PH dependent HSA binding molecules and methods of use

Abstract

Provided herein are pH dependent HSA binding molecules. Also provided herein 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

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 352,589, filed on June 15, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to pH-dependent human serum albumin (HSA) binding molecules and methods of making and using the same. Background Art

[0004] HSA is the most abundant plasma protein and plays an important role in the binding and transport of common therapeutic agents. Therefore, HSA is an interesting target for exploring and optimizing the pharmacokinetics and pharmacodynamics of therapeutic agents.

[0005] However, care is needed to keep HSA levels in the blood at healthy levels (normal range is 3.4 to 5.4 g / dL (34 to 54 g / L) because HSA maintains fluid balance in the body, helps prevent blood vessels from leaking too much, has a role in repairing tissues and helping the body grow, while transporting hormones, nutrients, and enzymes throughout the body.

[0006] Current techniques for extending the half-life of therapeutics using HSA binding are achieved through continuous binding to HSA (e.g., under all physiological conditions), thereby significantly increasing the overall size of the therapeutic (HSA is an approximately 66 kDa protein), which can affect efficacy and function through steric hindrance and reduced distribution to tissue and tumor penetration.

[0007] Therefore, 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

[0008] The present disclosure is broadly directed to HSA binding molecules and fusion proteins thereof that preferentially bind to HSA at acidic pH and have reduced or no binding to HSA at physiological pH.

[0009] 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 with 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 with HSA at pH 7.4 is at least 2.4 mM.

[0010] In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 5.5 is less than 0.03 mM, and the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 7.4 is at least 11.5 mM.

[0011] 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 times the binding affinity of the antigen binding molecule to HSA at pH 7.4. In some embodiments, the HSA binding molecule has a binding affinity for HSA at acidic pH that 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 greater than the binding affinity of the HSA binding molecule for HSA at physiological pH. In some embodiments, the acidic pH is pH 5.5 and / or pH 6.0. In some embodiments, physiological pH is pH 7.4.

[0012] In some embodiments, binding affinity is measured by surface plasmon resonance.

[0013] In some embodiments, the antigen binding molecule is selected from a Fab fragment, an sdAb, a scFv, an antibody mimetic, or an antigen binding fragment thereof. In some embodiments, the sdAb is a VHH fragment, which 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.

[0014] In one aspect, a fusion protein is provided, comprising an antigen binding molecule as described herein and a therapeutic protein.

[0015] In some embodiments, the therapeutic protein is a small peptide therapeutic.

[0016] In some embodiments, the therapeutic protein is an antibody or fragment thereof, optionally an Fc region or an antigen binding fragment.

[0017] 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, optionally 20 amino acids in length.

[0018] In some embodiments, the antigen binding molecule is fused to the antibody or fragment thereof via the IgG hinge region or a portion thereof.

[0019] 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.

[0020] Also provided are one or more isolated polynucleotides encoding any of the antigen binding molecules described herein or any of the fusion proteins described herein.

[0021] Also provided is an expression vector comprising any one or more of the isolated polynucleotides described herein.

[0022] Also provided is a host cell comprising any one or more of the isolated polynucleotides described herein or any expression vector described herein.

[0023] Also provided is a method for producing an antigen binding molecule or a fusion protein, the method comprising culturing a host cell as described herein under conditions that allow expression of the antigen binding molecule or the fusion protein.

[0024] 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.

[0025] Also provided is an antigen binding molecule as described herein, or a fusion protein as described herein, or a pharmaceutical composition thereof for use as a medicament.

[0026] Also provided is a method of increasing the serum half-life of a therapeutic protein, the method comprising fusing an antigen binding molecule as described herein to the therapeutic protein.

[0027] Also provided is the use of an antigen binding molecule as described herein for increasing the serum half-life of a therapeutic protein.

[0028] 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

[0029] Figure 1 is a series of sensorgrams showing the properties of selected clones 2H11 and 11C03 as monovalent and bivalent (VHH-Fc) molecules binding to HSA (pH 5.5) on Biacore. Association was performed at pH 5.5 for each run. Dissociation was performed at pH 5.5 or 7.4 to study the pH dependence shown.

[0030] Figure 2 Albumus mice were used to study TMSchematic representation of the PK and HSA binding scheme of Motavizumab (Mota)-Fab fusion protein.

[0031] Figure 3 Albumus mice TM A line graph showing the concentration of Mota-Fab fusion protein in serum versus time.

[0032] Figure 4 is a schematic diagram showing the potential mechanism of endocytic recycling in anti-HSA-VHH, where HSA is bound weakly at pH 7.4 and strongly at pH 5.5. DETAILED DESCRIPTION

[0033] The present disclosure provides engineered antigen binding molecules and fusion proteins thereof that specifically bind to HSA (HSA binding molecules) at acidic pH and do not bind (or weakly bind) HSA at physiological pH. Also provided herein are nucleic acids encoding such HSA binding molecules or fusion proteins, vectors, host cells, methods of manufacture, and methods of use thereof.

[0034] definition

[0035] As used herein, the term "antigen binding molecule" refers to any polypeptide that specifically binds to an antigen. Examples of antigen binding domains include polypeptides derived from antibodies, such as Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), single-chain Fv (scFv), CDR, VH domain (VH), VL domain (VL), single domain antibodies (sdAb), VHH fragments, camel antibodies, and antigen binding fragments of any of the above. The term also encompasses synthetic antigen binding proteins or antibody mimics, such as, for example, anticalins and DARPins.

[0036] 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.

[0037] As used herein, the term "HSA binding molecule" refers to an antigen binding molecule that specifically binds to HSA. In some embodiments, HSA comprises an amino acid sequence that is at least 95% identical to the amino acid sequence provided in GenBank Accession No.: AAA98797.1. In some embodiments, HSA comprises an amino acid sequence provided in GenBank Accession No.: AAA98797.1.

[0038] 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 a two-molecule binding complex to dissociate into two free molecules. Therefore, as the binding affinity increases, K D reduce.

[0039] As used herein, the term "specific binding" 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 may bind to other molecules, usually with lower affinity, as determined by, for example, immunoassays, BIAcore TM , KinExA 3000 instrument (Sapidyne Instruments, Boise, supra) or other assays known in the art. In a specific embodiment, a molecule that specifically binds to a given target has a K greater than that when the molecule non-specifically binds to another target. D K of at least 2 logs, 2.5 logs, 3 logs, 4 logs or less D Binds to antigen.

[0040] 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.

[0041] 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 a 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.

[0042] As used herein, the term "fusion protein" refers to a protein formed by fusing at least one antigen binding molecule described herein with at least one therapeutic protein (or a fragment or variant thereof).

[0043] As used herein, the term "fusion" refers to the connection of two peptides via a peptide bond or a peptide linker. In some embodiments, the two proteins are fused together directly and continuously via a peptide bond. In some embodiments, the two proteins are fused indirectly and non-continuously via a peptide linker. In some embodiments, one protein is fused to a peptide linker via a peptide bond at a first position, and the second protein is fused to a peptide linker via a peptide bond at a second position.

[0044] As used herein, the term "operably linked" refers to polynucleotide sequence elements being linked in a functional relationship. For example, when a polynucleotide sequence is in a functional relationship with another polynucleotide sequence, the polynucleotide sequence is operably linked. In some embodiments, if a transcriptional regulatory polynucleotide sequence (e.g., a promoter, enhancer, or other expression control element) affects the transcription of a polynucleotide sequence encoding a protein, the transcriptional regulatory polynucleotide sequence is operably linked to a polynucleotide sequence encoding the protein. Operably linked elements may be continuous or non-continuous.

[0045] As used herein, the term "therapeutic protein" refers to a protein, polypeptide, antibody, peptide, or fragment or variant thereof having 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 and fragments and variants thereof. Thus, the protein disclosed herein may contain at least one fragment or variant of a therapeutic protein, and / or at least one fragment or variant of an antibody. In addition, the term "therapeutic protein" may refer to an endogenous or naturally occurring relative of a therapeutic protein.

[0046] A protein that exhibits "therapeutic activity" or a protein with "therapeutic activity" refers to a protein that has one or more known biological and / or therapeutic activities associated with a therapeutic protein, such as one or more therapeutic proteins described herein or otherwise known in the art. As a non-limiting example, a "therapeutic protein" is a protein that can be used to treat, prevent, or improve a disease, condition, or illness. As a non-limiting example, a "therapeutic protein" can be a protein that specifically binds to a specific cell type (normal (e.g., lymphocytes) or abnormal (e.g., cancer cells)), and therefore can be used to specifically target a compound (drug or cytotoxic agent) to that cell type. Examples of "therapeutic proteins" include, but are not limited to, interferons, enzymes, hormones, growth factors, interleukins, coagulation factors, antibodies, and fragments and variants thereof.

[0047] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin S and Altschul SF, (1990) PNAS 87:2264-2268, as modified in Karlin S and Altschul SF, (1993) PNAS 90:5873-5877, each of which is 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, which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameter set (e.g., with score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameter set (e.g., with score = 50, word length = 3) to obtain amino acid sequences homologous to protein molecules described herein. In order to obtain gap alignments for comparison purposes, gap BLAST as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402 can be utilized, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterative search for distance relationships between detection molecules. Same as above. When utilizing BLAST, gap BLAST, and PSI BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17, which is incorporated herein by reference in its entirety. This 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.

[0048] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating the percent identity, typically only exact matches are counted.

[0049] As used herein, the term "antibody" or "antibodies" includes 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 chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody drug conjugates, single domain antibodies (sdAbs), 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-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the foregoing. The antibody can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or species (e.g., mouse IgG 2a or IgG 2b ).

[0050] 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 may be a wild-type Fc region (native Fc region) or a variant Fc region. The native Fc region is a homodimer. The Fc region may be derived from any natural immunoglobulin. In some embodiments, the Fc region is formed by IgA, IgD, IgE or IgG heavy chain constant regions. In some embodiments, the Fc region is formed by IgG heavy chain constant regions. 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 by an IgG1 heavy chain constant region. In some embodiments, the IgG1 heavy chain constant region comprises G1m1 (a), G1m2 (x), G1m3 (f) or G1m17 (z) allotypes. See, e.g., 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 entireties.

[0051] As used herein, the term "variant Fc region" refers to a variant of an Fc region having one or more changes relative to a native Fc region. The changes may include amino acid substitutions, additions and / or deletions, attachment of additional moieties, and / or changes in native glycans. The term encompasses heterodimeric Fc regions in which each of the constituent Fc domains is different. The term also encompasses single-chain Fc regions in which the constituent Fc domains are linked together by a linker moiety.

[0052] As used herein, the term "Fc domain" refers to a portion of a single immunoglobulin heavy chain that includes both the CH2 domain and the CH3 domain of an antibody. In some embodiments, the Fc domain includes at least a portion of the hinge region (e.g., upper, middle and / or lower hinge region), the CH2 domain, and the CH3 domain. In some embodiments, the Fc domain does not include a hinge region.

[0053] As used herein, the term "hinge region" refers to the portion of the heavy chain molecule that joins 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, the hinge region comprises about 11 to 17 amino acid residues and is flexible, thereby allowing 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: upper, middle and lower hinge domains. The antigen binding molecules or fusion proteins of the present disclosure may include all or any part of the hinge region. In some embodiments, the hinge region is from an IgG1 antibody. In some embodiments, the hinge region comprises the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO: 1).

[0054] As used herein, the term "FcRn" refers to the neonatal Fc receptor. Exemplary FcRn molecules include 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.

[0055] As used herein, the terms "treat", "treating", and "treatment" refer to therapeutic or preventive measures as described herein. The method of "treating" employs administering a polypeptide to a subject suffering from a disease or disorder or susceptible to such a disease or disorder to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or to prolong the survival of the subject beyond the expected survival in the absence of such treatment. In some embodiments, the method of "treating" employs administering a polypeptide to a subject suffering from a disease or disorder or susceptible to such a disease or disorder to prevent, cure, delay, reduce the severity of, or ameliorate the disease or disorder or recurring disease or disorder.

[0056] As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to that amount of the therapy that achieves the desired prophylactic or therapeutic effect.

[0057] As used herein, the term "dose" or "administration" refers to the amount of a pharmaceutical agent administered to a subject in a single administration.

[0058] As used herein, the term "equivalent dose" refers to the dose of a first therapeutic agent and a second therapeutic agent, wherein the number of molecules of the first agent and the second agent is approximately the same. In some embodiments, an equivalent dose is an equimolar dose. As used herein, the term "equimolar dose" refers to the dose of a first therapeutic agent and a second therapeutic agent, wherein the number of moles of the first agent and the second agent is the same. In some embodiments, the equivalent dose is calculated using the observed molecular weight of the first agent and the second agent. In some embodiments, the equivalent dose is calculated using the predicted molecular weight of the first agent and the second agent. 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.

[0059] As used herein, the terms "pharmacokinetics" and "PK" refer to the effect of an 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 metabolic rate and / or clearance rate of the therapeutic agent. As used herein, the terms "improved pharmacokinetics" or "improved PK" refer to an improvement in an organism's desired effect on a therapeutic agent administered to the organism. In some embodiments, improved pharmacokinetics include an improvement in the half-life (T / V) of the therapeutic agent in a subject. 1 / 2 ), clearance rate or area under the curve (AUC). In some embodiments, the therapeutic agent is a fusion protein or therapeutic protein as described herein.

[0060] 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.

[0061] As used herein, when referring to a measurable value, such as a dosage, the term "about" or "approximately" encompasses variations of ±20%, ±15%, ±10%, ±5%, ±1%, or ±0.1% of a given value or range, as long as it is suitable for performing the methods disclosed herein.

[0062] As used herein, the term "molecular weight" may refer to a "predicted molecular weight" or an "observed molecular weight". The "predicted molecular weight" of a protein is the sum of the molecular weights of all the amino acids in the protein. In some cases, the "predicted molecular weight" of a molecule may differ from the "observed molecular weight". In some embodiments, these differences may occur in a protein due to changes in glycosylation, glycanation, ubiquitination, phosphorylation, or proteolytic cleavage of the protein or other proteins in complex with a given protein.

[0063] Antigen Binding Molecules

[0064] In one aspect, the present disclosure provides antigen binding molecules (i.e., HSA binding molecules) that specifically bind to HSA. More specifically, the HSA binding molecules of the present disclosure preferentially bind to HSA at acidic pH, and do not bind or weakly bind 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 times the binding affinity of the HSA binding molecule to HSA at physiological pH. In some embodiments, the HSA binding molecule has a binding affinity for HSA at acidic pH that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 times greater 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, physiological pH is pH 7.4.

[0065] In some embodiments, the HSA binding molecule binds to HSA with an equilibrium dissociation constant of 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 at acidic pH. In some embodiments, the HSA binding molecule binds to HSA with an equilibrium dissociation constant of 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 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.

[0066] In some embodiments, the HSA binding molecule binds to HSA with an equilibrium dissociation constant of 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 at acidic pH and the HSA binding molecule is or 15.0 mM at a pH of 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.

[0067] In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with 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 with HSA at pH 7.4 is at least 2.4 mM. In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with 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 with HSA at pH 7.4 is at least 11.5 mM. In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with 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 with HSA at pH 7.4 is at least 11.5 mM. In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with 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 with HSA at pH 7.4 is at least 2.4 mM.

[0068] In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 5.5 is less than 0.140mM, and the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 7.4 is at least 2.4mM. In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 5.5 is less than 0.03mM, and the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 7.4 is at least 11.5mM. In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 5.5 is less than 0.140mM, and the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 7.4 is at least 11.5mM. In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 5.5 is less than 0.03mM, and the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 7.4 is at least 2.4mM.

[0069] In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 5.5 is less than 0.140mM, and the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 7.4 is at least 2.4mM. In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 5.5 is less than 0.03mM, and the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 7.4 is at least 11.5mM. In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 5.5 is less than 0.140mM, and the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 7.4 is at least 11.5mM. In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 5.5 is less than 0.03mM, and the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 7.4 is at least 2.4mM.

[0070] In some embodiments, the equilibrium dissociation constant of the antigen binding molecule with 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 with 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 with HSA at pH 5.5 is less than 0.03 to 0.140 mM, and the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 7.4 is at least 2.4 to 11.5 mM.

[0071] 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 are bound to HSA at pH 5.5 and / or pH 6.0, and at most 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 bound to HSA are free 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 are bound to HSA at pH 5.5 and / or pH 6.0, and at most 1%-25%, 2%-20%, or 3%-15% of the antigen binding molecules bound to HSA are free at pH 7.4.

[0072] In some embodiments, at least 80% of the antigen binding molecules that specifically bind to HSA are bound to HSA at pH 5.5 and / or pH 6.0, and at most 20% of the antigen binding molecules that bind to HSA are free at pH 7.4. In some embodiments, at least 95% of the antigen binding molecules that specifically bind to HSA are bound to HSA at pH 5.5 and / or pH 6.0, and at most 5% of the antigen binding molecules that bind to HSA are free at pH 7.4.

[0073] 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 not detectable at physiological pH. In some embodiments, physiological pH is pH 7.4.

[0074] The binding affinity of an HSA binding molecule to HSA can be measured by any known method. In some embodiments, the binding affinity of an HSA binding molecule to HSA is measured by surface plasmon resonance.

[0075] In some embodiments, the HSA binding molecule does not compete with FcRn for HSA binding.

[0076] In some embodiments, the antigen binding molecule is a polypeptide derived from an antibody, including but 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 antibody mimetic protein, including but not limited to anticalin or DARPin.

[0077] 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 for histidine (H), aspartic acid (D), glutamic acid (E) or alanine (A). In some embodiments, 1, 2 or 3 amino acids in one or more CDRs are independently substituted with H, D, E or A. In some embodiments, 1, 2 or 3 amino acids in CDR1 are independently substituted with H, D, E or A. In some embodiments, 1, 2 or 3 amino acids in CDR2 are independently substituted with H, D, E or A. In some embodiments, 1, 2 or 3 amino acids in CDR3 are independently substituted with H, D, E or A. In some embodiments, 1, 2 or 3 amino acids in HCDR1 are independently substituted with H, D, E or A. In some embodiments, 1, 2 or 3 amino acids in HCDR2 are independently substituted with H, D, E or A. In some embodiments, 1, 2 or 3 amino acids in HCDR3 are independently substituted with H, D, E or A. In some embodiments, 1, 2, or 3 amino acids in LCDR1 are independently substituted with H, D, E, or A. In some embodiments, 1, 2, or 3 amino acids in LCDR2 are independently substituted with H, D, E, or A. In some embodiments, 1, 2, or 3 amino acids in LCDR3 are independently substituted with H, D, E, or A.

[0078] In some embodiments, 1, 2, or 3 amino acids of each of CDR1 and CDR2 are independently substituted with H, D, E, or A. In some embodiments, 1, 2, or 3 amino acids of each of CDR2 and CDR3 are independently substituted with H, D, E, or A. In some embodiments, 1, 2, or 3 amino acids of each of CDR1 and CDR3 are independently substituted with H, D, E, or A. In some embodiments, 1, 2, or 3 amino acids of each of CDR1, CDR2, and CDR3 are independently substituted with H, D, E, or A.

[0079] In some embodiments, 1, 2, or 3 amino acids of each of HCDR1 and HCDR2 are independently substituted with H, D, E, or A. In some embodiments, 1, 2, or 3 amino acids of each of HCDR2 and HCDR3 are independently substituted with H, D, E, or A. In some embodiments, 1, 2, or 3 amino acids of each of HCDR1 and HCDR3 are independently substituted with H, D, E, or A. In some embodiments, 1, 2, or 3 amino acids of each of HCDR1, HCDR2, and HCDR3 are independently substituted with H, D, E, or A.

[0080] In some embodiments, 1, 2, or 3 amino acids of each of LCDR1 and LCDR2 are independently substituted with H, D, E, or A. In some embodiments, 1, 2, or 3 amino acids of each of LCDR2 and LCDR3 are independently substituted with H, D, E, or A. In some embodiments, 1, 2, or 3 amino acids of each of LCDR1 and LCDR3 are independently substituted with H, D, E, or A. In some embodiments, 1, 2, or 3 amino acids of each of LCDR1, LCDR2, and LCDR3 are independently substituted with H, D, E, or A.

[0081] 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 selected from A, AG, GG and PP added at the C-terminus. In some embodiments, the C-terminus of VHH is the amino acid sequence VTVSS (SEQ ID NO: 195). In some embodiments, the C-terminus of VHH consists of the amino acid sequence VTVSS (SEQ ID NO: 195).

[0082] In some embodiments, the antigen binding molecule specifically binds to HSA and is selected from a Fab fragment, a scFv, an sdAb, and an antigen binding fragment thereof. In some embodiments, the antigen binding domain specifically binds to HSA and is an 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 No.: AAA98797.1. In some embodiments, HSA comprises an amino acid sequence provided in GenBank Accession No.: AAA98797.1.

[0083] In some embodiments, the antigen binding domain is a VHH fragment comprising the following: 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.

[0084] In some embodiments, the antigen binding domain is a VHH fragment comprising or consisting of a combination of CDR1, CDR2, and CDR3, wherein 1, 2, 3, 4, or 5 amino acids differ in at least one of the amino acid sequences selected from: SEQ IDNO: 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.

[0085] In some embodiments, the antigen binding domain is a VHH fragment comprising or consisting of a combination of CDR1, CDR2, and CDR3 selected from the group consisting of SEQ ID NO: 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 65, 66, and 67; 68, 69, and 70; 71, 72, and 73; 74, 75, and 76; and 173, 174, and 175, wherein one or more amino acids within one or more of the CDRs are substituted with alanine or histidine.

[0086] In some embodiments, the antigen binding domain is a VHH fragment comprising or consisting of a combination of CDR1, CDR2, and CDR3 selected from the group consisting of: SEQ ID NO: 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 38; 33, 34 and 39; 33, 34 and 40; 33, 34 and 41; 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; 1 77, 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.

[0087] In some embodiments, the antigen binding domain is a VHH fragment comprising or consisting of an amino acid sequence 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 comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 15-20, 77-88, 90-115 and 186-194.

[0088] In some embodiments, the antigen binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 15-20, 77-88, 90-115 and 186-194, or a variant thereof, and one or more amino acids selected from A, AG, GG and PP added at the C-terminus.

[0089] In some embodiments, the antigen binding domain is a Fab or scFv comprising the HCDR1, HCDR2, and HCDR3 amino acid sequence of VH and comprising the LCDR1, LCDR2, and LCDR3 amino acid sequence of VL, wherein VH comprises the amino acid sequence of SEQ ID NO: 157, 159, 161, 163, 165, 167, 169 or 171, and the VL comprises the amino acid sequence of SEQ ID NO: 158, 160, 162, 164, 166, 168, 170 or 172.

[0090] In some embodiments, the antigen binding domain is a Fab or scFv comprising a combination of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of VH and VL, wherein 1, 2, 3, 4, or 5 amino acids differ in at least one of the amino acid sequences of the CDRs selected from SEQ ID NO: 157 and 158, 159 and 160, 161 162, 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 a combination of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NO: 157 and 158, 159 and 160, 161 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, or 171 and 172.

[0091] In some embodiments, the antigen binding domain is a Fab or scFv composed of a combination of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 of VH and VL, wherein 1, 2, 3, 4 or 5 amino acids are different in at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 157 and 158, 159 and 160, 161 162, 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 consisting of a combination of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of VH and VL selected from SEQ ID NO: 157 and 158, 159 and 160, 161 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, or 171 and 172.

[0092] In some embodiments, the antigen binding domain is a Fab or scFv comprising or consisting of a combination of HCDR1, HCDR2, and HCDR3 selected from: SEQ ID NO: 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 comprising or consisting of a combination of LCDR1, LCDR2, and LCDR3 selected from the group consisting of 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 comprising or consisting of a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 selected from the group consisting of 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.

[0093] In some embodiments, the antigen binding domain is a Fab or scFv comprising VH and VL, wherein VH comprises an amino acid sequence 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 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 wherein VL comprises an amino acid sequence selected from SEQ ID NO: 158, 160, 162, 164, 166, 168, 170 or 172.

[0094] In some embodiments, the antigen binding domain is a Fab or scFv comprising VH and VL, wherein VH and VL comprise 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: 157 and 158, 159 and 160, 161 162, 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 an amino acid sequence of SEQ ID NOs: 157 and 158, 159 and 160, 161 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, or 171 and 172.

[0095] Therapeutic proteins

[0096] 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, 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.

[0097] In some embodiments, the therapeutic protein is a therapeutic antibody or fragment thereof.

[0098] 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 fragments, scFv, or antibody mimics). In some embodiments, the therapeutic proteins disclosed herein comprise one or more antigen binding domains (e.g., VH, VL, sdAb, Fab fragments, scFv, or antibody mimics).

[0099] In general, therapeutic antibodies or fragments thereof are human immunoglobulins. However, it should be understood that therapeutic antibodies may be derived from immunoglobulins of any other mammalian species, including, for example, camel species, rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates (e.g., orangutans, macaques) species. In addition, therapeutic antibodies or fragments thereof may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3 and IgG4.

[0100] In order to enhance the manufacturability of the therapeutic antibodies and fusion proteins containing the same as disclosed herein, it is preferred that the constituent Fc region does not contain any non-disulfide bonded cysteine ​​residues. Thus, in one embodiment, the Fc region does not contain free cysteine ​​residues.

[0101] Fusion Protein

[0102] The present disclosure provides fusion proteins comprising at least one antigen binding molecule that specifically binds to HSA and at least one therapeutic protein. It has been advantageously found that fusing the HSA binding molecules of the present disclosure to therapeutic proteins increases the half-life of the therapeutic protein due to the ability of the HSA binding molecules to bind to HSA in endosomes (acidic pH) and thus recirculate with HSA. However, since the HSA binding molecules do not bind or weakly bind to HSA at physiological pH in the bloodstream, the fusion protein does not interfere with albumin function. Likewise, the fusion protein retains its efficacy because it is not bound by HSA in the bloodstream.

[0103] The therapeutic protein can be any therapeutic protein described herein or known in the art. The antigen binding molecule can be any antigen binding molecule 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.

[0104] 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.

[0105] In some embodiments, one antigen binding molecule is fused to the N-terminus of a therapeutic protein, and another antigen binding molecule is fused to the C-terminus of a therapeutic protein. In some embodiments, one antigen binding molecule is fused to a position other than the N-terminus or C-terminus of a therapeutic protein, and another antigen binding molecule is fused to the N-terminus of a therapeutic protein. In some embodiments, one antigen binding molecule is fused to a position other than the N-terminus or C-terminus of a therapeutic protein, and another antigen binding molecule is fused to the C-terminus of a therapeutic protein.

[0106] In some embodiments, a therapeutic protein is fused to the N-terminus of an antigen binding molecule, and another therapeutic protein is fused to the C-terminus of an antigen binding molecule. In some embodiments, a therapeutic protein is fused to a position other than the N-terminus or C-terminus of an antigen binding molecule, and another therapeutic protein is fused to the N-terminus of an antigen binding molecule. In some embodiments, a therapeutic protein is fused to a position other than the N-terminus or C-terminus of an antigen binding molecule, and another therapeutic protein is fused to the C-terminus of an antigen binding molecule.

[0107] In some embodiments, the therapeutic protein is a therapeutic antibody or fragment thereof comprising an Fc region. In some embodiments, the antigen binding molecule is fused to the C-terminus of one or two Fc domains in the Fc region. In some embodiments, the antigen binding molecule is fused to the N-terminus of one or two Fc domains in 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 fragments, 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.

[0108] In some embodiments, one antigen binding molecule is fused to the C-terminus of one of the Fc domains in the Fc region, and another antigen binding molecule is fused to the C-terminus of another Fc domain in the Fc region. In some embodiments, one antigen binding molecule is fused to the N-terminus of one of the Fc domains in the Fc region, and another antigen binding molecule is fused to the N-terminus of another Fc domain in the Fc region. In some embodiments, one antigen binding molecule is fused to the N-terminus of one of the Fc domains in the Fc region, and another antigen binding molecule is fused to the C-terminus of another Fc domain in 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 in the Fc region, and another antigen binding molecule is fused to the N-terminus of another Fc domain in 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 in the Fc region, and another antigen binding molecule is fused to the C-terminus of another Fc domain in the Fc region.

[0109] 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 a non-cleavable linker.

[0110] 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 joint. The joint can be any suitable joint, including those described herein.

[0111] Connectors

[0112] The antigen binding molecule can be fused to the N-terminus or C-terminus of the therapeutic protein (eg, antibody or fragment thereof).

[0113] 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 connected to the N-terminus or C-terminus of the therapeutic protein via a joint. In some embodiments, the joint is a non-cleavable joint.

[0114] 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 connected to the N-terminus or C-terminus of the Fc domain via a joint. In some embodiments, the joint is a non-cleavable joint. As used herein, the term "non-cleavable joint" refers to a joint that is not easily cut by one or more of a given enzyme, chemical agent or light irradiation. In some embodiments, the enzyme is a protease.

[0115] In some embodiments, the linker is a synthetic compound linker, such as, for example, a chemical cross-linking agent. Non-limiting examples of commercially available suitable 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-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES) and bis[2-(sulfosuccinimidyloxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0116] The Fc domain disclosed herein may include a part of the hinge region. Therefore, the antigen binding molecule can be connected to the N-terminus of the Fc domain via the hinge region. 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 to the N-terminus of the Fc domain via the hinge region or a portion thereof. In some embodiments, the hinge region is an IgG hinge region, such as a human IgG hinge region.

[0117] In some embodiments, the linker is a peptide linker. Examples of peptide linkers are well known, and one skilled in the art can select a suitable peptide linker for linking an antigen binding molecule to a therapeutic protein.

[0118] The peptide linker can be any length. In certain embodiments, the length and amino acid composition of the linker peptide sequence can be optimized to change the orientation and / or proximity of the antigen binding molecules and therapeutic proteins to each other, to achieve the desired activity of the fusion protein. In certain embodiments, the length of the peptide linker is between about 1 and about 100 amino acids, between about 8 and about 40 amino acids, or between about 15 amino acids and about 25 amino acids. In certain embodiments, the length of the peptide linker is between 1 and 100 amino acids, between 8 and 40 amino acids, or between 15 and 25 amino acids. 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.

[0119] In some embodiments, the peptide linker contains only glycine and / or serine residues (e.g., glycine-serine linker or GS linker). Examples of such peptide linkers include: Gly(x)Ser, wherein x is 0 to 6; or Ser Gly(x), wherein x is 0 to 6; (Gly Gly Gly Gly Ser)n, wherein n is an integer of 1 or greater; and (Ser Gly Gly Gly Gly)n, wherein n is an integer of 1 or greater. In some embodiments, the peptide linker includes an amino acid sequence selected from the group consisting of: (GGGGS)n and (SGGGG)n, wherein n is 1 to 8. In some embodiments, the linker peptide is modified so that the amino acid sequence GSG (which appears at the junction of the traditional Gly / Ser linker peptide repeats) is not present. For example, in some embodiments, the peptide linker includes an amino acid sequence selected from the group consisting of: (GGGXX)nGGGGS and GGGGS(XGGGS)n, wherein X is any amino acid that can be inserted into the sequence and does not produce a polypeptide comprising the sequence GSG, and n is 0 to 4. In some embodiments, the sequence of the linker peptide is (GGGX1X2)nGGGGS, and X1 is P and X2 is S and n is 0 to 4. In some other embodiments, the sequence of the linker peptide is (GGGX1X2)nGGGGS, and X1 is G and X2 is Q and n is 0 to 4. In some other embodiments, the sequence of the linker peptide is (GGGX1X2)nGGGGS, and X1 is G and X2 is A and n is 0 to 4. In yet other embodiments, the sequence of the linker peptide is GGGGS(XGGGS)n, and X is P and n is 0 to 4. In some embodiments, the linker peptide of the present disclosure comprises or consists of the amino acid sequence (GGGGA)2GGGGS. In some embodiments, the linker peptide comprises or consists of the amino acid sequence (GGGGQ)2GGGGS. In another embodiment, the linker peptide comprises or consists of the amino acid sequence (GGGPS)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).

[0120] In some embodiments, the peptide linker is a GS linker of about 20 or about 30 amino acids in length. In some embodiments, the peptide linker is a GS linker of 20 or 30 amino acids in length.

[0121] Polynucleotides, vectors and methods of producing

[0122] The present disclosure also provides polynucleotides encoding antigen binding molecules disclosed herein or fragments thereof. In some embodiments, polynucleotides encode therapeutic proteins of the present disclosure. In some embodiments, polynucleotides encode fusion proteins of the present disclosure. In some embodiments, polynucleotides encode one or more of antigen binding molecules, therapeutic proteins and joints. In some embodiments, polynucleotides encode antigen binding molecules and therapeutic proteins, and optionally joints. In some embodiments, polynucleotides encode one or more of antigen binding molecules, Fc regions and joints. In some embodiments, polynucleotides encode antigen binding molecules and Fc regions, and optionally joints. In some embodiments, polynucleotides encode fusion proteins comprising one or more antigen binding molecules and therapeutic proteins. In some embodiments, polynucleotides encode one or more of antigen binding molecules, Fc domains and joints. In some embodiments, polynucleotides encode antigen binding molecules and Fc domains, and optionally joints. In some embodiments, polynucleotides encode fusion proteins comprising one or more antigen binding molecules and one or more therapeutic proteins.

[0123] As used herein, "isolated" polynucleotides or nucleic acid molecules are polynucleotides or nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of nucleic acid molecules (e.g., in mice or humans). In addition, "isolated" nucleic acid molecules, such as cDNA molecules, can be substantially free of other cellular materials or culture media when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the wording "substantially free" includes preparations of polynucleotides or nucleic acid molecules with less than about 15%, 10%, 5%, 2%, 1%, 0.5% or 0.1% (particularly less than about 10%) of other substances (e.g., cellular materials, culture media, other nucleic acid molecules, chemical precursors and / or other chemicals). In one embodiment, the nucleic acid molecules encoding the polypeptides described herein are separated or purified.

[0124] On the one hand, provided herein are polynucleotides comprising nucleotide sequences encoding therapeutic proteins or fusion proteins as described herein. On the other hand, provided herein are polynucleotides comprising nucleotide sequences encoding antigen binding molecules as described herein. On the other hand, provided herein are polynucleotides comprising nucleotide sequences encoding fusion proteins as described herein.

[0125] The polynucleotide may include a nucleotide sequence encoding an sdAb (e.g., VHH fragment), a Fab fragment, a scFv, a VH or a VL comprising the FR and CDR of an antigen binding molecule described herein. The polynucleotide may also include a nucleotide sequence encoding an antibody mimetic as described herein. In some embodiments, the polynucleotide may include a nucleotide sequence encoding a VHH fragment comprising the FR and CDR of an antigen binding molecule described herein. In some embodiments, the polynucleotide may include a nucleotide sequence encoding a light chain comprising the VL FR and CDR of an antigen binding molecule described herein or a nucleotide sequence encoding a heavy chain comprising the VH FR and CDR of an antigen binding molecule described herein and / or an Fc domain as described herein. In one embodiment, the polynucleotide encodes the VH, VL, heavy chain and / or light chain of an antigen binding molecule described herein. In one embodiment, the polynucleotide encodes the first VH and the first VL of an antigen binding molecule described herein. In one embodiment, the polynucleotide encodes the second VH and the second VL of an antigen binding molecule described herein. In one embodiment, the polynucleotide encodes the first heavy chain and the first light chain of an antigen binding molecule described herein. In one embodiment, the polynucleotide encodes the second heavy chain and the second light chain of an antigen binding molecule described herein. In one embodiment, the polynucleotide encodes the VH and / or VL, or the heavy chain and / or light chain of the antigen binding molecule described herein.

[0126] 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 in different nucleic acid molecules. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are contained in the same nucleic acid molecule.

[0127] In some embodiments, the first nucleotide sequence and the second nucleotide sequence also encode antigen binding molecules.In some embodiments, the first nucleotide sequence and the second nucleotide sequence encode the same antigen binding molecules.In some embodiments, the first nucleotide sequence and the second nucleotide sequence encode different antigen binding molecules.In some embodiments, the first nucleotide sequence encodes the Fc domain and antigen binding molecules of therapeutic antibodies, and the second nucleotide sequence encodes the Fc domain and antigen binding domain of therapeutic antibodies.The antigen binding molecules encoded by the first nucleotide sequence and / or the second nucleotide sequence can be any antigen binding molecules described herein.

[0128] In some embodiments, the first nucleotide sequence and the second nucleotide sequence also encode a peptide joint. In some embodiments, the first nucleotide sequence and the second nucleotide sequence encode the same peptide joint. In some embodiments, the first nucleotide sequence and the second nucleotide sequence encode different peptide joints. In some embodiments, the first nucleotide sequence encodes Fc domains, peptide joints and antigen binding molecules, and the second nucleotide sequence encodes Fc domains and antigen binding domains from therapeutic antibodies. In some embodiments, the first nucleotide sequence encodes antigen binding molecules, peptide joints and Fc domains, and the second nucleotide sequence encodes Fc domains and antigen binding domains from therapeutic antibodies. The peptide joint encoded by the first nucleotide sequence and / or the second nucleotide sequence can be any peptide joint as described herein. In some embodiments, the joint comprises SEQ ID NO:2,3 or 4 amino acid sequences.

[0129] Also provided herein is a polynucleotide encoding a polypeptide as provided above, which is optimized, for example, by codon / RNA optimization, replacement with a heterologous signal sequence and elimination of mRNA instability elements. The method for generating an optimized nucleic acid for recombinant expression by introducing codon changes and / or eliminating the inhibitory zone in mRNA can be performed by adjusting accordingly, for example, U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and the optimization method described in 6,794,498, all of which are incorporated herein by reference in their entirety. For example, potential splicing sites and unstable elements (e.g., elements rich in A / T or A / U) in RNA can be mutated without changing the amino acid encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. These changes utilize the degeneracy of the genetic code, for example, using alternative codons of the same amino acid. In one embodiment, one or more codons may need to be changed to encode conservative mutations, for example, similar amino acids with chemical structures and properties and / or functions similar to the original amino acids.

[0130] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotides is determined. The nucleotide sequence encoding the protein described herein and the modified form of these antibodies can be determined using methods well known in the art, i.e., the nucleotide codons known to encode specific amino acids are assembled in a manner to generate nucleic acids encoding proteins. 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), in short, it involves synthesizing overlapping oligonucleotides containing sequence portions encoding antibodies, annealing and connection of these oligonucleotides, and then amplifying the connected oligonucleotides by PCR.

[0131] Alternatively, polynucleotides encoding proteins described herein can be generated from nucleic acids from suitable sources (e.g., hybridomas) 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 known sequences can be performed using genomic DNA obtained from hybridoma cells that produce a polypeptide of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising sequences encoding polypeptides. Amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning.

[0132] If a clone containing a nucleic acid encoding a particular polypeptide is not available, but the sequence of the polypeptide is known, the nucleic acid encoding the polypeptide can be chemically synthesized or obtained from a suitable source (e.g., a cDNA library generated from nucleic acid (preferably poly A+RNA) isolated from any tissue or cell expressing the polypeptides described herein) by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence to identify, for example, cDNA clones from a cDNA library encoding the polypeptide. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0133] DNA encoding the proteins described herein can be readily isolated and sequenced using conventional methods. Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell (e.g., from a CHO GS System TM (Lonza) CHO cells) or myeloma cells that do not otherwise produce the proteins described herein.

[0134] Also provided are polynucleotides that hybridize under high stringency, medium or lower stringency hybridization conditions to a polynucleotide encoding a protein described herein.

[0135] Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may include DNA hybridization bound to a filter in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50°C-65°C; hybridization under highly stringent conditions may involve nucleic acid hybridization bound to a filter in 6xSSC at about 45°C, followed by one or more washes in 0.1xSSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those skilled in the art and has been described, see, e.g., Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pp. 6.3.1-6.3.6 and 2.10.3, which are incorporated herein by reference in their entirety.

[0136] In one aspect, cells (e.g., host cells) expressing (e.g., recombinantly) proteins described herein are provided herein, as well as related polynucleotides and expression vectors. Vectors (e.g., expression vectors) comprising polynucleotides are provided herein, which comprise nucleotide sequences encoding proteins described herein for recombinant expression in host cells, preferably mammalian cells (e.g., CHO cells). Host cells comprising such vectors for recombinantly expressing proteins described herein are also provided herein. In one aspect, methods for producing proteins described herein are provided herein, comprising expressing polypeptides from host cells.

[0137] The recombinant expression of proteins described herein generally involves constructing an expression vector containing a polynucleotide encoding a polypeptide. Once a polynucleotide encoding a polypeptide described herein is obtained, a vector for producing a polypeptide can be produced by recombinant DNA technology using techniques well known in the art. Therefore, a method for preparing a protein by expressing a polynucleotide containing a polypeptide encoding nucleotide sequence is described herein. Methods well known to those skilled in the art can be used to construct an expression vector containing a polypeptide encoding sequence and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo gene recombination. A replicable vector is also provided, which comprises a nucleotide sequence encoding a polypeptide described herein that is operably linked to a promoter. Such vectors may, for example, include a nucleotide sequence encoding a constant region of a polypeptide (see, for example, International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464, which is incorporated herein by reference in its entirety), and the variable region of the polypeptide can be cloned into this vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy chain and the light chain.

[0138] In one embodiment, the vector comprises a polynucleotide encoding a sdAb, a Fab fragment, a scFv, a VHH fragment, a VH, a VL, a heavy chain and / or a light chain of a polypeptide described herein. In another embodiment, the vector comprises a polynucleotide encoding a VH and a VL of a polypeptide described herein. In another embodiment, the vector comprises a polynucleotide encoding a heavy chain and a light chain of a polypeptide described herein.

[0139] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce a polypeptide or fragment thereof as described herein. Therefore, provided herein is a host cell containing a polynucleotide encoding a polypeptide or fragment thereof as described herein, or a heavy chain or light chain thereof, or a fragment thereof, or a single-chain antibody as described herein, the polynucleotide being operably linked to a promoter for expressing such sequences in a host cell.

[0140] In one embodiment, the host cell comprises a polynucleotide containing one of the first nucleotide sequences and one of the second nucleotide sequences described above. In another embodiment, the host cell comprises a first polynucleotide containing one of the first nucleotide sequences described above and a second polynucleotide containing one of the first nucleotide sequences described above. In another embodiment, the host cell comprises a first vector containing one of the first nucleotide sequences described above and one of the second nucleotide sequences. In another embodiment, the host cell comprises a first vector containing one of the first nucleotide sequences described above and one of the second nucleotide sequences, and a second vector containing a second polynucleotide, the second polynucleotide comprising one of the first nucleotide sequences described above.

[0141] 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 host cell population comprising such a first host cell and such a second host cell.

[0142] In some embodiments, provided herein are vector populations comprising a first vector and a second vector, the first vector comprising a polynucleotide encoding an antigen binding molecule or a fusion protein, and the second vector comprising a polynucleotide encoding an antigen binding molecule or a fusion protein. In some embodiments, provided herein are vector populations comprising a first vector and a second vector, the first vector comprising a polynucleotide encoding a fusion protein, and the second vector comprising a polynucleotide encoding a therapeutic protein. In some embodiments, provided herein are vector populations comprising a first vector and a second vector, the first vector comprising a polynucleotide encoding a fusion protein, and the second vector comprising a polynucleotide encoding an antigen binding molecule. In some embodiments, provided herein are vector populations comprising a first vector and a second vector, the first vector comprising a polynucleotide encoding an antigen binding molecule, and the second vector comprising a polynucleotide encoding a therapeutic protein.

[0143] A variety of host-expression vector systems can be used to express the polypeptides described herein (see, e.g., U.S. Pat. No. 5,807,715, which is incorporated herein by reference in its entirety). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells that can express the polypeptides described herein in situ when transformed or transfected with the appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with, for example, recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing protein coding sequences; yeast (e.g., Saccharomyces and Pichia) transformed with, for example, recombinant yeast expression vectors containing protein coding sequences; insect cell systems infected with, for example, recombinant virus expression vectors (e.g., baculovirus) containing protein coding sequences; plant cell systems (e.g., green algae, such as Chlamydomonas reinhardtii) infected with, for example, recombinant virus expression vectors (e.g., cauliflower mosaic virus CaMV; tobacco mosaic virus TMV) or transformed with, for example, recombinant plasmid expression vectors (e.g., Ti plasmid) containing protein coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20 and BMT10 cells), which contain, for example, a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In one embodiment, the cell used to express the protein described herein is a Chinese hamster ovary (CHO) cell, such as a CHO GS System TM In one embodiment, the heavy chain and / or light chain of the antibody produced by the CHO cells may have an N-terminal glutamine or glutamic acid residue replaced by pyroglutamic acid. In one embodiment, the cell used to express the polypeptide described herein is a human cell, such as a human cell line. In one embodiment, the mammalian expression vector is pOptiVEC TMOr pcDNA3.3. In one embodiment, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells) are used to express recombinant polypeptides. For example, mammalian cells such as CHO cells, combined with vectors such as major intermediate early gene promoter elements from human cytomegalovirus, are effective expression systems for antibodies (Foecking MK and Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al., (1990) Biotechnology 8 (7): 662-7, each of which 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.

[0144] In bacterial systems, many expression vectors can be advantageously selected according to the intended use of the molecule to be expressed. For example, when a large number of such polypeptides are to be produced, in order to produce a pharmaceutical composition of an antibody molecule, it may be necessary to instruct a carrier for the high-level expression of a fusion protein product that is easily purified. Such carriers include, but are not limited to, the E. coli expression vector pUR278 (Ruether U and Mueller-Hill B (1983) EMBO J 2: 1791-1794), in which the coding sequence can be individually connected to a carrier in a framework with a lac Z coding region, thereby producing a fusion protein; pIN vectors (Inouye S and Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G and Schuster SM (1989) J Biol Chem 24: 5503-5509); etc., all of which are incorporated herein by reference in their entirety. For example, pGEX vectors can also be used to express exogenous polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, 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. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0145] For example, in insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence can be cloned individually into a non-essential region of the virus (e.g., a polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., a polyhedrin promoter).

[0146] In mammalian host cells, many virus-based expression systems can be used. In the case of using adenovirus as an expression vector, the coding sequence of interest can be connected to an adenovirus transcription / translation control complex, for example, a late promoter and a tripartite leader sequence. The chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., E1 region or E3 region) will produce a recombinant virus that is viable and capable of expressing the molecule in an infected host (see, e.g., Logan J and Shenk T (1984) PNAS 81 (12): 3655-9, which is incorporated herein by reference in its entirety). The effective translation of the inserted coding sequence may also require a specific start signal. These signals include an ATG start codon and adjacent sequences. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and start codons can have a variety of sources, including natural and synthetic. Expression efficiency may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, and the like (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153:516-544, which is incorporated herein by reference in its entirety).

[0147] In addition, host cell strains can be selected that regulate the expression of inserted sequences or modify and process gene products in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristics and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign proteins. For this reason, eukaryotic host cells with cellular machinery for appropriate processing of primary transcripts, glycosylation and phosphorylation of gene products 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, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In one embodiment, the proteins described herein are produced in mammalian cells such as CHO cells.

[0148] In one embodiment, the polypeptides described herein comprise a portion of an antibody having reduced fucose content or no fucose content. Such proteins can be produced using techniques known to those skilled in the art. For example, the protein can be expressed in cells lacking or lacking fucosylation ability. In one example, a cell line with two alleles of α1,6-fucosyltransferase knocked out can be used to produce antibodies with reduced fucose content. The ANTIBODYNAMIC® system (Lonza) is an example of such a system that can be used to generate antibodies with reduced fucose content.

[0149] For long-term, high-yield production of recombinant proteins, stable expressing cells can be generated. For example, cell lines that stably express the proteins described herein can be engineered. In one embodiment, cells provided herein stably express antigen binding molecules, fusion proteins, or therapeutic proteins that associate to form polypeptides described herein.

[0150] In some aspects, instead of using the expression vector containing the viral origin of replication, the host cell can be transformed with DNA and selectable markers that are controlled by suitable expression control elements (for example, promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.). After introducing foreign DNA / polynucleotide, the cell through through engineering approaches can be allowed to grow one to two days in enrichment medium, and then switch to selective medium. The selectable marker in the recombinant plasmid gives the resistance to selection, and allows the cell to stably integrate the plasmid into their chromosome and grow to form a focus, which can then be cloned and expanded in the cell line. The method can be advantageously used for the cell line of through engineering approaches expressing polypeptide as described herein or its segment. This type of cell line through through engineering approaches can be particularly used for screening and evaluating the compositions directly or indirectly interacting with the polypeptide.

[0151] A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH and Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) genes in tk cells, hgprt cells, or aprt cells, respectively, all of which are incorporated herein by reference in their entirety. In addition, antimetabolite resistance can be used as the basis for selection for the following genes: dhfr, which 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, which confers resistance to mycophenolic acid (Mulligan RC and Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY and 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 and Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ and Felgner PL (1993) Trends Biotechnol 11(5):211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-56), all of which are incorporated herein by reference in their entirety.Methods well known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clones, and 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 in Chapters 12 and 13, Dracopoli NC et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbère-Garapin F et al., (1981) J Mol Biol 150:1-14, all of which are incorporated herein by reference in their entirety.

[0152] The expression level of the polypeptide can be increased by vector amplification (for review, see, Bebbington CR and Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, pp. 163-188 in DNA Cloning, Vol. III, A Practical Approach. DM Glover (ed.) (Academic Press, New York, 1987), which is incorporated herein by reference in its entirety). When the marker in the vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the gene of interest, the yield of the polypeptide will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66, which is incorporated herein by reference in its entirety).

[0153] Host cells can be co-transfected with two or more expression vectors described herein. The two vectors can contain the same selectable marker, which enables polypeptides (such as the first heavy chain and the second heavy chain polypeptide) to be expressed equally. Host cells can be co-transfected with two or more expression vectors of different amounts. For example, host cells can be transfected with the first expression vector and the second expression vector of any one of the following ratios: 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.

[0154] Alternatively, a single vector encoding and capable of expressing two polypeptides can be used. The coding sequence can include cDNA or genomic DNA. The expression vector can be a monocistronic or polycistronic. The polycistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes / nucleotide sequences, or in the range of 2-5, 5-10 or 10-20 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 this expression vector, the transcription of the two genes can be driven by the promoter, and the translation of the mRNA from the first gene can be carried out by a cap-dependent scanning mechanism, and the translation of the mRNA from the second gene can be carried out by a cap-independent mechanism, for example, by IRES.

[0155] Once a polypeptide described herein is produced by recombinant expression, it can be purified by any method known in the art for purifying proteins, such as by chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen after protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In addition, the polypeptides described herein can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0156] In one embodiment, polypeptides described herein are separated or purified. In one embodiment, separated polypeptides are polypeptides that are substantially free of other polypeptides having different antigenic specificities from the separated polypeptides. For example, in certain embodiments, protein preparations described herein are substantially free of cellular material and / or chemical precursors. The wording "substantially free of cellular material" includes polypeptide preparations in which the polypeptide is separated from the cellular components of the cell that separates or recombinantly produces the polypeptide. Therefore, polypeptides substantially free of cellular material include polypeptide preparations with 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 polypeptide variants (e.g., different post-translational modified forms of polypeptides or other different versions of polypeptides (e.g., polypeptide fragments)). When the polypeptide is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10%, 2%, 1%, 0.5% or 0.1% of the volume of the protein preparation. When a polypeptide is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in protein synthesis. Thus, such protein preparations 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 polypeptide described herein is isolated or purified.

[0157] The polypeptides described herein can be produced by any method known in the art for synthesizing proteins, such as by chemical synthesis or by recombinant expression techniques. Unless otherwise indicated, the methods described herein employ 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 skill of the art. For example, these techniques are described in the references cited herein and are fully explained in the literature. See, e.g., 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, 2nd ed., 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 et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); B et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of which are incorporated herein by reference in their entirety.

[0158] In one embodiment, the polypeptides described herein are prepared, expressed, produced or isolated by any means involving production, e.g., via synthesis of a DNA sequence, genetic engineering. In one embodiment, such polypeptides comprise sequences (e.g., DNA sequences or amino acid sequences) that are not naturally present in the antibody germline repertoire in an animal or mammal (e.g., human).

[0159] Pharmaceutical composition

[0160] In one aspect, the present disclosure provides a pharmaceutical composition comprising a fusion protein as disclosed herein, which is used in a method for treating a disease or illness. In another aspect, the present disclosure provides a pharmaceutical composition comprising an antigen binding molecule as disclosed herein, which is used in a method for treating a disease or illness.

[0161] The formulations disclosed herein include bulk pharmaceutical compositions that can be used to make pharmaceutical compositions (e.g., compositions suitable for administration to subjects or patients), which can be used to prepare unit dosage forms. In one embodiment, the composition of the invention is a pharmaceutical composition. Such compositions comprise a preventive or therapeutically effective amount of one or more of the prophylactic or therapeutic agents of the invention (e.g., fusion proteins) (or other prophylactic or therapeutic agents) and a pharmaceutically acceptable carrier.

[0162] In some embodiments, the pharmaceutical compositions are formulated for administration to a subject by any suitable route of administration, including but not limited to intramuscular, intravenous, intradermal, intraperitoneal, subcutaneous, epidural, nasal, oral, rectal, topical, inhaled, buccal (e.g., sublingual), and transdermal administration. In one embodiment, the pharmaceutical compositions are formulated to be suitable for intravenous administration to a subject. In one embodiment, the pharmaceutical compositions are formulated to be suitable for subcutaneous administration to a subject.

[0163] method

[0164] 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 present invention to a therapeutic protein. The therapeutic protein can be any therapeutic protein described herein or known in the art.

[0165] In some embodiments, compared with the clearance rate of the therapeutic protein after a single therapeutic administration of the therapeutic protein, after a single therapeutic administration of the fusion protein (comprising the antigen binding molecules of the present invention and the therapeutic protein), the clearance rate of the fusion protein in the subject is reduced. In some embodiments, compared with the clearance rate of the therapeutic protein after a single therapeutic administration of the therapeutic protein, after a single therapeutic administration of the fusion protein, the clearance rate of the fusion protein in the subject is reduced by at least 1 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times or at least 20 times.

[0166] In some embodiments, compared with the clearance rate of the therapeutic protein after a single administration of an equivalent dose of therapeutic protein, after a single administration of fusion protein (comprising the antigen binding molecules of the present invention and therapeutic protein), the clearance rate of the fusion protein in the subject is reduced. In some embodiments, compared with the clearance rate of the therapeutic protein after a single administration of an equivalent dose of therapeutic protein, after a single administration of fusion protein, the clearance rate of the fusion protein in the subject is reduced by at least 1 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times or at least 20 times.

[0167] In some embodiments, the terminal half-life (t 1 / 2,z ), after a single therapeutic administration of a fusion protein (comprising an antigen binding molecule of the invention and a therapeutic protein), the t of the fusion protein in a subject is 1 / 2,z In some embodiments, the t of the therapeutic protein after a single therapeutic administration of the therapeutic protein is increased. 1 / 2,z In contrast, after a single therapeutic administration of the fusion protein, the t 1 / 2,z At least 0.5 times, at least 1 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times, or at least 20 times.

[0168] In some embodiments, the t of the therapeutic protein after a single administration of an equivalent dose of the therapeutic protein is 1 / 2,z In comparison, after a single administration of a fusion protein (comprising an antigen binding molecule of the invention and a therapeutic protein), the t 1 / 2,z In some embodiments, the t of the therapeutic protein after a single administration of an equivalent dose of the therapeutic protein is 1 / 2,z In comparison, after a single administration of the fusion protein, the t1 / 2,z At least 0.5 times, at least 1 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times, or at least 20 times.

[0169] In some embodiments, the efficacy of the fusion protein comprising the antigen binding molecules of the present invention and the therapeutic protein is the same or substantially the same as the efficacy of the therapeutic protein. In some embodiments, the efficacy of the fusion protein is reduced by no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% compared to the efficacy of the therapeutic protein.

[0170] In certain embodiments, the efficacy of the dosage of the fusion protein comprising the antigen binding molecules of the present invention and the therapeutic protein is the same or substantially the same as the efficacy of the therapeutic protein of the equivalent dose. In certain embodiments, the efficacy of the fusion protein dosage is reduced by no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% compared to the efficacy of the therapeutic protein of the equivalent dose.

[0171] In one embodiment, the fusion protein or antigen binding molecule does not antagonize the binding of FcRn to albumin.

[0172] In one embodiment, after administration of the fusion protein or antigen binding molecule, the level of albumin in the subject is not reduced compared to the baseline level of albumin. In one embodiment, a reduction in albumin of less than about 1%, 2%, 3%, 4% or 5% compared to the baseline albumin level is observed. In one embodiment, a reduction in albumin of less than about 10% compared to the baseline albumin level is observed.

[0173] 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.

[0174] In one embodiment, the fusion protein and another therapeutic agent are administered to the subject simultaneously or sequentially. In one embodiment, the other therapeutic agent is an anti-inflammatory agent. In one embodiment, the other therapeutic agent is a corticosteroid. In one embodiment, the other therapeutic agent is rituximab, daclizumab, basiliximab, muromonab-CD3, infliximab, adalimumab, omalizumab, efalizumab, natalizumab, tocilizumab, eculizumab, golimumab, canakinumab, ustekinumab or belimumab. In one embodiment, the other therapeutic agent is a leukocyte depleting agent.

[0175] In one embodiment, the additional therapeutic agent is a B cell depleting agent. In one embodiment, the B cell depleting agent is an antibody. In one embodiment, the B cell depleting 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.

[0176] 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.

[0177] 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.

[0178] Examples

[0179] The following examples are offered by way of illustration and not limitation.

[0180] Example 1: Generation of pH-dependent anti-albumin VHH / scFV

[0181] In brief, two llamas were immunized with humans and phage display libraries (VHH / scFv) were generated using standard procedures. Selection was performed with phage binding to HSA and MSA at pH 5.5 and eluted at pH 7.4 (using trypsin as a control). Screening of selected clones was performed using pH 5.5 or 7.4 by ELISA and Biacore (human, mouse and cynomolgus monkey serum albumin). Typically, pH-dependent clones were selected based on their reduced binding at pH 7.4. The characteristics of the clones selected are shown in Table S1.

[0182] Table S1. Binding and sequence characteristics of selected VHH clones.

[0183]

[0184] Sensorgrams showing the properties of selected clones 2H11 and 11C03 as monovalent (VHH) and bivalent (VHH-Fc) molecules binding to HSA (pH 5.5) on Biacore, as shown Figure 1 As shown. Association for each run was performed at pH 5.5. One clone, 2H11, showed good pH dependence, no cross-reactivity with mouse and cynomolgus serum albumin, did not bind to isolated DII, and did not compete with Alb23.

[0185] In order for the VHH to bind to albumin only in the endosome (acidic pH) and very limited (at most none) in the circulation (pH 7.4), the affinity of the VHH needs to be adjusted. In fact, the percentage of the complex VHH:albumin depends mainly on the affinity at a specific pH. Assuming that the albumin concentration in the circulation and in the endosome is between 34 and 54 mg / mL, it can be calculated that if the affinity of the VHH for albumin is about 2.4 mM (calculated for an albumin concentration of 40 mg / ml), 80% of the VHH will not bind (free). Similarly, in order for at least 80% of the VHH to bind to albumin (in the endosome), the affinity at pH 5.5 must be 0.12 mM (or lower). Ideally, less than 5% of VHHs bind to albumin at pH 7.4 (with an affinity of 11.5 mM and above), while more than 95% of VHHs bind to albumin at acidic pH (with an affinity of 30 nM and below).

[0186] To reduce its affinity at pH 7.4, all three CDRs of 2H11 were alanine scanned. The resulting variant VHH was generated as a two-arm Fc-ABDEG-20GS-C-terminal fusion and analyzed by FcRn ELISA QC; Biacore (3000) with human, cynomolgus monkey and mouse albumin on the chip (selection criteria: binding at pH 5.5 maintained, binding at pH 7.4 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).

[0187] Interestingly, mutations in the CDR3 region of 2H11 increased pH dependence by decreasing binding at pH 7.4 while maintaining good binding at pH 5.5.

[0188] Another clone, 11C03, selected for alanine scanning, cross-reacted with mouse and cynomolgus monkey serum albumin and also showed pH-dependent binding. For this clone, alanine mutations in CDR3 had no apparent effect on pH dependence. Thus, alanine scanning in CDR1 and CDR2 also altered binding to HSA. A set of variants that showed reduced binding and differential binding at pH 7.4 versus pH 5.5 were identified in binding to human (HSA) and cynomolgus monkey albumin (CSA, Table S3).

[0189] Data from selected 2H11 and 11C03 CDR3 variants are provided in Table S2.

[0190] Table S2: Analysis of 2H11 and 11C03 CDR3 variants fused to albumin at the C-terminus of Fc-ABDEG on chip (Biacore 3000).

[0191]

[0192] *Note the difference in dissociation rates.

[0193] Data from selected 2H11 and 11C03 CDR1-2 variants are provided in Table S3.

[0194] Table S3: Analysis of binding of 2H11 (CDR3) and 11C03 (CDR1-2) alanine variants fused to albumin at the C-terminus of Fc-ABDEG to human and cynomolgus monkey albumin on chip (Biacore 3000).

[0195]

[0196] *Note the difference in dissociation rates

[0197] The sequences of the 2H11 parental VHH and selected variants, 11C03 parental VHH and selected variants, additional VHH clones and scFv clones, and Alb23 VHH generated from the phage display library are provided in Tables S4-S8 below.

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206] Example 2: pH-dependent anti-albumin VHH prolongs Albumus mice TM Assessment of the half-life of Fab fragments in IgG independent of IgG-FcRn-mediated recycling.

[0207] Mota-Fab-VHH fusions were used to evaluate the affinity requirements for half-life extension of anti-albumin VHHs with varying degrees of pH dependence (strong binding at pH 5.5, weak binding at pH 7.4). Albumus mice that mimic physiological antibody clearance in humans TM (HSA / hFcRn) was injected intraperitoneally (IP single dose) with seven different test items to evaluate their clearance in this mouse model. The test items administered are described in detail as follows:

[0208] 1. Motavizumab-Fab fragment was used as a control treatment condition. This particular molecule has the same basic structure as the test item of interest; however, it is smaller in size (approximately 50 kDa).

[0209] 2. Motavizumab-Fab with an unrelated VHH 3Rab fused to the light chain at the C-terminus using a 20GS linker was used as a second control treatment condition. This particular molecule has a similar size to the test item of interest (approximately 65

[0210] kDa); however, it does not have any affinity for albumin.

[0211] 3. Motavizumab-Fab fragment with Alb23 was fused to the light chain at the C-terminus using a 20GS linker. Alb23 is an anti-albumin VHH. This treatment condition was included because this molecule appears to have a high affinity for albumin, but this affinity is not pH dependent.

[0212] 4. The Motavizumab-Fab fragment was fused to the light chain at the C-terminus with 2H11 using a 20GS linker. Previous in vitro studies have shown that this variant appears to have a high affinity for albumin at pH 5.5, however this affinity is slightly reduced at pH 7.4, further suggesting a pH-dependent affinity.

[0213] 5. The motavizumab-Fab fragment was fused to the light chain at the c-terminus with 2H11 mutants v3, v8, v9 or v15 using a 20GS linker. After alanine scanning and in vitro characterization, these 2H11 mutants carry alanine mutations in CDR3 (positions 3, 8, 9 and 15, respectively) and appear to have reduced affinity at both pH 5.5 and pH 7.4 compared to the parental molecule 2H11, but have a stronger pH-dependent affinity.

[0214] The doses of the tested items were 19 mg / kg (wt Fab) and 25 mg / kg (C-terminally fused Fab fragment), based on the equimolar dose of 30 mg / kg Fc-ABDEG-Alb23 used in previous studies as a reference.

[0215] For practical reasons, the experiment was divided into two parts that were performed consecutively:

[0216] Experiment 1: Treatment groups 1, 2, 3, 4

[0217] Experiment 2: Treatment groups 4, 5, 6, 7, 8

[0218] In total, forty-five (45) male and female Albumus mice TM Used in both experiments (treatment group 4 was included in both studies). Albumus mice approximately 14 to 15 weeks old TM Homozygous for the human FcRn transgene and the human albumin transgene. Mice were tail-tagged for identification and housed individually in well-ventilated polysulfone cages with HEPA-filtered air at a maximum density of up to 4 mice per cage. The normal temperature and relative humidity ranges for the animal room were 22°C ± 4°C and 50% ± 15%, respectively. Filtered tap water acidified to pH 2.5 to 3.0 and standard laboratory chow were provided ad libitum.

[0219] 1. Albumus Mice TM The mice were randomly divided into 8 groups, with 5 mice in each group.

[0220] 2. On day -3 (-3d), mice were preloaded with IVIg.

[0221] 3. On day 0, 0 hours, administer the test article IP to mice as follows:

[0222] · 19 mg / kg Mota-Fab, n=5, Group 1

[0223] · 25 mg / kg Mota-Fab-LC-3Rab (Mota-Fab-3Rab), n=5, Group 2

[0224] · 25 mg / kg Mota-Fab-LC-Alb23 (Mota-Fab-Alb23), n=5, Group 3

[0225] · 25 mg / kg Mota-Fab-LC-2H11 (Mota-Fab-2H11), n=5, Group 4

[0226] · 25 mg / kg Mota-Fab-LC-2H11v3 (Mota-Fab-2H11v3), n=5, Group 5

[0227] · 25 mg / kg Mota-Fab-LC-2H11v8 (Mota-Fab-2H11v8), n=5, Group 6

[0228] · 25 mg / kg Mota-Fab-LC-2H11v9 (Mota-Fab-2H11v9), n=5, Group 7

[0229] · 25 mg / kg Mota-Fab-LC-2H11v15 (Mota-Fab-2H11v15), n=5, Group 8

[0230] 4. According to the bleeding schedule shown in Table S9 below, 20 μL blood samples were collected from each mouse via the tail vein at 2h, 1d, 2d, 3d, 4d, 7d, and 8d.

[0231] 5. Serum samples were assessed in the following ELISA-based assays (in order of preference):

[0232] a. Test items PK ELISA

[0233] b. Human serum albumin ELISA

[0234] The program is also summarized in Figure 2 and Table S9. The collected mouse sera were read using PK and HSA assays.

[0235] Table S9. In vivo protocols

[0236]

[0237]

[0238] The serum concentrations of Mota-Fab-VHH were plotted as the mean values ​​for each group over time during the study and are shown in Figure 3 Data points show mean ± SD of 5 animals per group.

[0239] Mota-Fab-VHHs that do not bind albumin, such as Mota-Fab (approximately 50 kDa) and Mota-Fab-3Rab (approximately 65 kDa), showed relatively short half-lives with undetectable serum concentrations after 1 day. This data suggests that molecular weight is not the primary driver of serum persistence. Mota-Fab-2H11, which contains an inverse pH-dependent anti-albumin VHH, showed a comparable half-life to Mota-Fab-Alb23. Mota-Fab coupled to 2H11 alanine variants with reduced affinity to albumin at pH 7.4 (see Table S10, using Biacore 8K+ in a single cycle kinetic (SCK) protocol. Human serum albumin was immobilized on a CM5 chip and the Fab-VHH variants were injected in 1XHBS-EP+ pH 7.4 or citrate buffer pH 5.5 at a rate of 30 μl / min. in eight steps of 1.5-fold dilution series over 2 minutes) showed serum half-lives corresponding to their albumin binding affinity. Compared to Mota fusions with higher affinity for HSA, Mota-Fab-2H11-v9 and Mota-Fab-2H11-v15 showed shorter half-lives, but they still showed prolonged PK profiles. In contrast, Mota-Fab-2H11-v8 showed no detectable binding to albumin in SPR at both pH 5.5 and pH 7.4 (data not shown), and displayed a similar half-life to Mota-Fab and Mota-Fab-3Rab.

[0240] Table S10. Binding kinetics and theoretical anti-albumin:albumin complex formation

[0241]

[0242]

[0243] This suggests that Mota-Fab-2H11v9 and -2H11v15 have very low binding to HSA in serum and are therefore recycled only by binding to albumin in endosomes. Figure 4 As shown, anti-HSA-VHHs have reduced binding to HSA in serum but increased binding in endosomes, tending to lead to recycling of HSA and anti-HSA-VHHs without affecting the size of anti-HSA-VHHs. We have demonstrated clones that bind to HSA in a pH-dependent manner with a desirable half-life and are expected to have better biodistribution in tissues and tumors after administration.

[0244] ***

[0245] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to the modifications described, 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

1. An antigen binding molecule that specifically binds to HSA, wherein the equilibrium dissociation constant of the antigen binding molecule with 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 with HSA at pH 7.4 is at least 2.4 mM.

2. The antigen binding molecule according to claim 1, wherein the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 5.5 is less than 0.03 mM, and the equilibrium dissociation constant of the antigen binding molecule with HSA at pH 7.4 is at least 11.5 mM.

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 the binding affinity of the antigen binding molecule to HSA at pH 7.

4. 4 . The antigen binding molecule according to claim 1 , wherein the binding affinity is measured by surface plasmon resonance.

5. The antigen binding molecule according to any one of claims 1 to 4, wherein the antigen binding molecule is selected from a Fab fragment, sdAb, scFv, an antibody mimetic, HSA or an antigen binding fragment thereof. The antigen binding molecule according to claim 5 , wherein the sdAb is a VHH fragment. The antigen-binding molecule according to claim 6 , further comprising one or more amino acids added to the C-terminus of the VHH fragment.

8. The antigen binding molecule according to claim 7, wherein the one or more amino acids are selected from the group consisting of: a) A; b) AG; c) GG; and d)PP. 9 . A fusion protein comprising the antigen-binding molecule according to claim 1 and a therapeutic protein.

10. The fusion protein of claim 9, wherein the therapeutic protein is a small peptide therapeutic agent.

11. The fusion protein of claim 9, wherein the therapeutic protein is an antibody or a fragment thereof, optionally an Fc region or an antigen binding fragment.

12. The fusion protein according to any one of claims 9 to 11, wherein the antigen binding molecule is fused to the therapeutic protein via a linker. The fusion protein of claim 12 , wherein the linker is a non-cleavable linker. The fusion protein according to claim 12 , wherein the linker is a peptide linker.

15. The fusion protein of claim 14, wherein the peptide linker is a GS linker, optionally 8 to 40 amino acids in length, optionally 20 amino acids in length.

16. The fusion protein according to any one of claims 11 to 14, wherein the antigen binding molecule is fused to the antibody or fragment thereof via an IgG hinge region or a portion thereof.

17. The fusion protein of any one of claims 11 to 14, wherein the therapeutic protein is an Fc region comprising a first Fc domain and a second Fc domain, and wherein 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.

18. One or more isolated polynucleotides encoding an antigen binding molecule according to any one of claims 1 to 8 or a fusion protein according to any one of claims 9 to 17.

19. An expression vector comprising one or more isolated polynucleotides according to claim 18.

20. A host cell comprising one or more isolated polynucleotides according to claim 18 or an expression vector according to claim 18.

21. A method for producing an antigen binding molecule or a fusion protein, the method comprising culturing the host cell according to claim 19 under conditions that allow expression of the antigen binding molecule or the fusion protein.

22. A pharmaceutical composition comprising the antigen binding molecule according to any one of claims 1 to 8 or the fusion protein according to any one of claims 9 to 17 and at least one pharmaceutically acceptable carrier.

23. The antigen binding molecule according to any one of claims 1 to 8, or the fusion protein according to any one of claims 9 to 17, or the pharmaceutical composition according to claim 22, for use as a medicament.

24. A method for increasing the serum half-life of a therapeutic protein, the method comprising fusing the antigen binding molecule according to any one of claims 1 to 8 to the therapeutic protein.

25. Use of an antigen binding molecule according to any one of claims 1 to 8 for increasing the serum half-life of a therapeutic protein.

26. Use of the antigen binding molecule according to any one of claims 1 to 8 or the fusion protein according to any one of claims 9 to 17 for the manufacture of a medicament.

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