claims 1-10 or an albumin binding domain according to claim 18 for the manufacture of a medicament and/or for extending the half-life of a medicament.
20. The use according to claim 19, wherein the active molecule having a prophylactic or therapeutic effect is bound to the molecule having an albumin binding function or the albumin binding domain by a covalent bond, a non-covalent bond, or a linker molecule.
CN202210624226.3A 2021-06-03 2022-06-02 Nano antibody of targeted Human Serum Albumin (HSA) and application thereof Pending CN115433274A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118496355A (en) * 2024-07-12 2024-08-16 通化安睿特生物制药股份有限公司 Nanometer antibody against serum albumin, derivative and application thereof

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Publication number Priority date Publication date Assignee Title
CA3098165A1 (en) * 2018-04-24 2019-10-31 National Research Council Of Canada Serum albumin binding antibodies for tuneable half-life extension of biologics
CN111138537B (en) * 2018-11-06 2021-07-09 瑞阳(苏州)生物科技有限公司 Anti-human serum albumin antibody fragment, preparation method and application
CN111138536B (en) * 2018-11-06 2021-07-09 瑞阳(苏州)生物科技有限公司 Preparation and application of anti-human serum albumin single-domain antibody
CN112409480B (en) * 2019-08-20 2024-08-27 四川科伦博泰生物医药股份有限公司 Serum albumin binding proteins and uses thereof
CN113912730B (en) * 2021-12-14 2022-03-04 北京科诺信诚科技有限公司 Sustained-release anti-FcRn antibody or antigen-binding fragment and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118496355A (en) * 2024-07-12 2024-08-16 通化安睿特生物制药股份有限公司 Nanometer antibody against serum albumin, derivative and application thereof
CN118496355B (en) * 2024-07-12 2024-10-01 通化安睿特生物制药股份有限公司 Nanometer antibody against serum albumin, derivative and application thereof

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