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 filed on August 14, 2020, with application number 202010820234.6 and invention title "Non-pH-dependent long-acting anti-serum albumin nanobody and its application". Technical Field

[0002] This invention relates to the fields of biomedicine or biopharmaceutical technology, and more specifically to a pH-independent, long-acting antiserum albumin nanobody and its applications. Background Technology

[0003] As a crucial pharmacokinetic parameter, the half-life of an antibody comprehensively reflects its absorption and distribution in the body. Increasing the half-life can effectively enhance antibody efficacy, reduce dosage and frequency of administration, decrease potential side effects, and lessen the treatment burden on patients. Therefore, modifying the half-life of antibody drugs has long been a significant research direction in antibody engineering. Various factors influencing the half-life of antibody drugs include: molecular weight, FcRn binding, isoelectric point, glycosylation, target-mediated clearance, and anti-drug antibodies. Current strategies for extending the half-life of antibody drugs include glycosylation modification, polyethylene glycol conversion, albumin fusion, transferrin fusion, Fc fusion, inert protein fusion, and negatively charged protein fusion.

[0004] Human serum albumin (HSA) is the most abundant soluble protein in human blood plasma and a carrier for many endogenous factors and exogenous drugs. HSA is composed of 585 amino acids with a relative molecular mass of approximately 66.5 kDa. Because it does not readily cross the glomeruli under normal conditions, HSA has a relatively long half-life in plasma (14–20 days, with an average of about 19 days). It lacks enzymatic and immunological activity, is safe and non-toxic, has good biocompatibility, and is widely distributed in the body, making it an ideal drug carrier. Various long-acting drug delivery technologies based on HSA have been widely applied and developed, currently including the construction of HSA fusion proteins, covalent chemical bonding with HSA, and reversible non-covalent binding to HSA.

[0005] Nanobodies (Nb), or variable domain of heavy chain antibody (VHH), are naturally occurring heavy chain antibodies (HCAbs) lacking the light chain, found in camels. Cloning their variable domains yields single-domain antibodies consisting of only one heavy chain variable region, representing the smallest stable, fully functional antigen-binding unit currently available. Nanobodies possess characteristics such as high stability, good water solubility, simple humanization, high targeting, and strong penetration, playing an immense role in immunological experiments, diagnosis, and therapy. Ablynx, a global leader in nanobodies, was one of the first companies to utilize nanobodies in the development of therapeutic antibodies. It has over 45 proprietary and collaborative nanobodies, many of which are fusion proteins constructed using anti-human serum albumin as a long-acting factor, targeting targets such as IL-6R, TNF-α, RANKL, or IL-17A / IL17F.

[0006] Although several patent documents have reported on nanobodies targeting human serum albumin in the prior art, existing nanobodies still have some shortcomings in application, and there is a need in the field to develop new nanobodies with better functions. Summary of the Invention

[0007] The purpose of this invention is to provide a pH-independent, long-acting anti-serum albumin nanobody and its application.

[0008] Specifically, this invention provides four nanobodies that specifically bind to serum albumin, along with their coding sequences, preparation methods, and applications.

[0009] In a first aspect of the invention, an anti-serum albumin nanobody is provided, the nanobody being capable of specifically binding to serum albumin, and the complementarity-determining region (CDR) of the VHH chain of the nanobody being one or more selected from the group consisting of:

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

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

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

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

[0014] In another preferred embodiment, the antiserum albumin nanobody is a pH-independent, long-acting antiserum albumin nanobody.

[0015] In another preferred embodiment, the anti-serum albumin nanobody comprises a single variable domain of an immunoglobulin 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 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3;

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

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

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

[0022] In another preferred embodiment, any of the above-mentioned amino acid sequences further includes a derived sequence which has optionally been added, deleted, modified and / or substituted at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and retains the ability to bind to serum albumin in a pH-independent specific manner.

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

[0024] In another preferred embodiment, the serum albumin is a serum albumin from a human or non-human mammal.

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

[0026] In another preferred embodiment, the antiserum albumin nanobody is pH-independent.

[0027] In another preferred embodiment, the anti-serum albumin nanobody can bind to serum albumin under different pH conditions.

[0028] In another preferred embodiment, the anti-serum albumin nanobody can bind serum albumin under conditions of pH 3.0-10.0, more preferably pH 4.0-9.0, and even more preferably pH 5.0-8.0.

[0029] In another preferred embodiment, the monoclonal antibody exhibits a binding activity of A1 with serum albumin at approximately pH ≥ 7.4 (preferably ≥ 7.8, more preferably ≥ 8.0; and pH ≤ 8.5); and the monoclonal antibody exhibits a binding activity of A2 with serum albumin at approximately pH ≤ 5.0 (preferably ≤ 4.8, more preferably ≤ 4.5; and pH ≥ 4.0); then 0.5 ≤ A1 / A2 ≤ 2, preferably 0.7 ≤ A1 / A2 ≤ 1.5, and more preferably 0.8 ≤ A1 / A2 ≤ 1.2.

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

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

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

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

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

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

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

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

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

[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 combinations 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 combinations thereof.

[0041] In another preferred embodiment, the nanobody includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0042] In another preferred embodiment, the serum albumin-specific nanobody of the present invention further comprises an anti-serum albumin antibody molecule capable of binding human serum albumin with a VHH consisting of any of the amino acid sequences 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 invention, an anti-serum albumin antibody is provided, said antibody comprising one or more VHH chains of the nanobody described in the first aspect of the invention.

[0044] In another preferred embodiment, the antiserum albumin antibody may be a monomer, a bivalent antibody, and / or a multivalent antibody.

[0045] In a third aspect of the invention, a separated polynucleotide is provided, said polynucleotide encoding the anti-serum albumin nanobody described in the first aspect of the invention or the anti-serum albumin antibody described in the second aspect of the invention.

[0046] In another preferred embodiment, the polynucleotide is in combinatorial form.

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

[0048] In another preferred embodiment, the present invention relates to a nucleic acid molecule encoding the antiserum albumin nanobody of the present invention. The nucleic acid of the present invention may be RNA, DNA, or cDNA.

[0049] In a fourth aspect of the invention, an expression vector is provided that expresses the polynucleotide of the third aspect of the invention.

[0050] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0051] In another preferred embodiment, the viral vector includes a lentiviral vector, an adenovirus vector, an AAV viral vector, a retroviral vector, or a combination thereof.

[0052] In a fifth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the fourth aspect of the invention, or having the polynucleotide described in the third aspect of the invention integrated into its genome.

[0053] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0054] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0055] In a sixth aspect of the invention, a method for generating anti-serum albumin nanobodies is provided, comprising the steps of:

[0056] (a) Culturing the host cells described in the fifth aspect of the invention under conditions suitable for generating nanobodies, thereby obtaining a culture containing the said anti-serum albumin nanobodies; and

[0057] (b) Isolating or recovering the anti-serum albumin nanobody from the culture; and optionally...

[0058] (c) Purification and / or modification of the antiserum albumin nanobody obtained in step (b).

[0059] In another preferred embodiment, the antiserum albumin nanobody has an amino acid sequence as shown in SEQ ID NO:8, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53 or SEQ ID NO:59.

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

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

[0062] (b) The conjugate selected from the group consisting of: detectable markers, drugs, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0063] In another preferred embodiment, the components (a) and (b) are operably connected.

[0064] In another preferred embodiment, the coupling portion is a chemical marker and a biological marker.

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

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

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

[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 combinations thereof; and / or

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

[0070] In another preferred embodiment, the coupling portion is a detectable marker.

[0071] In another preferred embodiment, the coupling portion is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), or any form of nanoparticles.

[0072] In an eighth aspect of the invention, a combination is provided, the combination comprising:

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

[0074] (b) Modification markers selected from the following group: chemical markers and biological markers.

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

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

[0077] In a ninth aspect of the present invention, a multispecific antibody is provided, the multispecific antibody comprising: an antiserum albumin nanobody as described in the first aspect of the present invention, or an antiserum albumin antibody as described in the second aspect of the present invention.

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

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

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

[0081] In another preferred embodiment, the multispecific antibody further comprises the Fc segment of the 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 an 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 shown in Formula I:

[0086] IIB type I

[0087] in,

[0088] "-" indicates a peptide bond;

[0089] I represents an anti-IL4R nanobody;

[0090] B is the antiserum albumin nanobody as described in the first aspect of the present invention.

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

[0092] (i) Anti-serum albumin nanobody as described in the first aspect of the present invention, or anti-serum albumin antibody as described in the second aspect of the present invention;

[0093] (ii) Optional polypeptide molecules or fragments with therapeutic functions.

[0094] In another preferred embodiment, the therapeutic polypeptide molecule or fragment includes, but is not limited to, polypeptide molecules or fragments targeting IL-4R, IL-4Rα, TNF-α, 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 therapeutic polypeptide molecule or fragment includes, but is not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analog, albumin, antibody fragment, and cytokines.

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

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

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

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

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

[0101] (i) Anti-serum albumin nanobody as described in the first aspect of the present invention, anti-serum albumin antibody as described in the second aspect of the present invention, immunoconjugate as described in the seventh aspect of the present invention, conjugate as described in the eighth aspect of the present invention, multispecific antibody as described in the ninth aspect of the present invention, or fusion protein as described in the tenth aspect of the present invention.

[0102] (ii) Pharmaceutically acceptable carriers.

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

[0104] In a twelfth aspect of the present invention, there is provided an use of an active ingredient selected from the group consisting of: anti-serum albumin nanobodies as described in the first aspect of the present invention, anti-serum albumin antibodies as described in the second aspect of the present invention, immunoconjugates as described in the seventh aspect of the present invention, conjugates as described in the eighth aspect of the present invention, multispecific antibodies as described in the ninth aspect of the present invention, fusion proteins as described in the tenth aspect of the present invention, or combinations thereof, for the preparation of long-acting protein drugs.

[0105] In a thirteenth aspect of the invention, the use of anti-serum albumin nanobodies as described in the first aspect of the invention, anti-serum albumin antibodies as described in the second aspect of the invention, or immunoconjugates as described in the seventh aspect of the invention is provided: (a) for the preparation of reagents, detection plates, or kits for the detection of serum albumin; (b) for the preparation of pharmaceutical agents (long-acting protein drugs) that bind to serum albumin.

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

[0107] In another preferred embodiment, the reagent is used to detect serum albumin or fragments thereof in a sample.

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

[0109] In a fourteenth aspect of the present invention, an immunoadsorbent material for purifying serum albumin is provided, wherein the immunoadsorbent material comprises an anti-serum albumin nanobody as described in the first or second aspect, and a VHH chain of the anti-serum albumin nanobody as described in the first or second aspect of the present invention.

[0110] In another preferred embodiment, the immunoadsorption material further includes a carrier.

[0111] In another preferred embodiment, the carrier includes, but is not limited to, magnetic beads, agarose gel, silica microspheres, and porous materials.

[0112] In a fifteenth aspect of the present invention, a method for in vitro detection of serum albumin or fragments thereof in a sample is provided, the method comprising the steps of:

[0113] (1) In vitro, the sample is contacted with an antiserum albumin nanobody as described in the first aspect of the present invention, an antiserum albumin antibody as described in the second aspect of the present invention, or an immunoconjugate as described in the seventh aspect of the present invention.

[0114] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of serum albumin or its fragments in the sample.

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

[0116] According to a sixteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0117] (a) Culturing the host cells described in page five of this invention under suitable expression conditions;

[0118] (b) Isolating recombinant polypeptides from the culture, said recombinant polypeptides including antiserum albumin nanobodies as described in the first aspect of the present invention, antiserum albumin antibodies as described in the second aspect of the present invention, multispecific antibodies as described in the ninth aspect of the present invention, and fusion proteins as described in the tenth aspect of the present invention.

[0119] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0120] Figure 1A The binding activity of the albumin nanobody Nb1-15 to serum albumin from humans, mice, rats, and cynomolgus monkeys was demonstrated under different pH conditions.

[0121] Figure 1B The binding activity of the albumin nanobody Nb1-60 with serum albumin from humans, mice, rats, and cynomolgus monkeys was demonstrated under different pH conditions.

[0122] Figure 1C The binding activity of the albumin nanobody Nb1-83 with serum albumin from humans, mice, rats, and cynomolgus monkeys was demonstrated under different pH conditions.

[0123] Figure 1D The binding activity of the albumin nanobody Nb3-24 to serum albumin from humans, mice, rats, and cynomolgus monkeys was demonstrated under different pH conditions.

[0124] Figure 2A The binding activity of the IL4R nanobody fused with the humanized albumin nanobody HuNb1-15 to serum albumin of humans, mice, rats, and cynomolgus monkeys under different pH conditions was demonstrated.

[0125] Figure 2B The binding activity of IL4R nanobody fused with humanized albumin nanobody HuNb1-60 to serum albumin of humans, mice, rats and cynomolgus monkeys under different pH conditions was demonstrated.

[0126] Figure 2CThe binding activity of IL4R nanobody fused with humanized albumin nanobody HuNb1-83 to serum albumin of humans, mice, rats and cynomolgus monkeys under different pH conditions was demonstrated.

[0127] Figure 2D The binding activity of the IL4R nanobody fused with the humanized albumin nanobody HuNb3-24 to serum albumin in humans, mice, rats, and cynomolgus monkeys under different pH conditions was demonstrated.

[0128] Figure 3 The results show the half-life of the IL4R-HSA trivalent antibody in mice.

[0129] Figure 4 The results of half-life detection of the IL4R-HSA trivalent antibody in rats are shown.

[0130] Figure 5 The results show the half-life of the IL4R-HSA trivalent antibody in cynomolgus monkeys. Detailed Implementation

[0131] Through extensive and in-depth research and numerous screenings, the inventors successfully obtained a group of anti-serum albumin nanobodies. Experimental results show that the four anti-serum albumin nanobodies obtained in this invention can effectively bind to serum albumin. Based on this, the present invention was completed.

[0132] Specifically, this invention utilizes human serum albumin extracellular antigen protein to immunize camels, obtaining a high-quality immune nanobody gene library. Serum albumin molecules are then coupled to an ELISA plate to display the correct spatial structure of serum albumin. Using this antigen, phage display technology is employed to screen the immune nanobody gene library (camel heavy chain antibody phage display gene library), thereby obtaining serum albumin-specific nanobody genes.

[0133] As used herein, the terms "nanobody of the present invention," "anti-serum albumin nanobody of the present invention," and "anti-serum albumin nanobody of the present invention" are used interchangeably and all refer to nanobodies that specifically recognize and bind to serum albumin (including human serum albumin). Particularly preferred are nanobodies with the amino acid sequence of the VHH chain as shown in SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53, or SEQ ID NO:59.

[0134] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0135] As used herein, the terms "single domain antibody (sdAb, or VHH)" and "nanobody" have the same meaning: a nanobody constructed by cloning the variable region of an antibody heavy chain, consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0136] Nanobodies / single-domain antibodies are novel small-molecule antibody fragments cloned from the variable heavy chain region (VHH) of natural camel heavy-chain antibodies. Nanobodies (Nb) possess excellent biological properties, with a molecular weight of 12-15 kDa, about one-tenth the size of a complete antibody. They exhibit excellent tissue penetration, high specificity, and good water solubility. Due to their unique structural properties, they combine the advantages of traditional antibodies and small-molecule drugs, almost perfectly overcoming the shortcomings of traditional antibodies such as long development cycles, low stability, and demanding storage conditions. They are gradually becoming an emerging force in next-generation antibody therapy, showing broad application prospects in immunodiagnosis and treatment.

[0137] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are closely packed together through the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0138] As those skilled in the art will recognize, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens that bind to the described antiserum albumin antibody or fragments thereof.

[0139] As used in this article, the terms "heavy chain variable region" and "V" are used interchangeably. H "They can be used interchangeably."

[0140] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

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

[0142] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0143] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to serum albumin, such as proteins or peptides having a heavy chain variable region. They may or may not contain initiating methionine.

[0144] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0145] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0146] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having variable regions of antibody heavy chains with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0147] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0148] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0149] The antibody of this invention refers to a polypeptide having serum albumin-binding activity and including the aforementioned CDR region. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the antibody of this invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibody of this invention.

[0150] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

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

[0152] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

[0153] Table 1

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

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

[0156] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0157] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0158] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0159] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0160] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0161] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0162] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0163] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[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*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0165] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0166] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0167] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

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

[0169] Therapeutic agents that can bind to or conjugate with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. drug-activated enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0170] Pharmaceutical Composition

[0171] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-described antibody or its active fragment or fusion protein, a pharmaceutically acceptable carrier or excipient, and optionally other bioactive substances. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

[0172] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0173] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0174] Detection methods

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

[0176] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0177] Reagent test kit

[0178] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention or a detection plate. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0179] This invention also provides a test kit for detecting serum albumin levels. The kit includes an antibody that recognizes serum albumin, a lysis medium for dissolving the sample, and universal reagents and buffers required for the detection, such as various buffer solutions, detection labels, and detection substrates. This test kit can be used as an in vitro diagnostic device.

[0180] application

[0181] As described above, the nanobody of the present invention has broad biological and clinical application value. Its application involves the use of anti-serum albumin nanobody to prepare long-acting protein drugs, providing a research and development foundation for the development of long-acting protein drugs.

[0182] The main advantages of this invention include:

[0183] (a) The nanobody of the present invention can bind to serum albumin of humans, mice, rats and cynomolgus monkeys under different pH conditions.

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

[0185] (c) The nanobody of the present invention is suitable for prokaryotic and eukaryotic expression, and has the advantages of high solubility, non-aggregation, and resistance to denaturing conditions such as high temperature, strong acid, and strong alkali. It is suitable for laboratory and industrial development.

[0186] (d) The preparation of protein drugs using the nanobodies of the present invention will not affect the targeting and activity of the drugs.

[0187] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

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

[0189] One Bactrian camel from Xinjiang was immunized with human serum albumin. After seven immunizations, total RNA was isolated from the camel's peripheral blood. The VHH gene was amplified by reverse transcription and PCR, and then cloned into the phage vector pMECS. This pMECS was then transformed into TG1 host cells to construct a phage display library. The library was subsequently screened using phage display technology. After four rounds of adsorption-washing-enrichment, phages containing the antibody gene were obtained with a 30-fold enrichment. Forty hundred phage clones were randomly selected from the enriched phage clones for PE-ELISA identification. All positive clones were sequenced for identification. Antibodies with different sequences were used as candidates, and various antibodies were expressed and purified using *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 μg / mL with pH 8.2 NaHCO3 and coated onto ELISA plates, then incubated overnight at 4°C. The plates were washed with PBST, and each well was blocked with 300 μL of 1% skim milk at room temperature for 2 hours. The plates were then washed with PBST, and 100 μL of serially diluted test antibodies (each antibody diluted separately with PBS solutions at pH 5.0 and pH 7.4, starting at 300 μg / mL, 3-fold serial dilution) were added, and incubated at 37°C for 1 hour. The plates were then washed with PBST, and 100 μL of diluted Goat pAb to Nanobody (HRP) (diluted 1:1000 with PBS) was added, and incubated at 37°C for 1 hour. The plates were washed with PBST, and 100 μL of TMB chromogenic buffer was added. The reaction was incubated at room temperature in the dark for 5 minutes, then 50 μL of 2M sulfuric acid was added to stop the reaction. The absorbance was read at 450 nm using an ELISA reader. Results are as follows: Figures 1A-1DAs shown, four anti-serum albumin nanobodies can simultaneously bind to serum albumin from humans, mice, rats, and cynomolgus monkeys, and can bind to serum albumin from various species under different pH conditions (pH 5.0 and pH 7.4). Statistical analysis is shown in Table 2.

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

[0193]

[0194]

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

[0196] The amino acid sequences of the above four nanobodies were placed in a structural database for homology searches. The antibody sequences are shown in Table 3.

[0197] Table 3. Sequence numbers of antiserum albumin nanobodies

[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] Structures with high sequence isomorphism were selected and compared. Based on crystal structure resolution and the constructed phylogenetic tree, proteins including 3dwt were chosen for multi-template homology modeling of the target nanobody sequence. The structure with the lowest molpdf was then selected based on the scoring function. The solvent accessibility of the residues in the optimal modeled structure was calculated using the ProtSA server. The optimal modeled structure was then compared with DP-47, and residues exposed to the solvent were replaced. Finally, a humanized anti-serum albumin nanobody was determined. The corresponding humanized antibody sequence is shown in Table 4 below.

[0200] Table 4. Sequence numbers of humanized antiserum albumin nanobodies

[0201]

[0202]

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

[0204] IL4R nanobody was selected as a representative to construct a long-acting protein drug with humanized albumin nanobody, in order to verify the function of humanized albumin nanobody in prolonging the in vivo half-life of protein. The four candidate humanized albumin nanobodies were tandemly linked with IL4R nanobody (sequence derived from patent CN2019110547879) to form trivalent nanobodies, the structural sequences of which are shown in Table 5 below.

[0205] Table 5. Structure and sequence of multivalent antibodies

[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 is as follows: (1) The nanobody sequence bases shown in SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, and SEQ ID NO. 70 are constructed into the pPICZaA vector; (2) After linearization with Sac I restriction endonuclease, the nanobody is electroporated into X-33 competent cells; (3) The electroporated samples are plated on YPD plates containing different concentrations of bleomycin resistance and cultured in a 30°C incubator for 3 days. For specific implementation plans, please refer to the pPICZaA vector instruction manual provided by Invitrogen; (4) After single clones grow on the plate medium, single clones are picked and placed in BMGY medium. When the OD value of BMGY medium reaches about 20, the cells are collected and replaced with BMMY medium and cultured at 28°C and 250 rpm; (5) Methanol is added every 24 hours to a final volume of 1%; the induction is continued for 5 days, and the culture is ended; (6) The supernatant is purified by cation chromatography to remove the target antibody.

[0208] The purified antibodies were tested for binding to various albumins under different pH conditions. The detection method was the same as in Example 2, and the results are as follows. Figures 2A-2D As shown, the humanized trivalent antibody fused with human IL4R nanobody can still bind to serum albumin from different species, and its binding activity is independent of pH changes. Numerical statistics are shown in Table 6.

[0209] Table 6. Binding of humanized trivalent antibodies to serum albumin of different species.

[0210]

[0211]

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

[0213] 100 μg of a multivalent antibody expressed in yeast cells was intravenously injected into mice. Orbital blood samples were collected at 5 min, 3 h, 8 h, 24 h, 48 h, 96 h, and 144 h post-administration. Plasma was separated after centrifugation. Blood samples were collected at 5 min, 15 min, 30 min, 1.5 h, and 3 h post-administration of the control antibody MY8171. Pharmacokinetic analysis was then performed using ELISA. 1 μg / mL hIL4R protein was coated onto the microplate at 100 μL / well overnight; the plate was washed 5 times with PBST, and then blocked at room temperature for 2 hours with 300 μL of 0.3% Casein; the plate was washed 5 times with PBST, and diluted serum samples were added to the corresponding wells and incubated at 37°C for 1 hour; the plate was washed 5 times with PBST, and then Goat1 pAb to Nanobody-HRP (1:1000 dilution) was added to 100 μL / well and incubated at 37°C for 1 hour; the plate was washed 5 times with PBST, and then TMB chromogenic solution was added and allowed to stand at room temperature for 5 minutes to develop color; the reaction was terminated by adding 2M sulfuric acid, and the absorbance was read at 450 nm using a microplate reader. The detection results were converted according to the standard curve to obtain the time-drug concentration relationship graph, as shown in the figure. Figure 3 As shown in Table 7, the half-life of the bivalent antibody MY8171 without albumin nanobodies in mice is only about 1.86 hours, while the half-life of the trivalent antibody with anti-serum albumin nanobodies in mice is significantly prolonged (as shown in Table 7), with a maximum extension of about 22 times.

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

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

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

[0217] 400 μg of a multivalent antibody expressed in yeast cells was intravenously injected into rats. Orbital blood samples were collected at 5 min, 3 h, 8 h, 24 h, 48 h, 96 h, and 144 h post-administration. Plasma was separated after centrifugation. Blood samples were collected at 5 min, 15 min, 30 min, 1.5 h, and 3 h after administration of the control antibody MY8171. Pharmacokinetic analysis was then performed using ELISA. The detection method was the same as in Example 5. The results were converted according to a standard curve to obtain a time-drug concentration relationship graph. The results are shown below. Figure 4 As shown, the half-life of the bivalent antibody MY8171 without albumin nanobodies in rats is only about 0.82 hours, while the half-life of the trivalent antibody with anti-serum albumin nanobodies in mice is significantly prolonged (as shown in Table 8), with a maximum extension of about 63 times.

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

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

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

[0221] Two antibodies, MY8267 and MY8268, were intravenously injected into the left forelimb of cynomolgus monkeys at a dose of 2 mg / kg. Blood samples were collected 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, and 35 days after administration. Plasma was separated by centrifugation. Pharmacokinetic analysis was then performed using ELISA, following the same method as in Example 5. The results were converted to a standard curve to obtain a time-drug concentration relationship graph, as shown below. Figure 5 As shown, the half-life of trivalent antibodies carrying anti-serum albumin nanobodies in cynomolgus monkeys can reach approximately 9-15 days (Table 9).

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

[0223] Antibody number MY8267 MY8268 <![CDATA[T 1 / 2 (days)]]> 9.53 14.21

[0224] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-serum albumin nanobody, characterized in that, The nanobody can specifically bind to serum albumin, and the VHH chain of the nanobody has the following complementarity-determining region (CDR): CDR1 shown in SEQ ID NO:19, CDR2 shown in SEQ ID NO:20, and CDR3 shown in SEQ ID NO:

21.

2. The nanobody as described in claim 1, characterized in that, The VHH chain of the nanobody also includes a framework region FR, wherein the framework region FR is selected from the following group: (1) FR1 shown in SEQ ID NO: 22, FR2 shown in SEQ ID NO: 23, FR3 shown in SEQ ID NO: 24, and FR4 shown in SEQ ID NO:

25. (2) FR1 shown in SEQ ID NO: 49, FR2 shown in SEQ ID NO: 50, FR3 shown in SEQ ID NO: 51, and FR4 shown in SEQ ID NO:

52.

3. The nanobody as described in claim 1, characterized in that, The amino acid sequence of the VHH chain of the nanobody is selected from the following groups: SEQ ID NO: 26 and SEQ ID NO:

53.

4. A polynucleotide, characterized in that, The polynucleotide encodes the nanobody of claim 1.

5. The polynucleotide as described in claim 4, characterized in that, The nucleotide sequence of the polynucleotide is selected from the following groups: SEQ ID NO: 27 and SEQ ID NO:

54.

6. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 4.

7. A host cell, characterized in that, The host cell contains the expression vector of claim 6, or the polynucleotide of claim 4 is integrated into its genome.

8. A method for generating anti-serum albumin nanobodies, characterized in that, Includes the following steps: (a) Culturing the host cells of claim 7 under conditions suitable for the production of nanobodies, thereby obtaining a culture containing anti-serum albumin nanobodies; (b) Isolating and / or recovering the antiserum albumin nanobody from the culture; and (c) Purification and / or modification of the antiserum albumin nanobody obtained in step (b).

9. A complex comprising: (a) The anti-serum albumin nanobody as claimed in claim 1; and operably linked (b) Modification markers selected from the group consisting of chemical markers and biological markers.

10. A composition comprising: (i) The anti-serum albumin nanobody as described in claim 1 or the conjugate as described in claim 9; (ii) Pharmaceutically acceptable carriers.

11. Use of the anti-serum albumin nanobody as described in claim 1: (a) for preparing reagents, detection plates or kits for detecting serum albumin; (b) for preparing reagents that bind to serum albumin; (c) for preparing reagents that prolong the in vivo half-life of proteins.

12. An immunoadsorption material for purifying serum albumin, wherein, The immunoadsorption material comprises the antiserum albumin nanobody as described in claim 1.

13. A method for in vitro non-diagnostic detection of serum albumin or fragments thereof in a sample, the method comprising the steps of: (1) In vitro, the sample is contacted with the anti-serum albumin nanobody as described in claim 1; (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of serum albumin or its fragments in the sample.

14. A method for preparing a recombinant polypeptide, the method comprising: (a) Culturing the host cells as described in claim 7 under suitable expression conditions; (b) Isolating recombinant polypeptides from the culture, said recombinant polypeptides including the antiserum albumin nanobody as described in claim 1.

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