KR20140012094A - Hsa-related compositions and methods of use - Google Patents

Abstract

HSA compositions are provided having improved properties compared to natural human serum albumin (HSA).

Description

HSA-RELATED COMPOSITIONS AND METHODS OF USE}

1. Cross-Reference to Related Applications

This application claims priority to PCT Application No. PCT / US2011 / 24855, filed February 15, 2011, which is incorporated by reference in its entirety.

2. Reference to Sequence Listing

This application is a text file of file name “MED0554_PCT2_ST25”, dated August 7, 2011, which is 45,056 bytes in size, and includes by reference the Sequence Listing submitted with this application via EFS-Web.

3. Background of the Invention

The neonatal Fc receptor (FcRn) binds both molecules to the default lysosomal degradation pathway by specifically binding to both IgG and human serum albumin (HSA) at the acidic pH of the endosome and recycling them back to the cell surface. The pH dependent mechanism of bypassing from prolongs the lifespan of both IgG and human serum albumin (HSA). FcRn binding performance has been found to be inherent in domain III of albumin.

4. Summary of the Invention

The present disclosure provides HSA related compositions and methods of use. The present disclosure provides a chimeric polypeptide comprising a human serum albumin (HSA) moiety comprising a neonatal FcRn binding fragment and a heterologous polypeptide or a bioactive fragment thereof, as well as a composition comprising said chimeric polypeptide with a pharmaceutical carrier. Constructs useful for the preparation of such chimeric polypeptides are also disclosed. In addition, the present disclosure teaches methods for making such chimeric polypeptides and constructs encoding them. The present disclosure further provides a polypeptide comprising a human serum albumin (HSA) moiety comprising HSA domain III or a neonatal Fc receptor (FcRn) binding fragment thereof, wherein the HSA domain III is substituted with 1-18 amino acids. By increasing one or both of the affinity and serum half-life of the polypeptide relative to FcRn relative to a control polypeptide having an HSA moiety that does not include the amino acid substitution. The present disclosure also provides chimeric polypeptides comprising a heterologous protein and a human serum albumin (HSA) moiety comprising an HSA domain III or a neonatal Fc receptor (FcRn) binding fragment thereof, wherein the chimeric polypeptide is comprised of the heterologous protein. Capable of binding functional FcRn and binding to FcRn, wherein the HSA domain III contains one or more amino acid substitutions relative to the FcRn of the chimeric polypeptide relative to a control chimeric polypeptide having an HSA moiety that does not include the amino acid substitution. Increases one or both of Mars and serum half-life. In addition, disclosed herein are methods of using such chimeric polypeptides, for example, to increase the serum half-life of a protein. Methods and vectors are also disclosed that are useful for the generation of adenovirus libraries useful for screening large populations of polypeptides. This method is useful for screening and identifying HSA domain III amino acid substitutions that increase one or both of affinity for FcRn and serum half-life.

In some embodiments, the chimeric polypeptide has one or both of increased affinity and increased serum half-life for FcRn relative to a control polypeptide that does not comprise an HSA moiety. In some embodiments, the chimeric polypeptide has increased serum half life. In some embodiments, the chimeric polypeptide has both increased affinity for FcRn and increased serum half life. In some embodiments, the chimeric polypeptide has increased affinity for FcRn at acidic pH (eg, pH of approximately 5.5). In other embodiments, the chimeric polypeptide has increased affinity for FcRn at acidic pH (eg, a pH of approximately 5.5), but the chimeric polypeptide for FcRn at neutral pH (eg, a pH of approximately 7.4). The affinity of the polypeptide is substantially unaltered.

The present disclosure contemplates all combinations of any of the foregoing aspects and embodiments, as well as combinations of any of the embodiments described in the specification and examples.

5. Brief Description of Drawings
For purposes of explanation of the invention, some embodiments of the invention are shown in the drawings. However, the present invention is not limited to the exact alignment and means of the embodiments shown in the drawings.
1 provides kinetic and equilibrium analysis of human FcRn binding to domain III of human serum albumin (HSA). Here, SPR-induced binding, dissociation kinetics and equilibrium binding constants for human FcRn binding to immobilized domain III at pH 5.5 are shown. 1A shows a Coomassie stained PAGE gel demonstrating successful expression and purification of Domain III of HSA from Pichia Pastoris (indicated by arrow). FIG. 1B shows binding sensorgrams generated by injecting various concentrations of FcRn onto domain III immobilized on a CM5 chip. 1C shows a curve of equilibrium binding reaction versus FcRn concentration, fitted to a steady state affinity model.
2 not only schematically shows the various construct designs, but also provides information on purification and characterization of IgG fused to HSA and IgG fused to domain III. 2A shows the YTE variant as well as the DNA construct of the heavy chain of recombinant IgG-HSA or IgG-Domain III fusion protein. 2B shows SDS-PAGE analysis of purified fusion proteins (5 μg / lane) under reducing and non-reducing conditions. 2C shows analytical size exclusion chromatography of purified IgG-fusion proteins.
3 provides SPR-induced equilibrium constants for IgG fused to HSA and human FcRn binding to IgG fused to domain III. Equilibrium RU (Req) vs. human FcRn concentrations were plotted for each FcRn injection and fitted data to a steady-state affinity model to immobilize fixed IgG (FIG. 3A), IgG fused to HSA (FIG. 3B) and domain III. K _D was calculated for IgG fused to (FIG. 3C). The sensorgram shown in the inset shows the mass (resonance unit) of FcRn bound to the immobilized ligand on the Y axis after the blank subtraction versus the time on the X axis.
4 provides evidence suggesting that epitopes on HSA for FcRn are conformational epitopes. Sepharose (S) -HSA, S-IgG or S-tris treated in three different ways were incubated with human FcRn at pH 5.5. Bound FcRn was eluted and quantified by immunoblotting with anti-β2 microglobulin antibody. The position of the molecular weight marker (M, kD units) is indicated. Lane 1 contains 20 μg human FcRn, the amount added to all adsorption samples.
5 shows that HSA and domain III displayed on the surface of yeast cells ( S. Cerevisiae ) retain FcRn binding performance. FIG. 5A shows HSA or domain III on Saccharomyces cerevisiae cells transformed with galactose-induced pYD1 cell surface display plasmids detected by flow cytometry using FITC-conjugated anti-HSA antibodies. Results are shown. The cells were induced with galactose for the indicated time. FIG. 5B shows HSA or domains displayed on biotinylated human FcRn and Saccharomyces cerevisiae cells induced for 48 hours, visualized by anti-streptavidin phycoerythrin through the use of flow cytometry. Indicate binding of III. Yeast cells transformed with scfv were used as controls for background fluorescence against FITC and phycoerythrin. The experiment is expressed as a histogram of fluorescence intensity (log scale) versus cell number.
6 provides amino acid sequence alignments of domain III from various species (human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep, cat and horse). The alignment in FIGS. 6A-6D includes chickens, while FIGS. 6E-6H exclude chickens. Conserved amino acid residues between the various species are represented by straight lines, and conserved cysteine residues are indicated by dashed lines. The shaded amino acid residues are not conserved between species. It is recognized that the amino acid numbering of FIG. 6 is only indicated for human domain III rather than for numbering of domain III based on full length mature HSA.
FIG. 7 shows that fusion of IgG with HSA with wild type increases serum persistence to levels similar to those observed for IgG-YTE variants. The% versus time (1 to 240 hours) of the injected sample remaining in the serum is plotted.
8 shows a plasmid map of the scFv-Fc cell surface display library introduction vector. 8A shows a plasmid map of a pENDisplay vector containing an scFv-Fc-GPI-anchor cassette operably linked to a promoter (here CMV promoter) and terminated with a polyA sequence (here BGH polyA sequence). Indicates. The scFv moiety is flanked with SfiI and NotI restriction enzyme sites to facilitate cloning of various scFv sequences. The attLl and attL2 sites flank the scFv-Fc-GPI-anchor expression cassette. FIG. 8B shows a plasmid map of the pENDisplay-OriP vector based on the vector shown in FIG. 8A, but following the polyA tail of the scFv-Fc-GPI-anchor cassette (see FIG. 9C).
9 provides representative sequences for EBNA-1 and OriP. Amino acid sequences and nucleotide sequences for EBNA-1 are provided in FIGS. 9A and 9B, respectively. The sequence of OriP is provided in Figure 9c.
10 provides a schematic of representative general adenovirus expression vectors for expression of the protein (s) of interest. The vector shown may comprise a DNA sequence of interest that encodes one or more proteins of interest; OriP sequence; And optionally an EBNA-1 coding region. These components are optionally flanked by att recombination sites that may have been used to construct the vector. These components are flanking on one side by the adenovirus genome (in this case having deletions of the El and / or E3 genes) and the ITR sequences. 3 'and 5' ITR sequences are shown. The vector is a sequence diagram for replication in bacterial cells (e.g., E. coli origin of replication) and antibiotic selection (e.g. ampicillin resistance) for easy construction, proliferation and selection. And these components are positioned so that they are not introduced into the rescued adenovirus.
11 shows that HSA displayed on the surface of mammalian cells (293F cells) retains FcRn binding performance. FIG. 11A shows (G ₄ S) ₃ linker flanking CMV promoter (bold line), signal sequence (bold dashed line), N-terminal Flag tag (thin dashed line), HSA moiety (shaded parallel box) (FIG. Thin straight lines) and a mammalian expression construct named pEN-HSA-GPI comprising the DAF-GPI sequence. FIG. 11B shows FITC-conjugated HSA on 293F cell surface infected with adenovirus generated from pEN-HSA-GPI (FIG. 11A) after 16 and 24 hours, or from a control plasmid encoding a control scFv-Fc fusion protein. The result detected by flow cytometry using the HSA antibody is shown. 11C and 11D are displayed on biotinylated human FcRn (25 μg / ml and 5 μg / ml, respectively) and 293F cells visualized by anti-streptavidin phycoerythrin through the use of flow cytometry. Indicates binding of HSAs. The experiment is expressed as a histogram of fluorescence intensity (log scale) versus cell number.
FIG. 12 shows changes in binding profile of wild-type HSA (HSA-wt) and two HSA mutant libraries (HSA-DIII-lib1 and HSA-DIII-lib2) displayed on biotinylated human FcRn and 293F cells. . Panel A shows flow cytometry detection of HSA on 293F cell surface infected with wild type and mutant HSA-DIII libraries. Panel B shows flow cytometric detection of biotinylated human FcRn bound to HSA on the cell surface, visualized by anti-streptavidin phycoerythrin. The experiment is expressed as a histogram of fluorescence intensity (log scale) versus cell number.
FIG. 13 shows wild type HSA (HSA-wt, FIG. 13A) and two HSA mutant libraries (HSA-) stained with biotinylated human FcRn (10 μg / ml) detected with anti-streptavidin phycoerythrin. FACS sorting profiles of cells expressing DIII-lib1 and HSA-DIII-lib2, FIGS. 13B and 13C, respectively.
14 shows binding of biotinylated human FcRn with 293F cells expressing wild-type HSA (HSA-wt) and HSA-DIII-lib1 mutant libraries on their cell surface before sorting and after primary and secondary sorting. Show the change in profile. 14A shows flow cytometry detection of HSA on 293F cell surface expressing wild type HSA and HSA-DIII-lib1 prior to sorting and after primary and secondary sorting. 14B and 14C show binding of the same cell set with biotinylated human FcRn (1 μg / ml and 0.1 μg / ml, respectively), visualized by anti-streptavidin phycoerythrin through the use of flow cytometry. Indicates. The experiment is expressed as a histogram of fluorescence intensity (log scale) versus cell number.
15 shows that the binding of FcRn to mutant HSA displayed on the cell surface is pH dependent. Panels A and B are control scFv-Fc fusion proteins, wild type HSA and three representative mutants (see Table 5) at pH 5.5 (0.1 μg / ml FcRn, Panel A) and pH 7.2 (10 μg / ml, Panel B). Flow cytometry detection of biotinylated human FcRn detected with anti-streptavidin phycoerythrin on the surface of 293F cells expressing. Panel C shows flow cytometric detection of HSA on these cell surfaces through the use of FITC-conjugated anti-HSA antibodies.
16 shows that the majority of isolated HSA mutants have a higher affinity for FcRn as measured by flow cytometry. Panels of wild type HSA and selected mutants were analyzed by flow cytometry for binding to different concentrations of biotinylated human FcRn. Data is plotted as MFI for FcRn concentration.
17 shows the location of a number of variants on the resolved structure of HSA (PDB Accession No. 1BM0). Most of the structure is represented as a ribbon plot with residues L463, E495, T508, 1523 and K534, represented by bars and indicated by arrows. Loops 6 and 7, comprising residues 492 to 536 and helixes 7 and 8 are represented by circles. The majority of hotspots and preferred spots are found in this area.
18 shows the half-life curves of several combinatorial mutant HSAs in human FcRn transgenic mice. These results are also summarized in Table 11 of Example 8.14.

6. Detailed Description of the Invention

6.1. Introduction

The neonatal Fc receptor (FcRn) is pH dependent, bypassing both molecules from the default lysosomal degradation pathway by specifically binding to both IgG and human serum albumin (HSA) molecules at the acidic pH of the endosome and recycling them back to the cell surface. Mechanisms extend the lifespan of both IgG and human serum albumin (HSA). FcRn binding performance has been found to be inherent in domain III of albumin. As demonstrated herein, the addition of FcRn binding fragments of HSA can be used to increase serum half-life of proteins and / or FcRn binding affinity of therapeutic agents such as antibodies, antibody substitutes, proteins, protein scaffolds and peptides. have. In particular, as demonstrated herein, the RcRn binding affinity at acidic pH (eg, pH of approximately 5.5) is increased while the affinity at neutral pH (eg, pH of approximately 7.4) is substantially It does not change. Chimeric polypeptides comprising the FcRn binding domain variants of the present disclosure can increase the serum half-life or FcRn binding affinity of a protein even more than wild-type FcRn binding domains. The HSA variant polypeptides of the invention can act as scaffolds for binding to a therapeutic agent or can be coupled to a therapeutic agent.

The present disclosure provides variants of domain III. Such variants of domain III may be used alone or in combination with additional HSA sequences to increase serum half-life and / or FcRn binding affinity of heterologous proteins and / or nonprotein agents.

Chimeric polypeptides and HSA variants disclosed herein have numerous uses. It is recognized that proteins and other molecules are often removed relatively quickly from the human or animal body. Rapid elimination can weaken the ability to study proteins and other molecules in animal models and can weaken the ability to use them effectively for therapeutic purposes. In some cases, proteins are removed so quickly that they have no therapeutic effect. In other cases, the protein is removed at a rate that requires frequent dosing. Frequent dosing adds to the costs associated with treatment and also increases the risk of noncompliance with the therapy. In some cases, the protein is removed at a rate that requires higher dosages. Higher doses of active substance may increase the risk of side effects, including immune responses.

Chimeric polypeptides and variant HSA polypeptides of the present disclosure contribute to solving problems associated with fast or relatively fast protein removal by increasing serum half-life and / or affinity for FcRn. Similarly, nonprotein materials can be conjugated to variant HSA polypeptides of the present disclosure to increase serum half-life and / or affinity for FcRn.

6.2. Terms

Before continuing to describe the present invention in more detail, it should be understood that the present invention is not limited to specific compositions or process steps that may vary by themselves. As used in this specification and the appended claims, it is to be understood that the singular forms "a", "an" and "an" include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, for example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press); The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press); And the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary of the many terms used in the present invention to those skilled in the art.

Amino acids may be referred to herein by their commonly known 3-letter symbols, or by the 1-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly recognized one-letter codes. As used herein, “amino acid substitution” means replacing an amino acid with another amino acid at a specific position in the parent polypeptide sequence. For example, substitution L463N refers to a variant polypeptide wherein leucine at position 463 is substituted with asparagine.

Unless otherwise indicated, the numbering of amino acids in the variable domain, complementarity determining region (CDRs) and framework region (FR) of an antibody is described by Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Follow the Kabat definition as described in the National Institutes of Health, Bethesda, MD. (1991). When using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of the FR or CDR of the variable domain, or insertion into the FR or CDR. For example, the heavy chain variable domain may comprise a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat), and residues inserted after heavy chain FR residue 82 (eg, residues 82a, 82b according to Kabat and 82c, etc.). Kabat numbering of residues for a given antibody can be determined by alignment in the homology region of the sequence of said antibody with the "standard" Kabat numbered sequence. Maximum alignment of framework residues often requires the insertion of "spacer" residues in the numbering system to be used for the Fv region. In addition, the identity of some individual residues at any given Kabat site number may differ from antibody chain to antibody chain due to species or allele differences.

As used herein, the terms “antibody” and “antibodies” (also known as immunoglobulins) refer to monospecific antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific formed from two or more different epitope binding fragments. Enemy antibodies (eg, bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single chain Fvs (scFv), single chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F (ab ') ₂ fragments, antibody fragments exhibiting the desired biological activity (eg, antigen binding moieties), disulfide-linked Fvs (dsFv), anti-idiotype (anti-Id) antibodies (eg, the present invention) Epitope binding fragments of any of the above-described antibodies, including an anti-Id antibody to an antibody of), intrabodies, and the like. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie molecules containing one or more antigen binding sites. Immunoglobulin molecules can be of any isotype (eg, IgG, IgE, IgM, IgD, IgA, and IgY), sub-isotypes (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or allotypes ( allotype) (e.g. Gm, e.g. G1m (f, z, a or x), G2m (n), G3m (g, b or c), Am, Em, and Km (1, 2 or 3) Immunoglobulin molecule). The antibody may be derived from any mammal, or other animal, such as a bird (eg, a chicken), including but not limited to humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, and the like. .

As used herein, the term “full length HSA” refers to a mature full length human serum albumin protein or nucleotide sequence encoding such a protein. The full length HSA protein has approximately 585 amino acids (after removal of the N-terminal precursor sequence and pre-progenitor sequence). Mature full length HSA (also referred to as full length mature HSA) protein is described in SEQ ID NO: 2. In some embodiments, full length HSA refers to the mature full length form of HSA that does not have a precursor sequence. The sequence of the preliminary precursor HSA consists of 609 amino acids (before removal of the N terminal precursor sequence and the preliminary precursor sequence) and is described by GenBank Accession No. NP — 000468. In addition, the identity of some individual residues may differ from that set forth in SEQ ID NO: 2 due to allelic differences. Allelic variations that occur in domain III of HSA include the following variants when numbered based on their position in full-length mature HSA: R → C at residue 410; K → E at residue 466; E → K at residue 479; D → N at residue 494; E → K at residue 510; E → K at residue 505; V → M at residue 533; K → E at residue 536; K → E at residue 541; D → A or D → G at residue 550; K → E at residue 560; D → N at residue 563; E → K at residue 565; E → K at residue 570; K → E at residue 573; K → E at residue 574; GKKLVAASQAALGL → PTMRIRERK at residues 572 to 585; And LVAASQAALGL → TCCCKSSCLRLITSHLKASQPTMRIRERK at residues 575 to 585.

As used herein, the term “domain III of HSA” refers to the conventional domain III of the HSA or nucleotide sequence encoding such a protein spanning amino acids 381 to 585 (approximately 205 amino acids) of full length mature HSA. Domain III of HSA is abbreviated herein as domain III or simply DIII. The amino acid sequence for the domain III polypeptide is set forth in SEQ ID NO: 1. As mentioned above, the identity of some individual residues may differ from that set forth in SEQ ID NO: 1 due to allelic differences.

The term “chimeric polypeptide” as used herein refers to a polypeptide comprising two or more moieties that do not exhibit heterogeneity with respect to each other. For example, chimeric polypeptides, also referred to as fusion polypeptides or fusion proteins, include one or more HSA moieties linked to heterologous protein moieties. The HSA moiety and the heterologous protein moiety may themselves be fusions with, for example, an Fc or other moiety. The HSA moiety and the heterologous protein moiety can be linked through covalent or non-covalent interactions. For example, the HSA moiety and the heterologous protein moiety can be chemically conjugated to each other or can be fused recombinantly (eg, as an in-frame translation fusion).

As used herein, the term “heterologous protein” refers to all or part of a protein that is not HSA. Although the general term “heterologous protein” is used herein, the term is intended to encompass full length or substantially full length proteins as well as bioactive peptides of various lengths, including antibodies and antibody fragments. Preferred heterologous proteins can be used or studied for therapeutic purposes. Exemplary classes of heterologous proteins include, but are not limited to, enzymes, cytokines and growth factors.

As used herein, HSA polypeptides include various bioactive fragments and variants, fusion proteins, and modified forms of wild type HSA polypeptides. Such bioactive fragments or variants, fusion proteins, and altered forms of the HSA polypeptide have at least a portion of the amino acid sequence having substantial sequence identity to the native HSA protein and retain at least the FcRn binding activity of the native HSA protein. In some embodiments, a bioactive fragment, variant, or fusion protein of an HSA polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the HSA polypeptide. . As used herein, the term “fragment” is understood to include bioactive fragments or bioactive variants that exhibit FcRn binding activity. Suitable bioactive fragments can be used for the preparation of chimeric polypeptides, and such chimeric polypeptides can be used in any of the methods described herein.

As used herein, the terms “mutated”, “mutant” and the like, have one or more amino acids deleted, added or substituted through the use of well-known site-directed mutagenesis techniques or any other conventional method. Molecules, in particular HSA polypeptides.

6.3. HSA  domain III

In some embodiments, the present disclosure provides an HSA variant polypeptide comprising human serum albumin (HSA) domain III or a neonatal Fc receptor (FcRn) binding fragment thereof, wherein the variant polypeptide is capable of binding to FcRn and wherein the HSA Domain III contains 1-18 amino acid substitutions to increase serum half-life or increase the affinity of said variant polypeptides for FcRn relative to control HSA polypeptides lacking said amino acid substitutions.

In some embodiments, said 1-18 amino acid substitutions increase the affinity of the HSA variant polypeptide for FcRn. In some embodiments, said 1-18 amino acid substitutions increase the serum half-life of the HSA variant polypeptide. In some embodiments, said 1-18 amino acid substitutions increase both the affinity of the HSA variant polypeptide to FcRn and the serum half-life of the HSA variant polypeptide. In some embodiments, said 1-18 amino acid substitutions increase the affinity of the HSA variant polypeptide for FcRn at an acidic pH (eg, a pH of approximately 5.5). In some embodiments, said 1-18 amino acid substitutions increase the affinity of the HSA variant polypeptide for FcRn at an acidic pH (eg, a pH of approximately 5.5) but at a neutral pH (eg, a pH of approximately 7.4). ) Does not substantially alter the affinity of the HSA variant polypeptide to FcRn.

In some embodiments, the HSA variant binds to FcRn and has a dissociation rate or binding rate that is different from the dissociation rate or binding rate of said control HSA polypeptide. For example, in some embodiments, HSA variants bind to FcRn and have faster binding rates and / or slower dissociation rates. In other embodiments, the binding rate is slower and / or the dissociation rate is faster.

In some embodiments, the present disclosure provides a chimeric polypeptide comprising a HSA moiety comprising domain III or an FcRn binding portion thereof, and a heterologous protein, wherein the chimeric polypeptide retains the functional activity of the heterologous protein. In some embodiments, the HSA moiety comprises a whole HSA polypeptide, or a bioactive fragment comprising HSA domain III or a neonatal Fc receptor (FcRn) binding fragment thereof. In some embodiments, the HSA portion is HSA domain III or a neonatal Fc receptor (FcRn) binding fragment thereof, and at least a portion of another domain of HSA, eg, at least a portion of HSA domain I, at least a portion of HSA domain II Or at least a portion of HSA domains I and II. As used herein, when numbered based on location in full length mature HSA, HSA domain I comprises residues 1 to 197, HSA domain II comprises residues 189 to 385, and HSA domain III is residue 381 To 585.

In some embodiments, the chimeric polypeptide has one or both of an increased affinity for FcRn and an increased serum half-life relative to a control polypeptide that does not comprise an HSA moiety. In some embodiments, the chimeric polypeptide has increased affinity for FcRn. In some embodiments, the chimeric polypeptide has increased serum half life. In some embodiments, the chimeric polypeptide has both increased affinity for FcRn and increased serum half life. In some embodiments, the chimeric polypeptide has increased affinity for FcRn at acidic pH (eg, pH of approximately 5.5). In other embodiments, the chimeric polypeptide has increased affinity for FcRn at an acidic pH (eg, a pH of approximately 5.5), but at a neutral pH (eg, a pH of approximately 7.4) of the chimeric polypeptide for FcRn. Affinity is not substantially altered.

Further, as described herein, in some embodiments, domain III of the HSA portion of a polypeptide (eg, HSA variant or chimeric polypeptide) does not include such amino acid substitutions by including one to eighteen amino acid substitutions. It increases one or both of the affinity of the chimeric polypeptide to FcRn and the serum half-life of the chimeric polypeptide relative to the control chimeric polypeptide having the HSA moiety. In some embodiments, said 1-18 amino acid substitutions increase the affinity of the chimeric polypeptide for FcRn. In some embodiments, said 1-18 amino acid substitutions increase the serum half-life of the chimeric polypeptide. In some embodiments, said 1-18 amino acid substitutions increase both the affinity of the chimeric polypeptide for FcRn and the serum half-life of the chimeric polypeptide.

In some embodiments, domain III of the HSA portion of a polypeptide (eg, an HSA variant polypeptide or chimeric polypeptide) is one, two, three, four, five, six, seven, eight, nine Dog, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acid substitutions. Similarly, with respect to HSA variant polypeptides comprising domain III or its FcRn binding moiety, domain III has one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acid substitutions. In some embodiments, the amino acid substitution does not mean only a substitution of a single amino acid by another residue present in allelic variants.

In some embodiments, domain III of the HSA portion of the polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) comprises one or more amino acid substitutions at any of the following positions numbered based on the position in the full-length mature HSA Residue 381, residue 383, residue 391, residue 401, residue 402, residue 407, residue 411, residue 413, residue 414, residue 415, residue 416, residue 424, residue 426, residue 434, residue 442, residue 445, Residue 447, residue 450, residue 454, residue 455, residue 456, residue 457, residue 459, residue 463, residue 495, residue 506, residue 508, residue 509, residue 511, residue 512, residue 515, residue 516, residue 517 , Residue 519, residue 521, residue 523, residue 524, residue 525, residue 526, residue 527, residue 531, residue 535, residue 538, residue 539, residue 541, residue 557, residue 561, residue 566 and residue 569.

 In some embodiments, domain III of the HSA portion of the polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) comprises one or more amino acid substitutions at any of the following positions numbered based on the position in the full-length mature HSA (A) residues 383 and 413; (b) residues 401 and 523; (c) residues 407 and 447; (d) residues 407, 447 and 539; (e) residues 407 and 509; (f) residues 407 and 526; (g) residues 411 and 535; (h) residues 414 and 456; (i) residues 415 and 569; (j) residues 426 and 526; (k) residues 442, 450 and 459; (l) residues 463 and 508; (m) residues 508, 519 and 525; (n) residues 509 and 527; (o) residues 523 and 538; (p) residues 526 and 557; (q) residues 541 and 561; (r) residues 463 and 523; (s) residues 508 and 523; (t) residues 508 and 524; (u) residues 463, 508 and 523; (v) residues 463, 508 and 524; (w) residues 508, 523 and 524; (x) residues 463, 508, 523 and 524; (y) residues 463 and 524; (z) residues 523 and 524; And (aa) residues 463, 523 and 524.

In one embodiment, domain III of the HSA portion of the polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) is such that X ₁ is not leucine and / or X ₂ is not threonine and / or X ₃ is SEQ ID NO: 18 which is not isoleucine and / or X ₄ is not lysine. In some embodiments, X ₁ is cysteine, phenylalanine, glycine, histidine, isoleucine, asparagine, serine or glutamine; X ₂ is cysteine, glutamate, isoleucine, lysine, arginine or serine; X ₃ is alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine; X ₄ is alanine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, glutamine, threonine or valine. In other embodiments, X ₁ is phenylalanine or asparagine; X ₂ is arginine or serine; X ₃ is aspartate, glutamate, glycine, phenylalanine, lysine or arginine; X ₄ is leucine. In other embodiments, X ₁ is asparagine, X ₂ is arginine, X ₃ is glycine and / or X ₄ is leucine.

In some embodiments, increased affinity and / or serum half-life for FcRn is assessed against different controls. For example, the properties of the chimeric polypeptide can be assessed against the properties of the heterologous protein without the HSA moiety, or against the properties of the same or similar HSA moieties without the amino acid substitution and / or without the heterologous protein. Can be. Similarly, HSA variant polypeptides can be evaluated against HSA molecules that do not have amino acid substitutions.

In some embodiments, the chimeric polypeptide / HSA variant polypeptide binds FcRn with a higher affinity than the control polypeptide. In some embodiments, said 1-18 amino acid substitutions increase the affinity of the chimeric polypeptide / HSA variant polypeptide for FcRn at an acidic pH (eg, a pH of approximately 5.5). In some embodiments, said 1-18 amino acid substitutions increase the affinity of the chimeric polypeptide / HSA variant polypeptide for FcRn at an acidic pH (eg, a pH of approximately 5.5), but at a neutral pH (eg, Does not substantially alter the affinity of the chimeric polypeptide for FcRn at a pH of approximately 7.4). In some embodiments, the chimeric polypeptide binds to FcRn with higher affinity at acidic pH and has increased serum half-life.

In some embodiments, the chimeric polypeptide / HSA variant polypeptide binds to FcRn and has a dissociation rate or binding rate that is different from that of the control polypeptide. For example, in some embodiments, the chimeric polypeptide / HSA variant polypeptide binds to FcRn and has a faster binding rate and / or slower dissociation rate. In other embodiments, the binding rate is slower and / or the dissociation rate is faster.

In some embodiments, the HSA domain III of a polypeptide (eg, an HSA variant, and a chimeric polypeptide having an HSA moiety) comprises a control polypeptide having an HSA moiety that does not include the amino acid substitution by including 1 to 10 amino acid substitutions. Relatively increase the affinity of the polypeptide for FcRn and / or increase the serum half-life of the polypeptide. In some embodiments, the HSA domain III comprises one amino acid substitution. In some embodiments, the HSA domain III comprises two, three, four, five, six, seven, eight, nine or ten amino acid substitutions. In some embodiments, the HSA domain III comprises 11, 12, 13, 14, 15, 16, 17 or 18 amino acid substitutions. In some embodiments, the HSA domain III comprises one or more amino acid substitutions. In some embodiments, the HSA domain III comprises at least 10 amino acid substitutions.

Exemplary amino acid substitutions include (i) replacement with alanine; (ii) conservative amino acid substitutions; And (iii) non-conservative amino acid substitutions. The present disclosure contemplates that all of the amino acid substitutions within domain III of a given polypeptide may be members of one of these substitution categories, and that domain III of a given polypeptide (eg, HSA variants, and chimeric polypeptides having HSA moieties) It is also contemplated that each of the amino acid substitutions within may be selected individually and independently from these categories.

In some embodiments, a substitution (one, two, three, four, five, or six or more HSA domain III substitutions) is where the residues in the HSA are replaced with alanine. In some embodiments, a substitution (one, two, three, four, five, or six or more HSA domain III substitutions) replaces a given neutral amino acid residue in the HSA with another neutral amino acid residue. In some embodiments, substitutions (one, two, three, four, five or six or more HSA domain III substitutions) replace a given acidic amino acid residue in the HSA with another acidic amino acid residue. In some embodiments, substitutions (one, two, three, four, five or six or more HSA domain III substitutions) replace a given basic amino acid residue in the HSA with another basic amino acid residue. The present disclosure contemplates embodiments in which each substitution is independently selected from the above substitution classes. Polypeptides (eg, HSA variants, and chimeric polypeptides having HSA moieties) comprising any combination of the above substitution categories are specifically contemplated.

In some embodiments, a substitution (one, two, three, four, five or six or more HSA domain III substitutions) replaces one amino acid with another amino acid in the following group: Lysine (K; Lys) , Arginine (R; Arg) and histidine (H; His). In some embodiments, a substitution (one, two, three, four, five, or six or more HSA domain III substitutions) replaces one amino acid with another amino acid in the following group: Aspartate (D; Asp; Aspartic Acid) and Glutamate (E; Glu; Glutamic Acid). In some embodiments, a substitution (one, two, three, four, five or six or more HSA domain III substitutions) replaces one amino acid with another amino acid in the following group: Asparagine (N; Asn) , Glutamine (Q; Gln), serine (S; Ser), threonine (T; Thr) and tyrosine (Y; Tyr). In some embodiments, a substitution (1, 2, 3, 4, 5 or 6 or more HSA domain III substitutions) replaces one amino acid with another amino acid in the following group: Alanine (A; Ala) , Valine (V; Val), isoleucine (I; Ile), leucine (L; Leu), proline (P; Pro), phenylalanine (F; Phe), tryptophan (W; Trp), methionine (M; Met), Cysteine (C; Cys) and glycine (G; Gly). In some embodiments, a substitution (one, two, three, four, five or six or more HSA domain III substitutions) replaces one amino acid with another amino acid in the following group: phenylalanine, tryptophan and tyrosine. In some embodiments, a substitution (one, two, three, four, five or six or more HSA domain III substitutions) replaces one amino acid with another amino acid in the following group: cysteine, serine and threo Banging. In some embodiments, a substitution (one, two, three, four, five or six or more HSA domain III substitutions) replaces one amino acid with another amino acid in the following group: asparagine, glutamine, serine, Threonine, tyrosine, lysine, arginine, histidine, aspartate and glutamate. In some embodiments, a substitution (one, two, three, four, five or six or more HSA domain III substitutions) replaces one amino acid with another amino acid in the following group: glycine, serine, threo Nin, alanine, valine, leucine and isoleucine. The present disclosure contemplates embodiments in which each substitution is independently selected from the above substitution categories. Polypeptides comprising any combination of the above substitution classes (eg, HSA variants, and chimeric polypeptides having HSA moieties) are specifically contemplated.

In certain embodiments, domain III of the HSA portion of a polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) with increased affinity for FcRn and / or increased serum half-life is selected from the group consisting of the following amino acid substitutions One or more amino acid substitutions include: V381N, V381Q, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, K402A, K402G, K402I, K402L, K402VH, L402VH L407M, L407N, L407Q, L407R, L407W, L407Y, Y411Q, Y411N, K413C, K413S, K413T, K414S, K414T, V415C, V415L, V415S, V415T, Q416H, Q416P, V424A, V424D, V424G, V424I, V424L, V424L, V424L V424N, V424Q, V424W, V426D, V426E, V426F, V426H, V426L, V426N, V426P, V426Q, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, P447S, P447T, E450D, E450E, S454C, S454C, S454C, S454C S454T, V455N, V455Q, V456N, V456Q, L457F, L457W, L457Y, Q459K, Q459R, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T506F, T 506M, T506N, T506R, T506W, T506Y, T508C, T508E, T508I, T508K, T508R, T508S, F509C, F509D, F509G, F509I, F509L, F509M, F509P, F509V, F509W, F509Y, A511D, A511F, A511I, A511I, A511I A511T, A511V, A511W, A511Y, D512F, D512M, D512Q, D512W, D512Y, T515C, T515D, T515E, T515E, T515G, T515H, T515L, T515N, T515P, T515Q, T515S, T515W, T515Y, L516C, L516F, L516F, L516F, L516F L516T, L516W, L516Y, S517C, S517F, S517M, S517T, S517W, S517Y, K519A, K519G, K519I, K519L, K519V, R521F, R521H, R521M, R521Q, R521T, R521W, R521Y, I523A, I523C, I523D, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W, I523Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524M K524V, K525A, K525G, K525I, K525L, K525V, Q526A, Q526C, Q526F, Q526H, Q526L, Q526M, Q526P, Q526S, Q526T, Q526V, Q526Y, T527F, T527W, T527Y, E531A, E531G, E531I, E531L, E531 H535D, H535E, H535K, H535L, H535N, H535P, H535S, K538F, K538W, K538Y, A539I, A539L, A539V, K541F, K541W, K541Y, K557A, K557G, K557I, K557L, K557N, K557S, K557V, A561F, A561W, A561Y, T566F, T566W, T566Y, A569H and A569P. In some embodiments more than one (eg, two, three, four, five, etc.) amino acid substitutions in domain III or even all amino acid substitutions are selected from such substitutions.

In another specific embodiment, domain III of the HSA portion of the polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) comprises one or more amino acid substitutions selected from the group consisting of the following amino acid substitutions: V381N, E383G, N391V, Y401E , K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F, Q459R, L463C, L463C, L463C , L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C, S517W521 K519I , I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524A, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526M, Q526Y, Q526Y , E531I, H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W and A569P. In some embodiments more than one (eg, two, three, four, five, etc.) amino acid substitutions in domain III or even all amino acid substitutions are selected from such substitutions.

In another specific embodiment, domain III of the HSA portion of the polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) comprises one or more amino acid substitutions selected from the group consisting of the following amino acid substitutions: L407N, L407Y, V415T, V424I , V424Q, V426E, V426H, P447S, V455N, V456N, L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523K, I523K, I523 , Q526M, T527Y, H535P, and K557G. In some embodiments more than one (eg, two, three, four, five, etc.) amino acid substitutions in domain III or even all amino acid substitutions are selected from such substitutions.

In other specific embodiments, domain III of the HSA portion of the polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) comprises one or more amino acid substitutions selected from the group consisting of the following amino acid substitutions: L407Y, V415T, V424I, V424Q , P447S, V455N, V456N, L463N, E495D, T506Y, T508R, S517W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T527Y, H535P and K557G. In some embodiments more than one (eg, two, three, four, five, etc.) amino acid substitutions in domain III or even all amino acid substitutions are selected from such substitutions.

In some embodiments, domain III of the HSA portion of the polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) comprises residues 463 and 508; Residues 463 and 523; Residues 463 and 524; Residues 508 and 524; Residues 463, 508 and 523; Residues 463, 508 and 524; Or residues 508, 523 and 524; Or amino acid substitutions in HSA domain III at residues 463, 508, 523, and 524, wherein the substitutions at residues 463 are selected from L463C, L463F, L463G, L463H, L463I, L463N, L463S, and L463Q; Substitution at residue 508 is selected from T508C, T508E, T508I, T508K, T508R and T508S, and substitution at residue 523 is I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N , I523P, I523Q, I523R, I523S, I523T, I523V, 1523W and I523Y, and the substitution at residue 524 is selected from K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T and K524V.

In some embodiments, domain III of the HSA portion of the polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) comprises amino acid substitutions in HSA domain III selected from the group consisting of the following amino acid substitutions: (a) E383G / K413S ; (b) Y401E / I523G; (c) L407N / P447S; (d) L407N / P447S / A539I; (e) L407N / F509M; (f) L407Y / Q526T; (g) Y411Q / H535N; (h) K414S / V456N; (i) V415T / A569P; (j) V426H / Q526Y; (k) E442K / E450D / Q459R; (l) L463N / T508R; (m) T508R / K519I / K525V; (n) F509I / T527Y; (o) I523Q / K538Y; (p) Q526M / K557G; (q) K541F / A561F; (r) L463N / K524L; (s) T508R / I523G; (t) T508R / K524L; (u) L463N / T508R / I523G; (v) L463N / T508R / K524L; (w) T508R / I523G / K524L; (x) L463N / T508R / I523G / K524L; (y) L463N / I523G; (z) I523 G / K524L; And (aa) L463N / I523G / K524L. In some embodiments more than one (eg, two, three, four, five, etc.) amino acid substitutions in domain III or even all amino acid substitutions are selected from such substitutions.

In some embodiments, domain III of the HSA portion of the polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) comprises amino acid substitutions in HSA domain III selected from the group consisting of the following amino acid substitutions: (a) L407N / P447S ; (b) L407N / P447S / A539I; (c) L407N / F509M; (d) Y411Q / H535N; (e) K414S / V456N; (f) V426H / Q526Y; (g) L463N / T508R; (h) F509I / T527Y; (i) I523Q / K538Y; (j) Q526M / K557G; (k) K541F / A561F; (l) L463N / K524L; (m) T508R / I523 G; (n) T508R / K524L; (o) L463N / T508R / I523G; (p) L463N / T508R / K524L; (q) T508R / I523G / K524L; (r) L463N / T508R / I523G / K524L; (s) L463N / I523G; (t) I523G / K524L; And (u) L463N / I523G / K524L.

In some embodiments, domain III of the HSA portion of the polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) comprises amino acid substitutions in HSA domain III selected from the group consisting of the following amino acid substitutions: (a) L463N / T508R ; (b) L463N / K524L; (c) T508R / I523G; (d) T508R / K524L; (e) L463N / T508R / I523G; (f) L463N / T508R / K524L; (g) T508R / I523G / K524L; And (h) L463N / T508R / I523G / K524L.

In some embodiments, domain III of the HSA portion of the polypeptide (eg, HSA variant polypeptide or chimeric polypeptide) comprises amino acid substitutions in HSA domain III selected from the group consisting of the following amino acid substitutions: (a) L463N / K508R ; (b) T508R / I523G; (c) T508R / K524L; And (d) L463N / T508R / I523G.

In addition, the fragments or variants may be chemically synthesized through techniques known in the art, such as using conventional Merrifield solid phase f-Moc or t-Boc chemistry. The preparation or testing of such fragments or variants (recombinantly or chemically synthetic) can identify, for example, fragments or variants that can function as well as, or behave substantially similar to, native HSA proteins.

In some embodiments, the invention alters the structure of an HSA polypeptide for purposes such as improving the therapeutic or prophylactic efficacy, or stability (eg, serum half-life, in vitro shelf life, and resistance to proteolytic degradation in vivo). Consider doing it. Such altered HSA polypeptides have the same or substantially the same bioactivity as naturally occurring (ie, naturally or wild type) HSA polypeptides. The altered HSA polypeptide can be conjugated to other therapeutic moieties (eg, proteins and nonprotein materials) as described herein. Altered polypeptides can be produced, for example, by amino acid substitutions, deletions or additions. For example, isolated replacement of leucine by isoleucine or valine, isolated replacement of aspartate by glutamate, isolated replacement of threonine by serine or similar replacement of amino acids by structurally related amino acids (e.g. For example, it is reasonable to predict that conservative amino acid substitutions will not have a major impact on the biological activity of the resulting molecule. Conservative substitutions are those that occur within the amino acid family involved in their side chains.

In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are substitutions of residues that are conserved among multiple species. In some embodiments, all of said amino acid substitutions in HSA domain III are substitutions of residues that are conserved among multiple species. In some embodiments, one or more amino acid substitutions of the amino acid substitutions in HSA domain III are serum albumin proteins from humans, pigs, rats, mice, dogs, rabbits, cows, chickens, donkeys, sand mice, sheep, cats and horses. It is substitution of residue preserved in between. In some embodiments, all of said amino acid substitutions in HSA domain III are conserved between serum albumin proteins from humans, pigs, rats, mice, dogs, rabbits, cows, chickens, donkeys, sand rats, sheep, cats and horses Substitution of residues. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are substitutions of residues that are conserved between serum albumin proteins from highly conserved species, such as apes and monkeys, for humans. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are substitutions of residues that are conserved between serum albumin proteins from a non-mammalian. In some embodiments, all of said amino acid substitutions in HSA domain III are substitutions of residues that are conserved between serum albumin proteins from highly conserved species, such as apes and monkeys, for humans. In some embodiments, all of said amino acid substitutions in HSA domain III are substitutions of residues that are conserved between serum albumin proteins from non-mammalian. In some embodiments, one or more amino acid substitutions of the amino acid substitutions in HSA domain III are serum albumin from the majority of humans, pigs, rats, mice, dogs, rabbits, cows, chickens, donkeys, sand mice, sheep, cats and horses. Substitution of residues that are conserved between proteins. In some embodiments, two or more amino acid substitutions of the above amino acid substitutions in HSA domain III are selected from the group consisting of human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep, cat and horse It is the substitution of residues conserved between serum albumin proteins from the above species. In some embodiments, one or more amino acid substitutions of the amino acid substitutions in HSA domain III are selected from the group consisting of human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep, cat and horse It is the substitution of residues conserved between serum albumin proteins from the above species. In some embodiments, four or more amino acid substitutions of the amino acid substitutions in HSA domain III are selected from the group consisting of human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep, cat and horse It is the substitution of residues conserved between serum albumin proteins from the above species. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are selected from the group consisting of human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep, cat and horse It is the substitution of residues conserved between serum albumin proteins from the above species. In some embodiments, two or more amino acid substitutions of the above amino acid substitutions in HSA domain III are selected from the group consisting of human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep, cat and horse Substitution of residues conserved between serum albumin proteins from to five species. In some embodiments, all of the amino acid substitutions in HSA domain III are between serum albumin proteins from the majority of humans, pigs, rats, mice, dogs, rabbits, cows, chickens, donkeys, sand mice, sheep, cats and horses. It is substitution of the residue which is preserved. In some embodiments, all of said amino acid substitutions in HSA domain III are from two or more species selected from the group consisting of human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep, cat and horse. It is the substitution of residues that are conserved between serum albumin proteins. In some embodiments, all of said amino acid substitutions in HSA domain III are from three or more species selected from the group consisting of human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep, cat, and horse It is the substitution of residues that are conserved between serum albumin proteins. In some embodiments, all of said amino acid substitutions in HSA domain III are from four or more species selected from the group consisting of human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep, cat and horse. It is the substitution of residues that are conserved between serum albumin proteins. In some embodiments, all of said amino acid substitutions in HSA domain III are from at least 5 species selected from the group consisting of human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep, cat and horse. It is the substitution of residues that are conserved between serum albumin proteins. In some embodiments, two to five species all of said amino acid substitutions in HSA domain III are selected from the group consisting of human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep, cat, and horse Substitution of residues that are conserved between serum albumin proteins from. Polypeptides (eg, HSA variants and chimeric polypeptides) comprising any combination of the above amino acid substitution categories are also contemplated.

In some embodiments, one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on position in full length mature HSA: residue 383, residue 389, residue 391, residue 410, residue 417 , Residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571. In some embodiments, one in HSA domain III The above amino acid substitution is at any of the following positions numbered based on the position in full-length mature HSA: residue 417, residue 442, residue 499 and residue 502. In some embodiments, one or more amino acids in HSA domain III The substitution is at any of the following positions numbered based on the position in full length mature HSA: residue 392, residue 399, residue 403, residue 411, residue 412, residue 414, residue 416, residue 418, residue 420, residue 423, residue 434, residue 437, residue 438, residue 445, residue 448, residue 450, residue 453, residue 461, residue 476, residue 477, residue 484, residue 485, residue 487, residue 488, residue 494, Residue 497, residue 507, residue 509, residue 514, residue 529, residue 534, residue 537, residue 540, residue 551, residue 558, residue 559, residue 567, residue 568 and residue 572. In some embodiments, within domain III More than one (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18) amino acid substitutions or even all amino acid substitutions are present at the residues. Specifically contemplated are polypeptides (eg, HSA variants and chimeric polypeptides) that include all combinations of amino acid substitutions at any one or more of these residues.

In some embodiments, one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on position in full length mature HSA domain III: residue 383, residue 391, residue 411, residue 414, Residue 416, residue 434, residue 442, residue 445, residue 450 and residue 509.

In some embodiments, one or more amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions: E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y411Q, Y411N, K414S , K414T, Q416H, Q416P, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, E450D, E450E, F509C, F509D, F509G, F509I, F509L, F509M, F509P, F509V, F509W and F509Y. In some embodiments more than one (eg, two, three, four, five, etc.) amino acid substitutions in domain III or even all amino acid substitutions are selected from such substitutions.

In some embodiments, one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on position in full length mature HSA domain III: residue 380, residue 381, residue 384, residue 387, Residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441, residue 457, residue 458 , Residue 463, residue 464, residue 466, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547 and residue 576. In some embodiments, more than one (eg, two in domain III) , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18)Amino acid substitutions, or even all amino acid substitutions are present in the residue. Specifically contemplated are polypeptides (eg, HSA variants and chimeric polypeptides) that include all combinations of amino acid substitutions at any one or more of these residues.

In some embodiments, one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full length mature HSA domain III: residue 381, residue 401, residue 424, residue 457, Residue 463, residue 495, residue 508, residue 515, residue 516, residue 524, residue 525, residue 526, residue 531, residue 535 and residue 539. In some embodiments, a polypeptide (eg, an HSA variant polypeptide or a chimeric polypeptide Domains III of the HSA moiety of residues are residues 463 and 508; Residues 463 and 524; Residues 508 and 524; Or amino acid substitutions in domain III at residues 463, 508 and 524.

In some embodiments, one or more amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions: V381N, V381Q, Y401D, Y401E, V242A, V242G, V424I, V424L, V424N, V424Q, V424V, L457F, L457W , L457Y, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T508C, T508E, T508I, T508K, T508R, T508S, T515C, T515H, T515N, T515P, T515Q, T515S, L516F, L516F516 L516 , L516W, L516Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, E531A, E531G, , E531L, E531V, H535D, H535E, H535P, A539I, A539L and A539V. In some embodiments more than one (eg, two, three, four, five, etc.) amino acid substitutions in domain III or even all amino acid substitutions are selected from such substitutions.

In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are substitutions of residues that are not conserved among multiple species. In some embodiments, all of said amino acid substitutions in HSA domain III are substitutions of residues that are not conserved among multiple species. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are substitutions of residues that are not conserved between serum albumin proteins from humans, rats, dogs, rabbits, and cows. In some embodiments, all of said amino acid substitutions in HSA domain III are substitutions of residues that are not conserved between serum albumin proteins from humans, rats, dogs, rabbits, and cows. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are substitutions of residues that are not conserved between serum albumin proteins from humans, rats, dogs, rabbits, and cows. In some embodiments, all of said amino acid substitutions in HSA domain III are substitutions of residues that are not conserved between serum albumin proteins from humans, rats, dogs, rabbits, and cows. Also contemplated are polypeptides comprising any combination of amino acid substitutions of this category (eg, HSA variants, and chimeric polypeptides having HSA moieties).

In some embodiments, one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on position in full length mature HSA: residue 382, residue 385, residue 390, residue 397, residue 400 , Residue 402, residue 415, residue 429, residue 432, residue 435, residue 439, residue 440, residue 443, residue 444, residue 446, residue 447, residue 459, residue 471, residue 472, residue 478, residue 479, residue 483, residue 490, residue 492, residue 493, residue 503, residue 511, residue 517, residue 518, residue 519, residue 521, residue 538, residue 541, residue 542, residue 546, residue 549, residue 550, residue 552, Residue 554, residue 556, residue 560, residue 562, residue 563, residue 565 and residue 566. In some embodiments, more than one (eg, two, three, four, five, six, in domain III) Dogs, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18) amino acid substitutions or even all Acid substitutions are present in the residue. Specifically contemplated are polypeptides (eg, HSA variants, and chimeric polypeptides having HSA moieties) comprising any combination of amino acid substitutions at any one or more of these residues.

In some embodiments, one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on position in full length mature HSA: residue 402, residue 415, residue 447, residue 459, residue 511 , Residue 517, residue 519, residue 521, residue 538, residue 541 and residue 566.

In some embodiments, one or more amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions: K402A, K402G, K402I, K402L, K402V, V415C, V415S, V415T, P447S, P447T, Q459K, Q459R, L463N , L463Q, A511F, A511W, A511Y, S517C, S517F, S517M, S517T, S517W, S517Y, K519A, K519G, K519I, K519L, K519V, R521F, R521W, R521Y, K538F, K538W, K538Y, K541F, K541566, , T566W and T566Y. In some embodiments more than one (eg, two, three, four, five, etc.) amino acid substitutions in domain III or even all amino acid substitutions are selected from such substitutions.

In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are substitutions of surface accessible residues. In some embodiments, all of said amino acid substitutions in HSA domain III are substitutions of surface accessible residues. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are substitutions of residues that are surface accessible and conserved among multiple species. In some embodiments, all of said amino acid substitutions in HSA domain III are substitutions of residues that are surface accessible and conserved among multiple species. In some embodiments, one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on position in full length mature HSA: residue 383, residue 389, residue 391, residue 410, residue 417 , Residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571. In some embodiments, one in HSA domain III The above amino acid substitution is at any of the following positions numbered based on the position in full-length mature HSA: residue 417, residue 442, residue 499 and residue 502. In some embodiments, one or more amino acids in HSA domain III The substitution is at any of the following positions numbered based on the position in full length mature HSA: residue 383, residue 391 and residue 442. In some embodiments, more than one (in domain III) ( For example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen Or 18) amino acid substitutions or even all amino acid substitutions are present at the residues. Polypeptides including any combination of amino acid substitutions of the above categories (eg, HSA variants and chimeric polypeptides) are specifically contemplated. In some embodiments, one or more amino acid substitutions in HSA domain III are selected from the group consisting of E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, E442K, and E442R. In some embodiments more than one (eg, two, three, four, five, etc.) amino acid substitutions in domain III or even all amino acid substitutions are selected from such substitutions.

In some embodiments, one or more amino acid substitutions in HSA domain III are R410C; K466E; E479K; D494N; E501K; E505K; V533M; K536E536; K541E; D550A or D550G; K560E; D563N; E565K; E570K; K573E; Or not just a substitution of K574E.

In some embodiments, a polypeptide (eg, a HSA variant, and a chimeric polypeptide having an HSA moiety) comprises an HSA domain III comprising an amino acid sequence that is at least 80%, 85%, or 90% identical to SEQ ID NO: 1. In some embodiments, HSA domain III comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, HSA domain III comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1. In some embodiments, the HSA moiety comprises an amino acid sequence that is at least 80%, 85% or 90% identical to the corresponding part of SEQ ID NO: 2. In some embodiments, the HSA moiety comprises an amino acid sequence that is at least 95% identical to the corresponding part of SEQ ID NO: 2. In some embodiments, the HSA moiety comprises an amino acid sequence that is at least 98% identical to the corresponding moiety of SEQ ID NO: 2.

In some embodiments, the present disclosure provides that polypeptides (eg, HSA variants, and chimeric polypeptides having HSA moieties) will include one or more amino acid substitutions within the HSA site outside domain III in addition to one or more amino acid substitutions in HSA domain III. Consider that you can.

In some embodiments, the present disclosure provides that a polypeptide (eg, HSA variant, and a chimeric polypeptide having an HSA moiety) is one or more (eg, 1, 2) in domain III in addition to one or more amino acid substitutions in HSA domain III. Dog, three, four, five, six, seven or eight) amino acid deletions and / or insertions are also contemplated. If the HSA moiety contains one or more amino acid deletions and / or insertions, it is recognized that such inserted or deleted residues may be indicated through the use of letters so as not to destroy residue numbering based on residue numbering of the native HSA. do. For example, if an amino acid residue is inserted between residues 414 and 415, such residue may be designated as 414a. In some embodiments, inserted amino acid residues are inserted into surface accessible loops to increase the size of such loops. In some embodiments, inserted amino acid residues are inserted into helices to increase the size of these helices and / or alter the structure of such helices.

In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in loop 2 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 2 of HSA domain III. In some embodiments, HSA domain III comprises 1 to 5 (1, 2, 3, 4 or 5) amino acid substitutions, wherein the 1 to 5 amino acid substitutions are HSA domain III Is present in loop 2. In some embodiments, HSA domain III comprises 1 to 5 (1, 2, 3, 4 or 5) amino acid substitutions within loop 2 of HSA domain III and is not present in loop 2 One or more additional amino acid substitutions within HSA domain III. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in loop 3 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 3 of HSA domain III. In some embodiments, HSA domain III comprises 1 to 5 (1, 2, 3, 4 or 5) amino acid substitutions, wherein the 1 to 5 amino acid substitutions are HSA domain III Is present in loop 3. In some embodiments, HSA domain III comprises one to five (one, two, three, four or five) amino acid substitutions within loop 3 of HSA domain III and not present in loop 3 One or more additional amino acid substitutions within HSA domain III. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in loop 6 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 6 of HSA domain III. In some embodiments, HSA domain III comprises 1-18 amino acid substitutions (1, 2, 3, 4, 5, 6, etc.), wherein said 1-18 amino acid substitutions Is present in loop 6 of HSA domain III. In some embodiments, HSA domain III comprises 1-18 amino acid substitutions (1, 2, 3, 4, 5, 6, etc.) within loop 6 of HSA domain III, loop 6 Further comprises one or more additional amino acid substitutions in HSA domain III not present in. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in helix 7 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in helix 7 of HSA domain III. In some embodiments, HSA domain III comprises 1 to 6 (1, 2, 3, 4, 5 or 6) amino acid substitutions, wherein the 1 to 6 amino acid substitutions are It is present in helix 7 of HSA domain III. In some embodiments, HSA domain III comprises 1 to 6 (1, 2, 3, 4, 5 or 6) amino acid substitutions within helix 7 of HSA domain III, and in helix 7 One or more additional amino acid substitutions in HSA domain III that are not present. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in loop 7 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 7 of HSA domain III. In some embodiments, HSA domain III comprises one to three (one, two or three) amino acid substitutions, wherein said one to three amino acid substitutions are present in loop 7 of HSA domain III. . In some embodiments, HSA domain III comprises one to three (one, two or three) amino acid substitutions within loop 7 of HSA domain III and one or more in HSA domain III not present in loop 7 Further amino acid substitutions are included. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are in helix 8 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in helix 8 of HSA domain III. In some embodiments, HSA domain III comprises 1-18 amino acid substitutions (1, 2, 3, 4, 5, 6, etc.), wherein said 1-18 amino acid substitutions Is present in helix 8 of HSA domain III. In some embodiments, HSA domain III comprises one to six (1, 2, 3, 4, 5 or 6) amino acid substitutions within helix 8 of HSA domain III and in helix 8 One or more additional amino acid substitutions in HSA domain III that are not present. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in loop 8 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 8 of HSA domain III. In some embodiments, HSA domain III comprises 1 to 5 (1, 2, 3, 4 or 5) amino acid substitutions, wherein the 1 to 5 amino acid substitutions are HSA domain III Is present in loop 8. In some embodiments, HSA domain III comprises one to five (one, two, three, four or five) amino acid substitutions within loop 8 of HSA domain III and not present in loop 8 One or more additional amino acid substitutions within HSA domain III. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are in loop 9 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 9 of HSA domain III. In some embodiments, HSA domain III comprises 1 to 4 amino acid substitutions, wherein said 1 to 4 (1, 2, 3 or 4) amino acid substitutions are loop 9 of HSA domain III. Exists in In some embodiments, HSA domain III comprises one to five (1, 2, 3, 4) amino acid substitutions within loop 9 of HSA domain III and is not present in loop 9 Further comprises one or more additional amino acid substitutions in the. As described in detail above, the potential amino acid substitutions at each position independently are selected from any class of substitutions described above in detail (eg, substitutions with alanine, conservative substitutions, etc.). Also contemplated are polypeptides (eg, HSA variants and chimeric polypeptides) comprising any combination of amino acid substitutions of this category.

In some embodiments, said amino acid substitutions in HSA domain III are not present in loop 2 of HSA domain III. In some embodiments, said amino acid substitutions in HSA domain III are not in loop 3 of HSA domain III. In some embodiments, said amino acid substitutions in HSA domain III are not present in loop 6 of HSA domain III. In some embodiments, the amino acid substitutions are not present in helix 7 of HSA domain III. In some embodiments, the amino acid substitutions are not present in loop 7 of HSA domain III. In some embodiments, the amino acid substitutions are not present in helix 8 of HSA domain III. In some embodiments, said amino acid substitutions in HSA domain III are not present in loop 8 of HSA domain III. In some embodiments, said amino acid substitutions in HSA domain III are not in loop 9 of HSA domain III.

In some embodiments, said amino acid substitutions in HSA domain III are not present in at least two loops of HSA domain III selected from the group consisting of loops 2, 3, 6, 7, 8, and 9. In some embodiments, said amino acid substitutions in HSA domain III are not present in at least three loops of HSA domain III selected from the group consisting of loops 2, 3, 6, 7, 8, and 9. In some embodiments, said amino acid substitutions in HSA domain III are not in four or more loops of HSA domain III selected from the group consisting of loops 2, 3, 6, 7, 8, and 9. In some embodiments, said amino acid substitutions in HSA domain III are not present in helix 7 or 8.

In some embodiments, domain III comprises two or more amino acid substitutions, wherein the amino acid substitutions are two of HSA domain III selected from the group consisting of loops 2, 3, 6, 7, 8 and 9, and helixes 7 and 8 Above loops and / or spirals. In some embodiments, domain III comprises three or more amino acid substitutions, wherein the amino acid substitutions are two of HSA domain III selected from the group consisting of loops 2, 3, 6, 7, 8 and 9, and helixes 7 and 8 Above loops and / or spirals. In some embodiments, domain III comprises four or more amino acid substitutions, wherein the amino acid substitutions are four of the HSA domain III selected from the group consisting of loops 2, 3, 6, 7, 8 and 9, and helixes 7 and 8 Above loops and / or spirals. In some embodiments, domain III comprises five or more amino acid substitutions, wherein the amino acid substitutions are five of HSA domain III selected from the group consisting of loops 2, 3, 6, 7, 8 and 9, and helixes 7 and 8 Above loops and / or spirals. In some embodiments, domain III comprises six or more amino acid substitutions, wherein the amino acid substitutions are six of HSA domain III selected from the group consisting of loops 2, 3, 6, 7, 8 and 9, and helixes 7 and 8 Above loops and / or spirals. In some embodiments, domain III comprises five or more amino acid substitutions, said amino acid substitutions being in loops 2, 3, 6, 7, 8, and 9, respectively, of HSA domain III. In some embodiments, domain III comprises six or more amino acid substitutions, said amino acid substitutions being in loops 2, 3, 6, 7, 8, and 9, respectively, of HSA domain III. In some embodiments, domain III comprises two or more amino acid substitutions, said amino acid substitutions being in helix 7 and 8 of HSA domain III respectively.

In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are present in loop 2 and are selected from residues 415, residues 416, residues 417, residues 418 and residues 419. In some embodiments, more than one (2, 3, 4, 5, etc.) amino acid substitutions are in loop 2 and are selected from residues 415, residues 416, residues 417, residues 418 and residues 419. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III loop 2 are selected from V415C, V415S, V415T, Q416H, and Q416P. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are present in loop 3 and are selected from residues 439, residues 440, residues 441, residues 442 and 443. In some embodiments, more than one (2, 3, 4, 5, etc.) amino acid substitutions are in loop 3 and are selected from residues 439, residues 440, residues 441, residues 442 and residues 443. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III loop 3 are selected from E442K and E442R. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are present in loop 6 and include residue 492, residue 493, residue 494, residue 495, residue 496, residue 497, residue 498, residue 499, residue 500. , Residue 501, residue 502, residue 503, residue 504, residue 505, residue 506, residue 507, residue 508 and residue 509. In some embodiments, more than one (2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions are in loop 6 and residues 492, Residue 493, residue 494, residue 495, residue 496, residue 497, residue 498, residue 499, residue 500, residue 501, residue 502, residue 503, residue 504, residue 505, residue 506, residue 507, residue 508 and residue 509 Is selected from. In some embodiments, the amino acid substitutions of one or more of the amino acid substitutions in HSA domain III loop 6 are T506F, T506W, T506Y, T508C, T508E, T508I, T508K, T508R, T508S, F509C, F509I, F509L, F509M, F509V, F509W and Is selected from F509Y. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are in helix 7 and are selected from residue 510, residue 511, residue 512, residue 513, residue 514 and residue 515. In some embodiments, more than one (2, 3, 4, 5, 6, etc.) amino acid substitutions are in helix 7 and comprise residue 510, residue 511, residue 512, residue 513, residue 514 and Selected from residue 515. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III loop 7 are selected from A511F, A511W, A511Y, D512F, D512W, D512Y, T515C, T515H, T515N, T515P, T515Q, and T515S. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are present in loop 7 and are selected from residues 516, residues 517 and residues 518. In some embodiments, more than one (2, 3, etc.) amino acid substitutions are in loop 7 and are selected from residues 516, residues 517 and residues 518. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III loop 7 are selected from L516F, L516S, L516T, L516W, L516Y, S517C, S517F, S517M, S517T, S517W, and S517Y. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are in helix 8, and residue 519, residue 518, residue 519, residue 520, residue 521, residue 522, residue 523, residue 524, residue 525 , Residue 526, residue 527, residue 528, residue 529, residue 530, residue 531, residue 532, residue 533, residue 534, residue 535 and residue 536. In some embodiments, more than one (2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions are in helix 8 and residues 519, Residue 518, residue 519, residue 520, residue 521, residue 522, residue 523, residue 524, residue 525, residue 526, residue 527, residue 528, residue 529, residue 530, residue 531, residue 532, residue 533, residue 534 , Residue 535 and residue 536. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III helix 8 are selected from the following amino acid substitutions: K519A, K519G, K519I, K519L, K519V, R521F, R521W, R521Y, I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W, I523Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524M, K524M K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, T527F, T527W, T527Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E and H535P. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are present in loop 8 and are selected from residues 537, residues 538, residues 539, residues 540 and residues 541. In some embodiments, more than one (2, 3, 4, 5, etc.) amino acid substitutions are present in loop 8 and are selected from residues 537, residues 538, residues 539, residues 540 and residues 541. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are present in loop 9 and are selected from residues 561, residues 562, residues 563 and residues 564. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III loop 8 are selected from K538F, K538W, K538Y, A539I, A539L, A539V, K541F, K541W, and K541Y. In some embodiments, more than one (2, 3, 4, etc.) amino acid substitutions are in loop 9 and are selected from residues 561, residues 562, residues 563 and residues 564. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III loop 9 are selected from A561F, A561W, and A561Y.

For example, insertions or deletions in domain III that increase or decrease the length of the HSA domain III loop are also contemplated. In some embodiments, said insertion or deletion in HSA domain III changes the length of loop 2. In some embodiments, said insertion or deletion in HSA domain III alters the length of loop 3. In some embodiments, said insertion or deletion in HSA domain III alters the length of loop 6. In some embodiments, said insertion or deletion in HSA domain III changes the length of helix 7. In some embodiments, said insertion or deletion in HSA domain III alters the length of loop 7. In some embodiments, said insertion or deletion in HSA domain III alters the length of helix 8. In some embodiments, said insertion or deletion in HSA domain III changes the length of loop 8. In some embodiments, said insertion or deletion in HSA domain III alters the length of loop 9. In some embodiments, said insertion or deletion in domain III alters the length of at least two loops and / or helices selected from the group consisting of loops 2, 3, 6, 7, 8 and 9, and helixes 7 and 8. In some embodiments, said insertion or deletion in domain III alters the length of at least three loops and / or helices selected from the group consisting of loops 2, 3, 6, 7, 8 and 9, and helixes 7 and 8. In some embodiments, said insertion or deletion in domain III alters the length of at least four loops and / or helices selected from the group consisting of loops 2, 3, 6, 7, 8 and 9, and helixes 7 and 8. In some embodiments, said insertion or deletion in domain III alters the length of at least five loops and / or helices selected from the group consisting of loops 2, 3, 6, 7, 8 and 9, and helixes 7 and 8. In some embodiments, said insertion or deletion in HSA domain III changes the length of each of loops 2, 3, 6, 7, 8, and 9 of HSA domain III. In the event that multiple loops and / or helices are changed, the present disclosure provides that the loops and / or helixes are independently such that one or more loops / helixes can be increased by insertion and one or more loops / helixes can be reduced by deletion. Be aware of the change.

For embodiments in which domain III comprises insertion or deletion of amino acids, the present disclosure contemplates insertion or deletion of one amino acid. Also contemplated are insertions or deletions of more than one, such as two, three, four, five, six, seven, eight, nine or ten amino acids. In some embodiments, the present disclosure contemplates the insertion of more than 10 amino acid residues, such as 10-20, 20-40, 40-50, or 50-100 amino acids. Consistent with the chimeric polypeptides and HSA variant polypeptides of the disclosure, compositions comprising insertions or deletions are tested to confirm that they retain FcRn binding activity. Preferred compositions are those that provide relatively improved FcRn binding and / or serum half-life compared to the control.

For clarity, the present disclosure specifically contemplates any combination of any of the above or below aspects and embodiments. With respect to chimeric polypeptides comprising an HSA moiety as well as variant HSA polypeptides comprising an HSA moiety, such HSA moieties include domain III or FcRn binding moieties thereof. In addition, as described herein, domain III of the HSA moiety comprises 1-18 amino acid substitutions. In some embodiments, domain III of the HSA moiety is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, 14, 15, 16, 17 or 18 amino acid substitutions. Exemplary amino acid substitutions include (i) replacement with alanine; (ii) conservative amino acid substitutions; And (iii) non-conservative amino acid substitutions. The present disclosure contemplates that all amino acid substitutions in domain III of a given polypeptide may be a member of one of these amino acid substitution categories, and wherein each amino acid substitution in domain III of a given polypeptide is individually and independently from these categories. It is also contemplated that this may be chosen. In some embodiments, natural cysteine residues in domain III are maintained and unsubstituted. In some embodiments, natural proline residues in domain III are maintained and unsubstituted. In some embodiments, the natural cysteine residues and native proline residues in domain III are maintained and unsubstituted. In some embodiments, cysteine residues are not substituted (eg, cysteine is not used to replace natural residues). In some embodiments, proline residues are not substituted (eg, proline is not used to replace natural residues). In other embodiments, any one of the 20 amino acids is used to substitute a given natural residue.

Also contemplated are HSA variants and chimeric polypeptides comprising any combination of amino acid substitutions of this class.

The present invention encompasses variants and chimeric polypeptides having an HSA moiety comprising an amino acid sequence substantially identical to the amino acid sequence described herein. Amino acid sequences substantially identical to the sequences described herein include sequences comprising amino acid deletions and / or insertions as well as sequences comprising conservative amino acid substitutions. Conservative amino acid substitutions replace the first amino acid with a second amino acid having chemical and / or physical properties similar to the chemical and / or physical properties (eg, charge, structure, polarity, hydrophobicity / hydrophilicity) of the first amino acid. Refers to. Conservative amino acid substitutions include replacing one amino acid with another amino acid in the following groups: lysine (K), arginine (R) and histidine (H); Aspartate (D) and glutamate (E); Asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; Alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.

In some embodiments, the HSA variant polypeptides are substantially purified. In some embodiments, the chimeric polypeptide is substantially purified. In some embodiments, the present disclosure provides a composition comprising the HSA variant or chimeric polypeptide of the present disclosure and a pharmaceutically acceptable carrier. In some embodiments, the composition is a sterile composition. In some embodiments, the composition is a nonpyrogenic composition.

6.4. Combination domain III  Mutant

The present invention contemplates generating truncation mutants as well as combination sets of HSA moieties comprising domain III, which are particularly useful for identifying bioactive variant sequences. Combinationally derived variants with relatively selective efficacy relative to naturally occurring HSA polypeptides can be generated. Likewise, mutagenesis can result in variants with intracellular half-lives that differ markedly from the corresponding wild-type HSA polypeptide. For example, the altered protein may be more stable or less stable against other cellular processes that cause proteolytic degradation, or disruption of the protein of interest or other ways of inactivation. Such variants can be used to alter HSA polypeptide levels by modulating their half-life. There are many ways in which libraries of potential HSA variant sequences can be generated, for example, from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automated DNA synthesizer, after which the synthetic genes can be ligated into the appropriate genes for expression. The purpose of the degenerate gene set is to provide all sequences encoding the desired set of potential polypeptide sequences in one mixture. Synthesis of degenerate oligonucleotides is well known in the art (eg, Narang, SA (1983) Tetrahedron 39: 3); Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056) and Ike et al., (1983) Nucleic Acid Res. 11: 477. Such techniques have been used for directed evolution of other proteins (eg, Scott et al., (1990) Science 249: 386-390); Roberts et al., (1992) PNAS USA 89: 2429 2433); Devlin et al., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; and US Pat. Nos. 5,223,409, 5,198,346 And 5,096,815).

Alternatively, other forms of mutagenesis can be used to generate combinatorial libraries. For example, HSA polypeptide variants can be screened using, for example, alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochemistry 33: 1565-1572; Wang et al., (1994) J. Biol. Chem. 269: 3095-3099; Balint et al., (1993) Gene 137: 109-118; Grodberg et al., (1993) Eur. J. Biochem. 218: 597-601; Nagashima et al., (1993) J Biol.Chem. 268: 2888-2892; Lowman et al., (1991) Biochemistry 30: 10832-10838; and Cunningham et al., (1989) Science 244: 1081-1085); Linkage scanning mutagenesis (Gustin et al., (1993) Virology 193: 653-660; Brown et al., (1992) Mol. Cell Biol. 12: 2644-2652; McKnight et al., (1982) Science 232 : 316); Saturation mutagenesis (Meyers et al., (1986) Science 232: 613); PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol 1: 11-19); Or random mutagenesis, including chemical mutagenesis (Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994) Strategies in Mol Biol 7 : 32-34) can be generated and isolated from the library. Linkage scanning mutagenesis, particularly in combinatorial settings, is an interesting way to identify truncated (bioactive) forms of HSA polypeptide. Additional methods for generating and screening libraries of HSA polypeptide variants are provided herein (see, eg, paragraph 8 entitled "Examples"). In particular, see paragraphs 8.10 and 8.11 for specific methods useful for the generation and screening of combinatorial HSA domain III mutant libraries.

Any of the above embodiments described above for amino acid substitutions in the polypeptides of the present disclosure can be used to generate a library of peptides. In some embodiments, the combinationally derived variant is at a residue in HSA domain III. In some embodiments, domain III mutations are introduced and screened for one or more of the following conditions (i) to (iii): (i) variant domain III constructs alone; (ii) variant domain III constructs provided in the state of full-length HSA; Or (iii) a variant domain III construct provided in the state of a truncated HSA, or at least a chimeric polypeptide comprising domain III. In some embodiments, libraries of peptides are screened for variants having one or both of increased affinity for FcRn and increased serum half-life relative to the starting polypeptide. In some embodiments, variants are assessed through the use of standard in vitro assays (eg, flow cytometry) described herein. In some embodiments, variant (s) are identified that exhibit improved affinity for FcRn. In other embodiments, the variants are screened to verify that improved affinity for FcRn occurs only at acidic pH (eg, pH of approximately 5.5) and not at neutral pH (eg, pH of approximately 7.4). .

In some embodiments, a combination derived variant comprises two or more amino acid substitutions at any of the following positions numbered based on the position in full length mature HSA: residue 407, residue 415, residue 463, residue 495, residue 508, residue 509, residue 511, residue 512, residue 515, residue 516, residue 517, residue 521, residue 523, residue 524, residue 526, residue 527 and residue 557.

In some embodiments, the combination derived variant comprises two or more amino acid substitutions selected from the group consisting of the following amino acid substitutions: L407N, L407Y, V415T, L463N, L463F, E495D, T508R, T508S, F509M, F509W, F509I, A511F, D512Y, D512M, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523F, I523G, I523K, I523R, K524L, Q526A, Q526M, Q526Y, T527Y and T557G.

In some embodiments, the combinationally derived variant comprises amino acid substitutions in HSA domain III at a position selected from the group consisting of the following positions numbered based on position in full-length mature HSA: (a) residues 383 and 413 ; (b) residues 401 and 523; (c) residues 407 and 447; (d) residues 407, 447 and 539; (e) residues 407 and 509; (f) residues 407 and 526; (g) residues 411 and 535; (h) residues 414 and 456; (i) residues 415 and 569; (j) residues 426 and 526; (k) residues 442, 450 and 459; (l) residues 463 and 508; (m) residues 508, 519 and 525; (n) residues 509 and 527; (o) residues 523 and 538; (p) residues 526 and 557; (q) residues 541 and 561; (r) residues 463 and 523; (s) residues 508 and 523; (t) residues 508 and 524; (u) residues 463, 508 and 523; (v) residues 463, 508 and 524; (w) residues 508, 523 and 524; (x) residues 463, 508, 523 and 524; (y) residues 463 and 524; (z) residues 523 and 524; And (aa) residues 463, 523 and 524.

In some embodiments, combinatorially derived variants comprise (a) residues 463 and 508 numbered based on their position in full-length mature HSA; (b) residues 463 and 523; (c) residues 508 and 523; (d) residues 508 and 524; (e) residues 463, 508 and 523; (f) residues 463, 508 and 524; (g) residues 508, 523 and 524; (h) residues 463, 508, 523 and 524; (i) residues 463 and 523; And (j) amino acid substitutions in HSA domain III at a position selected from the group consisting of residues 523 and 524, wherein the substitution at residue 463 is selected from L463C, L463F, L463G, L463H, L463I, L463N, L463S and L463Q; , Substitution at residue 508 is selected from T508C, T508E, T508I, T508K, T508R and T508S, and substitution at residue 523 is I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, 1523W and I523Y, and the substitution at residue 524 is selected from K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T and K524V.

In some embodiments, combinatorially derived variants comprise (a) residues 463 and 524 numbered based on location in full-length mature HSA; (b) residues 508 and 523; (c) residues 508 and 524; And (d) amino acid substitutions in HSA domain III at a position selected from the group consisting of residues 463, 508, and 523, wherein the substitution at residue 463 is from L463C, L463F, L463G, L463H, L463I, L463N, L463S, and L463Q. Wherein the substitution at residue 508 is selected from T508C, T508E, T508I, T508K, T508R and T508S, and the substitution at residue 523 is I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, Is selected from I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W and I523Y and the substitution at residue 524 is selected from K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T and K524V do.

In some embodiments, the combinationally derived variant comprises an amino acid substitution in HSA domain III selected from the group consisting of: (a) E383G / K413S; (b) Y401E / I523G; (c) L407N / P447S; (d) L407N / P447S / A539I; (e) L407N / F509M; (f) L407Y / Q526T; (g) Y411Q / H535N; (h) K414S / V456N; (i) V415T / A569P; (j) V426H / Q526Y; (k) E442K / E450D / Q459R; (l) L463N / T508R; (m) T508R / K519I / K525V; (n) F509I / T527Y; (o) I523Q / K538Y; (p) Q526M / K557G; (q) K541F / A561F; (r) L463N / K524L; (s) T508R / I523G; (t) T508R / K524L; (u) L463N / T508R / I523G; (v) L463N / T508R / K524L; (w) T508R / I523G / K524L; (x) L463N / T508R / I523G / K524L; (y) L463N / I523G; (z) I523 G / K524L; And (aa) L463N / I523G / K524L.

In some embodiments, the combination derived variant comprises an amino acid substitution in HSA domain III selected from the group consisting of: (a) L463N / T508R; (b) L463N / K524L; (c) T508R / I523G; (d) T508R / K524L; (e) L463N / T508R / I523G; (f) L463N / T508R / K524L; (g) T508R / I523G / K524L; And (h) L463N / T508R / I523G / K524L.

In some embodiments, the combination derived variant comprises an amino acid substitution in HSA domain III selected from the group consisting of: (a) L463N / K508R; (b) T508R / I523G; (c) T508R / K524L; And (d) L463N / T508R / I523G.

In some embodiments, the combinatorially derived variant comprises SEQ ID NO: 18, wherein X ₁ is not leucine and / or X ₂ is not threonine and / or X ₃ is not isoleucine / no Or X ₄ is not lysine. In some embodiments, X ₁ is cysteine, phenylalanine, glycine, histidine, isoleucine, asparagine, serine or glutamine; X ₂ is cysteine, glutamate, isoleucine, lysine, arginine or serine; X ₃ is alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine; X ₄ is alanine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, glutamine, threonine or valine. In other embodiments, X ₁ is phenylalanine or asparagine; X ₂ is arginine or serine; X ₃ is aspartate, glutamate, glycine, phenylalanine, lysine or arginine; X ₄ is leucine. In other embodiments, X ₁ is asparagine; X ₂ is arginine and / or; X ₃ is glycine and / or; X ₄ is leucine.

In some embodiments, combinatorially derived variants occur at residues that are conserved between multiple species. In some embodiments, combinatorially derived variants occur at surface accessible residues. In some embodiments, combinatorially derived variants arise from residues that are surface accessible and conserved among multiple species. In some embodiments, combinatorially derived variants occur at residues that are conserved among multiple species but not conserved in chicken HSA.

In some embodiments, the present disclosure provides a library comprising a plurality of polypeptides, wherein each of the plurality of polypeptides comprises HSA domain III or an FcRn binding fragment thereof, wherein each of the plurality of polypeptides is human, swine, rat, One or more amino acid substitutions of residues in the HSA domain III that are conserved between serum albumin proteins from mice, dogs, rabbits, cows, chickens, donkeys, sand rats, sheep, cats and horses.

In some embodiments, the present disclosure provides a library comprising a plurality of polypeptides, wherein each of the plurality of polypeptides comprises HSA domain III or an FcRn binding fragment thereof, wherein each of the plurality of polypeptides is human, swine, rat, One or more amino acid substitutions of residues in the HSA domain III that are conserved between serum albumin proteins from mice, dogs, rabbits, cows, donkeys, sand rats, sheep, cats and horses and not in serum albumin from chickens Include independently.

In some embodiments, the present disclosure provides a library comprising a plurality of polypeptides, wherein each of the plurality of polypeptides comprises HSA domain III or an FcRn binding fragment thereof, wherein each of the plurality of polypeptides is a surface accessible residue. Independently one or more amino acid substitutions of residues in HSA domain III.

In some embodiments, the present disclosure provides a library comprising a plurality of polypeptides, wherein each of the plurality of polypeptides comprises HSA domain III or an FcRn binding fragment thereof, wherein each of the plurality of polypeptides is (i) surface accessible One of the residues in said HSA domain III that is a residue and (ii) conserved between serum albumin proteins from humans, pigs, rats, mice, dogs, rabbits, cows, chickens, donkeys, sand rats, sheep, cats and horses The above amino acid substitutions are included independently.

In some embodiments, said surface accessible moiety is in loop 2 of HSA domain III. In some embodiments, said surface accessible residue is in loop 3 of HSA domain III. In some embodiments, said surface accessible residue is in loop 6 of HSA domain III. In some embodiments, said surface accessible moiety is in helix 7 of HSA domain III. In some embodiments, said surface accessible residue is in loop 7 of HSA domain III. In some embodiments, said surface accessible moiety is in helix 8 of HSA domain III. In some embodiments, said surface accessible residue is in loop 8 of HSA domain III. In some embodiments, said surface accessible residue is in loop 9 of HSA domain III.

In some embodiments, one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on position in full length mature HSA: residue 383, residue 389, residue 391, residue 410, residue 417 , Residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571. In some embodiments, one in HSA domain III The above amino acid substitutions are at any of the following positions numbered based on the position in full length mature HSA: residue 417, residue 442, residue 499 and residue 502.

In some embodiments, one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on position in full length mature HSA: residue 392, residue 399, residue 403, residue 411, residue 412 , Residue 414, residue 416, residue 418, residue 420, residue 423, residue 434, residue 437, residue 438, residue 445, residue 448, residue 450, residue 453, residue 461, residue 476, residue 477, residue 484, residue 485, residue 487, residue 488, residue 494, residue 497, residue 507, residue 509, residue 514, residue 529, residue 534, residue 537, residue 540, residue 551, residue 558, residue 559, residue 567, residue 568 and Residue 572. In some embodiments, one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA domain III: residue 380, residue 381, residue 384, Residue 387, residue 396, residue 401, residue 404, residue 405, Residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441, residue 457, residue 458, residue 463, residue 464, residue 466, residue 469 , Residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547 and residue 576.

In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in loop 2 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 2 of HSA domain III. In some embodiments, HSA domain III comprises one or more amino acid substitutions of one or more of the above amino acid substitutions in HSA domain III present in loop 2 of HSA domain III and one or more additional amino acid substitutions in HSA domain III not present in loop 2 It includes more. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in loop 3 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 3 of HSA domain III. In some embodiments, HSA domain III comprises one or more amino acid substitutions within loop 3 of HSA domain III and further comprises one or more additional amino acid substitutions within HSA domain III not present in loop 3. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in loop 6 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 6 of HSA domain III. In some embodiments, the HSA domain comprises one or more amino acid substitutions in loop 6 of HSA domain III and further comprises one or more additional amino acid substitutions in HSA domain III not present in loop 6. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in helix 7 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in helix 7 of HSA domain III. In some embodiments, the HSA domain comprises one or more amino acid substitutions in helix 7 of HSA domain III and further comprises one or more additional amino acid substitutions in HSA domain III not present in helix 7. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in loop 7 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 7 of HSA domain III. In some embodiments, the HSA domain comprises one or more amino acid substitutions within loop 7 of HSA domain III and further comprises one or more additional amino acid substitutions within HSA domain III not present in loop 7. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are in helix 8 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in helix 8 of HSA domain III. In some embodiments, the HSA domain comprises one or more amino acid substitutions in helix 8 of HSA domain III and further comprises one or more additional amino acid substitutions in HSA domain III not present in helix 8. In some embodiments, one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in loop 8 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 8 of HSA domain III. In some embodiments, HSA domain III comprises one or more amino acid substitutions within loop 8 of HSA domain III and further comprises one or more additional amino acid substitutions within HSA domain III not present in loop 8. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are in loop 9 of HSA domain III. In some embodiments, all of said amino acid substitutions in HSA domain III are in loop 9 of HSA domain III. In some embodiments, HSA domain III comprises one or more amino acid substitutions in HSA domain III and further comprises one or more additional amino acid substitutions in HSA domain III not present in loop 9.

In some embodiments of any of the above or the following aspects or embodiments, the amino acid substitutions can be alterations of residues to alanine. In some embodiments of any of the above or the following aspects or embodiments, the amino acid substitutions can be conservative amino acid substitutions, wherein the residues are replaced with residues having similar charge and other properties. In some embodiments of any of the above or the following aspects or embodiments, the amino acid substitutions can be non-conservative amino acid substitutions, wherein the residues are replaced with residues that do not have similar charge or other properties. In some embodiments of any of the above or the following aspects or embodiments, the amino acid substitutions can be alterations of residues to any other residues. Where the HSA moiety comprises more than one amino acid residue, it is contemplated that the substitution may be included in any one or any combination of amino acid substitutions in the above categories. For example, all substitutions can be alterations to alanine, conservative amino acid substitutions, or non-conservative amino acid substitutions. Alternatively, amino acid substitutions may include any combination, eg, alteration of one residue with alanine, one conservative amino acid substitution, and one non-conservative amino acid substitution.

A wide variety of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations and for cDNA libraries for gene products with specific properties in this regard. Such techniques would be applicable for the rapid screening of gene libraries generally generated by combinatorial mutagenesis of HSA polypeptides. The most widely used techniques for the screening of large gene libraries typically include cloning the gene library into a replicable expression vector, transforming the appropriate cells into the resulting vector library, and detecting the desired activity of the product detected. Expressing the combination gene under conditions that facilitate relatively easy isolation of the vector encoding the gene. Each of the exemplary assays described below can be subjected to high efficiency analysis to screen a large number of degenerate sequences generated by combinatorial mutagenesis techniques as needed.

In some embodiments, the HSA polypeptide may comprise peptides and peptidomimetic. As used herein, the term "peptidomimetic" includes chemically modified peptides and peptide like molecules containing non-naturally occurring amino acids, peptoids, and the like. Peptidomimetic offers a variety of advantages over peptides, including improved stability when administered to a subject. Methods of identifying peptidomimetic are well known in the art and include screening of databases containing libraries of potential peptidomimetic. For example, the Cambridge structure database contains a collection of more than 300,000 compounds with known crystal structures (Allen et al., Acta Crystallogr. Section B, 35: 2331 (1979)). If the crystal structure of the target molecule is not available, for example, the structure can be generated using a program CONCORD (Rusinko et al., J. Chem. Inf. Comput. Sci. 29: 251). (1989)). Another database, the Available Chemicals Directory (Molecular Design Limited, Informations Systems; San Leandro, CA, USA) is commercially available and HSA polypeptide It contains about 100,000 compounds that may be searched to identify potential peptidomimetics.

In some embodiments, the HSA polypeptide may further comprise post-translational modifications. Exemplary post-translational protein alterations include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. As a result, the altered HSA polypeptide may contain nonamino acid elements such as lipids, polysaccharides or monosaccharides, and phosphates. The effect of these non-amino acid elements on the functionality of the HSA polypeptide can be tested for its biological activity, for example its ability to bind FcRn. As long as the native HSA polypeptide can be glycosylated, in some embodiments the HSA polypeptide used in the chimeric polypeptide according to the present disclosure is glycosylated. In some embodiments, the level and pattern of glycosylation is the same or substantially the same as the level and pattern of glycosylation of the native HSA polypeptide. In other embodiments, the level and / or pattern of glycosylation is different from the level and / or pattern of glycosylation of the native HSA polypeptide (eg, undersoglycosylated, hyperglycosylated or aglycosylated).

In some embodiments of the invention, a polypeptide (eg, HSA variant or chimeric polypeptide) comprising an HSA moiety can be conjugated to a nonprotein material. Such nonprotein materials include, but are not limited to, nucleic acid molecules, chemicals, organic molecules, and the like, each of which may be derived from natural sources such as natural product screening or may be chemically synthesized. In some embodiments, the HSA moiety is chemically conjugated to a nonprotein material.

In one particular embodiment of the invention, the HSA polypeptide may be altered by a nonproteinaceous polymer. In one particular embodiment, the polymer is polyethylene glycol (“PEG”), polypropylene glycol or poly in the manner described in US Pat. Oxyalkylene. PEG is a well known water soluble polymer that can be obtained commercially or prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Kara, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161).

In some embodiments, fragments or variants of the HSA polypeptide will preferably retain at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% biological activity associated with the native HSA polypeptide. . In some embodiments, the fragments or variants of the HSA polypeptide has a relatively improved half-life (t _1/2) compared to the half-life of the native protein. For embodiments where the half-life is enhanced, the half-life of the HSA fragment or variant is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 relative to the half-life of the native HSA protein. Up to%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500% or even up to 1000%. In some embodiments, protein half-life is measured in vitro, such as in buffered saline solution or serum. In other embodiments, the protein half-life is a half-life in vivo, such as the half-life of a protein in animal serum or other body fluids.

In some embodiments, a polypeptide comprising an HSA moiety can be a fusion protein further comprising one or more fusion domains. Well known examples of such fusion domains are polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, Protein A, which are particularly useful for isolation of fusion proteins by affinity chromatography. Protein G, immunoglobulin heavy chain constant region (Fc) and maltose binding protein (MBP) are but are not limited to these. For affinity purification, matrices suitable for affinity chromatography are used, such as glutathione-bonded resins, amylase-bonded resins and nickel-bonded or cobalt-bonded resins. Fusion domains also include “epitope tags”, which are typically short peptide sequences from which specific antibodies can be obtained. Well known epitope tags from which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags. In some cases, the fusion domain may be a protease cleavage site, such as a protease cleavage site for factor Xa or thrombin, which enables the involved protease to partially cleave the fusion protein to release the recombinant protein from the fusion protein. Has The released protein can then be isolated from the fusion domain by subsequent chromatographic separation. In some embodiments, an HSA polypeptide may contain one or more alterations that can stabilize this polypeptide. For example, such alterations enhance the in vitro half-life of the polypeptide, improve the circulating half-life of the polypeptide, or reduce proteolytic degradation of the polypeptide. Similarly, with respect to chimeric polypeptides, alterations of this type may be added or alternatively added to the heterologous protein portion of the chimeric polypeptide.

In some embodiments, a polypeptide comprising an HSA moiety can be a fusion protein having all or part of the Fc region of an immunoglobulin. In some embodiments, the fusion protein comprises an FcRn binding domain or fragment thereof. Similarly, in some embodiments, all or part of the Fc region of an immunoglobulin can be used as a linker that connects the HSA moiety with a heterologous protein. As is known, each immunoglobulin heavy chain constant region comprises four or five domains. The domains are sequentially named as follows: CH1-hinge-CH2-CH3 (-CH4). The DNA sequence of the heavy chain domain has cross homology between immunoglobulin classes (eg, the CH2 domain of IgG has homology to the CH2 domain of IgA and IgD, and the CH3 domain of IgM and IgE). The term “immunoglobulin Fc region” as used herein is understood to mean the carboxyl terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region or portion thereof. For example, an immunoglobulin Fc region may comprise 1) CH1 domain, CH2 domain and CH3 domain, 2) CH1 domain and CH2 domain, 3) CH1 domain and CH3 domain, 4) CH2 domain and CH3 domain, or 5) two or more Combinations of domains and immunoglobulin hinge regions. In a preferred embodiment, the immunoglobulin Fc region comprises at least an immunoglobulin hinge region, a CH2 domain and a CH3 domain, and preferably lacks a CH1 domain. In one embodiment, the class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igγ) (γ subclass 1, 2, 3 or 4). Other classes of immunoglobulins can be used, namely IgA (Igα), IgD (Igδ), IgE (Igε) and IgM (Igμ). The selection of appropriate immunoglobulin heavy chain constant regions is discussed in detail in US Pat. Nos. 5,541,087 and 5,726,044. The selection of specific immunoglobulin heavy chain constant region sequences from some immunoglobulin classes and subclasses to achieve certain results is considered to be within the skill of the art. The portion of the DNA construct encoding the immunoglobulin Fc region preferably comprises at least a portion of the hinge domain, preferably at least a portion of the CH3 domain of Fcγ, or at least a portion of a homologous domain in any IgA, IgD, IgE or IgM. Include. Furthermore, it is contemplated that substitutions or deletions of amino acids in immunoglobulin heavy chain constant regions may be useful in the practice of the present invention. One example is the introduction of amino acid substitutions into the upper CH2 region to produce Fc variants with reduced affinity for the Fc receptor (Cole et al. (1997) J. IMMUNOL. 159: 3613). One of ordinary skill in the art can make such constructs using well known molecular biology techniques. Similarly, with respect to chimeric polypeptides, alterations of this type may be added or alternatively added to the heterologous protein portion of the chimeric polypeptide.

In some embodiments, the HSA polypeptide can be a scaffold. In some embodiments, a protein is used to select or design a protein backbone that can specifically bind to a target. When designing proteins from scaffolds, amino acid residues important to the beneficial properties of the backbone are retained, while other residues may be altered. In some embodiments, the scaffold may have up to 50% amino acid residues of amino acid residues that are altered between protein derivatives with different properties and at least 50% of residues that are constant between such derivatives. In some embodiments, the scaffold is up to 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of amino acid residues that are altered between protein derivatives with different properties. Amino acid residues, and at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the residues that are constant between these derivatives. In some embodiments, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% amino acid residues of the amino acid residues that are altered between protein derivatives having different properties, And up to 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of residues that are constant between these derivatives. In some embodiments, these constant residues impart the same overall three dimensional fold to all variant domains regardless of their nature. In some embodiments, HSA polypeptide scaffolds may be altered or substituted as discussed in other aspects and embodiments of the disclosure. In some embodiments, the HSA polypeptide scaffold can be a therapeutic agent. In some embodiments, the HSA polypeptide scaffolds can exhibit an agonist effect on the target. In some embodiments, the HSA polypeptide scaffolds can exhibit an antagonist effect on the target.

6.5. chimera  Polypeptide

Polypeptides comprising the HSA moiety, including the HSA variant polypeptides of the disclosure, may be conjugated to any heterologous protein. In some embodiments, the heterologous protein is a therapeutic agent. In some embodiments, the therapeutic agent is an antibody or peptide. In some embodiments, the heterologous protein portion of the chimeric polypeptide comprises an antibody or antigen binding fragment thereof. In some embodiments, the chimeric polypeptide further comprises a constant region of an IgG immunoglobulin. In some embodiments, the heterologous protein comprises a non-antibody therapeutic protein. In some embodiments, the heterologous protein portion of the chimeric polypeptide comprises a growth factor or cytokine. In some embodiments, chimeric polypeptides further comprise epitopes, eg, epitopes useful for detection and / or purification (eg His tags, FLAG tags, etc.).

In some embodiments, the HSA moiety is chemically conjugated to a heterologous protein. In some embodiments, the HSA moiety is recombinantly conjugated to a heterologous protein. In some embodiments, chimeric polypeptides are produced through the use of recombinant vectors encoding both HSA moieties and heterologous proteins.

In some embodiments, HSA variants are produced in prokaryotic or eukaryotic cells. In some embodiments, the chimeric polypeptide is produced in prokaryotic or eukaryotic cells. In some embodiments, the eukaryotic cells are selected from yeast cells, algal cells, insect cells, and mammalian cells.

Chimeric polypeptides of the invention can be prepared in a variety of ways. In some embodiments, the C terminus of the HSA moiety can be linked to the N terminus of a heterologous protein (eg, an antibody or therapeutic peptide). Alternatively, the C terminus of the heterologous protein (eg, antibody or therapeutic peptide) may be linked to the N terminus of the HSA moiety. In some embodiments, the HSA moiety is conjugated to an internal amino acid of a heterologous protein. In some embodiments, the potential configuration includes the use of truncated portions of the heavy and light chain sequences of the antibody as needed to maintain functional integrity of the attached HSA portion and / or attached heterologous protein. In some other embodiments, the HSA moiety is HSA domain III or a neonatal Fc receptor (FcRn) binding fragment thereof; And at least a portion of HSA domain I, at least a portion of HSA domain II, or at least a portion of HSA domains I and II. In addition, the heterologous protein may be linked to exposed internal (non-terminal) residues of the HSA moiety or variant thereof. In further embodiments, any combination of HSA-heterologous protein constructs can be used to generate an HSA: heterologous protein ratio of greater than 1: 1 (eg, two HSA molecules to one heterologous protein).

The HSA moiety and the heterologous protein may be directly conjugated to each other. Alternatively, they may be linked to each other via a linker sequence that separates the HSA moiety and the heterologous protein by a distance sufficient to ensure that each domain is properly folded into its secondary and tertiary structures. In some embodiments, the linker is a cleavable linker. Preferred linking agents should (1) adopt flexible extended conformation, (2) exhibit no propensity to develop ordered secondary structures that can interact with the functional domains of HSA polypeptides or heterologous proteins, and (3) It must have a minimum of hydrophobic or charged properties that can facilitate its interaction with the protein domain.

In some embodiments, the linker length is at least 80 Angstroms, at least 100 Pa, at least 120 Pa, at least 140 Pa, at least 160 Pa, at least 180 Pa, or at least 200 Pa. In some embodiments, the linker length is from about 80 kPa to about 200 kPa, from about 100 kPa to about 180 kPa, or from about 120 kPa to about 160 kPa.

In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a peptide linker and the peptide linker has one or more of the following properties: a) the linker allows the heterologous protein sequence and the HSA moiety to rotate relative to each other; b) the linking agent is resistant to degradation by proteases; (c) the linking agent does not interact with the heterologous protein sequence or HSA moiety. Typical surface amino acids in the flexible protein region include Gly, Asn and Ser. Overmutation of amino acid sequences containing Gly, Asn and Ser is expected to meet the above criteria for linker sequences. Other amino acids, such as Thr and Ala, which are almost neutral, can also be used in the linker sequence. In some embodiments, each of the amino acids in the peptide linker is selected from the group consisting of Gly, Ser, Asn, Thr, and Ala. In some embodiments, the peptide linker comprises a Gly-Ser element. In certain embodiments, the peptide linker comprises one or more Gly-Gly-Gly-Gly-Ser repeats. In certain embodiments, the linking agent comprises one, two, three, four, five, six or seven Gly-Gly-Gly-Gly-Ser repeats. In certain embodiments, linker sequence lengths of about 20 amino acids may be used to provide for proper separation of functional protein domains, but longer or shorter linker sequences may also be used. The linker sequence separating the HSA polypeptide from the heterologous protein may be between 5 and 500 amino acids, more preferably between 5 and 100 amino acids. In some embodiments, the linker sequence is about 5 to 60 or about 5 to 30 amino acids in length. In some embodiments, the linker sequence is about 5 to about 20 amino acids in length and advantageously about 10 to about 30 amino acids. In other embodiments, the linking agent that connects the HSA moiety to the heterologous protein may be a constant domain of the antibody (eg, all or part of the Fc region of the antibody). In some embodiments, the linker is a cleavable linker.

In some embodiments, chimeric polypeptides of the invention can be generated through the use of well known crosslinking reagents and protocols. For example, there are a large number of chemical crosslinkers known to those skilled in the art and useful for crosslinking HSA polypeptides with heterologous proteins (eg, antibodies). For example, crosslinkers are heterobifunctional crosslinkers that can be used to link molecules in a stepwise manner. Heterofunctional crosslinkers provide the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrence of unwanted side reactions, such as the occurrence of homoprotein polymers. Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-succinimidyl (4-io Doacetyl) aminobenzoate (SIAB), succinimidyl 4- (p-maleimidophenyl) butyrate (SMPB), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 4 Succinimidyloxycarbonyl-a-methyl-a- (2-pyridyldithio) -toluene (SMPT), N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) and A wide variety of heterofunctional crosslinkers are known in the art, including succinimidyl 6- [3- (2-pyridyldithio) propionate] hexanoate (LC-SPDP). Crosslinkers with N-hydroxysuccinimide moieties can generally be obtained as N-hydroxysulfosuccinimide analogs with higher water solubility. In addition, crosslinkers having disulfide bridges in the linking chain may instead be synthesized as alkyl derivatives to reduce the amount of linker cleavage in vivo. In addition to the heterobifunctional crosslinking agents, there are a number of other crosslinking agents, including homobifunctional crosslinking agents and photoreactive crosslinking agents. Disuccinimidyl subcrate (DSS), bismaleimidohexane (BMH) and dimethyl pimeliimidate. 2HCl (DMP) are examples of useful homobifunctional crosslinkers and are bis- [B- (4-azidosalts). Lysylamido) ethyl] disulfide (BASED) and N-succinimidyl-6 (4'-azido-2'-nitrophenylamino) hexanoate (SANPAH) are useful photoreactive crosslinks for use in the present invention. This is an example. For a recent review of protein coupling techniques, see Means et al. (1990) Bioconjugate Chemistry. 1: 2-12, which is incorporated herein by reference.

One particularly useful class of heterobifunctional crosslinkers included above contains primary amine reactive groups, N-hydroxysuccinimides (NHS) or water soluble analogs N-hydroxysulfosuccinimides (sulfo-NHS) thereof. . At alkaline pH primary amines (lysine epsilon groups) are unprotonated and reacted by nucleophilic attack on NHS or sulfo-NHS esters. This reaction results in the formation of amide bonds and the release of NHS or sulfo-NHS as a byproduct. Another reactive group useful as part of a heterobifunctional crosslinker is a thiol reactive group. Common thiol reactive groups include maleimides, halogens and pyridyl disulfides. Maleimides specifically react with free sulfhydryl (cysteine residues) within minutes under mildly to neutral (pH 6.5-7.5) conditions. Halogen (iodoacetyl function) reacts with the --SH group at physiological pH. Both of these reactive groups lead to the formation of stable thioether bonds. The third component of the heterobifunctional crosslinker is a spacer arm or crosslink. The crosslinking is a structure connecting two reactive ends. The most prominent characteristic of this crosslinking is its effect on steric hindrance. In some cases, longer crosslinking can more easily widen the distance needed to connect two composite biomolecules.

Preparation of protein-conjugates using heterobifunctional reagents is a two step process involving an amine reaction and a sulfhydryl reaction. For the first step, the amine reaction, the selected protein must contain a primary amine. This may be a lysine epsilon amine or primary alpha amine found at the N terminus of most proteins. The protein should not contain free sulfhydryl groups. If both proteins to be conjugated contain a free sulfhydryl group, one protein can be altered such that all sulfhydryls are blocked, for example through the use of N-ethylmaleimide (see, eg, herein (Partis et al. (1983) J. Pro. Chem. 2: 263). Elman's Reagent can be used to calculate the amount of sulfhydryl in a particular protein (see, eg, Ellman et al. (1958) Arch. Biochem. Biophys. 74, incorporated herein by reference). : 443) and Riddles et al. (1979) Anal. Biochem. 94:75).

In some embodiments, chimeric polypeptides of the invention are standard protein chemistry techniques such as, for example, Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant GA (ed.), Synthetic Peptides: A User's Guide, WH Freeman and Company, New York (1992)). In addition, automated peptide synthesizers (eg, Advanced ChemTech Model 396; Milligen / Biosearch 9600) are commercially available. In any of the above crosslinking methods for chemical conjugation of HSA with a heterologous protein, cleavable domains or cleavable linking agents can be used. Cleavage allows for the separation of heterologous proteins and HSA polypeptides. For example, after infiltration of cells by chimeric polypeptides, cleavage of the cleavable linker will allow for the separation of HSA from the heterologous protein.

In some embodiments, chimeric polypeptides of the invention can be generated as fusion proteins containing an HSA moiety and a heterologous protein (eg, an antibody or therapeutic peptide) expressed as one continuous polypeptide chain. Such chimeric polypeptides are referred to herein as recombinantly conjugated chimeric polypeptides. In the preparation of such fusion proteins, a fusion gene is constructed comprising a nucleic acid encoding an HSA moiety and a heterologous protein, and optionally a peptide linker sequence for separating the HSA moiety and the heterologous protein. The use of recombinant DNA techniques to generate fusion genes, wherein the translation product is the desired fusion protein, is well known in the art. Both the coding sequence of the gene and its regulatory region can be redesigned to change the functionality of the protein product, the amount of protein produced, or the cell type in which the protein is produced. The coding sequence of a gene can be varied widely, for example, by fusing a portion thereof with the coding sequence of a different gene to create a novel hybrid gene encoding the fusion protein. Examples of methods for making fusion proteins include PCT / US87 / 02968, PCT / US89 / 03587 and PCT / US90 / 07335, which are incorporated herein by reference, as well as in Traunecker et al. (1989). Nature 339: 68). In essence, the ligation of various DNA fragments encoding different polypeptide sequences results in blunt or stagger ends for ligation, restriction enzyme digestion to provide appropriate ends, and cohesive ends, where appropriate. Is carried out according to conventional techniques using alkaline phosphatase treatment and enzyme ligation to avoid filling, undesirable linkage. Alternatively, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. In another method, PCR amplification of gene fragments is through the use of anchor primers that generate complementary overhangs between two consecutive gene fragments that can subsequently be annealed to generate chimeric gene sequences. (See, eg, Current Protocols in Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992). Chimeric polypeptides encoded by the fusion gene can be produced recombinantly through the use of various expression systems, as is well known in the art (see also below).

Recombinantly conjugated chimeric polypeptides include embodiments in which the HSA moiety is conjugated to the N terminus or C terminus of a heterologous protein.

In some embodiments, the immunogenicity of a chimeric polypeptide identifies candidate T cell epitopes within the junction region spanning the chimeric polypeptide and alters amino acids within the junction region as described in US Patent Publication 2003/0166877. Can be reduced.

The term chimeric protein refers to a protein comprising the HSA moiety (eg, HSA variant polypeptides) and the heterologous protein regardless of how the HSA moiety and the heterologous protein are interconnected (eg, chemical conjugation or recombinant conjugation). Will be used. Thus, the terms chimeric protein, fusion protein and conjugated protein will be used interchangeably.

Exemplary heterologous protein categories include, but are not limited to, enzymes, growth factors and cytokines. In some embodiments, the heterologous protein is an antibody.

The heterologous protein to be used in the chimeric polypeptide comprising the HSA moiety can be a therapeutic protein or fragment thereof, such as growth factors, enzymes, bone morphogenic proteins and soluble receptor fragments. Exemplary heterologous polypeptides include growth factors such as hepatocyte growth factor (HGF), nerve growth factor (NGF), epidermal growth factor (EGF; members of the EGF family of growth factors), fibroblast growth factor (FGF; FGF of growth factors) Members of the family), transforming growth factors (eg TGF-alpha, TGF-beta, TGF-beta2, TGF-beta3), vascular endothelial growth factor (VEGF; eg VEGF-2), interferon (Eg, INF-alpha, INF-beta), interleukin (eg, IL-1, IL-2), cytokines and insulin. Other exemplary heterologous proteins include enzymes. Other exemplary heterologous polypeptides include bone morphogenic proteins (BMP; members of the BMP family of proteins), erythropoietin (EPO), miyostatin, and tumor necrosis factor (eg, TNF-α). Other exemplary heterologous polypeptides include the extracellular domain of the transmembrane receptor, including any naturally occurring extracellular domain of the cellular receptor, as well as any variant thereof (including mutants, fragments and peptidomimetic forms).

The heterologous protein to be used in the chimeric polypeptide comprising the HSA moiety may be a therapeutic protein or fragment thereof comprising the antibody or antigen binding moiety thereof. Exemplary antibodies and antibody fragments include Humira®, Remicade®, Simponi®, Rituxan®, Herceptin®, Avastin®, Erbitux ( Erbitux®; Synagis®, Mylotarg®, Camppath®, TheraCIM®, Vectibix®, Tysabri®, Leopro (ReoPro) ®, Lucentis®, Cimzia®, and the like.

6.6. HSA  Related Nucleic Acids and Expression

In some embodiments, the present disclosure provides nucleic acid constructs comprising nucleotide sequences encoding any polypeptide of the present disclosure (eg, HSA variants, and chimeric polypeptides having HSA moieties). In addition, the present invention uses such nucleic acids to prepare chimeric polypeptides or HSA variant polypeptides (eg, including HSA moieties—bioactive fragments, variants, and fusions thereof). In some specific embodiments, the nucleic acid may further comprise DNA encoding a heterologous protein (eg, an antibody or therapeutic peptide) to prepare a recombinant chimeric protein of the invention. All these nucleic acids are collectively referred to as HSA nucleic acids.

In some embodiments, the present disclosure provides (i) a human serum albumin (HSA) domain III or FcRn binding fragment thereof comprising (i) 1 to 18 amino acid substitutions, operably linked to a nucleotide sequence encoding a heterologous protein. A nucleic acid construct comprising a nucleotide sequence encoding an HSA moiety comprising: wherein the nucleic acid construct encodes a chimeric polypeptide that retains the functional activity of a heterologous protein and is capable of binding to FcRn, wherein the chimeric polypeptide is the amino acid It has increased serum half-life and / or increased affinity for FcRn relative to control chimeric polypeptides having HSA moieties that do not include substitutions.

In some embodiments, the chimeric polypeptide encoded by the nucleic acid construct binds to FcRn with a higher affinity than the control chimeric polypeptide. In some embodiments, the chimeric polypeptide encoded by the nucleic acid construct has an increased serum half-life relative to the control chimeric polypeptide. In some embodiments, the chimeric polypeptide encoded by the nucleic acid construct has both of these properties.

In some embodiments, the nucleotide sequence of (i) is 1 to 18 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 Encoding a HSA moiety comprising human serum albumin (HSA) domain III or FcRn binding fragment thereof comprising amino acid substitutions of 12, 13, 14, 15, 16, 17 or 18) Nucleotide sequences.

In some embodiments, one or more amino acid substitutions of the amino acid substitutions in HSA domain III encoded by the nucleic acid construct are substitutions of residues that are conserved between multiple species. In some embodiments, all of said amino acid substitutions in HSA domain III are substitutions of residues that are conserved among multiple species. In some embodiments, one or more amino acid substitutions of the amino acid substitutions in HSA domain III are serum albumin proteins from humans, pigs, rats, mice, dogs, rabbits, cows, chickens, donkeys, sand mice, sheep, cats and horses. It is substitution of residue preserved in between. In some embodiments, all of said amino acid substitutions in HSA domain III are conserved between serum albumin proteins from humans, pigs, rats, mice, dogs, rabbits, cows, chickens, donkeys, sand rats, sheep, cats and horses Substitution of residues. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are substitutions of surface accessible residues. In some embodiments, all of said amino acid substitutions in HSA domain III are substitutions of surface accessible residues. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions in HSA domain III are substitutions of residues that are surface accessible and conserved among multiple species. In some embodiments, all of said amino acid substitutions in HSA domain III are substitutions of residues that are surface accessible and conserved among multiple species.

In some embodiments, the nucleotide sequence of (i) comprises a nucleotide sequence encoding HSA domain III that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the nucleotide sequence of (i) comprises a nucleotide sequence encoding HSA domain III at least 95% identical to SEQ ID NO: 1. In some embodiments, the nucleotide sequence of (i) comprises a nucleotide sequence encoding HSA domain III that is at least 98% identical to SEQ ID NO: 1.

In some embodiments, one or more amino acid substitutions of the above amino acid substitutions are present in loop 2 of HSA domain III. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions are present in loop 3 of HSA domain III. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions are present in loop 6 of HSA domain III. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions are in helix 7 of HSA domain III. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions are present in loop 7 of HSA domain III. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions are in helix 8 of HSA domain III. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions are present in loop 8 of HSA domain III. In some embodiments, one or more amino acid substitutions of the above amino acid substitutions are present in loop 9 of HSA domain III.

In some embodiments, the nucleotide sequence of (ii) comprises a nucleotide sequence encoding a heterologous protein comprising an antibody or antigen binding fragment thereof. In some embodiments, the nucleotide sequence of (ii) comprises a nucleotide sequence encoding a heterologous protein comprising a therapeutic protein.

In some embodiments, the nucleotide sequence further encodes a constant region of an IgG immunoglobulin.

In some embodiments, the nucleotide sequence of (ii) comprises a nucleotide sequence encoding a heterologous protein comprising a growth factor or cytokine. In some embodiments, the nucleic acid construct further comprises a nucleotide sequence encoding a linking agent.

The nucleic acid may be a single stranded or double stranded DNA or RNA molecule. In some embodiments, the present disclosure provides at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the same region of the HSA sequence (eg, SEQ ID NOs: 1 and 2). It relates to an isolated or recombinant nucleic acid sequence encoding the same HSA moiety. In further embodiments, HSA nucleic acid sequences may be isolated, recombinantly prepared, and / or may be fused with fusion nucleotide sequences or present in a DNA library.

In some embodiments, the HSA nucleic acid also includes a nucleotide sequence or its complement sequence that hybridizes to any of the native HSA nucleotide sequences mentioned above under high stringency conditions. One of ordinary skill in the art will readily understand that appropriate stringency conditions that promote DNA hybridization may be altered. For example, one skilled in the art can perform hybridization at 6.0 × sodium chloride / sodium citrate (SSC) at 45 ° C. and then wash 2.0 × SSC at 50 ° C. For example, the salt concentration in the washing step can be selected from low stringency (about 2.0 × SSC at 50 ° C.) to high stringency (about 0.2 × SSC at 50 ° C.). In addition, the temperature in the washing step can be increased from low stringency conditions of room temperature (about 22 ° C.) to high stringency conditions of about 65 ° C. Both temperature and salt can be changed, or the temperature or salt concentration can be kept constant while other variables are changed. In one embodiment, the present invention provides nucleic acids that hybridize under low stringency conditions of 6 x SSC at room temperature and then washed with 2 x SSC at room temperature.

Isolated nucleic acids that differ from native HSA nucleic acids due to the degeneracy of the genetic code are also within the scope of the present invention. For example, multiple amino acids are represented by more than one 3-base codon. Codons or synonyms that specify the same amino acid (eg, CAU and CAC are synonymous codons for histidine) can cause "silent" mutations that do not affect the amino acid sequence of the protein. However, it is predicted that DNA sequence polymorphisms causing changes in the amino acid sequence of the present protein will exist between mammalian cells. Those skilled in the art will recognize that these variations in one or more nucleotides (up to about 3% to 5% of nucleotides) of a nucleic acid encoding a particular protein may exist between individuals of a given species due to natural allelic variations. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.

In some embodiments, a recombinant HSA nucleic acid can be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences will generally be suitable for the host cell used for expression. Many types of suitable expression vectors and suitable regulatory sequences for various host cells are known in the art. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. It is not limited. Constitutive or inducible promoters known in the art are contemplated by the present invention. The promoters may be naturally occurring promoters or may be hybrid promoters that combine elements of more than one promoter. Expression constructs may be present in cells on episomes, such as plasmids, or expression constructs may be inserted into chromosomes. In a preferred embodiment, the expression vector contains a selectable marker gene that allows for the selection of transformed host cells. Selectable marker genes are well known in the art and will vary depending on the host cell used. In some embodiments, the present invention relates to an expression vector comprising a nucleotide sequence encoding an HSA polypeptide and operably linked to one or more regulatory sequences. Regulatory sequences are recognized in the art and are selected to direct expression of the encoded polypeptide. Thus, the term regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). It is to be understood that the design of the expression vector may depend on factors such as the selection of the host cell to be transformed and / or the type of protein desired to be expressed. Furthermore, the copy number of the vector, the ability to control the copy number, and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.

The invention also relates to a host cell transfected with a recombinant gene encoding the HSA polypeptide or chimeric polypeptide of the invention. The host cell can be any prokaryotic or eukaryotic cell. For example, an HSA polypeptide or chimeric polypeptide may be a bacterial cell, such as E. coli. It can be expressed in E. coli, insect cells (eg, using a baculovirus expression system), yeast or mammalian cells. Other suitable host cells are known to those skilled in the art.

In addition, the present invention relates to a method for preparing HSA polypeptides, heterologous proteins and / or chimeric polypeptides of the present invention. For example, host cells transfected with an expression vector encoding an HSA polypeptide or chimeric polypeptide may be cultured under conditions suitable for causing expression of the polypeptide. The polypeptide can be secreted and isolated from a mixture of cells and medium containing the polypeptide. Alternatively, the polypeptide may be retained in the cytoplasm or membrane fraction, cells may be harvested and lysed, and proteins may be isolated. Cell cultures include host cells, media and other byproducts. Suitable media for cell culture are well known in the art. Such polypeptides include ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification using antibodies specific for a particular epitope of the polypeptide (eg, HSA polypeptide). It can be isolated from cell culture medium, host cells or both through the use of known protein purification techniques. In a preferred embodiment, the polypeptide is a fusion protein containing a domain that facilitates its purification.

Recombinant HSA nucleic acids can be prepared by ligating a cloned gene or portion thereof into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, algae, insects or mammals) or both. Expression vehicles for the production of recombinant polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: prokaryotic cells, such as E. coli. Plasmids derived from pBR322, plasmids derived from pEMBL, plasmids derived from pEX, plasmids derived from pBTac and plasmids derived from pUC for expression in E. coli. Preferred mammalian expression vectors contain both prokaryotic sequences to facilitate propagation of said vector in bacteria, and one or more eukaryotic transcriptional units expressed in eukaryotic cells. Examples of mammalian expression vectors where vectors from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg are suitable for transfection of eukaryotic cells to be. Some of these vectors are altered with sequences of bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, derived from pREP and p205) can be used for transient expression of proteins in eukaryotic cells. have. Various methods used for the preparation of plasmids and for the transformation of host organisms are well known in the art. For other expression systems and general recombination procedures suitable for both prokaryotic and eukaryotic cells, see Molecular Cloning A Laboratory Manual, 2nd Ed., Ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17 ). In some cases, it may be desirable to express the recombinant polypeptide using a baculovirus expression system. Examples of such baculovirus expression systems are vectors derived from pVL (eg, pVL1392, pVL1393 and pVL941), vectors derived from pAcUW (eg pAcUW1) and vectors derived from pBlueBac (eg, β-gal containing pBlueBac III).

Techniques for making fusion genes are well known. In essence, ligation of various DNA fragments encoding different polypeptide sequences is necessary to avoid blunt or staggered ends for ligation, restriction enzyme digestion to provide appropriate ends, filling of cohesive ends if appropriate, and undesirable linkages. For alkaline phosphatase treatment and enzyme ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed by using anchor primers that generate complementary overhangs between two consecutive gene fragments that can subsequently be annealed to generate chimeric gene sequences (eg, , Current Protocols in Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992).

6.7. Treatment method

In some embodiments, the present disclosure provides a method of treating a condition that can be treated using heterologous proteins of the chimeric fusions of the disclosure. In some embodiments, the present disclosure provides a method of increasing the serum half-life of a protein in a subject and / or increasing the binding affinity of the protein for FcRn, comprising domain III or a neonatal Fc receptor (FcRn) binding fragment thereof. And conjugating an HSA moiety to the protein. In some embodiments, HSA domain III includes one to eighteen amino acid substitutions to increase one or both of the affinity and serum half-life of the chimeric polypeptide relative to FcRn relative to the control polypeptide. These methods include administering a therapeutically effective amount of the aforementioned chimeric or HSA variant polypeptide to the individual in need thereof. In some embodiments, the method comprises administering a chimeric polypeptide comprising (a) a HSA moiety or a bioactive fragment thereof, and (b) a heterologous protein. These methods are particularly aimed at the therapeutic and prophylactic treatment of animals, more particularly humans.

It is appreciated that the specific diseases and conditions that can be treated depend on the heterologous protein portion of the chimeric protein. In addition, the present disclosure contemplates that chimeric polypeptides comprising heterologous proteins suitable for the treatment of a particular disease or condition may be administered as part of a therapy in combination with one or more other compounds or other modes of treatment suitable for the treatment of a particular disease or condition. do. In addition, the present disclosure contemplates that the chimeric polypeptide is administered in a manner consistent with a medically appropriate treatment given the age, weight, health, severity of disease, and the like of the patient.

For example, if the heterologous protein is aft, the chimeric polypeptide can be used, for example, in the treatment of rheumatoid arthritis, psoriasis, juvenile arthritis and Crohn's disease. If the heterologous protein is insulin, the chimeric polypeptide can be used, for example, for the treatment of insulin related diseases.

The terms “treatment”, “treating” and the like are generally used herein to mean obtaining the desired pharmacological and / or physiological effect. The effect may be a prophylactic effect in terms of completely or partially preventing the disease, condition or symptoms thereof and / or partial or complete cure for the disease or condition and / or adverse effects resulting from the disease or condition. It may be a therapeutic effect in terms of. As used herein, “treatment” covers any treatment of a disease or condition in a mammal, particularly a human, and (a) may be vulnerable to a disease or condition but is not yet diagnosed as having said disease or condition. Preventing the development of a disease or condition; (b) inhibiting the disease or condition (eg, stopping its development); Or (c) alleviating the disease or condition (eg, causing regression of the disease or condition or providing amelioration of one or more symptoms). Improvements in any condition can be readily assessed according to standard methods and techniques known in the art. The population of subjects treated by a method of treating a disease includes not only a subject suffering from an undesirable condition or disease, but also a subject at risk of developing the condition or disease.

The term "therapeutically effective dosage" or "effective amount" means a dosage that, when administered, produces the desired effect. The exact dosage will depend on the therapeutic purpose and will be ascertained by one skilled in the art using known techniques (see, eg, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

In some embodiments, one or more chimeric or HSA variant polypeptides of the invention may be administered together (simultaneously) or may be administered at different times (sequential). In addition, chimeric or HSA variant polypeptides of the invention may be administered in combination with one or more additional compounds or therapies for treating the same disease or condition. For example, one or more chimeric or HSA variant polypeptides can be co-administered with one or more therapeutic compounds. Combination therapy may encompass simultaneous or alternation administration. Combination therapy may also encompass acute or chronic administration. Optionally, the chimeric or HSA variant polypeptides and additional compounds of the invention act in an additive or synergistic manner for the treatment of a disease or condition. Additional compounds to be used in the combination therapy include, but are not limited to, small molecules, polypeptides, antibodies, antisense oligonucleotides, and siRNA molecules. In addition, combination therapies also include the methods disclosed herein in conjunction with other non-pharmaceutical therapies. Depending on the nature of the combination therapy, administration of the chimeric or HSA variant polypeptides of the invention may continue, while other therapies are being administered and / or administered later. Administration of the chimeric or HSA variant polypeptide can be performed in a single dose or multiple doses. In some cases, administration of the chimeric or HSA variant polypeptide begins at least a few days before the administration of the other therapy, while in other cases the administration begins immediately before or at the time of administration of the other therapy.

6.8. Method of administration

In some embodiments, administration to said subject comprises systemic administration of HSA variant or chimeric polypeptide. In some embodiments, administering to said subject comprises orally administering an HSA variant or chimeric polypeptide. In some embodiments, administering to said subject comprises intravenously administering said HSA variant or chimeric polypeptide.

In some embodiments, the present disclosure provides a composition comprising the HSA variant or chimeric polypeptide of the present disclosure and a pharmaceutically acceptable carrier. In some embodiments, the composition is a sterile composition. In some embodiments, the composition is a nonpyrogenic composition.

Various delivery systems are known, such as encapsulation into liposomes, microparticles, microcapsules, recombinant cells capable of expressing compounds and receptor mediated endocytosis and the chimeric or HSA variant polypeptides of the disclosure are known. It can be used for administration (see, eg, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432). The method of introduction may be an enteral or parenteral route including, but not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, lung, intranasal, intraocular, epidural and topical routes, and oral route. . In certain embodiments, parenteral introduction comprises intramuscular, subcutaneous, intravenous, endovascular and intrapericardial administration.

The chimeric or HSA variant polypeptides may be administered by any convenient route, such as by irrigation or bolus injection, by incorporating absorption of epithelial or mucosal skin linings (eg, oral mucosa, rectal and intestinal mucosa, etc.) It may be administered in conjunction with other biologically active substances. Administration may be systemic or topical. Pulmonary administration can also be used, for example, by the use of inhalers or nebulizers and formulation with an aerosolizing agent.

In some embodiments, it may be desirable to topically administer the chimeric or HSA variant polypeptides of the invention to a region (eg, muscle) in need of treatment, which may be, for example, local irrigation, topical treatment, eg, during surgery For example, but not limited to, injection, catheter or implant, the implant may be porous, nonporous or gelatinous, including membranes such as siaastic membranes, fibers or commercial skin substitutes. It is a substance.

In other embodiments, chimeric or HSA variant polypeptides of the disclosure can be delivered in the form of vesicles, particularly liposomes (see Langer, 1990, Science 249: 1527-1533). In another embodiment, the chimeric or HSA variant polypeptides of the present disclosure can be delivered in the form of a controlled release system. In another embodiment, a pump may be used (see Langer, 1990, supra). In another embodiment, polymeric materials can be used (see, eg, Howard et al., 1989, J. Neurosurg. 71: 105). In some specific embodiments, chimeric or variant polypeptides of the present disclosure may be delivered intravenously.

In some embodiments, the chimeric or HSA variant polypeptides are administered by intravenous irrigation. In some embodiments, the chimeric or HSA variant polypeptides are irrigated over a time of at least 10 minutes, at least 15 minutes, at least 20 minutes, or at least 30 minutes. In other embodiments, the chimeric or HSA variant polypeptides are irrigated over a time period of 60 minutes, 90 minutes or 120 minutes or more. Regardless of irrigation time, the present disclosure contemplates that the chimeric or HSA variant polypeptide is part of an overall treatment plan administered on a regular schedule (eg, weekly, monthly, etc.).

6.9. Assessment Methods

The chimeric polypeptides of the present disclosure have increased FcRn relative to (i) substantially retaining the function of the heterologous protein, and (ii) a chimeric polypeptide conjugated to an unaltered HSA moiety or relative to another suitable control. Affinity for and / or increased serum half-life. The HSA variant polypeptides of the disclosure are characterized by having an increased affinity and / or increased serum half-life for FcRn relative to the native HSA moiety or relative to another suitable control. The nature of the chimeric polypeptide or HSA variant polypeptide can be assessed in any one or more suitable assays in vitro or in vivo.

For example, affinity (K _a and / or K _d ) for FcRn can be assessed in vitro, for example, by using any one or more of the assays described in the examples or other binding assays. have. Similarly, k _off and / or k _on can be assessed in vitro by using, for example, one or more of the assays described in the examples or other binding assays.

Determination of the affinity constants and specificity of binding between antigen and antibody is a pivotal factor in confirming the efficacy of therapeutic, diagnostic and research methods using anti-HSA antibodies. “Binding affinity” generally refers to the strength of the total sum of noncovalent interactions between a single binding site of a molecule (eg, an antibody, HSA moiety) and its binding partner (eg, an antigen, FcRn). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of the molecule X for its partner Y can be represented by the equilibrium dissociation constant (K _d or K _D ), which is generally calculated as the ratio k _off / k _on . See, eg, Chen, Y. et al., (1999) J. Mol Biol 293: 865-881. Affinity can be measured by common methods known in the art, including Biacore, including those described and illustrated herein. Low affinity antibodies generally show a tendency to bind slowly and readily dissociate, whereas high affinity antibodies generally tend to bind more quickly to an antigen and to remain bound longer. Various methods of measuring binding affinity are known in the art, and any of these methods can be used for the purposes of the present method.

In some embodiments, the chimeric polypeptide or variant HSA polypeptide has improved affinity for FcRn by up to approximately 1.5, 2, 2.5, 3, 4 or approximately 5 times relative to the control. In some embodiments, the chimeric polypeptide has improved affinity up to a level of more than 5 or even more than 10 times relative to the control. In some embodiments, the chimeric polypeptide or HSA variant polypeptide has improved affinity up to a level of 20, 25, 40 or 50 times relative to the control. In some embodiments, the chimeric polypeptide or HSA variant polypeptide is about 5 to 10 times, about 10 to 20 times, about 25 to 40 times, about 40 to 50 times, about 50 to 75 times relative to the control. Fold or improved affinity up to approximately 75 to 100 fold. When affinity is evaluated by the calculation of K _a , these improvements in affinity are interpreted as an increase in K _a (eg, 2 times, 5 times, 10 times, etc. as summarized above). When affinity is assessed by the calculation of K _d , these improvements in affinity translate to a decrease in K _d (eg, 2 times, 5 times, 10 times, etc. as summarized above).

In some embodiments, the affinity for FcRn is improved at low pH (eg, pH of about 5.5). In some other embodiments, the affinity for FcRn is improved at low pH, and the affinity at neutral pH (eg, pH of about 7.2) is not changed.

Further, for example, serum half-life can be measured in human or animal models. The increase in serum half-life in any animal model (including but not limited to transgenic animals with human FcRn) is sufficient to characterize chimeric or HSA variant polypeptides as having an increase in serum half-life relative to the control. .

In one embodiment, the chimeric polypeptide or HSA variant polypeptide has a half-life of at least 10 days, preferably at least about 14 days, and most preferably at least 50% of the half-life of the native serum albumin protein or homologue thereof in the blood. In another embodiment, the half-life of the chimeric polypeptide or HSA variant polypeptide is increased by approximately 1.5, 2, 2.5, 3, 4, or 5 times relative to the control polypeptide. In some embodiments, the half-life of a chimeric polypeptide or HSA variant polypeptide is increased to a level that is more than 5 times or even more than 10 times relative to the control polypeptide. In some embodiments, the half-life of a chimeric polypeptide or HSA variant polypeptide is increased by a level 20, 25, 40 or more than 50 times relative to the control polypeptide. In some embodiments, the half-life of a chimeric polypeptide or HSA variant polypeptide is approximately 5-10 times, approximately 10-20 times, approximately 25-40 times, approximately 40-50 times, approximately 50 relative to the control polypeptide. It is increased from fold to 75 times or approximately 75 to 100 times.

Assays suitable for assessing whether a chimeric polypeptide substantially retains the function of a heterologous protein will depend on the heterologous protein and its natural function. However, function can be assessed in any suitable in vitro or in vivo assay, including animal models. Exemplary functions include (i) the ability to bind specific receptors; (ii) the ability to induce or inhibit signaling through specific signaling pathways; (iii) the ability to induce or inhibit apoptosis; (iv) the ability to induce or inhibit angiogenesis; (v) the ability to stimulate or inhibit cell proliferation; (vi) the ability to promote or inhibit cell differentiation; (vii) the ability to promote cell survival; And (viii) the ability to promote or inhibit the secretion of another protein factor.

In some embodiments, chimeric polypeptides of the disclosure comprising a biologically active heterologous protein are more potent than the biologically active heterologous protein itself, eg, the biologically active heterologous protein that is not fused to the HSA moiety. For example, a chimeric polypeptide can be 2, 4, 5, 10, 25, 50, 100 or even 1000 times higher activity than a biologically active protein sequence alone, eg, single digit, double digit. Or even three orders of magnitude higher activity. Thus, in the embodiment to inhibit the biological activity peptide sequence is biologically active, IC ₅₀ of the chimeric polypeptide may be lower 10 times the IC ₅₀ of a single biologically active protein, 100-fold or even 1000-fold, biologically active protein sequences are biologically in embodiments that induce or promote the activity of the chimeric polypeptide EC ₅₀ it may be lower than 10 times the sole biologically active peptides EC _50, 100 fold or even 1000-fold. In embodiments wherein the biologically active protein sequence binding to a biological molecule, e.g., nucleic acids, peptides or carbohydrates, the chimeric polypeptide and this is the dissociation constant of the biological molecules binding K _d is 10 times higher than the K _d alone the biological molecule and the biologically active protein , 100 times or even 1000 times lower (eg, the combination of the two materials becomes increasingly prevalent over their dissociation).

6.10. Pharmaceutical composition

In some embodiments, the chimeric or HSA variant polypeptides of the present disclosure are formulated with a pharmaceutically acceptable carrier. One or more chimeric or HSA variant polypeptides may be administered alone or as a component of a pharmaceutical formulation (composition). The chimeric or HSA variant polypeptides may be formulated to be administered in any convenient manner for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, mold release agents, coatings, sweeteners, flavors, fragrances, preservatives and antioxidants may also be present in the compositions.

Formulations of the chimeric or HSA variant polypeptides include agents suitable for oral, nasal, topical, parenteral, rectal and / or vaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect.

In some embodiments, the methods of preparing these agents or compositions comprise combining another type of therapeutic agent and carrier, and optionally one or more accessory ingredients. In general, the formulations may be prepared by using liquid carriers or finely divided solid carriers, or both, and then the product may be molded if necessary.

Formulations for oral administration include capsules, cachets, pills, tablets, lozenges (using aromatic bases, usually sucrose and acacia or tragacanth), powders, each containing a predetermined amount of the present polypeptide therapeutic agent as an active ingredient, Granules, solutions or suspensions in aqueous or non-aqueous liquids, oil-in-water or water-in-oil liquid emulsions, elixirs or syrups, pastilles (using inert bases such as gelatin and glycerin, or sucrose and acacia) and / or mouthwashes May exist in the form. Suspensions in addition to the active compounds may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and these It may contain a mixture of.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more polypeptide therapeutic agents of the present invention may comprise one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and And / or may be mixed with any of the following materials: fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates, and sodium carbonate; (5) solution buffering agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; And (10) a colorant. In the case of capsules, tablets and pills, the pharmaceutical composition may also comprise a buffer. Solid compositions of a similar type may also be used as fillers in soft-filled gelatin capsules and hard-filled gelatin capsules using excipients such as lactose or lactose as well as high molecular weight polyethylene glycols and the like. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms are inert diluents commonly used in the art, in addition to the active ingredient, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate Fatty acids of propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, embryo oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and sorbitan Esters, and mixtures thereof. Oral compositions may include auxiliaries such as wetting agents, emulsifiers, suspending agents, sweetening agents, flavoring agents, coloring agents, fragrances and preservatives in addition to inert diluents.

Pharmaceutical compositions suitable for parenteral administration include antioxidants, buffers, bacteriostatic agents, and one or more pharmaceutically acceptable sterile isotonic agents which may contain solutes, suspensions or thickeners that render the formulation isotonic to the blood of the intended recipient. Aqueous or non-aqueous solutions; Dispersions; Suspensions or emulsions; Or one or more chimeric or HSA variant polypeptides with sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately prior to use. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (e.g. glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and Injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical formulation may be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

In some embodiments, compositions of the present disclosure, including pharmaceutical compositions, are nonpyrogenic compositions. In other words, in some embodiments, the composition is substantially free of pyrogen. In one embodiment, the formulation of the invention is a pyrogen-free formulation that is substantially free of endotoxins and / or related pyrogenic substances. Endotoxins include toxins that are confined within a microorganism and are released only when the microorganism is destroyed or killed. The pyrogenic substances also include pyrogen-inducing heat-stable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock when administered to humans. Because of the potential deleterious effects, even low amounts of endotoxins must be removed from the pharmaceutical drug solution administered intravenously. The Food and Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units (EU) per kg body weight in a single hour for intravenous drug application (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1)). : 223 (2000)). If the therapeutic protein is administered in amounts of hundreds or thousands of mg / kg body weight (which may be the case with antibodies), even trace amounts of harmful dangerous endotoxins should be eliminated. In some specific embodiments, the endotoxin and pyrogen levels in the composition are less than 10 EU / mg, less than 5 EU / mg, less than 1 EU / mg, less than 0.1 EU / mg, less than 0.01 EU / mg or less than 0.001 EU / mg. .

Injectable depot formulations are prepared by forming microcapsule matrices composed of one or more polypeptide therapeutic agents of a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the specific polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers are poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

In some embodiments, chimeric or HSA variant polypeptides of the disclosure are formulated according to routine procedures as pharmaceutical compositions suitable for intravenous administration to humans. If desired, the composition may also include solubilizers and local anesthetics such as lidocaine to relieve pain at the injection site. When the composition is administered by irrigation, the composition may be dispensed into an irrigation bottle containing sterile pharmaceutical grade water or saline. If the composition is administered by injection, an ampoule of sterile injectable water or saline may be provided so that the components can be mixed prior to administration.

The amount of chimeric or HSA variant polypeptides of the present disclosure that will be effective in the treatment of a tissue related condition or disease can be determined by standard clinical techniques. In addition, in vitro assays can optionally be used to help identify optimal dosage ranges. The exact dosage to be used in the formulation will also depend on the route of administration and the severity of the condition and should be determined by the physician's judgment and the environment of each subject. However, suitable dosage ranges for intravenous administration are generally from about 20 μg to 5000 μg active chimeric or HSA variant polypeptides per kg body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg to 1 mg / kg body weight. Effective dosages can be estimated from dose-response curves derived from in vitro or animal model test systems.

6.11. Products and Kit

In some embodiments, the present disclosure also provides a pharmaceutical package or kit comprising one or more containers filled with one or more chimeric or HSA variant polypeptides of the present disclosure. In certain embodiments, formulations of the present disclosure may be heterologous proteins, heterologous polypeptides, heterologous peptides, large molecules, small molecules, marker sequences, diagnostic or detectable substances, therapeutic moieties, drug moieties, radioactive metal ions, second antibodies and Chimeric or HSA variant polypeptides that are recombinantly fused or chemically conjugated to another moiety, including but not limited to a solid support. In certain embodiments, the formulations of the present disclosure are formulated as sterile liquid in a single dose vial. The formulations of the present disclosure may be provided in 3 cc USP Type Borosilicate Amber Vials (West Pharmaceutical Serice-Part No. 6800-0675) with a target volume of 1.2 ml. Exemplary containers include, but are not limited to, vials, bottles, prefilled syringes, IV bags, and blister packs (including one or more pills). Optionally, a notice in the form prescribed by a governmental authority that regulates the manufacture, use, or sale of a drug or biological product, reflecting the agency's approval for manufacture, use, or sale for human diagnosis and / or administration. This may be present with the container (s).

In some embodiments, kits are also provided comprising chimeric or HSA variant polypeptides useful for various purposes, such as increasing the serum half-life of a therapeutic agent or increasing FcRn binding affinity. For the isolation and purification of reagents, the kit may contain a polypeptide coupled to the beads (eg, sepharose beads). Kits containing polypeptides for detection and quantification of targets in vitro, for example in ELISA or Western blot, can be provided. Like the product, the kit includes a container and a label or package insert present on or with the container. The container holds a composition comprising one or more chimeric or HSA variant polypeptides of the present disclosure. Additional containers containing control diagnostic reagents such as diluents and buffers may be included. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.

6.12. Adenovirus  Vectors and methods

DNA vectors useful for generating recombinant adenovirus particles from host cells are well known in the art and commercial reagents are readily available (e.g., catalog numbers V493-20 from Invitrogen and See V494-20). The present disclosure provides a method of enhancing the development of recombinant adenoviruses by introducing OriP sequences into DNA vectors useful for the development of adenovirus particles (also referred to herein as adenovirus vectors). The OriP containing adenovirus vector may further comprise a sequence for the expression of the EBNA-1 protein, or alternatively host cells expressing the EBNA-1 protein are used for the generation of recombinant adenovirus particles. OriP containing adenovirus vectors are particularly useful for the generation of recombinant adenovirus populations that include a diverse / complex library of DNA sequences of interest (eg, DNA sequences encoding HSA domain III variants).

OriP sequences can be readily manipulated into any known adenovirus vector through the use of a number of techniques known in the art. For example, where a Gateway recombination system is used, the OriP sequence must be located between the att recombination sites of the introduction vector or adenovirus target vector. When adding a sequence to an adenovirus vector, care must be taken to avoid inserting the sequence into sites that would interfere with the replication or assembly of adenovirus DNA. Alternatively or optionally, adenovirus vectors containing OriP sequences can be engineered to also express EBNA-1 protein (see FIG. 10). By expressing the EBNA-1 protein directly from the adenovirus vector, the host cell does not need to express the EBNA-1 gene.

Adenovirus vectors of the invention comprise OriP sequences and adenovirus genomic sequences. Adenovirus vectors of the invention may further comprise one or more of the following elements:

(i) recombination site (s) (eg, attR1 and attR2) for recombination cloning with another vector (such sites are responsible for cloning the DNA sequence of interest for expression in recombinant adenoviruses generated from the vector). Useful to perform);

(ii) antibiotic / drug (eg chloramphenicol) resistance gene (s) and / or toxin expressing genes (eg ccdB gene) useful for screening and / or opposing screening;

(iii) a cloning site useful for subcloning the DNA sequence of interest (which may be a multicloning site);

(iv) a DNA of interest, which may include one or more genes of interest that encode one or more proteins of interest;

(v) promoters for expressing genes of interest in a wide variety of mammalian cells (eg, human cytomegalovirus (CMV) immediate early promoters), promoters which may be either constitutive or inducible promoters;

(vi) epitope tags (eg, His6X epitopes) for detection and / or purification of the protein of interest (the epitope tag may be present at the 5 'or 3' end of the recombinant protein of interest);

(vii) polyadenylation (polyA) sequences (eg, monkey 40 virus polyA sequence) for efficient transcription termination and polyadenylation of mRNA;

(viii) this. Origin of replication for high-copy replication and maintenance of plasmids in E. coli;

(ix) this. Antibiotic resistance genes for selection in E. coli;

(x) restriction enzyme site (s) (eg, PacI) for linearizing the vector, the restriction enzyme site capable of flanking elements (viii) and (ix); And

(xi) DNA sequence encoding EBNA-1 protein.

Epstein-Barr virus (EBV) origin of replication (OriP) of plasmid DNA synthesis efficiently supports DNA synthesis in a variety of higher eukaryotic cells. Representative OriP sequences are provided in FIG. 9C. This origin of replication uses only one viral protein EBNA-1, while all other factors are provided by the cell. In some embodiments, the EBNA-1 protein is provided by a host cell (eg, 293E cell). In other embodiments, the adenovirus vectors of the invention further comprise a DNA sequence encoding the element (xi) EBNA-1 protein. Representative EBNA-1 protein and DNA sequences are provided in FIGS. 9A and 9B, respectively.

Adenovirus genomic sequences (e.g., human adenovirus type 5 sequences) are genes and other elements (e.g., left and right inverted terminal repeats (ITRs), capsidization signals required for proper packaging and production of adenoviruses). It is specifically contemplated that it will encode sequences, late genes (Hitt et al., 1999, The Development of Human Gene Therapy, T. Friedmann, ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 61-86; Russell, 2000, J. Gen. Virol. 81, 2573-2604). The adenovirus genomic sequence may encode the complete adenovirus genome. Alternatively, the adenovirus genomic sequence may encode all proteins and other regulatory elements except for one or more proteins and / or regulatory elements provided in trans to produce non-replicating adenoviruses. For example, the El region encoding the El proteins (Ela and Elb) can be excluded from the adenovirus vectors of the invention (Russell, 2000, J. Gen. Virol. 81, 2573-2604). Then, missing proteins and / or other elements will generally be provided in trans by the host used to generate the adenovirus (eg, 293 cell lines contain genomic copies of the El region). In certain embodiments, the adenovirus genomic sequence comprises or consists essentially of human adenovirus type 5 sequences corresponding to wild type sequences 1 to 458 and 3513 to 35935.

Those skilled in the art will understand that some elements may be provided together and / or may introduce other elements, for example, element (iv) may introduce elements (v) through (vii). Also, some elements will be provided in the 5 'and / or 3' direction relative to other elements, for example, element (x) may be useful for linearization of adenovirus vectors and when introduced for linearization It will also be appreciated that x) should be provided in the 5 'direction for the 5' ITR and / or in the 3 'direction for the 3' ITR. Similarly, elements (viii) and (ix), when present, are generally positioned such that they are not introduced into the rescued adenovirus (eg, these elements are in the 5 'direction and 3' for the 5 'ITR). In the 3 'direction with respect to the ITR). OriP and elements (i) through (vii), if present, are flanked with adenovirus genomic sequences comprising 3 'ITRs on one side and flanked with 5' ITR sequences on the other side (see, eg, FIG. 10). ).

Representative adenovirus vectors of the invention introducing elements (i), (ii), (iv), (viii) to (x) and optionally (xi) are provided in FIG. 10.

7. Embodiment

1.A chimeric polypeptide comprising (a) a HSA portion comprising human serum albumin (HSA) domain III or a neonatal Fc receptor (FcRn) binding fragment thereof, and (b) a heterologous protein, wherein the chimeric polypeptide is the heterologous protein Of the chimeric polypeptide relative to a control chimeric polypeptide having an HSA moiety that does not include the amino acid substitution by having a functional activity of and capable of binding to FcRn and wherein the HSA domain III contains 1 to 18 amino acid substitutions A chimeric polypeptide that increases one or both of affinity for FcRn and serum half-life.

2. The chimeric polypeptide of embodiment 1, which binds FcRn with a higher affinity than the control chimeric polypeptide.

3. The chimeric polypeptide of embodiment 1 or 2, wherein the chimeric polypeptide binds FcRn with a higher affinity than the control chimeric polypeptide, wherein the affinity is measured at acidic pH.

4. The chimeric polypeptide of embodiment 3, wherein the acidic pH is 5.0 to 6.0.

5. The chimeric polypeptide of embodiment 4, wherein the acidic pH is 5.5 ± 0.2.

6. The method of any one of embodiments 1-3, which binds FcRn with a higher affinity than the control chimeric polypeptide at acidic pH but does not bind FcRn with a higher affinity than the control chimeric polypeptide at neutral pH. Does not contain chimeric polypeptide.

7. The chimeric polypeptide of embodiment 6 having a neutral pH of 6.9 to 7.9.

8. The chimeric polypeptide of embodiment 7, wherein the neutral pH is 7.4 ± 0.2.

9. The chimeric polypeptide according to any one of embodiments 1 to 6, which binds to FcRn and has a dissociation rate or binding rate different from the dissociation rate or binding rate of said control chimeric polypeptide.

10. Chimeric polypeptide of embodiment 9, which binds to FcRn and has an increased binding rate and / or reduced dissociation rate relative to the control polypeptide.

11. The chimeric polypeptide of embodiment 9, which binds to FcRn and has an increased dissociation rate relative to the control polypeptide.

12. The method of any of embodiments 1-11, wherein the HSA domain III comprises from 1 to 10 amino acid substitutions, thereby reducing the relative chimeric polypeptide relative to the control chimeric polypeptide having an HSA moiety that does not include said amino acid substitutions. Chimeric polypeptide to increase serum half-life.

13. The chimeric polypeptide according to any one of embodiments 1 to 12, wherein the amino acid substitution of at least one of said amino acid substitutions in HSA domain III is a substitution of a residue which is conserved between a plurality of species.

14. The chimeric polypeptide of embodiment 13, wherein all of the amino acid substitutions in HSA domain III are substitutions of residues that are conserved among multiple species.

15. The method of any one of embodiments 1-13, wherein one or more amino acid substitutions of the amino acid substitutions in HSA domain III are human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand mouse, sheep. , Chimeric polypeptide, which is a substitution of residues conserved between serum albumin proteins from cats and horses.

16. The method of embodiment 15, wherein all of said amino acid substitutions in HSA domain III are between serum albumin proteins from humans, pigs, rats, mice, dogs, rabbits, cows, chickens, donkeys, sand rats, sheep, cats and horses. A chimeric polypeptide which is a substitution of a residue preserved in.

17. A chimeric polypeptide according to any one of embodiments 1 to 15, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is selected from the amino acid substitutions listed in Table 5.

18. The method of any of embodiments 1-15, wherein one or more amino acid substitutions of the amino acid substitutions in HSA domain III are numbered based on the position in full-length mature HSA (SEQ ID NO: 2). Chimeric polypeptide present at position: residue 381, residue 383, residue 391, residue 401, residue 402, residue 407, residue 411, residue 413, residue 414, residue 415, residue 416, residue 424, residue 426, residue 434 , Residue 442, residue 445, residue 447, residue 450, residue 454, residue 455, residue 456, residue 457, residue 459, residue 463, residue 495, residue 506, residue 508, residue 509, residue 511, residue 512, residue 515, residue 516, residue 517, residue 519, residue 521, residue 523, residue 524, residue 525, residue 526, residue 527, residue 531, residue 535, residue 538, residue 539, residue 541, residue 557, residue 561, Residue 566 and residue 569.

19. A chimeric polypeptide according to any one of embodiments 1 to 15 comprising an amino acid substitution in HSA domain III at a position selected from the group consisting of the following positions numbered based on position in full length mature HSA: (a ) Residues 383 and 413; (b) residues 401 and 523; (c) residues 407 and 447; (d) residues 407, 447 and 539; (e) residues 407 and 509; (f) residues 407 and 526; (g) residues 411 and 535; (h) residues 414 and 456; (i) residues 415 and 569; (j) residues 426 and 526; (k) residues 442, 450 and 459; (l) residues 463 and 508; (m) residues 508, 519 and 525; (n) residues 509 and 527; (o) residues 523 and 538; (p) residues 526 and 557; (q) residues 541 and 561; (r) residues 463 and 523; (s) residues 508 and 523; (t) residues 508 and 524; (u) residues 463, 508 and 523; (v) residues 463, 508 and 524; (w) residues 508, 523 and 524; (x) residues 463, 508, 523 and 524; (y) residues 463 and 524; (z) residues 523 and 524; And (aa) residues 463, 523 and 524.

20. A chimeric polypeptide according to any one of embodiments 1 to 15 comprising an amino acid substitution in HSA domain III at a position selected from the group consisting of the following positions numbered based on position in full length mature HSA: (a ) Residues 463 and 508; (b) residues 463 and 523; (c) residues 508 and 523; (d) residues 508 and 524; (e) residues 463, 508 and 523; (f) residues 463, 508 and 524; (g) residues 508, 523 and 524; (h) residues 463, 508, 523 and 524; (i) residues 463 and 523; And (j) residues 523 and 524.

21. A chimeric polypeptide according to any one of embodiments 1 to 15 and 18 to 20, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is selected from the group consisting of the following amino acid substitutions: V381N, V381Q , E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, K402A, K402G, K402I, K402L, K402V, L407F, L407H, L407M, L407N, L407Q, L407N, L407 , Y411Q, Y411N, K413C, K413S, K413T, K414S, K414T, V415C, V415L, V415S, V415T, Q416H, Q416P, V424A, V424D, V424G, V424I, V424L, V424M, V424N, V424Q, V424W, V426D, V426E, V426E , V426H, V426L, V426N, V426P, V426Q, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, P447S, P447T, E450D, E450E, S454C, S454M, S454T, V455N, V455Q, V456N, V456Q, V456Q, V456Q , L457W, L457Y, Q459K, Q459R, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T506F, T506M, T506N, T506R, T506W, T506Y, T508C, T508E, T508I, T508K, T508R 8S, F509C, F509D, F509G, F509I, F509L, F509M, F509P, F509V, F509W, F509Y, A511D, A511F, A511I, A511R, A511T, A511V, A511W, A511Y, D512F, D512M, D512Q, D512W, D512Y, T515C T515D, T515E, T515E, T515G, T515H, T515L, T515N, T515P, T515Q, T515S, T515W, T515Y, L516C, L516F, L516S, L516T, L516W, L516Y, S517C, S517F, S517M, S517T, S517W, S517Y, K517 K519G, K519I, K519L, K519V, R521F, R521H, R521M, R521Q, R521T, R521W, R521Y, I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523P I523R, I523S, I523T, I523V, I523W, I523Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526A, Q526C, Q526F, Q526H Q526L, Q526M, Q526P, Q526S, Q526T, Q526V, Q526Y, T527F, T527W, T527Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535K, H535L, H535N, H535P, H535S, K538F538, K538W A539I, A539L, A539V, K541F, K541W, K541Y, K557A, K557G, K557I, K557L, K557N, K557S, K557V, A561F, A561W, A561Y, T566F, T566W, T5 66Y, A569H, and A569P.

22. Chimeric polypeptide according to any one of embodiments 1 to 15 and 18 to 20, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is selected from the group consisting of the following amino acid substitutions: V381N, E383G , N391V, Y401E, K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F , L463C, L463F, L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C , K519I, R521W, I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526M, Q526M , T527Y, E531I, H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W and A569P.

23. A chimeric polypeptide according to any one of embodiments 1 to 15 and 18 to 20, comprising one amino acid substitution in the HSA domain III selected from the group consisting of the following amino acid substitutions: V381N, E383G, N391V, Y401E, K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F, Q459R, L463C, L463C, L463C, L463C L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C, S517W, K519I, K519I I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526M, Q526Y, T527Y, T527Y H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W and A569P.

24. Chimeric polypeptide of embodiment 22, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is selected from the group consisting of the following amino acid substitutions: L407N, L407Y, V415T, V424I, V424Q, V426E, V426H, P447S, V455N, V456N, L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T535 and H5P27Y K557G.

25. A chimeric polypeptide according to embodiment 23 comprising one amino acid substitution in HSA domain III selected from the group consisting of the following amino acid substitutions: L407N, L407Y, V415T, V424I, V424Q, V426E, V426H, P447S, V455N, V456N , L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T527Y, H535P and K557G.

26. A chimeric polypeptide according to any one of embodiments 1 to 14 and 18 to 20, comprising an amino acid substitution in HSA domain III selected from the group consisting of: (a) E383G / K413S; (b) Y401E / I523G, (c) L407N / P447S; (d) L407N / P447S / A539I; (e) L407N / F509M; (f) L407Y / Q526T; (g) Y411Q / H535N; (h) K414S / V456N; (i) V415T / A569P; (j) V426H / Q526Y; (k) E442K / E450D / Q459R; (l) L463N / T508R; (m) T508R / K519I / K525V; (n) F509I / T527Y; (o) I523Q / K538Y; (p) Q526M / K557G; (q) K541F / A561F; (r) L463N / K524L; (s) T508R / I523G; (t) T508R / K524L; (u) L463N / T508R / I523G; (v) L463N / T508R / K524L; (w) T508R / I523G / K524L; (x) L463N / T508R / I523G / K524L; (y) L463N / I523G; (z) I523 G / K524L; And (aa) L463N / I523G / K524L.

27. A chimeric polypeptide according to embodiment 26 comprising an amino acid substitution in HSA domain III selected from the group consisting of: (a) L407N / P447S; (b) L407N / P447S / A539I; (c) L407N / F509M; (d) Y411Q / H535N; (e) K414S / V456N; (f) V426H / Q526Y; (g) L463N / T508R; (h) F509I / T527Y; (i) I523Q / K538Y; (j) Q526M / K557G; (k) K541F / A561F; (l) L463N / K524L; (m) T508R / I523 G; (n) T508R / K524L; (o) L463N / T508R / I523G; (p) L463N / T508R / K524L; (q) T508R / I523G / K524L; (r) L463N / T508R / I523G / K524L; (s) L463N / I523G; (t) I523G / K524L; And (u) L463N / I523G / K524L.

28. A chimeric polypeptide according to embodiment 26 comprising an amino acid substitution in HSA domain III selected from the group consisting of: (a) L463N / T508R; (b) L463N / K524L; (c) T508R / I523G; (d) T508R / K524L; (e) L463N / T508R / I523G; (f) L463N / T508R / K524L; (g) T508R / I523G / K524L; And (h) L463N / T508R / I523G / K524L.

29. A chimeric polypeptide according to embodiment 26 comprising an amino acid substitution in the HSA domain III selected from the group consisting of: (a) L463N / K508R; (b) T508R / I523G; (c) T508R / K524L; And (d) L463N / T508R / I523G.

30. The method of any one of embodiments 1-15, wherein the amino acid substitutions of one or more of the amino acid substitutions in HSA domain III are human, pig, rat, mouse, dog, rabbit, cow, donkey, sand mouse, sheep, cat. And a chimeric polypeptide that is a substitution of residues that are conserved among serum albumin proteins from horses but not conserved in chicken serum albumin.

31. The method of embodiment 30, wherein all of said amino acid substitutions in HSA domain III are conserved between serum albumin proteins from humans, pigs, rats, mice, dogs, rabbits, cattle, donkeys, sand rats, sheep, cats and horses. A chimeric polypeptide which is a substitution of a residue which has been made but not preserved in chicken serum albumin.

32. A chimeric polypeptide according to embodiment 15, wherein said at least one amino acid substitution in HSA domain III is at any of the following positions numbered based on the position in full length mature HSA (SEQ ID NO: 2): residues 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and Residue 571.

33. The chimeric polypeptide of embodiment 32, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 383, residue 391, Residue 434, residue 442, residue 445 and residue 450.

34. The chimeric polypeptide of embodiment 33, wherein the one or more amino acid substitutions in the amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions: V381N, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, E450D and E450E.

35. The chimeric polypeptide of embodiment 32, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 417, residue 442, Residue 499 and residue 502.

36. The chimeric polypeptide of embodiment 30, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 380, residue 381, Residue 384, residue 387, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441. , Residue 457, residue 458, residue 463, residue 464, residue 466, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547 and residue 576.

37. The chimeric polypeptide of embodiment 36, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 381, residue 401, Residue 424, residue 457, residue 463, residue 495, residue 508, residue 515, residue 516, residue 524, residue 525, residue 526, residue 531, residue 535 and residue 539.

38. The chimeric polypeptide of embodiment 37, wherein the HSA domain III comprises amino acid substitutions at a position selected from the group consisting of the following positions numbered based on position in full length mature HSA: residues 463 and 508; Residues 463 and 524; Residues 508 and 524; And residues 463, 508 and 524.

39. Chimeric polypeptide according to embodiment 37 or 38, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is selected from the group consisting of the following amino acid substitutions: V381N, V381Q, Y401D, Y401E, V424N, V424Q, L457F, L457W, L457Y, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T508C, T508E, T508I, T508K, T508R, T508S, T515C, T515H, T515N, T515P, T515Q, T515S, L516S, L516T, L516W, L516Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, E531A, E531A, E531A E531I, E531L, E531V, H535D, H535E, H535P, A539I, A539L and A539V.

40. The chimeric polypeptide of embodiment 32, wherein all of the amino acid substitutions are selected from the members of the group consisting of: residues 383, residues 389, residues 391, residues 410, residues 417, residues 425, residues 442 , Residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.

41. The chimeric polypeptide of embodiment 36, wherein all of the amino acid substitutions are selected from the members of the group consisting of the following residues: residue 380, residue 381, residue 384, residue 387, residue 396, residue 401, residue 404 , Residue 405, residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441, residue 457, residue 458, residue 463, residue 464, residue 466, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, Residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547 and residue 576.

42. Chimeric polypeptide of embodiment 32 or 36, wherein all of the amino acid substitutions are selected from the members of the group consisting of: residues 380, residues 381, residues 384, residues 383, residues 387, residues 389, Residue 391, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 410, residue 417, residue 419, residue 421, residue 422, residue 424, residue 425, residue 428, residue 430, residue 431 , Residue 433, residue 441, residue 442, residue 457, residue 458, residue 463, residue 464, residue 465, residue 466, residue 467, residue 468, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 486, residue 489, residue 491, residue 495, residue 499, residue 500, residue 502, residue 508, residue 510, residue 515, residue 516, residue 520, residue 524, residue 525, residue 526, residue 528, Residue 531, residue 532, residue 535, residue 536, residue 539, residue 543, residue 544, residue 547, residue 571 and residue 576.

43. The chimeric polypeptide of embodiment 42, wherein all of the amino acid substitutions are selected from the members of the group consisting of: residues 381, residues 383, residues 391, residues 401, residues 424, residues 442, residues 463 , Residue 495, residue 506, residue 508, residue 515, residue 516, residue 524, residue 525, residue 526, residue 531, residue 535 and residue 539.

44. Chimeric polypeptide of embodiment 43, wherein one or more amino acid substitutions of the amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions: V381N, V381Q, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, V424A, V424G, V424I, V424L, V424N, V424Q, E442K, E442R, L463C, L463F, L463G, L463H, L463M, L463N, L463Q, E495D T506Y, T508C, T508E, T508I, T508K, T508R, T508S, T515C, T515H, T515N, T515P, T515Q, T515S, L516S, L516T, L516W, L516Y, K524A, K524F, K524G, K524I, K524L, K524M, K524T, K524T, K524T K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535P, A539I, A539L and A539V.

45. A chimeric polypeptide according to any one of embodiments 1 to 44 wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are substitutions of surface accessible residues.

46. A chimeric polypeptide according to embodiment 45 wherein all of said amino acid substitutions in HSA domain III are substitutions of surface accessible residues.

47. A chimeric polypeptide according to any one of embodiments 1 to 12, wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are substitutions of residues that are surface accessible and conserved among multiple species.

48. A chimeric polypeptide according to embodiment 47 wherein all of said amino acid substitutions in HSA domain III are substitutions of residues that are surface accessible and conserved among multiple species.

49. Chimeric polypeptide of embodiment 47 or 48, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.

50. The chimeric polypeptide of embodiment 49, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 383, residue 391 and Residue 442.

51. A chimeric polypeptide according to embodiment 49 wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions: E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, E442K and E442R.

52. The chimeric polypeptide of embodiment 49, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 417, residue 442, Residue 499 and residue 502.

53. The chimeric polypeptide of any one of embodiments 1 to 52 wherein the HSA domain III comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.

54. The chimeric polypeptide of embodiment 53, wherein the HSA domain III comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.

55. A chimeric polypeptide according to embodiment 53 wherein the HSA domain III comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1.

56. A chimeric polypeptide according to any one of embodiments 1 to 52 wherein the HSA moiety comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2.

57. The chimeric polypeptide of embodiment 56, wherein the HSA moiety comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 2.

58. The chimeric polypeptide of embodiment 56, wherein the HSA moiety comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 2.

59. The chimeric polypeptide of any one of embodiments 1 to 58 wherein one or more amino acid substitutions of the amino acid substitutions in HSA domain III are in loop 2 of HSA domain III.

60. The loop of any of embodiments 1-23, 30-32, 35, 36, 40-42, 45-49, and 52-59, wherein all of said amino acid substitutions in HSA domain III are Chimeric polypeptide present in.

61. Chimeric polypeptide of embodiment 59 or 60, wherein the chimeric polypeptide comprises 1 to 5 amino acid substitutions in HSA domain III, wherein said 1 to 5 amino acid substitutions are present in loop 2 of HSA domain III. Polypeptide.

62. The chimeric polypeptide of any one of embodiments 1 to 59 wherein one or more amino acid substitutions of the amino acid substitutions in HSA domain III are in loop 3 of HSA domain III.

63. The chimeric polypeptide according to any one of embodiments 1 to 23, 30 to 36, 40 to 58 and 62, wherein all of said amino acid substitutions in HSA domain III are present in loop 3 of HSA domain III.

64. Chimeric polypeptide according to embodiment 62 or 63, wherein the chimeric polypeptide comprises 1 to 5 amino acid substitutions in HSA domain III, wherein said 1 to 5 amino acid substitutions are present in loop 3 of HSA domain III. Polypeptide.

65. A chimeric polypeptide according to any one of embodiments 1 to 59 and 62, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is in loop 6 of HSA domain III.

66. The method of any one of embodiments 1 to 23, 30 to 32, 35, 36 to 49, 52 to 58 and 65, wherein all of said amino acid substitutions in HSA domain III are present in loop 6 of HSA domain III Phosphorus chimeric polypeptide.

67. A chimeric polypeptide according to embodiment 65 or 66, wherein the chimeric polypeptide comprises 1 to 18 amino acid substitutions in HSA domain III, wherein said 1 to 18 amino acid substitutions are present in loop 6 of HSA domain III. Polypeptide.

68. A chimeric polypeptide according to any one of embodiments 1 to 59, 62 and 65, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is in helix 7 of HSA domain III.

69. The method of any one of embodiments 1 to 23, 30 to 32, 35, 36 to 49, 52 to 58 and 68, wherein all of said amino acid substitutions in HSA domain III are present in helix 7 of HSA domain III Phosphorus chimeric polypeptide.

70. A chimeric polypeptide according to embodiment 71 or 72, wherein the chimeric polypeptide comprises 1 to 3 amino acid substitutions in HSA domain III, wherein said 1 to 6 amino acid substitutions are present in helix 7 of HSA domain III. Polypeptide.

71. A chimeric polypeptide according to any one of embodiments 1 to 59, 62, 65 and 68, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is in loop 7 of HSA domain III.

72. The method of any one of embodiments 1 to 23, 30 to 32, 35, 36 to 49, 52 to 58 and 71, wherein all of said amino acid substitutions in HSA domain III are present in loop 7 of HSA domain III. Phosphorus chimeric polypeptide.

73. Chimeric polypeptide of embodiment 71 or 72, wherein the chimeric polypeptide comprises 1 to 3 amino acid substitutions in HSA domain III, wherein said 1 to 3 amino acid substitutions are present in loop 7 of HSA domain III. Polypeptide.

74. A chimeric polypeptide according to any one of embodiments 1 to 59, 62, 65, 68 and 71, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is in helix 8 of HSA domain III.

75. The method of any of embodiments 1-23, 30, 31, 41, 42, 45-48, 53-58, and 74, wherein all of said amino acid substitutions in HSA domain III are present in helix 8 of HSA domain III Chimeric polypeptide.

76. Chimeric polypeptide of embodiment 74 or 75, wherein the chimeric polypeptide comprises 1 to 5 amino acid substitutions in HSA domain III, wherein said 1 to 18 amino acid substitutions are in helix 8 of HSA domain III. Polypeptide.

77. The chimeric of any of embodiments 1 to 59, 62, 65, 68, 71, and 74, wherein the amino acid substitution of one or more of the amino acid substitutions in HSA domain III is present in loop 8 of HSA domain III. Polypeptide.

78. The compound of any one of embodiments 1 to 23, 30, 31, 41, 42, 45 to 48, 53 to 58 and 77, wherein all of said amino acid substitutions in HSA domain III are present in loop 8 of HSA domain III Chimeric polypeptide.

79. Chimeric polypeptide of embodiment 77 or 78, wherein the chimeric polypeptide comprises 1 to 5 amino acid substitutions in HSA domain III, wherein said 1 to 5 amino acid substitutions are present in loop 8 of HSA domain III. Polypeptide.

80. The method of any one of embodiments 1-59, 62, 65, 68, 71, 74, and 77, wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are present in loop 9 of HSA domain III. Phosphorus chimeric polypeptide.

81. A chimeric according to any one of embodiments 1 to 23, 30, 31, 45 to 48, 53 to 58 and 80, wherein all of said amino acid substitutions in HSA domain III are present in loop 9 of HSA domain III. Polypeptide.

82. Chimeric polypeptide of embodiment 80 or 81, wherein the chimeric polypeptide comprises 1 to 4 amino acid substitutions in HSA domain III, wherein said 1 to 4 amino acid substitutions are present in loop 9 of HSA domain III Polypeptide.

83. The chimeric polypeptide of any one of embodiments 1 to 82 wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing an amino acid residue with an alanine.

84. The chimeric polypeptide of any one of embodiments 1 to 83 wherein one or more amino acid substitutions of the amino acid substitutions comprise a conservative amino acid substitution.

85. The chimeric polypeptide of any one of embodiments 1 to 84 wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing a basic amino acid with another basic amino acid.

86. A chimeric polypeptide according to any one of embodiments 1 to 85 wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing an acidic amino acid with another acidic amino acid.

87. The chimeric polypeptide of any one of embodiments 1 to 86 wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing a neutral amino acid with another neutral amino acid.

88. The chimeric polypeptide of any one of embodiments 1 to 87 wherein the amino acid substitution of one or more of the amino acid substitutions comprises replacing one amino acid with another amino acid in the following group: lysine, arginine and histidine.

89. The chimeric polypeptide of any one of embodiments 1 to 88, wherein the amino acid substitution of one or more of the amino acid substitutions comprises replacing one amino acid with another amino acid in the following group: aspartate and glutamate.

90. Chimeric polypeptide of any one of embodiments 1 to 89, wherein the amino acid substitution of one or more of the amino acid substitutions comprises replacing one amino acid with another amino acid in the following group: asparagine, glutamine, serine, Threonine and tyrosine.

91. The chimeric polypeptide of any one of embodiments 1 to 90 wherein the amino acid substitution of one or more of the amino acid substitutions comprises replacing one amino acid with another amino acid in the group: alanine, valine, isoleucine, Leucine, Proline, Phenylalanine, Tryptophan, Methionine, Cysteine and Glycine.

92. The chimeric polypeptide of any one of embodiments 1 to 91, wherein the amino acid substitution of one or more of the amino acid substitutions comprises replacing one amino acid with another amino acid in the following group: phenylalanine, tryptophan and tyrosine.

93. The chimeric polypeptide of any one of embodiments 1 to 92, wherein the amino acid substitution of one or more of the amino acid substitutions comprises replacing one amino acid with another amino acid in the following group: cysteine, serine and threo Banging.

94. The chimeric polypeptide of any one of embodiments 1 to 93 wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing one amino acid with another amino acid in the following group: asparagine, glutamine, serine, Threonine, tyrosine, lysine, arginine, histidine, aspartate and glutamate.

95. A chimeric polypeptide of any one of embodiments 1 to 94 wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing one amino acid with another amino acid in the following group: glycine, serine, threo Nin, alanine, valine, leucine and isoleucine.

96. The chimeric polypeptide of any one of embodiments 1 to 95 wherein one or more amino acid substitutions of the amino acid substitutions comprise a non-conservative substitution.

97. The chimeric polypeptide of embodiment 83, wherein all of the amino acid substitutions comprise replacing an amino acid residue with an alanine.

98. The chimeric polypeptide of embodiment 84, wherein all of the amino acid substitutions comprise conservative amino acid substitutions independently at each position.

99. The chimeric polypeptide of embodiment 85, wherein all of the amino acid substitutions comprise independently at each position replacing the basic amino acid with another basic amino acid.

100. The chimeric polypeptide of embodiment 87, wherein all of the amino acid substitutions comprise independently at each position replacing an acidic amino acid with another acidic amino acid.

101. The chimeric polypeptide of embodiment 87, wherein all of the amino acid substitutions comprise independently at each position replacing the neutral amino acid with another neutral amino acid.

102. The chimeric polypeptide of embodiment 88, wherein all of the amino acid substitutions comprise independently at each position replacing one amino acid with another amino acid in the group: lysine, arginine and histidine.

103. The chimeric polypeptide of embodiment 89, wherein all of the amino acid substitutions comprise independently at each position replacing one amino acid with another amino acid in the group: aspartate and glutamate.

104. The chimeric polypeptide of embodiment 90, wherein all of the amino acid substitutions comprise independently at each position replacing one amino acid with another amino acid in the group: asparagine, glutamine, serine, threonine and tyrosine .

105. The chimeric polypeptide of embodiment 91, wherein all of the amino acid substitutions comprise independently at each position replacing one amino acid with another amino acid in the group: alanine, valine, isoleucine, leucine, proline, phenylalanine , Tryptophan, methionine, cysteine and glycine.

106. The chimeric polypeptide of embodiment 92, wherein all of the amino acid substitutions comprise independently at each position replacing one amino acid with another amino acid in the group: phenylalanine, tryptophan and tyrosine.

107. The chimeric polypeptide of embodiment 93, wherein all of the amino acid substitutions comprise independently at each position replacing one amino acid with another amino acid in the following group: cysteine, serine and threonine.

108. The chimeric polypeptide of embodiment 94, wherein all of the amino acid substitutions comprise independently at each position replacing one amino acid with another amino acid in the group: asparagine, glutamine, serine, threonine, tyrosine , Lysine, arginine, histidine, aspartate and glutamate.

109. The chimeric polypeptide of embodiment 95, wherein all of the amino acid substitutions comprise independently at each position replacing one amino acid with another amino acid in the following group: glycine, serine, threonine, alanine, valine , Leucine and isoleucine.

110. The chimeric polypeptide of embodiment 96, wherein all of the amino acid substitutions comprise non-conservative substitutions independently at each position.

111. The chimeric polypeptide of any one of embodiments 1 to 110, wherein the heterologous protein comprises an antibody or antigen binding fragment thereof.

112. The chimeric polypeptide of any one of embodiments 1-111 wherein the heterologous protein comprises a therapeutic protein.

113. The chimeric polypeptide of any one of embodiments 1-112 further comprising a constant region of an IgG immunoglobulin.

114. The chimeric polypeptide of any one of embodiments 1-113 wherein the HSA moiety is chemically conjugated to a heterologous protein.

115. The chimeric polypeptide of any one of embodiments 1-113 wherein the HSA moiety is recombinantly conjugated to a heterologous protein.

116. A chimeric polypeptide according to embodiment 115, prepared by using a recombinant vector encoding both an HSA moiety and a heterologous protein.

117. A chimeric polypeptide according to embodiment 115 or 116, produced in prokaryotic or eukaryotic cells.

118. The chimeric polypeptide of embodiment 117, wherein the eukaryotic cell is selected from yeast cells, algal cells, insect cells and mammalian cells.

119. The chimeric polypeptide of any one of embodiments 114-118 wherein the HSA moiety and the heterologous protein are directly conjugated to each other.

120. The chimeric polypeptide of any one of embodiments 114-118 wherein the HSA moiety and the heterologous protein are conjugated via a linking agent.

121. The chimeric polypeptide of embodiment 120, wherein the linker comprises one or more Gly-Gly-Gly-Gly-Ser repeats.

122. The chimeric polypeptide of any one of embodiments 1 to 121 wherein the HSA moiety is conjugated to the N terminal amino acid of the heterologous protein.

123. The chimeric polypeptide of any one of embodiments 1 to 121 wherein the HSA moiety is conjugated to the C-terminal amino acid of the heterologous protein.

124. The chimeric polypeptide of any one of embodiments 1 to 121 wherein the HSA moiety is conjugated to an internal amino acid of a heterologous protein.

125. The method of any of embodiments 1-124, wherein the HSA moiety is at least a portion of HSA domain I; At least a portion of HSA domain II; Or at least a portion of HSA domain I and at least a portion of HSA domain II.

126. The chimeric polypeptide of any one of embodiments 1 to 125, wherein the chimeric polypeptide is substantially purified.

127. A composition comprising a chimeric polypeptide of any one of embodiments 1 to 126 and a pharmaceutically acceptable carrier.

128. The composition of embodiment 127, which is a sterile composition.

129. The composition of embodiment 127 or 128 which is a nonpyrogenic composition.

130. A method of treating a subject in need of treatment comprising administering to the subject a chimeric polypeptide according to any one of embodiments 1 to 126 or a composition according to any of embodiments 127 to 129. Way.

131. A method of increasing the serum half-life of a protein in a subject in need of an increase in the serum half-life of the protein, the method comprising administering to the subject a chimeric polypeptide according to any one of embodiments 1 to 126.

132. The method of embodiment 130 or 131, wherein administering to the subject comprises administering the chimeric polypeptide systemically.

133. The method of embodiment 130 or 131, wherein administering to the subject comprises administering the chimeric polypeptide by a route selected from the group consisting of: intradermal, transdermal, intramuscular, intraperitoneal , Intravenous, intravascular, pericardial, subcutaneous, lung, intranasal, intraocular, epidural, topical and oral.

134. The method of embodiment 130 or 131, wherein administering to the subject comprises intravenously administering the chimeric polypeptide.

135. A nucleic acid construct comprising a nucleotide sequence encoding the chimeric polypeptide of any of embodiments 1-125.

136. An HSA variant polypeptide comprising human serum albumin (HSA) domain III or a neonatal Fc receptor (FcRn) binding fragment thereof, wherein the variant polypeptide can bind FcRn, wherein the HSA domain III is from 1 to 18 Incorporating amino acid substitutions to increase the affinity of said variant polypeptides for FcRn relative to a control HSA polypeptide lacking said substitutions.

137. The variant polypeptide of embodiment 136, wherein the variant polypeptide binds FcRn with a higher affinity than the control polypeptide, wherein the affinity is measured at acidic pH.

138. The variant polypeptide of embodiment 136 or 137, wherein the acidic pH is 5.0 to 6.0.

139. The variant polypeptide of embodiment 138, wherein the acidic pH is 5.5 ± 0.2.

140. The variant polypeptide of embodiment 136, which binds FcRn with a higher affinity than the control polypeptide at acidic pH but does not bind FcRn with a higher affinity than the control polypeptide at neutral pH.

141. The variant polypeptide of embodiment 140, wherein the neutral pH is between 6.9 and 7.9.

142. The variant polypeptide of embodiment 141, wherein the neutral pH is 7.4 ± 0.2.

143. The variant polypeptide of embodiment 136, having a serum half-life longer than the control HSA polypeptide.

144. The variant polypeptide of any one of embodiments 136 to 143, wherein the HSA domain III comprises 1 to 10 amino acid substitutions.

145. The variant polypeptide of any one of embodiments 136-144, wherein the amino acid substitution of one or more of said amino acid substitutions in HSA domain III is a substitution of a residue that is conserved between multiple species.

146. Variant polypeptide of embodiment 145, wherein all of said amino acid substitutions in HSA domain III are substitutions of residues conserved between multiple species.

147. The method of any of embodiments 136-144, wherein one or more amino acid substitutions of the amino acid substitutions in HSA domain III are human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand rat, sheep. And variant polypeptides which are residue substitutions conserved between serum albumin proteins from cats and horses.

148. The method of embodiment 147, wherein all of said amino acid substitutions in HSA domain III are between serum albumin proteins from humans, pigs, rats, mice, dogs, rabbits, cattle, chickens, donkeys, sand rats, sheep, cats, and horses. A variant polypeptide that is a substitution of a residue conserved in.

149. The variant polypeptide of any one of embodiments 136 to 147, wherein the amino acid substitutions of one or more of the amino acid substitutions in HSA domain III are selected from the amino acid substitutions listed in Table 5.

150. The method of any of embodiments 136-147, wherein one or more amino acid substitutions of the amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA Phosphorus variant polypeptides: residue 381, residue 383, residue 391, residue 401, residue 402, residue 407, residue 411, residue 413, residue 414, residue 415, residue 416, residue 424, residue 426, residue 434, residue 442, residue 445, residue 447, residue 450, residue 454, residue 455, residue 456, residue 457, residue 459, residue 463, residue 495, residue 506, residue 508, residue 509, residue 511, residue 512, residue 515, residue 516, Residue 517, residue 519, residue 521, residue 523, residue 524, residue 525, residue 526, residue 527, residue 531, residue 535, residue 538, residue 539, residue 541, residue 557, residue 561, residue 566 and residue 569 .

151. Variant polypeptide according to any one of embodiments 136 to 147 and 150, comprising an amino acid substitution in HSA domain III at a position selected from the group consisting of the following positions numbered based on the position in full-length mature HSA: (a) residues 383 and 413; (b) residues 401 and 523; (c) residues 407 and 447; (d) residues 407, 447 and 539; (e) residues 407 and 509; (f) residues 407 and 526; (g) residues 411 and 535; (h) residues 414 and 456; (i) residues 415 and 569; (j) residues 426 and 526; (k) residues 442, 450 and 459; (l) residues 463 and 508; (m) residues 508, 519 and 525; (n) residues 509 and 527; (o) residues 523 and 538; (p) residues 526 and 557; (q) residues 541 and 561; (r) residues 463 and 523; (s) residues 508 and 523; (t) residues 508 and 524; (u) residues 463, 508 and 523; (v) residues 463, 508 and 524; (w) residues 508, 523 and 524; (x) residues 463, 508, 523 and 524; (y) residues 463 and 524; (z) residues 523 and 524; And (aa) residues 463, 523 and 524.

152. Variant polypeptide according to embodiment 151, comprising amino acid substitutions in HSA domain III at a position selected from the group consisting of the following positions numbered based on position in full-length mature HSA: (a) residues 463 and 508; (b) residues 463 and 523; (c) residues 508 and 523; (d) residues 508 and 524; (e) residues 463, 508 and 523; (f) residues 463, 508 and 524; (g) residues 508, 523 and 524; (h) residues 463, 508, 523 and 524; (i) residues 463 and 524; (j) residues 523 and 524; And (k) residues 463, 523 and 524.

153. Variant polypeptide of any one of embodiments 136-148 and 150-152, wherein the amino acid substitution of at least one of said amino acid substitutions in HSA domain III is selected from the group consisting of the following amino acid substitutions: V381N, V381Q , E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, K402A, K402G, K402I, K402L, K402V, L407F, L407H, L407M, L407N, L407Q, L407N, L407 , Y411Q, Y411N, K413C, K413S, K413T, K414S, K414T, V415C, V415L, V415S, V415T, Q416H, Q416P, V424A, V424D, V424G, V424I, V424L, V424M, V424N, V424Q, V424W, V426D, V426E, V426E , V426H, V426L, V426N, V426P, V426Q, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, P447S, P447T, E450D, E450E, S454C, S454M, S454T, V455N, V455Q, V456N, V456Q, V456Q, V456Q , L457W, L457Y, Q459K, Q459R, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T506F, T506M, T506N, T506R, T506W, T506Y, T508C, T508E, T508I, T508K, T508K, T508 R, T508S, F509C, F509D, F509G, F509I, F509L, F509M, F509P, F509V, F509W, F509Y, A511D, A511F, A511I, A511R, A511T, A511V, A511W, A511Y, D512F, D512M, D512Q, D512W, D512Y T515C, T515D, T515E, T515E, T515G, T515H, T515L, T515N, T515P, T515Q, T515S, T515W, T515Y, L516C, L516F, L516S, L516T, L516W, L516Y, S517C, S517F, S517M, S517T, S517W, S517Y K519A, K519G, K519I, K519L, K519V, R521F, R521H, R521M, R521Q, R521T, R521W, R521Y, I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523N, I523N I523Q, I523R, I523S, I523T, I523V, I523W, I523Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526A, Q526C, Q526C, Q526C Q526H, Q526L, Q526M, Q526P, Q526S, Q526T, Q526V, Q526Y, T527F, T527W, T527Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535K, H535L, H535N, H535P, H535S538 K538 K538Y, A539I, A539L, A539V, K541F, K541W, K541Y, K557A, K557G, K557I, K557L, K557N, K557S, K557V, A561F, A561W, A561Y, T566F, T56 6 W, T566Y, A569H and A569P.

154. Variant polypeptide of any one of embodiments 136-148 and 150-153, wherein the amino acid substitution of at least one of said amino acid substitutions in HSA domain III is selected from the group consisting of the following amino acid substitutions: V381N, E383G , N391V, Y401E, K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F , L463C, L463F, L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C , K519I, R521W, I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524A, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526T , Q526Y, T527Y, E531I, H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W and A569P.

155. Variant polypeptide of any one of embodiments 136-148 and 150-153, wherein the variant polypeptide comprises one amino acid substitution in HSA domain III selected from the group consisting of the following amino acid substitutions: V381N, E383G, N391V, Y401E, K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F, Q459R, L463C, L463C, L463C, L463C L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C, S517W, K519I, K519I I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524A, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526M, Q526Y, T526 E531I, H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W and A569P.

156. Variant polypeptide of embodiment 154, wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions: L407N, L407Y, V415T, V424I, V424Q, V426E, V426H, P447S, V455N, V456N, L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T535 and H5P27Y K557G.

157. Variant polypeptide of embodiment 155, wherein the variant polypeptide comprises one amino acid substitution in HSA domain III selected from the group consisting of: amino acid substitutions: L407N, L407Y, V415T, V424I, V424Q, V426E, V426H, P447S, V455N, V456N , L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T527Y, H535P and K557G.

158. The variant polypeptide of any one of embodiments 136-147 and 150-152, wherein the variant polypeptide comprises an amino acid substitution in HSA domain III selected from the group consisting of the following amino acid substitutions: (a) E383G / K413S; (b) Y401E / I523G; (c) L407N / P447S; (d) L407N / P447S / A539I; (e) L407N / F509M; (f) L407Y / Q526T; (g) Y411Q / H535N; (h) K414S / V456N; (i) V415T / A569P; (j) V426H / Q526Y; (k) E442K / E450D / Q459R; (l) L463N / T508R; (m) T508R / K519I / K525V; (n) F509I / T527Y; (o) I523Q / K538Y; (p) Q526M / K557G; (q) K541F / A561F; (r) L463N / K524L; (s) T508R / I523G; (t) T508R / K524L; (u) L463N / T508R / I523G; (v) L463N / T508R / K524L; (w) T508R / I523G / K524L; (x) L463N / T508R / I523G / K524L; (y) L463N / I523G; (z) I523 G / K524L; And (aa) L463N / I523G / K524L.

159. The variant polypeptide of embodiment 158, comprising an amino acid substitution selected from the group consisting of the following amino acid substitutions in HSA domain III: (a) L407N / P447S; (b) L407N / P447S / A539I; (c) L407N / F509M; (d) Y411Q / H535N; (e) K414S / V456N; (f) V426H / Q526Y; (g) L463N / T508R; (h) F509I / T527Y; (i) I523Q / K538Y; (j) Q526M / K557G; (k) K541F / A561F; (l) L463N / K524L; (m) T508R / I523 G; (n) T508R / K524L; (o) L463N / T508R / I523G; (p) L463N / T508R / K524L; (q) T508R / I523G / K524L; (r) L463N / T508R / I523G / K524L; (r) L463N / T508R / I523G / K524L; (s) L463N / I523G; (t) I523G / K524L; And (u) L463N / I523G / K524L.

160. Variant polypeptide of embodiment 158, wherein the variant polypeptide comprises an amino acid substitution in the HSA domain III selected from the group consisting of: (a) L463N / T508R; (b) L463N / K524L; (c) T508R / I523G; (d) T508R / K524L; (e) L463N / T508R / I523G; (f) L463N / T508R / K524L; (g) T508R / I523G / K524L; And (h) L463N / T508R / I523G / K524L.

161. Variant polypeptide according to embodiment 158, comprising an amino acid substitution selected from the group consisting of the following amino acid substitutions in HSA domain III: (a) L463N / K508R; (b) T508R / I523G; (c) T508R / K524L; And (d) L463N / T508R / I523G.

162. The method of any of embodiments 136-147, wherein one or more amino acid substitutions of the amino acid substitutions in HSA domain III are human, pig, rat, mouse, dog, rabbit, cow, donkey, sand rat, sheep, cat. And variant polypeptides that are substitutions of residues that are conserved among serum albumin proteins from horses but not conserved in chicken serum albumin.

163. The antibody of embodiment 162, wherein all of said amino acid substitutions in HSA domain III are conserved between serum albumin proteins from humans, pigs, rats, mice, dogs, rabbits, cattle, donkeys, sand rats, sheep, cats, and horses. The variant polypeptide which is a substitution of the residue which is carried out but is not preserved in chicken serum albumin.

164. The variant polypeptide of embodiment 147, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA (SEQ ID NO: 2): residues 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and Residue 571.

165. Variant polypeptide of embodiment 164, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 383, residue 391, Residue 434, residue 442, residue 445 and residue 450.

166. Variant polypeptide of embodiment 165, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is selected from the group consisting of the following amino acid substitutions: V381N, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, E450D and E450E.

167. Variant polypeptide of embodiment 164, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 417, residue 442, Residue 499 and residue 502.

168. Variant polypeptide of embodiment 162, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 380, residue 381, Residue 384, residue 387, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441. , Residue 457, residue 458, residue 463, residue 464, residue 466, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547 and residue 576.

169. Variant polypeptide of embodiment 168, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 381, residue 401, Residue 424, residue 457, residue 463, residue 495, residue 508, residue 515, residue 516, residue 524, residue 525, residue 526, residue 531, residue 535 and residue 539.

170. Variant polypeptides of embodiment 169 wherein the HSA domain III comprises amino acid substitutions at a position selected from the group consisting of the following positions numbered based on position in full length mature HSA: residues 463 and 508; Residues 463 and 524; Residues 508 and 524; And residues 463, 508 and 524.

171. Variant polypeptide of embodiment 169 or 170, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is selected from the group consisting of the following amino acid substitutions: V381N, V381Q, Y401D, Y401E, V424N, V424Q, L457F, L457W, L457Y, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T508C, T508E, T508I, T508K, T508R, T508S, T515C, T515H, T515N, T515P, T515Q, T515S, L516S, L516T, L516W, L516Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, E531A, E531A, E531A E531I, E531L, E531V, H535D, H535E, H535P, A539I, A539L and A539V.

172. Variant polypeptide of embodiment 164, wherein all of the amino acid substitutions are selected from the members of the group consisting of: residues 383, residues 389, residues 391, residues 410, residues 417, residues 425, residues 442 , Residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.

173. Variant polypeptide of embodiment 168, wherein all of said amino acid substitutions are selected from members of the group consisting of: residues 380, residues 381, residues 384, residues 387, residues 396, residues 401, residues 404 , Residue 405, residue 406, residue 409, residue 419, residue 421, residue 422, residue 424, residue 428, residue 430, residue 431, residue 433, residue 441, residue 457, residue 458, residue 463, residue 464, residue 466, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 489, residue 491, residue 495, residue 500, residue 508, residue 510, residue 515, residue 516, residue 524, residue 525, Residue 526, residue 528, residue 531, residue 535, residue 539, residue 544, residue 547 and residue 576.

174. Variant polypeptide according to embodiment 164 or 168, wherein all of said amino acid substitutions are selected from members of the group consisting of: residues 380, residues 381, residues 384, residues 383, residues 387, residues 389, Residue 391, residue 396, residue 401, residue 404, residue 405, residue 406, residue 409, residue 410, residue 417, residue 419, residue 421, residue 422, residue 424, residue 425, residue 428, residue 430, residue 431 , Residue 433, residue 441, residue 442, residue 457, residue 458, residue 463, residue 464, residue 465, residue 466, residue 467, residue 468, residue 469, residue 470, residue 474, residue 475, residue 480, residue 481, residue 486, residue 489, residue 491, residue 495, residue 499, residue 500, residue 502, residue 508, residue 510, residue 515, residue 516, residue 520, residue 524, residue 525, residue 526, residue 528, Residue 531, residue 532, residue 535, residue 536, residue 539, residue 543, residue 544, residue 547, residue 571 and residue 576.

175. Variant polypeptide of embodiment 174, wherein all of said amino acid substitutions are selected from the members of the group consisting of: residues 381, residues 383, residues 391, residues 401, residues 424, residues 442, residues 463 , Residue 495, residue 508, residue 515, residue 516, residue 524, residue 525, residue 526, residue 531, residue 535 and residue 539.

176. Variant polypeptide of embodiment 175, wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions: V381N, V381Q, E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, V424A, V424G, V424I, V424L, V424N, V424Q, E442K, E442R, L463C, L463F, L463G, L463H, L463M, L463N, L463Q, E495D T506Y, T508C, T508E, T508I, T508K, T508R, T508S, T515C, T515H, T515N, T515P, T515Q, T515S, L516S, L516T, L516W, L516Y, K524A, K524F, K524G, K524I, K524L, K524M, K524T, K524T, K524T K525A, K525G, K525I, K525L, K525V, Q526C, Q526M, Q526S, Q526T, Q526Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535P, A539I, A539L and A539V.

177. The variant polypeptide of any one of embodiments 136 to 174, wherein one or more amino acid substitutions of the amino acid substitutions in HSA domain III are substitutions of surface accessible residues.

178. Variant polypeptide of embodiment 177, wherein all of said amino acid substitutions in HSA domain III are substitutions of surface accessible residues.

179. The variant polypeptide of any one of embodiments 136-174, wherein the amino acid substitution of one or more of said amino acid substitutions in HSA domain III is a substitution of a residue that is surface accessible and conserved among multiple species.

180. Variant polypeptide of embodiment 179, wherein all of said amino acid substitutions in HSA domain III are substitutions of residues that are surface accessible and conserved among multiple species.

181. Variant polypeptide of embodiment 179 or 180, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 383, residue 389, residue 391, residue 410, residue 417, residue 425, residue 442, residue 465, residue 467, residue 468, residue 486, residue 499, residue 502, residue 520, residue 532, residue 536, residue 543 and residue 571.

182. Variant polypeptide of embodiment 181, wherein the one or more amino acid substitutions in HSA domain III are at any of the following positions numbered based on the position in full-length mature HSA: residue 383, residue 391 and Residue 442.

183. Variant polypeptide of embodiment 182, wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions: E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, E442K and E442R.

184. The variant polypeptide of any one of embodiments 136 to 180, wherein the HSA domain III comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.

185. The variant polypeptide of embodiment 184, wherein the HSA domain III comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.

186. The variant polypeptide of any one of embodiments 177 to 185, wherein the HSA domain III comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1.

187. The variant polypeptide of any one of embodiments 136 to 186, wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are present in loop 2 of HSA domain III.

188. The compound of any of embodiments 136 to 155, 162 to 164, 168, 172 to 174, 177 to 181 and 184 to 187, wherein all of said amino acid substitutions in HSA domain III are present in loop 2 of HSA domain III Variant polypeptide.

189. Variant polypeptide of embodiment 187 or 188, wherein the variant polypeptide comprises 1 to 5 amino acid substitutions in HSA domain III, wherein said 1 to 5 amino acid substitutions are present in loop 2 of HSA domain III Polypeptide.

190. The variant polypeptide of any one of embodiments 136 to 187, wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are present in loop 3 of HSA domain III.

191. The variant polypeptide of any one of embodiments 136-155, 162-168, 172-186, and 190, wherein all of said amino acid substitutions in HSA domain III are present in loop 3 of HSA domain III.

192. The variant polypeptide of embodiment 190 or 191, wherein the variant polypeptide comprises 1 to 5 amino acid substitutions in HSA domain III, wherein said 1 to 5 amino acid substitutions are present in loop 3 of HSA domain III. Polypeptide.

193. The variant polypeptide of any one of embodiments 136 to 173 and 190, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is in loop 6 of HSA domain III.

194. The variant of any one of embodiments 136-155, 162-164, 167-181, 184-186, and 193, wherein all of said amino acid substitutions in HSA domain III are present in loop 6 of HSA domain III Polypeptide.

195. Variant polypeptide according to embodiment 193 or 194, wherein the variant polypeptide comprises 1 to 5 amino acid substitutions in HSA domain III, wherein said 1 to 5 amino acid substitutions are present in loop 6 of HSA domain III. Polypeptide.

196. The variant polypeptide of any one of embodiments 136 to 173, 190, and 193, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is in helix 7 of HSA domain III.

197. The variant of any one of embodiments 136-155, 162-164, 167-181, 184-186, and 196, wherein all of said amino acid substitutions in HSA domain III are present in helix 7 of HSA domain III Polypeptide.

198. Variant polypeptide of embodiment 196 or 197, wherein the variant polypeptide comprises one to three amino acid substitutions in HSA domain III, wherein said one to six amino acid substitutions are present in helix 7 of HSA domain III Polypeptide.

199. The variant polypeptide of any one of embodiments 136 to 173, 190, 193, and 196, wherein the amino acid substitution of one or more of said amino acid substitutions in HSA domain III is present in loop 7 of HSA domain III.

200. Variants according to any one of embodiments 136 to 155, 162 to 164, 167 to 181, 184 to 186, and 199, wherein all of said amino acid substitutions in HSA domain III are present in loop 7 of HSA domain III. Polypeptide.

201. Variant polypeptide according to embodiment 199 or 200, wherein the variant polypeptide comprising one to three amino acid substitutions in HSA domain III, wherein said one to three amino acid substitutions are present in loop 7 of HSA domain III. Polypeptide.

202. The variant polypeptide of any one of embodiments 136 to 173, 190, 193, 196, and 199, wherein the amino acid substitution of one or more of the amino acid substitutions in HSA domain III is in helix 8 of HSA domain III.

203. The compound of any of embodiments 136 to 155, 162 to 164, 173, 174, 177 to 181, 184 to 186, and 202, wherein all of said amino acid substitutions in HSA domain III are present in helix 8 of HSA domain III Variant polypeptide.

204. Variant polypeptide according to embodiment 202 or 203, wherein the variant polypeptide comprises 1 to 5 amino acid substitutions in HSA domain III, wherein said 1 to 18 amino acid substitutions are present in helix 8 of HSA domain III. Polypeptide.

205. Variants according to any of embodiments 136 to 173, 190, 193, 196, 199, and 202, wherein the amino acid substitution of one or more of the amino acid substitutions in HSA domain III is present in loop 8 of HSA domain III. Polypeptide.

206. The method of any of embodiments 136-155, 162-164, 173, 174, 177-181, 184-186, and 205, wherein all of said amino acid substitutions in HSA domain III are present in loop 8 of HSA domain III Variant polypeptide.

207. Variant polypeptide according to embodiment 205 or 206, wherein the variant polypeptide comprises 1 to 5 amino acid substitutions in HSA domain III, wherein said 1 to 5 amino acid substitutions are present in loop 8 of HSA domain III. Polypeptide.

208. The variant polypeptide of any one of embodiments 136 to 173, 190, 193, and 205, wherein the amino acid substitution of one or more of the amino acid substitutions in HSA domain III is present in loop 9 of HSA domain III.

209. The variant of any one of embodiments 136-155, 162, 163, 177-180, 184-186, and 208, wherein all of said amino acid substitutions in HSA domain III are present in loop 9 of HSA domain III Polypeptide.

210. Variant polypeptide of embodiment 208 or 209, wherein the variant polypeptide comprises 1 to 4 amino acid substitutions in HSA domain III, wherein said 1 to 4 amino acid substitutions are present in loop 9 of HSA domain III. Polypeptide.

211. The variant polypeptide of any one of embodiments 136-210, wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing an amino acid residue with an alanine.

212. The variant polypeptide of any one of embodiments 136 to 211, wherein at least one amino acid substitution of said amino acid substitutions comprises a conservative amino acid substitution.

213. The variant polypeptide of any one of embodiments 136 to 212, wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing a basic amino acid with another basic amino acid.

214. The variant polypeptide of any one of embodiments 136 to 213, wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing an acidic amino acid with another acidic amino acid.

215. The variant polypeptide of any one of embodiments 136 to 214, wherein the amino acid substitution of one or more of the amino acid substitutions comprises replacing a neutral amino acid with another neutral amino acid.

216. The variant polypeptides of any one of embodiments 136 to 215, wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing one amino acid with another amino acid in the group: lysine, arginine, and histidine.

217. Variant polypeptides according to any of embodiments 136 to 216, wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing one amino acid with another amino acid in the following group: aspartate and glutamate.

218. Variant polypeptide of any one of embodiments 136 to 217, wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing one amino acid with another amino acid in the group: asparagine, glutamine, serine, Threonine and tyrosine.

219. The variant polypeptide of any one of embodiments 136-218, wherein the amino acid substitution of one or more of the amino acid substitutions comprises replacing one amino acid with another amino acid in the group: alanine, valine, isoleucine, Leucine, Proline, Phenylalanine, Tryptophan, Methionine, Cysteine and Glycine.

220. Variant polypeptides according to any of embodiments 136 to 219, wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing one amino acid with another amino acid in the group: phenylalanine, tryptophan and tyrosine.

221. Variant polypeptide of any one of embodiments 136-220, wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing one amino acid with another amino acid in the group: cysteine, serine and threo Banging.

222. Variant polypeptide of any one of embodiments 136 to 221, wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing one amino acid with another amino acid in the group: asparagine, glutamine, serine, Threonine, tyrosine, lysine, arginine, histidine, aspartate and glutamate.

223. Variant polypeptide of any one of embodiments 136 to 222, wherein one or more amino acid substitutions of the amino acid substitutions comprise replacing one amino acid with another amino acid in the following group: glycine, serine, threo Nin, alanine, valine, leucine and isoleucine.

224. The variant polypeptide of any one of embodiments 136 to 223, wherein one or more amino acid substitutions of the amino acid substitutions comprise non-conservative substitutions.

225. The variant polypeptide of embodiment 211, wherein all of the amino acid substitutions comprise replacing an amino acid residue with an alanine.

226. The variant polypeptide of embodiment 212, wherein all of the amino acid substitutions comprise conservative amino acid substitutions independently at each position.

227. The variant polypeptide of embodiment 213, wherein all of the amino acid substitutions comprise independently replacing at each position a basic amino acid for another basic amino acid.

228. The variant polypeptide of embodiment 214, wherein all of the amino acid substitutions comprise independently replacing at each position an acidic amino acid for another acidic amino acid.

229. The variant polypeptide of embodiment 215, wherein all of the amino acid substitutions comprise independently at each position replacing a neutral amino acid with another neutral amino acid.

230. Variant polypeptides of embodiment 216, wherein all of the amino acid substitutions comprise independently replacing at each position replacing one amino acid with another amino acid in the group: lysine, arginine and histidine.

231. The variant polypeptides of embodiment 217, wherein all of the amino acid substitutions comprise independently replacing at each position replacing one amino acid with another amino acid in the following group: aspartate and glutamate.

232. The variant polypeptide of embodiment 218, wherein all of said amino acid substitutions comprise independently replacing at each position replacing one amino acid with another amino acid in the following group: asparagine, glutamine, serine, threonine and tyrosine .

233. Variant polypeptide of embodiment 219, wherein all of said amino acid substitutions comprise independently at each position replacing one amino acid with another amino acid in the group: alanine, valine, isoleucine, leucine, proline, phenylalanine , Tryptophan, methionine, cysteine and glycine.

234. The variant polypeptides of embodiment 220 wherein all of the amino acid substitutions comprise independently replacing at each position replacing one amino acid with another amino acid in the group: phenylalanine, tryptophan and tyrosine.

235. Variant polypeptides of embodiment 221 wherein all of said amino acid substitutions comprise independently replacing at each position replacing one amino acid with another amino acid in the following group: cysteine, serine and threonine.

236. The variant polypeptide of embodiment 222, wherein all of the amino acid substitutions comprise independently replacing at each position replacing one amino acid with another amino acid in the group: asparagine, glutamine, serine, threonine, tyrosine , Lysine, arginine, histidine, aspartate and glutamate.

237. The variant polypeptide of embodiment 223, wherein all of said amino acid substitutions comprise independently replacing at each position replacing one amino acid with another amino acid in the following group: glycine, serine, threonine, alanine, valine , Leucine and isoleucine.

238. The variant polypeptide of embodiment 224, wherein all of the amino acid substitutions comprise non-conservative substitutions independently at each position.

239. The method of any of embodiments 136 to 238, wherein the HSA moiety is at least a portion of HSA domain I; At least a portion of HSA domain II; Or at least a portion of HSA domain I and at least a portion of HSA domain II.

240. The variant polypeptide of any one of embodiments 136 to 239, wherein the variant polypeptide is substantially purified.

241. The variant polypeptide of any one of embodiments 136 to 240, further comprising a binding site for an epitope on the target.

242. The variant polypeptide of embodiment 241, wherein the binding site exhibits an antagonist effect on said target.

243. The variant polypeptide of embodiment 241, wherein the binding site exhibits an agonist effect on the target.

244. A composition comprising the variant polypeptide of any one of embodiments 136 to 243 and a pharmaceutically acceptable carrier.

245. The composition of embodiment 244, which is a sterile composition.

246. The composition of embodiment 244 or 245, which is a nonpyrogenic composition.

247. A method of increasing the serum half-life of a protein, comprising conjugating a variant polypeptide according to any one of embodiments 136 to 243 to said protein.

248. A method of treating a subject in need thereof, comprising administering to the subject a variant polypeptide according to any one of embodiments 241 to 243.

249. A nucleic acid construct comprising a nucleotide sequence encoding a variant polypeptide of any one of embodiments 136 to 239.

250. (b) an HSA moiety comprising (a) a human serum albumin (HSA) domain III comprising a 1-18 amino acid substitution, operably linked to a nucleotide sequence encoding a heterologous protein, or an FcRn binding fragment thereof A nucleic acid construct comprising a nucleotide sequence encoding, wherein said nucleic acid construct encodes a chimeric polypeptide capable of retaining the functional activity of said heterologous protein and capable of binding to FcRn, wherein said chimeric polypeptide does not comprise said amino acid substitution A nucleic acid construct having an increased serum half-life and / or increased affinity for FcRn relative to a control chimeric polypeptide having.

251. The nucleic acid construct of embodiment 250, wherein the chimeric polypeptide binds FcRn with a higher affinity than the control chimeric polypeptide.

252. The nucleic acid construct of embodiments 250 or 251 wherein the chimeric polypeptide binds FcRn with a higher affinity than the control chimeric polypeptide, wherein the affinity is measured at acidic pH.

253. The nucleic acid construct of embodiment 252 wherein the acidic pH is between 5.0 and 6.0.

254. The nucleic acid construct of embodiment 253 wherein the acidic pH is 5.5 ± 0.2.

255. The method of any one of embodiments 250 to 255, wherein the chimeric polypeptide binds FcRn with a higher affinity than the control chimeric polypeptide at acidic pH, but the chimeric polypeptide is higher than the control chimeric polypeptide at neutral pH. A nucleic acid construct that does not bind FcRn on Mars.

256. The nucleic acid construct of embodiment 255, wherein the neutral pH is between 6.9 and 7.9.

257. The nucleic acid construct of embodiment 256 wherein the neutral pH is 7.4 ± 0.2.

258. The nucleotide sequence of embodiment 250, wherein the nucleotide sequence of (a) comprises a nucleotide sequence encoding a HSA moiety comprising human serum albumin (HSA) domain III or an FcRn binding fragment thereof comprising 1 to 10 amino acid substitutions Nucleic acid construct.

259. The nucleic acid construct of any one of embodiments 250-258, wherein the amino acid substitution of one or more of said amino acid substitutions in HSA domain III is a substitution of a residue that is conserved between multiple species.

260. The nucleic acid construct of embodiment 259 wherein all of said amino acid substitutions in HSA domain III are substitutions of residues that are conserved among multiple species.

261. The method of any one of embodiments 250 to 258, wherein one or more amino acid substitutions of the amino acid substitutions in HSA domain III are human, pig, rat, mouse, dog, rabbit, cow, chicken, donkey, sand rat, sheep. , Nucleic acid construct that is a substitution of residues that are conserved between serum albumin proteins from cats and horses.

262. The method of embodiment 261, wherein all of the amino acid substitutions in HSA domain III are between serum albumin proteins from humans, pigs, rats, mice, dogs, rabbits, cattle, chickens, donkeys, sand rats, sheep, cats, and horses. A nucleic acid construct that is a substitution of a residue stored in.

263. The nucleic acid construct of any one of embodiments 250-262, wherein the amino acid substitutions of one or more of said amino acid substitutions in HSA domain III are selected from the amino acid substitutions listed in Table 5.

264. The method according to any one of embodiments 250 to 262, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is at any of the following positions numbered relative to the position in full-length mature HSA Phosphorous nucleic acid constructs: residue 381, residue 383, residue 391, residue 401, residue 402, residue 407, residue 411, residue 413, residue 414, residue 415, residue 416, residue 424, residue 426, residue 434, residue 442, residue 445, residue 447, residue 450, residue 454, residue 455, residue 456, residue 457, residue 459, residue 463, residue 495, residue 506, residue 508, residue 509, residue 511, residue 512, residue 515, residue 516, Residue 517, residue 519, residue 521, residue 523, residue 524, residue 525, residue 526, residue 527, residue 531, residue 535, residue 538, residue 539, residue 541, residue 557, residue 561, residue 566 and residue 569 .

265. The nucleic acid construct of any one of embodiments 250-262, wherein the HSA domain III comprises an amino acid substitution at a position selected from the group consisting of the following positions numbered based on the position in the full length mature HSA: (a) residues 383 and 413; (b) residues 401 and 523; (c) residues 407 and 447; (d) residues 407, 447 and 539; (e) residues 407 and 509; (f) residues 407 and 526; (g) residues 411 and 535; (h) residues 414 and 456; (i) residues 415 and 569; (j) residues 426 and 526; (k) residues 442, 450 and 459; (l) residues 463 and 508; (m) residues 508, 519 and 525; (n) residues 509 and 527; (o) residues 523 and 538; (p) residues 526 and 557; (q) residues 541 and 561; (r) residues 463 and 523; (s) residues 508 and 523; (t) residues 508 and 524; (u) residues 463, 508 and 523; (v) residues 463, 508 and 524; (w) residues 508, 523 and 524; (x) residues 463, 508, 523 and 524; (y) residues 463 and 524; (z) residues 523 and 524; And (aa) residues 463, 523 and 524.

266. The nucleic acid construct of any one of embodiments 250 to 262, wherein the HSA domain III comprises an amino acid substitution at a position selected from the group consisting of the following positions numbered based on the position in full-length mature HSA: (a) residues 463 and 508; (b) residues 463 and 523; (c) residues 508 and 523; (d) residues 508 and 524; (e) residues 463, 508 and 523; (f) residues 463, 508 and 524; (g) residues 508, 523 and 524; (h) residues 463, 508, 523 and 524; (i) residues 463 and 524; (j) residues 523 and 524; And (k) residues 463, 523 and 524.

267. The nucleic acid construct of any one of embodiments 250-262 and 264-266, wherein the one or more amino acid substitutions of said amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions: V381N, V381Q , E383A, E383G, E383I, E383L, E383V, N391A, N391G, N391I, N391L, N391V, Y401D, Y401E, K402A, K402G, K402I, K402L, K402V, L407F, L407H, L407M, L407N, L407Q, L407N, L407 , Y411Q, Y411N, K413C, K413S, K413T, K414S, K414T, V415C, V415L, V415S, V415T, Q416H, Q416P, V424A, V424D, V424G, V424I, V424L, V424M, V424N, V424Q, V424W, V426D, V426E, V426E , V426H, V426L, V426N, V426P, V426Q, G434C, G434S, G434T, E442K, E442R, R445F, R445W, R445Y, P447S, P447T, E450D, E450E, S454C, S454M, S454T, V455N, V455Q, V456N, V456Q, V456Q, V456Q , L457W, L457Y, Q459K, Q459R, L463C, L463F, L463G, L463H, L463I, L463N, L463S, L463Q, E495D, T506F, T506M, T506N, T506R, T506W, T506Y, T508C, T508E, T508I, T508K, T508R , F509C, F509D, F509G, F509I, F509L, F509M, F509P, F509V, F509W, F509Y, A511D, A511F, A511I, A511R, A511T, A511V, A511W, A511Y, D512F, D512M, D512Q, D512W, D512Y, T515C, T515C, T515 T515E, T515E, T515G, T515H, T515L, T515N, T515P, T515Q, T515S, T515W, T515Y, L516C, L516F, L516S, L516T, L516W, L516Y, S517C, S517F, S517M, S517T, S517W, S517Y, K519A, K519 K519I, K519L, K519V, R521F, R521H, R521M, R521Q, R521T, R521W, R521Y, I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523 I523S, I523T, I523V, I523W, I523Y, K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T, K524V, K525A, K525G, K525I, K525L, K525V, Q526A, Q526C, Q526L, Q526H, Q526H Q526M, Q526P, Q526S, Q526T, Q526V, Q526Y, T527F, T527W, T527Y, E531A, E531G, E531I, E531L, E531V, H535D, H535E, H535K, H535L, H535N, H535P, H535S, K538F, K538W5 K538 A539L, A539V, K541F, K541W, K541Y, K557A, K557G, K557I, K557L, K557N, K557S, K557V, A561F, A561W, A561Y, T566F, T566W, T566Y , A569H and A569P.

268. The nucleic acid construct of any one of embodiments 250-262 and 264-266, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is selected from the group consisting of the following amino acid substitutions: V381N, E383G , N391V, Y401E, K402A, L407N, L407Y, Y411Q, K414S, K413S, V415T, V415C, Q416P, V424I, V424Q, V426E, V426H, G434C, E442K, R445W, P447S, E450D, S454C, V455N, V456N, L457F , L463C, L463F, L463H, L463M, L463N, L463Q, E495D, T506Y, T508C, T508E, T508I, T508R, T508S, F509I, F509M, F509W, A511F, D512Y, T515P, T515Q, T515S, L516T, L516W, S517C , K519I, R521W, I523C, I523D, I523E, I523F, I523Q, I523H, I523K, I523L, I523N, I523P, I523G, I523R, I523Y, K524F, K524I, K524L, K524M, K524T, K524V, K525V, Q526T, Q526M, Q526M , T527Y, E531I, H535N, H535P, K538Y, A539I, K541F, K557G, A561F, T566W and A569P.

269. The nucleic acid construct of embodiment 268, wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions: L407N, L407Y, V415T, V424I, V424Q, V426E, V426H, P447S, V455N, V456N, L463N, E495D, T506Y, T508R, F509M, F509W, A511F, D512Y, T515Q, L516T, L516W, S517W, R521W, I523D, I523E, I523G, I523K, I523R, K524L, Q526M, T535 and H5P27Y K557G.

270. The nucleic acid construct of any of embodiments 250-262 and 264-269, wherein the chimeric polypeptide comprises an amino acid substitution in HSA domain III selected from the group consisting of the following amino acid substitutions: (a) E383G / K413S; (b) Y401E / I523G; (c) L407N / P447S; (d) L407N / P447S / A539I; (e) L407N / F509M; (f) L407Y / Q526T; (g) Y411Q / H535N; (h) K414S / V456N; (i) V415T / A569P; (j) V426H / Q526Y; (k) E442K / E450D / Q459R; (l) L463N / T508R; (m) T508R / K519I / K525V; (n) F509I / T527Y; (o) I523Q / K538Y; (p) Q526M / K557G; (q) K541F / A561F; (r) L463N / K524L; (s) T508R / I523G; (t) T508R / K524L; (u) L463N / T508R / I523G; (v) L463N / T508R / K524L; (w) T508R / I523G / K524L; (x) L463N / T508R / I523G / K524L; (y) L463N / I523G; (z) I523 G / K524L; And (aa) L463N / I523G / K524L.

271. The nucleic acid construct of embodiment 270, wherein the chimeric polypeptide comprises an amino acid substitution selected from the group consisting of the following amino acid substitutions in HSA domain III: (a) L407N / P447S; (b) L407N / P447S / A539I; (c) L407N / F509M; (d) Y411Q / H535N; (e) K414S / V456N; (f) V426H / Q526Y; (g) L463N / T508R; (h) F509I / T527Y; (i) I523Q / K538Y; (j) Q526M / K557G; (k) K541F / A561F; (l) L463N / K524L; (m) T508R / I523 G; (n) T508R / K524L; (o) L463N / T508R / I523G; (p) L463N / T508R / K524L; (q) T508R / I523G / K524L; (r) L463N / T508R / I523G / K524L; (s) L463N / I523G; (t) I523G / K524L; And (u) L463N / I523G / K524L.

272. The nucleic acid construct of embodiment 270 wherein the chimeric polypeptide comprises an amino acid substitution in HSA domain III selected from the group consisting of: (a) L463N / T508R; (b) L463N / K524L; (c) T508R / I523G; (d) T508R / K524L; (e) L463N / T508R / I523G; (f) L463N / T508R / K524L; (g) T508R / I523G / K524L; And (h) L463N / T508R / I523G / K524L.

273. The nucleic acid construct of embodiment 270, wherein the chimeric polypeptide comprises an amino acid substitution in HSA domain III selected from the group consisting of: (a) L463N / K508R; (b) T508R / I523G; (c) T508R / K524L; And (d) L463N / T508R / I523G

274. The nucleic acid construct of any of embodiments 250 to 262, wherein one or more amino acid substitutions of the amino acid substitutions in HSA domain III are substitutions of surface accessible residues.

275. The nucleic acid construct of embodiment 274 wherein all of said amino acid substitutions in HSA domain III are substitutions of surface accessible residues.

276. The nucleic acid construct of any one of embodiments 250-258, wherein the amino acid substitution of one or more of said amino acid substitutions in HSA domain III is a substitution of a residue that is surface accessible and conserved between multiple species.

277. The nucleic acid construct of embodiment 276 wherein all of said amino acid substitutions in HSA domain III are substitutions of residues that are surface accessible and conserved between multiple species.

278. The nucleic acid construct of any one of embodiments 250 to 277, wherein the nucleotide sequence of (a) comprises a nucleotide sequence encoding HSA domain III that is at least 90% identical to SEQ ID NO: 1.

279. The nucleic acid construct of embodiment 278, wherein the nucleotide sequence of (a) comprises a nucleotide sequence encoding HSA domain III that is at least 95% identical to SEQ ID NO: 1.

280. The nucleic acid construct of embodiment 279 wherein the nucleotide sequence of (a) comprises a nucleotide sequence that encodes an HSA domain III that is at least 98% identical to SEQ ID NO: 1.

281. The nucleic acid construct of any one of embodiments 250-280, wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are present in loop 2 of HSA domain III.

282. The nucleic acid construct of any one of embodiments 250 to 281, wherein the amino acid substitution of one or more of said amino acid substitutions in HSA domain III is in loop 3 of HSA domain III.

283. The nucleic acid construct of any one of embodiments 250 to 282 wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are present in loop 6 of HSA domain III.

284. The nucleic acid construct of any one of embodiments 250 to 272 wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are in helix 7 of HSA domain III.

285. The nucleic acid construct of any one of embodiments 250 to 284, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is in loop 7 of HSA domain III.

286. The nucleic acid construct of any one of embodiments 250 to 285 wherein the amino acid substitution of one or more of said amino acid substitutions in HSA domain III is in helix 8 of HSA domain III.

287. The nucleic acid construct of any one of embodiments 250 to 286 wherein one or more amino acid substitutions of said amino acid substitutions in HSA domain III are present in loop 8 of HSA domain III.

288. The nucleic acid construct of any one of embodiments 250 to 287, wherein at least one amino acid substitution of said amino acid substitutions in HSA domain III is in loop 9 of HSA domain III.

289. The nucleic acid construct of any one of embodiments 250 to 288, wherein the nucleotide sequence of (b) comprises a nucleotide sequence encoding a heterologous protein comprising an antibody or antigen binding fragment thereof.

290. The nucleic acid construct of any one of embodiments 250 to 289 further comprising a nucleotide sequence encoding a linking agent.

291. The nucleic acid construct of embodiment 290, wherein the nucleotide sequence encodes a linker comprising one or more Gly-Gly-Gly-Gly-Ser repeats.

292. The method of any one of embodiments 250 to 291, wherein the HSA moiety is at least a portion of HSA domain I; At least a portion of HSA domain II; Or at least a portion of HSA domain I and at least a portion of HSA domain II.

293. A library comprising a plurality of polypeptides, wherein each of the plurality of polypeptides comprises HSA domain III or an FcRn binding fragment thereof, wherein each of the plurality of polypeptides comprises human, pig, rat, mouse, dog, rabbit, bovine, And one or more amino acid substitutions of residues conserved between serum albumin proteins from chickens, donkeys, sand rats, sheep, cats and horses independently in the HSA domain III.

294. A library comprising a plurality of polypeptides, wherein each of the plurality of polypeptides comprises HSA domain III or an FcRn binding fragment thereof, wherein each of the plurality of polypeptides comprises human, pig, rat, mouse, dog, rabbit, bovine, A library comprising independently one or more amino acid substitutions in the HSA domain III of residues that are conserved between serum albumin proteins from donkeys, sand rats, sheep, cats and horses and not conserved in serum albumin from chickens .

295. A library comprising a plurality of polypeptides, wherein each of the plurality of polypeptides comprises HSA domain III or an FcRn binding fragment thereof, wherein each of the plurality of polypeptides replaces one or more amino acid substitutions of the surface accessible residues of the HSA domain III. A library contained independently within.

296. The library of embodiment 295, wherein said surface accessible residue is in loop 2 of HSA domain III.

297. The library of embodiment 295 or 296, wherein said surface accessible residue is in loop 3 of HSA domain III.

298. The library of any of embodiments 295-297, wherein said surface accessible residue is in loop 6 of HSA domain III.

299. The library of any of embodiments 295-273, wherein said surface accessible residue is in loop 7 of HSA domain III.

300. The library of any of embodiments 295-299, wherein said surface accessible residue is in loop 8 of HSA domain III.

301. The library of any of embodiments 295-300, wherein said surface accessible residue is in loop 9 of HSA domain III.

302. A library comprising a plurality of polypeptides, wherein each of the plurality of polypeptides comprises HSA domain III or an FcRn binding fragment thereof, wherein each of the plurality of polypeptides is (i) a surface accessible residue and (ii) human, swine Independently comprising one or more amino acid substitutions of residues conserved between serum albumin proteins from rats, mice, dogs, rabbits, cattle, chickens, donkeys, sand mice, sheep, cats and horses Library.

303. A library comprising a plurality of polypeptides, wherein each of the plurality of polypeptides comprises HSA domain III or an FcRn binding fragment thereof, wherein each of the plurality of polypeptides comprises pig, rat, mouse, dog, rabbit, cow, chicken, Independently comprising one or more amino acid residue substitutions in the HSA domain III with amino acids conserved between serum albumin proteins from two or more species except humans selected from the group consisting of donkeys, sand mice, sheep, cats and horses Library.

304. The library of embodiments 302 or 303, wherein the one or more amino acid substitutions are in loop 2 of HSA domain III.

305. The library of any of embodiments 302-304, wherein said one or more amino acid substitutions are in loop 3 of HSA domain III.

306. The library of any of embodiments 302-305, wherein said one or more amino acid substitutions are in loop 6 of HSA domain III.

307. The library of any of embodiments 302-306, wherein said one or more amino acid substitutions are in helix 7 of HSA domain III.

308. The library of any of embodiments 302-307, wherein said one or more amino acid substitutions are in loop 7 of HSA domain III.

309. The library of any of embodiments 302-308, wherein said one or more amino acid substitutions are in helix 8 of HSA domain III.

310. The library of any of embodiments 302-309, wherein said one or more amino acid substitutions are in loop 8 of HSA domain III.

311. The library of any of embodiments 302-310, wherein said one or more amino acid substitutions are in loop 9 of HSA domain III.

312. The method of any one of embodiments 293 to 311, wherein each of the plurality of polypeptides comprises at least a portion of HSA domain I; At least a portion of HSA domain II; Or at least a portion of HSA domain I and at least a portion of HSA domain II.

313. The library of any of embodiments 293-312, which is a display library.

314. The library of embodiment 313, wherein the type of display library is selected from the group consisting of yeast, phage, and mammal.

315. A method of screening a library of any one of embodiments 293 to 314, comprising: (a) selecting a plurality of polypeptides for screening; (b) screening for polypeptides with increased binding affinity or increased serum half-life for FcRn; And (c) identifying the sequence of the polypeptide having increased binding affinity or increased serum half-life for FcRn.

8. Example

Although the invention has been described in general, it will be more readily understood by reference to the following examples which are included solely for the purpose of illustrating some aspects and embodiments of the invention and are not intended to limit the invention. For example, certain constructs and experimental designs disclosed herein represent exemplary means and methods for demonstrating proper function. Thus, it will be readily apparent that any of the specific constructs and experimental designs disclosed may be substituted within the scope of the present disclosure. Furthermore, it will be appreciated that the specific listings or descriptions of the specific devices and reagents used, sizes, manufacturers, etc., should not be considered as limiting the invention, unless specifically stated as limiting the invention. It will also be appreciated that other devices and reagents that can act similarly can be readily substituted.

8.1. Example  1: human serum albumin ( HSA ) 'S domain III Human being coupled to FcRn Dynamics and affinity analysis

This example uses surface plasmon resonance (SPR) to determine the binding, dissociation and equilibrium affinity constants of domain III of HSA to human FcRn. Domain III (also abbreviated as “DIII”) is a fragment of human serum albumin protein spanning amino acid residues 381-585. The amino acid sequence of domain III is set forth in SEQ ID NO: 1. The amino acid sequence of full length mature HSA is set forth in SEQ ID NO: 2. For use in these experiments, domain III was expressed and purified from Pichia pastoris.

8.1.1. Recombinant Protein Expression and Purification

Recombinant plasmids encoding domain III genes were obtained from Geneart AG (Regensburg, Germany). The domain III gene was cleaved from the vector provided by the supplier using restriction enzymes EcoRI and NotI and cloned into the pPICZ-alpha-A Pichia expression vector (Invitrogen, catalog number V195-20). Recombinant domain III protein was expressed in the manner outlined in the manufacturer's instructions. Recombinant domain III protein was secreted into the medium and purified by hydrophobic interaction chromatography on a HiTrap butyl-Sepharose rapid flow column from GE Healthcare (Cat. No. 17 5197 01). In summary, the salinity and pH of the culture medium were first adjusted with 1.5 M ammonium sulfate and 50 mM sodium phosphate, pH 7.0. The culture medium was filtered and passed through the butyl sepharose column, and bound domain III was eluted using low salt sodium phosphate buffer (pH 7.0). Fractions of purified protein were degreased by passing through a hydroxyaloxifyl dextran (Sigma-Aldrich, Catalog No. H6258) column with a circulating cold water bath maintained at 50 ° C. The purity of both degreased and non-degreased forms was 99% when visualized by 4% to 12% SDS-PAGE by Coomassie Blue staining (FIG. 1A). Protein concentration was measured as A ₂₈₀ . Near-ultraviolet CD, which is closely correlated with previously published measurements (see, eg, Giancola et al. International Journal of Biological Macromolecules 20 (1997) 193-204), for proper folding of purified domain III and Ultraviolet CD measurement confirmed.

8.1.2. Dynamics measurement

Equilibrium, binding, and dissociation rate constants were measured on a BIAcore T100 instrument (BIAcore, Inc, Upsala, Sweden) at 25 ° C. and the Biacore T100 evaluation software v. Data were analyzed using 1.1 (Biacore Inc., Upsala, Sweden). Both the degreasing and degreasing forms of domain III were either CM4 chips (catalog number BR-1005-39) or CM5 chips at standard coupling amine coupling (Biacore Handbook, 2002) with coupling densities of 1016 RU and 1184 RU, respectively. (Co-catalog number BR-1000-14). One of the flow cells was mock coupled using the same fixation protocol using no protein for use as a blank. All injections were performed in 50 mM phosphate and 150 mM NaCl buffer at pH 5.5 and the chip surface was regenerated with phosphate buffered saline (PBS) at pH 7.4 between injections. In order to determine the binding constant (k _on ), dissociation constant (k _off ) and equilibrium dissociation constant (K _D ) in a single experiment, increasing concentrations of human FcRn (39 nM to 40 μM) on immobilized domain III protein Inject at a rate of 50 μl / min (FIG. 1B, left panel). The binding reached equilibrium, and the kinetics constants k _on and k _off were derived by simultaneously fitting both the binding phase (4 min) and dissociation phase (1 min) of the curve to the 1: 1 Langmuir model. K _D was induced by fitting a curve of equilibrium binding response (Req) versus analyte concentration to a stationary affinity model using nonlinear regression analysis (FIG. 1B, right panel). Both degreased and non-degreased forms of domain III showed similar binding sensorgrams and Req versus ligand concentration curves.

The interaction of FcRn with domain III shows rapid binding (k _on of about 7e ³ ) and dissociation (k _off of about 4e ⁻² ) kinetics (Table 1). The K _D of FcRn for domain III is 5 μΜ to 8 μΜ approximately 7 times higher (eg, greater dissociation constant) than K _D of full length HSA for FcRn (Table 1). This difference in K _D is mainly due to k _off for domain III, which is relatively faster than HSA, whereas k _on for the two molecules is similar. The K _D derived from k _on and k _off corroborates the kinetic measurement by exactly matching the experimentally obtained values. Moreover, two different sensor chips (CM4 and CM5) with variable carboxy moieties provided similar affinity. Kinetic constants and equilibrium constants are similar between the degreased and non-degreased forms of domain III, suggesting that lipid molecules do not mediate or facilitate the binding of FcRn to domain III.

8.2. Example  2: battlefield HSA  Or domain III Having only The fused product FcRn Human IgG To improve the affinity of the.

This experiment demonstrated that the fusion of domain III of HSA with a therapeutic protein or antibody, or variant thereof, enhances the affinity of the therapeutic protein / antibody for FcRn at acidic pH without affecting the pH dependence of the interaction. . This increase in affinity for FcRn at pH 5.5 will translate into increased serum half-life (eg, improved lifetime in vivo or in an appropriate model system). For example, in a transgenic mouse expressing a single copy of the human FcRn gene (but lacking a murine FcRn), a pharmacokinetic half-life measurement of the HSA moiety comprising domain III fused to a therapeutic protein is performed to determine the wild type or variant domain. The effect on the half-life of the fusion with the moiety comprising III can be assessed.

8.2.1. Construct Design

Human IgG1 was used as a representative protein to compare FcRn affinity between IgG alone and IgG fused to HSA or domain III. HSA or domain III was fused to the C terminus of the human IgG1 heavy chain via a linker comprising four Gly-Gly-Gly-Gly-Ser repeat units (FIG. 2A). The length of the linker was designed based on the distance between the IgG binding site on the FcRn and the HSA binding site to allow the two ligands to bind to their respective binding sites simultaneously. HSA and domain III fusion versions of previously known known high affinity IgG variants (IgG-YTE, Dall'Acqua, et al., 2002) showing a 10-fold improved affinity for FcRn relative to native IgG. , J Immunol., 169: 5171-5180). Thus, the following constructs were prepared and used: IgG; IgG (YTE); IgG- (G ₄ S) ₄ -HSA; IgG- (G ₄ S) ₄ -domain III; IgG (YTE)-(G ₄ S) ₄ -HSA; And IgG (YTE)-(G ₄ S) ₄ -domain III.

8.2.2. Tablet and Characterization

In summary, all six constructs were cloned into expression vectors and proteins were purified by transient expression in 293F cells (GIBCO, catalog number R79007). IgG1 and fusion proteins secreted into the culture medium were purified using GE Healthcare's HighTrap ™ Protein A affinity column (Catalog No. 17-0403-03). Purified proteins were resolved by SDS-PAGE under reducing and non-reducing conditions and observed 99% purity when visualized by Coomassie staining (FIG. 2B).

IgG- (G ₄ S) ₄ -HSA whereas the molecular weight of 284 kilodaltons (KDa) estimation of the fusion _{protein, IgG- (G 4 S) 4} - The estimated molecular weight of domain III is a 196 KDa. The molecular weight observed was very closely correlated with these estimates. To assess whether these fusion proteins form aggregates due to their large size or altered physicochemical properties, the fusion proteins were analyzed by size exclusion chromatography (SEC) using Agilent Technologies 1200 Series SEC (FIG. 2c). IgG- (G ₄ S) ₄ -HSA and IgG- (G ₄ S) ₄ - domain III was both a beam of a single peak corresponding to a monomer A on the curve ₂₈₀ dae retention time (minutes), which any of the above fusion protein to It does not imply aggregation to a measurable degree of. SEC profiles of the fusion proteins for IgG-YTE variants could not be distinguished from IgG- (G ₄ S) ₄ -HSA or IgG- (G ₄ S) ₄ -HSA chimeric proteins.

8.2.3. Equilibrium Coupling Constant (K _D )

The affinity (K _D ) of human FcRn for the fusion proteins was measured on a Biacore T100 instrument (Biacore Inc., Upsala, Sweden). , Using standard amino coupling chemistry, human _{IgG, IgG- (G 4 S)} 4 , as summarized by the device manufacturer hageondae Summary domain _{III, IgG- (G 4 S)} 4 -HSA, IgG- High density fixation of YTE, IgG-YTE- (G ₄ S) ₄ -domain III and IgG-YTE- (G ₄ S) ₄ -HSA on separate flow cells on two Series 5 sensor chips (GE Healthcare) I was. Final surface IgG densities were 5116 RU, 5258 RU, 6097 RU, 5256 RU, 5561 RU and 5531 RU, respectively. A reference flow cell was also prepared on each sensor chip without any protein using the same fixation protocol. Two-fold serial dilutions of human FcRn (5.86 nM to 3000 nM) in 50 mM P0 ₄ and 150 mM NaCl buffer (pH 5.5) were injected onto the protein coupled cell reference and the reference cell surface at a flow rate of 5 μl / min. It was. Binding data was collected for 50 minutes and then regenerated using multiple 60-second injections of phosphate buffered saline at pH 7.4 containing 0.05% Tween 20. Equilibrium binding response (Req) versus concentration for each injection was plotted and the Biacore T100 evaluation software v. Equilibrium binding constant K _D was derived by fitting to a stationary affinity model (FIG. 3) using 1.1 (Biacore Inc., Upsala, Sweden). Inset shows the binding of immobilized ligand with various concentrations of FcRn. K _D for IgG-YTE variants and corresponding fusion proteins were also induced in the same manner (data not shown).

IgG- (G ₄ S) ₄ -HSA the K _D is 183 nM, _{and, IgG- (G 4 S) 4} - domain III of the K _D is the 305 nM compared to 1.15 μM for IgG alone, which is the domain or HSA III And the fusion of and IgG demonstrate FcRn affinity up to 10 and 5 fold, respectively (Table 2). A similar but less pronounced trend is observed for YTE variants, where IgG-YTE- (G ₄ S) ₄ -HSA shows a 3.8-fold improvement (42.5 nM) of affinity compared to IgG-YTE and IgG-YTE -(G ₄ S) ₄ -Domain III shows a 2.5-fold (65.1 nM) affinity improvement relative to IgG-YTE (Table 2).

However, the improvement of affinity at pH 5.5 does not affect FcRn binding at neutral pH. This was tested by injecting 1 μM FcRn on the same fixed surface at pH 7.2. No measurable difference in binding of FcRn with any of the surfaces to which the fusion protein was coupled was detected compared to the IgG / IgG-YTE control (data not shown).

The binding of FcRn with HSA-fusion at acidic pH (about 5.5-6.0) and release at neutral pH (about 7.4) are correlated with in vivo efficacy because these properties mimic in vivo binding. Accordingly, preferred HSA variants are (i) variants with improved affinity relative to native HSA, or conjugates comprising native HSA, and (ii) variants with improved affinity observed at acidic pH. Furthermore, variants with increased binding affinity for FcRn at neutral pH may impair efficacy and reduce the beneficial effect of increased affinity at acidic pH.

8.3. Example  3: battlefield HSA Fusion with human IgG Improves serum persistence.

This experiment demonstrated that the fusion of HSA with the antibody increased the serum half-life of the antibody. As shown in FIG. 7, the serum duration of the IgG-HSA fusions described in Example 1 was increased compared to IgG alone. The increase in serum half-life was similar to the increase observed for IgG-YTE variants. However, the addition of HSA to the IgG-YTE variant did not appear to cause a significant improvement over YTE alone in this study.

PK study using human FcRnC57BL / 6 transgenic mice (JAX laboratories), 4 to 5 months old, in which the mouse neonatal Fc receptor (mFcRn) is replaced with a single copy of human FcRn (huFcRn). Was performed. A 15 mg / kg dose of the appropriate protein diluted in phosphate buffered saline at pH 7.2 was injected into the mice via the tail vein. One hour after infusion, all animals were bled (75 μl) from the post-orbital plexus and serum was collected to determine the actual amount injected into the circulatory system. Serum samples were then collected and stored at −80 ° C. 24 hours, 72 hours, 168 hours and 240 hours after injection. The amount of labeled protein remaining in the serum was analyzed by ELISA. In summary, various IgG fusion constructs were captured using anti-HSA coated plates and detected using anti-kappa detection antibodies. For IgG and YTE constructs, IgG was captured using plates coated with antigen and detected using anti-heavy chain detection antibody. The percentage of protein remaining in the serum was plotted as fraction of time injected amount (1 hour sample).

8.4. Example  4: FcRn For HSA  top The epitope It is a conformational epitope .

When visualized by immunoblotting with anti-β2 microglobulin antibody, it was observed that human FcRn binds well to native HSA in a concentration dependent manner. However, similar results were not observed when using denatured HSAs tested under similar experimental conditions.

Human serum albumin (HSA; Catalog No. A-8763), Sigma-Aldrich's human IgG (hIgG; Catalog No. I-4506) and Tris buffer were CNBr-activated Sepharose 4B (concentrations of 10 mg protein / ml Sepharose). GE Healthcare. Sepharose-Tris was prepared by blocking the reactive groups of CNBr-activated Sepharose 4B with 0.1 M Trizma base and 0.5 M NaCl (pH 8). Sepharose beads (20 μl equivalent to about 180 μg linked protein) linked to HSA, hIgG or Tris were reduced (1% 2-mercaptoethanol) or SDS containing sample buffer (60 mM Tris, pH 6.8, 2.3% SDS, 10% glycerol, 0.01% bromophenol blue) was boiled or left untreated for 10 minutes. The treated protein-coupled or tris-coupled beads contained 0.1% fish gelatin (BIOFX Laboratories Inc, Catalog No. PFGP-1000-01) at pH 5.5. Washed with 50 mM sodium phosphate and 150 mM NaCl buffer and then incubated with 200 μl of various concentrations of human FcRn (0 μg to 20 μg) in pH 5.5 buffer for 2 hours at room temperature. Unbound protein was removed by washing with pH 5.5 buffer. The bound protein was eluted by boiling with SDS containing sample buffer containing 1% 2-mercaptoethanol and analyzed on an SDS polyacrylamide gel, followed by anti-β2 microglobulin antibody (Abcam, catalog number Ab6608). ) Was immunoblotted.

The binding of human FcRn with Sepharose-HSA was maximal for native HSA over the entire FcRn concentration range. However, when HSA was denatured under reducing and non-reducing conditions, the binding was drastically reduced, suggesting that the epitope on the HSA to FcRn is very likely to be a conformational epitope (FIG. 4). As expected, Sepharose-IgG bound to human FcRn, but tris-blocked beads did not bind to FcRn under any conditions.

8.5. HSA and domain III can be displayed on the surface of yeast cells, and the displayed protein retains FcRn binding performance.

This example demonstrates that both HSA and domain III can be successfully expressed on the surface of yeast cells as assessed by altered flow cytometry performed at acidic pH and that these displayed proteins bind FcRn in a pH dependent manner. Thus, expression on yeast cells can be screened for constructs that contain alterations in domain III (eg, domain III alone, full length HSA, truncated HSA, or at least chimeric polypeptide comprising domain III), thereby (i) FcRn And (ii) assessing the ability of such constructs to bind FcRn, for example, with increased affinity relative to non-variant constructs.

8.5.1. Yeast cell surface display

HSA, domain III or single-chain Fv fragment (scfv) was cloned into pYD1 yeast display vector (Invitrogen, catalog number V835-01) and S. cerevisiae ( S. cerevisiae ) for presentation on yeast cell surface. Transformed into. pYD1 displays a protein of interest as a C-terminal fusion with Saccharomyces cerevisiae protein Aga2p under the control of a galactose inducible promoter. All experimental procedures were performed as described in the supplier's manual. Transformed cells were selected using the ureotropic selection markers uracil and tryptophan and incubated in an appropriate selection medium (Technova Inc., Catalog No. C8140). Cultures were induced with galactose for up to 48 hours to express Aga2p fusion proteins. Cells were sampled at 0 hours, 24 hours and 48 hours. Cell samples were washed, blocked with PBS at pH 7.2 containing 0.1% fish gelatin, stained with FITC-conjugated rabbit polyclonal anti-HSA antibody (Abcam Inc., Catalog No. AB34669) and by flow cytometry Analyzed.

Although no cell surface expression of HSA or domain III was observed at 0 hours, expression of the two proteins on yeast cells was observed at 24 hours. This expression was maintained 48 hours after induction. Expression was visualized by positive FITC staining. 5A shows that both HSA and domain III can be successfully expressed on the surface of yeast cells. Cells transformed with scfv were not stained with anti-HSA as expected (eg negative). Thus, cells transformed with scfv served as negative controls.

8.5.2. On the surface  Expressed HSA  Or domain III of FcRn  Combined performance

Yeast cells expressing HSA, domain III or scfv and induced with galactose for 48 hours were treated with 50 mM sodium phosphate and 150 mM NaCl buffer (also referred to as FACS buffer) at pH 5.5 containing 0.1% fish gelatin. Blocked. The cells were then incubated with biotinylated FcRn (70 μM) in phosphate buffer at pH 5.5 and bound FcRn was replaced with streptavidin phycoerythrin (PE) (Invitrogen Incorporated). Visualized. The cells thus stained were analyzed by flow cytometry using 50 mM sodium phosphate and 150 mM NaCl buffer at pH 5.5 instead of commercially available PBS.

As can be seen from the displacement in the histogram compared to the displacement for scfv, cells expressing HSA and domain III stained positive for PE, but negative control scfv expressing cells were not stained (FIG. 5B), which It is demonstrated that HSA and domain III expressed on the yeast cell surface have functionality since they retain FcRn binding performance. In a separate experiment, the cells were treated similarly to low pH flow cytometry for FcRn binding and analyzed using the HyperCyt® system (IntelliCyt Corporation), a high efficiency sampling technique, to obtain similar results. It was.

8.6. Example  6: to generate a library with high diversity OriP Containing Adenovirus  Mammalian cell surface display vector

Mammalian surface display libraries using glycosylphosphatidylinositol (GPI) -anchors for surface display of scFv-Fc proteins were constructed in a Gateway® transduction vector engineered to contain an scFv-Fc expression cassette designated as pENDisplay ( See FIG. 8A). Libraries of different scFv sequences were easily inserted into the Sfi / NotI site. The library in the pENDisplay vector was combined with the pAd / PL-DEST ™ vector (Invitrogen, catalog number V494-20) according to the manufacturer's instructions to generate an adenovirus expression library and a total of approximately 5 × 10 ⁶ colony forming units (cfu) were generated. Obtained. To generate adenoviruses, 293A cells (Invitrogen, Catalog No. R70507) containing stably inserted copies of the E1 gene that feed trans to the E1 proteins (E1a and E1b) necessary to generate the recombinant adenovirus Were transfected with adenovirus expression libraries linearized to expose left and right inverted terminal repeats (ITRs). FACS analysis confirmed that at least 50% of 293A cells of 293A cells directly transfected with the linearized adenovirus library display antibodies on their surface. However, less than 50 plaques per 110 mm (diameter) dish were obtained when the linearized adenovirus library was transfected into 293A cells for production of adenovirus. Adenovirus was harvested on day 10, viral DNA was isolated, PCR was used to amplify the scFv coding region to be recloned back into the pENDisplay vector, and 96 colonies were picked and sequenced. Only 14 unique VH sequences of the 96 clones analyzed were identified. The low efficiency of plaque recovery can be due to the degradation of the linearized adenovirus vector and results in a significant reduction in the complexity of the adenovirus library.

Epstein-Barr nuclear antigen 1 (EBNA-1) contains a nuclear localization signal (NLS) and binds to an OriP containing nucleic acid such as a plasmid. EBNA-1 protein (see FIG. 9A) may contribute to translocation of OriP containing nucleic acids to the nucleus via NLS and may improve episome maintenance. Although retained episomes are not considered necessary for the adenovirus structure, the OriP sequence (see FIG. 9C) was introduced after the polyA sequence of the scFv-Fc cassette between the attL1 and attL2 sequences of the pENDisplay vector. The new vector named pENDisplay-OriP is shown in FIG. 8B. The library in pENDisplay-OriP was combined with the pAd / PL-DEST ™ vector (Invitrogen, catalog number V494-20) vector to generate a second adenovirus expression library with about 5 × 10 ⁶ cfu. The library generated from the pENDisplay-OriP vector was linearized and transfected into 293E cells (Invitrogen, Catalog No. R620-07) stably expressing Epstein-Barr virus nuclear antigen (EBNA-1) and adenovirus E1a protein. More than 10,000 plaques per 110 mm (diameter) dish. The virus was harvested on day 7 and 96 clones were analyzed as described above. In contrast to the small number of unique clones isolated from the first library, which did not have an OriP site, all 96 VH sequences analyzed were unique. These results demonstrate that the addition of the OriP sequence to the adenovirus expression library vector greatly improved both the structure of the adenovirus from the cells expressing EBNA-1 and the diversity of the adenovirus library.

The addition of an OriP sequence (eg, FIG. 9C) to the adenovirus vector enhances the efficiency with which recombinant adenovirus particles are generated from the host cell in the presence of EBNA-1 protein. When building adenovirus expression libraries, the improved efficiency of virus development maintains the diversity / complexity of the library by reducing the number of clones lost. Example 7 below details the construction and screening of a mammalian surface display library expressing an HSA domain III variant which introduces the OriP sequence into the adenovirus expression vector essentially as described above.

10 provides a schematic of representative general adenovirus expression vectors for expression of the protein (s) of interest. In this example, a mutant adenovirus genome is provided in which the E1 and / or E3 portions are deleted. Missing viral genes are provided in trans by the host cell used for viral rescue. The deletion interferes with the replication of the adenovirus in the host cell used for expression of the protein (s) of interest. The DNA of interest contains all components necessary for expression of the protein (s), including but not limited to coding sequence (s), promoter sequence (s), termination signal (s), polyA sequence (s), and the like. Will include. The protein (s) of interest may be a soluble protein or may comprise a sequence, such as a transmembrane domain or a GPI-anchor signal, to adhere the protein (s) to the cell surface. As exemplified herein, adenovirus vectors can be engineered to express a library of variant proteins. The adenovirus expression vector shown in FIG. 10 provides the location of the att recombination site that will be generated by the Gateway ™ introduction and use of the desired plasmid for construction. One possible position at which the EBNA-1 DNA sequence can be located is also indicated. Other positions and orientations of the vector components are contemplated. Those skilled in the art will appreciate that the orientation and / or relative position of the vector region may vary.

8.7. Example 7: Use of Other Display Platforms for HSA and Domain III

Phage display and mammalian cell surface display techniques were also evaluated as potential display platforms for HSA and domain III. Phage display platform presumably failed to express HSA and domain III on the surface of bacterial cells because of the abundant disulfide bonds in these molecules (data not shown). However, mammals using transient surface expression in 293F cells mediated via glycosylphosphatidylinositol (GPI) -anchor signals from decay accelerating factors (e.g., mutated DAFs described in US Patent Publication No. 2007/0111260). Animal cell display systems have been successful in displaying HSA and domain III. The displayed protein retained FcRn binding performance (data not shown).

An additional epitope tag (e.g. a Flag tag) is added for double staining and a linker is added to both 5 'and 3' of the HSA to increase the flexibility of the fusion protein and to facilitate binding of the HSA and FcRn. A mammalian expression construct (named pEN-HSA-GPI) was generated (FIG. 11A), and this construct was used directly for transient transfection, or an adenovirus expression vector (pAd-HSA-GPI) that also introduced the OriP sequence. Was designated as). Functional HSA was expressed on the surface of mammalian cells (eg, 293F cells) from this construct in both transient transfection assays (data not shown) and use of adenovirus expression systems. FIG. 11 shows displacements generated in histograms of cells stained with anti-HSA (FIG. 11B) or FcRn at 25 μg / ml and 5 μg / ml (FIGS. 11C and 11D, respectively). Thus, the ability to express HSA and domain III variants and to screen these variants for (i) binding to FcRn and (ii) binding to FcRn, for example, with increased affinity relative to non-variant constructs. There are potential systems.

On the cell surface Transfection of 293F Cells to Display HSA : 1.5 μg of plasmid and 2.25 μl of 293 pectin were added to 100 μl Omtimem medium (Invitrogen) in a separate tube and incubated at room temperature for 5 minutes. After treatment, the two components were combined together. After incubation for an additional 20 minutes at room temperature, the mixture was added to 2 ml 293F cells present at a density of 1 × 10 ⁶ cells / ml in 24 deep well plates. Transfected cells were grown for 24 hours at 250 rpm in the presence of 8% CO ₂ .

Generation and Use of Adenovirus Expression Vectors : Adenovirus expression vectors were generated by recombining HSA in the introduction vector (pEN-HSA-GPI) with an invitrogen target vector using Gateway® technology (Invitrogen). In summary, a total of 10 μl of reaction mixture was added with 150 ng of pEN-HSA-GPI vector, 300 ng of pAd / PL-DEST (Invitrogen), 2 μl of LR Clonase II (Invitrogen) and TE buffer. Was added. After incubation overnight at 25 ° C., 2 μl of the reaction mixture was used according to the manufacturer's protocol to transform One-shot TOP 10 transfecting cells (Invitrogen). Transformed TOP 10 cells were plated on ampicillin plates and incubated overnight at 37 ° C. Single colonies were imprinted into LB medium to make plasmids. HSA gene containing adenovirus expression vectors were linearized with PacI prior to transfection to generate adenoviruses. Adenoviruses were generated by transfecting 293E cells using 2 μg linearized adenovirus vector and 6 μl lipopecattine-2000. Seven days after transfection, adenovirus was released from the transfected cells by performing two or three alternations of freezing (at -80 ° C) and thawing (at 37 ° C). Cell debris was removed by centrifugation at 3000 rpm for 10 minutes, and the adenovirus containing supernatant was aliquoted into a new tube and stored at -80 ° C. Virus titers were measured using the Adeno-XTM Rapid Titer Kit (Clontech: PT3651-2) according to the manufacturer's instructions. The adenovirus was used at MOI of 1 for expression of HSA constructs.

8.8. Example 8: Alanine Scanning Mutagenesis of Surface Exposed Loops on DIII to Identify FcRn Binding Epitopes

This example illustrates the role of surface exposed loops on domain III in mediating the binding of FcRn to HSA.

Domain III consists of 205 amino acid residues and encodes 10 helices linked through 9 loops and is stabilized through 6 disulfide bonds (Sugio et al. 1999, Protein Eng. 12: 439-46 and PDB: 1BM0 ). The positions of the amino acid residues that make up these loops and the length of each loop are listed below. Amino acid numbering is based on the location of these loops in full length mature HSA protein (SEQ ID NO: 2).

Loop 1: residues 398 to 400 = 3 amino acids

Loop 2: residues 415 to 419 = 5 amino acids

Loop 3: residues 439 to 443 = 5 amino acids

Loop 4: residues 468-470 = 3 amino acids

Loop 5: residues 480-482 = 3 amino acids

Loop 6: residues 492-509 = 18 amino acids

Loop 7: residues 516-517 = 2 amino acids

Loop 8: residues 537 to 541 = 5 amino acids

Loop 9: residues 561 to 564 = 4 amino acids

Loops numbered 2, 3, 6, 8 and 9 are solvent accessible and exposed on the surface of the molecule (Sugio et al. Ibid, and PDB: 1BM0).

In some examples, the amino acids of each individual surface accessible loop were alternately mutated to alanine (except for proline and cysteine, which are not mutated), where odd numbered residues were mutated in one set and numbered evenly Residues were mutated in another set. Two such mutant sets were generated per loop except for loop 9, which required only one construct to fit the experimental design. A total of nine such constructs were generated in vector for cell surface display (eg, pYD1 yeast display vector) and evaluated for FcRn binding performance. Variants can be evaluated using standard in vitro assays (eg, flow cytometry) described herein. Variant (s) were identified which showed improved affinity for FcRn. In addition, each variant could be screened to ensure that improved affinity for FcRn occurs only at acidic pH and not at neutral pH. (i) variant domain III constructs alone; (ii) variant domain III constructs provided in the state of full-length HSA; Or (iii) the variant domain III construct provided in the state of truncated HSA, or at least a chimeric polypeptide comprising domain III, exhibits an improved affinity for FcRn at acidic pH and no such affinity at neutral pH. Could try. The constructs were compared with wild type domain III, wild type full length HSA, or chimeric polypeptide without mutation.

In some examples, the information obtained from the screens identified residues in surface accessible loops that could easily be changed while maintaining (or even improving) FcRn binding performance. A series of variants in which these identified positions were mutated to each of the other 20 amino acids was constructed and these variants were also screened. Additional variants comprising mutations at more than one position were later constructed and screened.

In some examples, libraries of variants were generated and evaluated.

8.9. Example 9: Alanine Scanning Mutagenesis of Conserved and Surface Exposed Residues

Surface accessible amino acid residues conserved in domain III between 13 different animal species were identified by amino acid sequence alignment. Mutating these conserved residues alone with alanine confirmed their role in FcRn binding.

The domain III amino acid sequence of HSA was compared with serum albumin domain III sequences from 12 different species, including rats, mice, cattle, dogs, rabbits, pigs, chickens, donkeys, sand mice, sheep, cats and horses. The residues conserved between all these species were identified (FIGS. 6A-6D). Because chicken HSA is different from mammalian HSA protein, a second alignment of only mammalian species is provided (FIGS. 6E-6H). Serum albumin from pigs, rats, mice, dogs, sheep, rabbits and cows has already been shown to bind to human FcRn by ELISA, immunoblotting and SPR (data not shown). In a separate analysis, surface exposed residues in domain III were identified using GETAREA 1.0 beta software available on the Internet (http://curie.utmb.edu/getarea.html.). The software calculates the accessible surface area of individual atoms and their gradients and scores each amino acid residue against the probability of surface accessibility expressed as "i" or "o", indicating inaccessible and accessible, respectively. (Table 3). The software calculated and confirmed by manual inspection of the HSA crystal structure identified the amino acids that are conserved and surface exposed between all of these different species (marked in boxes in Table 3).

All 18 amino acid residues thus identified were mutated alone with alanine and the effect on FcRn binding was assessed using cell surface display (eg, pYD1 yeast display system). Domain III mutations were introduced and screened into one or more states of domain III alone, full length HSA protein, truncated HSA, or at least chimeric polypeptide comprising domain III. Conserved and surface exposed cysteine and proline were not included in this analysis.

In another embodiment, all 18 amino acid residues (or fewer than 18 if the alanine experiment indicates a specific position that cannot accept a substitution) are mutated alone to each of the other 19 amino acid residues The effect on FcRn binding was assessed using the pYD1 yeast display system (or another display system).

In another example, variants comprising a combination of mutations were constructed and evaluated. Variants can be evaluated using standard in vitro assays (eg, flow cytometry) described herein. Variant (s) were identified which showed improved affinity for FcRn. In addition, each variant could be screened to confirm that improved affinity for FcRn occurs only at acidic pH and not at neutral pH. (i) variant domain III constructs alone; (ii) variant domain III constructs provided in the state of full-length HSA; And (iii) at least one of the variant domain III constructs provided in the form of truncated HSA, or at least a chimeric polypeptide comprising domain III, exhibits improved affinity for FcRn at acidic pH and such improved affinity at neutral pH. Was tested. The constructs were compared with wild type domain III, wild type full length HSA, or chimeric polypeptide without mutation.

[Table 3]

8.10. Example 10 Domain to All Possible Amino Acids for Generating a Library of Single Amino Acid Mutants III  top Residue  Each mutagenesis

Library of mutants by mutating all amino acids in domain III except cysteine and proline to all 20 amino acids (ie wild type amino acids and all 19 non-wild type amino acids) so that each individual mutant has a single mutation in only one position Generated. Since the entire length of 205 amino acids was covered in the library construction, 184 residues were individually mutated to generate a total library diversity of 3496. Domain III mutations were introduced and screened into one or more states of domain III alone, full length HSA protein, truncated HSA, and chimeric protein comprising at least domain III. Standard mutagenesis methods can be used to generate libraries of domain III mutants. Optionally or alternatively, libraries of domain III mutants are prepared by commercial facilities, such as Gene Art AG (Germany). Cloning the library of mutants into a display vector comprising an OriP sequence for improved development of recombinant adenovirus, such as the pYD1 yeast display vector or mammalian display vector pEN-HSA-GPI described above (generic introduction comprising OrIP) For a schematic of the vector, see FIG. 10), FcRn binding performance was screened using the standard in vitro assays described herein (eg, flow cytometry). Variant (s) were identified which showed improved affinity for FcRn. In addition, each variant could be screened to confirm that improved affinity for FcRn occurs only at acidic pH and not at neutral pH. (i) variant domain III constructs alone; (ii) variant domain III constructs provided in the state of full-length HSA; Or (iii) variant domain III constructs provided in the form of chimeric polypeptides were tested for improved affinity for FcRn at acidic pH and no improved affinity at neutral pH. The constructs were compared with wild type domain III, wild type full length HSA, or chimeric polypeptide without mutation. The experimental design enabled the identification of binding epitopes with mutation (s) that improved affinity for FcRn.

Synthetic domain III (DIII) libraries with ⁶ × 10e ⁴ independent transformants were generated as described above. The library was designed such that each individual mutant had a single mutation only in one position, but multiple double mutations or even triple mutations occurred. The synthetic DIII library was amplified by PCR and assembled with DI and DII by overlapping PCR to form a full length HSA library. PCR products were cleaved with SfiI and EcoRI and similarly cut into the vector pEN-HSA-GPI (enhanced mammalian display gateway ™ transduction vector comprising OriP sequence as described above) (see, eg, FIGS. 8B and 10). Cloned. The primers used to amplify the DIII library had six (6) wild-type amino acid residues at the N and C termini, thus eliminating the diversity in these 12 amino acids in domain III. Two libraries were generated in the pEN-HSA-GPI vector and the corresponding adenovirus library was essentially as described above (except that 12 μg of PAC I linearized adenovirus expression vector was used for library generation). Generated. The size of each library is listed in Table 4. The diversity of the pEN-HSA-GPI library was examined, with about 50% of the clones in library 1 wild type, while about 30% of the clones in library 2 wild type.

Cells infected with adenovirus expressing wild type HSA, HSA-DIII library 1 or HSA-DIII library 2 were essentially detected with anti-HSA-FITC antibody and FcRn-biotin (SA-PE) as described in Example 5 above. ) And analyzed / screened by FACS as described below. Expression levels of wild type HSA and the two libraries were similar (Panel A in FIG. 12). Only cell populations expressing the HSA-DIII library showed displacement in the histogram when stained with 10 μg / ml of FcRn (Panel B of FIG. 12). 13 shows the corresponding double staining FACS profile. The enriched cell population was recovered from the sorting, amplified and concentrated to the secondary sorting or used to isolate individual clones as described below. As can be seen from the histogram of FIG. 14A, expression levels of wild type HSA, starting library and sorted library were similar. However, staining with FcRn results in cells expressing HSA-DIII mutants capable of binding FcRn in which primary and secondary sorted libraries are present at low concentrations (1 μg / ml and even 0.1 μg / ml). It was concentrated against (Figs. 14B and 14C).

Multiple individual clones were isolated (as described below) and the individual clones were screened for pH dependent FcRn binding essentially as described in Example 2 above except that the experiment was performed at pH 7.2. FIG. 15 shows representative histograms for control cells, wild type HSA and several representative clones at pH 5.5 (Panel A) and pH 7.2 (Panel B). The expression level between these HSA mutants and wild type HSA was similar (Panel C of FIG. 15). As shown in FIG. 16, 16 clones identified as clones with pH dependent binding (ie, predominantly binding at low pH) were sequenced, and various FcRn concentrations (eg, with wild type HSA and control cells) The relative affinity of the mutations to FcRn was analyzed by further analysis with FACS using 0.1 μg / ml, 1 μg / ml and 10 μg / ml of FcRn-biotin. An additional about 1100 clones from the enriched population were sequenced before any other characterization. Table 5 provides a summary of amino acid substitutions isolated from the libraries and / or identified in the sequenced clones. The position indicated in bold font indicates that a substitution was found at that position between about 1% to 5% of the clones and may be referred to as a "preferred spot". The position shown in bold and underlined indicates that a substitution was found at that position in more than 5% of the clones and may be referred to as a "hot spot." Amino acid substitutions indicated in italics (“substituted AA” columns) were identified only in the state of double / triple mutations. Amino acid substitutions (“substituted AA” columns) found in five or more clones are shown in bold typeface. Combinations found in three or more clones are also indicated in bold typeface with the number of identified clones displayed within the landscape. Amino acid numbering is based on the mature full length HSA provided in SEQ ID NO: 2. Multiple clones containing the same amino acid substitutions at the same position and / or different amino acid substitutions at the same position were isolated (see Table 5), suggesting that these positions may represent mutant hotspots.

The locations of various preferred spots and / or hot spots on the resolved structure of the HSA are shown in FIG. 17. The majority of hotspots and preferred spots except amino acids 407, 415, and 463 are loops 6 and 7 (including residues 492 to 509 and 516 to 518, respectively) and helixes 7 and 8, respectively, indicated by circles in FIG. 510-515 and 519-536).

[Table 5]

Several mutants thus identified were generated as soluble proteins by site-directed mutagenesis and purified for further analysis as described below. Binding affinity (K _D at pH 5.5) and pH dependence (7.2 vs 5.5) were measured with ProteOn (as described below) and / or Biacore (essentially as described above). Table 6 provides a summary of binding studies and includes protein densities for these studies. L407Y / Q526T shown in bold in Table 6; L463N / T508R; I523G; V424Q; L83N / 128R / I143G; And a number of mutants, including K144, have improved binding at pH 5.5. It was also found that all of the variants tested that showed improved binding at pH 5.5 retained pH dependent binding (see the last column of Table 6). Several mutants analyzed were found to have unchanged or even increased K _D (ie, unchanged or worsened binding) at pH 5.5. There are several possible reasons why these clones could be identified, for example, these mutations can improve protein expression or stability in the display system. The GPI anchor used in the display may have exerted some effects on binding that could be complemented by these mutants, but it is also possible that the effects were not duplicated when mutant HSAs were expressed as soluble molecules. Furthermore, the design of screening methods optimized to capture dissociation rates stabilized the mutants, which may or may not be interpreted as an improvement in overall affinity. This may reflect that these mutations provide an improvement when combined with one or more other mutations. Multiple mutations were found in combination (see Table 5).

Cell staining and FACS analysis and sorting : 30 million 293F cells at a density of 1 × 10 ⁶ cells / ml were infected with the HSA adenovirus library at MOI = 1. After 16 hours from transduction, the cells were harvested by centrifugation, washed with cold FACS buffer and resuspended to a density of about 1 × 10 ⁷ cells / ml. 10 μg / ml (212 nM) biotinylated FcRn was added for the primary sorting. After incubation at 4 ° C. for 60 minutes, the cells were washed twice with FACS buffer and resuspended in streptavidin-PE at a 1: 500 dilution. After incubation for 30 min at 4 ° C., cells were washed once with FACS buffer and sorted for FcRn binding to concentrate cells binding to FcRn at pH 5.5. Sorted cells were amplified for further screening. For secondary sorting, the enriched cell population was sorted essentially as described above (except that 1 μg / ml (21.2 nM) of biotinylated FcRn was used) to further add high affinity HSA mutants. Concentrated. Further analysis of the concentrated library was also performed at 0.1 μg / ml (2.12 nM) (see, eg, FIG. 14). In some screens, a “deselection” step was introduced into the primary or secondary screening that sorted the enriched cell population to remove cells that bind FcRn at neutral pH (pH 7.4). To identify individual clones, viral DNA was extracted from the enriched cell population and the DIII HSA variant was cloned into a mammalian expression vector for transient transfection of 293F cells. Individual clones were screened for pH dependent binding by flow cytometry at pH 5.5 and pH 7.4 essentially as described above.

Development and Expression of HSA-DIII Mutants : Using a Standard Protocol (QuikChange® II XL Site-Specified Mutagenesis Kit, Agilent Catalog No. 200521) in mammalian expression vectors using specific mutagenic primers Wild type HSA was mutated to generate several DIII mutants. Mutants were expressed in 293F cells with transient transfection using a 293fectin ™ transfection reagent (Invitrogen, catalog number Sku12347-019) according to the manufacturer's protocol, and the mutants were expressed using anti-FLAG M2 using standard procedures. Purification was carried out on an affinity gel (Sigma-Aldrich Catalog No. A2220). All purified mutants were thus analyzed by SDS-PAGE and size exclusion chromatography to assess purity and aggregation. All mutants were judged to have about 99% purity without significant aggregation (greater than 95% monomer) (data not shown).

Proteon Assay : The affinity (K _D ) of human FcRn for HSA variants was measured on the Proteon XPR36 Protein Interaction Array System (Biorad). In summary, the proteon amine coupling kit was used as outlined by the manufacturer to fix the HSA variants at high density on a separate flow surface on the proteon GLM sensor chip. Final surface HSA density was 3740 RU to 3950 RU. A reference flow surface was also prepared on this chip without any protein using the same fixation protocol. Two-fold serial dilutions of human FcRn (5.86 nM to 10000 nM) in 50 mM P0 ₄ and 150 mM NaCl buffer (pH 5.5) were applied on both the protein-coupled surface and the reference cell surface at a flow rate of 25 μl / min. Injected. Binding data was collected for 8 minutes and then regenerated by multiple 60-second injections of phosphate buffered saline at pH 7.4 containing 0.05% Tween 20. Equilibrium binding response (Req) versus concentration for each injection was plotted and equilibrium binding constant K _D was derived by fitting to a stationary affinity model using the Proteon Manager software.

Based on this analysis, further analysis of the identified combination mutations was performed to assess the activity of each individual mutation. In addition, additional screening was performed to confirm that different combinations of mutations (eg, constructs with mutations at more than one position not directly identified on the screen) provide improved affinity for FcRn.

8.11. Example 11: Combination Mutagenesis of Selected Residues on Domain III

To examine the combination of the most frequent mutations identified in Example 10 (above), residues 407, 415, 463, 495, 508, 509, 511, 512, such that each individual mutant has two to four mutations Synthetic domain III libraries were generated to generate libraries of mutants by mutating 515, 516, 517, 521, 523, 524, 526, 527 and 557 to the residues indicated in Table 7. Alternatively, each listed residue may be mutated to all 20 amino acids (ie, wild type amino acids and all 19 non-wild type amino acids) to generate the library to examine a broader set of combinations.

Domain III combinatorial mutations were introduced and screened in the state of one or more of domain III alone, full length HSA protein, truncated HSA, and chimeric protein comprising at least domain III. Standard mutagenesis methods can be used to generate libraries of domain III mutants. Optionally or alternatively, libraries of domain III mutants are prepared by commercial facilities, such as Gene Art AG (Germany).

Libraries of combinatorial mutants were cloned into display vectors, such as the above-described pYD1 yeast display vector or mammalian display vector pEN-HSA-GPI, using standard in vitro assays (eg, flow cytometry) described herein. Screened for FcRn binding performance. Positive and / or negative screening methods such as those described in Example 10 could be used. Combination variant (s) were identified which showed improved affinity for FcRn. In addition, by screening each combination variant, it was possible to confirm that improved affinity for FcRn occurred only at acidic pH and not at neutral pH. (i) variant domain III constructs alone; (ii) variant domain III constructs provided in the state of full-length HSA; Or (iii) variant domain III constructs provided in the form of chimeric polypeptides were tested for improved affinity for FcRn at acidic pH and no improved affinity at neutral pH. The constructs could be compared to wild type domain III, wild type full length HSA, or chimeric polypeptide without mutation. Alternatively or optionally, combinatorial mutations can be compared to domain III, full-length HSA or chimeric polypeptides comprising each mutation alone to determine whether the combination further enhances affinity and / or serum half-life for FcRn. . The experimental design enabled the identification of combinatorial mutations that improved affinity for FcRn and / or improved serum half-life.

8.12. Example 12 Mutagenesis of residues on domain III to generate single amino acid mutants to be screened for improved FcRn affinity

Eighteen single amino acids were selected from conserved amino acids in domain III and mutated alone with alanine using the standard methods described herein so that each variant has a single mutation only in one position. Eighteen variants were introduced and screened in the state of full-length HSA protein, or alternatively truncated HSA or chimeric protein comprising at least domain III. Eighteen variants were screened for FcRn binding performance using the standard in vitro assays described herein. Variant (s) were identified which showed improved affinity for FcRn. In addition, the screening of each variant confirmed that improved affinity for FcRn occurred only at acidic pH and not at neutral pH. (i) variant domain III constructs alone; (ii) variant domain III constructs provided in the state of full-length HSA; Or (iii) variant domain III constructs provided in the form of chimeric polypeptides were tested for improved affinity for FcRn at acidic pH and no improved affinity at neutral pH. The constructs were compared with wild type domain III, wild type full length HSA, or chimeric polypeptide without mutation.

Based on this analysis, additional screening was performed to confirm that combinations of mutations (eg, constructs with mutations in more than one position) provide improved affinity for FcRn.

8.13. Add " Hot spot " Mutant  And combination Mutant

Positions 463, 508, 523, and 524 were mutated separately to all 19 non-wild type amino acids, and a set of 76 individual point mutations were essentially as described above (see paragraph "Generation and Expression of HSA-DIII Mutants" above). ) Cloned and expressed. Biacore analysis was performed using anti-HSA (non-domain III binder) coupled to the chip to capture HSA variants from the cell supernatant. Human FcRn was then passed on the chip to enable binding at pH 5.5 and binding and dissociation were measured at pH 5.5. In this format, the dissociation constant k _d (1 / s) will be more accurate than the coupling constant. Binding constants for single mutants with dissociation rates that are at least 2.5 times slower than wild type HSA are provided in Table 8. As can be seen from Table 8, many of the substitutions present at these positions resulted in slower dissociation rates and improved binding to FcRn compared to wild type HSA.

A set of double, triple and quadruple combinatorial HSA mutations was generated with each of the "hotspot" mutations identified in Example 10 (above). "Hotspot" combinatorial mutants were cloned and expressed and equilibrium binding constants and pH dependent FcRn binding (human and cynomolgus monkeys) were essentially as described above (paragraph "Generation and Expression of HSA-DIII Mutants" above). And “kinetic measurements”). The pH dependence of FcRn binding was calculated by injecting 3 μM FcRn at pH 5.5 and pH 7.4 at a flow rate of 5 μl / min and inducing the ratio of equilibrium response at pH 7.4 to equilibrium response at pH 5.5. As summarized in Table 9, each "hotspot" combinatorial mutation showed a two to three or more improvement in binding affinity, with quadruple mutations showing the greatest improvement (20 to 75 times). In addition, some of the mutants maintained nearly wild-type levels of pH dependent FcRn binding (L463N / T128R; L463N / K524L; T508R / I523G; and L463N / T508R / I523G). The SPR-induced kinetics constants and equilibrium constants of the various “hotspot” combination HSA variants are provided in Table 10. In addition, the SPR-induced equilibrium constants and pH dependencies of the various “hotspot” combination variants for cynomolgus monkey FcRn (Table 10) are similar to the SPR-induced equilibrium constants and pH dependence observed for human FcRn. It was confirmed (Table 9).

8.14. Half-life measurement

The only mouse model currently available to demonstrate the concept that improved FcRn binding HSA mutants will have an extended half-life is huFcRn tgn mice (homozygous KO for mouse genes and heterozygous KI for human genes) (Petkova SB et al., (2006) International Immunology, Vol. 18, No. 12, pp. 1759-1769. This model has an important limitation in terms of measuring the half-life of HSA (very high circulating concentration of mouse serum albumin (600 μM) that binds human FcRn with 10-fold higher affinity than wild type HSA. However, this model could be used if the HSA variant to be tested could successfully compete with murine serum albumin (MSA).

In vitro Biacore competition experiments were designed to predict whether mutants showing improved affinity could successfully compete with murine serum albumin (MSA) for huFcRn binding. This experiment was performed by testing the ability of MSA to compete with HSA variants for FcRn binding by premixing 1 μM FcRn with MSA at various concentrations (41 nM to 100 μM) and injecting on mutant HSA surfaces. IC ₅₀ was calculated by fitting the data to a single class competitive binding site model.

Three mutants L463N / K524L, T508R / I523G and L463N / T508R / I523G were tested first, each of which showed improved affinity for FcRn and maintained pH dependence (see Table 8). T508R / I523G and L463N / K524L were found to have IC ₅₀ values 10 to 15 times higher than wild type HSA or MSA (Table 11). These IC ₅₀ values are consistent with dissociation rate measurements, suggesting that T508R / I523G and L463N / K524L form a more stable complex with FcRn.

The half-life of each of these three mutants was then measured in vivo in huFcRn tgn mice (see PK analysis below). As shown in Table 11, in this model system mutant L463N / K524L shows a 5 hour improvement in half-life and mutant T508R / I523G shows a 3 hour improvement. However, the mutant L463N / T508R / I523G was eliminated faster than the wild type in this model. Faster removal of the L463N / T508R / I523G may be an artificial phenomenon due to the limitations of the mouse model as described above. Alternatively, the mutations may affect the stability of the protein in vivo or may affect the biodistribution of the protein. Half-life values for L463N / K524L and T508R / I523G were consistent with the dissociation rate profile of these mutants. In addition, these data were consistent with IC ₅₀ measurements obtained using competition experiments using MSA.

[Table 11]

PK analysis : Wild type HSA (Wt HSA) bearing N terminal His10 tag, or L463N / T508R / I523G, T508R / I523G or L463N / K524L variants were generated. All proteins were expressed in 293F cells as described above and purified using a HisTrap HP column from GE Healthcare (Cat. No. 17-5248-02) according to the manufacturer's instructions. 15 mg / kg of Wt HSA, or L463N / T508R / I523G, T508R / I523G, or L463N / K524L variants were intravenously irrigated through the tail vein into huFcRn tgn mice, 1 hour using 5 mice / times / group, Serum samples were collected at 16 hours, 24 hours, 48 hours, 72 hours, 96 hours and 120 hours. Serum samples collected from the mice were analyzed by ELISA. In summary, ELISA plates were coated with anti-His mAb (in-house), then incubated with appropriate dilutions of serum samples, and the amount of HSA / variant remaining in the serum was determined by rabbit anti-HSA polyclonal antibody ( Abcam, catalog number AB60191) and donkey anti-rabbit-IgG HRP (Jackson Immunoresearch, catalog number 711-035-152). All animal studies were approved by the Institutional Review Board.

Noncompartmental analyzes were performed to determine pharmacokinetic parameters from the mean serum protein concentration-time profile using WinNonlin Professional (v5.2 Pharsight Corp.). Algebraic trapezoids using an area exceeding C _{p , last} , estimated from the _last measured value (C _{p, last} ) / λ _z , where λ _z is the slope of the line fitted by the least squares to the end of elimination ( The area under the curve (AUC) was calculated using the logarithmic trapezoidal method. The clearance (CL) value for the protein was calculated from CL = dose / AUC, with the dose being irrelevant cpm. Half-life (t _1/2 ) was calculated from ln 2 / λ _z .

9. Sequence

6 provides an alignment of domain III of serum albumin proteins from various species.

The wild type amino acid sequence for domain III of rat serum albumin is described as SEQ ID NO: 3 in FIG. 6. The wild type amino acid sequence for domain III of mouse serum albumin is described as SEQ ID NO: 4 in FIG. The wild type amino acid sequence for domain III of bovine serum albumin is described as SEQ ID NO: 5 in FIG. 6. The wild type amino acid sequence for domain III of human serum albumin is described as SEQ ID NO: 2 in FIG. 6. The wild type amino acid sequence for domain III of dog serum albumin is described as SEQ ID NO: 6 in FIG. The wild type amino acid sequence for domain III of rabbit serum albumin is described as SEQ ID NO: 7 in FIG. 6. The wild type amino acid sequence for domain III of porcine serum albumin is described as SEQ ID NO: 8 in FIG. The wild type amino acid sequence for domain III of chicken serum albumin is described as SEQ ID NO: 9 in FIG. 6. The wild type amino acid sequence for domain III of donkey serum albumin is described as SEQ ID NO: 10 in FIG. The wild type amino acid sequence for domain III of sand rat serum albumin is described as SEQ ID NO: 11 in FIG. 6. The wild type amino acid sequence for domain III of both serum albumin is described as SEQ ID NO: 12 in FIG. The wild type amino acid sequence for domain III of feline serum albumin is described as SEQ ID NO: 13 in FIG. The wild type amino acid sequence for domain III of horse serum albumin is described as SEQ ID NO: 14 in FIG. 6.

All publications and patents mentioned herein are incorporated herein by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In addition, U.S. Provisional Patent Application 61 / 304,954, filed Feb. 16, 2010, and U.S. Provisional Patent Application No. 61 / 364,503, filed July 15, 2010, are incorporated by reference in their entirety for all purposes.

While certain embodiments of the invention have been discussed, the foregoing specification is intended to be illustrative, and not restrictive. Many modifications of the invention will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the invention should be determined by reference to the following claims (with full scope of equivalents thereof) and the specification (with such modifications).

                         SEQUENCE LISTING <110> MedImmune LLC   <120> HSA-Related Compositions and Methods of Use <130> MED0554.PCT2 <150> PCT / US11 / 24855 <151> 2011-02-15 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 205 <212> PRT <213> Homo sapiens <400> 1 Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu 1 5 10 15 Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr             20 25 30 Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg         35 40 45 Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys     50 55 60 Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu 65 70 75 80 Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys                 85 90 95 Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu             100 105 110 Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr         115 120 125 Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys     130 135 140 Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr 145 150 155 160 Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu                 165 170 175 Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly             180 185 190 Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu         195 200 205 <210> 2 <211> 585 <212> PRT <213> Homo sapiens <400> 2 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln             20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu         35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys     50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro                 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu             100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His         115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg     130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala                 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser             180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu         195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro     210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp                 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser             260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His         275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser     290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg                 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Arg Leu Ala Lys Thr             340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu         355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro     370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro                 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys             420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys         435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His     450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr                 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp             500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala         515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu     530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val                 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu             580 585 <210> 3 <211> 205 <212> PRT <213> Rattus norvegicus <400> 3 Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys Glu Leu Tyr 1 5 10 15 Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Val Leu Val Arg Tyr             20 25 30 Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ala Ala         35 40 45 Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu Pro Glu Ala     50 55 60 Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn Arg 65 70 75 80 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val Thr Lys                 85 90 95 Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe Ser Ala Leu             100 105 110 Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala Glu Thr Phe         115 120 125 Thr Phe His Ser Asp Ile Cys Thr Leu Pro Asp Lys Glu Lys Gln Ile     130 135 140 Lys Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His Lys Pro Lys Ala 145 150 155 160 Thr Glu Asp Gln Leu Lys Thr Val Met Gly Asp Phe Ala Gln Phe Val                 165 170 175 Asp Lys Cys Cys Lys Ala Ala Asp Lys Asp Asn Cys Phe Ala Thr Glu             180 185 190 Gly Pro Asn Leu Val Ala Arg Ser Lys Glu Ala Leu Ala         195 200 205 <210> 4 <211> 205 <212> PRT <213> Mus musculus <400> 4 Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys Asp Leu Tyr 1 5 10 15 Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ale I Leu Val Arg Tyr             20 25 30 Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ala Ala         35 40 45 Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu Pro Glu Asp     50 55 60 Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn Arg 65 70 75 80 Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His Val Thr Lys                 85 90 95 Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe Ser Ala Leu             100 105 110 Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala Glu Thr Phe         115 120 125 Thr Phe His Ser Asp Ile Cys Thr Leu Pro Glu Lys Glu Lys Gln Ile     130 135 140 Lys Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His Lys Pro Lys Ala 145 150 155 160 Thr Ala Glu Gln Leu Lys Thr Val Met Asp Asp Phe Ala Gln Phe Leu                 165 170 175 Asp Thr Cys Cys Lys Ala Ala Asp Lys Asp Thr Cys Phe Ser Thr Glu             180 185 190 Gly Pro Asn Leu Val Thr Arg Cys Lys Asp Ala Leu Ala         195 200 205 <210> 5 <211> 205 <212> PRT <213> Bos taurus <400> 5 Leu Val Asp Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Asp Gln Phe 1 5 10 15 Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val Arg Tyr             20 25 30 Thr Arg Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser         35 40 45 Arg Ser Leu Gly Lys Val Gly Thr Arg Cys Cys Thr Lys Pro Glu Ser     50 55 60 Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu Asn Arg 65 70 75 80 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val Thr Lys                 85 90 95 Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu             100 105 110 Thr Pro Asp Glu Thr Tyr Val Pro Lys Ala Phe Asp Glu Lys Leu Phe         115 120 125 Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Lys Gln Ile     130 135 140 Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro Lys Ala 145 150 155 160 Thr Glu Glu Gln Leu Lys Thr Val Met Glu Asn Phe Val Ala Phe Val                 165 170 175 Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Ala Cys Phe Ala Val Glu             180 185 190 Gly Pro Lys Leu Val Val Ser Thr Gln Thr Ala Leu Ala         195 200 205 <210> 6 <211> 205 <212> PRT <213> Canis familiaris <400> 6 Leu Val Asp Glu Pro Gln Asn Leu Val Lys Thr Asn Cys Glu Leu Phe 1 5 10 15 Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Leu Val Arg Tyr             20 25 30 Thr Lys Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser         35 40 45 Arg Lys Leu Gly Lys Val Gly Thr Lys Cys Cys Lys Lys Pro Glu Ser     50 55 60 Glu Arg Met Ser Cys Ala Glu Asp Phe Leu Ser Val Val Leu Asn Arg 65 70 75 80 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Arg Val Thr Lys                 85 90 95 Cys Cys Ser Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Gly Leu             100 105 110 Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe         115 120 125 Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Ala Glu Lys Gln Val     130 135 140 Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro Lys Ala 145 150 155 160 Thr Asp Glu Gln Leu Lys Thr Val Met Gly Asp Phe Gly Ala Phe Val                 165 170 175 Glu Lys Cys Cys Ala Ala Glu Asn Lys Glu Gly Cys Phe Ser Glu Glu             180 185 190 Gly Pro Lys Leu Val Ala Ala Ala Gln Ala Ala Leu Val         195 200 205 <210> 7 <211> 205 <212> PRT <213> Oryctolagus cuniculus <400> 7 Leu Val Asp Glu Pro Lys Asn Leu Val Lys Gln Asn Cys Glu Leu Tyr 1 5 10 15 Glu Gln Leu Gly Asp Tyr Asn Phe Gln Asn Ala Leu Leu Val Arg Tyr             20 25 30 Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ile Ser         35 40 45 Arg Ser Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala     50 55 60 Glu Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Val Val Leu Asn Arg 65 70 75 80 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val Thr Lys                 85 90 95 Cys Cys Ser Glu Ser Leu Val Asp Arg Arg Pro Cys Phe Ser Ala Leu             100 105 110 Gly Pro Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe         115 120 125 Thr Phe His Ala Asp Ile Cys Thr Leu Pro Glu Thr Glu Arg Lys Ile     130 135 140 Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro His Ala 145 150 155 160 Thr Asn Asp Gln Leu Lys Thr Val Val Gly Glu Phe Thr Ala Leu Leu                 165 170 175 Asp Lys Cys Cys Ser Ala Glu Asp Lys Glu Ala Cys Phe Ala Val Glu             180 185 190 Gly Pro Lys Leu Val Glu Ser Ser Lys Ala Thr Leu Gly         195 200 205 <210> 8 <211> 205 <212> PRT <213> Sus scrofa <400> 8 Leu Val Asp Glu Pro Lys Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe 1 5 10 15 Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val Arg Tyr             20 25 30 Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ala         35 40 45 Arg Lys Leu Gly Leu Val Gly Ser Arg Cys Cys Lys Arg Pro Glu Glu     50 55 60 Glu Arg Leu Ser Cys Ala Glu Asp Tyr Leu Ser Leu Val Leu Asn Arg 65 70 75 80 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val Thr Lys                 85 90 95 Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu             100 105 110 Thr Pro Asp Glu Thr Tyr Lys Pro Lys Glu Phe Val Glu Gly Thr Phe         115 120 125 Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Asp Glu Lys Gln Ile     130 135 140 Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro His Ala 145 150 155 160 Thr Glu Glu Gln Leu Arg Thr Val Leu Gly Asn Phe Ala Ala Phe Val                 165 170 175 Gln Lys Cys Cys Ala Ala Pro Asp His Glu Ala Cys Phe Ala Val Glu             180 185 190 Gly Pro Lys Phe Val Ile Glu Ile Arg Gly Ile Leu Ala         195 200 205 <210> 9 <211> 198 <212> PRT <213> Gallus gallus <400> 9 His Ile Lys Glu Thr Gln Asp Val Val Lys Thr Asn Cys Asp Leu Leu 1 5 10 15 His Asp His Gly Glu Ala Asp Phe Leu Lys Ser Ile Leu Ile Arg Tyr             20 25 30 Thr Lys Lys Met Pro Gln Val Pro Thr Asp Leu Leu Leu Glu Thr Gly         35 40 45 Lys Lys Met Thr Thr Ile Gly Thr Lys Cys Cys Gln Leu Gly Glu Asp     50 55 60 Arg Arg Met Ala Cys Ser Glu Gly Tyr Leu Ser Ile Val Ile His Asp 65 70 75 80 Thr Cys Arg Lys Gln Glu Thr Thr Pro Ile Asn Asp Asn Val Ser Gln                 85 90 95 Cys Cys Ser Gln Leu Tyr Ala Asn Arg Arg Pro Cys Phe Thr Ala Met             100 105 110 Gly Val Asp Thr Lys Tyr Val Pro Pro Pro Phe Asn Pro Asp Met Phe         115 120 125 Ser Phe Asp Glu Lys Leu Cys Ser Ala Pro Ala Glu Glu Arg Glu Val     130 135 140 Gly Gln Met Lys Leu Leu Ile Asn Leu Ile Lys Arg Lys Pro Gln Met 145 150 155 160 Thr Glu Glu Gln Ile Lys Thr Ile Ala Asp Gly Phe Thr Ala Met Val                 165 170 175 Asp Lys Cys Cys Lys Gln Ser Asp Ile Asn Thr Cys Phe Gly Glu Glu             180 185 190 Gly Ala Asn Leu Ile Val         195 <210> 10 <211> 205 <212> PRT <213> Equus asinus <400> 10 Leu Val Glu Glu Pro Lys Ser Leu Val Lys Lys Asn Cys Asp Leu Phe 1 5 10 15 Glu Glu Val Gly Glu Tyr Asp Phe Gln Asn Ala Leu Ile Val Arg Tyr             20 25 30 Thr Lys Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ile Gly         35 40 45 Arg Thr Leu Gly Lys Val Gly Ser Arg Cys Cys Lys Leu Pro Glu Ser     50 55 60 Glu Arg Leu Pro Cys Ser Glu Asn His Leu Ala Leu Ala Leu Asn Arg 65 70 75 80 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Ile Thr Lys                 85 90 95 Cys Cys Thr Asp Ser Leu Ala Glu Arg Arg Pro Cys Phe Ser Ala Leu             100 105 110 Glu Leu Asp Glu Gly Tyr Ile Pro Lys Glu Phe Lys Ala Glu Thr Phe         115 120 125 Thr Phe His Ala Asp Ile Cys Thr Leu Pro Glu Asp Glu Lys Gln Ile     130 135 140 Lys Lys Gln Ser Ala Leu Ala Glu Leu Val Lys His Lys Pro Lys Ala 145 150 155 160 Thr Lys Glu Gln Leu Lys Thr Val Leu Gly Asn Phe Ser Ala Phe Val                 165 170 175 Ala Lys Cys Cys Gly Ala Glu Asp Lys Glu Ala Cys Phe Ala Glu Glu             180 185 190 Gly Pro Lys Leu Val Ala Ser Ser Gln Leu Ala Leu Ala         195 200 205 <210> 11 <211> 198 <212> PRT Meriones unguiculatus <400> 11 Leu Val Glu Glu Pro Gln Asn Leu Val Lys Ser Asn Cys Glu Leu Tyr 1 5 10 15 Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Val Leu Val Arg Tyr             20 25 30 Thr Lys Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ala Ala         35 40 45 Arg Ser Leu Gly Arg Val Gly Thr His Cys Cys Ala Leu Pro Glu Lys     50 55 60 Lys Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn Arg 65 70 75 80 Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu Gln Val Thr Lys                 85 90 95 Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe Ser Ala Leu             100 105 110 Pro Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala Glu Thr Phe         115 120 125 Thr Phe His Ala Asn Ile Cys Thr Leu Pro Glu Lys Glu Lys Gln Met     130 135 140 Glu Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His Lys Pro Gln Ala 145 150 155 160 Thr Glu Glu Gln Leu Lys Lys Val Met Gly Asp Phe Ala Glu Phe Leu                 165 170 175 Glu Lys Cys Cys Lys Gln Glu Asp Lys Glu Ala Cys Phe Ser Thr Glu             180 185 190 Gly Pro Lys Leu Val Ala         195 <210> 12 <211> 198 <212> PRT <213> Ovis aries <400> 12 Leu Val Asp Glu Pro Gln Asn Leu Ile Lys Lys Asn Cys Glu Leu Phe 1 5 10 15 Glu Lys His Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val Arg Tyr             20 25 30 Thr Arg Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ile Ser         35 40 45 Arg Ser Leu Gly Lys Val Gly Thr Lys Cys Cys Ala Lys Pro Glu Ser     50 55 60 Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu Asn Arg 65 70 75 80 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val Thr Lys                 85 90 95 Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Asp Leu             100 105 110 Thr Leu Asp Glu Thr Tyr Val Pro Lys Pro Phe Asp Glu Lys Phe Phe         115 120 125 Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Lys Gln Ile     130 135 140 Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro Lys Ala 145 150 155 160 Thr Asp Glu Gln Leu Lys Thr Val Met Glu Asn Phe Val Ala Phe Val                 165 170 175 Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Gly Cys Phe Val Leu Glu             180 185 190 Gly Pro Lys Leu Val Ala         195 <210> 13 <211> 198 <212> PRT <213> Felis catus <400> 13 Leu Val Glu Glu Pro His Asn Leu Val Lys Thr Asn Cys Glu Leu Phe 1 5 10 15 Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Leu Val Arg Tyr             20 25 30 Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser         35 40 45 Arg Ser Leu Gly Lys Val Gly Ser Lys Cys Cys Thr His Pro Glu Ala     50 55 60 Glu Arg Leu Ser Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Arg 65 70 75 80 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Arg Val Thr Lys                 85 90 95 Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu             100 105 110 Gln Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Ser Ala Glu Thr Phe         115 120 125 Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Ala Glu Lys Gln Ile     130 135 140 Lys Lys Gln Ser Ala Leu Val Glu Leu Leu Lys His Lys Pro Lys Ala 145 150 155 160 Thr Glu Glu Gln Leu Lys Thr Val Met Gly Asp Phe Gly Ser Phe Val                 165 170 175 Asp Lys Cys Cys Ala Ala Glu Asp Lys Glu Ala Cys Phe Ala Glu Glu             180 185 190 Gly Pro Lys Leu Val Ala         195 <210> 14 <211> 198 <212> PRT <213> Equus caballus <400> 14 Leu Val Glu Glu Pro Lys Ser Leu Val Lys Lys Asn Cys Asp Leu Phe 1 5 10 15 Glu Glu Val Gly Glu Tyr Asp Phe Gln Asn Ala Leu Ile Val Arg Tyr             20 25 30 Thr Lys Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu Ile Gly         35 40 45 Arg Thr Leu Gly Lys Val Gly Ser Arg Cys Cys Lys Leu Pro Glu Ser     50 55 60 Glu Arg Leu Pro Cys Ser Glu Asn His Leu Ala Leu Ala Leu Asn Arg 65 70 75 80 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Ile Thr Lys                 85 90 95 Cys Cys Thr Asp Ser Leu Ala Glu Arg Arg Pro Cys Phe Ser Ala Leu             100 105 110 Glu Leu Asp Glu Gly Tyr Val Pro Lys Glu Phe Lys Ala Glu Thr Phe         115 120 125 Thr Phe His Ala Asp Ile Cys Thr Leu Pro Glu Asp Glu Lys Gln Ile     130 135 140 Lys Lys Gln Ser Ala Leu Ala Glu Leu Val Lys His Lys Pro Lys Ala 145 150 155 160 Thr Lys Glu Gln Leu Lys Thr Val Leu Gly Asn Phe Ser Ala Phe Val                 165 170 175 Ala Lys Cys Cys Gly Arg Glu Asp Lys Glu Ala Cys Phe Ala Glu Glu             180 185 190 Gly Pro Lys Leu Val Ala         195 <210> 15 <211> 641 <212> PRT <213> Human herpesvirus 4 <400> 15 Met Ser Asp Glu Gly Pro Gly Thr Gly Pro Gly Asn Gly Leu Gly Glu 1 5 10 15 Lys Gly Asp Thr Ser Gly Pro Glu Gly Ser Gly Gly Ser Gly Pro Gln             20 25 30 Arg Arg Gly Gly Asp Asn His Gly Arg Gly Arg Gly Arg Gly Arg Gly         35 40 45 Arg Gly Gly Gly Arg Pro Gly Ala Pro Gly Gly Ser Gly Ser Gly Pro     50 55 60 Arg His Arg Asp Gly Val Arg Arg Pro Gln Lys Arg Pro Ser Cys Ile 65 70 75 80 Gly Cys Lys Gly Thr His Gly Gly Thr Gly Ala Gly Ala Gly Ala Gly                 85 90 95 Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly             100 105 110 Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly         115 120 125 Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala     130 135 140 Gly Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly 145 150 155 160 Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly                 165 170 175 Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly             180 185 190 Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly Gly Ala Gly         195 200 205 Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala     210 215 220 Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala 225 230 235 240 Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly                 245 250 255 Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly             260 265 270 Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly         275 280 285 Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly     290 295 300 Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly 305 310 315 320 Gly Ala Gly Ala Gly Gly Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly                 325 330 335 Arg Gly Arg Gly Gly Ser Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly             340 345 350 Arg Arg Gly Arg Gly Arg Glu Arg Ala Arg Gly Gly Ser Arg Glu Arg         355 360 365 Ala Arg Gly Arg Gly Arg Gly Arg Gly Glu Lys Arg Pro Arg Ser Pro     370 375 380 Ser Ser Gln Ser Ser Ser Ser Gly Ser Pro Pro Arg Arg Pro Pro Pro 385 390 395 400 Gly Arg Arg Pro Phe Phe His Pro Val Gly Glu Ala Asp Tyr Phe Glu                 405 410 415 Tyr His Gln Glu Gly Gly Pro Asp Gly Glu Pro Asp Val Pro Pro Gly             420 425 430 Ala Ile Glu Gln Gly Pro Ala Asp Asp Pro Gly Glu Gly Pro Ser Thr         435 440 445 Gly Pro Arg Gly Gln Gly Asp Gly Gly Arg Arg Lys Lys Gly Gly Trp     450 455 460 Phe Gly Lys His Arg Gly Gln Gly Gly Ser Asn Pro Lys Phe Glu Asn 465 470 475 480 Ile Ala Glu Gly Leu Arg Ala Leu Leu Ala Arg Ser His Val Glu Arg                 485 490 495 Thr Thr Asp Glu Gly Thr Trp Val Ala Gly Val Phe Val Tyr Gly Gly             500 505 510 Ser Lys Thr Ser Leu Tyr Asn Leu Arg Arg Gly Thr Ala Leu Ala Ile         515 520 525 Pro Gln Cys Arg Leu Thr Pro Leu Ser Arg Leu Pro Phe Gly Met Ala     530 535 540 Pro Gly Pro Gly Pro Gln Pro Gly Pro Leu Arg Glu Ser Ile Val Cys 545 550 555 560 Tyr Phe Met Val Phe Leu Gln Thr His Ile Phe Ala Glu Val Leu Lys                 565 570 575 Asp Ala Ile Lys Asp Leu Val Met Thr Lys Pro Ala Pro Thr Cys Asn             580 585 590 Ile Arg Val Thr Val Cys Ser Phe Asp Asp Gly Val Asp Leu Pro Pro         595 600 605 Trp Phe Pro Pro Met Val Glu Gly Ala Ala Ala Glu Gly Asp Asp Gly     610 615 620 Asp Asp Gly Asp Glu Gly Gly Asp Gly Asp Glu Gly Glu Glu Gly Gln 625 630 635 640 Glu      <210> 16 <211> 1926 <212> DNA <213> Human herpesvirus 4 <400> 16 atgtctgacg aggggccagg tacaggacct ggaaatggcc taggagagaa gggagacaca 60 tctggaccag aaggctccgg cggcagtgga cctcaaagaa gagggggtga taaccatgga 120 cgaggacggg gaagaggacg aggacgagga ggcggaagac caggagcccc gggcggctca 180 ggatcagggc caagacatag agatggtgtc cggagacccc aaaaacgtcc aagttgcatt 240 ggctgcaaag ggacccacgg tggaacagga gcaggagcag gagcgggagg ggcaggagca 300 ggaggggcag gagcaggagg aggggcagga gcaggaggag gggcaggagg ggcaggaggg 360 gcaggagggg caggagcagg aggaggggca ggagcaggag gaggggcagg aggggcagga 420 ggggcaggag caggaggagg ggcaggagca ggaggagggg caggaggggc aggagcagga 480 ggaggggcag gaggggcagg aggggcagga gcaggaggag gggcaggagc aggaggaggg 540 gcaggagggg caggagcagg aggaggggca ggaggggcag gaggggcagg agcaggagga 600 ggggcaggag caggaggggc aggaggggca ggaggggcag gagcaggagg ggcaggagca 660 ggaggagggg caggaggggc aggaggggca ggagcaggag gggcaggagc aggaggggca 720 ggagcaggag gggcaggagc aggaggggca ggaggggcag gagcaggagg ggcaggaggg 780 gcaggagcag gaggggcagg aggggcagga gcaggaggag gggcaggagg ggcaggagca 840 ggaggagggg caggaggggc aggagcagga ggggcaggag gggcaggagc aggaggggca 900 ggaggggcag gagcaggagg ggcaggaggg gcaggagcag gaggaggggc aggagcagga 960 ggggcaggag caggaggtgg aggccggggt cgaggaggca gtggaggccg gggtcgagga 1020 ggtagtggag gccggggtcg aggaggtagt ggaggccgcc ggggtagagg acgtgaaaga 1080 gccagggggg gaagtcgtga aagagccagg gggagaggtc gtggacgtgg agaaaagagg 1140 cccaggagtc ccagtagtca gtcatcatca tccgggtctc caccgcgcag gccccctcca 1200 ggtagaaggc catttttcca ccctgtaggg gaagccgatt attttgaata ccaccaagaa 1260 ggtggcccag atggtgagcc tgacgtgccc ccgggagcga tagagcaggg ccccgcagat 1320 gacccaggag aaggcccaag cactggaccc cggggtcagg gtgatggagg caggcgcaaa 1380 aaaggagggt ggtttggaaa gcatcgtggt caaggaggtt ccaacccgaa atttgagaac 1440 attgcagaag gtttaagagc tctcctggct aggagtcacg tagaaaggac taccgacgaa 1500 ggaacttggg tcgccggtgt gttcgtatat ggaggtagta agacctccct ttacaaccta 1560 aggcgaggaa ctgcccttgc tattccacaa tgtcgtctta caccattgag tcgtctcccc 1620 tttggaatgg cccctggacc cggcccacaa cctggcccgc taagggagtc cattgtctgt 1680 tatttcatgg tctttttaca aactcatata tttgctgagg ttttgaagga tgcgattaag 1740 gaccttgtta tgacaaagcc cgctcctacc tgcaatatca gggtgactgt gtgcagcttt 1800 gacgatggag tagatttgcc tccctggttt ccacctatgg tggaaggggc tgccgcggag 1860 ggtgatgacg gagatgacgg agatgaagga ggtgatggag atgagggtga ggaagggcag 1920 gagtga 1926 <210> 17 <211> 1976 <212> DNA <213> Human herpesvirus 4 <400> 17 cctttatgtg taactcttgg ctgaagctct tacaccaatg ctgggggaca tgtacctccc 60 aggggcccag gaagactacg ggaggctaca ccaacgtcaa tcagaggggc ctgtgtagct 120 accgataagc ggaccctcaa gagggcatta gcaatagtgt ttataaggcc cccttgttaa 180 ccctaaacgg gtagcatatg cttcccgggt agtagtatat actatccaga ctaaccctaa 240 ttcaatagca tatgttaccc aacgggaagc atatgctatc gaattagggt tagtaaaagg 300 gtcctaagga acagcgatat ctcccacccc atgagctgtc acggttttat ttacatgggg 360 tcaggattcc acgagggtag tgaaccattt tagtcacaag ggcagtggct gaagatcaag 420 gagcgggcag tgaactctcc tgaatcttcg cctgcttctt cattctcctt cgtttagcta 480 atagaataac tgctgagttg tgaacagtaa ggtgtatgtg aggtgctcga aaacaaggtt 540 tcaggtgacg cccccagaat aaaatttgga cggggggttc agtggtggca ttgtgctatg 600 acaccaatat aaccctcaca aaccccttgg gcaataaata ctagtgtagg aatgaaacat 660 tctgaatatc tttaacaata gaaatccatg gggtggggac aagccgtaaa gactggatgt 720 ccatctcaca cgaatttatg gctatgggca acacataatc ctagtgcaat atgatactgg 780 ggttattaag atgtgtccca ggcagggacc aagacaggtg aaccatgttg ttacactcta 840 tttgtaacaa ggggaaagag agtggacgcc gacagcagcg gactccactg gttgtctcta 900 acacccccga aaattaaacg gggctccacg ccaatggggc ccataaacaa agacaagtgg 960 ccactctttt ttttgaaatt gtggagtggg ggcacgcgtc agcccccaca cgccgccctg 1020 cggttttgga ctgtaaaata agggtgtaat aacttggctg attgtaaccc cgctaaccac 1080 tgcggtcaaa ccacttgccc acaaaaccac taatggcacc ccggggaata cctgcataag 1140 taggtgggcg ggccaagata ggggcgcgat tgctgcgatc tggaggacaa attacacaca 1200 cttgcgcctg agcgccaagc acagggttgt tggtcctcat attcacgagg tcgctgagag 1260 cacggtgggc taatgttgcc atgggtagca tatactaccc aaatatctgg atagcatatg 1320 ctatcctaat ctatatctgg gtagcatagg ctatcctaat ctatatctgg gtagcatatg 1380 ctatcctaat ctatatctgg gtagtatatg ctatcctaat ttatatctgg gtagcatagg 1440 ctatcctaat ctatatctgg gtagcatatg ctatcctaat ctatatctgg gtagtatatg 1500 ctatcctaat ctgtatccgg gtagcatatg ctatcctaat agagattagg gtagtatatg 1560 ctatcctaat ttatatctgg gtagcatata ctacccaaat atctggatag catatgctat 1620 cctaatctat atctgggtag catatgctat cctaatctat atctgggtag cataggctat 1680 cctaatctat atctgggtag catatgctat cctaatctat atctgggtag tatatgctat 1740 cctaatttat atctgggtag cataggctat cctaatctat atctgggtag catatgctat 1800 cctaatctat atctgggtag tatatgctat cctaatctgt atccgggtag catatgctat 1860 cctcatgcat atacagtcag catatgatac ccagtagtag agtgggagtg ctatcctttg 1920 catatgccgc cacctcccaa gggggcgtga attttcgctg cttgtccttt tcctgc 1976 <210> 18 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> synthetic human HSA domain III <220> <221> residue463 <83> (83) <223> any amino acid residue <220> <221> residue508 &Lt; 222 > (128) <223> any amino acid residue <220> <221> residue523 &Lt; 222 > (143) .. (143) <223> any amino acid residue <220> <221> residue524 <144> (144) <223> any amino acid residue <400> 18 Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu 1 5 10 15 Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr             20 25 30 Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg         35 40 45 Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys     50 55 60 Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu 65 70 75 80 Cys Val Xaa His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys                 85 90 95 Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu             100 105 110 Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Xaa         115 120 125 Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Xaa Xaa     130 135 140 Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr 145 150 155 160 Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu                 165 170 175 Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly             180 185 190 Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu         195 200 205

Claims (32)

A polypeptide comprising an HSA moiety comprising human serum albumin (HSA) domain III or a neonatal Fc receptor (FcRn) binding fragment thereof, wherein said HSA domain III or a neonatal Fc receptor (FcRn) binding fragment is expressed in full-length mature HSA. An amino acid substitution at a position selected from the group consisting of the following positions numbered by position, wherein the substitution is relative to the FcRn of the polypeptide relative to a control polypeptide in which the HSA moiety does not include the amino acid substitution And increasing one or both of serum half-lives:
(a) residues 463 and 508;
(b) residues 463 and 523;
(c) residues 463 and 524;
(d) residues 508 and 523;
(e) residues 508 and 524;
(f) residues 523 and 524;
(g) residues 463, 508 and 523;
(h) residues 463, 508 and 524;
(i) residues 463, 523 and 524;
(j) residues 508, 523 and 524; And
(k) residues 463, 508, 523 and 524. A polypeptide comprising SEQ ID NO: 18 wherein X ₁ is not leucine, X ₂ is not threonine, X ₃ is not isoleucine, or X ₄ is not lysine, and X ₁ is A polypeptide having one or both of an increase in affinity for FcRn and an increase in serum half-life relative to a control polypeptide that is leucine, X ₂ is threonine, X ₃ is isoleucine, and X ₄ is lysine. The compound of claim 2, wherein X ₁ is cysteine, phenylalanine, glycine, histidine, isoleucine, asparagine, serine or glutamine; X ₂ is cysteine, glutamate, isoleucine, lysine, arginine or serine; X ₃ is alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine; A polypeptide wherein X ₄ is alanine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, glutamine, threonine or valine. The polypeptide of claim 2, wherein X ₁ is asparagine, X ₂ is arginine, X ₃ is glycine and / or X ₄ is leucine. The polypeptide of claim 1, which binds to FcRn with a higher affinity than the control polypeptide. The polypeptide of claim 1, wherein the polypeptide binds to FcRn with a higher affinity than the control polypeptide, wherein the affinity is measured at acidic pH. 7. The method of claim 1, wherein the substitution at residue 463 is selected from L463C, L463F, L463G, L463H, L463I, L463N, L463S and L463Q; The substitution at residue 508 is selected from T508C, T508E, T508I, T508K, T508R, and T508S; Substitution at residue 523 is selected from I523A, I523C, I523D, I523E, I523F, I523G, I523H, I523I, I523K, I523L, I523M, I523N, I523P, I523Q, I523R, I523S, I523T, I523V, I523W and I523Y; The polypeptide at residue 524 is selected from K524A, K524F, K524G, K524H, K524I, K524L, K524M, K524Q, K524T and K524V. The compound of claim 7, wherein the substitution at residue 463 is selected from L463F and L463N; The substitution at residue 508 is selected from T508R and T508S; The substitution at residue 523 is selected from I523D, I523E, I523F, I523G, I523K and I523R; The polypeptide of substitution at residue 524 is K524L. The polypeptide of claim 7 or 8, wherein the amino acid substitutions in HSA domain III are selected from the group consisting of the following amino acid substitutions:
(a) L463N / T508R;
(b) L463N / I523G;
(c) L463N / K524L;
(d) T508R / I523G;
(e) T508R / K524L;
(f) I523G / K524L;
(g) L463N / T508R / I523G;
(h) L463N / T508R / K524L;
(i) L463N / I523G / K524L;
(j) T508R / I523G / K524L; And
(k) L463N / T508R / I523G / K524L. The polypeptide of claim 9, wherein the amino acid substitution in HSA domain III is L463N / T508R. The polypeptide of claim 9, wherein the amino acid substitution in HSA domain III is L463N / I523G. The polypeptide of claim 9, wherein the amino acid substitution in HSA domain III is L463N / K524L. The polypeptide of claim 9, wherein the amino acid substitution in HSA domain III is T508R / I523G. The polypeptide of claim 9, wherein the amino acid substitution in HSA domain III is T508R / K524L. The polypeptide of claim 9, wherein the amino acid substitution in HSA domain III is L463N / T508R / I523G. The polypeptide of claim 9, wherein the amino acid substitution in HSA domain III is L463N / T508R / K524L. The polypeptide of claim 9, wherein the amino acid substitution in HSA domain III is L463N / I523G / K524L. The polypeptide of claim 9, wherein the amino acid substitution in HSA domain III is T508R / I523G / K524L. The polypeptide of claim 9, wherein the amino acid substitution in HSA domain III is L463N / T508R / I523G / K524L. A polypeptide comprising the amino acid sequence of SEQ ID NO. 16. A polypeptide according to any one of the preceding claims comprising a heterologous protein, retaining the functional activity of said heterologous protein and capable of binding to FcRn. The polypeptide of claim 21, wherein the heterologous protein comprises an antibody or antigen binding fragment thereof. The polypeptide of claim 21 or 22, wherein the heterologous protein comprises a therapeutic protein. 24. The polypeptide of any one of claims 21 to 23 wherein the heterologous protein is chemically or recombinantly conjugated to the HSA moiety. The polypeptide of any one of claims 1 to 15, wherein the polypeptide is conjugated to a nonprotein agent and retains the functional activity of the heterologous nonprotein material and is capable of binding to FcRn. The polypeptide of claim 21, wherein the heterologous protein or nonprotein material is conjugated to the N terminal amino acid of the HSA moiety, the C terminal amino acid of the HSA moiety, or the internal amino acid of the HSA moiety. 27. The method of any one of claims 1 to 26, wherein the HSA moiety comprises at least a portion of HSA domain I; Or at least part of HSA domain II; Or at least a portion of HSA domain I and at least a portion of HSA domain II. A composition comprising the polypeptide of any one of claims 1 to 27 and a pharmaceutically acceptable carrier. A method of treating a subject in need thereof, comprising administering to the subject a polypeptide according to any one of claims 1 to 22 or a composition according to claim 28. A nucleic acid construct comprising a nucleotide sequence encoding a polypeptide of any one of claims 1-27. A method of increasing one or both of affinity of a protein to FcRn and serum half-life, the method comprising conjugating the protein to a polypeptide of any one of claims 1 to 15 and 27. 28. A method of increasing one or both of affinity for a FcRn and a serum half-life of a nonprotein material, comprising: conjugating the nonprotein material to a polypeptide of any one of claims 1 to 15 and 27. Way.

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