CN115043940A

CN115043940A - Anti-human serum albumin antibody and application thereof

Info

Publication number: CN115043940A
Application number: CN202210227069.2A
Authority: CN
Inventors: 张大挺; 马海立; 刘恒
Original assignee: Tianchen Biomedical Suzhou Co ltd
Current assignee: Tianchen Biomedical Suzhou Co ltd
Priority date: 2021-03-09
Filing date: 2022-03-08
Publication date: 2022-09-13

Abstract

The present application relates to anti-human serum albumin antibodies raised against human serum albumin with a K of about 9E-09M or less and uses thereof _D The values specifically bind to human serum albumin. After the anti-human serum albumin antibody is combined with the human serum albumin, the combination of the human serum albumin and a neonatal receptor can not be reduced or inhibited.

Description

Anti-human serum albumin antibody and application thereof

Technical Field

The application relates to the field of biomedicine, in particular to an anti-human serum albumin antibody and application thereof.

Background

Human Serum Albumin (HSA) is the major plasma protein, consisting of about 591 amino acids, with a molecular weight of about 67 Kd. In the preparation process of the antibody, the antibody is used as a carrier protein for coupling hapten and is also commonly used as a molecular weight standard protein for electrophoresis or chromatographic chromatography. Holt et al have extended the half-life of short-term drugs through the use of anti-serum albumin domain antibodies (Holt et al, Protein Engineering, Design and Selection 21(2008)283S 28288).

Currently, known anti-human serum albumin antibodies also have the defects of low selectivity, low affinity and the like. Therefore, it is necessary to develop a novel anti-human serum albumin antibody having high affinity and high specificity for human serum albumin.

Disclosure of Invention

The present application provides an isolated antigen binding protein having one or more of the following properties: 1) in the Octet assay, with a K of about 9E-09M or less _D Values specifically bind to human serum albumin; 2) in the Octet assay, with a K of about 3.5E-08M or less _D Specifically binding to cynomolgus serum albumin; and 3) does not reduce or inhibit the binding of human serum albumin to neonatal receptors after binding to human serum albumin.

In certain embodiments, the isolated antigen binding protein comprises HCDR3, wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID No. 5 or SEQ ID No. 6.

In certain embodiments, the isolated antigen binding protein comprises HCDR2 and HCDR2 comprises the amino acid sequence set forth in SEQ ID No. 3 or SEQ ID No. 4.

In certain embodiments, the isolated antigen binding protein comprises HCDR1, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID No. 1 or SEQ ID No. 2.

In certain embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH comprising the HCDR1, HCDR2, and HCDR3, the HCDR1 comprises the amino acid sequence set forth in SEQ ID No. 1 or SEQ ID No. 2, the HCDR2 comprises the amino acid sequence set forth in SEQ ID No. 3 or SEQ ID No. 4, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID No. 5 or SEQ ID No. 6.

In certain embodiments, the HCDR1, HCDR2 and HCDR3 in the isolated antigen binding protein comprise an amino acid sequence selected from any one of the group consisting of:

a) HCDR 1: 1, HCDR 2: 3, and HCDR 3: 5, SEQ ID NO; and

b) HCDR 1: 2, HCDR 2: 4, and HCDR 3: 6 in SEQ ID NO.

In certain embodiments, the isolated antigen binding protein comprises H-FR1, the C-terminus of H-FR1 is linked directly or indirectly to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8.

In certain embodiments, the isolated antigen binding protein comprises 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.

In certain embodiments, the isolated antigen binding protein comprises 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.

In certain embodiments, the isolated antigen binding protein comprises H-FR4, the N-terminus of H-FR4 is linked directly or indirectly to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO 13 or SEQ ID NO 14.

In certain embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8; the H-FR2 comprises an amino acid sequence shown as SEQ ID NO. 9 or SEQ ID NO. 10; the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 11 or SEQ ID NO. 12; and the H-FR4 contains an amino acid sequence shown in SEQ ID NO. 13 or SEQ ID NO. 14.

In certain embodiments, the isolated antigen binding protein comprises an amino acid sequence selected from any one of H-FR1, H-FR2, H-FR3, and H-FR 4:

a) H-FR 1: 7, H-FR 2: 9, H-FR 3: 11 and H-FR 4: 13 in SEQ ID NO; and

b) H-FR 1: 8, H-FR 2: 10, H-FR 3: 12 and H-FR 4: 14 in SEQ ID NO.

In certain embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO. 15 or SEQ ID NO. 16.

In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.

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

In certain embodiments, the isolated antigen binding protein comprises a VHH or an antigen binding fragment thereof.

In certain embodiments, the antibody is selected from the group consisting of: monoclonal antibodies, chimeric antibodies and fully human antibodies.

In certain embodiments, the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO. 15 or SEQ ID NO. 16.

In another aspect, the present application provides one or more polypeptides comprising the isolated antigen binding protein.

In another aspect, the present application provides one or more immunoconjugates comprising the isolated antigen binding protein or the polypeptide.

In certain embodiments, the immunoconjugate further comprises a pharmaceutically acceptable therapeutic agent.

In certain embodiments, the therapeutic agent is selected from the group consisting of: cytotoxic agents and cytostatic agents.

In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the isolated antigen binding protein, or the polypeptide.

In another aspect, the present application provides one or more vectors comprising the isolated nucleic acid molecules.

In another aspect, the present application provides one or more cells comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, and/or the vector.

In another aspect, the present application provides a method of making the isolated antigen binding protein and/or the polypeptide, the method comprising culturing the cell under conditions such that the isolated antigen binding protein and/or the polypeptide is expressed.

In another aspect, the present application provides one or more pharmaceutical compositions comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and/or a pharmaceutically acceptable adjuvant and/or excipient.

In another aspect, the present application provides a method for detecting human serum albumin, comprising: administering said isolated antigen binding protein or said polypeptide.

In another aspect, the present application provides a kit for the detection of human serum albumin comprising said isolated antigen binding protein or said polypeptide.

In another aspect, the present application provides a use of the isolated antigen binding protein or the polypeptide in the preparation of a kit.

In another aspect, the present application provides the use of the isolated antigen binding protein and/or the polypeptide in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder.

In another aspect, the present application provides the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for preventing, ameliorating and/or treating a disease or disorder.

In another aspect, the present application provides a method of preventing and/or treating a disease or disorder comprising administering to a subject in need thereof an effective amount of the isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and/or the cell.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The drawings are briefly described as follows:

FIG. 1 shows the binding activity of an antigen binding protein described herein to HSA.

Figure 2 shows that the antigen binding proteins described herein are capable of binding to HSA after binding to FcRn.

Figure 3 shows that the antigen binding proteins described herein are capable of binding to HSA after binding to FcRn.

FIG. 4 shows HAS after negative control failed to bind to FcRn.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "isolated" generally refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" does not exclude the presence of other impurities which do not affect the activity of the substance, mixed with artificial or synthetic substances.

In the present 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 the present application, the term "antigen binding protein" may include an "antibody" or an "antigen binding fragment". For example, the antibody may comprise an immunoglobulin of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and may include any molecule comprising an antigen-binding portion thereof. 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 antibody fragments that bind to an antigen (e.g., 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 derivatives thereof described herein. In the present application, the "antibody" may include a single domain antibody.

In the present application, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that function to specifically bind antigen. The antigen binding function of an antibody can be achieved by a full-length fragment of the antibody. The antigen binding function of an antibody can also be achieved by: a heavy chain comprising a fragment of Fv, scFv, dsFv, Fab 'or F (ab') 2, or a light chain comprising a fragment of Fv, scFv, dsFv, Fab 'or F (ab') 2. (1) Fab fragments, typically monovalent fragments consisting of VL, VH, CL and CH domains; (2) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (3) an Fd fragment consisting of the VH and CH domains; (4) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (5) dAb fragments consisting of VH domains (Ward et al, (1989) Nature 341: 544-546); (6) an isolated Complementarity Determining Region (CDR), and (7) a combination of two or more isolated CDRs which may optionally be joined by a linker. For example, there may also be included a monovalent single chain molecule formed by pairing VL and VH, fv (scFv) (see Bird et al (1988) Science 242: 423-. For example, one class of antibody VHHs can also be included that lacks the antibody light chain and has only the heavy chain variable region (see, e.g., Shenzhen et al, BioEngineers, 2018,34(12): 1974-. The "antigen binding portion" may also include a fusion protein comprising a binding domain selected from the group consisting of: (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 a CH2 constant region.

In the present application, the term "single domain antibody" generally refers to a class of antibodies that lacks the light chain of the antibody and has only the variable region of the heavy chain. It has been found that, among bactrian, unimodal, alpaca and llama, there is a heavy chain antibody (hcAb) which is composed of heavy chain only and has complete function, and its variable domains of the hcAb (VHH) has molecular weight of 1/10 which is only that of conventional antibody, and is the smallest molecular fragment with complete antibody function which can be obtained at present, and is called single domain antibody (sdAb). Compared with other antibodies, the single domain antibody has the advantages of low immunogenicity, small molecules, strong penetrating power and the like, so that the single domain antibody has wide application prospects in the fields of basic research, drug development, disease treatment and the like. For example, the single domain antibody may be from an alpaca. Single domain antibodies may be composed of heavy chain variable regions (VH). The term "heavy chain variable region" generally refers to the amino-terminal domain of the heavy chain of an antigen-binding fragment. The heavy chain variable regions can be further distinguished as hypervariable regions, called Complementarity Determining Regions (CDRs), interspersed with more conserved regions that become Framework Regions (FRs). Each heavy chain variable region may be composed of three CDRs and four FR regions, which may be arranged from amino-terminus to carboxy-terminus in the following order: H-FR1, HCDR1, H-FR2, HCDR2, H-FR3, HCDR3, and H-FR 4. 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 according to different systems. The system described by Kabat (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of antigen binding Proteins, but also provides precise residue boundaries defining CDRs which may 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 to have substantial diversity at the amino acid sequence level, although some of the sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, these sub-portions are designated L1, L2 and L3 or H1, H2 and H3, where these sub-portions may be referred to as "L" H "and" H "regions overlapping with other CDRs" H "133. the CDRs are referred to" Haia and "Haia" Hayata "1995. Hayata et al And MacCallum (J Mol Biol 262 (5): 732-45 (1996)). In addition, other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with the Kabat CDRs, although they may be shortened or lengthened in light of predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. In the present application, the CDRs may be defined using the Kabat numbering system. In the present application, the term "single domain antibody" is used interchangeably with "nanobody", "VHH".

In the present application, the term "monoclonal antibody" generally refers to a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. For example, the monoclonal antibodies can be prepared by hybridoma technology or produced in bacterial, eukaryotic animal or plant cells by using recombinant DNA methods. Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as those described in Clackson et al, Nature,352: 624-.

In the present application, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to a corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class, while the remaining segments of the chain are homologous to corresponding sequences in another species. For example, the variable regions of both the light and heavy chains are derived from the variable region of an antibody from one animal species (e.g., mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (e.g., human). For example, to obtain a chimeric antibody, B cells or hybridoma cells of non-human origin can be used to produce the variable region, in combination with a constant region from a human. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region with which it is combined. Also, since the constant region of a chimeric antibody may be of human origin, the possibility of the antibody eliciting an immune response upon injection of the chimeric antibody may be lower than if the constant region were of non-human origin.

In this application, the term "murine antibody" generally refers to an antibody in which the variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains constant regions, it is also derived from mouse germline immunoglobulin sequences. The murine antibodies of the present application may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, such as mutations introduced by random or point mutations in vitro or by somatic mutations in vivo. However, the term "murine antibody" does not include antibodies having CDR sequences from other mammalian species inserted into the mouse framework sequences.

In the present application, the terms "HSA protein" or "HSA antigen" are used interchangeably and include any functionally active fragment, variant and homologue of HSA, which is expressed naturally by a cell or on a cell transfected with the HSA gene. In the present application, HSA may have an accession number NP _000468.1 at NCBI. For example, the "functionally active fragment" may include a fragment that retains the endogenous function of at least one naturally occurring protein (e.g., binds to an antigen binding protein described herein). For example, the "functionally active fragment" may include a domain that binds to an antigen binding protein of the present application.

In addition to the specific proteins and nucleotides mentioned herein, the present application may also include functionally active fragments, derivatives, analogs, homologs and fragments thereof.

The term "functionally active fragment" refers to a polypeptide having substantially the same amino acid sequence as a naturally occurring sequence or encoded by substantially the same nucleotide sequence and capable of having one or more of the activities of a naturally occurring sequence. In the context of this application, a functionally active fragment of any given sequence refers to a sequence in which the particular 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. The sequence encoding a functionally active fragment may be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide, so long as the original functional activity is maintained.

In the present application, the term "derivative" generally refers to a polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and/or addition of one (or more) amino acid residues from/to the sequence, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

In the present application, the term "analog" generally with respect to a polypeptide or polynucleotide includes any mimetic of a polypeptide or polynucleotide, i.e., a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics.

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

In the present application, the term "homologue" generally refers to an amino acid sequence or a nucleotide sequence having a certain homology with a naturally occurring sequence. The term "homology" may be equivalent to sequence "identity". A homologous sequence can include an amino acid sequence that can be 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, homologues will comprise the same active site etc. as the subject amino acid sequence. Homology may be considered in terms of similarity (i.e., amino acid residues having similar chemical properties/functions), or may be expressed in terms of sequence identity. In the present application, a sequence having a percent identity of any one of SEQ ID NOs of the referenced amino acid sequence or nucleotide sequence refers to a sequence having said percent identity over the entire length of the referenced SEQ ID NOs. To determine sequence identity, sequence alignments can be performed, which can be performed by various means known to those skilled in the art, e.g., using BLAST, BLAST-2, ALIGN, needlet, or megalign (dnastar) software, etc. One skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment over the full-length sequences being compared.

The proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues which produce silent changes and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with similar hydrophilicity values without an electrically polar head group include asparagine, glutamine, serine, threonine, and tyrosine.

In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating (e.g., covalently linking via a linking molecule) the additional therapeutic agent to the isolated antigen binding protein, which conjugate can deliver the additional therapeutic agent to a target cell (e.g., a tumor cell) via specific binding of the isolated antigen binding protein to an antigen on the target cell. The immunoconjugate is then internalized by the internalization, eventually entering the interior of the target cell (e.g., into the lysosomal thylakoid capsule), at which point the linking molecule in the immunoconjugate can cleave, releasing the other agent to exert its cytotoxic effect. Furthermore, the antigen may be secreted by the target cell and located in a space outside the target cell.

In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

In the present application, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length, isolated from their natural environment or artificially synthesized.

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

In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or may already contain a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing a polypeptide described herein or an antigen binding protein described herein. The cell may comprise progeny of a single cell. Due to natural, accidental, or deliberate mutation, the progeny cells may not be identical in morphology or in genome to the original parent cell, but may be capable of expressing the polypeptide or antigen binding protein described herein. The cells can be obtained by in vitro transfection of cells using the vectors described herein. The cell may be a prokaryotic cell (e.g., E.coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese Hamster Ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NS0 cell, or a myeloma cell). In some embodiments, the cell may be an immune cell. For example, the immune cell 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.

In the present application, the term "treatment" generally means: (i) preventing the occurrence of a disease, disorder, or condition in a patient who may be predisposed to the disease, disorder, and/or condition, but has not yet been diagnosed as having the disease; (ii) inhibiting, i.e., arresting the development of, the disease, disorder or condition; and (iii) ameliorating the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and/or condition and/or symptoms associated with the disease, disorder, and/or condition.

In the present application, the terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and generally refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may comprise: disulfide bond formation, glycosylation, lipidation (acetylation), acetylation, phosphorylation, or any other manipulation (e.g., binding to a labeling component). The term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine as well as D and L optical isomers, as well as amino acid analogs and peptidomimetics.

In the present application, the terms "polynucleotide", "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. The polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci (one locus) defined according to ligation analysis, exons, introns, messenger RNA (mrna), transfer RNA, ribosomal RNA, short interfering RNA (sirna), short hairpin RNA (shrna), micro-RNA (mirna), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure, if present, may be performed before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeled component.

In this application, the term "K _D "(likewise," K) _D "or" KD ") is generally referred to as the" affinity constant "or" equilibrium dissociation constant ", and is referred to in a titration measurement at equilibrium, or by relating the dissociation rate constant (k) to the equilibrium _d ) Divided by the binding rate constant (k) _a ) The obtained value. Using the binding Rate constant (k) _a ) Dissociation rate constant (k) _d ) And equilibrium dissociation constant (K) _D ) Refers to the binding affinity of a binding protein (e.g., an isolated antigen binding protein described herein) for an antigen (e.g., HSA protein). Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine the sample at equilibrium in physiological buffer. For example, the K can be determined by Octet _D Values, other experimental pathways and instruments such as BIAcore (biomolecular interaction analysis) assays (e.g., instruments available from BIAcore international ab, algehalthica, Uppsala, sweden) may also be used. In addition, it is also possibleThe K was determined using KinExA (dynamic exclusion assay) available from SapidyneInstructions (Boise, Idaho) _D Value, or measuring the K by using Surface Plasmon Resonance (SPR) _D The value is obtained. In the present application, the K can be determined by an amine coupling kit _D The value is obtained.

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

In the present application, the term "comprising" is generally intended to include the explicitly specified features, but not to exclude other elements. In some cases, "comprising" also covers the case where only the specified components are included. For example, inclusive is also meant to also mean "consisting of … …".

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 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 the stated value.

In this application, the term "comprising" is used in a generic sense to mean including, summarizing, containing or encompassing. In some cases, the meaning of "is", "consisting of … …" is also indicated.

Detailed Description

In one aspect, the present application provides an isolated antigen binding protein that can have a K of about 9E-09M or less in an Octet assay _D Value (e.g., the K) _D A value of not greater than about 9E-09M, not greater than about 8.5E-09M, not greater than about 8E-09M, not greater than about 7.5E-09M, not greater than about 7E-09M, not greater than about 6.5E-09M, not greater than about 6E-09M, not greater than about 5E-09M, not greater than about 4E-09M, not greater than about 3E-09M, not greater than about 2E-09M, not greater than about 1E-09M, not greater than about 9E-10M, not greater than about 8E-10M, not greater than 7E-10M, not greater than about 6E-10M, not greater than about 5E-10M, not greater than about 4E-10M, not greater than about 3E-10M, not greater than about 2E-10M, or not greater than about 1E-10M or less) with human serum albumin.

In the present application, the isolated antigen binding protein may have a K of about 3.5E-08M or less _D Value (e.g., the K) _D Values no greater than about 3E-08M, no greater than about 2.5E-08M, no greater than about 2E-08M, no greater than about 1.5E-08M, no greater than about 1E-08M, no greater than about 9E-09M, no greater than about 8E-09M, no greater than about 7E-09M, no greater than about 6E-09M, no greater than about 5E-09M, no greater than about 4E-09M, no greater than about 3E-09M, no greater than about 2E-9M, or no greater than about 1E-9M or less) specifically bind to cynomolgus monkey albumin.

In the present application, the isolated antigen binding proteins do not generally reduce or inhibit the binding of human serum albumin to neonatal receptors after binding to human serum albumin.

For example, an antigen binding protein of the present application can be first bound to HSA, and after saturation, binding of HSA to FcRn can be detected by the Octet system (e.g., via K) _D Values determine whether HSA binds to FcRn).

In one aspect, the present application provides an isolated antigen binding protein, which may comprise at least one CDR in a VH of an antibody heavy chain variable region, which VH may comprise the amino acid sequence shown in SEQ ID No. 15 or SEQ ID No. 16.

In the present application, the HCDR of the isolated antigen binding protein may be divided in any form, and any form of divided HCDR may fall within the scope of the present application, as long as the VH is identical to the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO: 16.

The CDRs of an antibody, also known as complementarity determining regions, are part of the variable region. The amino acid residues of this region may be in contact with an antigen or an antigenic epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, consensus Kabat/Chothia, and the like. These coding systems are known in the art and can be found in particular, for example, http:// www.bioinf.org.uk/abs/index. One skilled in the art can determine the CDR regions using different coding systems depending on the sequence and structure of the antibody. The CDR regions may differ using different coding systems. In the present application, the CDR encompasses CDR sequences divided according to any CDR division manner; variants thereof are also contemplated, the variants comprising the amino acid sequence of the CDR substituted, deleted and/or added with one or more amino acids. E.g., 1-30, 1-20 or 1-10, further e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or insertions; also encompassed are homologs thereof, 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 more) sequence homology to the amino acid sequence of the CDR. In certain embodiments, the isolated antigen binding protein described herein is defined by the Kabat coding system.

In the present application, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise at least one, two or three of HCDR1, HCDR2 and HCDR 3.

In the present application, the HCDR3 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 5. In the present application, the HCDR3 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 6. For example, the sequence of HCDR3 of the antigen binding protein can be defined by the Kabat coding system.

In the present application, the HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 3. In the present application, the HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 4. For example, the sequence of HCDR2 of the antigen binding protein can be defined by the Kabat coding system.

In the present application, the HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 1. In the present application, the HCDR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 2. For example, the sequence of HCDR1 of the antigen binding protein can be defined by the Kabat coding system.

For example, the HCDR1 of the antigen binding protein can comprise the amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2; the HCDR2 of the antigen binding protein can comprise the amino acid sequence shown as SEQ ID NO. 3 or SEQ ID NO. 4; and the HCDR3 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.

For example, the HCDR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO. 1; the HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 3; and the HCDR3 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 5. For example, the antigen binding protein may include single domain antibody LB21 or an antigen binding protein (e.g., a single domain antibody) having the same HCDR3 as it (e.g., having the same HCDR1-3 as it).

For example, the HCDR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO. 2; the HCDR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 4; and the HCDR3 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 6. For example, the antigen binding protein may include single domain antibody LB22 or an antigen binding protein (e.g., a single domain antibody) having the same HCDR3 as it (e.g., having the same HCDR1-3 as it).

For example, the VH of the antigen binding protein may comprise the framework regions H-FR1, H-FR2, H-FR3 and H-FR 4.

In the present application, H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 7.

In the present application, H-FR1 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 8.

In the present application, H-FR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 9.

In the present application, H-FR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 10.

In the present application, H-FR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 11.

In the present application, H-FR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 12.

In the present application, H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 13.

In the present application, H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 14.

In the present application, H-FR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO. 7 or SEQ ID NO. 8; H-FR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO 9 or SEQ ID NO 10; H-FR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 11 or SEQ ID NO. 12; and H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 13 or SEQ ID NO. 14.

In the present application, H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 7; H-FR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 9; H-FR3 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 11; and H-FR4 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO. 13. For example, the antigen binding protein may include the single domain antibody LB21 or an antigen binding protein having the same H-FR 1-4.

In the present application, H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 8; H-FR2 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 10; H-FR3 of the antigen binding protein can comprise the amino acid sequence shown in SEQ ID NO. 12; and H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 14. For example, the antigen binding protein may include the single domain antibody LB22 or an antigen binding protein having the same H-FR 1-4.

In the present application, the antigen binding protein may comprise a heavy chain variable region which may comprise HCDR1-3 and H-FR 1-4. For example, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 1; the HCDR2 can comprise the amino acid sequence shown as SEQ ID NO. 3; the HCDR3 can comprise the amino acid sequence shown as SEQ ID NO. 5; the H-FR1 can comprise an amino acid sequence shown as SEQ ID NO. 7; the H-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 9; the H-FR3 can comprise an amino acid sequence shown as SEQ ID NO. 11; the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO. 14. For example, the antigen binding protein may include single domain antibody LB21 or an antigen binding protein (e.g., a single domain antibody) having the same HCDR as it (e.g., the same HCDR1-3 as it). For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO. 15. For example, the nucleotide sequence encoding the antigen binding protein can be shown in SEQ ID NO 17. In certain instances, the antigen binding protein may include an antigen binding protein (e.g., a single domain antibody) having the same VH as the LB 21.

In the present application, the antigen binding protein may comprise a heavy chain variable region which may comprise HCDR1-3 and H-FR 1-4. For example, the HCDR1 can comprise the amino acid sequence shown in SEQ ID NO. 2; the HCDR2 can comprise the amino acid sequence shown as SEQ ID NO. 4; the HCDR3 can comprise an amino acid sequence shown as SEQ ID NO. 6; the H-FR1 can comprise an amino acid sequence shown as SEQ ID NO. 8; the H-FR2 can comprise an amino acid sequence shown as SEQ ID NO. 10; the H-FR3 can comprise the amino acid sequence shown in SEQ ID NO. 12; the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO. 14. For example, the antigen binding protein may include single domain antibody LB21 or an antigen binding protein (e.g., a single domain antibody) having the same HCDR as it (e.g., the same HCDR1-3 as it). For example, the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO 16. For example, the nucleotide sequence encoding the antigen binding protein can be shown in SEQ ID NO 18. In certain instances, the antigen binding protein may include an antigen binding protein (e.g., a single domain antibody) having the same VH as the LB 22.

In the present application, the isolated antigen binding protein may compete for binding to human serum albumin with a reference antibody, which may comprise a heavy chain variable region VH, which may comprise at least one, two or three of HCDR1, HCDR2 and HCDR 3.

In the present application, the HCDR3 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 5. In the present application, the HCDR3 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 6. For example, the sequence of HCDR3 of the reference antibody can be defined by the Kabat coding system.

In the present application, the HCDR2 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 3. In the present application, the HCDR2 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 4. For example, the sequence of HCDR2 of the reference antibody can be defined by the Kabat coding system.

In the present application, the HCDR1 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 1. In the present application, the HCDR1 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 2. For example, the sequence of HCDR1 of the reference antibody can be defined by the Kabat coding system.

For example, the HCDR1 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 1; the HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO. 3; and the HCDR3 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 5. For example, the reference antibody can include single domain antibody LB21 or an antigen-binding protein (e.g., a single domain antibody) having the same HCDR3 as it (e.g., having the same HCDR1-3 as it).

For example, the HCDR1 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 2; the HCDR2 of the reference antibody can comprise the amino acid sequence set forth in SEQ ID NO. 4; and the HCDR3 of the reference antibody can comprise the amino acid sequence shown in SEQ ID NO. 6. For example, the reference antibody can include single domain antibody LB22 or an antigen binding protein (e.g., a single domain antibody) having the same HCDR3 as it (e.g., having the same HCDR1-3 as it).

In the present application, the heavy chain variable region of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO. 15.

In the present application, the heavy chain variable region of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO 16.

Polypeptides and immunoconjugates

In another aspect, the present application provides one or more polypeptides that can comprise an isolated antigen binding protein of the present application. For example, the polypeptide may comprise a fusion protein. For example, the polypeptide can include a multispecific antibody (e.g., bispecific antibody).

In another aspect, the present application provides one or more immunoconjugates that can comprise an isolated antigen binding protein of the present application. In certain embodiments, the immunoconjugate may further comprise a pharmaceutically acceptable therapeutic agent, label, and/or detection agent.

Nucleic acids, vectors, cells and pharmaceutical compositions

In another aspect, the present application also provides one or more nucleic acid molecules that can encode an isolated antigen binding protein described herein. For example, each of the one or more nucleic acid molecules may encode the entire antigen binding protein or a portion thereof (e.g., one or more of HCDR1-3, heavy chain variable region).

For example, where the nucleic acid molecules encode a portion of the antigen binding proteins, respectively, the products encoded by the nucleic acid molecules taken together can form a functional (e.g., can bind HSA) isolated antigen binding protein of the present application.

The nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) in vitro amplified, e.g., by Polymerase Chain Reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, e.g., by enzymatic cleavage and gel electrophoresis fractionation, or (iv) synthesized, e.g., by chemical synthesis. For example, the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA techniques.

In the present application, nucleic acids encoding the isolated antigen binding proteins described herein can be prepared by a variety of methods known in the art, including, but not limited to, the use of reverse transcription PCR and PCR to obtain the nucleic acid molecules of the isolated antigen binding proteins described herein.

In another aspect, the present application provides one or more vectors comprising one or more of the nucleic acid molecules described herein. One or more of the nucleic acid molecules may be included in each vector. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may contain expression control elements that allow for the proper expression of the coding region 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 that regulate gene transcription or mRNA translation, among others. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but typically comprises 5 ' non-transcribed sequences and 5 ' and 3 ' non-translated sequences, such as TATA box, capping sequences, CAAT sequences, etc., which are involved in initiation of transcription and translation, respectively. For example, the 5' non-transcribed expression control sequence can comprise a promoter region that can comprise a promoter sequence for transcription control functionally linked to the nucleic acid. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerase, the human U6RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (e.g., CMV), wherein a portion of the promoter may be fused to a portion of the promoter of other cellular proteins (e.g., human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) genes, which may or may not contain additional introns. One or more of the nucleic acid molecules described herein can be operably linked to the expression control element.

The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector 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. The viral vector may be administered directly to the patient (in vivo) or may be administered to the patient (ex vivo), for example, by treating the cells with the virus in vitro and then administering the treated cells to the patient. Viral vector technology is well known in the art and is 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, adenoviral vectors, adeno-associated viral vectors and herpes simplex viral vectors for gene transfer. In some cases, retroviral, lentiviral and adeno-associated viral approaches can be used to integrate gene transfer into the host genome, allowing long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers. The lentiviral vector may comprise a long terminal repeat 5 'LTR and a truncated 3' LTR, an RRE, a rev response element (cPPT), a Central Termination Sequence (CTS) and/or a post-translational regulatory element (WPRE). The vectors described herein can be introduced into cells.

In another aspect, the present application provides a cell. The cell may comprise an isolated antigen binding protein as described herein, a polypeptide as described herein, an immunoconjugate as described herein, one or more nucleic acid molecules, and/or one or more vectors as described herein. For example, each or every cell may comprise one or more nucleic acid molecules or vectors as described herein. For example, each or every cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors described herein. For example, a vector described herein can be introduced into the host cell, e.g., a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS/0 cell, an HEK293T cell, a 293F cell, or an HEK293A cell, or other eukaryotic cell, such as a plant-derived cell, a fungal or yeast cell, and the like. The vectors described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like. For example, the cells may comprise yeast cells. For example, the cells may comprise E.coli cells. For example, the cells may comprise mammalian cells. For example, the cells may comprise immune cells.

The cells may comprise immune cells. In some cases, the cells may include immune cells. For example, the cells can include T cells, B cells, Natural Killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells.

In another aspect, the present application provides a pharmaceutical composition. The pharmaceutical composition may comprise an isolated antigen binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and/or a pharmaceutically acceptable adjuvant and/or excipient described herein. In the present application, the pharmaceutically acceptable adjuvant may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counter ion, a metal complex, and/or a non-ionic surfactant. Except insofar as any conventional media or agent is incompatible with the cells described herein, its use in the pharmaceutical compositions of the present application is contemplated. In the present application, the pharmaceutically acceptable excipient may include an additive other than the main drug in the pharmaceutical preparation, and may also be referred to as an auxiliary material. For example, the excipients may include binders, fillers, disintegrants, lubricants in tablets. For example, the excipients may include wine, vinegar, medicinal juices, etc. in a traditional Chinese medicine pill. For example, the excipients may include the base portion of a semisolid formulation ointment, cream. For example, the excipients may include preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, tonicity adjusting agents, colorants in the liquid formulation.

Detection method, kit, use and method

In another aspect, the present application provides a method for detecting the presence and/or amount of human serum albumin, which may comprise administering said isolated antigen binding protein or said polypeptide.

In the present application, the methods may include in vitro methods, ex vivo methods, methods of non-diagnostic or non-therapeutic interest.

For example, the method may comprise a method for detecting the presence and/or amount of human serum albumin for non-diagnostic purposes, which may comprise the steps of:

1) contacting a sample with an antigen binding protein of the present application; and

2) detecting the presence and/or amount of said antigen binding protein bound by the sample to determine the presence and/or expression level of human serum albumin in the sample obtained from the subject.

For example, the sample may comprise plasma.

In another aspect, the present application provides a kit comprising the isolated antigen binding protein or the polypeptide.

In the present application, the kit may further comprise instructions describing a method for detecting the presence and/or amount of human serum albumin. For example, the methods may include in vitro methods, ex vivo methods, methods of non-diagnostic or non-therapeutic interest.

In another aspect, the present application provides the use of said isolated antigen binding protein or said polypeptide in the preparation of a kit for use in a method for detecting the presence and/or amount of human serum albumin. For example, the methods may include in vitro methods, ex vivo methods, methods of non-diagnostic or non-therapeutic interest.

In another aspect, the present application provides a use of the isolated antigen binding protein and/or the polypeptide in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder.

In another aspect, the present application provides an isolated antigen binding protein, a polypeptide, an immunoconjugate, an isolated nucleic acid molecule, a vector, a cell and/or a pharmaceutical composition for preventing, ameliorating and/or treating a disease or disorder.

The present application also provides the following specific embodiments:

1. an isolated antigen binding protein having one or more of the following properties:

a) in the Octet assay, with a K of about 9E-09M or less _D Values specifically bind to human serum albumin;

b) in the Octet assay, with a K of about 3.5E-08M or less _D Specifically binding to cynomolgus serum albumin; and

c) binding to human serum albumin does not reduce or inhibit the binding of human serum albumin to neonatal receptors.

2. The isolated antigen binding protein of embodiment 1, comprising HCDR3, wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID No. 5 or SEQ ID No. 6.

3. The isolated antigen binding protein of any of embodiments 1-2, comprising HCDR2, the HCDR2 comprising SEQ ID No. 3 or the amino acid sequence set forth in SEQ ID No. 3.

4. The isolated antigen binding protein of any of embodiments 1-3, comprising HCDR1, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO 1 or SEQ ID NO 2.

5. The isolated antigen binding protein of any of embodiments 1-4, comprising a heavy chain variable region VH, said VH comprising the HCDR1, HCDR2, and HCDR3, said HCDR1 comprising the amino acid sequence set forth in SEQ ID NO 1 or SEQ ID NO 2, said HCDR2 comprising the amino acid sequence set forth in SEQ ID NO 3 or SEQ ID NO 4, and said HCDR3 comprising the amino acid sequence set forth in SEQ ID NO 5 or SEQ ID NO 6.

6. The isolated antigen binding protein of embodiment 5, wherein the HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from any one of the group consisting of:

a) HCDR 1: 1, HCDR 2: 3, and HCDR 3: 5, SEQ ID NO; and

b) HCDR 1: 2, HCDR 2: 4, and HCDR 3: 6 in SEQ ID NO.

7. The isolated antigen binding protein of any one of embodiments 1-6, comprising H-FR1, the C-terminus of H-FR1 being linked directly or indirectly to the N-terminus of HCDR1, and the H-FR1 comprising the amino acid sequence set forth in SEQ ID NO 7 or SEQ ID NO 8.

8. The isolated antigen binding protein of any one of embodiments 1-7, comprising H-FR2, the H-FR2 being located between the HCDR1 and the HCDR2, and the H-FR2 comprising the amino acid sequence set forth in SEQ ID NO 9 or SEQ ID NO 10.

9. The isolated antigen binding protein of any one of embodiments 1-8, comprising H-FR3, the H-FR3 being located between the HCDR2 and the HCDR3, and the H-FR3 comprising the amino acid sequence set forth in SEQ ID NO 11 or SEQ ID NO 12.

10. The isolated antigen binding protein of any one of embodiments 1-9, comprising H-FR4, the N-terminus of H-FR4 being linked directly or indirectly to the C-terminus of HCDR3, and the H-FR4 comprising the amino acid sequence set forth in SEQ ID No. 13 or SEQ ID No. 14.

11. The isolated antigen binding protein of any one of embodiments 1-10, comprising H-FR1, H-FR2, H-FR3, and H-FR4, said H-FR1 comprising the amino acid sequence set forth in SEQ ID NO 7 or SEQ ID NO 8; the H-FR2 comprises an amino acid sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10; the H-FR3 comprises an amino acid sequence shown as SEQ ID NO. 11 or SEQ ID NO. 12; and the H-FR4 contains an amino acid sequence shown in SEQ ID NO. 13 or SEQ ID NO. 14.

12. The isolated antigen binding protein of embodiment 11, wherein said H-FR1, H-FR2, H-FR3, and H-FR4 comprise an amino acid sequence selected from any one of the group consisting of:

a) H-FR 1: 7, H-FR 2: 9, H-FR 3: 11 and H-FR 4: 13 is SEQ ID NO; and

b) H-FR 1: 8, H-FR 2: 10, H-FR 3: 12 and H-FR 4: 14 in SEQ ID NO.

13. The isolated antigen binding protein of any one of embodiments 1-12, comprising a heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID No. 15 or SEQ ID No. 16.

14. The isolated antigen binding protein of any one of embodiments 1-13, comprising an antibody or antigen binding fragment thereof.

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

16. The isolated antigen binding protein of any one of embodiments 1-15, comprising a VHH or an antigen binding fragment thereof.

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

18. The isolated antigen binding protein of any one of embodiments 1-17, comprising the amino acid sequence set forth in SEQ ID NO 15 or SEQ ID NO 16.

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

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

21. The immunoconjugate according to embodiment 20, further comprising a pharmaceutically acceptable therapeutic agent.

22. The immunoconjugate according to embodiment 21, wherein the therapeutic agent is selected from the group of: cytotoxic agents and cytostatic agents.

23. An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of embodiments 1-18, or the polypeptide of embodiment 19.

24. A vector comprising the isolated nucleic acid molecule of embodiment 23.

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 vector of embodiment 24.

26. A method of making the isolated antigen binding protein of any one of embodiments 1-18 and/or the polypeptide of embodiment 19, the method comprising culturing the cell of embodiment 25 under conditions such that the isolated antigen binding protein and/or the polypeptide is expressed.

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

28. A method for detecting human serum albumin, comprising: administering the isolated antigen binding protein of any one of embodiments 1-18 or the polypeptide of embodiment 19.

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

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

31. Use of an isolated antigen binding protein of any one of embodiments 1-18 and/or a polypeptide of embodiment 19 in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder.

32. 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 vector of embodiment 24, and/or the cell of embodiment 25 for use in the prevention and/or treatment of a disease or disorder.

33. A method of preventing and/or treating a disease or disorder comprising administering to a subject in need thereof 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 vector of embodiment 24, and/or the cell of embodiment 25.

Without wishing to be bound by any theory, the following examples are only intended to illustrate the antigen binding proteins, preparation methods and uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.

Examples

EXAMPLE 1 preparation of anti-Human Serum Albumin (HSA) antibody

1.1 alpaca immunization

Natural HSA (abcam cat # ab205808) was immunized 500ug subcutaneously every 7 days for a total of 4 immunizations, and peripheral blood of alpaca was collected at the end of the third and fourth immunizations, centrifuged, and the supernatant was diluted 10-fold and diluted 10-fold ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 、10 ^-8 、10 ^-9 、10 ^-10 The supernatant was diluted and the anti-HSA antibody Titer (Titer) in alpaca serum was measured by ELISA.

1.2 measurement of the titer of alpaca serum and Human Serum Albumin (HSA) by ELISA

HSA was coated at a concentration of 1ug/ml onto ELISA plates overnight at 4 ℃. 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; collecting blood to obtain alpaca serum, adding 15ml centrifuge tube, centrifuging at 4000g for 10 min, collecting supernatant 10ul for detecting antibody titer (titer) of alpaca serum against HSA, diluting alpaca serum with PBSM to 10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 、10 ^-8 、10 ^-9 、10 ^-10 Adding 100ul serum diluent into ELISA plate coated with HSA in each well, making 2 wells (multiple wells) in each dilution gradient, and combining at 37 deg.C for 1 hr; after the serum dilutions were bound, each well was washed 3 times with 0.1% Tween 20 in pbs (pbst);

after washing, an anti-alpaca antibody labeled with HRP (Goat PAb to Llama IgG (HRP), abcam cat # ab112786) 1: 10000 diluted for use, 100ul per well, and incubating for 30 minutes at room temperature; the plate was washed again 3 times with PBST, developed 5-10 minutes by adding TMB substrate, and read rapidly by OD450nm after the reaction was stopped by adding stop solution.

Example 2 Human Serum Albumin (HSA) monoclonal antibody VHH variable region Gene library construction and screening

2.1 alpaca PBMC isolation and bacterial clone Bank preparation

In vitro collecting alpaca peripheral blood after 4 times of immunization with HSA, separating alpaca peripheral mononuclear lymphocytes (PBMC) by using lymphocyte separation tube (Dake, cat # 7922021), lysing PBMC to extract total RNA (RNeasy plus Mini kit, cat # 74134, Qiagen), and reverse transcribing the total RNA into cDNA (Superscript) ^TM IV First-strand synthinsis system, Invirogen, cat #: 18091050), using cDNA as a template, amplifying a VHH region in alpaca B cells by Polymerase Chain Reaction (PCR), purifying and connecting to a phage display vector (pComb3XTT), transferring the phage display vector inserted with the VHH into TG1 cells by electrotransformation to construct a VHH bacterial clone bank.

The VHH bacterial clone bank is inoculated into 2XYT culture medium containing aminobenzene antibiotic and 2% glucose to be cultured on a constant temperature shaker at 37 ℃, when the bacterial concentration OD600 reaches 0.4-0.8, M13K07 (product number N0315S, NEB) auxiliary phage is added, standing incubation is carried out for 30 minutes at 37 ℃, the bacterial body is centrifugally collected, the bacterial body is suspended by 2XYT culture medium containing kanamycin antibiotic and 1mM IPTG, and the bacterial body is cultured overnight at 30 ℃ and 250 rpm.

The overnight culture was centrifuged at 9000g for 15 minutes at 4 ℃ and the supernatant collected, sterilized through a 0.22um filter and added 1/5 volumes of PEG-NaCl to precipitate the phage particles, incubated on ice for 1 hour, the phage suspension was centrifuged at 9000g for 15 minutes at 4 ℃ and the supernatant discarded as clean as possible, resuspended with PBS buffer and transferred to a fresh sterile tube. The obtained phage display library can be stored at 4 ℃ for a short time or added with glycerol with the final concentration of 20 percent, and is frozen and stored in a refrigerator at-80 ℃ for a long time.

2.2 phage display screening of antigen binding proteins of the present application

Biotin-labeled Human Serum Albumin (HSA) and cynomolgus serum albumin (albumin-macaca, abb.) were used to screen antigen binding proteins of the present application against both HSA and cynomolgus serum albumin. The first round was incubated with 50nM biotin-labeled cynomolgus monkey serum albumin and phage display library for 2 hours at room temperature in PBS containing 2.5% milk, followed by addition of M-270 magnetic beads (cat # 65305, Invitrogen) to capture the biotin-labeled cynomolgus monkey serum albumin and phage-displayed antigen-binding proteins of the present application that bind to cynomolgus monkey serum albumin, and incubation for 30 minutes at room temperature. M-270 magnetic beads were adsorbed by a magnetic frame, washed 10 times with PBST and 10 times with PBS, after which 0.25mg/ml Trypsin solution (Trypsin, cat # 25200-072, Gibco) was added and incubated at room temperature for 30 minutes with shaking to elute the phage. Subsequently, 4mg/ml AEBSF (AEBSF protease inhibitor, Sigma, 78431) solution was added at a ratio of 20:1 to eliminate the enzyme activity. The eluted phage was the first round of phage display library. Fresh TG1 bacteria with OD ═ 0.4 to 0.5 were infected with the first round of phage pool and left for 30 minutes. 50ml of 2XYT medium containing an aminobenzyl antibiotic and 2% glucose was inoculated, shaken at 225rpm for 1 to 2 hours at 37 ℃ and centrifuged at 9000g for 15 minutes. The cells were resuspended in 2XYT medium containing kanamycin antibiotic and 1mM IPTG, 10-100ul of the suspension was taken for dilution counting, helper phage was added, and incubated overnight at 30 ℃ and 250 rpm.

The second round used 20nM biotin-labeled HSA with the phage from the first round of screening. The screening method is the same as the first round of screening to obtain a second round of phage display library, and then a third round of phage display library is obtained by sequentially screening one round.

And after the third round of phage display library infection TG1 is mixed evenly, the mixture is diluted and coated on an agar plate containing the aminobenzyl antibiotic, the single clone of the selected bacteria is inoculated into a sterile 96-hole deep-hole plate containing the aminobenzyl antibiotic and a 2XYT culture medium on the next day, the bacteria is inoculated at the temperature of 37 ℃ and 225rpm, when the bacteria grow to the logarithmic phase, 100ul of the bacteria are added into a new 96-hole plate, M13K07 auxiliary phage is added, the incubation is carried out for 30 minutes, and the bacteria are shaken at the temperature of 30 ℃ overnight.

The next day, the obtained phage-containing culture supernatants were verified by phage elisa (phage elisa) to determine whether they specifically bind to HSA and cynomolgus monkey serum albumin.

2.3 phage ELISA (Phage ELISA) screening of Positive clones

Coating HSA and cynomolgus monkey serum albumin 1ug/ml in Stripwell ^TM Microplate (cat # 42592, Costar) was coated overnight at 4 ℃. The next day the plates were washed and blocked with 2.5% mik in PBS for 1 hour at room temperature. After washing, 100ul of the supernatant from the centrifugation in a 96-well plate was added to each well and incubated at room temperature for 1 hour. Plates were washed again with anti-M13 HRP detection antibody (M13 Bacteriophage antibody (HRP) cat # 11973MM05T-HH013JA1501, Sino Bio) and incubated for 30 min at room temperature. The plate was washed 5 times, then 1-step Ultra TMB-ELISA reagent (Thermo Scientific, cat #34029) was added and the color change of each well was observed. When the color was appropriate, the reaction was stopped with 2M sulfuric acid and the value was read at OD450 nm. And (3) selecting positive clone bacteria expressing positive phage according to the result of ELISA reaction, and sequencing to obtain the gene sequence of the antigen binding protein of the application.

Example 3 expression, purification and binding experiments of antibodies

3.1 antibody expression and purification

By sequencing we obtained 2 sequences encoding the antigen binding proteins of the present application, designated LB21 and LB22, respectively, whose amino acid sequences are shown in table 1, and the sequence information of the CDRs, FRs and heavy chain variable regions of the antigen binding proteins of the present application are shown in table 2.

TABLE 1 sequences of antigen binding proteins

TABLE 2 sequence information of CDR, FR and heavy chain variable regions of the antigen binding proteins of the present application

The gene sequence encoding the antigen binding protein of the present application is cloned, inserted into a mammalian cell expression vector, and a signal peptide is added to the N-terminus of the antigen binding protein of the present application (MGWSCIILFLVATATGVHS,19) and adding 6xHis-tags and HA-tags to the C-terminus of the antigen binding protein of the present application, using Lipofectamine 2000(Thermofisher, cat # stock: 11668030) transfection reagents, transiently transferred to 293FT cells, and cultured for 96 hours. A VHH single domain antibody (see WO 2004/062551A 2) simultaneously expressing MSA21 was used as a control. Using a Nickel column (His Trap) ^TM excel cargo number: 17-3712-05, GE Healthcare) VHH antibodies were purified by changing the buffer with PBS.

3.2 antibody binding to HSA and cynomolgus serum albumin

3.2.1ELISA experiments to verify the binding of the antigen binding proteins of the present application to HSA

4 ℃ overnight coating with 1ug/ml HSA in Stripwell ^TM Microplate (cat # 42592, Costar) plate. The next day the plates were washed and blocked with 2.5% mik in PBS for 1 hour at room temperature. LB21 and LB22 were diluted in a gradient starting at 1ug/ml and 3-fold down the gradient to give 7 concentration points and 1 negative control point. Diluted LB21 and LB22 were added to the blocked ELISA plates at 100 ul/well as primary antibody and incubated at room temperature for one hour. After washing the plate secondary antibody (anti-His HRP) was added and the color change per well was observed. When the color was appropriate, the reaction was stopped with 2M sulfuric acid and the value was read at OD450 nm. The results are shown in FIG. 1, where the antigen binding proteins of the present application are all capable of binding to HSA.

3.2.2Octet detection of affinity between HSA and cynomolgus serum albumin and the antigen binding proteins of the present application

Biotin-labeled HSA or cynomolgus serum albumin was loaded onto SA probes (SA Biosensors, cat # 18-5019, Forte bio) and affinity values were determined by kinetic relationships between HSA and LB21 and LB 22. The SA probe is firstly balanced for 60s at the baseline, the SA probe is uploaded (captured) with biotin-labeled HSA or cynomolgus monkey serum albumin for 90s, the SA probe is balanced for 60s and respectively combined with LB21 and LB22 for 150s, the SA probe is dissociated for 300s in a reaction system buffer (NB buffer), and finally the SA probe is regenerated. The affinity of the antigen-binding protein of the present application for HSA is shown in table 3, and the affinity for cynomolgus serum albumin is shown in table 4.

TABLE 3 affinity of the antigen binding proteins of the present application for HSA

Name of antibody	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

TABLE 4 affinity of antigen binding proteins of the present application for cynomolgus monkey serum albumin

Name of antibody	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

Example 4 non-competitive validation of antibodies with FcRn

To verify whether FcRn and the antigen binding protein of the present invention competitively bind to Human Serum Albumin (HSA), the assay was performed by Octet system by first loading a biotin-labeled human FcRn (FcRn-biotin, beps biosome, Cat: FCM-H82W4) protein onto an SA probe (SA Biosensors, Cat No. 18-5019, Forte bio), then reloading with HSA under acidic conditions, and finally examining whether the antigen binding protein of the present invention can bind to HSA again. Specifically, SA probes were used to load 100nM FcRn-biotin 120s, equilibrate 60s, load 1000nM HSA 150s, and reload 50nM of the binding protein of the invention or control for 200 s. The reactions were all performed in citrate buffer at pH 5.4.

The results are shown in fig. 2-4, and show that HSA bound to human FcRn can still bind to the antigen binding protein of the present invention, demonstrating that the antigen binding protein of the present invention and human FcRn can bind to HSA simultaneously, and there is no competition relationship between them.

Sequence listing

<110> Shichen biopharmaceutical (Suzhou) Co., Ltd

<120> anti-human serum albumin antibody and application thereof

<130> 0125-PA-025

<160> 19

<170> PatentIn version 3.5

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<213> Artificial Sequence (Artificial Sequence)

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<223> LB21 HCDR1

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Val Tyr Asn Leu Ala

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Val Tyr Ala Met Gly

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Ser Ile Thr Ser Gly Leu Ser Thr Asp Tyr Ala Thr Thr Val Lys Gly

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Leu Ile Thr Ser Thr Ser Asp Thr Arg Tyr Ala Asp Ser Val Lys Gly

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Asn Ser Arg Thr Thr Trp Tyr Tyr

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Gly Asn Ser Trp Gly Ala Thr Val Gln Ala Met Cys Lys Ser Asp Tyr

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Asp Tyr

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ile Ile Phe Asp

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser

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Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala

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Trp Tyr Arg Gln Ala Pro Gly Lys Asp Cys Glu Leu Val Gly

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<213> Artificial Sequence (Artificial Sequence)

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<223> LB21 H-FR3

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Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln

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Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ala

20 25 30

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<223> LB22 H-FR3

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Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Lys Thr Val Tyr Leu Gln

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Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met Ala

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<223> LB21 H-FR4

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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

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<213> Artificial Sequence (Artificial Sequence)

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<223> LB22 H-FR4

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Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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<213> Artificial Sequence (Artificial Sequence)

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<223> LB21 amino acid sequence

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ile Ile Phe Asp Val Tyr

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Asn Leu Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val

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Ala Ser Ile Thr Ser Gly Leu Ser Thr Asp Tyr Ala Thr Thr Val Lys

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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

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Ala Asn Ser Arg Thr Thr Trp Tyr Tyr Trp Gly Gln Gly Thr Leu Val

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Thr Val Ser Ser

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<223> LB22 amino acid sequence

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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Val Tyr

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Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Asp Cys Glu Leu Val

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Gly Leu Ile Thr Ser Thr Ser Asp Thr Arg Tyr Ala Asp Ser Val Lys

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Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Lys Thr Val Tyr Leu

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Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Met

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Ala Gly Asn Ser Trp Gly Ala Thr Val Gln Ala Met Cys Lys Ser Asp

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Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<223> LB21 nucleotide sequence

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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 (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

gactccgtga agggccgatt ctccatctcc agagacaatg ccaagaaaac ggtgtatctg 240

caaatgaaca ggctgaaacc tgaggacacg gccgtgtatt actgtatggc aggcaattcc 300

tggggggcta ctgttcaggc tatgtgtaag agtgactatg actactgggg ccaggggacc 360

caggtcactg tctcctca 378

<210> 19

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<223> Signal peptide

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Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

Claims

b) in the Octet assay, with a K of about 3.5E-08M or less _D Specifically binding to cynomolgus monkey serum albumin;

2. The isolated antigen binding protein of claim 1, wherein the HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from any one of the group consisting of:

a) HCDR 1: 1, HCDR 2: 3, and HCDR 3: 5, SEQ ID NO; and

b) HCDR 1: 2, HCDR 2: 4, and HCDR 3: 6 in SEQ ID NO.

3. A polypeptide comprising the isolated antigen binding protein of any one of claims 1-2.

4. An immunoconjugate comprising the isolated antigen binding protein of any one of claims 1-2 or the polypeptide of claim 3.

5. An isolated nucleic acid molecule encoding the isolated antigen binding protein of any one of claims 1-2, or the polypeptide of claim 3.

6. A vector comprising the isolated nucleic acid molecule of claim 5.

7. A cell comprising the isolated antigen binding protein of any one of claims 1-2, the polypeptide of claim 3, the immunoconjugate of claim 4, the isolated nucleic acid molecule of claim 5, and/or the vector of claim 6.

8. A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-2, the polypeptide of claim 3, the immunoconjugate of claim 4, the isolated nucleic acid molecule of claim 5, the vector of claim 6, the cell of claim 7, and/or a pharmaceutically acceptable adjuvant and/or excipient.

9. A kit for the detection of human serum albumin comprising the isolated antigen binding protein of any one of claims 1-2 or the polypeptide of claim 3.

10. Use of an isolated antigen binding protein of any one of claims 1-2 and/or a polypeptide of claim 3 in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder.