pH-Dependent HSA-Binding Molecules and Methods of Use

Abstract

pH-dependent HSA-binding molecules are provided herein. Also provided are fusion proteins comprising a pH-dependent HSA-binding molecule and a therapeutic protein. Also provided herein are polynucleotides, vectors, host cells, methods of use, and methods of production.

Description

【Technical Field】 【0001】 Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 352,589, filed Jun. 15, 2022, the content of which is incorporated herein by reference in its entirety. 【0002】 The present disclosure relates to pH-dependent human serum albumin (HSA) binding molecules, and methods of making and using them. 【Background Art】 【0003】 HSA is the most abundant plasma protein and plays an important role in the binding and transport of common therapeutic agents. Thus, HSA is a target of interest for exploring and optimizing the pharmacokinetics and pharmacodynamics of therapeutic agents. 【0004】 However, since HSA has the role of maintaining fluid balance in the body, promoting the prevention of excessive vascular leakage, transporting hormones, nutrients, and enzymes throughout the body, repairing tissues, and promoting body growth, it is necessary to consider keeping the level of HSA in the blood at a healthy level (the normal range is 3.4 - 5.4 g / dL (34 - 54 g / L)). 【0005】 Current techniques that use HSA binding to extend the half-life of therapeutic agents do so by continuous binding to HSA (e.g., under all physiological conditions), thereby significantly increasing the total size of the therapeutic agent (HSA is a protein of about 66 kDa), which can affect efficacy and function due to steric hindrance of tissue and tumor penetration and reduced distribution. 【0006】 Accordingly, there is a need in the art for improved albumin binding molecules that extend the half-life of therapeutic agents without interfering with serum albumin function or therapeutic efficacy. 【Summary of the Invention】 【0007】 The present disclosure broadly relates to HSA-binding molecules that preferentially bind to HSA at acidic pH and have reduced binding or no binding to HSA at physiological pH, and fusion proteins thereof. 【0008】 In one aspect, provided herein is an antigen-binding molecule that specifically binds to HSA, wherein the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 and / or pH 6.0 is less than 0.140 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 2.4 mM. 【0009】 In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 is less than 0.03 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 11.5 mM. 【0010】 In another aspect, provided herein is an antigen-binding molecule that specifically binds to HSA, wherein the binding affinity of the antigen-binding molecule to HSA at pH 5.5 and / or pH 6.0 is at least 10-fold higher than the binding affinity of the antigen-binding molecule to HSA at pH 7.4. In some embodiments, the binding affinity of the HSA-binding molecule to HSA at acidic pH is at least about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 175-fold, 200-fold, 225-fold, 250-fold, 275-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold higher than the binding affinity of the HSA-binding molecule to HSA at physiological pH. In some embodiments, the acidic pH is pH 5.5 and / or pH 6.0. In some embodiments, the physiological pH is pH 7.4. 【0011】 In some embodiments, the binding affinity is measured by surface plasmon resonance. 【0012】 In some embodiments, the antigen-binding molecule is selected from a Fab fragment, an sdAb, an scFv, an antibody mimetic, or an antigen-binding fragment thereof. In some embodiments, the sdAb is a VHH fragment and optionally further comprises one or more amino acids added to the C-terminus of the VHH fragment. In some embodiments, the one or more amino acids are selected from the group consisting of A, AG, GG, and PP. 【0013】 In one aspect, provided is a fusion protein comprising an antigen-binding molecule described herein and a therapeutic protein. 【0014】 In some embodiments, the therapeutic protein is a small molecule peptide therapeutic. 【0015】 In some embodiments, the therapeutic protein is an antibody or a fragment thereof, optionally an Fc region or an antigen-binding fragment. 【0016】 In some embodiments, the antigen-binding molecule is fused to the therapeutic protein via a linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a peptide linker, optionally a GS linker, optionally 8 to 40 amino acids in length, and optionally 20 amino acids in length. 【0017】 In some embodiments, the antigen-binding molecule is fused to an antibody or a fragment thereof via an IgG hinge region or a portion thereof. 【0018】 In some embodiments, the therapeutic protein is an Fc region comprising a first Fc domain and a second Fc domain, and the antigen-binding molecule is fused to the first Fc domain or the second Fc domain via an IgG hinge region or a portion thereof. 【0019】 Also provided is an isolated one or more polynucleotides encoding any antigen-binding molecule described herein or any fusion protein described herein. 【0020】 Also provided is an expression vector comprising any one or more isolated polynucleotides described herein. 【0021】 Also provided is a host cell comprising any one or more isolated polynucleotides described herein or any expression vector described herein. 【0022】 Also provided is a method for producing an antigen-binding molecule or a fusion protein, the method comprising culturing a host cell described herein under conditions that allow for the expression of the antigen-binding molecule or the fusion protein. 【0023】 Also provided is a pharmaceutical composition comprising any antigen-binding molecule described herein or any fusion protein described herein and at least one pharmaceutically acceptable carrier. 【0024】 Also provided is an antigen-binding molecule described herein or a fusion protein described herein, or a pharmaceutical composition thereof, for use as a medicament. 【0025】 Also provided is a method for increasing the serum half-life of a therapeutic protein, the method comprising fusing an antigen-binding molecule described herein to the therapeutic protein. 【0026】 Also provided is the use of an antigen-binding molecule described herein for increasing the serum half-life of a therapeutic protein. 【0027】 Also provided is the use of any antigen-binding molecule described herein or any fusion protein described herein for the manufacture of a medicament. BRIEF DESCRIPTION OF THE DRAWINGS 【0028】 【Figure 1】A series of sensorgrams showing the characteristics of clones 2H11 and 11C03, selected as monovalent and bivalent (VHH-Fc) molecules that bind to HSA (pH 5.5) on Biacore. Association was performed for each run at pH 5.5. Dissociation was performed at either pH 5.5 or 7.4 to investigate the pH-dependence shown. 【0029】 【Figure 2】 A schematic diagram showing a protocol for the study of PK and HSA binding of Mota-Fab fusion protein in Albumus mice (trademark). 【0030】 【Figure 3】 A line graph showing the concentration of Mota-Fab fusion protein over time in the serum of Albumus mice (trademark). 【0031】 【Figure 4】 A schematic diagram showing a potential mechanism of endocytic recycling in anti-HSA-VHH with weaker binding to HSA at pH 7.4 and stronger binding at pH 5.5. 【DETAILED DESCRIPTION OF THE INVENTION】 【0032】 The present disclosure provides engineered antigen-binding molecules (HSA-binding molecules) that specifically bind to HSA at acidic pH and do not bind (or bind weakly) to HSA at physiological pH, and fusion proteins thereof. Also provided herein are nucleic acids, vectors, host cells, methods of manufacture, and methods of use thereof that encode such HSA-binding molecules or fusion proteins. 【0033】 Definitions As used herein, the term "antigen-binding molecule" refers to any polypeptide that specifically binds to an antigen. Examples of antigen-binding domains include antibodies, such as Fab fragments, F(ab’)2 fragments, disulfide-linked Fv (sdFv), single-chain Fv (scFv), CDRs, VH domains (VH), VL domains (VL), single-domain antibodies (sdAb), VHH fragments, camelid antibodies, and polypeptides derived from any of the foregoing antigen-binding fragments. The term also encompasses synthetic antigen-binding proteins or antibody mimetic proteins, such as anticalins and DARPins. 【0034】 In some embodiments, the antigen-binding molecule is a VHH fragment. In some embodiments, the VHH fragment has one or more additional amino acids at its C-terminus. In some embodiments, the one or more additional amino acids are selected from the group consisting of A, AG, GG, and PP. 【0035】 As used herein, the term "HSA-binding molecule" refers to an antigen-binding molecule that specifically binds to HSA. In some embodiments, the HSA comprises an amino acid sequence that is at least 95% identical to the amino acid sequence provided in GenBank accession number AAA98797.1. In some embodiments, the HSA comprises the amino acid sequence provided in GenBank accession number AAA98797.1. 【0036】 As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. As used herein, the term "equilibrium dissociation constant" or "K D " refers to the tendency of the binding complex of two molecules to dissociate into two free molecules. Thus, as the binding affinity increases, K D decreases. 【0037】 As used herein, the term "specifically binds" refers to the ability of any molecule to preferentially bind to a given target. For example, a molecule that specifically binds to a given target can bind to other molecules and generally bind with a lower affinity, as determined by, for example, an immunoassay, BIAcore™, a KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a specific embodiment, a molecule that specifically binds to a given target has a K D or K D less than when binding to an antigen by at least 2 logs, 2.5 logs, 3 logs, 4 logs. 【0038】 As used herein, the term "acidic pH" refers to a pH less than 7.0, including but not limited to a pH of about 5.5, 5.8, 6.0, 6.2, 6.5, or 6.8. In some embodiments, "acidic pH" refers to endosomal pH. In some embodiments, "acidic pH" refers to a pH of about 5.5 to about 6.0. In some embodiments, "acidic pH" refers to a pH of 5.5 to 6.0. In some embodiments, "acidic pH" refers to a pH of about 5.5 or about 6.0. In some embodiments, "acidic pH" refers to a pH of 5.5 or 6.0. 【0039】 As used herein, the term "physiological pH" refers to a pH of 7.2 to 7.6, including but not limited to a pH of about 7.2, 7.3, 7.4, 7.5, or 7.6. In some embodiments, "physiological pH" refers to the pH present in the bloodstream. In some embodiments, "physiological pH" refers to a pH of about 7.35 to about 7.45. In some embodiments, "physiological pH" refers to a pH of 7.35 to 7.45. In some embodiments, "physiological pH" refers to a pH of about 7.4. In some embodiments, "physiological pH" refers to a pH of 7.4. 【0040】 As used herein, the term "fusion protein" refers to a protein formed by fusing at least one antigen-binding molecule described herein to at least one therapeutic protein (or a fragment or variant thereof). 【0041】 As used herein, the term "fused" refers to the linkage of two peptides by a peptide bond or a peptide linker. In some embodiments, the two proteins are fused directly and continuously together by a peptide bond. In some embodiments, the two proteins are fused indirectly and discontinuously via a peptide linker. In some embodiments, one protein is fused to the peptide linker at a first position by a peptide bond, and a second protein is fused to the peptide linker at a second position by a peptide bond. 【0042】 As used herein, the term "operably linked" refers to the linkage of polynucleotide sequence elements that are in a functional relationship. For example, a polynucleotide sequence is operably linked when it is placed in a functional relationship with another polynucleotide sequence. In some embodiments, a transcriptional regulatory polynucleotide sequence, such as a promoter, enhancer, or other expression control element, is operably linked to a polynucleotide sequence encoding a protein if it affects the transcription of the polynucleotide sequence encoding the protein. The operably linked elements can be continuous or discontinuous. 【0043】 As used herein, the term "therapeutic protein" refers to a protein, polypeptide, antibody, peptide, or fragment or variant thereof that has one or more therapeutic and / or biological activities. Therapeutic proteins include, but are not limited to, proteins, polypeptides, peptides, antibodies, and biologics. The terms peptide, protein, and polypeptide are used interchangeably herein. The term "therapeutic protein" encompasses antibodies, as well as fragments and variants thereof. Thus, the proteins disclosed herein may contain at least fragments or variants of therapeutic proteins and / or at least fragments or variants of antibodies. Additionally, the term "therapeutic protein" may refer to endogenous or naturally occurring correlates of therapeutic proteins. 【0044】 A protein that exhibits "therapeutic activity" or is "therapeutically active" means that the protein has one or more known biological and / or therapeutic activities associated with one or more therapeutic proteins, such as those described herein or otherwise known in the art. By way of non-limiting example, a "therapeutic protein" is a protein that is useful for treating, preventing, or alleviating a disease, condition, or disorder. By way of non-limiting example, a "therapeutic protein" can be one that specifically binds to a particular cell type (normal (e.g., lymphocyte) or abnormal (e.g., cancer cell)), and thus can be used for a compound (drug or cytotoxic agent) to specifically target this cell type. Examples of "therapeutic proteins" include, but are not limited to, interferons, enzymes, hormones, growth factors, interleukins, blood clotting factors, antibodies, as well as fragments and variants thereof. 【0045】 The determination of the "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms. Specific non-limiting examples of mathematical algorithms used for comparing two sequences are the algorithms of Karlin S & Altschul SF, (1990) PNAS 87:2264-2268, as modified as in Karlin S & Altschul SF, (1993) PNAS 90:5873-5877, each of these references being incorporated herein by reference in its entirety. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403, this reference being incorporated herein by reference in its entirety. A BLAST nucleotide search can be performed with the NBLAST nucleotide program parameter set, e.g., score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed with the XBLAST program parameter set, e.g., score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402, this reference being incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterative search for detecting distant relationships between molecules. Id. When utilizing the BLAST, gapped BLAST, and PSI BLAST programs, the default parameters of each program (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov).Another specific non-limiting example of a mathematical algorithm used for array comparison is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used. 【0046】 Percent identity between two sequences can be determined using techniques similar to those above, with or without allowing gaps. In computing percent identity, typically only exact matches are counted. 【0047】 As used herein, the terms “antibody” and “antibodies” include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH domains (VH), or VL domains (VL). Examples of antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies (sdAb), monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelid antibodies, affibody molecules, VHH fragments, Fab fragments, F(ab’)2 fragments, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the foregoing. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or species (e.g., mouse IgG2a or IgG 2b can be of an immunoglobulin molecule of (). 【0048】 As used herein, the term "Fc region" refers to the portion of an immunoglobulin formed by the Fc domains of its two heavy chains. The Fc region can be a wild-type Fc region (native Fc region) or a variant Fc region. The native Fc region is a homodimer. The Fc region can be derived from any natural immunoglobulin. In some embodiments, the Fc region is formed from an IgA, IgD, IgE, or IgG heavy chain constant region. In some embodiments, the Fc region is formed from an IgG heavy chain constant region. In some embodiments, the IgG heavy chain is an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the Fc region is formed from an IgG1 heavy chain constant region. In some embodiments, the IgG1 heavy chain constant region includes the G1m1(a), G1m2(x), G1m3(f), or G1m17(z) allotype. See, for example, Jefferis and Lefranc (2009) mAbs 1(4):332-338, and de Taeye et al., (2020) Front Immunol. 11:740, which are incorporated herein by reference in their entirety. 【0049】 As used herein, the term "variant Fc region" refers to a variant of the Fc region that has one or more changes relative to the native Fc region. The modifications can include amino acid substitutions, additions and / or deletions, ligation of additional moieties, and / or modification of native glycans. This term encompasses heterodimeric Fc regions in which each of the constituent Fc domains is different. This term also encompasses single-chain Fc regions in which the constituent Fc domains are linked together by a linker moiety. 【0050】 As used herein, the term "Fc domain" refers to the portion of a single immunoglobulin heavy chain that includes both the CH2 and CH3 domains of an antibody. In some embodiments, the Fc domain includes at least a portion of the hinge (e.g., upper, middle, and / or lower hinge region) region, the CH2 domain, and the CH3 domain. In some embodiments, the Fc domain does not include the hinge region. 【0051】 As used herein, the term "hinge region" refers to the portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. In some embodiments, the hinge region is up to 70 amino acid residues in length. In some embodiments, this hinge region contains approximately 11-17 amino acid residues, is flexible, and thus allows the two N-terminal antigen-binding regions to move independently. In some embodiments, the hinge region is 12 amino acid residues in length. In some embodiments, the hinge region is 15 amino acid residues in length. In some embodiments, the hinge region is 62 amino acid residues in length. The hinge region can be subdivided into three different domains: the upper, middle, and lower hinge domains. The antigen-binding molecules or fusion proteins of the present disclosure can include all or any part of the hinge region. In some embodiments, the hinge region is derived from an IgG1 antibody. In some embodiments, the hinge region includes the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO: 1). 【0052】 As used herein, the term "FcRn" refers to the neonatal Fc receptor. Exemplary FcRn molecules include the human FcRn encoded by the FCGRT gene as shown in RefSeq NM 004107. The amino acid sequence of the corresponding protein is shown in RefSeq NP_004098. 【0053】 As used herein, the terms "treat", "treating", and "treatment" refer to the therapeutic or prophylactic measures described herein. A "treatment" method is used to prevent, cure, delay, reduce the severity of, or improve one or more symptoms of a disease or disorder, or a recurrent disease or disorder, or to extend the lifespan of a subject beyond that expected in the absence of such treatment, by administering a polypeptide to a subject having or susceptible to such a disease or disorder. In some embodiments, a "treatment" method uses the administration of a polypeptide to a subject having or predisposed to have such a disease or disorder to prevent, cure, delay, reduce the severity of, or remit such a disease or disorder, or a recurrent disease or disorder. 【0054】 As used herein, in the context of performing a therapy on a subject, the term "effective amount" refers to the amount of the therapy that achieves the desired prophylactic or therapeutic effect. 【0055】 As used herein, the term "dosage" or "dose" refers to the amount of a drug administered to a subject in a single administration. 【0056】 As used herein, the term "equivalent dose" refers to the doses of the first and second therapeutic agents where the number of molecules of the first and second therapeutic agents is approximately the same. In some embodiments, the equivalent dose is an equimolar dose. As used herein, the term "equimolar dose" refers to the doses of the first and second therapeutic agents where the number of moles of the first and second agents is the same. In some embodiments, the equivalent dose is calculated using the observed molecular weight of the first and second agents. In some embodiments, the equivalent dose is calculated using the predicted molecular weight of the first and second agents. In some embodiments, the equivalent dose is calculated using the observed molecular weight of the first agent and the predicted molecular weight of the second agent. In some embodiments, the equivalent dose is calculated using the predicted molecular weight of the first agent and the observed molecular weight of the second agent. 【0057】 As used herein, the terms "pharmacokinetics" and "PK" refer to the effect of a living organism on a therapeutic agent administered to the organism. In some embodiments, the effect is the metabolism and / or clearance of the therapeutic agent. In some embodiments, PK refers to the rate of metabolism and / or clearance of the therapeutic agent. As used herein, the term "improved pharmacokinetics" or "improved PK" refers to an improvement in the desired effect of a living organism on a therapeutic agent administered to the organism. In some embodiments, improved pharmacokinetics includes an increase in the half-life (T 1 / 2 )), clearance, or area under the curve (AUC) of the therapeutic agent in a subject. In some embodiments, the therapeutic agent is a fusion protein or a therapeutic protein described herein. 【0058】 As used herein, the term "subject", or "patient", or "participant" includes any human or non-human animal. In one embodiment, the subject, or patient, or participant is a human or non-human mammal. In one embodiment, the subject, or patient, or participant is a human. 【0059】 As used herein, the terms "about" or "approximately" when referring to measurable values such as dosages, encompass variations of ±20%, ±15%, ±10%, ±5%, ±1%, or ±0.1% of a given value or range as appropriate to practice the methods disclosed herein. 【0060】 As used herein, the term "molecular weight" can refer to "predicted molecular weight" or "observed molecular weight". The "predicted molecular weight" of a protein is the sum of the molecular weights of all amino acids in the protein. In certain situations, the "predicted molecular weight" can differ from the "observed molecular weight" of the molecule. In some embodiments, these differences can occur in a protein due to changes in glycosylation, glycanation, ubiquitination, phosphorylation, or protein cleavage of the protein or an additional protein complex with a given protein. 【0061】 Antigen-binding molecule In one aspect, an antigen-binding molecule that specifically binds to HSA (i.e., an HSA-binding molecule) is provided by the present disclosure. More specifically, the HSA-binding molecules of the present disclosure preferentially bind to HSA at acidic pH and do not have, or have weak binding to, HSA at physiological pH. In some embodiments, the binding affinity of the HSA-binding molecule to HSA at acidic pH is at least about 100-fold higher than the binding affinity of the HSA-binding molecule to HSA at physiological pH. In some embodiments, the binding affinity of the HSA-binding molecule to HSA at acidic pH is at least about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 175-fold, 200-fold, 225-fold, 250-fold, 275-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold higher than the binding affinity of the HSA-binding molecule to HSA at physiological pH. In some embodiments, the acidic pH is pH 5.5 and / or pH 6.0. In some embodiments, the physiological pH is pH 7.4. 【0062】 In some embodiments, the equilibrium dissociation constant for the binding of the HSA-binding molecule to HSA at acidic pH is less than 0.001, 0.005, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085, 0.090, 0.095, 0.10, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.3, 0.4, or 0.5 mM. In some embodiments, the equilibrium dissociation constant for the binding of the HSA-binding molecule to HSA at physiological pH is at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 13.0, 14.0, or 15.0 mM. In some embodiments, the acidic pH is pH 5.5 and / or pH 6.0. In some embodiments, the physiological pH is pH 7.4. 【0063】 In some embodiments, the equilibrium dissociation constant for the binding of the HSA-binding molecule to HSA at acidic pH is less than 0.001, 0.005, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085, 0.090, 0.095, 0.10, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.3, 0.4, or 0.5 mM, and the equilibrium dissociation constant for the binding of the HSA-binding molecule to HSA at physiological pH is at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 13.0, 14.0, or 15.0 mM. In some embodiments, the acidic pH is pH 5.5 and / or pH 6.0. In some embodiments, the physiological pH is pH 7.4. 【0064】 In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 and / or pH 6.0 is less than 0.140 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 2.4 mM. In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 and / or pH 6.0 is less than 0.03 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 11.5 mM. In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 and / or pH 6.0 is less than 0.140 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 11.5 mM. In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 and / or pH 6.0 is less than 0.03 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 2.4 mM. 【0065】 In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 is less than 0.140 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 2.4 mM. In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 is less than 0.03 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 11.5 mM. In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 is less than 0.140 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 11.5 mM. In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 is less than 0.03 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 2.4 mM. 【0066】 In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 is less than 0.140 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 2.4 mM. In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 is less than 0.03 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 11.5 mM. In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 is less than 0.140 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 11.5 mM. In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 is less than 0.03 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 2.4 mM. 【0067】 In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 and / or pH 6.0 is less than 0.03 to 0.140 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 2.4 to 11.5 mM. In some embodiments, the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 is less than 0.03 to 0.140 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 2.4 to 11.5 mM. 【0068】 In some embodiments, at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the antigen-binding molecules that specifically bind to HSA bind to HSA at pH 5.5 and / or pH 6.0, and up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25% of the antigen-binding molecules to HSA are released at pH 7.4. In some embodiments, at least 75-99, 80-98, or 85-97% of the antigen-binding molecules that specifically bind to HSA bind to HSA at pH 5.5 and / or pH 6.0, and up to 1-25, 2-20, or 3-15% of the antigen-binding molecules to HSA are released at pH 7.4. 【0069】 In some embodiments, at least 80% of the antigen-binding molecules that specifically bind to HSA bind to HSA at pH 5.5 and / or pH 6.0, and up to 20% of the antigen-binding molecules to HSA are released at pH 7.4. In some embodiments, at least 95% of the antigen-binding molecules that specifically bind to HSA bind to HSA at pH 5.5 and / or pH 6.0, and up to 5% of the antigen-binding molecules to HSA are released at pH 7.4. 【0070】 In some embodiments, the HSA-binding molecule does not bind to HSA at physiological pH. In some embodiments, the binding of the HSA-binding molecule to HSA is undetectable at physiological pH. In some embodiments, the physiological pH is pH 7.4. 【0071】 The binding affinity of the HSA-binding molecule to HSA can be measured by any known method. In some embodiments, the binding affinity of the HSA-binding molecule to HSA is measured by surface plasmon resonance. 【0072】 In some embodiments, the HSA-binding molecule does not compete for HSA binding with FcRn. 【0073】 In some embodiments, the antigen-binding molecule is a polypeptide derived from an antibody, and the antibody includes, but is not limited to, sdAb (e.g., VHH fragment), Fab fragment, scFv, VH, or VL. In some embodiments, the antigen-binding molecule is a synthetic antigen-binding protein or an antibody mimetic protein, and the synthetic antigen-binding protein or antibody mimetic protein includes, but is not limited to, anticalin or DARPin. 【0074】 In some embodiments, the pH-dependent binding of the HSA-binding molecule to HSA can be enhanced by substituting one or more amino acids in one or more CDRs with histidine (H), aspartic acid (D), glutamic acid (E), or alanine (A). In some embodiments, one, two, or three amino acids in one or more CDRs are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in CDR1 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in CDR2 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in CDR3 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in HCDR1 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in HCDR2 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in HCDR3 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in LCDR1 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in LCDR2 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in LCDR3 are independently substituted with H, D, E, or A. 【0075】 In some embodiments, one, two, or three amino acids in each of CDR1 and CDR2 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in each of CDR2 and CDR3 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in each of CDR1 and CDR3 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in each of CDR1, CDR2, and CDR3 are independently substituted with H, D, E, or A. 【0076】 In some embodiments, one, two, or three amino acids in each of HCDR1 and HCDR2 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in each of HCDR2 and HCDR3 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in each of HCDR1 and HCDR3 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in each of HCDR1, HCDR2, and HCDR3 are independently substituted with H, D, E, or A. 【0077】 In some embodiments, one, two, or three amino acids in each of LCDR1 and LCDR2 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in each of LCDR2 and LCDR3 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in each of LCDR1 and LCDR3 are independently substituted with H, D, E, or A. In some embodiments, one, two, or three amino acids in each of LCDR1, LCDR2, and LCDR3 are independently substituted with H, D, E, or A. 【0078】 In some embodiments, the antigen-binding molecule further comprises one or more amino acids added at its C-terminus. In some embodiments, the antigen-binding molecule further comprises one or more amino acids added at the C-terminus, and the one or more amino acids are selected from A, AG, GG, and PP. In some embodiments, the C-terminus of the VHH is the amino acid sequence VTVSS (SEQ ID NO: 195). In some embodiments, the C-terminus of the VHH consists of the amino acid sequence VTVSS (SEQ ID NO: 195). 【0079】 In some embodiments, the antigen-binding molecule specifically binds to HSA and is selected from Fab fragments, scFv, sdAb, and antigen-binding fragments thereof. In some embodiments, the antigen-binding domain specifically binds to HSA and is a sdAb, such as a VHH fragment. In some embodiments, HSA comprises an amino acid sequence that is at least 95% identical to the amino acid sequence provided in GenBank accession number AAA98797.1. In some embodiments, HSA comprises the amino acid sequence provided in GenBank accession number AAA98797.1. 【0080】 In some embodiments, the antigen-binding domain is a VHH fragment, and the VHH fragment comprises the CDR1, CDR2, and CDR3 amino acid sequences of a VHH fragment comprising an amino acid sequence selected from SEQ ID NOs: 15-20, 77-88, 90-115, and 186-194. 【0081】 In some embodiments, the antigen-binding domain is a VHH fragment, and the VHH fragment comprises or consists of a combination of CDR1, CDR2, and CDR3, and 1, 2, 3, 4, or 5 amino acids are different in at least one of the amino acid sequences selected from SEQ ID NOs: 5, 6, and 8; 5, 6, and 9; 5, 6, and 10; 5, 6, and 11; 5, 6, and 12; 5, 6, and 13; 5, 6, and 21; 5, 6, and 22; 5, 6, and 23; 5, 6, and 24; 5, 6, and 25; 5, 6, and 26; 5, 6, and 27; 5, 6, and 28; 5, 6, and 29; 5, 6, and 30; 5, 6, and 31; 5, 6, and 32; 33, 34, and 36; 33, 34, and 37; 33, 34, and 38; 33, 34, and 39; 33, 34, and 40; 33, 34, and 41; 33, 34, and 42; 33, 34, and 43; 33, 34, and 44; 33, 34, and 45; 33, 34, and 46; 33, 34, and 47; 33, 34, and 48; 33, 34, and 49; 33, 34, and 50; 33, 34, and 51; 33, 34, and 52; 33, 34, and 53; 177, 34, and 35; 178, 34, and 35; 33, 179, and 35; 33, 180, and 35; 33, 181, and 35; 33, 182, and 35; 33, 183, and 35; 33, 184, and 35; 33, 185, and 35; 54, 55, and 56; 54, 57, and 58; 59, 60, and 61; 62, 63, and 64; 65, 66, and 67; 68, 69, and 70; 71, 72, and 73; 74, 75, and 76; and 173, 174, and 175. 【0082】 In some embodiments, the antigen-binding domain is a VHH fragment, and the VHH fragment comprises or consists of a combination of CDR1, CDR2, and CDR3 selected from SEQ ID NOs: 5, 6, and 8; 5, 6, and 9; 5, 6, and 10; 5, 6, and 11; 5, 6, and 12; 5, 6, and 13; 5, 6, and 21; 5, 6, and 22; 5, 6, and 23; 5, 6, and 24; 5, 6, and 25; 5, 6, and 26; 5, 6, and 27; 5, 6, and 28; 5, 6, and 29; 5, 6, and 30; 5, 6, and 31; 5, 6, and 32; 33, 34, and 36; 33, 34, and 37; 33, 34, and 38; 33, 34, and 39; 33, 34, and 40; 33, 34, and 41; 33, 34, and 42; 33, 34, and 43; 33, 34, and 44; 33, 34, and 45; 33, 34, and 46; 33, 34, and 47; 33, 34, and 48; 33, 34, and 49; 33, 34, and 50; 33, 34, and 51; 33, 34, and 52; 33, 34, and 53; 177, 34, and 35; 178, 34, and 35; 33, 179, and 35; 33, 180, and 35; 33, 181, and 35; 33, 182, and 35; 33, 183, and 35; 33, 184, and 35; 33, 185, and 35; 54, 55, and 56; 54, 57, and 58; 59, 60, and 61; 62, 63, and 64; 65, 66, and 67; 68, 69, and 70; 71, 72, and 73; 74, 75, and 76; and 173, 174, and 175, and one or more amino acids within one or more of the CDRs are substituted with alanine or histidine. 【0083】 In some embodiments, the antigen-binding domain is a VHH fragment, and the VHH fragment comprises, or consists of, a combination of CDR1, CDR2, and CDR3 selected from SEQ ID NOs: 5, 6, and 8; 5, 6, and 9; 5, 6, and 10; 5, 6, and 11; 5, 6, and 12; 5, 6, and 13; 5, 6, and 21; 5, 6, and 22; 5, 6, and 23; 5, 6, and 24; 5, 6, and 25; 5, 6, and 26; 5, 6, and 27; 5, 6, and 28; 5, 6, and 29; 5, 6, and 30; 5, 6, and 31; 5, 6, and 32; 33, 34, and 36; 33, 34, and 37; 33, 34, and 38; 33, 34, and 39; 33, 34, and 40; 33, 34, and 41; 33, 34, and 42; 33, 34, and 43; 33, 34, and 44; 33, 34, and 45; 33, 34, and 46; 33, 34, and 47; 33, 34, and 48; 33, 34, and 49; 33, 34, and 50; 33, 34, and 51; 33, 34, and 52; 33, 34, and 53; 177, 34, and 35; 178, 34, and 35; 33, 179, and 35; 33, 180, and 35; 33, 181, and 35; 33, 182, and 35; 33, 183, and 35; 33, 184, and 35; 33, 185, and 35; 54, 55, and 56; 54, 57, and 58; 59, 60, and 61; 62, 63, and 64; 65, 66, and 67; 68, 69, and 70; 71, 72, and 73; 74, 75, and 76; and 173, 174, and 175. 【0084】 In some embodiments, the antigen-binding domain is a VHH fragment, and the VHH fragment comprises, or consists of, an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 15-20, 77-88, 90-115, and 186-194. In some embodiments, the antigen-binding domain is a VHH fragment, and the VHH fragment comprises, or consists of, an amino acid sequence selected from SEQ ID NOs: 15-20, 77-88, 90-115, and 186-194. 【0085】 In some embodiments, the antigen-binding domain comprises any one of the amino acid sequences of SEQ ID NOs: 15-20, 77-88, 90-115, and 186-194 or a variant thereof, and one or more amino acids added at the C-terminus, and the one or more amino acids are selected from A, AG, GG, and PP. 【0086】 In some embodiments, the antigen-binding domain is a Fab or scFv, and the Fab or scFv comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH comprising the amino acid sequence of SEQ ID NO: 157, 159, 161, 163, 165, 167, 169, or 171, and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL comprising the amino acid sequence of SEQ ID NO: 158, 160, 162, 164, 166, 168, 170, or 172. 【0087】 In some embodiments, the antigen-binding domain is a Fab or scFv, and the Fab or scFv comprises a combination of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of VH and VL, and 1, 2, 3, 4, or 5 amino acids are different in at least one of the amino acid sequences of the CDRs selected from SEQ ID NOs: 157 and 158, 159 and 160, 161 and 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, or 171 and 172. In some embodiments, the antigen-binding domain is a Fab or scFv, and the Fab or scFv comprises a combination of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 157 and 158, 159 and 160, 161 and 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, or 171 and 172. 【0088】 In some embodiments, the antigen-binding domain is a Fab or scFv, the Fab or scFv consists of a combination of VH and VL's VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3, and 1, 2, 3, 4, or 5 amino acids differ in at least one of the amino acid sequences selected from SEQ ID NOs: 157 and 158, 159 and 160, 161 and 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, or 171 and 172. In some embodiments, the antigen-binding domain is a Fab or scFv, the Fab or scFv consists of a combination of VH and VL's VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 157 and 158, 159 and 160, 161 and 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, or 171 and 172. 【0089】 In some embodiments, the antigen-binding domain is a Fab or scFv, and the Fab or scFv comprises or consists of a combination of HCDR1, HCDR2, and HCDR3 selected from SEQ ID NOs: 116, 117, and 118; 119, 120, and 121; 122, 120, and 121; 123, 124, and 125; 126, 127, and 128; 129, 130, and 131; 132, 133, and 134; and 135, 136, and 137. In some embodiments, the antigen-binding domain is a Fab or scFv, and the Fab or scFv comprises or consists of a combination of LCDR1, LCDR2, and LCDR3 selected from SEQ ID NOs: 138, 139, and 140; 141, 142, and 143; 144, 145, and 146; 141, 142, and 143; 147, 148, and 149; 150, 151, and 152; 153, 148, and 154; and 155, 151, and 156. In some embodiments, the antigen-binding domain is a Fab or scFv, and the Fab or scFv comprises or consists of a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 selected from SEQ ID NOs: 116, 117, 118, 138, 139, and 140; 119, 120, 121, 141, 142, and 143; 122, 120, 121, 144, 145, and 146; 123, 124, 125, 141, 142, and 143; 126, 127, 128, 147, 148, and 149; 129, 130, 131, 150, 151, and 152; 132, 133, 134, 153, 148, and 154; and 135, 136, 137, 155, 151, and 156. 【0090】 In some embodiments, the antigen-binding domain is a Fab or scFv comprising VH and VL, wherein VH comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NO: 157, 159, 161, 163, 165, 167, 169, or 171, and VL comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NO: 158, 160, 162, 164, 166, 168, 170, or 172. In some embodiments, the antigen-binding domain is a Fab or scFv comprising VH and VL, wherein VH comprises an amino acid sequence selected from SEQ ID NO: 157, 159, 161, 163, 165, 167, 169, or 171, and VL comprises an amino acid sequence selected from SEQ ID NO: 158, 160, 162, 164, 166, 168, 170, or 172. 【0091】 In some embodiments, the antigen-binding domain is a Fab or scFv comprising VH and VL, wherein VH and VL comprise amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acid sequences selected from SEQ ID NO: 157 and 158, 159 and 160, 161 and 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, or 171 and 172. In some embodiments, the antigen-binding domain is a Fab or scFv comprising VH and VL, wherein VH and VL comprise amino acid sequences selected from SEQ ID NO: 157 and 158, 159 and 160, 161 and 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, or 171 and 172. 【0092】 Therapeutic protein Therapeutic proteins can be fused to the HSA-binding molecules of the present disclosure to extend their half-life. Examples of therapeutic proteins include, but are not limited to, interferons, enzymes, hormones, growth factors, interleukins, blood coagulation factors, antibodies, and fragments and variants thereof. In some embodiments, the therapeutic protein is a human protein or is derived from a human protein. 【0093】 In some embodiments, the therapeutic protein is a therapeutic antibody or a fragment thereof. 【0094】 In some embodiments, the therapeutic proteins disclosed herein comprise one or more Fc regions or fragments thereof in combination with one or more antigen-binding domains (e.g., VH, VL, sdAb, Fab fragment, scFv, or antibody mimetic). In some embodiments, the therapeutic proteins disclosed herein comprise one or more antigen-binding domains (e.g., VH, VL, sdAb, Fab fragment, scFv, or antibody mimetic). 【0095】 Generally, therapeutic antibodies or fragments thereof are human immunoglobulins. However, it is understood that therapeutic antibodies can be derived from immunoglobulins of any other mammalian species, including, for example, camelid species, rodents (e.g., mice, rats, rabbits, guinea pigs), or non-human primate (e.g., chimpanzee, macaque) species. Also, therapeutic antibodies or fragments thereof can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. 【0096】 To enhance the manufacturability of the therapeutic antibodies and fusion proteins containing them disclosed herein, the Fc region, which is a component, preferably does not contain any non-disulfide-bonded cysteine residues. Thus, in one embodiment, the Fc region does not contain free cysteine residues. 【0097】 Fusion protein The present disclosure provides a fusion protein comprising at least one antigen-binding molecule that specifically binds to HSA and at least one therapeutic protein. Advantageously, fusing the HSA-binding molecule of the present disclosure to a therapeutic protein has been found to increase the half-life of the therapeutic protein due to the ability of the HSA-binding molecule to bind to HSA in endosomes (acidic pH) and thus be recycled with HSA. However, since the HSA-binding molecule does not bind or binds weakly to HSA at physiological pH in the bloodstream, the fusion protein does not interfere with albumin function. Similarly, since the fusion protein is unbound in the bloodstream by HSA, its efficacy is retained. 【0098】 The therapeutic protein can be any of the therapeutic proteins described herein or any therapeutic protein known in the art otherwise. The antigen-binding molecule can be any of the antigen-binding molecules described herein. In some embodiments, the fusion protein comprises only one antigen-binding molecule. In some embodiments, the fusion protein comprises two or more antigen-binding molecules. In some embodiments, the fusion protein comprises only one therapeutic protein. In some embodiments, the fusion protein comprises two or more therapeutic proteins. In some embodiments, the fusion protein comprises one antigen-binding molecule and one therapeutic protein. 【0099】 In some embodiments, the antigen-binding molecule is fused to the C-terminus of the therapeutic protein. In some embodiments, the antigen-binding molecule is fused to the N-terminus of the therapeutic protein. 【0100】 In some embodiments, one antigen-binding molecule is fused to the N-terminus of the therapeutic protein, and another antigen-binding molecule is fused to the C-terminus of the therapeutic protein. In some embodiments, one antigen-binding molecule is fused at a position other than the N-terminus or C-terminus of the therapeutic protein, and another antigen-binding molecule is fused to the N-terminus of the therapeutic protein. In some embodiments, one antigen-binding molecule is fused at a position other than the N-terminus or C-terminus of the therapeutic protein, and another antigen-binding molecule is fused to the C-terminus of the therapeutic protein. 【0101】 In some embodiments, one therapeutic protein is fused to the N-terminus of the antigen-binding molecule, and another therapeutic protein is fused to the C-terminus of the antigen-binding molecule. In some embodiments, one therapeutic protein is fused at a position other than the N-terminus or C-terminus of the antigen-binding molecule, and another therapeutic protein is fused to the N-terminus of the antigen-binding molecule. In some embodiments, one therapeutic protein is fused at a position other than the N-terminus or C-terminus of the antigen-binding molecule, and another therapeutic protein is fused to the C-terminus of the antigen-binding molecule. 【0102】 In some embodiments, the therapeutic protein is a therapeutic antibody or a fragment thereof that includes an Fc region. In some embodiments, the antigen-binding molecule is fused to the C-terminus of one or both of the Fc domains of the Fc region. In some embodiments, the antigen-binding molecule is fused to the N-terminus of one or both of the Fc domains of the Fc region. In some embodiments, the antigen-binding molecule "replaces" one or both of the antigen-binding domains (e.g., VHH, VH, VL, Fab fragment, scFv, etc.) of the therapeutic antibody. In some embodiments, the antigen-binding molecule is fused to the N-terminus of the antigen-binding domain of the therapeutic antibody. 【0103】 In some embodiments, one antigen-binding molecule is fused to the C-terminus of one of the Fc domains of the Fc region, and another antigen-binding molecule is fused to the C-terminus of the other Fc domain of the Fc region. In some embodiments, one antigen-binding molecule is fused to the N-terminus of one of the Fc domains of the Fc region, and another antigen-binding molecule is fused to the N-terminus of the other Fc domain of the Fc region. In some embodiments, one antigen-binding molecule is fused to the N-terminus of one of the Fc domains of the Fc region, and another antigen-binding molecule is fused to the C-terminus of the other Fc domain of the Fc region. In some embodiments, one antigen-binding molecule is fused to a position other than the N-terminus or C-terminus of one of the Fc domains of the Fc region, and another antigen-binding molecule is fused to the N-terminus of the other Fc domain of the Fc region. In some embodiments, one antigen-binding molecule is fused to a position other than the N-terminus or C-terminus of one of the Fc domains of the Fc region, and another antigen-binding molecule is fused to the C-terminus of the other Fc domain of the Fc region. 【0104】 In some embodiments, the antigen-binding molecule can be directly fused to the N-terminus or C-terminus of the therapeutic protein. In some embodiments, the antigen-binding molecule is fused to the N-terminus or C-terminus of the therapeutic protein via a linker. In some embodiments, the linker is an uncleavable linker. 【0105】 In some embodiments, the antigen-binding molecule can be directly fused to the N-terminus or C-terminus of the Fc domain. In some embodiments, the antigen-binding molecule is fused to the N-terminus or C-terminus of the Fc domain via a linker. The linker can be any suitable linker including those described herein. 【0106】 Linker The antigen-binding molecule can be fused to the N-terminus or C-terminus of a therapeutic protein (e.g., an antibody or a fragment thereof). 【0107】 In some embodiments, the antigen-binding molecule can be directly fused to the N-terminus or C-terminus of the therapeutic protein. In some embodiments, the antigen-binding molecule is linked to the N-terminus or C-terminus of the therapeutic protein via a linker. In some embodiments, the linker is a non-cleavable linker. 【0108】 In some embodiments, the antigen-binding molecule can be directly fused to the N-terminus or C-terminus of the Fc domain. In some embodiments, the antigen-binding molecule is linked to the N-terminus or C-terminus of the Fc domain via a linker. In some embodiments, the linker is a non-cleavable linker. As used herein, the term "non-cleavable linker" refers to a linker that is not readily cleaved by one or more of a given enzyme, chemical agent, or light irradiation. In some embodiments, the enzyme is a protease. 【0109】 In some embodiments, the linker is a synthetic compound linker, such as a chemical cross-linking agent. Non-limiting examples of suitable commercially available cross-linking agents include N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl] sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES). 【0110】 The Fc domain disclosed in this specification may include a portion of the hinge region. Thus, the antigen-binding molecule may be linked via this hinge region to the N-terminus of the Fc domain. In some embodiments, one or more amino acids are included between the C-terminus of the antigen-binding molecule and the N-terminus of the Fc domain. In some embodiments, the one or more amino acids included between the C-terminus of the antigen-binding molecule and the N-terminus of the Fc domain are amino acids of the native hinge region. In some embodiments, the C-terminus of the antigen-binding molecule is fused via a hinge region or a portion thereof to the N-terminus of the Fc domain. In some embodiments, the hinge region is an IgG hinge region, such as a human IgG hinge region. 【0111】 In some embodiments, the linker is a peptide linker. Examples of peptide linkers are well known, and those skilled in the art can select a suitable peptide linker for use in linking an antigen-binding molecule, such as a therapeutic protein, to an antigen-binding molecule. 【0112】 The peptide linker can be of any length. In some embodiments, the length and amino acid composition of the linker peptide sequence can be optimized to vary the orientation and / or proximity of the antigen-binding molecule and the therapeutic protein to each other to achieve the desired activity of the fusion protein. In some embodiments, the peptide linker is about 1 to about 100 amino acids in length, about 8 to about 40 amino acids in length, or about 15 amino acids to about 25 amino acids in length. In some embodiments, the peptide linker is 1 to 100 amino acids in length, 8 to 40 amino acids in length, or 15 to 25 amino acids in length. In some embodiments, the peptide linker is about 8 amino acids in length, about 9 amino acids in length, about 10 amino acids in length, about 11 amino acids in length, about 12 amino acids in length, about 13 amino acids in length, about 14 amino acids in length, about 15 amino acids in length, about 16 amino acids in length, about 17 amino acids in length, about 18 amino acids in length, about 19 amino acids in length, about 20 amino acids in length, about 21 amino acids in length, about 22 amino acids in length, about 23 amino acids in length, about 24 amino acids in length, about 25 amino acids in length, about 26 amino acids in length, about 27 amino acids in length, about 28 amino acids in length, about 29 amino acids in length, about 30 amino acids in length, about 31 amino acids in length, about 32 amino acids in length, about 33 amino acids in length, about 34 amino acids in length, about 35 amino acids in length, about 36 amino acids in length, about 37 amino acids in length, about 38 amino acids in length, about 39 amino acids in length, or about 40 amino acids in length. In some embodiments, the peptide linker is 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, 20 amino acids in length, 21 amino acids in length, 22 amino acids in length, 23 amino acids in length, 24 amino acids in length, 25 amino acids in length, 26 amino acids in length, 27 amino acids in length, 28 amino acids in length, 29 amino acids in length, 30 amino acids in length, 31 amino acids in length, 32 amino acids in length, 33 amino acids in length, 34 amino acids in length, 35 amino acids in length, 36 amino acids in length, 37 amino acids in length, 38 amino acids in length, 39 amino acids in length, or 40 amino acids in length. 【0113】 In some embodiments, the peptide linker contains only glycine residues and / or serine residues (e.g., glycine-serine linker or GS linker). Examples of such peptide linkers include Gly(x)Ser (where x is from 0 to 6), or Ser Gly(x) (where x is from 0 to 6), (Gly Gly Gly Gly Ser)n (where n is an integer of 1 or more), and (Ser Gly Gly Gly Gly)n (where n is an integer of 1 or more). In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of (GGGGS)n and (SGGGG)n, where n is from 1 to 8. In some embodiments, the linker peptide is modified such that the amino acid sequence GSG (which occurs at the junction of conventional Gly / Ser linker peptide repeats) is absent. For example, in some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of (GGGXX)nGGGGS and GGGGS(XGGGS)n (where X is any amino acid that can be inserted into the sequence and does not result in a polypeptide containing the sequence GSG, and n is from 0 to 4). In some embodiments, the sequence of the linker peptide is (GGGX1X2)nGGGGS, where X1 is P, X2 is S, and n is from 0 to 4. In some other embodiments, the sequence of the linker peptide is (GGGX1X2)nGGGGS, where X1 is G, X2 is Q, and n is from 0 to 4. In some other embodiments, the sequence of the linker peptide is (GGGX1X2)nGGGGS, where X1 is G, X2 is A, and n is from 0 to 4. In yet other embodiments, the sequence of the linker peptide is GGGGS(XGGGS)n, where X is P, and n is from 0 to 4. In some embodiments, the linker peptide of the present disclosure comprises or consists of the amino acid sequence (GGGG A)2GGGGS. In some embodiments, the linker peptide comprises or consists of the amino acid sequence (GGGG Q)2GGGGS. In another embodiment, the linker peptide comprises or consists of the amino acid sequence (GGGP S)2GGGGS. In another embodiment, the linker peptide comprises or consists of the amino acid sequence GGGGS(PGGGS)2.In yet another embodiment, the linker peptide comprises or consists of the amino acid sequence GSGGS or SGGSGS. In some embodiments, the linker peptide comprises or consists of the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 2), GGGGSGGGGS (SEQ ID NO: 3), or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4). 【0114】 In some embodiments, the peptide linker is a GS linker that is about 20 or about 30 amino acids in length. In some embodiments, the peptide linker is a GS linker that is 20 or 30 amino acids in length. 【0115】 Polynucleotides, Vectors, and Production Methods The present disclosure also provides polynucleotides encoding the antigen-binding molecules or fragments thereof disclosed herein. In some embodiments, the polynucleotide encodes a therapeutic protein of the present disclosure. In some embodiments, the polynucleotide encodes a fusion protein of the present disclosure. In some embodiments, the polynucleotide encodes one or more of an antigen-binding molecule, a therapeutic protein, and a linker. In some embodiments, the polynucleotide encodes an antigen-binding molecule and a therapeutic protein, and optionally encodes a linker. In some embodiments, the polynucleotide encodes one or more of an antigen-binding molecule, an Fc region, and a linker. In some embodiments, the polynucleotide encodes an antigen-binding molecule and an Fc region, and optionally encodes a linker. In some embodiments, the polynucleotide encodes a fusion protein comprising one or more antigen-binding molecules and a therapeutic protein. In some embodiments, the polynucleotide encodes one or more of an antigen-binding molecule, an Fc domain, and a linker. In some embodiments, the polynucleotide encodes an antigen-binding molecule and an Fc domain, and optionally encodes a linker. In some embodiments, the polynucleotide encodes a fusion protein comprising one or more antigen-binding molecules and one or more therapeutic proteins. 【0116】 As used herein, an "isolated" polynucleotide or nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule (e.g., in a mouse or a human). Also, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the expression "substantially free of" means that a preparation of a polynucleotide or nucleic acid molecule has less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular, less than about 10%) of other materials, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In one embodiment, the nucleic acid molecule(s) encoding the polypeptide(s) described herein is isolated or purified. 【0117】 In one aspect, a polynucleotide is provided herein that comprises a nucleotide sequence encoding a therapeutic protein or fusion protein described herein. In another aspect, a polynucleotide is provided herein that comprises a nucleotide sequence encoding an antigen-binding molecule described herein. In another aspect, a polynucleotide is provided herein that comprises a nucleotide sequence encoding a fusion protein described herein. 【0118】 The polynucleotide can comprise a nucleotide sequence encoding an sdAb (e.g., a VHH fragment), a Fab fragment, a scFv, a VH, or a VL that includes the FRs and CDRs of the antigen-binding molecules described herein. The polynucleotide can also comprise a nucleotide sequence encoding an antibody mimetic described herein. In some embodiments, the polynucleotide can comprise a nucleotide sequence encoding a VHH fragment that includes the FRs and CDRs of the antigen-binding molecules described herein. In some embodiments, the polynucleotide can comprise a nucleotide sequence encoding a light chain that includes the VL FRs and CDRs of the antigen-binding molecules described herein, or a nucleotide sequence encoding a heavy chain that includes the VH FRs and CDRs, and / or the Fc domain described herein. In one embodiment, the polynucleotide encodes a VH, VL, heavy chain, and / or light chain of the antigen-binding molecule described herein. In one embodiment, the polynucleotide encodes a first VH and a first VL of the antigen-binding molecule described herein. In one embodiment, the polynucleotide encodes a second VH and a second VL of the antigen-binding molecule described herein. In one embodiment, the polynucleotide encodes a first heavy chain and a first light chain of the antigen-binding molecule described herein. In one embodiment, the polynucleotide encodes a second heavy chain and a second light chain of the antigen-binding molecule described herein. In one embodiment, the polynucleotide encodes a VH and / or VL, or a heavy chain and / or light chain of the antigen-binding molecule described herein. 【0119】 In some embodiments, the polynucleotide comprises a nucleotide sequence encoding two or more Fc domains. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding two Fc domains. In some embodiments, the polynucleotide comprises a first nucleotide sequence encoding a first Fc domain and a second nucleotide sequence encoding a second Fc domain. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are contained within different nucleic acid molecules. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are contained within the same nucleic acid molecule. 【0120】 In some embodiments, the first and second nucleotide sequences also encode an antigen-binding molecule. In some embodiments, the first and second nucleotide sequences encode the same antigen-binding molecule. In some embodiments, the first and second nucleotide sequences encode different antigen-binding molecules. In some embodiments, the first nucleotide sequence encodes an Fc domain and an antigen-binding molecule, and the second nucleotide sequence encodes an Fc domain and an antigen-binding domain of a therapeutic antibody. The antigen-binding molecule encoded by the first and / or second nucleotide sequence can be any of those described herein. 【0121】 In some embodiments, the first and second nucleotide sequences also encode a peptide linker. In some embodiments, the first and second nucleotide sequences encode the same peptide linker. In some embodiments, the first and second nucleotide sequences encode different peptide linkers. In some embodiments, the first nucleotide sequence encodes an Fc domain, a peptide linker, and an antigen-binding molecule, and the second nucleotide sequence encodes an Fc domain and an antigen-binding domain from a therapeutic antibody. In some embodiments, the first nucleotide sequence encodes an antigen-binding molecule, a peptide linker, and an Fc domain, and the second nucleotide sequence encodes an Fc domain and an antigen-binding domain from a therapeutic antibody. The peptide linker encoded by the first and / or second nucleotide sequence can be any of those described herein. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 2, 3, or 4. 【0122】 Also provided herein is a polynucleotide encoding the polypeptide provided above, which is optimized, for example, by codon / RNA optimization, replacement with a heterologous signal sequence, and elimination of mRNA instability elements. Thus, methods for generating optimized nucleic acids for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in mRNA can be carried out, for example, by adapting the optimization methods described in U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498, all of which are hereby incorporated by reference in their entirety. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) within the RNA can be mutated without changing the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. The changes use the degeneracy of the genetic code and, for example, alternative codons for the same amino acid are used. In one embodiment, it may be desirable to change one or more codons to encode a conservative mutation, e.g., a similar amino acid having a similar chemical structure and properties and / or function as the original amino acid. 【0123】 A polynucleotide can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide can be determined by any method known in the art. The nucleotide sequences encoding the proteins described herein, and modified versions of these antibodies, can be determined using methods well known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled to generate a nucleic acid encoding the protein. Such polynucleotides encoding proteins can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994) BioTechniques 17:242-6, which is incorporated herein by reference in its entirety), briefly, this involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, the annealing and ligation of these oligonucleotides, and then the amplification of the ligated oligonucleotides by PCR. 【0124】 Alternatively, the polynucleotides encoding the proteins described herein can be generated from nucleic acids from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells that produce the polypeptide of interest. Such PCR amplification methods can be used to obtain a nucleic acid containing the sequence encoding the polypeptide. The amplified nucleic acid can be cloned into a vector for expression and further cloning in a host cell. 【0125】 Although clones containing nucleic acids encoding a specific polypeptide are not available, if the sequence of the polypeptide is known, the nucleic acid encoding the polypeptide can be obtained from a suitable source (e.g., a cDNA library generated from any tissue or cell expressing the polypeptides described herein, or a nucleic acid isolated therefrom, preferably polyA+RNA), by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence, or by using an oligonucleotide probe specific for a particular gene sequence, e.g., by cloning to identify a cDNA clone from a cDNA library encoding the polypeptide, and may be chemically synthesized or obtained. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art. 【0126】 The DNA encoding the proteins described herein can be readily isolated and sequenced using conventional procedures. Hybridoma cells can function as a source of such DNA. When isolated, the DNA can be placed within an expression vector, which can then be transfected into a host cell, e.g., E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or otherwise myeloma cells that do not produce the proteins described herein. 【0127】 Also provided are polynucleotides that hybridize to a polynucleotide encoding a protein described herein under high stringency, intermediate, or low stringency hybridization conditions. 【0128】 Hybridization conditions are described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions can include hybridization to filter-bound DNA in 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2× SSC / 0.1% SDS at about 50-65° C., and hybridization under highly stringent conditions can include hybridization to filter-bound nucleic acid in 6× SSC at about 45° C., followed by one or more washes in 0.1× SSC / 0.2% SDS at about 68° C. Hybridization under other stringent hybridization conditions is known to and described by those skilled in the art, for example, see Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pages 6.3.1-6.3.6 and 2.10.3, which is hereby incorporated by reference in its entirety. 【0129】 In one aspect, provided herein are cells (e.g., host cells), as well as related polynucleotides and expression vectors, that express (e.g., recombinantly express) the proteins described herein. Also provided herein are vectors (e.g., expression vectors) for recombinant expression in a host cell, preferably a mammalian cell (e.g., a CHO cell), that contain a polynucleotide that includes a nucleotide sequence encoding a protein described herein. Also provided herein are host cells for recombinant expression of a protein described herein that contain such a vector. In one aspect, provided herein is a method for producing a protein described herein, the method including expressing a polypeptide from a host cell. 【0130】 The recombinant expression of the proteins described herein generally involves constructing an expression vector containing a polynucleotide encoding a polypeptide. When a polynucleotide encoding a polypeptide described herein is obtained, a vector for the production of the polypeptide can be produced by recombinant DNA techniques using techniques well known in the art. Accordingly, methods for preparing a protein by expressing a polynucleotide containing a polypeptide encoding a nucleotide sequence are described herein. Methods well known to those skilled in the art can be used to construct an expression vector containing a polypeptide coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Also provided is a replicable vector comprising a nucleotide sequence encoding a polypeptide described herein, wherein the nucleotide sequence is operably linked to a promoter. Such vectors can include, for example, a nucleotide sequence encoding a constant region of a polypeptide (see, for example, WO 86 / 05807 and WO 89 / 01036, and U.S. Patent No. 5,122,464, which are hereby incorporated by reference in their entirety), and the variable region of the polypeptide can be cloned in such vectors for the expression of the entire heavy chain, the entire light chain, or both the entire heavy chain and the entire light chain. 【0131】 In one embodiment, the vector comprises a polynucleotide encoding an sdAb, Fab fragment, scFv, VHH fragment, VH, VL, heavy chain, and / or light chain of a polypeptide described herein. In another embodiment, the vector comprises a polynucleotide encoding VH and VL of a polypeptide described herein. In another embodiment, the vector comprises a polynucleotide encoding the heavy chain and light chain of a polypeptide described herein. 【0132】 An expression vector can be transferred into a cell (e.g., a host cell) by conventional techniques, and then the obtained cell can be cultured by conventional techniques to produce the polypeptide or a fragment thereof described herein. Accordingly, provided herein is a host cell containing a polynucleotide encoding the polypeptide or a fragment thereof described herein, or a heavy or light chain thereof, or a fragment thereof, or a single-chain antibody described herein, wherein the polynucleotide is operably linked to a promoter for expression of such a sequence in the host cell. 【0133】 In one embodiment, the host cell contains a polynucleotide comprising one of the above first nucleotide sequences and one of the above second nucleotide sequences. In another embodiment, the host cell contains a first polynucleotide comprising one of the above first nucleotide sequences and a second polynucleotide comprising one of the above first nucleotide sequences. In another embodiment, the host cell contains a first vector comprising one of the above first nucleotide sequences and one of the above second nucleotide sequences. In another embodiment, the host cell contains a first vector comprising one of the above first nucleotide sequences and one of the above second nucleotide sequences, and a second vector containing a second polynucleotide comprising one of the above first nucleotide sequences. 【0134】 In some embodiments, the antigen-binding molecule or fusion protein expressed by the first host cell associates with the antigen-binding molecule or fusion protein expressed by the second host cell to form a dimer. In some embodiments, provided herein is a population of host cells comprising such a first host cell and such a second host cell. 【0135】 In some embodiments, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding an antigen-binding molecule or a fusion protein, and a second vector comprising a polynucleotide encoding an antigen-binding molecule or a fusion protein. In some embodiments, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding a fusion protein, and a second vector comprising a polynucleotide encoding a therapeutic protein. In some embodiments, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding a fusion protein, and a second vector comprising a polynucleotide encoding an antigen-binding molecule. In some embodiments, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding an antigen-binding molecule, and a second vector comprising a polynucleotide encoding a therapeutic protein. 【0136】 A variety of host expression vector systems can be used to express the polypeptides described herein (see, e.g., U.S. Patent No. 5,807,715, which is hereby incorporated by reference in its entirety). Such host expression systems represent a medium in which the desired coding sequence can be produced and then purified, and also represent cells that can express the polypeptides described herein in situ when transformed or transfected with the appropriate nucleotide coding sequence. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis), e.g., bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing a protein coding sequence; yeast (e.g., Saccharomyces and Pichia), e.g., yeast transformed with recombinant yeast expression vectors containing a protein coding sequence; insect cell lines, e.g., insect cell lines infected with recombinant virus expression vectors (e.g., baculovirus) containing a protein coding sequence; plant cell lines (e.g., green algae, e.g., Chlamydomonas reinhardtii), e.g., plant cell lines infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing a protein coding sequence, or plant cell lines transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a protein coding sequence; or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, NIH 3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20, and BMT10 cells), e.g., mammalian cell lines carrying recombinant expression constructs containing a promoter derived from the genome of a mammalian cell (e.g., the metallothionein promoter) or a promoter derived from a mammalian virus (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter), but are not limited thereto.In one embodiment, the cells for expressing the proteins described herein are Chinese hamster ovary (CHO) cells, e.g., CHO cells from the CHO GS System™ (Lonza). In one embodiment, the heavy and / or light chains of the antibody produced by the CHO cells may have an N-terminal glutamine or glutamate residue replaced by pyroglutamic acid. In one embodiment, the cells for expressing the polypeptides described herein are human cells, e.g., a human cell line. In one embodiment, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In one embodiment, bacterial cells, e.g., Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used for the expression of recombinant polypeptides. For example, mammalian cells, e.g., CHO cells, together with a vector, e.g., a major immediate early gene promoter element from human cytomegalovirus, are an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45:101-5, and Cockett MI et al., (1990) Biotechnology 8(7):662-7, each of these references is incorporated herein by reference in its entirety). In one embodiment, the polypeptides described herein are produced by CHO cells or NS0 cells. In one embodiment, the expression of the nucleotide sequence encoding the polypeptides described herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter. 【0137】 In the bacterial system, several expression vectors can be advantageously selected depending on the intended use for the expression of molecules. For example, when large amounts of such polypeptides are produced for the generation of pharmaceutical compositions of antibody molecules, a vector that directs the expression of a high level of fusion protein product that is easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2:1791-1794), in which the coding sequence can be individually ligated into the vector in-frame with the lac Z coding region, whereby a fusion protein is produced, pUR278; and pIN vectors (Inouye S & Inouye M (1985) Nuc Acids Res 13:3101-3109, Van Heeke G & Schuster SM (1989) J Biol Chem 24:5503-5509), etc. All of these references are hereby incorporated by reference in their entirety. Also, for example, the pGEX vector can be used to express a foreign polypeptide as a fusion protein with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads, followed by elution in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or factor Xa protease cleavage site, whereby the cloned target gene product can be released from the GST moiety. 【0138】 In the insect system, for example, the Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequences can be individually cloned into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter). 【0139】 In mammalian host cells, several virus-based expression systems can be used. When adenovirus is used as an expression vector, the coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, such as a late promoter and a tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable and capable of expressing the molecule in the infected host (see, for example, Logan J & Shenk T (1984) PNAS 81(12):3655-9, which is incorporated herein by reference in its entirety). Also, specific initiation signals may be required for efficient translation of the inserted coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in the same reading frame as the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by including appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see, for example, Bitter G et al., (1987) Methods Enzymol. 153:516-544, which is incorporated herein by reference in its entirety). 【0140】 In addition, a host cell line can be selected that regulates the expression of the inserted array or modifies and processes the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the foreign protein being expressed. For this purpose, eukaryotic host cells having cell machinery for the appropriate processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In one embodiment, the proteins described herein are produced in mammalian cells, e.g., CHO cells. 【0141】 In one embodiment, the polypeptides described herein include a portion of an antibody having a reduced fucose content or no fucose content. Such proteins can be produced using techniques known to those of skill in the art. For example, the protein can be expressed in cells that are deficient or lacking in the ability to fucosylate. In one example, a cell line having a knockout of both alleles of α1,6-fucosyltransferase can be used to produce an antibody having a reduced fucose content. The Potelligent® system (Lonza) is an example of such a system that can be used to produce antibodies having a reduced fucose content. 【0142】 For the long-term high-yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines that stably express the proteins described herein can be manipulated. In one embodiment, the cells provided herein stably express an antigen-binding molecule, a fusion protein, or a therapeutic protein, and the antigen-binding molecule, fusion protein, or therapeutic protein associates to form the polypeptides described herein. 【0143】 In certain embodiments, rather than using an expression vector containing a viral origin of replication, the host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introduction of the foreign DNA / polynucleotide, the engineered cells can be allowed to grow in enriched medium for 1-2 days and then replaced with selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and grown into cell lines. This method can advantageously be used to engineer cell lines that express the polypeptides or fragments thereof described herein. Such engineered cell lines can be particularly useful in screening and evaluating compositions that interact directly or indirectly with the polypeptide. 【0144】 Several selection systems can be used, each of which includes, but is not limited to, the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1):223-32), hypoxanthine-guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) genes in tk, hgprt, or aprt cells, and all of these references are incorporated herein by reference in their entirety. Also, metabolic antagonist resistance can be used as the basis for selection for the following genes, dhfr that confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6):3567-70, O’Hare K et al., (1981) PNAS 78:1527-31); gpt that confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); neo that confers resistance to aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3:87-95, Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596, Mulligan RC (1993) Science 260:926-932, and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62:191-217, Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5):211-5); and hygro that confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3):147-56), and all of these references are incorporated herein by reference in their entirety.Methods generally known in the art of recombinant DNA technology are typically applied to select the desired recombinant clones, such methods are described, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993), Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990), and Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Colbere-Garapin F et al., (1981) J Mol Biol 150:1-14, and all of these references are incorporated herein by reference in their entirety. 【0145】 The expression level of the polypeptide can be increased by vector amplification (for a review, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, p. 163-188. DNA Cloning, Vol III, A Practical Approach. D.M. Glover (ed.) (Academic Press, New York, 1987), which reference is incorporated herein by reference in its entirety). When the marker in the vector system is amplifiable, an increase in the level of the inhibitor present in the culture of the host cells increases the number of copies of the marker gene. Also, since the amplified region is related to the gene of interest, the production of the polypeptide increases (Crouse GF et al., (1983) Mol Cell Biol 3:257-66, which reference is incorporated herein by reference in its entirety). 【0146】 The host cell can be co-transfected with two or more expression vectors described herein. The two vectors can contain the same selectable marker, and the same selectable marker enables equal expression of polypeptides, such as a first heavy chain and a second heavy chain polypeptide. The host cell can be co-transfected with different amounts of two or more expression vectors. For example, the host cell can be transfected with any one of the following ratios of a first expression vector and a second expression vector: about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. 【0147】 Alternatively, a single vector that can encode and express both polypeptides can be used. The coding sequence can include cDNA or genomic DNA. The expression vector can be monocistronic or polycistronic. The polycistronic nucleic acid construct can encode in the range of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes / nucleotide sequences, or in the range of 2 to 5, 5 to 10, or 10 to 20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct can include a promoter, a first gene, and a second gene in the following order. In such an expression vector, transcription of both genes can be driven by the promoter, translation of the mRNA from the first gene can be by a cap-dependent scanning mechanism, and translation of the mRNA from the second gene can be by a cap-independent mechanism, such as an IRES. 【0148】 The polypeptides described herein, when produced by recombinant expression, can be purified by any of the methods known in the art for protein purification, e.g., chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Further, the polypeptides described herein can be fused to the heterologous polypeptide sequences described herein, or otherwise heterologous polypeptide sequences known in the art, to facilitate purification. 【0149】 In one embodiment, the polypeptides described herein are isolated or purified. In one embodiment, an isolated polypeptide is substantially free of other polypeptides having antigenic specificities different from that of the isolated polypeptide. For example, in certain embodiments, preparations of the proteins described herein are substantially free of cellular material and / or chemical precursors. The phrase "substantially free of cellular material" includes preparations of a polypeptide wherein the polypeptide is isolated from the cellular components of the cell from which it was isolated or recombinantly produced. Thus, a polypeptide substantially free of cellular material has less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or variants of the polypeptide, e.g., polypeptides in different post-translational modification forms or other different versions of the polypeptide (e.g., polypeptide fragments). Also, when recombinantly produced, the polypeptide is generally substantially free of the culture medium, i.e., the culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When produced by chemical synthesis, the polypeptide is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from the chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such preparations of the protein have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the molecule of interest. In one embodiment, the polypeptides described herein are isolated or purified. 【0150】 The polypeptides described herein can be produced by any method known in the art for the synthesis of antibodies, e.g., by chemical synthesis or recombinant expression techniques. The methods described herein use conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the art, unless otherwise indicated. These techniques are described, for example, in the references cited herein and are fully explained in the literature.For example, reference is made to Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates), Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates), Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press, Birren B et al., (ed.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and the entire contents of all of these references are hereby incorporated by reference into this specification. 【0151】 In one embodiment, the polypeptides described herein are prepared, expressed, produced, or isolated by any means including making, for example, by synthesis of a DNA sequence, through genetic manipulation. In one embodiment, such polypeptides contain sequences (e.g., DNA sequences or amino acid sequences) that do not naturally occur within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo. 【0152】 Pharmaceutical composition In one aspect, the present disclosure provides a pharmaceutical composition comprising a fusion protein disclosed herein for use in a method of treating a disease or disorder. In another aspect, the present disclosure provides a pharmaceutical composition comprising an antigen-binding molecule disclosed herein for use in a method of treating a disease or disorder. 【0153】 The formulations disclosed herein include bulk pharmaceutical compositions useful in the manufacture of pharmaceutical compositions (e.g., compositions suitable for administration to a subject or patient) that can be used in the preparation of unit dosage forms. In one embodiment, the compositions of the invention are pharmaceutical compositions. Such compositions include a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents of the invention (e.g., a fusion protein) (or other prophylactic or therapeutic agent) and a pharmaceutically acceptable carrier. 【0154】 In some embodiments, the pharmaceutical composition is formulated for administration via any suitable route of administration to a subject, and the routes of administration include, but are not limited to, intramuscular, intravenous, intradermal, intraperitoneal, subcutaneous, epidural, nasal, oral, rectal, topical, inhalation, buccal (e.g., sublingual), and transdermal administration. In one embodiment, the pharmaceutical composition is formulated to be suitable for intravenous administration to a subject. In one embodiment, the pharmaceutical composition is formulated to be suitable for subcutaneous administration to a subject. 【0155】 Method The present disclosure provides a method for increasing the serum half-life of a therapeutic protein, the method comprising fusing an antigen-binding molecule of the invention to the therapeutic protein. The therapeutic protein can be any therapeutic protein described herein or any therapeutic protein known in the art otherwise. 【0156】 In some embodiments, the clearance of a fusion protein comprising an antigen-binding molecule of the invention and a therapeutic protein is decreased in a subject, following a single therapeutic administration of the fusion protein, as compared to the clearance of the therapeutic protein following a single therapeutic administration of the therapeutic protein. In some embodiments, the clearance of the fusion protein is decreased in a subject, following a single therapeutic administration of the fusion protein, by at least 1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 15-fold, or at least 20-fold as compared to the clearance of the therapeutic protein following a single therapeutic administration of the therapeutic protein. 【0157】 In some embodiments, the clearance of a fusion protein comprising an antigen-binding molecule of the invention and a therapeutic protein is decreased in a subject, following a single administration of the fusion protein, as compared to the clearance of the therapeutic protein following a single administration of an equivalent dose of the therapeutic protein. In some embodiments, the clearance of the fusion protein is decreased in a subject, following a single administration of the fusion protein, by at least 1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 15-fold, or at least 20-fold as compared to the clearance of the therapeutic protein following a single administration of an equivalent dose of the therapeutic protein. 【0158】 In some embodiments, the terminal half-life (t 1 / 2,z ) of a fusion protein comprising an antigen-binding molecule of the invention and a therapeutic protein is increased in a subject, following a single therapeutic administration of the fusion protein, as compared to the t 1 / 2,z of the therapeutic protein following a single therapeutic administration of the therapeutic protein. In some embodiments, the t 1 / 2,z of the fusion protein is increased in a subject, following a single therapeutic administration of the fusion protein, as compared to the t 1 / 2,zIncreases by at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 15-fold, or at least 20-fold compared to 【0159】 In some embodiments, t of the fusion protein, which comprises an antigen-binding molecule of the invention and a therapeutic protein 1 / 2,z In a subject, after a single administration of the fusion protein, is increased compared to t of the therapeutic protein after a single administration of an equivalent dose of the therapeutic protein 1 / 2,z In some embodiments, t of the fusion protein 1 / 2,z In a subject, after a single administration of the fusion protein, is increased compared to t of the therapeutic protein after a single administration of an equivalent dose of the therapeutic protein 1 / 2,z Increases by at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 15-fold, or at least 20-fold compared to 【0160】 In some embodiments, the potency of the fusion protein, which comprises an antigen-binding molecule of the invention and a therapeutic protein, is the same as or essentially the same as the potency of the therapeutic protein. In some embodiments, the potency of the fusion protein is decreased by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less compared to the potency of the therapeutic protein. 【0161】 In some embodiments, the potency of one dose of the fusion protein, which comprises an antigen-binding molecule of the invention and a therapeutic protein, is the same as or essentially the same as the potency of an equivalent dose of the therapeutic protein. In some embodiments, the potency of one dose of the fusion protein is decreased by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less compared to the potency of an equivalent dose of the therapeutic protein. 【0162】 In one embodiment, the fusion protein or antigen-binding molecule does not antagonize the binding of FcRn to albumin. 【0163】 In one embodiment, the level of albumin in the subject does not decrease after administration of the fusion protein or antigen-binding molecule compared to the baseline level of albumin. In one embodiment, an albumin reduction of less than about 1%, 2%, 3%, 4%, or 5% is observed compared to the baseline albumin level. In one embodiment, an albumin reduction of less than about 10% is observed compared to the baseline albumin level. 【0164】 The present disclosure also provides a method for treating a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a fusion protein according to the present disclosure or a pharmaceutical composition comprising the same. 【0165】 In one embodiment, the fusion protein is administered to the subject simultaneously or sequentially with an additional therapeutic agent. In one embodiment, the additional therapeutic agent is an anti-inflammatory agent. In one embodiment, the additional therapeutic agent is a corticosteroid. In one embodiment, the additional therapeutic agent is rituximab, daclizumab, basiliximab, muromonab-CD3, infliximab, adalimumab, omalizumab, efalizumab, natalizumab, tocilizumab, eculizumab, golimumab, canakinumab, ustekinumab, or belimumab. In one embodiment, the additional therapeutic agent is a leukocyte depletion agent. 【0166】 In one embodiment, the additional therapeutic agent is a B cell depletion agent. In one embodiment, the B cell depletion agent is an antibody. In one embodiment, the B cell depletion antibody is an antibody that specifically binds to CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD70, CD72, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, or CD86. 【0167】 In some embodiments, the fusion protein is administered intravenously. In some embodiments, the fusion protein is administered intravenously once a week, once every two weeks, once every three weeks, once every four weeks, once a month, or once every six weeks. 【0168】 In some embodiments, the fusion protein is administered subcutaneously. In some embodiments, the fusion protein is administered subcutaneously once a week, once every two weeks, once every three weeks, once every four weeks, once a month, or once every six weeks. 【Examples】 【0169】 The following examples are provided by way of illustration and not limitation. 【0170】 Example 1: Generation of pH-Dependent Anti-Albumin VHH / scFV Briefly, two llamas were immunized with humans using standard procedures to generate a phage display library (VHH / scFv). Selection was performed by phage binding to HSA and MSA at pH 5.5 and elution at pH 7.4 (using trypsin as a control). Screening of the selected clones was performed by ELISA and Biacore (human, mouse, and cynomolgus monkey serum albumin) using pH 5.5 or 7.4. Typically, pH-dependent clones are selected based on their reduced binding at pH 7.4. The characteristics of the selected clones generated are shown in Table S1. Table S1. Binding and sequence characteristics of selected VHH clones. 【Table 1】 【0171】 Sensorgrams showing the characteristics of selected clones 2H11 and 11C03 as monovalent (VHH) and bivalent (VHH-Fc) molecules binding to HSA (pH 5.5) on Biacore are shown in Figure 1. Association was performed for each run at pH 5.5. One clone, 2H11, shows good pH-dependence, does not show cross-reactivity with mouse and cynomolgus monkey serum albumin, does not bind to isolated DII, and does not compete with Alb23. 【0172】 Since the binding of VHH to albumin is only present in endosomes (acidic pH) and very restricted in circulation (pH 7.4) (at most, no binding), it is necessary to adjust the affinity of VHH. In fact, the percentage of VHH:albumin complex mainly depends on the affinity at a specific pH. Assuming that the concentration of albumin is 34 - 54 mg / mL in circulation and endosomes, when the affinity of VHH for albumin is approximately 2.4 mM (calculated for an albumin concentration of 40 mg / ml), it can be calculated that 80% of VHH does not bind (is free). Similarly, the affinity must be 0.12 mM (or less) at pH 5.5 in order to obtain that at least 80% of VHH binds to albumin (in endosomes). Ideally, less than 5% of VHH binds to albumin at pH 7.4 (affinity of 11.5 mM or more), and more than 95% of VHH binds to albumin at acidic pH (affinity of 30 nM or less). 【0173】 To reduce its affinity at pH 7.4, 2H11 was subjected to alanine scanning of all three CDRs. The resulting variant VHHs were produced as a two-arm type Fc-ABDEG-20GS-C-terminal fusion and analyzed by FcRn ELISA QC, and Biacore (3000) with human, cynomolgus monkey, and mouse albumin on the chip (selection criteria: binding at pH 5.5 remains, binding at pH 7.4 is reduced), and Biacore T200 with Fc-ABDEG-VHH on the chip (selection criteria: lowest affinity at pH 7.4, highest affinity at pH 5.5). 【0174】 Interestingly, the mutations in the CDR3 region of 2H11 increased the pH-dependence by maintaining good binding at pH 5.5 while reducing binding at pH 7.4. 【0175】 Another clone selected for alanine scanning, 11C03, was cross-reactive with mouse and cynomolgus monkey serum albumin and also showed pH-dependent binding. For this clone, there was no significant effect of alanine mutations in CDR3 on pH-dependence. Thus, binding to HSA also varied by alanine scanning in CDR1 and CDR2. A panel of variants showing reduced binding and different binding at pH 7.4 versus pH 5.5 was identified in binding to human (HSA) and cynomolgus monkey albumin (CSA, Table S3). 【0176】 Data from the selected 2H11 and 11C03 CDR3 variants are provided in Table S2. Table S2: Analysis of 2H11 and 11C03 CDR3 variants fused to albumin on chip at the C-terminus of Fc-ABDEG (Biacore 3000). 【Table 2】 *Describe the difference in off-rate. 【0177】 Data from the selected 2H11 and 11C03 CDR1-2 variants are provided in Table S3. Table S3: Analysis of 2H11 (CDR3) and 11C03 (CDR1-2) alanine variants fused to albumin on chip at the C-terminus of Fc-ABDEG that bind to human and cynomolgus monkey albumin (Biacore 3000). 【Table 3】 *Describe the difference in off-rate 【0178】 The sequences of 2H11 parental VHHs and selected variants, 11C03 parental VHHs and selected variants, additional VHH clones, and scFv clones generated from a phage display library, as well as the sequence for Alb23 VHH, are provided in Tables S4 - S8 below. [Table 4] TIFF2025518971000006.tif242170TIFF2025518971000007.tif248170 [Table 5] TIFF2025518971000009.tif248170TIFF2025518971000010.tif248170TIFF2025518971000011.tif249170 [Table 6] [Table 7] [Table 8] TIFF2025518971000015.tif248170 【0179】 Example 2: Evaluation of pH - dependent anti - albumin VHHs for extending the half - life of Fab fragments in Albumus mice™ independently of IgG - FcRn - mediated recycling. The affinity requirements for extending the half - life with anti - albumin VHHs having different degrees of pH - dependence (stronger binding at pH 5.5, weaker binding at pH 7.4) were evaluated with Mota - Fab - VHH fusions. Seven different test substances were intraperitoneally injected (IP single dose) into Albumus mice™ (HSA / hFcRn), which can mimic physiological antibody clearance in humans, to evaluate their clearance in this mouse model. The administered test substances are described in detail below. 1. The motavizumab-Fab fragment was used as a control treatment condition. This specific molecule has the same basic structure as the test article of interest but is smaller in size (about 50 kDa). 2. Motavizumab-Fab with an irrelevant VHH 3Rab fused at the C-terminus to the light chain using a 20GS linker was used as a second control treatment condition. This specific molecule has a similar size (about 65 kDa) to the test article of interest but has no affinity for albumin. 3. Motavizumab-Fab fragment with Alb23 fused at the C-terminus to the light chain using a 20GS linker. Alb23 is an anti-albumin VHH. This molecule shows high affinity for albumin, and since this affinity is pH-dependent, this treatment condition was included. 4. Motavizumab-Fab fragment with 2H11 fused at the C-terminus to the light chain using a 20GS linker. Previous in vitro studies have shown that this variant has high affinity for albumin at pH 5.5, but this affinity is slightly reduced at pH 7.4, indicating further pH-dependent affinity. 5. Motavizumab-Fab fragment with v3, v8, v9, or v15, which are 2H11 variants, fused at the C-terminus to the light chain using a 20GS linker. After alanine scanning and in vitro characterization, these 2H11 variants carry alanine mutations at CDR3 (positions 3, 8, 9, and 15 respectively) and have reduced affinity compared to the parental molecule 2H11 at both pH 5.5 and pH 7.4, but show a stronger pH-dependent affinity. 【0180】 The doses for the test articles were 19 mg / kg (wt Fab) and 25 mg / kg (C-terminal fusion Fab fragment) according to the equimolar dosing of 30 mg / kg of Fc-ABDEG-Alb23 used as a reference in previous studies. 【0181】 For practical reasons, the experiment was divided into two parts and the two parts were carried out consecutively. ● Experiment 1: Treatment groups 1, 2, 3, 4 ● Experiment 2: Treatment groups 4, 5, 6, 7, 8 【0182】 In total, 45 male and female Albumus mice (trademark) were used for the two experiments (treatment group 4 was included in both studies). Albumus mice (trademark) approximately 14 - 15 weeks of age were homozygous for the human FcRn transgene and the human albumin transgene. The mice were marked on the tail for identification and individually housed at a maximum density of 4 mice per cage in polysulfone cages actively ventilated with HEPA-filtered air. The normal temperature and relative humidity ranges in the animal room were 22 ± 4°C and 50 ± 15%, respectively. Filtered tap water acidified to a pH of 2.5 - 3.0 and standard laboratory solid feed were provided ad libitum. 1. Albumus mice (trademark) were randomly assigned to 8 groups of 5 mice / group. 2. On day - 3 (-3d), the mice were pre-loaded with IVIg. 3. At 0 hours on day 0, the test articles were administered IP to the mice as follows. ● Mota-Fab at 19 mg / kg, n = 5, group 1 ● Mota-Fab-LC-3Rab (Mota-Fab-3Rab) at 25 mg / kg, n = 5, group 2 ● Mota-Fab-LC-Alb23 (Mota-Fab-Alb23) at 25 mg / kg, n = 5, group 3 ● Mota-Fab-LC-2H11 (Mota-Fab-2H11) at 25 mg / kg, n = 5, group 4 ● Mota-Fab-LC-2H11v3 (Mota-Fab-2H11v3) at 25 mg / kg, n = 5, group 5 ● Mota-Fab-LC-2H11v8 (Mota-Fab-2H11v8) at 25 mg / kg, n = 5, group 6 ● Mota-Fab-LC-2H11v9 (Mota-Fab-2H11v9) at 25 mg / kg, n = 5, group 7 ● Mota-Fab-LC-2H11v15 (Mota-Fab-2H11v15) at 25 mg / kg, n = 5, group 8 4.20 μL of blood samples were collected from each mouse via the tail vein at 2 h, 1 d, 2 d, 3 d, 4 d, 7 d, and 8 d according to the blood collection schedule shown in Table S9 below. 5. Serum samples were evaluated in the following ELISA-based assays (in order of priority). a. Test article PK ELISA b. Human serum albumin ELISA 【0183】 Also, the protocol is summarized in Figure 2 and Table S9. Readouts in the collected mouse sera were performed using PK and HSA assays. Table S9. In vivo protocol 【Table 9】 【0184】 The serum concentration of Mota-Fab-VHH was plotted over time as the mean per group during the course of the study. The serum concentration of Mota-Fab-VHH is shown in Figure 3. Data points represent the mean ± SD of 5 animals per group. 【0185】 Mota-Fab-VHHs that did not bind to albumin, such as Mota-Fab (∼50 kDa) and Mota-Fab-3Rab (∼65 kDa), showed relatively short half-lives and had undetectable serum concentrations after 1 day. This data indicates that molecular weight is not the main driver of serum persistence. Mota-Fab-2H11, which contains a reverse pH-dependent anti-albumin VHH, showed a half-life equivalent to that of Mota-Fab-Alb23. Mota-Fab bound to the 2H11 alanine variant, which has a reduced affinity for albumin at pH 7.4 (see Table S10 using Biacore 8K+ in a single-cycle kinetics (SCK) protocol. Human serum albumin protein was immobilized on a CM5 chip and the Fab-VHH variants were injected at 30 μl / min for 2 min in solution in a 8-step 1.5-fold dilution series in 1× HBS-EP+ at pH 7.4 or citrate buffer at pH 5.5.). showed serum half-lives corresponding to their albumin binding affinities. Mota-Fab-2H11-v9 and Mota-Fab-2H11-v15 showed reduced half-lives compared to Mota fusions with higher affinity for HSA but still showed an extended PK profile. In contrast, Mota-Fab-2H11-v8 did not show detectable binding to albumin at both pH 5.5 and pH 7.4 in SPR (data not shown) and showed a half-life similar to that of Mota-Fab and Mota-Fab-3Rab. Table S10. Binding kinetic properties and theoretical anti-albumin: albumin complex formation 【Table 10】 【0186】 This suggests that Mota-Fab-2H11v9 and -2H11v15 have very low binding to HSA in serum and are thus recycled only through binding to albumin in endosomes. As shown in Figure 4, anti-HSA-VHHs with reduced binding to HSA in serum but increased binding in endosomes tend to result in the recycling of both HSA and anti-HSA-VHH without affecting the size of the anti-HSA-VHH. The inventors have shown that clones that bind to HSA in a pH-dependent manner are expected to have a desirable half-life and better in vivo distribution in tissues and tumors upon administration. *** 【0187】 The invention, to the extent, is not limited by the specific embodiments described herein. Indeed, various modifications of the invention will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

[Claim 1] An antigen-binding molecule that specifically binds to HSA, wherein the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 and / or pH 6.0 is less than 0.140 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 2.4 mM. [Claim 2] The antigen-binding molecule according to claim 1, wherein the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 5.5 is less than 0.03 mM, and the equilibrium dissociation constant of the antigen-binding molecule to HSA at pH 7.4 is at least 11.5 mM. [Claim 3] An antigen-binding molecule that specifically binds to HSA, wherein the binding affinity of the antigen-binding molecule to HSA at pH 5.5 and / or pH 6.0 is at least 100 times higher than the binding affinity of the antigen-binding molecule to HSA at pH 7.

4. [Claim 4] The antigen-binding molecule according to claim 1 or 3, wherein the binding affinity is measured by surface plasmon resonance. [Claim 5] The antigen-binding molecule according to claim 1 or 3, wherein the antigen-binding molecule is selected from a Fab fragment, sdAb, scFv, antibody mimetic, HSA, or an antigen-binding fragment thereof. [Claim 6] The antigen-binding molecule according to claim 5, wherein the sdAb is a VHH fragment. [Claim 7] The antigen-binding molecule according to claim 6, further comprising one or more amino acids added to the C-terminus of the VHH fragment. [Claim 8] The one or more amino acids mentioned above a) A, b) AG, c) GG, and d) The antigen-binding molecule according to claim 7, selected from the group consisting of PP. [Claim 9] A fusion protein comprising an antigen-binding molecule according to claim 1 or 3 and a therapeutic protein. [Claim 10] The fusion protein according to claim 9, wherein the therapeutic protein is a low molecular weight peptide therapeutic agent. [Claim 11] The fusion protein according to claim 9, wherein the therapeutic protein is an antibody or a fragment thereof, and optionally an Fc region or an antigen-binding fragment. [Claim 12] The fusion protein according to claim 9, wherein the antigen-binding molecule is fused to the therapeutic protein via a linker. [Claim 13] The fusion protein according to claim 12, wherein the linker is an inclementable linker. [Claim 14] The fusion protein according to claim 12, wherein the linker is a peptide linker. [Claim 15] The fusion protein according to claim 14, wherein the peptide linker is a GS linker, optionally having a length of 8 to 40 amino acids, and optionally having a length of 20 amino acids. [Claim 16] The fusion protein according to claim 11, wherein the antigen-binding molecule is fused to the antibody or a fragment thereof via an IgG hinge region or a portion thereof. [Claim 17] The fusion protein according to claim 11, wherein the therapeutic protein is an Fc region comprising a first Fc domain and a second Fc domain, and the antigen-binding molecule is fused to the first Fc domain or the second Fc domain via an IgG hinge region or a portion thereof. [Claim 18] One or more isolated polynucleotides encoding the antigen-binding molecule described in claim 1 or 3. [Claim 19] An expression vector comprising one or more isolated polynucleotides as described in claim 18. [Claim 20] A host cell comprising one or more isolated polynucleotides as described in claim 18. [Claim 21] A method for producing an antigen-binding molecule, comprising culturing the host cells described in claim 20 under conditions that enable the expression of the antigen-binding molecule or fusion protein. [Claim 22] A pharmaceutical composition comprising an antigen-binding molecule according to claim 1 or 3 and at least one pharmaceutically acceptable carrier. [Claim 23] A pharmaceutical composition according to claim 22 for use as a medicine. [Claim 24] A method for increasing the serum half-life of a therapeutic protein, comprising fusing the antigen-binding molecule described in claim 1 or 3 to the therapeutic protein. [Claim 25] Use of the antigen-binding molecule according to claim 1 or 3 to increase the serum half-life of a therapeutic protein. [Claim 26] Use of the antigen-binding molecule according to claim 1 or 3 for the manufacture of a pharmaceutical product.

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