Human serum albumin variants and uses thereof

Abstract

A serum albumin variant or functional fragment thereof comprising one or more amino acid substitutions selected from the group consisting of: (i) glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine substituting glutamine at position (522); (ii) valine substituting alanine at position (552); and (iii) alanine, glutamic acid, histidine, serine, lysine, and arginine substituting glycine at position (572).

Description

[Technical Field]

[0001] Related application data This application claims priority to Australian Patent Application No. 2017904211, entitled "Human serum albumin variants and uses thereof," filed on 18 October 2017, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, the contents of which are incorporated herein by reference in their entirety.

[0003] Field The present disclosure relates to human serum albumin variants and uses thereof. [Background technology]

[0004] Serum albumin is the most abundant naturally occurring protein in human plasma, which plays a major role in maintaining the osmotic pressure of the blood as well as the transport of various substances in the bloodstream. Serum albumin is known to bind to many proteins in vitro, including fetal Fc receptor (FcRn), and this interaction is known to be important for albumin's plasma half-life. FcRn is a membrane-bound protein expressed in many cell and tissue types (e.g., endothelial cells) and is constantly internalized and recycled. Albumin does not bind to FcRn at neutral pH, but once internalized, albumin binds to FcRn under the acidic conditions of the endosome. FcRn-bound albumin is rescued from degradation and recycled back to the cell surface, where it dissociates from FcRn at physiological pH.

[0005] Albumin has a long plasma half-life of approximately 19 days. This long half-life has led to its use to extend the half-life of pharmaceutical compounds. For example, albumin has been fused to human blood clotting factor IX (FIX), resulting in an extended half-life of FIX (IDELVION®). Albumin has also been conjugated to chemotherapy compounds (such as paclitaxel) to enhance drug half-life and drug accumulation (e.g., Abraxane®). Human serum albumin variants with one or more amino acid substitutions that result in improved binding or affinity for FcRn compared to native serum albumin have previously been described (eg, WO2011051489).

[0006] However, it is clear to those skilled in the art that there remains a need in the art for serum albumin variants with improved pharmacokinetic properties, such as, for example, high affinity for FcRn, long plasma half-life, and / or reduced clearance. There is also a need in the art to develop methods for controlling the plasma half-life of drugs in plasma in order to optimize drug administration and accumulation. Summary of the Invention

[0007] The present disclosure is based on the inventors' identification that specific amino acid substitutions in serum albumin improve or enhance its binding to fetal Fc receptors (FcRn). Serum albumin variants with specific amino acid substitutions are capable of increased plasma half-life. The inventors have determined that residues 522, 552, and 572 of SEQ ID NO: 1 are important for binding to FcRn, and have further identified that substitution of these residues with specific amino acids increases or promotes binding to FcRn at acidic pH.

[0008] The inventors' findings provide the basis for serum albumin variants containing one or more amino acid substitutions at residues corresponding to amino acids 522, 552, or 572 of SEQ ID NO: 1. The inventors' findings also provide the basis for methods for treating disorders, such as bleeding disorders, in a subject.

[0009] The present disclosure provides the following: (i) an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine substituting for glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1; (ii) a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO: 1; (iii) an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine, and arginine substituting for glycine at a position corresponding to amino acid 572 of SEQ ID NO:1; and (iv) combinations thereof; The present invention provides a serum albumin variant or functional fragment thereof comprising one or more amino acid substitutions selected from the group consisting of:

[0010] In one example, the serum albumin variant or functional fragment thereof comprises an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine and leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1.

[0011] In one example, the serum albumin variant or functional fragment thereof comprises a glycine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises an isoleucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises a lysine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises a methionine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises a phenylalanine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises a tryptophan substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1.

[0012] In one example, the serum albumin variant or functional fragment thereof comprises a tyrosine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises a valine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises an alanine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine and arginine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises an alanine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1.

[0013] In one example, the serum albumin variant or functional fragment thereof comprises a glutamic acid substituting for a glycine at the position corresponding to amino acid 572 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises a histidine substituting for a glycine at the position corresponding to amino acid 572 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises a serine substituting for a glycine at the position corresponding to amino acid 572 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises a lysine substituting for a glycine at the position corresponding to amino acid 572 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises an arginine substituting for a glycine at the position corresponding to amino acid 572 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises a valine substituting for a glycine at a position corresponding to amino acid 572 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises the amino acid substitutions set out above at positions corresponding to amino acid 522 of SEQ ID NO:1, amino acid 552 of SEQ ID NO:1, and amino acid 572 of SEQ ID NO:1.

[0014] In one example, a serum albumin variant or functional fragment thereof optionally comprises one or more amino acid substitutions, deletions, or insertions in addition to the amino acid substitutions set forth above at positions corresponding to amino acid 522 of SEQ ID NO: 1, amino acid 552 of SEQ ID NO: 1, and amino acid 572 of SEQ ID NO: 1. Additional amino acid substitutions suitable for use in the present disclosure will be apparent to those of skill in the art and include naturally occurring substitutions and genetically engineered substitutions, such as those described in WO2011051489.

[0015] In one example, the serum albumin variants or functional fragments thereof of the present disclosure have improved pharmacokinetic properties compared to serum albumin set forth in SEQ ID NO: 1. Pharmacokinetic properties of serum albumin will be apparent to those skilled in the art and include, for example, affinity for binding to FcRn, plasma half-life, and / or plasma clearance rate. Methods for measuring the pharmacokinetic properties of serum albumin variants of the present disclosure will be apparent to those skilled in the art and / or are described herein.

[0016] In one example, a serum albumin variant or functional fragment thereof of the present disclosure binds with higher affinity to FcRn compared to serum albumin set forth in SEQ ID NO: 1. Methods for measuring the affinity of a serum albumin variant or functional fragment thereof for FcRn will be apparent to those skilled in the art and / or are described herein. In one example, the binding affinity of a serum albumin variant or functional fragment thereof for FcRn is measured by flow cytometry. For example, CHO cells stably expressing a serum albumin variant or functional fragment thereof are stained at acidic (pH 5.5) and neutral (pH 7.4) pH with alexa-488-labeled FcRn / β2m (to detect target binding) and anti-myc-alexa647 (to detect expression) and analyzed by flow cytometry. In one example, the affinity of a serum albumin variant or functional fragment thereof for FcRn / β2m is measured by calculating the mean fluorescence intensity relative to unmodified serum albumin (e.g., set forth in SEQ ID NO: 1). In one example, the affinity of a serum albumin variant or functional fragment thereof is measured by biosensor analysis (e.g., using a surface plasmon resonance (SPR) assay). For example, the binding affinity (i.e., strength of interaction) of a serum albumin variant or functional fragment thereof to immobilized FcRn is determined at pH 5.4 and / or pH 7.4 and 37°C. In another example, the binding affinity (i.e., strength of interaction) of an immobilized serum albumin variant or functional fragment thereof to FcRn is measured at pH 5.4 and / or pH 7.4 and 37°C. In one example, the affinity constant (K D), dissociation constant (Kd) and association constant (ka) are measured. For example, affinity constant (K D ) is the ratio of the dissociation constant (Kd) to the association constant (ka) (i.e., K D =Kd / ka).

[0017] In one example, the binding affinity is measured at an acidic pH. For example, an acidic pH is a pH lower than pH 6.0, such as pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, or pH 5.0. In one example, a serum albumin variant or functional fragment thereof of the present disclosure binds to FcRn with higher affinity at an acidic pH compared to serum albumin set forth in SEQ ID NO: 1. For example, a serum albumin variant or functional fragment thereof of the present disclosure binds to FcRn with higher affinity at a low pH, e.g., about pH 6.0, facilitating binding within endosomes. In one example, a serum albumin variant or functional fragment thereof binds to FcRn with higher affinity at a pH of about 6.0 compared to its affinity at about pH 7.4, which facilitates re-release of the serum albumin variant into the blood following cellular refolding. The amino acid substitutions of the present disclosure are useful for extending the half-life of proteins by enhancing FcRn-mediated refolding and, consequently, reducing clearance from the blood.

[0018] In one example, the binding level of a serum albumin variant or functional fragment thereof of the present disclosure to FcRn is enhanced at pH 5.4 compared to the serum albumin set forth in SEQ ID NO: 1, as measured by a cell binding assay. In one example, the binding level of a serum albumin variant or functional fragment thereof of the present disclosure to FcRn is enhanced at pH 5.4 compared to its affinity at pH 7.4, as measured by a cell binding assay. In one example, the binding level of a serum albumin variant or functional fragment thereof of the present disclosure to FcRn is enhanced at pH 5.4 compared to its affinity at pH 7.4, as measured by SPR analysis. In one example, the binding level of a serum albumin variant or functional fragment thereof of the present disclosure to FcRn is enhanced at pH 5.4 compared to its affinity at pH 7.4, as measured by flow cytometry.

[0019] In one example, the binding level of a serum albumin variant or functional fragment thereof of the present disclosure to FcRn is enhanced by at least about 2-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, or at least about 15-fold compared to serum albumin set forth in SEQ ID NO: 1. In one example, the binding level of the serum albumin variant to FcRn is enhanced by about 2-fold to about 5-fold compared to serum albumin set forth in SEQ ID NO: 1. For example, the binding level of the serum albumin variant to FcRn is enhanced by about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3-fold, about 3.25-fold, about 3.5-fold, about 3.75-fold, about 4-fold, about 4.25-fold, about 4.5-fold, about 4.75-fold, or about 5-fold compared to serum albumin set forth in SEQ ID NO: 1. In one example, the binding level of the serum albumin variant to FcRn is enhanced by about 2.4-fold, about 2.8-fold, about 3.1-fold, about 3.4-fold, about 3.7-fold, about 3.9-fold, about 4-fold, or about 4.8-fold compared to serum albumin defined in SEQ ID NO:1.

[0020] In one example, the binding level of the serum albumin variant to FcRn is enhanced by about 5- to about 15-fold compared to serum albumin defined by SEQ ID NO: 1. For example, the binding level of the serum albumin variant to FcRn is enhanced by about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 10.5-fold, about 11-fold, about 11.5-fold, about 12-fold, about 12.5-fold, about 13-fold, about 13.5-fold, about 14-fold, about 14.5-fold, or about 15-fold. In one example, the binding level of the serum albumin variant to FcRn is enhanced by about 7.6-fold, about 8.6-fold, or about 13.8-fold compared to serum albumin defined by SEQ ID NO: 1.

[0021] In one example, the binding level of the serum albumin variant to FcRn is enhanced by about 15- to about 50-fold compared to serum albumin defined as SEQ ID NO: 1. For example, the binding level of the serum albumin variant to FcRn is enhanced by about 15-, about 20-, about 25-, about 30-, about 35-, about 40-, about 45-, or about 50-fold compared to serum albumin defined as SEQ ID NO: 1. In one example, the binding level of the serum albumin variant to FcRn is enhanced by about 42-, about 43-, about 47-, or about 48-fold compared to serum albumin defined as SEQ ID NO: 1.

[0022] In another example, the binding level of the serum albumin variant to FcRn is enhanced by about 50 to 100 times compared to serum albumin defined as SEQ ID NO: 1. For example, the binding level of the serum albumin variant to FcRn is enhanced by about 50 times, about 55 times, about 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 85 times, about 90 times, about 95 times, or about 100 times compared to serum albumin defined as SEQ ID NO: 1. In one example, the binding level of the serum albumin variant to FcRn is enhanced by about 57 times or about 58 times compared to serum albumin defined as SEQ ID NO: 1.

[0023] In a further example, the binding level of the serum albumin variant to FcRn is enhanced by about 100 to about 250 times compared to serum albumin defined as SEQ ID NO: 1. For example, the binding level of the serum albumin variant to FcRn is enhanced by about 100 to about 250 times compared to serum albumin defined as SEQ ID NO: 1. In one example, the level of binding of the serum albumin variant to FcRn is enhanced by about 100-fold, about 110-fold, about 120-fold, about 130-fold, about 140-fold, about 150-fold, about 160-fold, about 170-fold, about 180-fold, about 190-fold, about 200-fold, about 210-fold, about 220-fold, about 230-fold, about 240-fold, or about 250-fold. This is enhanced by about 180-fold or about 240-fold compared to serum albumin defined in SEQ ID NO:1.

[0024] In one example, the binding level of the serum albumin variant to FcRn is enhanced by at least 250-fold compared to the serum albumin defined in SEQ ID NO: 1. For example, the binding level of the serum albumin variant to FcRn is enhanced by about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, or about 500-fold compared to the serum albumin defined in SEQ ID NO: 1. In one example, the binding level of the serum albumin variant to FcRn is enhanced by about 410-fold compared to the serum albumin defined in SEQ ID NO: 1.

[0025] In one example, the serum half-life of the serum albumin variant is extended compared to the serum albumin set forth in SEQ ID NO: 1. For example, the serum half-life of the serum albumin variant of the present disclosure is extended by at least about 1.5-fold compared to the serum albumin set forth in SEQ ID NO: 1. In one example, the serum half-life of the serum albumin variant of the present disclosure is extended by about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, or about 10-fold. In one example, the serum half-life of the serum albumin variant of the present disclosure is extended by about 5 days to about 10 days or more. For example, the serum half-life of the serum albumin variants of the present disclosure is enhanced by about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, or about 50 days. Methods for measuring the half-life of serum albumin variants will be apparent to those skilled in the art and / or are described herein. In one example, the half-life of serum albumin variants is measured using an in vivo assay. In one example, serum albumin concentration is measured by enzyme-linked immunosorbent assay (ELISA) using a human serum albumin-specific antibody. For example, ELISA is performed using commercially available methods. In another example, the serum albumin variant is intravenously injected into a mouse or cynomolgus monkey, and the plasma concentration is measured periodically as a function of time. In one example, the plasma concentration of the serum albumin variant is measured 3 minutes to 72 hours after injection. In one example, the plasma concentration of the serum albumin variant is measured up to 60 days after injection. In one example, the serum albumin variant is radiolabeled. In one example, the in vitro half-life of the serum albumin variant or functional fragment thereof is measured by calculating the clearance rate in the beta phase. In one example, the in vitro half-life of the serum albumin variant or functional fragment thereof is compared to the in vitro half-life of unmodified serum albumin (e.g., as set forth in SEQ ID NO: 1).

[0026] In one example, the rate of clearance (i.e., refolding and uptake) of the serum albumin variant is reduced compared to the serum albumin set forth in SEQ ID NO: 1. Methods for measuring the rate of clearance (i.e., refolding and uptake) of a serum albumin variant will be apparent to those skilled in the art and / or are described herein. In one example, confocal fluorescence microscopy is used to measure if a serum albumin variant is refolded. For example, to determine whether a serum albumin variant is refolded, a fluorescently labeled serum albumin variant is incubated with cells expressing human FcRn receptors on the cell surface and visualized by confocal fluorescence microscopy.

[0027] In one example, the serum albumin variant or functional fragment thereof of the present disclosure has the following structure: (i) an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine substituting glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, and valine substituting alanine at position 552 of SEQ ID NO:1; or (ii) an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine substituting for glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1, and an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine, and arginine substituting for glycine at position 572 of SEQ ID NO:1; or

[0028] (iii) an amino acid selected from the group consisting of valine substituting for alanine at a position corresponding to amino acid 552 of SEQ ID NO:1, and alanine, glutamic acid, histidine, serine, lysine, and arginine substituting for glycine at position 572 of SEQ ID NO:1; or (iv) an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine, which substitutes for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1; a valine which substitutes for alanine at position 552 of SEQ ID NO:1; and an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine, and arginine, which substitutes for glycine at position 572 of SEQ ID NO:1; Includes:

[0029] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine substituting for glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1, and valine substituting for alanine at position 552 of SEQ ID NO:1.

[0030] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1; and an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine, and arginine substituting for glycine at position 572 of SEQ ID NO:1.

[0031] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises an amino acid selected from the group consisting of valine substituting for alanine at a position corresponding to amino acid 552 of SEQ ID NO: 1, alanine substituting for glycine at position 572 of SEQ ID NO: 1, glutamic acid, histidine, serine, lysine, and arginine.

[0032] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting for alanine at position 552 of SEQ ID NO:1, and an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine, and arginine substituting for glycine at position 572 of SEQ ID NO:1.

[0033] In one example, a serum albumin variant or functional fragment thereof of the present disclosure further comprises a tyrosine substituting for a lysine at a position corresponding to amino acid 573 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine and leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, and a tyrosine substituting for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1.

[0034] In one example, the serum albumin variant or functional fragment thereof comprises a valine substituted for an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1, and a tyrosine substituted for a lysine at a position corresponding to amino acid 573 of SEQ ID NO:1. In one example, the serum albumin variant or functional fragment thereof comprises an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine and arginine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1, and tyrosine substituting for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1.

[0035] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting for alanine at position 552 of SEQ ID NO:1, and a tyrosine substituting for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1.

[0036] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine, and arginine substituting for glycine at position 572 of SEQ ID NO:1, and a tyrosine substituting for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1.

[0037] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1, an alanine substituting a glycine at position 572 of SEQ ID NO:1, an amino acid selected from the group consisting of glutamic acid, histidine, serine, lysine and arginine, and a tyrosine substituting a lysine at a position corresponding to amino acid 573 of SEQ ID NO:1.

[0038] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting for alanine at position 552 of SEQ ID NO:1, an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine, and arginine substituting for glycine at position 572 of SEQ ID NO:1, and a tyrosine substituting for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1.

[0039] In one example, the serum albumin variant or functional fragment thereof of the present disclosure has the following structure: (i) a leucine substituting for glutamine at a position corresponding to amino acid 522 of SEQ ID NO: 1; and / or (ii) a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO: 1; and / or (iii) an arginine substituting a glycine at a position corresponding to amino acid 572 of SEQ ID NO: 1; Includes:

[0040] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a leucine substituting for glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises an arginine substituting for a glycine at a position corresponding to amino acid 572 of SEQ ID NO:1.

[0041] In one example, the serum albumin variant or functional fragment thereof of the present disclosure has the following structure: (i) a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, and an arginine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1; or (ii) a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, and a valine substituting for alanine at the position corresponding to amino acid 552 of SEQ ID NO:1; or (iii) a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1, and an arginine substituting a glycine at a position corresponding to amino acid 572 of SEQ ID NO:1; or

[0042] (iv) a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, and a tyrosine substituting for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1; or (v) a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1, and a tyrosine substituting a lysine at a position corresponding to amino acid 573 of SEQ ID NO:1; or (vi) an arginine substituting a glycine at a position corresponding to amino acid 572 of SEQ ID NO:1, and a tyrosine substituting a lysine at a position corresponding to amino acid 573 of SEQ ID NO:1; or (vii) a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting for alanine at the position corresponding to amino acid 552 of SEQ ID NO:1, and an arginine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1; or

[0043] (viii) a leucine substituting a glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting an alanine at the position corresponding to amino acid 552 of SEQ ID NO:1, and a tyrosine substituting a lysine at the position corresponding to amino acid 573 of SEQ ID NO:1; or (ix) a leucine substituted for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, an arginine substituted for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1, and a tyrosine substituted for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1; or (x) a leucine substituting glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting alanine at position 552 of SEQ ID NO:1, an arginine substituting glycine at the position corresponding to amino acid 572 of SEQ ID NO:1, and a tyrosine substituting lysine at the position corresponding to amino acid 573 of SEQ ID NO:1; Includes:

[0044] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, and an arginine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a leucine substituting for glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1, and a valine substituting for alanine at a position corresponding to amino acid 552 of SEQ ID NO:1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1, and an arginine substituting a glycine at a position corresponding to amino acid 572 of SEQ ID NO:1.

[0045] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, and a tyrosine substituting for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1, and a tyrosine substituting a lysine at a position corresponding to amino acid 573 of SEQ ID NO:1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises an arginine substituting for a glycine at a position corresponding to amino acid 572 of SEQ ID NO:1, and a tyrosine substituting for a lysine at a position corresponding to amino acid 573 of SEQ ID NO:1.

[0046] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting for alanine at the position corresponding to amino acid 552 of SEQ ID NO:1, and an arginine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a leucine substituting a glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1, and a tyrosine substituting a lysine at a position corresponding to amino acid 573 of SEQ ID NO:1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, an arginine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1, and a tyrosine substituting for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1.

[0047] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a leucine substituting a glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting an alanine at position 552 of SEQ ID NO:1, an arginine substituting a glycine at a position corresponding to amino acid 572 of SEQ ID NO:1, and a tyrosine substituting a lysine at a position corresponding to amino acid 573 of SEQ ID NO:1. The present disclosure provides a serum albumin conjugate comprising a serum albumin variant or functional fragment thereof and a compound according to the present disclosure. In one example, a serum albumin conjugate of the present disclosure has a longer serum half-life compared to a serum albumin conjugate comprising the serum albumin set forth in SEQ ID NO: 1. Examples of extended serum half-lives and assays for measuring serum half-life are described herein and should be read mutatis mutandis to this example of the disclosure.

[0048] In another example, a serum albumin conjugate of the present disclosure has enhanced binding affinity to FcRn compared to a serum albumin conjugate comprising serum albumin set forth in SEQ ID NO: 1. For example, the binding level of the serum albumin conjugate to FcRn is enhanced by at least about 2-fold, 4-fold, 5-fold, or 10-fold. For example, the binding level to FcRn is enhanced by at least about 2-fold, 10-fold, 40-fold, 100-fold, or 150-fold. High affinity for FcRn and assays for measuring same are described herein, mutatis mutandis, in this example of the disclosure.

[0049] In one example, the serum albumin variant or functional fragment thereof is conjugated to and / or encapsulates another compound. Compounds contemplated by the present disclosure may take any of a variety of forms, including naturally occurring compounds, small chemical compounds, or biological compounds.

[0050] In one example, a serum albumin variant or functional fragment thereof of the present disclosure is conjugated to a compound, which is directly or indirectly bound to the serum albumin variant or functional fragment thereof.

[0051] Exemplary compounds include proteins, proteins including antibody variable regions, antibody mimetics, domain antibodies, toxins, radioisotopes, detectable labels, peptides, polypeptides, colloids, chemotherapeutic drugs, nucleic acids, small molecules, antisense oligonucleotides, short hairpin RNAs (shRNAs), siRNAs, interfering RNAs (RNAi), ribozymes, microRNAs, microRNA-adapted shRNAs (shRNAmirs), DNA enzymes (DNAzymes), and mixtures thereof. In one example, the detectable label is an imaging agent.

[0052] In one example, the compound is a protein-based compound, such as a peptide, polypeptide, or protein. In one example, the protein is a therapeutic protein. In another example, the compound is a protein (e.g., a therapeutic protein) that comprises a non-antibody antigen-binding domain, such as, for example, an adnectin, an affibody, an atrimer, an evasin, a designed ankyrin repeat protein (DARPin), or an anticalin.

[0053] In one example, the compound is a protein (e.g., a therapeutic protein) that comprises a variable region fragment (Fv). For example, the protein may be: (i) single-chain Fv fragment (scFv); (ii) dimeric scFv (di‐scFv); or (iii) diabodies; (iv) triabodies; (v) tetrabodies; (vi)Fab; (vii) F(ab')2; (viii) Fv; or (ix) the constant region of an antibody, the Fc or heavy chain constant domain (C H )2 and / or C H One of (i) to (viii) linked to 3, is selected from the group consisting of:

[0054] In one example, the protein is an antibody or antigen-binding fragment. In one example, an antibody or antigen-binding fragment of the disclosure is recombinant, chimeric, CDR-grafted, humanized, synhumanized, primatized, deimmunized, or human.

[0055] In one example, the present disclosure provides a serum albumin variant or a functional fragment thereof conjugated to von Willebrand factor or a modified form thereof. For example, the compound is von Willebrand factor or a modified form thereof. In one example, the von Willebrand factor comprises a D'D3 domain.

[0056] In one example, the disclosure provides a serum albumin variant or functional fragment thereof conjugated to a complement inhibitor or a modified form thereof. For example, the compound is a complement inhibitor or a modified form thereof. In one example, the complement inhibitor is selected from the group consisting of factor I (fI), factor H (fH), C4b-binding protein (C4bp), soluble CD55 (decay-accelerating factor (DAF)), C1 inhibitor (C1-INH or C1 esterase inhibitor); soluble CD35 (sCR1); soluble CD46 (membrane cofactor protein (MCP)), soluble CD59 (protectin), TT30 (CR2-fH), cobra venom factor (CVF), and functional fragments or variants thereof.

[0057] In one example, the complement inhibitor is a soluble complement inhibitor, such as sCR1, or a functional fragment or variant thereof. In one example, the complement inhibitor is a mutant or modified sCR1. In one example, the complement inhibitor is C1 inhibitor (i.e., C1-INH), or a functional fragment or variant thereof. In one example, the complement inhibitor is a mutant or modified C1-INH.

[0058] In one example, the present disclosure provides a serum albumin variant or functional fragment thereof conjugated to a blood clotting factor. In another example, the disclosure provides a serum albumin variant or functional fragment thereof conjugated to a compound that binds to a blood clotting factor, e.g., the compound is or binds to a blood clotting factor.

[0059] In one example, the blood clotting factor is selected from the group consisting of factor I, factor II (prothrombin) / thrombin, factor III, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, and activated forms of any of the above. For example, the blood clotting factor is factor IX and / or factor IXa. In another example, the blood clotting factor is factor X and / or factor Xa. In a further example, the blood clotting factor is factor IX / IXa and factor X / Xa. In one example, the blood clotting factor is factor VII and / or factor VIIa. In another example, the blood clotting factor is factor VIII and / or factor VIIIa.

[0060] The present disclosure also provides compositions comprising the disclosed serum albumin conjugates and a pharmaceutical carrier and / or excipient. In one example, the composition has enhanced binding affinity to FcRn compared to a composition comprising a serum albumin conjugate comprising serum albumin comprising the sequence set forth in SEQ ID NO:1.

[0061] In one example, the composition has an increased serum half-life compared to a composition comprising a serum albumin conjugate comprising serum albumin comprising the sequence set forth in SEQ ID NO:1. The present disclosure also provides a method of treating or preventing a disease or condition in a subject, the method comprising administering a serum albumin conjugate or composition comprising a serum albumin variant or functional fragment thereof of the present disclosure, in one example, to the subject in need of treatment for said disease.

[0062] In one example, the disclosure provides a serum albumin variant or functional fragment thereof, or a serum albumin conjugate or composition comprising the serum albumin variant or functional fragment thereof, for use in treating or preventing a disease or condition in a subject. In one example, the present disclosure provides the use of a composition comprising a serum albumin conjugate of the present disclosure, or a serum albumin variant or functional fragment thereof of the present disclosure, in the manufacture of a medicament for the treatment or prevention of a disease or condition in a subject.

[0063] In one example, the disease or condition is a bleeding disorder.

[0064] In one example, the subject is suffering from a bleeding disorder. In one example, the subject has been diagnosed with a bleeding disorder. In one example, the subject is being treated for a bleeding disorder.

[0065] In one example of any of the methods described herein, a serum albumin conjugate or composition comprising a serum albumin variant of the present disclosure is administered before or after the onset of a bleeding disorder. In one example of any of the methods described herein, a serum albumin conjugate or composition comprising a serum albumin variant of the present disclosure is administered before the onset of a bleeding disorder. In one example of any of the methods described herein, a serum albumin conjugate or composition comprising a serum albumin variant of the present disclosure is administered after the onset of a bleeding disorder.

[0066] In one example of any of the methods described herein, a serum albumin conjugate or composition comprising a serum albumin variant of the present disclosure is administered before or after the onset of a bleeding event. In one example, a serum albumin conjugate or composition comprising a serum albumin variant of the present disclosure is administered before the onset of a bleeding event. In another example, a serum albumin conjugate or composition comprising a serum albumin variant of the present disclosure is administered after the onset of a bleeding event.

[0067] Bleeding events are known to those skilled in the art and include, for example, minor and / or major bleeding events. In one example, the bleeding event is a major bleeding event. For example, a major bleeding event is any episode of bleeding that causes reduced hemoglobin of ≧5 g / dL or an absolute decrease in hematocrit of ≧15% or more. In one example, the bleeding event is a minor bleeding event. For example, a minor bleeding event is any episode of bleeding that causes reduced hemoglobin of ≦4 g / dL or an absolute decrease in hematocrit of ≧10% or more.

[0068] In one example, the subject is at risk of developing bleeding disorder.For example, the subject at risk of developing bleeding disorder includes, but is not limited to, the subject with mutation, deletion or rearrangement of blood coagulation factor, such as Factor VIII, or the subject with platelet disorder.In one example, the subject has a relative who has developed bleeding disorder.For example, bleeding disorder is hereditary.In one example, bleeding disorder is acquired.

[0069] In one example, a serum albumin conjugate or composition comprising a serum albumin variant is administered before or after the onset of symptoms of a bleeding disorder. In one example, a serum albumin conjugate or composition comprising a serum albumin variant is administered before the onset of symptoms of a bleeding disorder. In one example, a serum albumin conjugate or composition comprising a serum albumin variant is administered after the onset of symptoms of a bleeding disorder. In one example, a serum albumin conjugate or composition comprising a serum albumin variant of the present disclosure is administered at a dose that alleviates or relieves one or more symptoms of a bleeding disorder.

[0070] Symptoms of bleeding disorders will be apparent to those skilled in the art and include, for example, the following: easy bruising; bleeding gums; Heavy bleeding from small cuts or dental scars; Nosebleeds of unknown origin; Heavy menstrual bleeding; Hemarthrosis; and / or excessive bleeding after surgery, Includes:

[0071] In one example, bleeding disorder is caused by blood coagulation disorder.For example, blood coagulation disorder is hemophilia, von Willebrand's disease, factor I deficiency, factor II deficiency, factor V deficiency, combined factor V / factor VIII deficiency, factor VII deficiency, factor X deficiency, factor XI deficiency or factor XIII deficiency.In one example, hemophilia is hemophilia A or hemophilia B.In one example, the subject has a condition that requires preventive treatment.

[0072] In one example, a serum albumin conjugate or composition comprising a serum albumin variant of the present disclosure is administered to a subject in an amount that reduces the severity of bleeding in the subject.

[0073] In one example of any of the methods described herein, the subject is a mammal, for example a primate, such as a human.

[0074] The methods of treatment described herein may additionally include administering an additional compound to alleviate, treat, or prevent the effects of a bleeding disorder.

[0075] The present disclosure also provides a composition comprising a serum albumin variant or a functional fragment thereof that binds to a blood coagulation factor for use in treating or preventing a bleeding disorder.

[0076] The present disclosure also provides the use of a composition comprising a serum albumin variant or functional fragment thereof that binds to a blood coagulation factor in the manufacture of a medicament for treating or preventing a bleeding disorder.

[0077] The present disclosure also provides kits comprising at least one serum albumin conjugate or composition comprising a serum albumin variant or functional fragment thereof of the present disclosure that binds to a blood coagulation factor, packaged with instructions for use in treating or preventing a bleeding disorder in a subject. Optionally, the kit additionally comprises a therapeutically active compound or drug.

[0078] The present disclosure also provides kits comprising at least one of the serum albumin conjugates or compositions comprising a serum albumin variant or functional fragment thereof of the present disclosure that binds to a blood coagulation factor, optionally in combination with a therapeutically active compound or drug, packaged with instructions for administering the conjugate or composition to a subject suffering from or at risk of suffering from a bleeding disorder.

[0079] The exemplary effects of the serum albumin conjugates or compositions of the present disclosure that bind to blood coagulation factors are to be considered as applying mutatis mutandis to the examples of the present disclosure described herein and presented in the preceding five paragraphs. [Brief explanation of the drawings]

[0080] [Figure 1] FIG. 1 is a graphical representation showing serum HSA concentrations (μg / mL; mean±SEM) of HSA variants injected into mice expressing the human FcRn receptor. DETAILED DESCRIPTION OF THE INVENTION

[0081] Key points of the sequence listing SEQ ID NO: 1 Amino acid sequence of human serum albumin SEQ ID NO: 2 Amino acid sequence of human blood coagulation factor VIII SEQ ID NO: 3 Amino acid sequence of human blood coagulation factor IX SEQ ID NO: 4 Amino acid sequence of human blood coagulation factor X SEQ ID NO: 5 Amino acid sequence of human blood coagulation factor VII SEQ ID NO: 6 Amino acid sequence of human von Willebrand factor

[0082] Detailed Description General Provisions Throughout this specification, unless expressly stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be deemed to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps or group of compositions of matter.

[0083] Those skilled in the art will recognize that there are possibilities for changes and modifications other than those specifically described in the present disclosure. It should be understood that the present disclosure includes all such changes and modifications. The present disclosure also includes all of the steps, features, compositions and compounds referred to or indicated herein, individually or collectively, and any combination of any two or more of the steps or features.

[0084] The scope of the present disclosure is not limited by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0085] Any example of the present disclosure herein shall be deemed to apply mutatis mutandis to any other example of the present disclosure, unless otherwise specified. In other words, any embodiment of the present disclosure may be combined with any other embodiment of the present disclosure (except where mutually exclusive).

[0086] Any instance of the present disclosure disclosing a particular feature or group of features or method or method steps is to be considered as giving explicit support for the particular feature or group of features or method or method steps, disclaiming the same.

[0087] Unless otherwise defined, all technical and scientific terms used herein shall be assumed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the art of cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0088] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988) (including all current editions), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all current editions) and other sources.

[0089] The descriptions and definitions of variable regions and portions thereof, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J. Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol. Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883, 1989 and / or Al-Lazikani et al., J. Mol. Biol. 273, 927-948, 1997.

[0090] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be deemed to give explicit endorsement of both meanings or either meaning.

[0091] Throughout this specification the word "comprise" or variations thereof, such as "comprises" or "comprising", will be understood to imply the inclusion of the specified element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0092] As used herein, the term "derived from" shall be deemed to indicate that the specified integer can be obtained from a particular source, not necessarily directly from that source.

[0093] Selective Definition Serum albumin, or serum albumin, is the most abundant blood protein and functions as a carrier protein for steroids, fatty acids, and thyroid hormones in the blood, as well as playing a major role in stabilizing extracellular fluid volume. For nomenclature purposes only, and not for limitation, an exemplary sequence of human serum albumin is provided in NCBI GenBank Accession ID: AEE60908 and SEQ ID NO: 1. References to "serum albumin" or "albumin" should be understood to include preproalbumin, which includes the N-terminal peptide, proalbumin, and secreted albumin. Amino acid positions are referred to herein by reference to the 585-amino acid secreted albumin protein (e.g., as provided in SEQ ID NO: 1). Albumin contains three homologous domains, each of which is a product of two subdomains with a common structural motif. Domains I, II, and III can be defined with respect to human serum albumin (as defined in SEQ ID NO: 1). For example, Domain I comprises amino acids 1 (±1 to 15 amino acids) to 194 (±1 to 15 amino acids) of SEQ ID NO: 1, Domain II comprises amino acids 192 (±1 to 15 amino acids) to 387 (±1 to 15 amino acids) of SEQ ID NO: 1, and Domain III comprises amino acid residues 381 (±1 to 15 amino acids) to 585 (±1 to 15 amino acids) of SEQ ID NO: 1. The phrase "±1 to 15 amino acids" means that the amino acid residues may deviate by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids to the C-terminus and / or N-terminus of the recited amino acid positions. Exemplary domains I, II, and III are described by Dockal et al. (The Journal of Biological Chemistry, 1999, Vol. 274(41): 29303-29310) and Kjeldsen et al. (Protein Expression and Purification, 1998, Vol. 13: 163-169).

[0094] Additional sequences of serum albumins from other species (e.g., primate serum albumins (e.g., chimpanzee serum albumin, gorilla serum albumin, etc.), rodent serum albumins (e.g., hamster serum albumin, guinea pig serum albumin, mouse albumin, and rat serum albumin, etc.), bovine serum albumin, horse serum albumin, donkey serum albumin, rabbit serum albumin, goat serum albumin, sheep serum albumin, dog serum albumin, chicken serum albumin, and porcine serum albumin) can be determined using the sequences provided herein and / or within publicly available databases and / or (e.g., Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley‐Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press). (1989)).

[0095] As used herein, the phrase "corresponding to" when referring to an amino acid position within SEQ ID NO: 1 should be understood as a reference to an amino acid residue or position within an albumin sequence, and not necessarily a sequence that includes SEQ ID NO: 1. For example, a reference to "a position corresponding to amino acid 522 of SEQ ID NO: 1" in an albumin sequence that includes a 10 amino acid N-terminal truncation would necessarily refer to amino acid 512. In one example, serum albumin includes the sequence set forth in SEQ ID NO: 1.

[0096] Reference to a "functional fragment" of serum albumin should be understood as a reference to a fragment of serum albumin that retains and exhibits the functionality of serum albumin (i.e., the ability to bind to FcRn). The fragment may comprise or consist of another domain of albumin, a fragment of such a domain, or a combination thereof.

[0097] As used herein, an "amino acid substitution" refers to the replacement of an amino acid at a particular position in a polypeptide sequence with another amino acid. As used herein, the term "FcRn" refers to the fetal Fc receptor, also known as the Brambell receptor, which is a heterodimer of a truncated heavy chain of the major histocompatibility complex class I-like Fc receptor (FCGRT) and beta-2-microglobulin.

[0098] As used herein, the terms "variant" or "mutant" or "mutation" refer to serum albumin that has undergone one or more amino acid substitutions using well-known techniques for site-directed mutagenesis or other conventional methods. As used herein, the term "binds" with respect to the interaction of FcRn and serum albumin means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on a cell or protein.

[0099] As used herein, the phrases "enhanced affinity," "enhanced binding," or "higher levels of binding" in relation to the interaction of a serum albumin variant with FcRn are understood to mean that the serum albumin variant or functional fragment thereof binds or associates with FcRn more frequently, more rapidly, with longer persistence and / or with higher affinity (e.g., 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, 150-fold, or 200-fold) compared to human serum albumin set forth in SEQ ID NO: 1. For purposes of clarity, and as will be apparent to one of skill in the art based on the examples provided herein, references to "affinity" in this specification are references to the interaction, binding, or association of the serum albumin variant with FcRn.

[0100] For purposes of clarity, and as will be apparent to one of ordinary skill in the art based on the description herein, reference to "at least about an affinity" is understood to mean that the affinity is equal to or greater than the recited value (i.e., the recited value for affinity is less), i.e., an affinity of 2 nM is greater than an affinity of 3 nM. Alternatively stated, the term can be "an affinity of less than or equal to X," where X is a value recited herein.

[0101] As used herein, the term "serum half-life" or "plasma half-life," in the context of the present disclosure, refers to the time required for the concentration or amount of serum albumin in the body to be reduced by 50% (i.e., half) due to, for example, degradation and / or clearance or sequestration by natural mechanisms. Those skilled in the art will recognize that the serum half-life of a subject's serum albumin depends on various physiological conditions (e.g., health status, body size / weight). In a healthy human subject, the serum half-life of serum albumin is 19 days. Methods for measuring the serum half-life of serum albumin are known in the art and include, for example, pharmacokinetic analysis. For purposes of the present disclosure, a "prolonged" or "improved" serum half-life refers to an increase or prolongation of the time it takes for the serum concentration of a serum albumin variant to be reduced by 50% compared to serum albumin set forth in SEQ ID NO: 1.

[0102] The term "recombinant" shall be understood to mean the product of artificial genetic recombination. Thus, in the context of recombinant proteins comprising antibodies or antigen-binding domains thereof, this term does not encompass antibodies that occur naturally within a subject's body, which are the product of natural recombination that occurs during B-cell maturation. However, when such antibodies are isolated, they should be considered isolated proteins comprising an antibody antigen-binding domain. Similarly, when a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody antigen-binding domain. Recombinant proteins also encompass proteins expressed by artificial recombinant means when present within a cell, tissue, or subject, e.g., within the cell, tissue, or subject in which they are expressed.

[0103] The term "protein" shall be considered to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other covalently or non-covalently (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0104] It will be understood from the previous paragraph that the term "polypeptide" or "polypeptide chain" means a series of consecutive amino acids linked by peptide bonds.

[0105] As used herein, the term "antigen-binding domain" is understood to mean the region of an antibody capable of specifically binding to an antigen, i.e., a VH, or a VL, or an Fv comprising both a VH and a VL. The antigen-binding domain need not be in the context of a whole antibody, but can, for example, be in isolated form or in another form (such as an scFv, as described herein).

[0106] For purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to one or several closely related antigens (e.g., blood clotting factors) because the antigen-binding domain is contained in the Fv. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant antibodies, or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synhumanized antibodies, half antibodies, and bispecific antibodies). Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, IgY), or class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), or subclass. In one example, the antibody is a rodent (mouse or rat) antibody or a primate (e.g., human) antibody. In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is a humanized antibody, a synhumanized antibody, a chimeric antibody, a CDR-grafted antibody, or a deimmunized antibody.

[0107] As used herein, "variable region" refers to the portion of the light and / or heavy chain of an antibody defined herein that is capable of specifically binding to an antigen and includes the amino acid sequences of the complementarity-determining regions (CDRs), i.e., CDR1, CDR2, and CDR3, and the framework regions (FRs).

[0108] As used herein, the term "Fv" refers to the variable region of the light chain (V), whether composed of multiple polypeptides or a single polypeptide. L ) and the variable region of the heavy chain (V H and V associate to form a complex having an antigen-binding domain, i.e., capable of specifically binding to an antigen. H and V LThe V may be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of this disclosure (and any protein of this disclosure) may have multiple antigen-binding domains that may or may not bind to the same antigen. This term shall be understood to encompass fragments derived directly from antibodies and proteins corresponding to such fragments produced using recombinant means. In some examples, the V H is the heavy chain constant domain (C H ) 1 and / or V L is the light chain constant domain (C L Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetrabodies or higher order complexes, or constant regions or domains thereof, such as C H 2 or C H An "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digestion of whole antibody with the enzyme papain to yield a fragment consisting of an intact light chain and a portion of the heavy chain, or can be produced using recombinant means. An "Fab' fragment" of an antibody consists of an intact light chain and a portion of the heavy chain. H Fab' fragments can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of a portion of the heavy chain and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. Fab' fragments can also be produced by recombinant means. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments associated by two disulfide bonds and can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. "Fab2" fragments can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction, to yield a molecule consisting of a portion of the heavy chain containing a single constant domain, e.g., a leucine zipper or C HA "single-chain Fv" or "scFv" is a recombinant fragment containing two Fab fragments linked together using three domains. A "single-chain Fv" or "scFv" is a recombinant molecule containing an antibody variable region fragment (Fv) in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0109] An "antigen-binding fragment" of an antibody contains one or more variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, half antibodies, and multispecific antibodies formed from antibody fragments.

[0110] As used herein, the term "pathology" refers to a disruption or interference with normal function and is not limited to any particular pathology, but includes diseases or disorders.

[0111] As used herein, the term "bleeding condition" or "bleeding disorder" refers to a condition in which there is abnormal blood clotting, e.g., reduced or insufficient blood clotting ability, and / or abnormal bleeding (internal and / or external), e.g., excessive bleeding.

[0112] As used herein, "clotting factor" refers to a factor associated with the formation of a blood clot, i.e., blood clotting. In one example, a clotting factor has procoagulant activity. Clotting factors are known in the art and include, but are not limited to, Factor I, Factor II, Factor III, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, and Factor XIII, or activated forms of any of the above. The term also includes recombinant forms of clotting factors and / or modified forms thereof, for example, as known in the art and / or as described herein.

[0113] As used herein, a subject "at risk" of developing, or experiencing, or recurring with, a disease or condition may or may not have detectable disease or disease symptoms and may or may not exhibit detectable disease or disease symptoms prior to treatment according to the present disclosure. "At risk" denotes that the subject has one or more risk factors, which are measurable parameters that correlate with development of a disease or condition known in the art and / or described herein.

[0114] As used herein, the terms "treating," "treat," or "treatment" include administering a serum albumin variant conjugate described herein to reduce or eliminate at least one symptom of the specified disease or condition, or slow the progression of the disease or condition.

[0115] As used herein, the terms "preventing," "prevent" or "prevention" include providing prophylaxis against the occurrence or recurrence of a bleeding disorder or symptoms of a bleeding disorder in an individual who may be predisposed to or at risk of developing the disease or a recurrence of the disease, but who has not yet been diagnosed with the disease or recurrence.

[0116] An "effective amount" refers to an amount effective to achieve a desired result, at least at the dosage and for the duration necessary. For example, the desired result can be a therapeutic result or a prophylactic result. An effective amount can be administered in one or more administrations. In some examples of the present disclosure, the term "effective amount" refers to the amount necessary to achieve treatment of the disease or condition described above. In some examples of the present disclosure, the term "effective amount" refers to the amount necessary to achieve a change in a factor associated with the disease or condition described above. For example, an effective amount can be sufficient to achieve a change in coagulation levels. An effective amount can vary depending on the disease or condition to be treated or the factor to be changed, as well as on the body weight, age, ethnic background, sex, health and / or physical condition, and other factors associated with the mammal being treated. Typically, an effective amount falls within a relatively broad range (e.g., a "dosage" range) that can be determined by routine testing and experimentation by a physician. Therefore, this term should not be construed to limit the present disclosure to a specific amount of binding protein. An effective amount can be administered in a single administration or in one or several repeated administrations over a treatment period.

[0117] A "therapeutically effective amount" is the minimum concentration required to achieve at least a measurable improvement in a particular disease or condition. The therapeutically effective amount herein may vary depending on factors such as the patient's condition, age, sex, and weight, as well as the ability of the albumin conjugate to elicit a desired response in an individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the albumin conjugate are outweighed by the therapeutically beneficial effects. In one example, a therapeutically effective amount shall be considered to mean an amount of albumin conjugate sufficient to alleviate or suppress one or more symptoms of a bleeding disorder or its complications.

[0118] As used herein, the term "prophylactically effective amount" shall be taken to mean an amount of albumin conjugate sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a bleeding disorder or its complications.

[0119] As used herein, the term "subject" shall be taken to mean any animal, including humans, e.g., mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0120] Serum albumin variants The present disclosure provides serum albumin variants or functional fragments thereof having specified amino acid substitutions compared to the sequence set forth in SEQ ID NO: 1. In one example, the serum albumin variants or fragments thereof of the present disclosure comprise a sequence that is at least about 85%, 90%, 95%, 97%, 98%, or 99% identical to a sequence disclosed herein, wherein the serum albumin variants or fragments thereof bind to FcRn as described herein according to any of the Examples.

[0121] The present disclosure provides the following: (i) an amino acid selected from the group consisting of glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine, and leucine substituting for glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1; (ii) a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO: 1; (iii) an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine, and arginine substituting for glycine at a position corresponding to amino acid 572 of SEQ ID NO:1; and (iv) combinations thereof; The present invention provides a serum albumin variant or functional fragment thereof comprising one or more amino acid substitutions selected from the group consisting of:

[0122] In one example, the serum albumin variant or functional fragment thereof further comprises a tyrosine substituting for a lysine at a position corresponding to amino acid 573 of SEQ ID NO:1. For example, the inventors have identified several amino acid residues in the sequence set forth in SEQ ID NO: 1 that can be substituted without loss of function or that result in improved function. In one example, a serum albumin variant or functional fragment thereof is It comprises one to three amino acid substitutions at positions corresponding to amino acids 522, 552 and / or 572 compared to the sequence set forth in SEQ ID NO: 1. For example, the serum albumin variant or functional fragment thereof comprises one, two or three amino acid substitutions at positions corresponding to amino acids 522, 552 and / or 572 compared to the sequence set forth in SEQ ID NO: 1.

[0123] Optionally, the serum albumin variant or functional fragment thereof additionally comprises one amino acid substitution at the position corresponding to amino acid 573 compared to the sequence set forth in SEQ ID NO:1.

[0124] In one example, the serum albumin variant or functional fragment thereof comprises one to four amino acid substitutions at positions corresponding to amino acids 522, 552, and / or 572, and optionally amino acid 573, compared to the sequence set forth in SEQ ID NO:1. For example, the serum albumin variant or functional fragment thereof may be: It contains 1, 2, 3 or 4 amino acid substitutions at positions corresponding to amino acids 522, 552 and / or 5723, and optionally amino acid 573, relative to the sequence set forth in SEQ ID NO:1.

[0125] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a glycine at the position corresponding to amino acid 522 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least an isoleucine at the position corresponding to amino acid 522 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a lysine at the position corresponding to amino acid 522 of SEQ ID NO: 1.

[0126] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a methionine at the position corresponding to amino acid 522 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a phenylalanine at the position corresponding to amino acid 522 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a tryptophan at a position corresponding to amino acid 522 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a tyrosine at the position corresponding to amino acid 522 of SEQ ID NO: 1.

[0127] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a valine at a position corresponding to amino acid 522 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a leucine at the position corresponding to amino acid 522 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least an alanine at the position corresponding to amino acid 522 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a valine at the position corresponding to amino acid 552 of SEQ ID NO: 1.

[0128] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least an alanine at the position corresponding to amino acid 572 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a glutamic acid at a position corresponding to amino acid 572 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a histidine at the position corresponding to amino acid 572 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a serine at the position corresponding to amino acid 572 of SEQ ID NO: 1.

[0129] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a lysine at the position corresponding to amino acid 572 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least an arginine at the position corresponding to amino acid 572 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a valine at the position corresponding to amino acid 572 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a leucine at a position corresponding to amino acid 522 of SEQ ID NO: 1 and an arginine at a position corresponding to amino acid 572 of SEQ ID NO: 1.

[0130] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a leucine at a position corresponding to amino acid 522 of SEQ ID NO: 1 and a valine at a position corresponding to amino acid 552 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a leucine at a position corresponding to amino acid 522 of SEQ ID NO: 1 and a tyrosine at a position corresponding to amino acid 573 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a valine at a position corresponding to amino acid 552 of SEQ ID NO: 1 and a tyrosine at a position corresponding to amino acid 573 of SEQ ID NO: 1.

[0131] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least an arginine at a position corresponding to amino acid 572 of SEQ ID NO: 1 and a tyrosine at a position corresponding to amino acid 573 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a leucine at the position corresponding to amino acid 522 of SEQ ID NO: 1, a valine at the position corresponding to amino acid 552 of SEQ ID NO: 1, and an arginine at the position corresponding to amino acid 572 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a leucine at a position corresponding to amino acid 522 of SEQ ID NO: 1, a valine at a position corresponding to amino acid 552 of SEQ ID NO: 1, and a tyrosine at a position corresponding to amino acid 573 of SEQ ID NO: 1.

[0132] In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO: 1, wherein the variant sequence comprises at least a leucine at the position corresponding to amino acid 522 of SEQ ID NO: 1, an arginine at the position corresponding to amino acid 572 of SEQ ID NO: 1, and a tyrosine at the position corresponding to amino acid 573 of SEQ ID NO: 1. In one example, a serum albumin variant or functional fragment thereof of the present disclosure comprises a variant of the sequence set forth in SEQ ID NO:1, wherein the variant sequence comprises at least a leucine at the position corresponding to amino acid 522 of SEQ ID NO:1, a valine at the position corresponding to amino acid 552 of SEQ ID NO:1, an arginine at the position corresponding to amino acid 572 of SEQ ID NO:1, and a tyrosine at the position corresponding to amino acid 573 of SEQ ID NO:1.

[0133] Exemplary methods for making variant forms of serum albumin are described herein or known in the art and include the following: DNA mutagenesis (Thie et al., Methods Mol. Biol. 525: 309-322, 2009) or RNA mutagenesis (Kopsidas et al., Immunol. Lett. 107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7: 18, 2007; and WO1999 / 058661); introducing the nucleic acid encoding the polypeptide into mutagenized cells, such as XL-1Red bacterial cells, XL-mutS bacterial cells, and XL-mutS-Kanr bacterial cells (Stratagene); DNA shuffling, as disclosed, for example, in Stemmer, Nature 370: 389-91, 1994; and site-directed mutagenesis, as described, for example, in Dieffenbach (ed.) and Dveksler (ed.) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995); Includes:

[0134] Exemplary methods for measuring the biological activity of the disclosed serum albumin variants or functional fragments thereof will be apparent to those skilled in the art and are described herein, e.g., FcRn affinity. Methods for measuring the affinity of serum albumin variants or functional fragments thereof, including affinity, association, dissociation, and therapeutic efficiency, are described herein.

[0135] For example, the inventors have identified several amino acid residues in the sequence set forth in SEQ ID NO: 1 that can be substituted to extend the half-life of serum albumin. For example, a serum albumin variant or functional fragment thereof contains one or more amino acid substitutions that enhance the affinity of albumin for fetal Fc receptor (FcRn). In one example, the variant or functional fragment thereof binds with higher affinity to FcRn compared to serum albumin set forth in SEQ ID NO: 1. For example, the serum albumin variant or functional fragment thereof has a higher affinity for FcRn at a lower pH, e.g., about pH 6.0, thereby facilitating albumin / FcRn binding in endosomes. In one example, albumin has increased affinity for FcRn at about pH 6 compared to its affinity at pH 7.4, which facilitates re-release of albumin into the blood after refolding in cells. These amino acid substitutions are useful for extending the half-life of proteins by reducing clearance from the blood.

[0136] In one example, exemplary amino acid substitutions include Q522G, Q522I, Q522K, Q522M, Q522L, Q522F, Q522W, Q522Y, Q522V, A552V, G572A, G572E, G572H, G572S, G572K and G572R. In another example, exemplary amino acid substitutions include Q522G, Q522I, Q522K, Q522M, Q522L, Q522F, Q522W, Q522Y, Q522V, Q522A, A552V, G572A, G572E, G572H, G572S, G572K, G572V and G572R.

[0137] Conjugates In one example, the serum albumin variants or functional fragments thereof of the present disclosure are conjugated to and / or encapsulate another compound, for example, the compound selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a therapeutic protein, an imaging agent, a colloid, a toxin, a nucleic acid, a peptide, a protein, a small molecule, an antisense oligonucleotide, a short hairpin RNA (shRNA), an siRNA, an interfering RNA (RNAi), a ribozyme, a microRNA, a microRNA-adapted shRNA (shRNAmir), a DNA enzyme, and mixtures thereof.

[0138] The compound can be directly or indirectly bound to the serum albumin variant or functional fragment (e.g., a linker can be included in the case of indirect binding). Examples of compounds include radioisotopes (e.g., iodine-131, yttrium-90, or indium-111), detectable labels (e.g., fluorophores, fluorescent nanocrystals, or quantum dots), therapeutic compounds or proteins (e.g., chemotherapeutic agents, anti-inflammatory agents, or coagulation factors), colloids (e.g., gold), toxins (e.g., ricin or tetanus toxoid), nucleic acids, proteins (e.g., proteins containing the antigen-binding domain of an antibody), and mixtures thereof. In one example, the serum albumin variant or functional fragment thereof is conjugated to a coagulation factor.

[0139] For example, the compound is a protein and is conjugated to a serum albumin variant or a functional fragment thereof via an amine bond. In one example, the disclosure provides a fusion protein comprising a serum albumin variant or functional fragment thereof and a compound (e.g., a therapeutic protein, such as a clotting factor), where the compound is positioned at the N-terminus of the serum albumin variant or functional fragment thereof, at the C-terminus of the serum albumin variant or functional fragment thereof, inserted within a loop of the serum albumin variant or functional fragment thereof, or any combination thereof.

[0140] Exemplary compounds that can be conjugated to the serum albumin variants of the present disclosure, and methods for such conjugation, are known in the art and further described herein.

[0141] radioactive isotope In one example, the present disclosure provides a serum albumin variant or functional fragment thereof conjugated to a radioisotope.

[0142] Suitable radioisotopes for use in the present disclosure will be apparent to those of skill in the art, and include, for example, iodine-123 ( 123 I), iodine-125( 125 I), iodine-130( 130 I), iodine-133( 133 I), iodine-135( 135 I), Scandium-47( 47 Sc), arsenic-72( 72 As), Scandium-72( 72 Sc), Yttrium-90( 90 Y), yttrium-88( 88 Y), Ruthenium-97( 97 Ru), Palladium-100 ( 100 Pd), rhodium-101m( 101 mRh), antimony-119( 119 Sb), Barium-128( 128 Ba), mercury-197( 197Hg), astatine-211( 211 At), Bismuth-212( 212 Bi), samarium-153( 153 Sm), europium-169( 169 Eu), lead-212( 212 Pb), Palladium-109( 109 Pd), Indium 111 ( 111 In), 67 Gu, 68 Gu, copper-67( 67 Cu), Bromine-75( 75 Br), Bromine-76( 76 Br), Bromine-77( 77 Br), Technetium 99m ( 99 mTc), carbon-11 ( 11 C), nitrogen-13( 13 N), oxygen-15( 15 O), iodine-18( 18 I), rhenium-188( 188 Re), lead-203( 203 Pb), Copper 64( 64 Cu), Rhodium-105( 105 Rh), Gold-198( 198 Au), Argon-199( 199 Ag) or lutetium-177( 177 Lu).

[0143] Detectable Label In one example, the disclosure provides a serum albumin variant or functional fragment thereof conjugated to a detectable label, for example, the detectable label is a fluorophore, a fluorescent nanocrystal, or a quantum dot.

[0144] The term "fluorophore" refers to a chemical that absorbs light of a particular wavelength and either fluoresces or re-emits light at a longer wavelength. Fluorophores can fluoresce in the ultraviolet spectrum (10 nm to 400 nm), the visible spectrum (400 nm to 700 nm), or the near-infrared range (680 nm to 100,000 nm).

[0145] Suitable fluorophores for use in the present disclosure will be apparent to the skilled artisan and include, for example, indocyanine green, IRDye78, IRDye80, IRDye38, IRDye40, IRDye41, IRDye700, IRDye800, IRDye800CW, Cy5, Cy5.5, Cy7, IR-786, DRAQ5NO, Licor NIR, Alexa Fluor 488, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, La Jolla Blue, R-phycoerythrin (PE), hydroxycoumarin, methoxycoumarin, aminocoumarin, Fluorescein FITC, Rhodamine Red-X, Texas Red, Allophycocyanin (APC) and analogs thereof.

[0146] In one example, detectable label is quantum dot.Quantum dot is semiconductor nanocrystal with size-dependent optical and electronic properties.The exemplary material suitable for use as quantum dot includes ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaN, GaP, GaAs, GaSb, InP, InAs, Sb, AlS, AlP, AlAs, AlSb, PbS, PbSe, Ge and Si, and its ternary and quaternary mixtures.

[0147] In one example, the detectable label is a fluorescent protein. Suitable fluorescent proteins for use in the present disclosure will be clear to those skilled in the art, and include, for example, Renilla luciferase, green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFB) and its derivatives.

[0148] biological entities clotting factors The present disclosure provides a serum albumin variant or functional fragment thereof conjugated to a clotting factor.

[0149] Blood coagulation occurs through a series of steps involving the release of several clotting factors, ultimately resulting in the formation of a clot containing insoluble fibrin. Exemplary clotting factors include, but are not limited to, Factor I (fibrinogen), Factor II (prothrombin / thrombin), Factor III (tissue factor), Factor V (labile factor), Factor VII (proconvertin), Factor VIII (antihemophilic factor), Factor IX (Christmas factor), Factor X (Stuart-Blauer factor), Factor XI (plasma thromboplastin precursor), Factor XII (Hageman (contact) factor), and Factor XIII (fibrin-stabilizing factor / prekallikrein (Fletcher) factor / HMWK (Fitzgerald) factor).

[0150] For example, the compound is a clotting factor and is conjugated to a serum albumin variant or functional fragment thereof by an amine bond. In one example, the disclosure provides a fusion protein comprising a serum albumin variant or functional fragment thereof and a clotting factor, for example, the clotting factor is located at the N-terminus of the serum albumin variant or functional fragment thereof, at the C-terminus of the serum albumin variant or functional fragment thereof, inserted within a loop of the serum albumin variant or functional fragment thereof, or any combination thereof.

[0151] In one example, the coagulation factor is Factor VIII. For purposes of nomenclature only, and not limitation, an exemplary sequence of human Factor VIII is provided in NCBI Ref Seq ID NP_000123, protein accession number NM_000132.3 and SEQ ID NO:2. In one example, the clotting factor is Factor IX. For purposes of nomenclature only, and not limitation, an exemplary sequence of human Factor IX is provided in GenBank ID AAA98726.1 and SEQ ID NO:3.

[0152] In one example, the clotting factor is Factor X. For purposes of nomenclature only, and not limitation, an exemplary sequence of human Factor X is provided in Gene ID: 2159 and SEQ ID NO: 4. In one example, the clotting factor is Factor VII. For purposes of nomenclature only, and not limitation, an exemplary sequence of human Factor VII is provided in Ref Seq ID NM_00131 and SEQ ID NO:5.

[0153] For purposes of nomenclature only, and not limitation, exemplary sequences of human Factor I are provided in NCBI Ref Seq ID NM_000508 (alpha chain) and NM_005141 (beta chain), exemplary sequences of human Factor II are provided in Ref Seq ID NM_000506, exemplary sequences of human Factor III are provided in Ref Seq ID NM_001993, exemplary sequences of human Factor V are provided in Ref Seq ID NM_000130, exemplary sequences of human Factor XI are provided in Ref Seq ID NM_000128, exemplary sequences of human Factor XII are provided in Ref Seq ID NM_000505, and exemplary sequences of human Factor XIII are provided in Ref Seq ID NM_000129 (A chain) and NM_001994 (B chain).

[0154] Additional sequences of coagulation factors can be determined using the sequences presented herein and / or in publicly available databases and / or can be determined using standard techniques (e.g., as described in Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988) (including all current editions) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

[0155] Exemplary clotting factors can be plasma or recombinant proteins derived from a donor, e.g., the clotting factors are plasma-derived or recombinant clotting factor proteins. For example, the therapeutic protein can be factor I (fibrinogen), factor II ((prothrombin) / thrombin), factor III (tissue factor), factor V (labile factor), factor VII (proconvertin), factor VIIa (e.g., NovoSeven®), factor VIII (antihemophilic factor; e.g., single-chain recombinant factor VIII as described in Zollner et al., Thromb Res. 132:280-287, 2013); or plasma-derived factor VIII products such as FEIBA®, Monoclate-P®, or Biostate®; or factor VIII products such as Advate®, Eloctate®, Recombinate®, Kogenate Fs®, Helixate® Fs, Helixate®, Xyntha® / Refacto®, or other recombinant factor VIII products. recombinant factor VIII products such as Ab®, Hemofil-M®, Monarc-M®, Alphanate®, Koate-Dvi®, Nuwiq® or Hyate:C®), factor IX (Christmas factors, plasma-derived factor IX products such as Berinin® P, MonoFIX® or Mononine®; or Alphanine SD®, Alprolix®, Bebulin®, Bebulin V H(recombinant factor IX products such as Benefix®, Benefix®, Ixinity®, Profilnine SD®, Proplex T®, or Rixubis®), factor X (Stuart-Blauer factor), factor XI (plasma thromboplastin precursor), factor XII (Hageman (contact) factor), and factor XIII (fibrin-stabilizing factor / prekallikrein (Fletcher) factor / HMWK (Fitzgerald) factor; e.g., Fibrogammin® P, Corifact®, Cluvot®, or Cluviat®). In one example, the therapeutic protein is a von Willebrand factor / FVIII complex (e.g., Humate-P®, Haemate®-P, Biostate®, or Voncento®). In an alternative example, the therapeutic protein is a prothrombin complex (e.g., Beriplex® P / N, Confidex®, or Kcentra®). In another example, the therapeutic protein is fibrinogen (e.g., RiaSTAP®, Haemocomplettan® P).

[0156] von Willebrand factor The present disclosure provides a serum albumin variant or functional fragment thereof conjugated to von Willebrand factor.

[0157] As used herein, the term "von Willebrand factor" (vWF) includes naturally occurring (native) vWF, but also includes variants thereof that retain the biological activity of naturally occurring vWF, such as fragments, fusion proteins, or conjugates, or sequence variants in which one or more residues are inserted, deleted, or substituted. For nomenclature purposes only, and not for limitation, an exemplary sequence of human native vWF is provided in NCBI Ref Seq ID: NP_000543.2 and SEQ ID NO: 6. Those skilled in the art will understand that native vWF contains multiple domains. For purposes of this disclosure, the following annotation has been established: D1-D2-D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK (wherein the D' domain consists of amino acids 764-865 of SEQ ID NO:6, the D3 domain consists of amino acids 866-1242 of SEQ ID NO:6, and the C1 domain consists of amino acids 2255-2328 of SEQ ID NO:6).

[0158] In one example, the vWF is a modified, mutant, or variant vWF. For example, the modified vWF for use in the present disclosure includes the D'D3 domain and modified forms thereof, such as truncated or mutated forms thereof. For example, the modified vWF includes amino acids 764 to 1242 of SEQ ID NO: 6. In one example, the von Willebrand factor is recombinant von Willebrand factor.

[0159] Soluble complement inhibitors The present disclosure provides a serum albumin variant or functional fragment thereof conjugated to a soluble complement inhibitor.

[0160] The complement system includes many cell surface and soluble proteins that play a role in the elimination of foreign microorganisms and also protect the host from complement-associated damage. The three pathways of the complement system include the classical pathway (involving C1q, C1r, C1s, C4, C2, and C3 components), the lectin pathway, and the alternative pathway. Four complement receptors have been described: CR1 (CD35), CR2 (CD21), CR3 (CD11b / CD18), and CR4 (CD11c / CD18). CR1 is the primary regulator of complement system activation of plasma proteins. In one example, the serum albumin variant or functional fragment thereof is conjugated to a soluble complement inhibitor or a modified (ie, mutant) form thereof.

[0161] Suitable complement inhibitors for use in the present disclosure will be apparent to those of skill in the art and include, for example, Factor I (fI), Factor H (fH), C4b-binding protein (C4bp), soluble CD55 (decay accelerating factor (DAF)), C1 inhibitor (C1-INH or C1 esterase inhibitor); soluble CD35 (sCR1); soluble CD46 (membrane cofactor protein (MCP)), soluble CD59 (protectin), TT30 (CR2-fH), cobra venom factor (CVF) and functional fragments or variants thereof.

[0162] In one example, complement inhibitors are soluble complement receptor type 1 (sCR1), also known as type 1 complement receptor; CD35; C3BR; C3b / C4b receptor and TP10.For example, the soluble complement inhibitors for use in the present disclosure are modified or mutated sCR1.sCR1 and mutated sCR1 molecules are clear to those skilled in the art and are described in, for example, WO1991016437, WO1994000571 and WO1997031944.

[0163] In one example, the complement inhibitor is C1 inhibitor (C1-INH), also known as C1 esterase inhibitor, serpin family G member 1 (SERPING1), HAE1, HAE2, C1NH, and C1IN. For example, a complement inhibitor for use in the present disclosure is a modified or mutated C1-INH. In one example, the C1-INH is plasma-derived C1-INH. In another example, the C1-INH is recombinant C1-INH. C1-INH and mutated C1-INH molecules will be apparent to those skilled in the art and include, for example, Berinert®. Other suitable C1-INH and mutated C1-INH molecules are described, for example, in WO2016070156.

[0164] toxin The present disclosure provides a serum albumin variant or functional fragment thereof conjugated to a toxin.

[0165] Suitable toxins for use in the present disclosure will be apparent to those of skill in the art and include, for example, ricin, abrin, diphtheria toxin, tetanus toxoid, Pseudomonas exotoxin A (PE), and ribosomal inactivating proteins such as gelonin, pokeweed antiviral protein, and saporin.

[0166] chemotherapy compounds The present disclosure provides a serum albumin variant or functional fragment thereof conjugated to a chemotherapeutic compound.

[0167] Suitable chemotherapeutic compounds for use in the present disclosure will be apparent to those of skill in the art and include, for example, caboplatin, cisplatin, cyclophosphamide, decetaxel, doxorubicin, erlotinib, etoposide, fluorouracil, irinotecan, methotrexate, paclitaxel, topotecan, vincristine, vinblastine, methotrexate, l-asparaginase, vincristine, doxorubicin, danorubicin, cytarabine, idarubicin, mitoxantrone, cyclophosphamide, fludarabine, chlorambucil, and derivatives thereof.

[0168] Antibodies or antigen-binding fragments In one example, the disclosure provides a serum albumin variant or functional fragment thereof conjugated to an antibody or antigen-binding fragment.

[0169] Exemplary antibodies or antigen-binding fragments thereof for use in the present disclosure are described herein or known in the art and include: Humanized antibodies or fragments thereof, e.g., proteins comprising human-like variable regions, comprising CDRs from antibodies from non-human species (e.g., mouse, rat, or non-human primate) grafted or inserted into FRs from a human antibody (e.g., produced by the methods described in US5225539, US6054297, US7566771, or US5585089).

[0170] Human antibodies or fragments thereof, e.g., antibodies having variable regions, and optionally constant antibody regions, found in humans, e.g., human germline or somatic cells, or from libraries generated using such regions. "Human" antibodies may include amino acid residues not encoded by human sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro (e.g., produced by the methods described in U.S. Pat. No. 5,565,332), and affinity-matured forms of such antibodies.

[0171] A synhumanized antibody or fragment thereof, for example, an antibody (e.g., produced by the methods described in WO2007019620) comprising a variable region comprising FRs from a New World primate antibody variable region and CDRs from a non-New World primate antibody variable region. Primatized antibodies or fragments thereof, e.g., comprising antibody variable region(s) from antibodies generated following immunization of a non-human primate (e.g., a cynomolgus macaque) (e.g., generated by the methods described in U.S. Pat. No. 6,113,898).

[0172] Chimeric antibodies or chimeric antigen-binding fragments, for example antibodies or fragments in which one or more of the variable domains are derived from a particular species (e.g., murine, such as mouse or rat) or belong to a particular antibody class or subclass, and the remainder of the antibody or fragment is derived from another species (e.g., human or non-human primate) or belongs to another antibody class or subclass (e.g., produced by the methods described in US6331415; US5807715; US4816567 and US4816397).

[0173] Deimmunized antibodies or antigen-binding fragments thereof, for example, antibodies and fragments having one or more epitopes, e.g., B-cell epitopes or T-cell epitopes, removed (i.e., mutated) to thereby reduce the likelihood that a subject will mount an immune response against the antibody or protein (e.g., as described in WO2000034317 and WO2004108158).

[0174] Bispecific antibodies or fragments thereof, for example antibodies comprising two types of antibodies or antibody fragments (e.g. two half antibodies) with specificity for different antigens or epitopes (e.g. as described in US5731168).

[0175] Additional exemplary antibody fragments for use in the present disclosure are described herein or known in the art and include: Single domain antibodies (domain antibodies or dAbs), for example, a single peptide chain comprising all or part of the heavy chain variable domain of an antibody.

[0176] diabodies, triabodies, tetrabodies or higher order protein complexes (for example those described in WO98 / 044001 and / or WO94 / 007921). Single-chain Fv (scFv) fragments, e.g., V in a single polypeptide chain H and V LThe scFv forms the desired structure for antigen binding (i.e., the V domain of a single polypeptide chain). H and V L V allows V to associate with each other to form Fv H and V L and a polypeptide linker therebetween. Half antibodies or half molecules, e.g., proteins containing a single heavy chain and a single light chain.

[0177] The present disclosure also provides (i) minibodies, e.g., as described in US5837821; (ii) heteroconjugate proteins, for example those described in US 4,676,980; (iii) heteroconjugate proteins prepared using chemical crosslinkers, for example, as described in US4676980, and (iv) Fab3 (e.g., as described in EP19930302894), Other antibodies and antibody fragments such as are also contemplated.

[0178] Protein backbone In one example, the disclosure provides a serum albumin variant or functional fragment thereof conjugated to a protein scaffold, for example, the protein scaffold is an immunoglobulin or immunoglobulin fragment.

[0179] Suitable protein scaffolds for use in the present disclosure are described herein or will be apparent to those of skill in the art and include: Heavy chain immunoglobulins, such as immunoglobulins (e.g. antibodies) that do not contain light chains (e.g. as described in WO9404678, WO9749805 and WO9749805). V-like proteins, such as T cell receptors having two V domains combined in a structure similar to the Fv module of an antibody (e.g., as described in Novotny et al., Proc Natl Acad Sci USA 88: 8646-8650, 1991, WO1999045110 or WO2011107595).

[0180] Adnectins, for example immunoglobulins based on the tenth fibronectin type III (10Fn3) domain of human fibronectin in which the loop regions have been modified to provide antigen binding (e.g., as described in US20080139791 or WO2005056764).

[0181] Anticalins, for example lipocalin-derived immunoglobulins (e.g., as described in US7250297 or US20070224633) that have a rigid beta-sheet secondary structure with multiple loops at the open end of a conical structure that can be modified to bind antigens.

[0182] Affibodies, for example scaffolds derived from the Z domain (antigen binding domain) of Staphylococcus aureus protein A (for example as described in EP1641818), which can be engineered to bind to an antigen.

[0183] Avimers, for example multidomain proteins derived from the A-domain scaffold family (for example as described in WO2002088171). Ankyrin repeat proteins (DARPins), such as those from the ankyrin family of proteins that mediate the attachment of essential membrane proteins to the cytoskeleton, which can be modified to bind different target antigens (e.g., as described in US20040132028).

[0184] small molecule In another example, the binding molecule is a small molecule. Such small molecules can be isolated from libraries. Chemical small molecule libraries can be obtained commercially or can be prepared using methods known in the art (e.g., the methods described in U.S. Pat. No. 5,463,564).

[0185] Techniques for synthesizing small organic compounds will vary widely depending on the compound, but such methods will be known to those skilled in the art.

[0186] In one example, informatics is used to select appropriate chemical building blocks from known compounds to create combinatorial libraries. For example, the QSAR (quantitative structure-activity relationship) modeling approach uses linear regression or regression trees of compound structures to determine suitability. Software from Chemical Computing Group, Inc. (Montreal, Canada) uses experimental data from high-throughput screening of active and inactive compounds to create probabilistic QSAR models, which are then used to select lead compounds. The binomial QSAR method relies on three characteristic properties of compounds that form "descriptors" of the likelihood that a particular compound will or will not meet a desired function: partial charge, molar refractive index (binding interactions), and logP (lipophilicity of the molecule). Each atom in a molecule has a surface area, which contains these three properties associated with the atom. All atoms in a compound with a range of partial charges are identified, and the surface areas (van der Waals surface area descriptors) are summed. The binomial QSAR model is then used to create activity or ADMET models, which are then used to build combinatorial libraries. Thus, lead compounds identified in the initial screen can be used to expand the list of compounds screened, thereby identifying highly active compounds.

[0187] Nucleic acid-based agents In one example, the present disclosure provides a serum albumin variant or a functional fragment thereof conjugated to a nucleic acid-based agent. Suitable agents will be apparent to those skilled in the art, and include, for example, antisense oligonucleotides, short hairpin RNA (shRNA), siRNA, interfering RNA (RNAi), ribozymes, microRNAs, and DNA enzymes.

[0188] antisense oligonucleotides In one example, the nucleic acid-based agent is an antisense oligonucleotide or an antisense nucleic acid.

[0189] The term "antisense nucleic acid" refers to DNA or RNA or its derivatives (e.g., LNA or PNA), or a combination thereof, that are complementary to at least a portion of a specific mRNA molecule encoding a polypeptide described herein in any example of the present disclosure and are capable of interfering with post-transcriptional events such as mRNA translation. The use of antisense methods is well known in the art (see, for example, Hartmann and Endres (editors), Manual of Antisense Methodology, Kluwer (1999)).

[0190] The antisense nucleic acids of the present disclosure hybridize to a target nucleic acid under physiological conditions. Antisense nucleic acids include sequences corresponding to structural genes or coding regions, or sequences that affect gene expression or splicing control. For example, the antisense nucleic acid may correspond to a target coding region of a nucleic acid, or the 5'-untranslated region (UTR) or 3'-UTR, or a combination thereof. The antisense nucleic acid may be partially complementary to, for example, an intronic sequence that may be spliced ​​out during or after transcription to only exon sequences of the target gene. The length of the antisense sequence should be at least 19 contiguous nucleotides of the nucleic acid, e.g., at least 50 nucleotides, such as at least 100, 200, 500, or 1000 nucleotides. Full-length sequences complementary to the entire gene transcript may also be used. The length may be 100-2000 nucleotides. The degree of identity of the antisense sequence to the target transcript should be at least 90%, e.g., 95-100%.

[0191] catalytic nucleic acid In one example, the nucleic acid-based agent is a catalytic nucleic acid.

[0192] The term "catalytic nucleic acid" refers to a DNA or DNA-containing molecule (also known in the art as a "deoxyribozyme" or "DNAzyme") or an RNA or RNA-containing molecule (also known as a "ribozyme" or "RNAzyme") that specifically recognizes a particular substrate and catalyzes the chemical modification of that substrate. The nucleobases in a catalytic nucleic acid can be the bases A, C, G, T (and U for RNA).

[0193] Typically, catalytic nucleic acids contain an antisense sequence for specific recognition of a target nucleic acid and a nucleic acid cleavage enzyme activity (also referred to herein as a "catalytic domain"). Types of ribozymes useful in the present disclosure are hammerhead ribozymes and hairpin ribozymes.

[0194] RNA interference In one example, the nucleic acid-based agent is a small interfering RNA ("siRNA") molecule.

[0195] RNA interference (RNAi) is useful for specifically inhibiting the production of specific proteins.Without wishing to be bound by any particular theory, this technology relies on the existence of dsRNA molecules that contain essentially identical sequences or parts of the mRNA of target gene.Advantageously, dsRNA can be produced from a single promoter in recombinant vector host cell, where sense and antisense sequences are flanked by non-related sequences that allow sense and antisense sequences to hybridize and form dsRNA molecules with non-related sequences that form loop structures.The design and creation of suitable dsRNA molecules for the present disclosure are within the capabilities of those skilled in the art.

[0196] The length of the hybridizing sense and antisense sequences should be at least 19 contiguous nucleotides, e.g., at least 30 or 50 nucleotides, such as at least 100, 200, 500, or 1000 nucleotides. Full-length sequences complementary to the entire gene transcript may be used. The length may be 100 to 2000 nucleotides. The degree of identity of the sense and antisense sequences to the target transcript should be at least 90%, e.g., at least 85%, e.g., 95 to 100%.

[0197] Exemplary small interfering RNA (siRNA) molecules contain a nucleotide sequence identical to about 19-21 contiguous nucleotides of a target mRNA. For example, the siRNA sequence begins with the dinucleotide AA, contains about 30-70% (e.g., 30-60%, 40-60%, about 45%-55%, etc.) GC content, and does not share a high percentage of identity with any nucleotide sequence other than the target in the genome of the subject into which it is introduced, as determined, for example, by a standard BLAST search.

[0198] Assay of serum albumin mutant activity Serum albumin variants of the present disclosure are readily screened for biological activity, for example, as described below.

[0199] Affinity determination Optionally, the dissociation constant (Kd), binding constant (Ka) or affinity constant (K) of the serum albumin variant or functional fragment thereof D ) is determined. Affinity measurements can be measured by standard methodologies, such as immunoassays, surface plasmon resonance (SPR; e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) (Rich and Myszka Curr. Opin. Biotechnol 11: 54, 2000; Englebienne Analyst. 123: 1599, 1998)), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art.

[0200] In some instances, the serum albumin variant or functional fragment thereof has a similar K D or improved K D (i.e., a lower K D The binding affinity to FcRn can also be determined non-quantitatively using flow cytometry. For example, CHO cells stably expressing a serum albumin variant or a functional fragment thereof are stained at acidic (pH 5.5) and neutral (pH 7.4) pH with alexa-488-labeled FcRn / β2m (to detect target binding) and anti-myc-alexa647 (to detect expression) and analyzed by flow cytometry. Relative binding to FcRn / β2m is determined, for example, by calculating the mean fluorescence intensity relative to unmodified serum albumin (e.g., as defined in SEQ ID NO: 1).

[0201] Determination of half-life The serum albumin variants or functional fragments thereof encompassed by the present disclosure have improved half-lives, for example, are modified to have a longer half-life compared to serum albumin set forth in SEQ ID NO: 1 (i.e., unmodified serum albumin). Methods for identifying serum albumin variants or functional fragments thereof with extended half-lives will be apparent to those skilled in the art. For example, the ability of the serum albumin variant or functional fragment thereof to bind to fetal Fc receptor (FcRn) is assessed. In this regard, increased binding affinity to FcRn increases the serum half-life of the serum albumin variant or functional fragment thereof (see, for example, Kim et al., Eur J Immunol., 24:2429, 1994).

[0202] The half-life of the CD131 binding proteins of the present disclosure can also be measured by pharmacokinetic studies, for example, according to the method described in Kim et al., Eur J of Immunol 24:542, 1994. According to this method, radiolabeled CD131 binding proteins are intravenously injected into mice, and their plasma concentrations are measured periodically as a function of time (e.g., from 3 minutes to 72 hours after injection). The resulting clearance curve should be biphasic, i.e., have an α-phase and a β-phase. To determine the in vivo half-life of the protein, the clearance rate in the β-phase is calculated and compared to the clearance rate of the wild-type or unmodified protein (i.e., as defined in SEQ ID NO: 1).

[0203] In vitro cell assay A variety of in vitro assays are available for assessing the ability of a serum albumin variant or functional fragment thereof to treat the diseases or conditions described herein. In one example, the uptake and recycling of serum albumin variants or functional fragments thereof is tested in an in vitro cellular assay.

[0204] Methods for assessing cellular uptake and regeneration are known in the art and / or are exemplified herein. For example, fluorescently labeled serum albumin variants or their functional fragments are incubated with cells that express human FcRn receptors on their cell surface. After adding labeled serum albumin variants, the progress of protein regeneration can be tracked by a method incorporating flow cytometry and fluorescence microscopy (e.g., confocal fluorescence microscopy) and compared with unmodified serum albumin protein. The alterations in the normal regeneration pathway for specific serum albumin variants can be identified and characterized.

[0205] Serum albumin variants or functional fragments thereof that are found to be effectively refolded are identified as variants of the present disclosure.

[0206] Pharmacokinetic analysis In one example, the pharmacokinetic (PK) properties of a serum albumin variant or functional fragment thereof are evaluated. Methods for evaluating PK properties are known in the art and / or are exemplified herein.For example, serum albumin variants are injected into transgenic mice that express human FcRn receptors or other suitable mammalian hosts (e.g., rats, cynomolgus monkeys).In one example, the transgenic mice that express human FcRn receptors are "hFcRn Tg32" homozygous conjugate mice (i.e., B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ; The Jackson Laboratory stock number No.014565; or as described in Roopenian et al., J. Immunol 2003;170:3528-3533).The plasma level of serum albumin is evaluated by ELISA using commercially available methods.

[0207] Pharmaceutical Composition Suitably, in compositions or methods for administering the disclosed serum albumin variants or functional fragments thereof to a subject, the serum albumin variants or functional fragments thereof (i.e., the serum albumin variants or functional fragments thereof conjugated to a compound) are combined with a pharmaceutically acceptable carrier, as understood in the art. Thus, in one example of the present disclosure, a composition (e.g., a pharmaceutical composition) is provided comprising the disclosed serum albumin conjugates in combination with a pharmaceutically acceptable carrier.

[0208] Generally, the term "carrier" refers to a solid or liquid filler, binder, diluent, encapsulating substance, emulsifier, wetting agent, solvent, suspending agent, coating agent, or lubricant that can be safely administered to any subject, for example, a human. Depending on the specific route of administration, various acceptable carriers known in the art can be used, for example, as described in Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ USA, 1991).

[0209] The serum albumin conjugates of the present disclosure are useful for parenteral, topical, oral, local, aerosol, or transdermal administration for prophylactic or therapeutic treatment. In one example, the serum albumin conjugates are administered parenterally, for example, subcutaneously or intravenously. For example, the serum albumin conjugates are administered intravenously.

[0210] The formulation of the serum albumin conjugate to be administered will vary depending on the route of administration and formulation (e.g., solution, emulsion, capsule) selected. Suitable pharmaceutical compositions containing the serum albumin conjugate to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including, for example, saline and buffered media. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Various appropriate aqueous carriers are known to those skilled in the art and include water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution, and glycine. Intravenous vehicles may contain various additives, preservatives, or fluid, nutrient, or electrolyte replenishers (see generally Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions may optionally contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents and toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate, as needed to approximate physiological conditions. The serum albumin conjugates may be stored in a liquid state, or lyophilized for storage according to art-known lyophilization and reconstitution techniques and reconstituted in a suitable carrier prior to use.

[0211] Conditions being treated As discussed herein, the present disclosure provides a method of treating or preventing a disease or condition in a subject, the method comprising administering a serum albumin conjugate of the present disclosure or a composition of the present disclosure to a subject in need thereof. In one example, the present disclosure provides a method of treating a disease or condition in a subject in need thereof.

[0212] The present disclosure also provides a use of a serum albumin conjugate of the present disclosure for treating or preventing a disease or condition in a subject, comprising administering a serum albumin conjugate of the present disclosure or a composition of the present disclosure to a subject in need thereof. In one example, the present disclosure provides a use of a serum albumin conjugate of the present disclosure for treating a disease or condition in a subject in need thereof. In one example, the disease or condition is a bleeding disorder.

[0213] In one example, the subject suffers from a bleeding disorder. The bleeding disorder may be inherited or acquired. For example, the subject suffering from a bleeding disorder may, for example, have one of the following: easy bruising; bleeding gums; Heavy bleeding from small cuts or dental scars; Nosebleeds of unknown origin; Heavy menstrual bleeding; Hemarthrosis; and / or excessive bleeding after surgery, Suffering from bleeding disorders such as

[0214] In one example, a subject is at risk for developing a bleeding disorder. A subject is at risk if they have a higher risk of developing a bleeding disorder than a control population. The control population may have angina, stroke, and / or Or it may include one or more subjects randomly selected from the general population (e.g., matched by age, sex, race and / or ethnicity) who have not suffered from a heart attack or have a family history of angina, stroke and / or heart attack. A subject can be considered to be at risk for a bleeding disorder if a "risk factor" associated with a bleeding disorder is found to be associated with the subject. Risk factors can include, for example, any activity, trait, event or characteristic associated with a given disorder through statistical or epidemiological studies on a population of subjects. Thus, a subject can be classified as at risk for a bleeding disorder even if the study identifying potential risk factors does not specifically include the subject. For example, a subject with excessive bleeding is at risk for developing a bleeding disorder because the frequency of bleeding disorders is increased in a population of subjects with excessive bleeding compared to a population of subjects without excessive bleeding.

[0215] In one example, the subject is at risk of developing a bleeding disorder, and the serum albumin conjugate is administered before or after the onset of symptoms of the bleeding disorder. In one example, the serum albumin conjugate is administered before the onset of symptoms of the bleeding disorder. In one example, the serum albumin conjugate is administered after the onset of symptoms of the bleeding disorder. In one example, the serum albumin conjugate of the present disclosure is administered at a dose that alleviates or reduces one or more symptoms of the bleeding disorder in the at-risk subject.

[0216] The methods of the present disclosure can be readily applied to any form of bleeding disorder in a subject.

[0217] The methods of the present disclosure may also include the co-administration of a serum albumin conjugate according to the present disclosure in conjunction with the administration of another therapeutically active agent for the prevention or treatment of a bleeding disorder.

[0218] In one example, the serum albumin conjugate of the present disclosure is used in combination with at least one additional known compound or therapeutic protein that is currently being used or under development for the prevention or treatment of bleeding disorders.The compounds currently used for the treatment of bleeding disorders are known in the art.Exemplary therapeutic proteins can be donor-derived plasma or recombinant proteins.For example, therapeutic proteins are plasma-derived or recombinant coagulation factor proteins. For example, the therapeutic protein can be factor I, factor II ((prothrombin) / thrombin), factor III, factor V, factor VII, factor VIIa (e.g., NovoSeven®), factor VIII (e.g., single-chain recombinant factor VIII as described in Zollner et al., Thromb Res. 132:280-287, 2013; or plasma-derived factor VIII products such as FEIBA®, Monoclate-P®, or Biostate®; or Advate®, Eloctate®, Recombinate®, Kogenate Fs®, Helixate® Fs, Helixate®, Xyntha® / Refacto®, or other recombinant factor VIII products. recombinant factor VIII products such as Ab®, Hemofil-M®, Monarc-M®, Alphanate®, Koate-Dvi®, Nuwiq®, or Hyate:C®), factor IX (e.g., plasma-derived factor IX products such as Berinin® P, MonoFIX®, or Mononine®; or Alphanine SD®, Alprolix®, Bebulin®, Bebulin V, H(Recombinant factor IX products such as Fibrogammin®, Benefix®, Ixinity®, Profilnine SD®, Proplex T®, or Rixubis®), factor X, factor XI, factor XII, and factor XIII (e.g., Fibrogammin® P, Corifact®, Cluvot®, or Cluviat®). In one example, the therapeutic protein is a von Willebrand factor / FVIII complex (e.g., Humate-P®, Haemate®-P, Biostate®, or Voncento®). In an alternative example, the therapeutic protein is a prothrombin complex (e.g., Beriplex® P / N, Confidex®, or Kcentra®). In another example, the therapeutic protein is fibrinogen (e.g., RiaSTAP®, Haemocomplettan® P). In one example, the therapeutic protein is a modified form of a clotting factor, such as a modified form of a clotting factor described herein.

[0219] As is apparent from the above, the present disclosure provides a method of combination therapeutic treatment of a subject, comprising administering to a subject in need thereof effective amounts of a first agent and a second agent, wherein the first agent is a serum albumin conjugate of the present disclosure, and the second agent is also for the prevention or treatment of a bleeding disorder.

[0220] As used herein, the term "combination," as in the phrase "combined therapeutic treatment," includes administering a first agent in the presence of a second agent. Combination therapeutic treatment methods include methods in which first, second, third, or additional agents are co-administered. Combination therapeutic treatment methods also include methods in which a first or additional agent is administered in the presence of a second or additional agent, e.g., the second or additional agent has been previously administered. Combination therapeutic treatment may be performed stepwise by different actors. For example, one actor may administer a first agent to a subject, and a second actor may administer a second agent to a subject, and the administering steps may be performed simultaneously, nearly simultaneously, or at distant times, so long as the first agent (and / or additional agent) is in the presence of the second agent (and / or additional agent) after administration. The actor and subject may be the same entity (e.g., a human).

[0221] The optimum concentration of the active ingredient(s) in the chosen vehicle can be determined experimentally according to procedures known to those skilled in the art and will depend on the final pharmaceutical formulation desired.

[0222] The dosage range for administration of the binding proteins of the present disclosure is large enough to produce the desired effect. For example, the composition comprises an effective amount of serum albumin conjugate. In one example, the composition comprises a therapeutically effective amount of serum albumin conjugate. In another example, the composition comprises a prophylactically effective amount of serum albumin conjugate.

[0223] The dosage should not be so large as to cause adverse side effects such as paradoxical bleeding and the development of inhibitors. Generally, the dosage varies depending on the age, condition, sex and degree of disease of the patient, and can be determined by those skilled in the art. In the event of any complications, the dosage can be adjusted by an individual physician.

[0224] Dosages can vary from about 0.1 mg / kg to about 300 mg / kg, for example from about 0.2 mg / kg to about 200 mg / kg, for example from about 0.5 mg / kg to about 20 mg / kg, in one or more administrations daily over one or several days.

[0225] In some instances, the serum albumin conjugate is administered at an initial (or loading) dose that is higher than subsequent (maintenance) doses. For example, v is administered at an initial dose of about 10 mg / kg to about 30 mg / kg. The binding protein is then administered at maintenance doses of about 0.0001 mg / kg to about 10 mg / kg. Maintenance doses can be administered every 7 to 35 days, for example, every 7, 14, or 28 days.

[0226] In some instances, a dose escalation regimen is used in which the serum albumin conjugate is initially administered at a lower dose than is used in subsequent administrations, which regimen is useful if the subject initially experiences an adverse event.

[0227] In subjects who do not respond adequately to treatment, multiple doses per week can be administered. Alternatively, or additionally, increasing doses can be administered.

[0228] The subject can be re-treated with the serum albumin conjugate by two or more exposures or administrations, e.g., at least about two exposures of the binding protein, e.g., about 2 to 60 exposures, more particularly about 2 to 40 exposures, and most particularly about 2 to 20 exposures.

[0229] In one example, optional re-treatment can occur if signs or symptoms of the disease, such as bleeding episodes, recur.

[0230] In another example, optional retreatments can be administered at defined intervals. For example, subsequent exposures can be administered at various intervals, such as about 24 to 28 weeks, or 48 to 56 weeks, or longer. For example, such exposures can be administered at intervals of about 24 to 26 weeks, or about 38 to 42 weeks, or about 50 to 54 weeks.

[0231] In subjects who do not respond adequately to treatment, multiple doses per week can be administered. Alternatively, or additionally, increasing doses can be administered.

[0232] In another example, for subjects experiencing adverse reactions, the initial (or loading) dose can be split over several days in a week or over several consecutive days.

[0233] Administration of serum albumin conjugates according to the disclosed methods may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration may be essentially continuous over a preselected period of time, or may be in a series of spaced doses, for example, either at or after the onset of a pathological condition.

[0234] Kits and other compositions of matter In another example of the present disclosure, a kit is provided that includes a serum albumin conjugate of the present disclosure that is useful for treating or preventing bleeding disorders such as those described above.

[0235] In one example, the kit includes (a) a container with a serum albumin conjugate, optionally in a pharmaceutically acceptable carrier or diluent, and (b) a package insert containing instructions for treating or preventing a bleeding disorder in a subject.

[0236] In one example, the kit includes (a) at least one serum albumin conjugate, (b) instructions for using the kit to treat or prevent a bleeding disorder in a subject, and (c) optionally, at least one additional therapeutically active compound or drug.

[0237] According to this example of the disclosure, a package insert is attached to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials, such as glass or plastic. The container houses or contains a composition effective for treating or preventing a bleeding disorder and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a serum albumin conjugate. The label or package insert indicates that the composition is used to treat a subject intended for treatment, such as a subject with or predisposed to developing a bleeding disorder, and provides specific guidance regarding the dosage and interval of administration of the serum albumin conjugate and any other agents. The kit may further include additional containers containing pharmaceutically acceptable diluents, such as sterile water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0238] The kit optionally further comprises a container holding a second agent, wherein the serum albumin conjugate is the first agent, and the article further comprises instructions on the package insert for treating a subject with an effective amount of the second agent, which can be a therapeutic protein as described above. The present disclosure includes the following non-limiting examples. [Example]

[0239] Example 1: Creation of human serum albumin mutants A library of randomly mutated variants of human serum albumin (HSA) was generated using standard error-prone PCR of human serum albumin. Truncating and frameshift variants were removed using intein-based open reading frame (ORF) selection as previously described in Gerth et al. (Protein Eng Des Sel 2004 17(7):595-602).

[0240] The HSA mutant clones were then subcloned and transfected into Flp-In™ CHO cells (Invitrogen) to generate a library of transfected stable cell lines, each containing a single copy of the mutant albumin fusion protein. Briefly, a Flp-In™ CHO host cell line containing an integrated Flp recombination target (FRT) site was obtained. The HSA mutant library was cloned into the pcDNA5 / FRT expression plasmid, which contains an in-frame platelet-derived growth factor receptor (PDGF®) transmembrane domain and a Myc tag under the control of a human CMV promoter. The pcDNA5 / FRT vector containing the HSA library and the pOG44 plasmid, which constitutively expresses Flp recombinase under the control of a human CMV promoter, were cotransfected into the Flp-In™ host cell line, resulting in all transfected cells successfully undergoing Flp-catalyzed recombination to carry a single copy of the mutant albumin fusion protein. Expression of the HSA mutant as a fusion protein with the PDGFR transmembrane domain enabled extracellular display of the membrane-tethered mutant on the surface of mammalian cells. Stably transfected cells were cultured in selective medium for approximately 14 days to remove non-transfected cells.

[0241] Example 2: Identification of residues associated with high affinity for FcRn / β2m To measure the binding affinity of the HSA variants to the fetal Fc receptor (FcRn / β2m), individual pools of CHO cells stably expressing the HSA variants were incubated at pH 5.4 and pH 7.3. Cells were stained with alexa-488-labeled FcRn / β2m (10 μg / ml at pH 5.4 and 100 μg / ml at pH 7.3; to detect target binding) and anti-myc-alexa647 (to detect construct expression) and analyzed by flow cytometry. The amount of labeled FcRn bound to cells expressing each HSA variant was quantified as the geometric mean fluorescence intensity (geoMFI) of the stained cells, and background fluorescence due to nonspecific binding of labeled FcRn to the cells was subtracted to determine relative binding to FcRn / β2m compared with wild-type HSA and the HSA variant K573Y (described in Anderson et al. (2014) J Biol Chem 289:13492).

[0242] Two clones (clone 3.9 and clone 5.6) were selected for further analysis based on affinity, and individual mutations within each clone were excised to determine the contribution of each mutation to the overall affinity of the parent clone.

[0243] FcRn binding to each HSA mutant was measured by flow cytometry in two independent experiments. The amount of labeled FcRn bound to cells expressing the HSA mutant was quantified as the geometric mean fluorescence intensity (geoMFI) of stained cells. Background fluorescence due to nonspecific binding of labeled FcRn to the cells was subtracted from the obtained values. Improved binding to FcRn / β2m over wild-type was observed at pH 5.4 for the positive control HSA mutant K573Y and HSA mutants with single substitutions Q522L, G572R, and A552V. As shown in Table 1, many clones, including T133I, K162N, K317E, F377I, I388T, F157L, P282S, and Y452S, did not show improved binding over wild-type. No clones showed any significant binding to FcRn / β2m at pH 7.3.

[0244] Example 3: Creation of HSA mutants with single amino acid substitutions Amino acid residues Q522, G572, and A552 were selected as residues of interest. A panel of HSA mutants was created using single substitutions of any amino acid except cysteine ​​at residue Q522 or G572 and stably expressed in CHO cells. Binding of the HSA mutants to FcRn / β2m was measured by flow cytometry in four independent experiments as described above. The amount of labeled FcRn bound to cells expressing each HSA mutant was quantified as the geometric mean fluorescence intensity (geoMFI) data for stained cells. Background fluorescence due to nonspecific binding of labeled FcRn to the cells was subtracted, and upper and lower confidence intervals (95% CI) for each experimentally determined mean were calculated.

[0245] The FcRn binding intensities (geoMFI) of all HSA variants at both pH 5.5 and pH 7.4 were normalized separately to adjust for systematic differences in measured intensities between experiments. For both pH conditions, replicate measurements of binding for a given HSA variant were adjusted to the average across the replicates (similar to the intensity normalization of microarray data described by Dudoit et al. 2002 Statistica Sinica 12, 111-139). For FcRn binding measurements at pH 5.5, there were clear nonlinear differences between replicates across the range of geoMFI values ​​measured for all tested variants. A quadratic function was fitted by robust regression to the binding intensity of each replicate versus the average binding (across four replicates) using all geoMFI values ​​on the log2 scale. This adjustment replaced each predicted experiment-specific binding intensity with the average binding intensity across the experiments, while the residues for each variant remained the same. For measurements of FcRn binding at pH 7.4, a linear function was fitted (i.e., the adjustment step is equivalent to subtracting an experiment-specific (small) intercept constant and adding division by an experiment-specific scaling constant).

[0246] Nine Q522 mutants: Q522G, Q522I, Q522K, Q522M, Q522F, Q522W, Q522Y, Q522V, and Q522L demonstrated statistically significant enhancement of FcRn / β2m binding at acidic pH 5.5 relative to wild-type HSA (Table 2). Six G572 mutants: G572A, G572E, G572H, G572S, G572K, and G572R demonstrated statistically significant enhancement of FcRn / β2m binding at acidic pH 5.5 relative to wild-type HSA (Table 3). No significant binding to FcRn / β2m was observed at pH 7.4 with any of the HSA mutants. Substitutions with Q522L, A552V, G572R, and K573Y showed the most significant increase in binding to FcRn / β2m at acidic pH 5.5 compared to wild-type HSA (Table 4).

[0247] Example 4: Creation of HSA mutants with multiple amino acid substitutions To test the additive or synergistic effects of mutations on FcRn / β2m binding, HSA mutants carrying double, triple, and quadruple amino acid substitutions were generated as previously described.

[0248] As shown in Table 5, the double HSA mutants Q522L / A552V, Q522L / G572R, and A552V / G572R demonstrated significantly improved FcRn / β2m binding at pH 5.5 over wild-type HSA and the corresponding HSA mutants with single amino acid substitutions.

[0249] Double HSA mutants carrying the K573Y mutation (Q522L / K573Y, A552V / K573Y, and G572R / K573Y) showed significantly improved FcRn / β2m binding at pH 5.5 over wild-type HSA and the single HSA mutants Q522L A552V, G572R. The double HSA mutants Q552L / K573Y and A552V / K573Y (but not G572R / K573Y) also showed significantly improved FcRn / β2m binding at pH 5.5 over the HSA mutant K573Y.

[0250] The triple HSA mutant Q522L / A522V / G572R demonstrated not only significantly improved FcRn / β2m binding at pH 5.5 over wild-type HSA and the corresponding HSA mutants with single and double amino acid substitutions, but also approximately twofold higher binding levels compared to the HSA mutant K573Y (Table 5).

[0251] The triple HSA mutants Q522L / A552V / K573Y and Q522L / G572R / K573Y demonstrated significantly improved FcRn / β2m binding at pH 5.5 over wild-type HSA and the corresponding HSA mutants with single amino acid substitutions, as well as significantly improved binding over the HSA mutant K573Y.

[0252] The quadruple HSA mutant Q522L / A552V / G572R / K573Y demonstrated significantly improved FcRn / β2m binding at pH 5.5 over wild-type HSA and the corresponding HSA mutant with a single amino acid substitution. However, the quadruple mutant also demonstrated significantly enhanced FcRn / β2m binding at pH 7.4 over wild-type HSA.

[0253] Example 5: Binding kinetics of HSA variants with single, double, and triple amino acid substitutions To test the effect of mutations on FcRn / β2m binding, single, double, and triple HSA mutants were generated as previously described. Binding kinetics were measured using SPR with a BIAcore™ 4000 (GE Healthcare Life Sciences). Recombinant hFcRn / β2M was chemically biotinylated and tethered to a GE streptavidin sensor chip (GE Healthcare Life Sciences). Briefly, human FcRn / β2M was The protein was chemically modified (i.e., minimally biotinylated) using an extended biotin linker (Sulfo NHS-LC-LC Biotin) prepared at a molar ratio of 1:0.25 in PBS for 2 hours on ice. The reaction mix was desalted using a spin column (10,000 NMW) according to the manufacturer's instructions.

[0254] HSA variants were injected at concentrations of 5, 1.7, 0.6, and 0 μM. Assays were performed in quadruplicate at pH 6.0, 37°C, in 10 mM HEPES; 150 mM NaCl. Association and dissociation phases were observed for 100 s, and each cycle was terminated with a 90 s regeneration step at neutral pH 7.4. Sensograms were subtracted in duplicate from the reference buffer injection (spot 3) and blank buffer injection within each run.

[0255] Rate constants and overall affinity rates were obtained for all HSA variants screened against minimally biotinylated human FcRn at pH 6. Data obtained from 1.7 μM and 0.6 μM injections were well fitted to a 1:1 binding model, and data obtained from 5 μM injections were used to fit a model with a saturation response.

[0256] As shown in Table 6, eight Q522 mutants: Q522F, Q522G, Q522M, Q522K, Q522Y, Q522I, Q522A, and Q522L demonstrated enhanced FcRn / β2m binding at acidic pH 6.0 compared to wild-type HSA. Six G572 mutants: G572R, G572K, G572V, G572A, G572S, and G572H demonstrated enhanced FcRn / β2m binding at acidic pH 6.0 compared to wild-type HSA (Table 6). The double HSA mutants A522V / G572R, Q522L / A552V and triple HSA mutant Q522L / A552V / G572R all demonstrated enhanced FcRn / β2m binding at acidic pH 6.0 compared to wild-type HSA (Table 6).

[0257] Example 6: Binding kinetics of HSA variants with single, double, and triple amino acid substitutions The binding kinetics of single, double, and triple HSA mutants to FcRn / β2m binding were measured using SPR with a BIAcore™ 8000 (GE Healthcare Life Sciences). HSA[H464Q] was used as a negative control.

[0258] Recombinant hFcRn / β2M was chemically biotinylated and tethered to a GE streptavidin sensor chip as previously described. HSA variants were injected at concentrations ranging from 2 to 0.015 μM. Assays were performed in triplicate at 37°C in 10 mM HEPES; 150 mM NaCl (pH 6.0). The association phase was observed for 120 seconds and the dissociation phase for 240 seconds. Each cycle was terminated with a 90-second regeneration step under basic conditions (i.e., pH 8.0). Sensograms were subtracted in duplicate from reference and blank buffer injections within each run.

[0259] Data obtained under acidic conditions fit well to a 1:1 binding model. Rate constants and binding affinities were determined as previously described.

[0260] As shown in Table 7, most HSA mutants exhibited improved binding affinities (K D ) showed improvements (up to 3-fold) in binding affinity to hFcRn / β2m. The double HSA mutants [Q522L / A552V], [Q522L / G572R], and [A552V / G572R] showed 10-fold improved binding affinity to hFcRn / β2m compared to recombinant wild-type HSA. The triple HSA mutant [Q522L / A552V / G572R] had the strongest binding at approximately 25 nM. The negative control HSA [H464Q] did not bind to hFcRn / β2m.

[0261] Example 7: Binding affinity of selected HSA variants to mammalian FcRn / β2m at acidic and neutral pH The single HSA mutants G572R and K573Y, the double HSA mutants Q522L / A552V, Q522L / G572R and A552V / G572R, and the triple HSA mutant Q522L / A552V / G572R were generated as previously described, and binding kinetics were measured using SPR with a BIAcore™ 4000 (GE Healthcare Life Sciences).

[0262] HSA variants were diluted to 1 μg / mL and directly immobilized to two surface sites of a carboxymethyldextran (CM-5) sensor chip (GE Healthcare Life Sciences) using amine coupling chemistry (NHS / EDC). HSA variants were immobilized at 100–500 RU in the outer spots (1 and 5) of each flow cell and 200–1000 RU in the inner spots (2 and 4). Spot 3 of each flow cell was used for activation / deactivation and baseline subtraction. The FcRn / β2m flow rate was maintained constant at 30 μL / min.

[0263] Binding of soluble recombinant human, cynomolgus monkey ("cyno"), rat, and mouse FcRn / β2m to immobilized HSA variants was tested at acidic (pH 6.0) and neutral (pH 7.3) pH. Concentration ranges of 2–0.007 μM (pH 6.0) and 20–0.078 μM (pH 7.3) were used, and the association and dissociation phases were observed for 180 and 600 s, respectively. Surface regeneration was performed as previously described. Sensogram data obtained under acidic conditions were adequately fitted to a 1:1 binding model. However, sensograms obtained at pH 7.3 exhibited off-rates near the instrument's detection limit and required a steady-state binding model for analysis.

[0264] As shown in Table 8, human and cyno FcRn / β2m exhibited nanomolar binding affinities at pH 6.0. Human and cyno FcRn / β2m exhibited a 20-fold improved affinity for HSA K573Y and an approximately 5-fold improved affinity for HSA G572R compared to wild-type HSA at pH 6. The affinities of the double HSA mutants Q522L / A552V, Q522L / G572R, and A552V / G572R were approximately 50 nM, 27 nM, and 27 nM for human FcRn / β2m, respectively, and approximately 84 nM, 49 nM, and 48 nM for cyno FcRn / β2m, respectively.

[0265] In comparison, rat and mouse FcRn / β2m showed such weak binding that they did not bind under the same conditions. Rat FcRn / β2m exhibited 100-fold weaker affinity for the HSA mutants and did not bind to wild-type HSA, HSA mutant Q522L / A552V, or HSA mutant G572R. Rat FcRn / β2m bound to HSA K573Y at approximately 1 μM under these conditions, but no binding was detected for HSA G572R. Mouse FcRn / β2m did not bind to any of the HSA mutants tested.

[0266] As shown in Table 8, human and cyno FcRn / β2m did not bind to wild-type HSA and showed weak binding (>50 μM) to HSA K573Y at pH 7.3. Binding affinities to other HSA mutants ranged from 7 to 63 μM. Rat and mouse FcRn / β2m did not bind to any of the HSA mutants tested at pH 7.3.

[0267] Example 8: Pharmacokinetic (PK) properties of HSA mutants in mice expressing human FcRn To evaluate the PK properties of HSA mutants, transgenic mice expressing the human FcRn receptor (hFcRn Tg32) homozygous mice (i.e., B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr / DcrJ) were used. Mixed-sex and age-matched animals (10-14 weeks old; three animals per time point) were intravenously injected with 10 mg / kg wild-type HSA, the single HSA mutant K573Y, the double HSA mutant Q522L / A552V, or the triple HSA mutant Q522L / A552V / G572R.

[0268] Blood was collected and plasma levels of human serum albumin were assessed using a human albumin ELISA kit (Bethyl Laboratories, cat no. E88-129) according to the manufacturer's instructions, except that each test article was used to generate an individual reference curve. Data were averaged for each time point before analysis, and the data were analyzed by nonlinear (Marquandt-Levenberg) minimization fitted using the custom MATLAB program NCAPKfit. Model fitting was performed using a 1 / Y^2 weighted fit using least squares, and the following equation was used: PK2(t)=A * exp(‐log(2) * t / T1)+B * exp(‐log(2) * t / T2) was used.

[0269] The mean residence time (MRT), area under the curve (AUC) and clearance rate were calculated using standard statistical formulas.

[0270] As shown below in Figure 1, Tables 9 and 10, the pharmacokinetic properties of wild-type HSA were significantly different from all three HSA mutants, with wild-type HSA having a shorter mean residence time (MRT), a smaller area under the curve (AUC), and faster clearance. The single HSA mutant K573Y, the double HSA mutant Q522L / A552V, and the triple HSA mutant Q522L / A552V / G572R all had significantly better pharmacokinetics based on all three criteria (i.e., MRT, AUC, and clearance) compared to wild-type HSA.

[0271] [Table 1]

[0272] [Table 2]

[0273] [Table 3]

[0274] [Table 4]

[0275] [Table 5-1] [Table 5-2]

[0276] [Table 6]

[0277] [Table 7]

[0278] Table 8

[0279] Table 9

[0280] Table 10

Claims

1. below: (i) an amino acid selected from the group consisting of leucine, glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, and valine substituting for glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1; (ii) a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1; (iii) an amino acid selected from the group consisting of alanine, histidine, serine, and lysine substituting for glycine at a position corresponding to amino acid 572 of SEQ ID NO:1; (iv) an amino acid selected from the group consisting of leucine, glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, and valine substituting for glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1, and valine substituting for alanine at position 552 of SEQ ID NO:1; (v) an amino acid selected from the group consisting of leucine, glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, and valine substituting for glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1, and an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine, and arginine substituting for glycine at position 572 of SEQ ID NO:1; (vi) a valine substituting for alanine at a position corresponding to amino acid 552 of SEQ ID NO:1, and an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine, and arginine substituting for glycine at position 572 of SEQ ID NO:1; (vii) an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, and lysine and arginine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1, and a tyrosine substituting for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1; and (viii) an amino acid selected from the group consisting of leucine, glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, and valine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1; a valine substituting for alanine at position 552 of SEQ ID NO:1; and an amino acid selected from the group consisting of alanine, glutamic acid, histidine, serine, lysine, and arginine substituting for glycine at position 572 of SEQ ID NO:1; A soluble human serum albumin variant or a fragment of said soluble human serum albumin variant that retains the ability to bind to FcRn, comprising one or more amino acid substitutions selected from the group consisting of: the soluble human serum albumin variant or the fragment of the soluble human serum albumin variant binds to FcRn with higher affinity than serum albumin defined in SEQ ID NO: 1; wherein the binding affinity to FcRn is measured at acidic pH, the soluble human serum albumin variant or the fragment of the soluble human serum albumin variant comprises a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 1; The soluble human serum albumin variant or a fragment of the soluble human serum albumin variant that retains the ability to bind to FcRn.

2. The soluble human serum albumin variant or the fragment of the soluble human serum albumin variant described in claim 1, wherein the soluble human serum albumin variant or the fragment of the soluble human serum albumin variant has an extended serum half-life compared to serum albumin defined in SEQ ID NO:

1.

3. The soluble human serum albumin variant of claim 1 or 2, or the fragment of the soluble human serum albumin variant that maintains the ability to bind to FcRn, further comprising a tyrosine substituting for a lysine at the position corresponding to amino acid 573 of SEQ ID NO:

1.

4. below: (i) a leucine substituting for glutamine at a position corresponding to amino acid 522 of SEQ ID NO:1; and / or (ii) a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO: 1; and / or (iii) an arginine substituting a glycine at a position corresponding to amino acid 572 of SEQ ID NO:1; The soluble human serum albumin variant or the fragment of the soluble human serum albumin variant that maintains the ability to bind to FcRn according to any one of claims 1 to 3, comprising:

5. below: (i) a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, and an arginine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1; or (ii) a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, and a valine substituting for alanine at the position corresponding to amino acid 552 of SEQ ID NO:1; or (iii) a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1, and an arginine substituting a glycine at a position corresponding to amino acid 572 of SEQ ID NO:1; or (iv) a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, and a tyrosine substituting for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1; or (v) a valine substituting an alanine at a position corresponding to amino acid 552 of SEQ ID NO:1, and a tyrosine substituting a lysine at a position corresponding to amino acid 573 of SEQ ID NO:1; or (vi) an arginine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1, and a tyrosine substituting for lysine at the position corresponding to amino acid 573 of SEQ ID NO:1; or (vii) a leucine substituting for glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting for alanine at the position corresponding to amino acid 552 of SEQ ID NO:1, and an arginine substituting for glycine at the position corresponding to amino acid 572 of SEQ ID NO:1; or (viii) a leucine substituting glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting alanine at the position corresponding to amino acid 552 of SEQ ID NO:1, and a tyrosine substituting lysine at the position corresponding to amino acid 573 of SEQ ID NO:1; or (ix) a leucine substituting glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, an arginine substituting glycine at the position corresponding to amino acid 572 of SEQ ID NO:1, and a tyrosine substituting lysine at the position corresponding to amino acid 573 of SEQ ID NO:1; or (x) a leucine substituting glutamine at the position corresponding to amino acid 522 of SEQ ID NO:1, a valine substituting alanine at the position corresponding to amino acid 552 of SEQ ID NO:1, an arginine substituting glycine at the position corresponding to amino acid 572 of SEQ ID NO:1, and a tyrosine substituting lysine at the position corresponding to amino acid 573 of SEQ ID NO:1; The soluble human serum albumin variant or the fragment of the soluble human serum albumin variant that maintains the ability to bind to FcRn according to any one of claims 1 to 4, comprising:

6. A serum albumin conjugate comprising the soluble human serum albumin variant of any one of claims 1 to 5 or the fragment of the soluble human serum albumin variant that retains the ability to bind to FcRn, and a compound.

7. 7. The serum albumin conjugate of claim 6, wherein the serum albumin conjugate has a longer serum half-life compared to a serum albumin conjugate comprising the serum albumin defined in SEQ ID NO: 1, and / or has an enhanced binding affinity to FcRn compared to a serum albumin conjugate comprising the serum albumin defined in SEQ ID NO:

1.

8. 8. The serum albumin conjugate of claim 6, wherein the compound is one or more selected from the group consisting of a protein, a protein comprising an antibody variable region, an antibody mimetic, a single domain antibody, a toxin (the toxin is selected from the group consisting of ricin, abrin, diphtheria toxin, tetanus toxoid, Pseudomonas exotoxin A (PE), gelonin, pokeweed antiviral protein, and saporin), a compound comprising a radioisotope, a detectable label, a peptide, a polypeptide, a colloidal particle, a chemotherapeutic drug, a nucleic acid, an antisense oligonucleotide, a short hairpin RNA (shRNA), an siRNA, a ribozyme, a microRNA, a microRNA-adapted shRNA (shRNAmir), and a DNA enzyme (DNAzyme).

9. The serum albumin conjugate of claim 8, wherein the compound is an Fv-containing protein.

10. The protein is one of the following: (i) single chain Fv fragments (scFv); (ii) dimeric scFv (di-scFv); (iii) diabodies; (iv) triabodies; (v) tetrabodies; (vi) Fab; (vii) F(ab')2; (viii) Fv; and (ix) the constant region of an antibody, Fc or heavy chain constant domain (C Ｈ ) 2 and / or C Ｈ one of (i) to (viii) linked to 3; 10. The serum albumin conjugate of claim 9, selected from the group consisting of:

11. 9. The serum albumin conjugate of claim 8, wherein the compound is a therapeutic protein.

12. (i) the compound is von Willebrand factor; or (ii) the compound is a complement inhibitor; or (iii) the compound is or binds to a blood clotting factor; The serum albumin conjugate according to any one of claims 6 to 8.

13. (i) the von Willebrand factor comprises a D'D3 domain; or (ii) the blood coagulation factor is selected from the group consisting of factor I, factor II (prothrombin), factor III, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, and activated forms of any of the above; The serum albumin conjugate of claim 12.

14. A composition comprising the soluble human serum albumin variant of any one of claims 1 to 5 or the fragment of the soluble human serum albumin variant that retains the ability to bind to FcRn, or the serum albumin conjugate of any one of claims 6 to 13, and a pharmaceutical carrier and / or excipient.

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