Anti-human serum albumin antibodies and their applications

Abstract

This application relates to an anti-human serum albumin antibody and its application, wherein the anti-human serum albumin antibody is K with a concentration of about 9E-09M or less. D The antibody specifically binds to human serum albumin. After binding to human serum albumin, the antibody does not reduce or inhibit the binding of human serum albumin to the neonatal receptor.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to an anti-human serum albumin antibody and its applications. Background Technology

[0002] Human serum albumin (HSA) is a major plasma protein composed of approximately 591 amino acids with a molecular weight of approximately 67 kDa. In antibody preparation, it is used as a carrier protein for hapten conjugation and is also commonly used as a molecular weight standard protein in electrophoresis or chromatographic chromatography. Holt et al. have extended the half-life of short-term drugs by using antibodies against the serum albumin domain (Holt et al., Protein Engineering, Design and Selection 21(2008)283S28288).

[0003] Currently known anti-human serum albumin antibodies suffer from drawbacks such as poor selectivity and weak affinity. Therefore, it is necessary to develop novel anti-human serum albumin antibodies with high affinity and specificity for human serum albumin. Summary of the Invention

[0004] This application provides an isolated antigen-binding protein having one or more of the following properties: 1) in Octet assay, with a K content of about 9E-09M or less. D 1) The value specifically binds to human serum albumin; 2) In the Octet assay, K is used at approximately 3.5E-08M or less. D The value specifically binds to cynomolgus monkey serum albumin; and 3) after binding to human serum albumin, it does not reduce or inhibit the binding of human serum albumin to neonatal receptors.

[0005] In some embodiments, the isolated antigen-binding protein comprises HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6.

[0006] In some embodiments, the isolated antigen-binding protein comprises HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

[0007] In some embodiments, the isolated antigen-binding protein comprises HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0008] In some embodiments, the isolated antigen-binding protein comprises a heavy chain variable region VH, the VH comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6.

[0009] In some embodiments, the isolated antigen-binding proteins HCDR1, HCDR2, and HCDR3 comprise amino acid sequences selected from any of the following groups:

[0010] a) HCDR1: SEQ ID NO:1, HCDR2: SEQ ID NO:3, and HCDR3: SEQ ID NO:5; and

[0011] b) HCDR1: SEQ ID NO:2, HCDR2: SEQ ID NO:4, and HCDR3: SEQ ID NO:6.

[0012] In some embodiments, the isolated antigen-binding protein comprises H-FR1, the C-terminus of which is directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8.

[0013] In some embodiments, the isolated antigen-binding protein comprises H-FR2, which is located between HCDR1 and HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:10.

[0014] In some embodiments, the isolated antigen-binding protein comprises H-FR3, which is located between HCDR2 and HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:12.

[0015] In some embodiments, the isolated antigen-binding protein comprises H-FR4, the N-terminus of which is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:14.

[0016] In some embodiments, the isolated antigen-binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8; H-FR2 comprises the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:10; H-FR3 comprises the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:12; and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:14.

[0017] In some embodiments, the H-FR1, H-FR2, H-FR3, and H-FR4 of the isolated antigen-binding protein comprise an amino acid sequence selected from any of the following groups:

[0018] a) H-FR1: SEQ ID NO:7, H-FR2: SEQ ID NO:9, H-FR3: SEQ ID NO:11 and H-FR4: SEQ ID NO:13; and

[0019] b) H-FR1: SEQ ID NO:8, H-FR2: SEQ ID NO:10, H-FR3: SEQ ID NO:12 and H-FR4: SEQ ID NO:14.

[0020] In some embodiments, the isolated antigen-binding protein comprises a heavy chain variable region VH, the VH comprising the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16.

[0021] In some embodiments, the isolated antigen-binding protein includes an antibody or an antigen-binding fragment thereof.

[0022] In some embodiments, the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0023] In some embodiments, the isolated antigen-binding protein includes VHH or its antigen-binding fragment.

[0024] In some embodiments, the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, and fully human antibodies.

[0025] In some embodiments, the isolated antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16.

[0026] On the other hand, this application provides one or more polypeptides comprising the isolated antigen-binding protein.

[0027] On the other hand, this application provides one or more immune conjugates comprising the isolated antigen-binding protein or the polypeptide.

[0028] In some embodiments, the immune conjugate further includes a pharmaceutically acceptable therapeutic agent.

[0029] In some embodiments, the therapeutic agent is selected from the group consisting of cytotoxic agents and cell inhibitors.

[0030] On the other hand, this application provides one or more isolated nucleic acid molecules that encode the isolated antigen-binding protein or the polypeptide.

[0031] On the other hand, this application provides one or more vectors containing the isolated nucleic acid molecules.

[0032] On the other hand, this application provides one or more cells that contain the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, and / or the carrier.

[0033] On the other hand, this application provides a method for preparing the isolated antigen-binding protein and / or the polypeptide, the method comprising culturing the cells under conditions that cause the isolated antigen-binding protein and / or the polypeptide to be expressed.

[0034] On the other hand, this application provides one or more pharmaceutical compositions comprising the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and / or a pharmaceutically acceptable adjuvant and / or excipient.

[0035] On the other hand, this application provides a method for detecting human serum albumin, comprising: administering the isolated antigen-binding protein or the polypeptide.

[0036] On the other hand, this application provides a human serum albumin detection kit, which contains the isolated antigen-binding protein or the polypeptide.

[0037] On the other hand, this application provides the use of the isolated antigen-binding protein or the polypeptide in the preparation of a kit.

[0038] On the other hand, this application provides the use of the isolated antigen-binding protein and / or the polypeptide in the preparation of medicaments for the prevention and / or treatment of diseases or conditions.

[0039] On the other hand, this application provides the isolated antigen-binding protein, the polypeptide, the immune conjugate, the isolated nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition, for the prevention, relief, and / or treatment of diseases or conditions.

[0040] On the other hand, this application provides a method for preventing and / or treating a disease or condition, comprising administering to a subject in need an effective amount of the isolated antigen-binding protein, the polypeptide, the immune conjugate, the isolated nucleic acid molecule, the carrier, and / or the cell.

[0041] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0042] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0043] Figure 1 The image shows the binding activity of the antigen-binding protein described in this application with HSA.

[0044] Figure 2 The image shows that the antigen-binding protein described in this application can bind to HSA that has been bound to FcRn.

[0045] Figure 3 The image shows that the antigen-binding protein described in this application can bind to HSA that has been bound to FcRn.

[0046] Figure 4 The image shows that the negative control cannot bind to HAS after it has bound to FcRn. Detailed Implementation

[0047] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0048] Terminology Definition

[0049] In this application, the term "isolated" generally refers to something obtained artificially from its natural state. If a substance or component is found in nature as an "isolated" substance, it may be due to an alteration of its natural environment, the isolation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0050] In this application, the term "antigen-binding protein" generally refers to a polypeptide molecule capable of specifically recognizing and / or neutralizing a particular antigen. For example, in this application, the term "antigen-binding protein" may include an "antibody" or an "antigen-binding fragment." For example, an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and may include any molecule containing its antigen-binding portion. The term "antibody" may include monoclonal antibodies, antibody fragments, or antibody derivatives, including but not limited to murine antibodies, human antibodies (fully human antibodies), chimeric antibodies, single-chain antibodies (e.g., scFv), and antigen-binding antibody fragments (e.g., Fab, Fab', VHH, and (Fab)2 fragments). The term "antibody" may also include all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and their derivatives described herein. In this application, the term "antibody" may include single-domain antibodies.

[0051] In this application, the term "antigen-binding fragment" generally refers to one or more fragments in an antibody that perform the function of specifically binding antigens. The antigen-binding function of an antibody can be achieved by the full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by a heavy chain comprising a fragment including Fv, ScFv, dsFv, Fab, Fab', or F(ab')2, or a light chain comprising a fragment including Fv, scFv, dsFv, Fab, Fab', or F(ab')2. (1) Fab fragment, typically a monovalent fragment consisting of VL, VH, CL and CH domains; (2) F(ab')2 fragment, a divalent fragment consisting of two Fab fragments linked by disulfide bonds at the hinge region; (3) Fd fragment consisting of VH and CH domains; (4) Fv fragment consisting of VL and VH domains of an antibody single arm; (5) dAb fragment consisting of VH domain (Ward et al., (1989) Nature 341: 544-546); (6) separate complementarity-determining regions (CDRs) and (7) combinations of two or more separate CDRs optionally linked by a linker. For example, it may also include a monovalent single-chain molecule Fv (scFv) formed by pairing VL and VH (see Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85: 5879-5883). For example, it may also include a class of antibodies VHH that lacks the antibody light chain and only has the variable region of the heavy chain (see, for example, Kang Xiaozhen et al., Chinese Journal of Biotechnology, 2018, 34(12):1974-1984). The “antigen-binding portion” may also include a fusion protein containing a binding domain selected from the following: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.

[0052] In this application, the term "single-domain antibody" generally refers to a class of antibodies that lack the antibody light chain and only possess the variable region of the heavy chain. Studies have found that Bactrian camels, dromedary camels, alpacas, and llamas possess a type of heavy chain antibody (hcAb) that consists only of the heavy chain but has complete function. The variable domains of the hcAb (VHH) have a molecular weight only 1 / 10 that of conventional antibodies, making it the smallest molecular fragment with complete antibody function currently available; this is called a single-domain antibody (sdAb). Compared to other antibodies, single-domain antibodies have advantages such as low immunogenicity, small molecular size, and strong penetrability, thus showing broad application prospects in basic research, drug development, and disease treatment. For example, single-domain antibodies can be derived from alpacas. Single-domain antibodies can be composed of the variable region (VH) of the heavy chain. The term "variable region of the heavy chain" generally refers to the amino-terminal domain of the heavy chain of the antigen-binding fragment. The heavy chain variable region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are scattered within more conserved regions known as framework regions (FRs). Each heavy chain variable region may consist of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: H-FR1, HCDR1, H-FR2, HCDR2, H-FR3, HCDR3, and H-FR4. The heavy chain variable region contains a binding domain that interacts with an antigen (e.g., HSA). The exact boundaries of the CDRs have been defined differently depending on the system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) provides not only a definitive residue numbering system applicable to any variable region of antigen-binding proteins, but also precise residue boundaries defining CDRs. These CDRs can be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (1989)) found that despite great diversity at the amino acid sequence level, certain sub-regions within Kabat CDRs take almost identical peptide backbone conformations. These sub-regions are named L1, L2, and L3 or H1, H2, and H3, where “L” and “H” refer to the light and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, the Chothia CDRs being the most common CDRs in the world. CDRs have boundaries that overlap with Kabat CDRs.Other boundaries of defined CDRs overlapping with Kabat CDRs have been described by Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Additionally, other CDR boundary definitions may not strictly follow one of the aforementioned systems but will still overlap with Kabat CDRs, although they may be shortened or lengthened according to the prediction or experimental findings of antigen binding, regardless of the specific residue or group of residues or even the entire CDR. In this application, CDRs can be defined using the Kabat numbering system. In this application, the term "single-domain antibody" is used interchangeably with "nanobody" and "VHH".

[0053] In this application, the term "monoclonal antibody" generally refers to a group of substantially homologous antibodies, meaning that the individual antibodies comprising the group are identical except for the possibility of naturally occurring mutations present in trace amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site directly. For example, said monoclonal antibodies can be prepared by hybridoma technology or produced in bacterial, eukaryotic, or plant cells using recombinant DNA methods. Monoclonal antibodies can also be obtained from phage antibody libraries using techniques described, for example, those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991).

[0054] In this application, the term "chimeric antibody" generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to, or belongs to, a corresponding amino acid sequence from an antibody of a specific species, while the remaining segments of that chain are homologous to a corresponding sequence from another species. For example, the variable regions of both the light and heavy chains may originate from the variable region of an antibody from an animal species (e.g., mouse, rat, etc.), while the constant region is homologous to an antibody sequence from another species (e.g., human). For instance, to obtain a chimeric antibody, the variable region can be generated using non-human B cells or hybridoma cells, with the constant region combined with it derived from human cells. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region it is combined with. Furthermore, since the constant region of a chimeric antibody can be derived from humans, the likelihood of the chimeric antibody eliciting an immune response upon injection is lower than with antibodies using a non-human-derived constant region.

[0055] In this application, the term "mouse antibody" generally refers to an antibody whose variable frame and CDR regions are derived from mouse germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, it is also derived from a mouse germline immunoglobulin sequence. The mouse antibodies of this application may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, for example, mutations introduced through in vitro random or point mutations or through in vivo somatic mutations. However, the term "mouse antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the mouse frame sequence.

[0056] In this application, the terms "HSA protein" or "HSA antigen" are used interchangeably and include any functionally active fragment, variant, and homolog of HSA, which is naturally expressed in cells or expressed on cells transfected with the HSA gene. In this application, the NCBI accession number for HSA can be NP_000468.1. For example, the "functionally active fragment" may include a fragment that retains the endogenous function of at least one naturally occurring protein (e.g., binding to the antigen-binding protein described in this application). For example, the "functionally active fragment" may include a domain that binds to the antigen-binding protein of this application.

[0057] In addition to the specific proteins and nucleotides mentioned herein, this application may also include their functionally active fragments, derivatives, analogs, homologs, and fragments thereof.

[0058] The term "functionally active fragment" refers to a polypeptide that has a substantially identical amino acid sequence or is encoded by a substantially identical nucleotide sequence to the naturally occurring sequence and is capable of possessing one or more activities of the naturally occurring sequence. In the context of this application, a functionally active fragment of any given sequence refers to a sequence in which a specific sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one endogenous function. Sequences encoding functionally active fragments can be obtained by adding, deleting, substituting, modifying, replacing, and / or mutating at least one amino acid residue and / or nucleotide residue present in naturally occurring proteins and / or polynucleotides, as long as the original functional activity is maintained.

[0059] In this application, the term "derivative" generally refers to any substitution, variation, modification, replacement, deletion and / or addition of one (or more) amino acid residues of the self / pair sequence for the polypeptide or polynucleotide of this application, provided that the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

[0060] In this application, the term "analyte" generally refers to a polypeptide or polynucleotide, including any mimic of a polypeptide or polynucleotide, i.e., a chemical compound having at least one endogenous function of the polypeptide or polynucleotide that the mimic mimic has.

[0061] Typically, amino acid substitutions can be made, such as substitutions of at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20), as long as the modified sequence substantially retains the desired activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0062] In this application, the term "homologous" generally refers to an amino acid or nucleotide sequence that has a certain degree of homology with a naturally occurring sequence. The term "homology" can be equivalent to sequence "identity." Homologous sequences can include amino acid sequences that are at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence. Typically, homologous sequences will contain the same active sites, etc., as the subject amino acid sequence. Homology can be considered based on similarity (i.e., amino acid residues with similar chemical properties / functions) or can be expressed in terms of sequence identity. In this application, a sequence having a percentage identity with any of the referenced amino acid or nucleotide sequences in SEQ ID NO means a sequence having said percentage identity across the entire length of the referenced SEQ ID NO. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0063] The proteins or peptides used in this application may also have deletions, insertions, or substitutions of amino acid residues, which produce silent changes and result in functionally equivalent proteins. Intentional amino acid substitutions can be made based on the similarity of the residues' polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphoteric properties, as long as the endogenous function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids containing non-polarized head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0064] In this application, the term "immunoconjugate" generally refers to a conjugate formed by the conjugation (e.g., covalently linked by a linker molecule) of the other therapeutic agent to the isolated antigen-binding protein. This conjugate can deliver the other therapeutic agent to the target cell (e.g., tumor cells) through the specific binding of the isolated antigen-binding protein to the antigen on the target cell. The immunoconjugate is then internalized and eventually enters the target cell interior (e.g., into vesicles such as lysosomes), where the linker molecule in the immunoconjugate can cleave, releasing the other agent to exert its cytotoxic effect. Furthermore, the antigen may also be secreted by the target cell and located in the extracellular space.

[0065] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0066] In this application, the term "nucleic acid molecule" generally refers to any length of isolated nucleotide, deoxyribonucleotide, or ribonucleotide or analogue, isolated from its natural environment or synthesized artificially.

[0067] In this application, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. The vector can transfer an inserted nucleic acid molecule into cells and / or between cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into suitable cells. Typically, by culturing suitable cells containing the vector, the vector can produce a desired expression product.

[0068] In this application, the term "cell" generally refers to an individual cell, cell line, or cell culture that may contain or already contains a plasmid or vector including the nucleic acid molecules described in this application, or that is capable of expressing the polypeptides or antigen-binding proteins described in this application. The cell may include progeny cells of a single cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they must be capable of expressing the polypeptides or antigen-binding proteins described in this application. The cell can be obtained by in vitro transfection of cells using the vector described in this application. The cell can be a prokaryotic cell (e.g., *Escherichia coli*) or a eukaryotic cell (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells). In some embodiments, the cell may be an immune cell. For example, the immune cells may be selected from the group consisting of: T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

[0069] In this application, the term “treatment” generally means: (i) preventing a patient who may be susceptible to a disease, condition and / or symptom but has not yet been diagnosed with the disease, from developing such a disease, condition or symptom; (ii) suppressing such a disease, condition or symptom, i.e., curbing its development; and (iii) alleviating such a disease, condition or symptom, i.e., achieving the relief of such a disease, condition and / or symptom and / or symptoms associated with such a disease, condition and / or symptom.

[0070] In this application, the terms “polypeptide,” “peptide,” “protein,” and “protein protein” are used interchangeably and generally refer to a polymer having amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also cover polymers containing modified amino acids. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeled component). The term “amino acid” includes natural and / or non-natural or synthetic amino acids, including glycine and its D and L optical isomers, as well as amino acid analogs and peptide mimics.

[0071] In this application, the terms "polynucleotide," "nucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) as defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure may be modified before or after polymer assembly. The sequence of a nucleotide may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.

[0072] In this application, the term "K" D (Similarly, "K") D "KD" usually refers to the "affinity constant" or "equilibrium dissociation constant," and is used in titration measurements at equilibrium, or by expressing the dissociation rate constant (k... d Divide by the binding rate constant (k) a The value obtained using the binding rate constant (k) a ), dissociation rate constant (k d ) and equilibrium dissociation constant (K D The term K represents the binding affinity of a binding protein (e.g., the isolated antigen-binding protein described in this application) to an antigen (e.g., an HSA protein). Methods for determining binding and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. For example, the K can be determined by Octet. D The value can also be determined using other experimental methods and instruments, such as BIAcore (Biomolecular Interaction Analysis) (e.g., instruments available from BIAcore International AB, aGE Healthcare Company, Uppsala, Sweden). Alternatively, the K value can be determined using KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho). DThe value, or the K value, can be determined using a surface plasmon resonance (SPR) spectrometer. D Value. In this application, the K value can be determined using an amine coupling kit. D value.

[0073] In this application, the term "and / or" should be understood to mean any one of the options or both of the options.

[0074] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements. In some cases, "comprising" also covers the situation where only the specified components are included. For example, "comprising" also means "consisting of".

[0075] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0076] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of". Invention Details

[0078] On the one hand, this application provides an isolated antigen-binding protein that, in the Octet assay, can be expressed at a K0 of approximately 9E-09M or less. D Value (e.g., the K) D Values ​​not higher than approximately 9E-09M, not higher than approximately 8.5E-09M, not higher than approximately 8E-09M, not higher than approximately 7.5E-09M, not higher than approximately 7E-09M, not higher than approximately 6.5E-09M, not higher than approximately 6E-09M, not higher than approximately 5E-09M, not higher than approximately 4E-09M, not higher than approximately 3E-09M, not higher than approximately 2E-09M, not higher than approximately 1E-09M, not higher than approximately 9E-10M, not higher than approximately 8E-10M, not higher than 7E-10M, not higher than approximately 6E-10M, not higher than approximately 5E-10M, not higher than approximately 4E-10M, not higher than approximately 3E-10M, not higher than approximately 2E-10M, or not higher than approximately 1E-10M or below specifically bind to human serum albumin.

[0079] In this application, the isolated antigen-binding protein may be at a K0 of about 3.5E-08M or less. D Value (e.g., the K) DValues ​​not higher than approximately 3E-08M, not higher than approximately 2.5E-08M, not higher than approximately 2E-08M, not higher than approximately 1.5E-08M, not higher than approximately 1E-08M, not higher than approximately 9E-09M, not higher than approximately 8E-09M, not higher than approximately 7E-09M, not higher than approximately 6E-09M, not higher than approximately 5E-09M, not higher than approximately 4E-09M, not higher than approximately 3E-09M, not higher than approximately 2E-9M, or not higher than approximately 1E-9M or below specifically bind to cynomolgus monkey serum albumin.

[0080] In this application, the isolated antigen-binding protein, after binding to human serum albumin, generally does not reduce or inhibit the binding of human serum albumin to neonatal receptors.

[0081] For example, the antigen-binding protein of this application is first used to bind HSA. After saturation, the binding of HSA to FcRn can be detected using the Octet system (e.g., by K...). D The value determines whether HSA binds to FcRn.

[0082] On the one hand, this application provides an isolated antigen-binding protein that may contain at least one CDR in the variable region VH of the antibody heavy chain, wherein the VH may contain the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16.

[0083] In this application, the HCDR of the isolated antigen-binding protein can be divided in any form, as long as VH is the same as the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16. The HCDR obtained by dividing in any form can fall within the protection scope of this application.

[0084] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the term CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants of the CDR, including amino acid sequences with substitutions, deletions, and / or additions of one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; homologs are also included, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology with the amino acid sequence of the CDR. In some embodiments, the isolated antigen-binding protein described in this application is defined by the Kabat coding system.

[0085] In this application, the isolated antigen-binding protein may contain a heavy chain variable region VH, which may contain at least one, two, or three of HCDR1, HCDR2, and HCDR3.

[0086] In this application, the HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:5. In this application, the HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:6. For example, the sequence of the HCDR3 of the antigen-binding protein can be defined using the Kabat coding system.

[0087] In this application, the HCDR2 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:3. In this application, the HCDR2 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:4. For example, the sequence of the HCDR2 of the antigen-binding protein can be defined using the Kabat coding system.

[0088] In this application, the HCDR1 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1. In this application, the HCDR1 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:2. For example, the sequence of the HCDR1 of the antigen-binding protein can be defined by the Kabat coding system.

[0089] For example, the HCDR1 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; the HCDR2 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4; and the HCDR3 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6.

[0090] For example, the HCDR1 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1; the HCDR2 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:3; and the HCDR3 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:5. For example, the antigen-binding protein may include a single-domain antibody LB21 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., having the same HCDR1-3).

[0091] For example, the HCDR1 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:2; the HCDR2 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:4; and the HCDR3 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:6. For example, the antigen-binding protein may include a single-domain antibody LB22 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., having the same HCDR1-3).

[0092] For example, the VH of the antigen-binding protein may include frame regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0093] In this application, the H-FR1 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:7.

[0094] In this application, the H-FR1 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:8.

[0095] In this application, the H-FR2 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:9.

[0096] In this application, the H-FR2 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:10.

[0097] In this application, the H-FR3 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:11.

[0098] In this application, the H-FR3 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:12.

[0099] In this application, the H-FR4 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:13.

[0100] In this application, the H-FR4 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:14.

[0101] In this application, the H-FR1 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8; the H-FR2 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:10; the H-FR3 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:12; and the H-FR4 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:14.

[0102] In this application, the H-FR1 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:7; the H-FR2 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:9; the H-FR3 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:11; and the H-FR4 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:13. For example, the antigen-binding protein may include a single-domain antibody LB21 or an antigen-binding protein having the same H-FR1-4.

[0103] In this application, the H-FR1 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:8; the H-FR2 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:10; the H-FR3 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:12; and the H-FR4 of the antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:14. For example, the antigen-binding protein may include a single-domain antibody LB22 or an antigen-binding protein having the same H-FR1-4.

[0104] In this application, the antigen-binding protein may include a heavy chain variable region, which may include HCDR1-3 and H-FR1-4. For example, HCDR1 may include the amino acid sequence shown in SEQ ID NO:1; HCDR2 may include the amino acid sequence shown in SEQ ID NO:3; HCDR3 may include the amino acid sequence shown in SEQ ID NO:5; H-FR1 may include the amino acid sequence shown in SEQ ID NO:7; H-FR2 may include the amino acid sequence shown in SEQ ID NO:9; H-FR3 may include the amino acid sequence shown in SEQ ID NO:11; and H-FR4 may include the amino acid sequence shown in SEQ ID NO:14. For example, the antigen-binding protein may include a single-domain antibody LB21 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR (e.g., having the same HCDR1-3). For example, the heavy chain variable region of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:15. For example, the nucleotide sequence encoding the antigen-binding protein may be as shown in SEQ ID NO:17. In some cases, the antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) that has the same VH as the LB21.

[0105] In this application, the antigen-binding protein may include a heavy chain variable region, which may include HCDR1-3 and H-FR1-4. For example, HCDR1 may include the amino acid sequence shown in SEQ ID NO:2; HCDR2 may include the amino acid sequence shown in SEQ ID NO:4; HCDR3 may include the amino acid sequence shown in SEQ ID NO:6; H-FR1 may include the amino acid sequence shown in SEQ ID NO:8; H-FR2 may include the amino acid sequence shown in SEQ ID NO:10; H-FR3 may include the amino acid sequence shown in SEQ ID NO:12; and H-FR4 may include the amino acid sequence shown in SEQ ID NO:14. For example, the antigen-binding protein may include a single-domain antibody LB21 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR (e.g., having the same HCDR1-3). For example, the heavy chain variable region of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:16. For example, the nucleotide sequence encoding the antigen-binding protein may be as shown in SEQ ID NO:18. In some cases, the antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) that has the same VH as the LB22.

[0106] In this application, the isolated antigen-binding protein can compete with a reference antibody for binding to human serum albumin. The reference antibody may contain a heavy chain variable region VH, and the VH may contain at least one, two, or three of HCDR1, HCDR2, and HCDR3.

[0107] In this application, the HCDR3 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:5. In this application, the HCDR3 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:6. For example, the sequence of the HCDR3 of the reference antibody can be defined using the Kabat coding system.

[0108] In this application, the HCDR2 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:3. In this application, the HCDR2 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:4. For example, the sequence of the HCDR2 of the reference antibody can be defined using the Kabat coding system.

[0109] In this application, the HCDR1 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:1. In this application, the HCDR1 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:2. For example, the sequence of the HCDR1 of the reference antibody can be defined using the Kabat coding system.

[0110] For example, the HCDR1 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:1; the HCDR2 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:3; and the HCDR3 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:5. For example, the reference antibody may include a single-domain antibody LB21 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., having the same HCDR1-3).

[0111] For example, the HCDR1 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:2; the HCDR2 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:4; and the HCDR3 of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:6. For example, the reference antibody may include a single-domain antibody LB22 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., having the same HCDR1-3).

[0112] In this application, the heavy chain variable region of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:15.

[0113] In this application, the heavy chain variable region of the reference antibody may contain the amino acid sequence shown in SEQ ID NO:16.

[0114] Peptides and Immunoconjugates

[0115] On the other hand, this application provides one or more polypeptides that may comprise the isolated antigen-binding protein of this application. For example, the polypeptide may comprise a fusion protein. For example, the polypeptide may comprise a multispecific antibody (e.g., a bispecific antibody).

[0116] On the other hand, this application provides one or more immunoconjugates that may contain the isolated antigen-binding protein of this application. In some embodiments, the immunoconjugate may also contain pharmaceutically acceptable therapeutic agents, markers, and / or detection agents.

[0117] Nucleic acid, vector, cell and drug composition

[0118] On the other hand, this application also provides one or more nucleic acid molecules that can encode the antigen-binding protein isolated in this application. For example, each of the one or more nucleic acid molecules can encode the complete antigen-binding protein or a portion thereof (e.g., one or more of HCDR1-3 and the heavy chain variable region).

[0119] For example, when nucleic acid molecules encode a portion of the antigen-binding protein, the products encoded by the nucleic acid molecules can come together to form a functional (e.g., HSA-binding) isolated antigen-binding protein of this application.

[0120] The nucleic acid molecules described in this application can be isolated. For example, they can be produced or synthesized by: (i) in vitro amplification, such as by polymerase chain reaction (PCR), (ii) clonal recombination, (iii) purification, such as separation by enzyme digestion and gel electrophoresis, or (iv) synthesis, such as by chemical synthesis. For example, the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA technology.

[0121] In this application, nucleic acids encoding the isolated antigen-binding protein can be prepared by a variety of methods known in the art, including but not limited to reverse transcription PCR and PCR to obtain the nucleic acid molecules of the isolated antigen-binding protein described in this application.

[0122] On the other hand, this application provides one or more vectors containing one or more nucleic acid molecules described in this application. Each vector may contain one or more of the nucleic acid molecules described herein. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. In some embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on the species or cell type and function, but typically includes 5' non-transcriptional sequences and 5' and 3' non-translational sequences, respectively, involved in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcriptional expression control sequence may contain a promoter region, which may contain a promoter sequence for transcriptionally controlling functionally linked nucleic acids. The expression control sequence may also include enhancer sequences or upstream activator sequences. In this application, suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, human U6RNA promoters, CMV promoters, and their artificial hybrid promoters (such as CMV), wherein a portion of the promoter may be fused to a portion of a promoter of another cellular protein (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, and may or may not contain additional introns. One or more nucleic acid molecules described in this application may be operatively linked to the expression control element.

[0123] The vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. For example, the vector may be a viral vector. Viral vectors can be administered directly to a patient (in vivo) or indirectly, for example, by treating cells with a virus in vitro and then administering the treated cells to a patient (ex vivo). Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Conventional virus-based systems may include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors for gene transfer. In some cases, gene transfer can be integrated into the host genome using retroviral, lentiviral, and adeno-associated virus methods, resulting in long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing high viral titers. Lentiviral vectors may contain long terminal repeat sequences (5'LTRs) and truncated 3'LTRs, RREs, rev response elements (cPPTs), central termination sequences (CTSs), and / or post-translational regulatory elements (WPREs). The vectors described in this application can be introduced into cells.

[0124] On the other hand, this application provides a cell. The cell may contain the isolated antigen-binding protein, the polypeptide, the immunoconjugate, one or more nucleic acid molecules, and / or one or more vectors as described in this application. For example, each or every cell may contain one or more nucleic acid molecules or vectors as described in this application. For example, each or every cell may contain multiple (e.g., two or more) or more (e.g., two or more) nucleic acid molecules or vectors as described in this application. For example, the vectors as described in this application can be introduced into the host cell, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS / O cells, HEK293T cells, 293F cells, or HEK293A cells, or other eukaryotic cells, such as cells from plants, fungi, or yeast cells. The vectors as described in this application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc. For example, the cell may include yeast cells. For example, the cell may include Escherichia coli cells. For example, the cell may include mammalian cells. For example, the cell may include immune cells.

[0125] The cells may include immune cells. In some cases, the cells may include immune cells. For example, the cells may include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and / or peripheral blood mononuclear cells.

[0126] On the other hand, this application provides a pharmaceutical composition. The pharmaceutical composition may comprise the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and / or pharmaceutically acceptable adjuvants and / or excipients described in this application. In this application, the pharmaceutically acceptable adjuvant may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or nonionic surfactants. Unless incompatible with the cell described in this application, any conventional media or reagent may be considered for use in the pharmaceutical composition of this application. In this application, the pharmaceutically acceptable excipient may include additives other than the active pharmaceutical ingredient in a pharmaceutical formulation, also referred to as excipients. For example, the excipient may include binders, fillers, disintegrants, and lubricants in tablets. For example, the excipient may include wine, vinegar, or medicinal juice in traditional Chinese medicine pills. For example, the excipient may include the base portion in semi-solid preparations such as ointments or creams. For example, the excipients may include preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid formulations.

[0127] Detection methods, kits, uses and methods

[0128] On the other hand, this application provides a method for detecting the presence and / or content of human serum albumin, which may include administering the isolated antigen-binding protein or the polypeptide.

[0129] In this application, the method may include in vitro methods, ex vivo methods, and methods for non-diagnostic or non-therapeutic purposes.

[0130] For example, the method may include a method for detecting the presence and / or content of human serum albumin for non-diagnostic purposes, which may include the following steps:

[0131] 1) Contact the sample with the antigen-binding protein of this application; and

[0132] 2) Detect the presence and / or amount of the antigen-binding protein bound to the sample to determine the presence and / or expression level of human serum albumin in the sample obtained from the subject.

[0133] For example, the sample may include plasma.

[0134] On the other hand, this application provides a kit that may contain the isolated antigen-binding protein or the polypeptide.

[0135] In this application, the kit may also include instructions for use describing a method for detecting the presence and / or content of human serum albumin. For example, the method may include in vitro methods, ex vivo methods, and methods for non-diagnostic or non-therapeutic purposes.

[0136] On the other hand, this application provides the use of the isolated antigen-binding protein or the polypeptide in the preparation of a kit, which can be used as a method for detecting the presence and / or content of human serum albumin. For example, the method may include in vitro methods, ex vivo methods, and methods for non-diagnostic or non-therapeutic purposes.

[0137] On the other hand, this application provides the use of the isolated antigen-binding protein and / or the polypeptide in the preparation of a medicament that can be used to prevent and / or treat diseases or conditions.

[0138] On the other hand, this application provides the isolated antigen-binding protein, the polypeptide, the immune conjugate, the isolated nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition thereof for the prevention, relief, and / or treatment of diseases or conditions.

[0139] On the other hand, this application provides a method for preventing and / or treating a disease or condition, comprising administering to a subject in need an effective amount of the isolated antigen-binding protein, the polypeptide, the immune conjugate, the isolated nucleic acid molecule, the carrier, and / or the cell.

[0140] This application also provides the following specific implementation schemes:

[0141] 1. An isolated antigen-binding protein having one or more of the following properties:

[0142] a) In Octet determination, K is approximately 9E-09M or less. D The value specifically binds to human serum albumin;

[0143] b) In Octet determination, with K at approximately 3.5E-08M or less. D The value specifically binds to serum albumin in cynomolgus monkeys; and

[0144] c) After binding to human serum albumin, it does not reduce or inhibit the binding of human serum albumin to neonatal receptors.

[0145] 2. The isolated antigen-binding protein according to embodiment 1, comprising HCDR3, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6.

[0146] 3. The isolated antigen-binding protein according to any one of embodiments 1-2, comprising HCDR2, wherein the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:3.

[0147] 4. The isolated antigen-binding protein according to any one of embodiments 1-3, comprising HCDR1, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0148] 5. The isolated antigen-binding protein according to any one of embodiments 1-4, comprising a heavy chain variable region VH, wherein the VH comprises the HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:6.

[0149] 6. The isolated antigen-binding protein according to embodiment 5, wherein HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from any of the following groups:

[0150] a) HCDR1: SEQ ID NO:1, HCDR2: SEQ ID NO:3, and HCDR3: SEQ ID NO:5; and

[0151] b) HCDR1: SEQ ID NO:2, HCDR2: SEQ ID NO:4, and HCDR3: SEQ ID NO:6.

[0152] 7. The isolated antigen-binding protein according to any one of embodiments 1-6, comprising H-FR1, wherein the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8.

[0153] 8. The isolated antigen-binding protein according to any one of embodiments 1-7, comprising H-FR2 located between HCDR1 and HCDR2, and wherein H-FR2 comprises the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:10.

[0154] 9. The isolated antigen-binding protein according to any one of embodiments 1-8, comprising H-FR3, said H-FR3 being located between said HCDR2 and said HCDR3, and said H-FR3 comprising the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:12.

[0155] 10. The isolated antigen-binding protein according to any one of embodiments 1-9, comprising H-FR4, wherein the N-terminus of H-FR4 is directly or indirectly linked to the C-terminus of HCDR3, and wherein H-FR4 comprises the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:14.

[0156] 11. The isolated antigen-binding protein according to any one of embodiments 1-10, comprising H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8; H-FR2 comprises the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:10; H-FR3 comprises the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:12; and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:14.

[0157] 12. The isolated antigen-binding protein according to embodiment 11, wherein H-FR1, H-FR2, H-FR3, and H-FR4 comprise an amino acid sequence selected from any of the following groups:

[0158] a) H-FR1: SEQ ID NO:7, H-FR2: SEQ ID NO:9, H-FR3: SEQ ID NO:11 and H-FR4: SEQ ID NO:13; and

[0159] b) H-FR1: SEQ ID NO:8, H-FR2: SEQ ID NO:10, H-FR3: SEQ ID NO:12 and H-FR4: SEQ ID NO:14.

[0160] 13. The isolated antigen-binding protein according to any one of embodiments 1-12, comprising a heavy chain variable region VH, said VH comprising the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16.

[0161] 14. An isolated antigen-binding protein according to any one of embodiments 1-13, comprising an antibody or an antigen-binding fragment thereof.

[0162] 15. The isolated antigen-binding protein according to embodiment 14, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0163] 16. An isolated antigen-binding protein according to any one of embodiments 1-15, comprising VHH or an antigen-binding fragment thereof.

[0164] 17. The isolated antigen-binding protein according to any one of embodiments 14-16, wherein the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, and fully human antibodies.

[0165] 18. The isolated antigen-binding protein according to any one of embodiments 1-17, comprising the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16.

[0166] 19. A polypeptide comprising the isolated antigen-binding protein of any one of embodiments 1-18.

[0167] 20. An immunoconjugate comprising the isolated antigen-binding protein of any one of embodiments 1-19 or the polypeptide of embodiment 19.

[0168] 21. The immunoconjugate according to embodiment 20 further includes a pharmaceutically acceptable therapeutic agent.

[0169] 22. The immune conjugate according to embodiment 21, wherein the therapeutic agent is selected from the group consisting of cytotoxic agents and cell inhibitors.

[0170] 23. An isolated nucleic acid molecule encoding an isolated antigen-binding protein as described in any one of embodiments 1-18, or a polypeptide as described in embodiment 19.

[0171] 24. A vector comprising the isolated nucleic acid molecule as described in embodiment 23.

[0172] 25. A cell comprising the isolated antigen-binding protein of any one of embodiments 1-18, the polypeptide of embodiment 19, the immunoconjugate of any one of embodiments 20-22, the isolated nucleic acid molecule of embodiment 23, and / or the carrier of embodiment 24.

[0173] 26. A method for preparing the isolated antigen-binding protein of any one of embodiments 1-18 and / or the polypeptide of embodiment 19, the method comprising culturing the cells of embodiment 25 under conditions that cause the isolated antigen-binding protein and / or the polypeptide to be expressed.

[0174] 27. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of embodiments 1-18, the polypeptide of embodiment 19, the immunoconjugate of any one of embodiments 20-22, the isolated nucleic acid molecule of embodiment 23, the carrier of embodiment 24, the cell of embodiment 25, and / or a pharmaceutically acceptable adjuvant and / or excipient.

[0175] 28. A method for detecting human serum albumin, comprising: administering an isolated antigen-binding protein as described in any one of embodiments 1-18 or a polypeptide as described in embodiment 19.

[0176] 29. A human serum albumin detection kit comprising the isolated antigen-binding protein of any one of embodiments 1-18 or the polypeptide of embodiment 19.

[0177] 30. Use of the isolated antigen-binding protein of any one of embodiments 1-18 or the polypeptide of embodiment 19 in the preparation of the kit.

[0178] 31. Use of the isolated antigen-binding protein of any one of embodiments 1-18 and / or the polypeptide of embodiment 19 in the preparation of a medicament for the prevention and / or treatment of a disease or condition.

[0179] 32. The isolated antigen-binding protein of any one of embodiments 1-18, the polypeptide of embodiment 19, the immune conjugate of any one of embodiments 20-22, the isolated nucleic acid molecule of embodiment 23, the carrier of embodiment 24, and / or the cell of embodiment 25, for the prevention and / or treatment of diseases or conditions.

[0180] 33. A method for preventing and / or treating a disease or condition, comprising administering to a subject in need an effective amount of the isolated antigen-binding protein of any one of embodiments 1-18, the polypeptide of embodiment 19, the immunoconjugate of any one of embodiments 20-22, the isolated nucleic acid molecule of embodiment 23, the carrier of embodiment 24, and / or the cell of embodiment 25.

[0181] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the antigen-binding protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0182] Example

[0183] Example 1: Preparation of anti-human serum albumin (HSA) antibody

[0184] 1.1 Alpaca Immunity

[0185] Natural HSA (abcam product number: ab205808), 500ug subcutaneously per immunization, with immunization intervals of 7 days, for a total of 4 immunizations. Peripheral blood is collected from alpacas after the third and fourth immunizations, centrifuged, and the supernatant is diluted 10-fold. -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10 Dilute the supernatant and use ELISA to measure the antibody titer (titer) against HSA in alpaca serum.

[0186] 1.2 ELISA method for determining the serum albumin (HSA) titers of alpaca and human serum.

[0187] HSA was coated onto an ELISA plate at a concentration of 1 μg / ml and incubated overnight at 4°C. The next day, the HSA-coated ELISA plate was removed and blocked with PBS containing 2.5% skim milk (hereinafter referred to as PBSM) for 1 hour. Alpaca serum was collected, added to a 15 ml centrifuge tube, centrifuged at 4000g for 10 minutes, and 10 μl of the supernatant was used to detect the antibody titer of anti-HSA in the alpaca serum. The alpaca serum was diluted to 10 μL with PBSM. -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10Add 100 μL of serum diluent to each well of an HSA-coated ELISA plate. Perform two replicates for each dilution gradient. Incubate at 37°C for 1 hour. After the serum diluents have bound, wash each well three times with PBS containing 0.1% Tween 20 (PBST).

[0188] After washing, add HRP-labeled anti-alpaca antibody (Goat PAb to Llama IgG (HRP), abcam product number: ab112786) diluted 1:10000, 100 μL per well, and incubate at room temperature for 30 minutes; wash three times again with PBST, add TMB substrate for color development for 5-10 minutes, add stop solution to stop the reaction, and quickly read the plate at OD 450 nm.

[0189] Example 2: Library construction and screening of the VHH variable region gene of anti-human serum albumin (HSA) monoclonal antibody

[0190] 2.1 Isolation of alpaca PBMCs and preparation of bacterial clone library

[0191] Peripheral blood was collected from alpacas after four HSA immunizations. Peripheral mononuclear lymphocytes (PBMCs) were isolated using lymphocyte separation tubes (Dakow, catalog number: 7922021). Total RNA was extracted from the PBMCs using the RNeasy plus Mini kit (Qiagen, catalog number: 74134). The total RNA was then reverse transcribed into cDNA using Superscript. TM IV First-strand Synthwsis System (Invirogen, catalog number: 18091050) uses cDNA as a template to amplify the VHH region in alpaca B cells via polymerase chain reaction (PCR). After purification, the region is ligated into a phage display vector (pComb3XTT). The phage display vector containing the VHH region is then transformed into TG1 cells via electroporation to construct a VHH bacterial clone library.

[0192] The VHH bacterial clone library was inoculated into 2xYT medium containing ampicillin and 2% glucose and cultured on a shaker at 37°C. When the bacterial concentration OD600 reached 0.4-0.8, M13K07 (product number N0315S, NEB) helper phage was added, and the culture was incubated at 37°C for 30 minutes. The bacterial cells were collected by centrifugation and resuspended in 2xYT medium containing kanamycin and 1 mM IPTG. The culture was then incubated overnight at 30°C and 250 rpm.

[0193] Overnight cultures were centrifuged at 9000g for 15 minutes at 4°C, and the supernatant was collected. After sterilization by filtering through a 0.22µm filter, 1 / 5 volume of PEG-NaCl was added to precipitate the phage particles. The mixture was incubated on ice for 1 hour. The phage suspension was then centrifuged at 9000g for 15 minutes at 4°C, and the supernatant was discarded as completely as possible. The phage particles were resuspended in PBS buffer and transferred to a new sterile tube. The resulting phage display library can be stored at 4°C for a short period or frozen at -80°C with the addition of 20% glycerol for long-term storage.

[0194] 2.2 Phage display screening of antigen-binding proteins in this application

[0195] The antigen-binding protein of this application, which is resistant to both HSA and cynomolgus monkey serum albumin (albumin-macaca, abbreviated alb-ma), was screened using biotin-labeled human serum albumin (HSA) and cynomolgus monkey serum albumin (albumin-macaca, abbreviated alb-ma). In the first round, 50 nM biotin-labeled cynomolgus monkey serum albumin and the phage display library were incubated together in PBS containing 2.5% milk at room temperature for 2 hours. Then, M-270 magnetic beads (catalog number: 65305, Invitrogen) were added to capture the biotin-labeled cynomolgus monkey serum albumin and the antigen-binding protein of this application displayed by the phages that could bind to cynomolgus monkey serum albumin, and incubated together at room temperature for 30 minutes. The M-270 magnetic beads were then adsorbed using a magnetic rack, washed 10 times with PBST, and then washed 10 times with PBS. Finally, 0.25 mg / ml Trypsin solution (Trypsin, catalog number 25200-072, Gibco) was added, and the mixture was incubated with shaking at room temperature for 30 minutes to elute the phages. Subsequently, 4 mg / ml AEBSF (AEBSF protease inhibitor, Sigma, 78431) solution was added at a ratio of 20:1 to eliminate enzyme activity. The eluted phages constituted the first-round phage display library. Fresh TG1 bacteria with OD = 0.4-0.5 were infected with the first-round phage library and incubated for 30 minutes. The bacteria were then inoculated into 50 ml of 2xYT medium containing ampicillin and 2% glucose, shaken at 37°C and 225 rpm for 1-2 hours, and centrifuged at 9000g for 15 minutes. The bacteria were resuspended in 2xYT medium containing kanamycin and 1 mM IPTG, and 10-100 μl of the suspension was used for dilution and counting. Helper phages were added, and the culture was incubated overnight at 30°C and 250 rpm.

[0196] In the second round, 20 nM biotin-labeled HSA was used in conjunction with the phages obtained in the first round of screening. The screening method was the same as in the first round to obtain the second round of phage display libraries. This process was then repeated to obtain the third round of phage display libraries.

[0197] After the third round of phage display library infection with TG1 was mixed, it was diluted and spread onto agar plates containing ampicillin. The next day, single colonies of bacteria were picked and inoculated into sterile 96-well deep-well plates containing 2xYT medium containing ampicillin. The plates were incubated at 37°C and 225 rpm until the bacteria reached the logarithmic growth phase. 100 μL of the inoculated medium was added to a new 96-well plate and M13K07 helper phage was added. The plates were incubated for 30 minutes and then shaken overnight at 30°C.

[0198] The following day, the culture supernatant containing the phage was used to verify whether it could specifically bind to HSA and cynomolgus monkey serum albumin.

[0199] 2.3 Phage ELISA screening for positive clones

[0200] HSA and cynomolgus monkey serum albumin 1ug / ml were coated in Stripwell. TM Microplate (catalog number: 42592, Costa) was used for overnight coating at 4°C. The following day, after washing, the plates were blocked with PBS containing 2.5% milk at room temperature for 1 hour. After washing, 100 μL of supernatant from a 96-well plate was added to each well, and the plates were incubated at room temperature for 1 hour. After washing again, anti-M13 HRP detection antibody (M13Bacteriophage antibody (HRP) catalog number: 11973MM05T-HH013JA1501, Sino Bio) was added, and the plates were incubated at room temperature for 30 minutes. The plates were washed 5 times, and then 1-step Ultra TMB-ELISA reagent (Thermo Scientific, catalog number: #34029) was added. The color change in each well was observed. When the color was appropriate, the reaction was terminated with 2M sulfuric acid, and the value was read at OD450 nm. Based on the ELISA results, positive clones expressing positive bacteriophages were selected, and the gene sequence encoding the antigen-binding protein of this application was obtained by sequencing.

[0201] Example 3: Antibody Expression, Purification, and Binding Experiments

[0202] 3.1 Antibody Expression and Purification

[0203] Through sequencing, we obtained two sequences encoding the antigen-binding protein of this application, named LB21 and LB22, respectively. Their amino acid sequences are shown in Table 1. The sequence information of the CDR, FR and heavy chain variable region of the antigen-binding protein of this application is shown in Table 2.

[0204] Table 1. Sequences of antigen-binding proteins

[0205]

[0206] Table 2 Sequence information of the CDR, FR and heavy chain variable region of the antigen-binding protein in this application.

[0207]

[0208] The gene sequence encoding the antigen-binding protein of this application was cloned and inserted into a mammalian cell expression vector. A signal peptide (MGWSCIILFLVATATGVHS, SEQ ID NO:19) was added to the N-terminus of the antigen-binding protein, and 6x His-tags and HA-tags were added to the C-terminus. The cells were transiently transfected into 293FT cells using Lipofectamine 2000 (Thermofisher, catalog number: 11668030) transfection reagent and cultured for 96 hours. Simultaneously, an MSA21 VHH single-domain antibody (see patent WO 2004 / 062551 A2) was expressed as a control. A nickel column (His Trap) was used. TM Excel (Catalog No.: 17-3712-05, GE Healthcare) Purify VHH antibody and change the buffer with PBS buffer.

[0209] 3.2 Antibody binding to HSA and cynomolgus monkey serum albumin

[0210] 3.2.1 ELISA assay to verify the binding of the antigen-binding protein of this application to HSA

[0211] 4°C overnight encapsulation with 1ug / ml HSA in Stripwell TM Microplates (catalog number: 42592, Costa) were used. The plates were washed the following day and blocked with PBS containing 2.5% milk at room temperature for 1 hour. LB21 and LB22 were serially diluted, starting at 1 μg / ml and decreasing 3-fold downwards, resulting in 7 concentration spots and 1 negative control spot. The diluted LB21 and LB22 were used as primary antibodies, added at 100 μl / well to the blocked ELISA plate and incubated at room temperature for 1 hour. After washing, secondary antibody (anti-HisHRP) was added, and the color change in each well was observed. When the color was appropriate, the reaction was stopped with 2M sulfuric acid, and the values ​​were read at OD450 nm. Results are as follows: Figure 1 As shown, the antigen-binding proteins in this application can all bind to HSA.

[0212] 3.2.2 Octet assay to detect the affinity between HSA and cynomolgus monkey serum albumin and the antigen-binding protein of this application.

[0213] Biotin-labeled HSA or cynomolgus serum albumin was loaded onto SA probes (SA Biosensors, catalog number 18-5019, Forte bio). Affinity values ​​were determined by the kinetic relationship between HSA and LB21 and LB22. The SA probes were first equilibrated to baseline for 60 seconds, then loaded (captured) biotin-labeled HSA or cynomolgus serum albumin onto the SA probes for 90 seconds, equilibrated to baseline for 60 seconds, and then bound to LB21 and LB22 for 150 seconds respectively. The probes were then dissociated in the reaction buffer (NB buffer) for 300 seconds, and finally regenerated. The affinity of the antigen-binding protein of this application for HSA is shown in Table 3, and the affinity for cynomolgus serum albumin is shown in Table 4.

[0214] Table 3. Affinity of the antigen-binding protein of this application to HSA.

[0215] Antibody name KD(M) kon(1 / Ms) kd(1 / s) LB21 1.24E-09 5.54E+05 6.87E-04 LB22 1.09E-10 1.29E+06 1.40E-04

[0216] Table 4. Affinity of the antigen-binding protein of this application to cynomolgus monkey serum albumin.

[0217] Antibody name KD(M) kon(1 / Ms) kd(1 / s) LB21 2.17E-10 1.24E+05 2.69E-05 LB22 7.08E-10 9.13E+04 6.47E-05

[0218] Example 4: Non-competitive validation experiment of antibody and FcRn

[0219] To verify whether FcRn and the antigen-binding protein of this invention competitively bind to human serum albumin (HSA), the Octet system was used for detection. First, biotin-labeled human FcRn (FcRn-biotin, Biosensors, Cat:FCM-H82W4) protein was loaded onto an SA probe (SA Biosensors, catalog number 18-5019, Forte bio). Then, HSA was loaded under acidic conditions. Finally, the antigen-binding protein of this invention was tested to see if it could re-bind HSA. Specifically, 100 nM FcRn-biotin was loaded onto the SA probe for 120 seconds, equilibrated for 60 seconds, 1000 nM HSA was loaded for 150 seconds, and then 50 nM of the binding protein of this invention or a control was loaded for 200 seconds. All reactions were performed in citrate buffer at pH 5.4.

[0220] The results are as follows Figure 2-4 As shown, the results indicate that HSA bound to human FcRn can still bind to the antigen-binding protein of the present invention, proving that the antigen-binding protein of the present invention and human FcRn can bind to HSA simultaneously, and there is no competitive relationship between the two. sequence list <110> Tianchen Biopharmaceutical (Suzhou) Co., Ltd. <120> Anti-human serum albumin antibodies and their applications <130> 0125-PA-025 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> LB21 HCDR1 <400> 1 Val Tyr Asn Leu Ala 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> LB22 HCDR1 <400> 2 Val Tyr Ala Met Gly 1 5 <210> 3 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> LB21 HCDR2 <400> 3 Ser Ile Thr Ser Gly Leu Ser Thr Asp Tyr Ala Thr Thr Val Lys Gly 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> LB22 HCDR2 <400> 4 Leu Ile Thr Ser Thr Ser Asp Thr Arg Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> LB21 HCDR3 <400> 5 Asn Ser Arg Thr Thr Trp Tyr Tyr 1 5 <210> 6 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> LB22 HCDR3 <400> 6 Gly Asn Ser Trp Gly Ala Thr Val Gln Ala Met Cys Lys Ser Asp Tyr 1 5 10 15 Asp Tyr <210> 7 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> LB21 H‑FR1 <400> 7 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ile Ile Phe Asp 20 25 30 <210> 8 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> LB22 H-FR1 <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> LB21 H-FR2 <400> 9 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> LB22 H-FR2 <400> 10 Trp Tyr Arg Gln Ala Pro Gly Lys Asp Cys Glu Leu Val Gly 1 5 10 <210> 11 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> LB21 H‑FR3 <400> 11 Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ala 20 25 30 <210> 12 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> LB22 H‑FR3 <400> 12 Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Lys Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met Ala 20 25 30 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LB21 H‑FR4 <400> 13 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LB22 H‑FR4 <400> 14 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 15 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> LB21 Amino Acid Sequence <400> 15 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ile Ile Phe Asp Val Tyr 20 25 30 Asn Leu Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Ser Ile Thr Ser Gly Leu Ser Thr Asp Tyr Ala Thr Thr Val Lys 50 55 60 Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Ala Asn Ser Arg Thr Thr Trp Tyr Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 16 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> LB22 amino acid sequence <400> 16 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Val Tyr 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Asp Cys Glu Leu Val 35 40 45 Gly Leu Ile Thr Ser Thr Ser Asp Thr Arg Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Lys Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met 85 90 95 Ala Gly Asn Ser Trp Gly Ala Thr Val Gln Ala Met Cys Lys Ser Asp 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 17 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> LB21 nucleotide sequence <400> 17 caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60 tcctgtgcag cctctagaat aatcttcgat gtctataatt tggcctggtt ccgccaggct 120 ccagggaaag agcgcgagtt ggtcgcaagt attactagtg gtctgagcac agactatgcg 180 acaaccgtga agggccgatt catcatctcc agagacaacg ccaagaacac ggtgtatctg 240 caaatgaaca acctgaaacc tgaggatacg gccgtctatt actgcactgc taatagcaga 300 actacctggt actactgggg ccaggggacc ctggtcaccg tctcctca 348 <210> 18 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> LB22 nucleotide sequence <400> 18 caggtgcagc tgcaggagtc tgggggaggc gtggtgcagc ctggggggtc tctgagactc 60 tcctgtacag cctctggatt caccttcagt gtctatgcca tgggctggta ccgccaggct 120 ccagggaagg actgcgagtt ggtcggactt attactagta ctagtgacac acgctatgct 180 ccagggaagg actgcgagtt ggtcggactt attactagta ctagtgacac acgctatgct 180 gactccgtga agggccgatt ctccatctcc agagacaatg ccaagaaaac ggtgtatctg 240 gactccgtga agggccgatt ctccatctcc agagacaatg ccaagaaaac ggtgtatctg 240 caaatgaaca ggctgaaacc tgaggacacg gccgtgtatt actgtatggc aggcaattcc 300 caaatgaaca ggctgaaacc tgaggacacg gccgtgtatt actgtatggc aggcaattcc 300 tggggggcta ctgttcaggc tatgtgtaag agtgactatg actactgggg ccaggggacc 360 tggggggcta ctgttcaggc tatgtgtaag agtgactatg actactgggg ccaggggacc 360 caggtcactg tctcctca 378 caggtcactg tctcctca 378 <210> 19<210> 19 <211> 19<211> 19 <212> PRT<212> PRT <213> 人工序列(Artificial Sequence)<213> Artificial Sequence <220> <220> <223> 信号肽 <223> Signal peptide <400> 19 <400> 19 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 1 5 10 15 Val His Ser Val His Ser

Claims

1. A single domain antibody comprising a HCDR1, a HCDR2 and a HCDR3, the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are the amino acid sequences selected from any one of the following groups: a) HCDR1: SEQ ID NO: 1, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 5; and b) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 4, and HCDR3: SEQ ID NO: 6, which is capable of specifically binding to human serum albumin and / or cynomolgus serum albumin.

2. The single domain antibody of claim 1, comprising a H-FR1, the C-terminus of the H-FR1 is directly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO:

8.

3. The single domain antibody of claim 1, comprising a H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO:

10.

4. The single domain antibody of claim 1, comprising a H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO:

12.

5. The single domain antibody of claim 1, comprising a H-FR4, the N-terminus of the H-FR4 is directly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO:

14.

6. The single domain antibody of claim 1, comprising a H-FR1, a H-FR2, a H-FR3 and a H-FR4, the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8; the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10; the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12; and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO:

14.

7. The single domain antibody of claim 6, wherein the H-FR1, the H-FR2, the H-FR3 and the H-FR4 comprise the amino acid sequences selected from any one of the following groups: a) H-FR1: SEQ ID NO: 7, H-FR2: SEQ ID NO: 9, H-FR3: SEQ ID NO: 11 and H-FR4: SEQ ID NO: 13; and b) H-FR1: SEQ ID NO: 8, H-FR2: SEQ ID NO: 10, H-FR3: SEQ ID NO: 12 and H-FR4: SEQ ID NO:

14. ​ b) H-FR1 : SEQ ID NO: 8, H-FR2: SEQ ID NO: 10, H-FR3: SEQ ID NO: 12 and H-FR4: SEQ ID NO:

14.

8. The single-domain antibody of claim 1, comprising a heavy chain variable region VHH comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO:

16.

9. An isolated nucleic acid molecule encoding the single-domain antibody of any one of claims 1-8.

10. A vector comprising the isolated nucleic acid molecule of claim 9.

11. A cell comprising the single-domain antibody of any one of claims 1-8, the isolated nucleic acid molecule of claim 9, and / or the vector of claim 10, the cell not being a plant cell.

12. A method of making the single-domain antibody of any one of claims 1-9, the method comprising culturing the cell of claim 11 under conditions such that the single-domain antibody is expressed.

13. A pharmaceutical composition comprising the single-domain antibody of any one of claims 1-8, the isolated nucleic acid molecule of claim 9, the vector of claim 10, the cell of claim 11, and / or a pharmaceutically acceptable adjuvant and / or excipient.

14. A test kit for human serum albumin comprising the single-domain antibody of any one of claims 1-8.

15. Use of the single-domain antibody of any one of claims 1-8 in the manufacture of a kit for detecting the presence and / or amount of human serum albumin and / or cynomolgus serum albumin in a sample.

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