Non-pH dependent long-acting anti-serum albumin nanobodies and uses thereof

Abstract

The application provides a non-pH-dependent long-acting anti-serum albumin nanobody and application thereof. Specifically, the application provides amino acid sequences of VHH chain framework regions FR and complementarity determining regions CDR of the non-pH-dependent long-acting anti-serum albumin nanobody. The application also provides nucleotide sequences encoding the nanobody. The anti-serum albumin nanobody provided by the application can bind to human, mouse, rat and cynomolgus serum albumin under different pH conditions, and can significantly prolong the half-life of a protein drug, thereby providing a research basis for long-acting protein drug development.

Description

[0001] This application is a divisional application of the invention patent application with the application date of August 14, 2020, the application number of 202010820234.6, and the invention name of "Non-pH-dependent long-acting anti-serum albumin nanobody and its application". TECHNICAL FIELD

[0002] The present application relates to the field of biomedical or biopharmaceutical technology, more particularly to a non-pH-dependent long-acting anti-serum albumin nanobody and its application. BACKGROUND

[0003] As an important parameter of pharmacokinetics, the half-life of antibodies comprehensively reflects the process of absorption and distribution of antibodies in the body. Improving the half-life can effectively improve the efficacy of antibodies, reduce the dosage and frequency of administration, reduce possible side effects, and reduce the treatment burden of patients. Therefore, how to change the half-life of antibody drugs has always been an important research direction in the field of antibody engineering. Various factors affecting the half-life of antibody drugs include molecular weight, FcRn binding, isoelectric point, glycosylation, target-mediated clearance, anti-drug antibodies, etc. Current strategies for extending the half-life of antibody drugs include glycosylation modification, pegylation, albumin fusion, transferrin fusion, Fc fusion, inert protein fusion, and negative electric protein fusion, etc.

[0004] Human serum albumin is the most abundant soluble protein in human plasma and also the carrier of many endogenous factors and exogenous drugs. Human serum albumin is composed of 585 amino acids with a relative molecular mass of about 66.5 kDa. Since it is not easy to penetrate the glomerulus under normal circumstances, human serum albumin has a long half-life in plasma (up to 14-20 days, with an average of about 19 days), no enzymatic and immunological activity, safety, good biocompatibility, and very wide distribution in the body, making it an ideal drug carrier. Various long-acting technologies based on human serum albumin have been widely applied and developed, including the construction of human serum albumin fusion proteins, the coupling of human serum albumin through covalent chemical bonds, and the reversible binding of human serum albumin through non-covalent bonds.

[0005] Nanobody (Nb), i.e. variable domain of heavy chain of heavy-chain antibody (HCAb) - There are heavy-chain antibodies (HCAb) naturally lacking light chains in camelids. The single-domain antibody consisting of only one heavy chain variable region cloned from the variable region is the smallest unit with complete function and stable antigen-binding that can be obtained at present. Nanobody has the characteristics of high stability, good water solubility, simple humanization, high targeting, strong penetration, etc., and plays an unimaginably huge function in immunological experiments, diagnosis and treatment. Ablynx Company, as a global leader in the field of nanobody, is the first enterprise to use nanobody for therapeutic antibody development. It has more than 45 proprietary and cooperative nanobodies, among which there are several products constructed as fusion proteins with human serum albumin as long-acting factors, such as products targeting IL-6R, TNF-α, RANKL or IL-17A / IL17F.

[0006] Although there are many patent literatures in the prior art reporting nanobodies against human serum albumin, the existing nanobodies still have some deficiencies in application, and the field still needs to develop new nanobodies with better functions. SUMMARY

[0007] The purpose of the present application is to provide a non-pH-dependent long-acting anti-serum albumin nanobody and its application.

[0008] Specifically, the present application provides four strains of nanobodies that specifically bind to serum albumin, as well as the coding sequence, preparation method and application of the nanobodies.

[0009] In the first aspect of the present application, an anti-serum albumin nanobody is provided, which can specifically bind to serum albumin, and the complementarity determining region (CDR) of the VHH chain of the nanobody is one or more selected from the group consisting of:

[0010] (1) CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3;

[0011] (2) CDR1 as shown in SEQ ID NO: 10, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12;

[0012] (3) CDR1 as shown in SEQ ID NO: 19, CDR2 as shown in SEQ ID NO: 20, and CDR3 as shown in SEQ ID NO: 21; and

[0013] (4) CDR1 as shown in SEQ ID NO: 28, CDR2 as shown in SEQ ID NO: 29, and CDR3 as shown in SEQ ID NO: 30.

[0014] In another preferred embodiment, the anti-serum albumin Nanobody is a non-pH dependent long-acting anti-serum albumin Nanobody.

[0015] In another preferred embodiment, the anti-serum albumin Nanobody comprises one immunoglobulin single variable domain that specifically binds to serum albumin.

[0016] In another preferred embodiment, the anti-serum albumin Nanobody comprises two or more immunoglobulin single variable domains that specifically bind to serum albumin.

[0017] In another preferred embodiment, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 selected from the group consisting of:

[0018] (1) CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3;

[0019] (2) CDR1 as shown in SEQ ID NO: 10, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12;

[0020] (3) CDR1 as shown in SEQ ID NO: 19, CDR2 as shown in SEQ ID NO: 20, and CDR3 as shown in SEQ ID NO: 21; and

[0021] (4) CDR1 as shown in SEQ ID NO: 28, CDR2 as shown in SEQ ID NO: 29, and CDR3 as shown in SEQ ID NO: 30.

[0022] In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence that optionally has at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid added to, deleted from, modified in, and / or substituted for, and that retains the ability to specifically bind to serum albumin in a non-pH dependent manner.

[0023] In another preferred embodiment, said CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of a VHH chain.

[0024] In another preferred embodiment, said serum albumin is a human or non-human mammalian serum albumin.

[0025] In another preferred embodiment, said serum albumin is a human, mouse, rat or cynomolgus serum albumin.

[0026] In another preferred embodiment, said anti-serum albumin nanobody is pH-independent.

[0027] In another preferred embodiment, said anti-serum albumin nanobody binds serum albumin under different pH conditions.

[0028] In another preferred embodiment, said anti-serum albumin nanobody binds serum albumin at a pH between 3.0 and 10.0, preferably between 4.0 and 9.0, more preferably between 5.0 and 8.0.

[0029] In another preferred embodiment, said monoclonal antibody has a binding activity A1 to serum albumin at a pH of about > 7.4 (preferably > 7.8, more preferably > 8.0; and a pH of < 8.5); said monoclonal antibody has a binding activity A2 to serum albumin at a pH of about < 5.0 (preferably < 4.8, more preferably < 4.5; and a pH of > 4.0); and 0.5 < A1 / A2 < 2, preferably 0.7 < A1 / A2 < 1.5, more preferably 0.8 < A1 / A2 < 1.2.

[0030] In another preferred embodiment, the framework regions of said nanobody comprise FR1, FR2, FR3 and FR4 selected from the group consisting of:

[0031] (1) FR1 of SEQ ID NO: 4, FR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 6, and FR4 of SEQ ID NO: 7;

[0032] (2) FR1 of SEQ ID NO: 13, FR2 of SEQ ID NO: 14, FR3 of SEQ ID NO: 15, and FR4 of SEQ ID NO: 16;

[0033] (3) FR1 of SEQ ID NO: 22, FR2 of SEQ ID NO: 23, FR3 of SEQ ID NO: 24, and FR4 of SEQ ID NO: 25;

[0034] (4) FR1 of SEQ ID NO: 31, FR2 of SEQ ID NO: 32, FR3 of SEQ ID NO: 33, and FR4 of SEQ ID NO: 34;

[0035] (5) FR1 of SEQ ID NO: 37, FR2 of SEQ ID NO: 38, FR3 of SEQ ID NO: 39, and FR4 of SEQ ID NO: 40;

[0036] (6) FR1 of SEQ ID NO: 43, FR2 of SEQ ID NO: 44, FR3 of SEQ ID NO: 45, and FR4 of SEQ ID NO: 46;

[0037] (7) FR1 of SEQ ID NO: 49, FR2 of SEQ ID NO: 50, FR3 of SEQ ID NO: 51, and FR4 of SEQ ID NO: 52; and

[0038] (8) FR1 of SEQ ID NO: 55, FR2 of SEQ ID NO: 56, FR3 of SEQ ID NO: 57, and FR4 of SEQ ID NO: 58.

[0039] In another preferred embodiment, the amino acid sequence of the VHH chain in the antibody is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 59, or a combination thereof.

[0040] In another preferred embodiment, the amino acid sequence of the VHH chain in the antibody is selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53, SEQ ID NO: 59, or a combination thereof.

[0041] In another preferred embodiment, the nanobody comprises a humanized antibody, a camelid-derived antibody, a chimeric antibody.

[0042] In another preferred embodiment, the serum albumin specific Nanobody of the application also encompasses an anti-serum albumin antibody molecule capable of binding to human serum albumin by a VHH consisting of the amino acid sequence of any one of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53, or SEQ ID NO: 59.

[0043] In a second aspect of the application, an anti-serum albumin antibody is provided, said antibody comprising one or more VHH chains of the Nanobody of the first aspect of the application.

[0044] In another preferred embodiment, the anti-serum albumin antibody can be a monomer, a bivalent antibody, and / or a multivalent antibody.

[0045] In a third aspect of the application, an isolated polynucleotide is provided, said polynucleotide encoding the anti-serum albumin Nanobody of the first aspect of the application or the anti-serum albumin antibody of the second aspect of the application.

[0046] In another preferred embodiment, the polynucleotide is in a combination.

[0047] In another preferred embodiment, the polynucleotide has a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 60, or a combination thereof.

[0048] In another preferred embodiment, the application relates to a nucleic acid molecule encoding the anti-serum albumin Nanobody of the application. The nucleic acid of the application can be RNA, DNA or cDNA.

[0049] In a fourth aspect of the application, an expression vector is provided, said expression vector expressing the polynucleotide of the third aspect of the application.

[0050] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof.

[0051] In another preferred embodiment, the viral vector comprises a lentivirus vector, an adenovirus vector, an AAV virus vector, a retrovirus vector, or a combination thereof.

[0052] In a fifth aspect of the present application, a host cell is provided, said host cell comprising the expression vector of the fourth aspect of the present application, or having integrated into its genome the polynucleotide of the third aspect of the present application.

[0053] In another preferred embodiment, said host cell comprises a prokaryotic cell or a eukaryotic cell.

[0054] In another preferred embodiment, said host cell is selected from the group consisting of E. coli, a yeast cell, a mammalian cell.

[0055] In a sixth aspect of the present application, a method for producing an anti-serum albumin nanobody is provided, comprising the steps of:

[0056] (a) culturing the host cell of the fifth aspect of the present application under conditions suitable for production of a nanobody, thereby obtaining a culture comprising said anti-serum albumin nanobody; and

[0057] (b) isolating or recovering said anti-serum albumin nanobody from said culture; and optionally

[0058] (c) purifying and / or modifying the anti-serum albumin nanobody obtained in step (b).

[0059] In another preferred embodiment, said anti-serum albumin nanobody has an amino acid sequence as set forth in SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53, or SEQ ID NO: 59.

[0060] In a seventh aspect of the present application, an immunoconjugate is provided, said immunoconjugate comprising:

[0061] (a) an anti-serum albumin nanobody as defined in the first aspect of the present application, or an anti-serum albumin antibody as defined in the second aspect of the present application; and

[0062] (b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

[0063] In another preferred embodiment, said components (a) and (b) are operatively linked.

[0064] In another preferred embodiment, said conjugating moiety is a chemical label and a biological label.

[0065] In another preferred embodiment, said chemical label is an isotope, an immunotoxin and / or a chemical drug.

[0066] In another preferred embodiment, the biomarker is biotin, avidin or an enzymatic label.

[0067] In another preferred embodiment, the radionuclide comprises:

[0068] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0069] (ii) a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0070] In another preferred embodiment, the coupling moiety is a detectable label.

[0071] In another preferred embodiment, the coupling moiety is selected from the group consisting of a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biological toxin, a cytokine (e.g., IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or benzophenone hydrolase-like protein (BPHL)), or any form of nanoparticle.

[0072] In an eighth aspect of the application, a conjugate is provided, the conjugate comprising:

[0073] (a) an anti-serum albumin nanobody as described in the first aspect of the application, or an anti-serum albumin antibody as described in the second aspect of the application; and operatively linked to

[0074] (b) a modification label selected from the group consisting of a chemical label and a biological label.

[0075] In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0076] In another preferred embodiment, the biological label is biotin, avidin, or an enzyme label.

[0077] In a ninth aspect of the present application, a multispecific antibody is provided, said multispecific antibody comprising: an anti-serum albumin nanobody according to the first aspect of the present application, or an anti-serum albumin antibody according to the second aspect of the present application.

[0078] In another preferred embodiment, the multispecific antibody further comprises a second antigen binding region targeting a target selected from the group consisting of IL-4R, IL-4R alpha, TNF-alpha, VEGF, PD-1, PD-L1, 4-1BB, CD47, TIM3, CTLA4, IL-17A, CD19, CD22, CD38, IL-5, TSLP, BCMA, GLP-1, Trop2, TIGIT, or a combination thereof.

[0079] In another preferred embodiment, the second antigen binding region is a nanobody.

[0080] In another preferred embodiment, the multispecific antibody comprises one or more second antigen binding regions.

[0081] In another preferred embodiment, the multispecific antibody further comprises an Fc fragment of an antibody.

[0082] In another preferred embodiment, the multispecific antibody is a bispecific antibody targeting serum albumin and IL4R.

[0083] In another preferred embodiment, the multispecific antibody comprises one anti-serum albumin nanobody and two anti-IL4R nanobodies.

[0084] In another preferred embodiment, the multispecific antibody is a trivalent antibody.

[0085] In another preferred embodiment, the bispecific antibody has the structure of Formula I:

[0086] I-I-B Formula I

[0087] wherein,

[0088] “-” is a peptide bond;

[0089] I is an anti-IL4R nanobody;

[0090] B is an anti-serum albumin nanobody according to the first aspect of the present application.

[0091] In a tenth aspect of the present application, a fusion protein is provided, said fusion protein comprising:

[0092] (i) the anti-serum albumin Nanobody according to the first aspect of the application, or the anti-serum albumin antibody according to the second aspect of the application;

[0093] (ii) an optionally therapeutically functional polypeptide molecule or fragment.

[0094] In another preferred embodiment, the therapeutically functional polypeptide molecule or fragment includes, but is not limited to, a polypeptide molecule or fragment targeting IL-4R, IL-4R alpha, TNF-alpha, VEGF, PD-1, PD-L1, 4-1BB, CD47, TIM3, CTLA4, IL-17A, CD19, CD22, CD38, IL-5, TSLP, BCMA, GLP-1, Trop2 or TIGIT.

[0095] In another preferred embodiment, the therapeutically functional polypeptide molecule or fragment includes, but is not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptide, incretin analog, albumin, antibody fragment, cytokine.

[0096] In another preferred embodiment, the therapeutically functional polypeptide molecule or fragment includes a single-chain antibody (scFv), a diabody, a monoclonal antibody, or a chimeric antibody.

[0097] In another preferred embodiment, the fusion protein further comprises a tag sequence to facilitate expression and / or purification.

[0098] In another preferred embodiment, the tag sequence is selected from the group consisting of 6His tag, GGGS sequence, FLAG tag.

[0099] In another preferred embodiment, the fusion protein includes a bispecific antibody, a chimeric antibody.

[0100] In an eleventh aspect of the application, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0101] (i) the anti-serum albumin Nanobody according to the first aspect of the application, the anti-serum albumin antibody according to the second aspect of the application, the immunoconjugate according to the seventh aspect of the application, the conjugate according to the eighth aspect of the application, the multispecific antibody according to the ninth aspect of the application, or the fusion protein according to the tenth aspect of the application;

[0102] (ii) a pharmaceutically acceptable carrier.

[0103] In another preferred embodiment, the pharmaceutical composition further comprises other biologically active substances, such as drugs for treating tumors.

[0104] In a twelfth aspect of the present application, there is provided a use of an active ingredient selected from the group consisting of an anti-serum albumin Nanobody as defined in the first aspect of the present application, an anti-serum albumin antibody as defined in the second aspect of the present application, an immunoconjugate as defined in the seventh aspect of the present application, a conjugate as defined in the eighth aspect of the present application, a multispecific antibody as defined in the ninth aspect of the present application, a fusion protein as defined in the tenth aspect of the present application, or a combination thereof, for the manufacture of a long-acting protein drug.

[0105] In a thirteenth aspect of the present application, there is provided a use of an anti-serum albumin Nanobody as defined in the first aspect of the present application, an anti-serum albumin antibody as defined in the second aspect of the present application, or an immunoconjugate as defined in the seventh aspect of the present application: (a) for the manufacture of a reagent, an assay plate, or a kit for detecting serum albumin; (b) for the manufacture of an agent (a long-acting protein drug) that binds to serum albumin.

[0106] In another preferred embodiment, the reagent is a diagnostic reagent.

[0107] In another preferred embodiment, the agent is for detecting serum albumin or a fragment thereof in a sample.

[0108] In another preferred embodiment, the agent comprises an immunoconjugate as defined in the seventh aspect of the present application, a conjugate as defined in the eighth aspect of the present application, a multispecific antibody as defined in the ninth aspect of the present application, or a fusion protein as defined in the tenth aspect of the present application.

[0109] In a fourteenth aspect of the present application, there is provided an immunoabsorbent material for purifying serum albumin, wherein the immunoabsorbent material comprises an anti-serum albumin Nanobody as defined in the first aspect or the second aspect, or a VHH chain of an anti-serum albumin Nanobody as defined in the first aspect or the second aspect of the present application.

[0110] In another preferred embodiment, the immunoabsorbent material further comprises a carrier.

[0111] In another preferred embodiment, the carrier comprises, but is not limited to, a magnetic bead, a sepharose gel, a silica gel microsphere, a porous material.

[0112] In a fifteenth aspect of the present application, there is provided a method for detecting serum albumin or a fragment thereof in a sample in vitro, the method comprising the steps of:

[0113] (1) contacting, in vitro, the sample with an anti-serum albumin Nanobody as defined in the first aspect of the present application, an anti-serum albumin antibody as defined in the second aspect of the present application, or an immunoconjugate as defined in the seventh aspect of the present application;

[0114] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of serum albumin or a fragment thereof in the sample.

[0115] In another preferred embodiment, the detection is diagnostic or non-diagnostic.

[0116] In a sixteenth aspect of the application, a method for producing a recombinant polypeptide is provided, the method comprising:

[0117] (a) culturing the host cell of the fifth aspect of the application under conditions suitable for expression;

[0118] (b) isolating the recombinant polypeptide from the culture, the recombinant polypeptide comprising an anti-serum albumin Nanobody according to the first aspect of the application, an anti-serum albumin antibody according to the second aspect of the application, a multi-specific antibody according to the ninth aspect of the application, a fusion protein according to the tenth aspect of the application.

[0119] It should be understood that, within the scope of the present application, the above-mentioned technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0120] Figure 1A Binding activity of albumin Nanobody Nb1-15 to human, mouse, rat, cynomolgus serum albumin under different pH conditions.

[0121] Figure 1B Binding activity of albumin Nanobody Nb1-60 to human, mouse, rat, cynomolgus serum albumin under different pH conditions.

[0122] Figure 1C Binding activity of albumin Nanobody Nb1-83 to human, mouse, rat, cynomolgus serum albumin under different pH conditions.

[0123] Figure 1D Binding activity of albumin Nanobody Nb3-24 to human, mouse, rat, cynomolgus serum albumin under different pH conditions.

[0124] Figure 2A Binding activity of IL4R Nanobody fused with humanized albumin Nanobody HuNb1-15 to human, mouse, rat, cynomolgus serum albumin under different pH conditions.

[0125] Figure 2B Binding activity of IL4R Nanobody fused with humanized albumin Nanobody HuNb1-60 to human, mouse, rat, cynomolgus serum albumin under different pH conditions.

[0126] Figure 2CBinding activity of IL4R nanobody to human, mouse, rat, cynomolgus serum albumin under different pH conditions after fusing with humanized albumin nanobody HuNb1-83.

[0127] Figure 2D Binding activity of IL4R nanobody to human, mouse, rat, cynomolgus serum albumin under different pH conditions after fusing with humanized albumin nanobody HuNb3-24.

[0128] Figure 3 Results of half-life detection of IL4R-HSA trivalent antibody in mice.

[0129] Figure 4 Results of half-life detection of IL4R-HSA trivalent antibody in rats.

[0130] Figure 5 Results of half-life detection of IL4R-HSA trivalent antibody in cynomolgus monkeys. DETAILED DESCRIPTION

[0131] The present inventors, through extensive and in-depth research, after a large number of screening, successfully obtained a group of anti-serum albumin nanobodies, and experimental results showed that the four strains of anti-serum albumin nanobodies obtained by the present application can effectively bind to serum albumin. On this basis, the present application was completed.

[0132] Specifically, the present application uses human serum albumin extracellular segment antigen protein to immunize camels, and obtains high-quality immune nanobody gene library. Then the serum albumin molecule is coupled on the enzyme-labeled plate to display the correct spatial structure of serum albumin. In this form of antigen, the immune nanobody gene library (camel heavy chain antibody phage display gene library) is screened by phage display technology, so as to obtain serum albumin specific nanobody gene.

[0133] As used herein, the terms "nanobody of the present application", "anti-serum albumin nanobody of the present application", "anti-serum albumin nanobody of the present application" are used interchangeably, and all refer to a nanobody that specifically recognizes and binds to serum albumin (including human serum albumin). It is particularly preferred that the amino acid sequence of the VHH chain is as shown in SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53 or SEQ ID NO: 59.

[0134] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains varies among the different immunoglobulin isotypes. There are also interchain disulfide bonds at regular intervals along the chains. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions. At one end of each light chain is a variable region (VL) followed by a constant region; the constant region of the light chain is aligned with the first constant region of the heavy chain, and the variable region of the light chain is aligned with the variable region of the heavy chain. Particular amino acid residues form an interface between the variable regions of the light and heavy chains.

[0135] As used herein, the terms "single domain antibody" (sdAb), or "VHH", "nanobody" have the same meaning, referring to the variable region of the heavy chain of a cloned antibody, constructing a nanobody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Usually, after obtaining an antibody naturally lacking a light chain and a heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0136] Nanobody / single domain antibody (Nanobody) is a new type of small molecule antibody fragment, which is cloned from the heavy chain variable region (VHH) of the heavy chain antibody of camelids. Nanobody (Nb) has excellent biological properties, with a molecular weight of 12-15 kDa, which is one tenth of the complete antibody, has good tissue penetration, high specificity, and good water solubility. Due to its special structural properties, it has the advantages of traditional antibodies and small molecule drugs, almost perfectly overcoming the defects of long development cycle, low stability, and harsh storage conditions of traditional antibodies, and gradually becoming a new force in the new generation of antibody therapy, showing broad application prospects in immunodiagnosis and treatment.

[0137] As used herein, the term "variable" refers to certain portions of the variable region of an antibody that differ in sequence among antibodies and are responsible for the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable regions of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the light chain and the heavy chain variable regions. The more conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, largely β-sheet in structure, connected by three CDRs that form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, form the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0138] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates of drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules with the antibodies or fragments thereof of the present application. The present application also includes cell surface markers or antigens conjugated to the anti-serum albumin antibodies or fragments thereof.

[0139] As used herein, the term "heavy chain variable region" is used interchangeably with "V H H."

[0140] As used herein, the term "variable region" is used interchangeably with "complementarity determining region" (CDR).

[0141] In a preferred embodiment of the present application, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.

[0142] In a preferred embodiment of the present application, the heavy chain of the antibody comprises the heavy chain variable region described above and a heavy chain constant region.

[0143] In the present application, the terms "antibody of the present application", "protein of the present application", or "polypeptide of the present application" are used interchangeably and refer to a polypeptide that specifically binds to serum albumin, such as a protein or polypeptide having a heavy chain variable region. They can or can not contain the initial methionine.

[0144] The present application also provides other proteins or fusion expression products having the antibodies of the present application. In particular, the present application includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugates and fusion expression products) having a heavy chain comprising a variable region, provided that the variable region is identical to or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibodies of the present application.

[0145] Generally, the antigen binding properties of an antibody can be described by three specific regions of the variable region of the heavy chain, called the complementarity determining regions (CDRs), which are interspersed with four framework regions (FRs) whose amino acid sequences are relatively conserved and do not directly participate in binding interactions. The CDRs form loops or "hot spots" on the surface of the variable region, and are in close proximity to each other through the FRs that form a beta sheet. The CDRs on the heavy chain and the corresponding CDRs on the light chain make up the antigen binding site of the antibody. It is possible to determine which amino acids make up the FR or CDR regions by comparing the amino acid sequences of antibodies of the same class.

[0146] The variable regions of the heavy chains of the antibodies of the present application are of particular interest because at least some of them are involved in binding the antigen. Thus, the present application includes those molecules having the variable region of the heavy chain of the antibody with the CDRs, provided that the CDRs are at least 90% homologous, preferably at least 95% homologous, and most preferably at least 98% homologous to the CDRs identified herein.

[0147] The present application includes not only intact antibodies, but also fragments of the antibodies that are immunologically active or fusion proteins of the antibodies with other sequences. Thus, the present application also includes fragments, derivatives and analogs of the antibodies.

[0148] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present application. The polypeptide fragments, derivatives or analogs of the present application can be (i) polypeptides having one or more conservative or non-conservative amino acid substitutions (preferably conservative amino acid substitutions) of the amino acid residues, where such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) polypeptides having a substitution group at one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide to another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing additional amino acid sequences to the polypeptide sequence (such as a leader or secretion sequence, or a sequence or protein for purification of the polypeptide, or a proprotein sequence, or a fusion protein with a 6His tag). These fragments, derivatives and analogs are within the purview of one of skill in the art in light of the teachings herein.

[0149] The term "antibody" of the present application refers to a polypeptide having serum albumin binding activity comprising the CDR regions described above. The term also includes variants of polypeptides comprising the CDR regions described above which have the same function as the antibody of the present application. These variants include, but are not limited to, deletion, insertion and / or substitution of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (typically within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with similar or identical amino acids often does not change the function of the protein. For another example, addition of one or several amino acids at the C-terminus and / or N-terminus also often does not change the function of the protein. The term also includes active fragments and active derivatives of the antibody of the present application.

[0150] The variants of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA hybridizing to the DNA encoding the antibody of the present application under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present application.

[0151] The present application also provides other polypeptides, such as fusion proteins comprising a nanobody or a fragment thereof. In addition to the almost full-length polypeptides, the present application also includes fragments of the nanobody of the present application. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, most preferably at least about 100 contiguous amino acids, of the antibody of the present application.

[0152] In the present application, "conservative variants of the antibody of the present application" refer to polypeptides in which up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3, amino acids are replaced by similar or identical amino acids compared to the amino acid sequence of the antibody of the present application. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table 1.

[0153] Table 1

[0154] Original residue Representative substitution Preferred substitution Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe Leu Leu (L) lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Leu; Val; lie; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; Ala Leu

[0155] The present application also provides polynucleotide molecules encoding the antibody or fragment thereof or fusion protein thereof described above. The polynucleotide of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0156] Polynucleotides encoding the mature polypeptides of the present application include: coding sequences that encode only the mature polypeptide; coding sequences that encode the mature polypeptide and various additional coding sequences; coding sequences that encode the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0157] The term "polynucleotide encoding a polypeptide" can be a polynucleotide that includes only coding sequence that encodes the polypeptide, or it can be a polynucleotide that includes additional coding and / or non-coding sequences.

[0158] The present application also relates to polynucleotides that hybridize to the sequences described above and that have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application particularly relates to polynucleotides that hybridize to the polynucleotides of the present application under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2 x SSC, 0.1% SDS at 60°C; or (2) hybridization in the presence of a denaturing agent, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll at 42°C; or (3) hybridization only when the identity between the two sequences is at least 90%, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0159] The nucleotide full-length sequence of the antibody of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. One possible method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is short. Generally, a long fragment can be obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0160] Once the relevant sequence is obtained, it can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then transforming it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules that exist in an isolated form.

[0161] At present, it is possible to obtain the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present application by chemical synthesis.

[0162] The present application also relates to vectors that contain the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express the protein.

[0163] The host cell can be a prokaryote, such as a bacterial cell, or a eukaryote, such as a yeast cell, or a higher eukaryote, such as a mammalian cell. Representative examples of useful host cells include: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0164] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation, if necessary, followed by electroporation. When the host is a eukaryote, transformation can be effected by the use of techniques such as calcium phosphate precipitation, conventional mechanical procedures such as microinjection, electroporation, lipofection, etc.

[0165] The resulting transformant can be cultured in conventional nutrient media to express the polypeptide encoded by the gene of the application. The culture conditions, such as temperature, pH and the like, are those previously determined to be appropriate for the host cell used. When the host cell has reached an appropriate cell density, the selected promoter is induced by the appropriate method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period.

[0166] The recombinant polypeptide of the above method can be expressed intracellularly, on the cell membrane, or secreted from the cell. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0167] The antibodies of the application can be used alone or in combination or conjugation with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties, or any combination of these.

[0168] Detectable labels for diagnostic purposes include, but are not limited to: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing detectable products.

[0169] Therapeutic agents that can be conjugated or coupled to the antibodies of the present application include, but are not limited to: 1. Radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2 and the like; 4. Gold nanoparticles / nanorods; 5. Viral particles; 6. Liposomes; 7. Magnetic nanoparticles; 8. Drug-activating enzymes (e.g., DT-diaphorase (DTD) or benzyl-hydrolase-like protein (BPHL)); 9. Therapeutic agents (e.g., cisplatin) or any form of nanoparticles and the like.

[0170] Pharmaceutical composition

[0171] The present application also provides a composition. Preferably, the composition is a pharmaceutical composition, which comprises the above-mentioned antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier or excipient, and optionally other biologically active substances. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value can vary depending on the nature of the formulated substance and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including but not limited to intraperitoneal, intravenous, or topical administration.

[0172] The pharmaceutical composition of the present application comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned antibody (or conjugate thereof) of the present application and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as a needle, solution is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present application can also be used with other therapeutic agents.

[0173] When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dose should also take into account the administration route, the patient's health status and the like, which are within the skill of the skilled physician.

[0174] Detection method

[0175] The present application also relates to a method for detecting serum albumin. The method comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of serum albumin protein in the dissolved sample.

[0176] In the detection method of the present application, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.

[0177] Kit

[0178] The present application also provides a kit containing the antibody (or fragment thereof) or detection plate of the present application. In a preferred embodiment of the present application, the kit further comprises a container, an instruction manual, a buffer, etc.

[0179] The present application also provides a detection kit for detecting the level of serum albumin, which comprises an antibody recognizing serum albumin, a lysis medium for dissolving a sample, and general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0180] Application

[0181] As described above, the nanobody of the present application has wide biological and clinical application values, and its application involves the use of anti-serum albumin nanobody for preparing long-acting protein drugs, thereby providing a research and development basis for the development of long-acting protein drugs.

[0182] The main advantages of the present application include:

[0183] (a) The nanobody of the present application can bind to human, mouse, rat, and cynomolgus serum albumin under different pH conditions.

[0184] (b) The nanobody of the present application can significantly prolong the half-life of protein drugs, thereby providing a research and development basis for the development of long-acting protein drugs.

[0185] (c) The nanobody of the present application is suitable for prokaryotic expression and eukaryotic expression, has very high solubility, is not prone to aggregation, can resist denaturing conditions such as high temperature, strong acid, and strong base, and is suitable for laboratory and industrial development.

[0186] (d) The use of the nanobody of the present application for preparing protein drugs does not affect the targeting and activity of the drugs.

[0187] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate but not to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturers. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0188] Example 1: Screening and expression of anti-serum albumin nanobody

[0189] One Xinjiang Bactrian camel was immunized with human serum albumin protein. After 7 times of immunization, total RNA was isolated from the peripheral blood of the camel, and VHH gene was amplified by reverse transcription and PCR, and then cloned into phage vector pMECS, transformed into TG1 host cells to construct a phage display library. Subsequently, phage display technology was used for library screening, and after 4 rounds of "adsorption-washing-enrichment" screening process, the phage containing antibody gene was finally enriched 30 times. 400 phage clones were randomly selected from the above enriched phage clones for PE-ELISA identification, and all the positive clones obtained were sequenced and identified. All the antibodies with different sequences were used as candidates, and various antibodies were expressed and purified by E. coli for subsequent identification.

[0190] Example 2: Binding of anti-serum albumin nanobody to albumin under different pH conditions

[0191] Mouse, rat, cynomolgus monkey serum and human serum albumin were diluted to 10 ug / mL with pH8.2 NaHCO3 and coated onto an enzyme-labeled plate at 4°C overnight; the enzyme-labeled plate was washed with PBST, 300uL of 1% skim milk was added to each well, and the plate was incubated at room temperature for 2 hours; the enzyme-labeled plate was washed with PBST, and 100uL of gradient-diluted antibody to be tested (each antibody was diluted with pH5.0 and pH7.4 PBS solution, and 3-fold gradient dilution was started from 300ug / mL) was added, and the plate was incubated at 37°C for 1 hour; the enzyme-labeled plate was washed with PBST, 100uL of diluted Goat pAb to Nanobody (HRP) (diluted with PBS at a ratio of 1:1000) was added, and the plate was incubated at 37°C for 1 hour; the enzyme-labeled plate was washed with PBST, 100uL of TMB color developing solution was added, and the plate was reacted at room temperature in the dark for 5 minutes, then 50uL of 2M sulfuric acid was added to stop the color development, and the absorbance value was read at 450nm wavelength by an enzyme-labeled instrument. The results are as follows Figures 1A-1DAs shown: four anti-serum albumin nanobodies can bind human, mouse, rat, cynomolgus serum albumin at the same time, and can bind various species serum albumin under different pH conditions (pH 5.0 and pH 7.4). Numerical statistics are shown in Table 2.

[0192] Table 2 Binding of anti-serum albumin nanobodies to different species of albumin

[0193]

[0194]

[0195] Example 3: Humanization of anti-serum albumin nanobodies

[0196] The amino acid sequences of the above four nanobodies were placed in the structure database for homology structure search, and the antibody sequences are shown in Table 3.

[0197] Table 3 Anti-serum albumin nanobody sequence number

[0198] Antibody number Nb1-15 Nb1-60 Nb1-83 Nb3-24 CDR1 SEQ ID NO. 1 SEQ ID NO. 10 SEQ ID NO. 19 SEQ ID NO. 28 CDR2 SEQ ID NO. 2 SEQ ID NO. 11 SEQ ID NO. 20 SEQ ID NO. 29 CDR3 SEQ ID NO. 3 SEQ ID NO. 12 SEQ ID NO. 21 SEQ ID NO. 30 FR1 SEQ ID NO. 4 SEQ ID NO. 13 SEQ ID NO. 22 SEQ ID NO. 31 FR2 SEQ ID NO. 5 SEQ ID NO. 14 SEQ ID NO. 23 SEQ ID NO. 32 FR3 SEQ ID NO. 6 SEQ ID NO. 15 SEQ ID NO. 24 SEQ ID NO. 33 FR4 SEQ ID NO. 7 SEQ ID NO. 16 SEQ ID NO. 25 SEQ ID NO. 34 Full length amino acid sequence SEQ ID NO. 8 SEQ ID NO. 17 SEQ ID NO. 26 SEQ ID NO. 35 Full length base sequence SEQ ID NO. 9 SEQ ID NO. 18 SEQ ID NO. 27 SEQ ID NO. 36

[0199] Among them, the structure with higher sequence homology was selected for alignment, and according to the size of the crystal structure resolution and the constructed phylogenetic tree, the protein including 3dwt was finally selected for multi-template homology modeling of the target nanobody sequence, and then according to the height of the scoring function, the structure with the lowest molpdf was selected; Then, using the ProtSA server to calculate the solvent accessibility of the residues of the optimal structure of the modeling, and comparing the sequences of the optimal structure of the modeling and DP-47, replacing the corresponding residues exposed to the solvent. Finally, a humanized anti-serum albumin nanobody was determined, and the antibody sequence after humanization is as follows in Table 4:

[0200] Table 4 Humanized anti-serum albumin nanobody sequence number

[0201]

[0202]

[0203] Example 4: Construction and expression of HSA-IL4R trivalent nanobodies

[0204] IL4R nanobodies were selected as representatives and humanized albumin nanobodies to construct long-acting protein drugs to verify the function of humanized albumin nanobodies to extend the in vivo half-life of proteins. The above four candidate humanized albumin nanobodies were respectively connected in series with IL4R nanobodies (the sequence is derived from patent CN2019110547879) to form trivalent nanobodies, and the structure sequence is as follows in Table 5:

[0205] Table 5 Multivalent antibody structure and sequence

[0206] Antibody number Structure Amino acid sequence Base sequence MY8154 IL4R Nb103-Nb103-HSA HuNb1-15 SEQ ID NO. 61 SEQ ID NO. 62 MY8267 IL4R Nb103-Nb103-HSA HuNb1-60 SEQ ID NO. 63 SEQ ID NO. 64 MY8162 IL4R Nb103-Nb103-HSA HuNb1-83 SEQ ID NO. 65 SEQ ID NO. 66 MY8268 IL4R Nb103-Nb103-HSA HuNb3-24 SEQ ID NO. 67 SEQ ID NO. 68 MY8171 IL4R Nb103-Nb103 SEQ ID NO. 69 SEQ ID NO. 70

[0207] The above multivalent antibodies were expressed using Pichia pastoris. Briefly, the expression method was as follows: (1) the nanobody sequence shown in SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70 was constructed into a pPICZaA vector; (2) linearized with Sac I restriction endonuclease and then electroporated into X-33 competent cells; (3) the electroporated sample was plated on YPD plate medium containing different concentrations of blasticidin resistance and incubated in a 30°C incubator for 3 days. For specific embodiments, refer to the pPICZaA vector instruction provided by Invitrogen; (4) after single colonies grew on the plate medium, the single colonies were picked and placed in BMGY medium. When the OD value of the BMGY culture reached about 20, the bacterial cells were collected and replaced into BMMY medium and cultured at 28°C and 250 rpm; (5) then 1% methanol was added every 24 h. Continuous induction was performed for 5 days and the culture was ended; (6) the obtained supernatant was purified by cation chromatography to obtain the target antibody.

[0208] The above expressed and purified antibodies were placed under different pH conditions to detect their binding to various genus albumins. The detection method was the same as that in Example 2, and the results are shown in Table 5. Figures 2A-2D The humanized trivalent antibody fused with the human IL4R nanobody can still bind to serum albumins of different species, and does not change its binding activity depending on pH changes. The numerical statistics are shown in Table 6.

[0209] Table 6 Binding of humanized trivalent antibody to serum albumins of different species

[0210]

[0211]

[0212] Example 5: Half-life of anti-serum albumin nanobody in mice

[0213] Respectively, 100 ug of the multivalent antibody expressed by yeast cells was injected intravenously into mice, and after administration, the orbital blood was taken at 5 min, 3 h, 8 h, 24 h, 48 h, 96 h, 144 h, and the plasma was separated after centrifugation. The blood sampling time points of the control antibody MY8171 after administration were 5 min, 15 min, 30 min, 1.5 h and 3 h. Then, ELISA detection was used for PK analysis. 1 ug / mL hIL4R protein was coated on an enzyme-labeled plate with 100 uL / well overnight; the plate was washed with PBST for 5 times, 300 uL of 0.3% Casein was added for blocking at room temperature for 2 hours; the plate was washed with PBST for 5 times, the diluted serum sample was added to the corresponding well, and incubated at 37°C for 1 hour; the plate was washed with PBST for 5 times, Goat 1 pAb to Nanobody-HRP (1:1000 dilution) was added at 100 uL / well, and incubated at 37°C for 1 hour; the plate was washed with PBST for 5 times, TMB color developing liquid was added for color development at room temperature for 5 minutes, 2M sulfuric acid was added to terminate the reaction, and the absorbance value was read on a microplate reader at 450 nm wavelength. The detection results were converted according to the standard curve to obtain the time-drug concentration relationship graph, and the results are shown in Figure 3 Table 7 Half-life of different anti-serum albumin nanobodies in mice

[0214] Table 7 Half-life of different anti-serum albumin nanobodies in mice

[0215] Antibody Number MY8171 MY8154 MY8267 MY8162 MY8268 T 1 / 2 (hours)]]> 1.86 16.50 30.35 8.98 40.99

[0216] Example 6: Half-life of anti-serum albumin nanobodies in rats

[0217] Respectively, 400 ug of the multivalent antibody expressed by yeast cells was injected intravenously into rats, and after administration, the orbital blood was taken at 5 min, 3 h, 8 h, 24 h, 48 h, 96 h, 144 h, and the plasma was separated after centrifugation. The blood sampling time points of the control antibody MY8171 after administration were 5 min, 15 min, 30 min, 1.5 h and 3 h. Then, ELISA detection was used for PK analysis. The detection method was the same as in Example 5. The detection results were converted according to the standard curve to obtain the time-drug concentration relationship graph, and the results are shown in Figure 4 Table 8 Half-life of different anti-serum albumin nanobodies in rats

[0218] Table 8 Half-life of different anti-serum albumin nanobodies in rats

[0219] Antibody Number MY8171 MY8154 MY8267 MY8268 T 1 / 2 (hours)]]> 0.82 41.63 51.96 51.42

[0220] Example 7: Half-life of anti-serum albumin nanobodies in cynomolgus monkeys

[0221] MY8267 and MY8268 were injected intravenously into the left forelimb of cynomolgus monkeys at a dose of 2 mg / kg. Blood samples were taken from the right forelimb at 5 min, 20 min, 1 h, 2 h, 4 h, 8 h, 16 h, 1 day, 2 days, 4 days, 6 days, 8 days, 11 days, 14 days, 17 days, 20 days, 23 days, 26 days, 29 days, 32 days, 35 days after administration, and the plasma was separated by centrifugation. The PK analysis was then performed by ELISA. The detection method was the same as in Example 5. The results were converted according to the standard curve to obtain the time-drug concentration relationship graph, as shown in Figure 9. Figure 5 The half-life of the trivalent antibodies with anti-serum albumin nanobodies in cynomolgus monkeys was about 9-15 days (Table 9).

[0222] Table 9: Half-life of different anti-serum albumin nanobodies in cynomolgus monkeys

[0223] Antibody Number MY8267 MY8268 T 1 / 2 (days) 9.53 14.21

[0224] All documents referred to in this disclosure are incorporated by reference into this application as if each document were individually incorporated. In addition, it is to be understood that various alterations and modifications can be made to this application without departing from the scope thereof as defined by the appended claims.

Claims

1. An anti-serum albumin nanobody, characterized in that, The nanobody is capable of specifically binding to serum albumin, and the VHH chain of the nanobody has complementarity determining regions (CDRs) as follows: CDR1 as set forth in SEQ ID NO: 28, CDR2 as set forth in SEQ ID NO: 29, and CDR3 as set forth in SEQ ID NO:

30.

2. The Nanobody of claim 1, wherein The VHH chain of the nanobody further comprises framework regions (FRs) selected from the group consisting of: (1) FR1 as set forth in SEQ ID NO: 31, FR2 as set forth in SEQ ID NO: 32, FR3 as set forth in SEQ ID NO: 33, and FR4 as set forth in SEQ ID NO: 34; and (2) FR1 as set forth in SEQ ID NO: 55, FR2 as set forth in SEQ ID NO: 56, FR3 as set forth in SEQ ID NO: 57, and FR4 as set forth in SEQ ID NO:

58.

3. The Nanobody of claim 1, wherein The VHH chain of the nanobody has an amino acid sequence selected from the group consisting of SEQ ID NO: 35 and SEQ ID NO:

59.

4. An anti-serum albumin antibody, which comprises the VHH chain of the nanobody of claim 1.

5. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-4. The polynucleotide encodes a protein selected from the group consisting of the nanobody of claim 1 or the antibody of claim 4.

6. The polynucleotide of claim 5, wherein, The polynucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO:

60.

7. An expression vector, characterized by, The expression vector contains the polynucleotide of claim 5.

8. A host cell, characterized in that, The host cell contains the expression vector of claim 7, or has integrated into its genome the polynucleotide of claim 5.

9. A method of producing anti-serum albumin nanobodies, characterized in that, comprising the steps of: (a) culturing the host cell of claim 8 under conditions suitable for production of the nanobody, thereby obtaining a culture containing anti-serum albumin nanobodies; (b) isolating and / or recovering the anti-serum albumin nanobodies from the culture; and (c) optionally, purifying and / or modifying the anti-serum albumin nanobodies obtained in step (b).

10. A fusion protein, characterized in that, The fusion protein has: (i) the nanobody of claim 1; (ii) optionally, a polypeptide molecule or fragment having a therapeutic function.

11. An immunoconjugate, which comprises: (a) the anti-serum albumin nanobody of claim 1, or the anti-serum albumin antibody of claim 4; and (b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a viral coat protein or VLP, or a combination thereof.

12. The immunoconjugate of claim 11, wherein, The conjugating moiety is a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, or a cytokine.

13. A conjugate, which comprises: (a) the anti-serum albumin nanobody of claim 1, or the anti-serum albumin antibody of claim 4; and operatively linked to (b) a modifying tag selected from the group consisting of a chemical tag and a biological tag.

14. A multispecific antibody, which comprises the anti-serum albumin nanobody of claim 1, or the anti-serum albumin antibody of claim 4.

15. A composition for non-therapeutic purposes, said composition comprising: (i) an anti-serum albumin Nanobody according to claim 1, an anti-serum albumin antibody according to claim 4, an immunoconjugate according to claim 11, a conjugate according to claim 13, a multispecific antibody according to claim 14, or a fusion protein according to claim 10; (ii) a pharmaceutically acceptable carrier.

16. Use of an anti-serum albumin Nanobody according to claim 1, an anti-serum albumin antibody according to claim 4, or an immunoconjugate according to claim 11 for the preparation of a reagent, a plate or a kit for detecting serum albumin.

17. An immunoadsorbent material for the purification of serum albumin, wherein, The immunoabsorbent material comprises an anti-serum albumin Nanobody according to claim 1, or an anti-serum albumin antibody according to claim 4, or a VHH chain of an anti-serum albumin Nanobody according to claim 1.

18. An in vitro non-diagnostic method for detecting serum albumin or fragments thereof in a sample, said method comprising the steps of: (1) contacting, in vitro, said sample with an anti-serum albumin Nanobody according to claim 1, an anti-serum albumin antibody according to claim 4, or an immunoconjugate according to claim 11; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of serum albumin or fragments thereof in the sample.

19. A method for producing a recombinant polypeptide, said method comprising: (a) culturing the host cell of claim 8 under conditions suitable for expression; (b) isolating the recombinant polypeptide from the culture, said recombinant polypeptide comprising an anti-serum albumin Nanobody according to claim 1, an anti-serum albumin antibody according to claim 4, a multispecific antibody according to claim 14, or a fusion protein according to claim 10.

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