Single-domain serum albumin-binding protein

Abstract

Provided are single domain albumin binding proteins that can be used to extend the half-life of therapeutic molecules. [Solution] Disclosed herein are single-domain serum albumin binding proteins with improved thermostability, binding affinity, and robust aggregation profiles. Also described are multispecific binding proteins comprising the single-domain serum albumin binding proteins of the present disclosure. Pharmaceutical compositions comprising the binding proteins disclosed herein and methods of using such formulations are provided.

Description

[Technical Field]

[0001] This application claims the benefits of U.S. Provisional Application No. 62 / 339,682, filed on 20 May 2016, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing, which was filed electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the above ASCII file, created on 18 May 2017, has the filename 47517-703_601_SEQ.txt and is 22,218 bytes in size.

[0003] Embedding by citation All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually designated to be incorporated by reference. [Background technology]

[0004] Albumin is the most abundant plasma protein, highly soluble, very stable, and has a very long circulating half-life. Albumin can be used in various ways to increase the circulating half-life of therapeutic molecules. This disclosure provides a single-domain albumin-binding protein that can be used to extend the half-life of therapeutic molecules. [Overview of the project]

[0005] In one embodiment, a single-domain serum albumin-binding protein comprising complementarity-determining regions CDR1, CDR2, and CDR3 is provided herein, where (a) the amino acid sequence of CDR1 is as described in GFX1X2X3X4FGMS (SEQ ID NO. 1), where X1 is threonine, arginine, lysine, serine, or proline, X2 is phenylalanine or tyrosine, X3 is serine, arginine, or lysine, and X4 is serine, lysine, arginine, or alanine; (b) the amino acid sequence of CDR2 is SISGSGX5X6TLYAX7SX8K (SEQ ID NO. 1). As described in NO.2), X5 is serine, arginine, threonine, or alanine, X6 is aspartic acid, histidine, valine, or threonine, X7 is aspartic acid, histidine, arginine, or serine, and X8 is valine or leucine; and (c) the amino acid sequence of CDR3 is GGSLX9X 10 As described in (SEQ ID NO.3), X9 is serine, arginine, threonine, or lysine, and X 10 X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10These are not simultaneously threonine, phenylalanine, serine, serine, serine, aspartic acid, aspartic acid, valine, serine, and arginine. In some embodiments, the single-domain serum albumin-binding protein comprises the following formula: f1-r1-f2-r2-f3-r3-f4, where r1 is SEQ ID NO. 1, r2 is SEQ ID NO. 2, and r3 is SEQ ID NO. 3, where f1, f2, f3, and f4 are selected framework residues such that the above protein is at least 80 percent identical to the amino acid sequence described in SEQ ID NO: 10. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r1 comprises SEQ ID NO. 14, SEQ ID NO. 15, or SEQ ID NO. 16. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r2 comprises SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r3 comprises SEQ ID NO. 23 or SEQ ID NO. 24. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r1 comprises SEQ ID NO. 14. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r1 comprises SEQ ID NO. 15, r2 comprises SEQ ID NO. 17, and r3 comprises SEQ ID NO. 23. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r1 comprises SEQ ID NO. 16 and r3 comprises SEQ ID NO. 23. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r1 comprises SEQ ID NO. 15 and r2 comprises SEQ ID NO. 18.In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r1 comprises SEQ ID NO. 14 and r3 comprises SEQ ID NO. 23. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r1 comprises SEQ ID NO. 15, r2 comprises SEQ ID NO. 19, and r3 comprises SEQ ID NO. 24. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r1 comprises SEQ ID NO. 14 and r2 comprises SEQ ID NO. 20. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r1 comprises SEQ ID NO. 15 and r2 comprises SEQ ID NO. 21. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence, where r1 comprises SEQ ID NO. 15, r2 comprises SEQ ID NO. 22, and r3 comprises SEQ ID NO. 24. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence selected from SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 25, SEQ ID NO. 26, and SEQ ID NO. 27. In some embodiments, the single-domain serum albumin-binding protein includes the amino acid sequence described as SEQ ID NO. 4. In some embodiments, the single-domain serum albumin-binding protein includes the amino acid sequence described as SEQ ID NO. 7. In some embodiments, the single-domain serum albumin-binding protein includes the amino acid sequence described as SEQ ID NO. 9. In some embodiments, the single-domain serum albumin-binding protein includes the amino acid sequence described as SEQ ID NO. 26. In some embodiments, the single-domain serum albumin-binding protein includes the amino acid sequence described as SEQ ID NO. 27.In some embodiments, the single-domain serum albumin-binding protein includes the amino acid sequence described as SEQ ID NO. 5. In some embodiments, the single-domain serum albumin-binding protein includes the amino acid sequence described as SEQ ID NO. 6. In some embodiments, the single-domain serum albumin-binding protein includes the amino acid sequence described as SEQ ID NO. 8. In some embodiments, the single-domain serum albumin-binding protein includes the amino acid sequence described as SEQ ID NO. 25.

[0006] In some embodiments, the single-domain serum albumin-binding protein binds to serum albumin selected from human serum albumin, cynomolgus monkey serum albumin, and mouse serum albumin. In some embodiments, the single-domain serum albumin-binding protein binds to human serum albumin and cynomolgus monkey serum albumin having equivalent binding affinity (Kd). In some embodiments, the single-domain serum albumin-binding protein binds to mouse serum albumin having a binding affinity (Kd) about 1.5 to about 20 times weaker than the binding affinity (Kd) of the protein to human and cynomolgus monkey serum albumin. In some embodiments, the single-domain serum albumin-binding protein binds to human serum albumin having a human Kd (hKd) between about 1 nM and about 100 nM, and to cynomolgus monkey serum albumin having a cynomolgus monkey Kd (cKd) between 1 nM and 100 nM. In some embodiments, the hKd and cKd of a single-domain serum albumin-binding protein are between 1 nM and about 5 nM, or about 5 nM and about 10 nM. In some embodiments, the hKd and cKd of a single-domain serum albumin-binding protein are between about 1 nM to about 2 nM, about 2 nM to about 3 nM, about 3 nM to about 4 nM, about 4 nM to about 5 nM, about 5 nM to about 6 nM, about 6 nM to about 7 nM, about 7 nM to about 8 nM, about 8 nM to about 9 nM, or about 9 nM to about 10 nM. In some embodiments, the ratio of hKd to cKd (hKd:cKd) of a single-domain serum albumin-binding protein ranges from about 20:1 to about 1:2.

[0007] In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence described as SEQ ID NO. 4, where hKd and cKd are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence described as SEQ ID NO. 5, where hKd and cKd are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence described as SEQ ID NO. 6, where hKd and cKd are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence described as SEQ ID NO. 7, where hKd and cKd are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence described as SEQ ID NO. 8, where hKd and cKd are between approximately 5 nM and approximately 10 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence described as SEQ ID NO. 9, where hKd and cKd are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence described as SEQ ID NO. 22, where hKd and cKd are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence described as SEQ ID NO. 23, where hKd and cKd are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence described as SEQ ID NO. 24, where hKd and cKd are between approximately 1 nM and approximately 5 nM.

[0008] In some embodiments, the single-domain serum albumin-binding protein has an elimination half-life of at least 12 hours, at least 20 hours, at least 25 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, or at least 100 hours.

[0009] In another embodiment, a single-domain serum albumin-binding protein is provided comprising CDR1, CDR2, and CDR3, including the sequence described as SEQ ID NO. 10, wherein one or more amino acid residues selected from amino acid positions 28, 29, 30, or 31 of CDR1; positions 56, 57, 62, or 64 of CDR2; or positions 103 and 104 of CDR3 are substituted, where amino acid position 28 is substituted with arginine, lysine, serine, or proline, amino acid position 29 is substituted with tyrosine, amino acid position 30 is substituted with arginine or The amino acid position 31 is substituted with lysine, amino acid position 56 is substituted with arginine, threonine, or alanine, amino acid position 57 is substituted with histidine, valine, or threonine, amino acid position 62 is substituted with histidine, arginine, glutamic acid, or serine, amino acid position 64 is substituted with leucine, amino acid position 103 is substituted with arginine, threonine, or lysine, and amino acid position 104 is substituted with lysine, valine, proline, or asparagine. In some embodiments, the single-domain serum albumin binding includes one or more additional substitutions at amino acid positions other than positions 28, 29, 30, 31, 56, 57, 62, 64, 103, and 104. In some embodiments, the single-domain serum albumin binding protein includes a substitution at position 29. In some embodiments, the single-domain serum albumin binding protein includes a substitution at position 31. In some embodiments, the single-domain serum albumin-binding protein includes a substitution at position 56. In some embodiments, the single-domain serum albumin-binding protein includes a substitution at position 62. In some embodiments, the single-domain serum albumin-binding protein includes a substitution at position 64. In some embodiments, the single-domain serum albumin-binding protein includes a substitution at position 104. In some embodiments, the single-domain serum albumin-binding protein includes substitutions at amino acid positions 31 and 62.In some embodiments, the single-domain serum albumin-binding protein includes an amino acid sequence in which position 31 is substituted with arginine. In some embodiments, the single-domain serum albumin-binding protein includes an amino acid sequence in which position 31 is substituted with arginine and amino acid position 62 is substituted with glutamic acid. In some embodiments, the single-domain serum albumin-binding protein includes substitutions at amino acid positions 31, 56, 64, and 104. In some embodiments, the single-domain serum albumin-binding protein includes an amino acid sequence in which position 31 is substituted with lysine, amino acid position 56 is substituted with alanine, amino acid position 64 is substituted with leucine, and amino acid position 104 is substituted with lysine. In some embodiments, the single-domain serum albumin-binding protein includes substitutions at amino acid positions 29 and 104. In some embodiments, the single-domain serum albumin-binding protein includes an amino acid sequence in which amino acid position 29 is substituted with tyrosine and amino acid position 104 is substituted with lysine. In some embodiments, the single-domain serum albumin-binding protein includes substitutions at amino acid positions 31 and 56. In some embodiments, the single-domain serum albumin-binding protein includes an amino acid sequence in which amino acid position 31 is replaced with lysine and amino acid position 56 is replaced with threonine. In some embodiments, the single-domain serum albumin-binding protein includes substitutions at amino acid positions 31, 56, and 62. In some embodiments, the single-domain serum albumin-binding protein includes an amino acid sequence in which amino acid position 31 is replaced with lysine, amino acid position 56 is replaced with threonine, and amino acid position 62 is replaced with glutamic acid. In some embodiments, the single-domain serum albumin-binding protein includes substitutions at amino acid positions 31 and 104. In some embodiments, the single-domain serum albumin-binding protein includes an amino acid sequence in which amino acid position 31 is replaced with arginine and amino acid position 104 is replaced with lysine. In some embodiments, the single-domain serum albumin-binding protein includes substitutions at amino acid positions 31, 56, and 104.In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 31 is substituted with lysine, amino acid position 56 is substituted with arginine, and amino acid position 104 is substituted with valine. In some embodiments, the single-domain serum albumin-binding protein comprises substitutions at amino acid positions 31, 56, 62, and 104. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 31 is substituted with lysine, amino acid position 56 is substituted with arginine, amino acid position 62 is substituted with glutamic acid, and amino acid position 104 is substituted with valine. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 31 is substituted with arginine, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 31 is substituted with arginine, amino acid position 62 is substituted with glutamic acid, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 31 is substituted with lysine, amino acid position 56 is substituted with alanine, amino acid position 64 is substituted with leucine, amino acid position 104 is substituted with lysine, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 29 is substituted with tyrosine, amino acid position 104 is substituted with lysine, and the hKd and cKd are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 31 is substituted with lysine, amino acid position 56 is substituted with threonine, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 1 nM and approximately 5 nM.In some embodiments, the single-domain serum albumin comprises an amino acid sequence in which amino acid position 31 is substituted with lysine, amino acid position 56 is substituted with threonine, amino acid position 62 is substituted with glutamic acid, and the hKd and cKd are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 31 is substituted with arginine, amino acid position 104 is substituted with lysine, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 5 nM and approximately 10 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 31 is substituted with lysine, amino acid position 56 is substituted with arginine, amino acid position 104 is substituted with valine, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 31 is substituted with lysine, amino acid position 56 is substituted with arginine, amino acid position 62 is substituted with glutamic acid, amino acid position 104 is substituted with valine, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 1 nM and approximately 5 nM.

[0010] In another embodiment, a single-domain serum albumin-binding protein is provided herein, comprising at least one mutation in CDR1, CDR2, or CDR3, wherein CDR1 comprises the sequence as described in SEQ ID NO:11, CDR2 comprises the sequence as described in SEQ ID NO:12, and CDR3 comprises the sequence as described in SEQ ID NO:13, wherein at least one mutation is not at amino acid positions 1, 2, 7, 8, 9, or 10 of SEQ ID NO:11, positions 1, 3, 6, 10, or 11 of SEQ ID NO:12, or position 1 or 2 of SEQ ID NO:13. In some embodiments, the single-domain serum albumin-binding protein contains at least one mutation at amino acid positions selected from positions 3, 4, 5, and 6 of CDR1 (SEQ ID NO: 11), amino acid positions 7, 8, 13, and 15 of CDR2 (SEQ ID NO: 12), and amino acid positions 5 and 6 of CDR3 (SEQ ID NO: 13). In some embodiments, the single-domain serum albumin-binding protein contains one or more additional substitutions at amino acid positions other than positions 3, 4, 5, and 6 of CDR1 (SEQ ID NO: 11), amino acid positions 7, 8, 13, and 15 of CDR2 (SEQ ID NO: 12), and amino acid positions 5 and 6 of CDR3 (SEQ ID NO: 13). In some embodiments, the single-domain serum albumin-binding protein contains a mutation at amino acid position 6 of CDR1 (SEQ ID NO: 11). In some embodiments, the single-domain serum albumin-binding protein contains mutations at amino acid position 6 of CDR1 (SEQ ID NO: 11) and amino acid position 13 of CDR2 (SEQ ID NO: 12). In some embodiments, the single-domain serum albumin-binding protein contains mutations at amino acid position 6 of CDR1 (SEQ ID NO: 11), amino acid positions 7 and 15 of CDR2 (SEQ ID NO: 12), and amino acid position 6 of CDR3 (SEQ ID NO: 13).In some embodiments, the single-domain serum albumin-binding protein contains mutations at amino acid position 4 of CDR1 (SEQ ID NO: 11) and amino acid position 6 of CDR3 (SEQ ID NO: 13). In some embodiments, the single-domain serum albumin-binding protein contains mutations at amino acid position 6 of CDR1 (SEQ ID NO: 11) and amino acid position 7 of CDR2 (SEQ ID NO: 12). In some embodiments, the single-domain serum albumin-binding protein contains mutations at amino acid position 6 of CDR1 (SEQ ID NO: 11) and amino acid positions 7 and 13 of CDR2 (SEQ ID NO: 12). In some embodiments, the single-domain serum albumin-binding protein contains mutations at amino acid position 6 of CDR1 (SEQ ID NO: 11) and amino acid position 6 of CDR3 (SEQ ID NO: 13). In some embodiments, the single-domain serum albumin-binding protein contains mutations at amino acid position 6 of CDR1 (SEQ ID NO: 11), amino acid position 7 of CDR2 (SEQ ID NO: 12), and amino acid position 6 of CDR3 (SEQ ID NO: 13). In some embodiments, the single-domain serum albumin-binding protein contains mutations at amino acid position 6 of CDR1 (SEQ ID NO: 11), amino acid positions 7 and 13 of CDR2 (SEQ ID NO: 12), and amino acid position 6 of CDR3 (SEQ ID NO: 13). In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 6 of CDR1 (SEQ ID NO: 11) is mutated to arginine, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 6 of CDR1 (SEQ ID NO: 11) is mutated to arginine, and amino acid position 13 of CDR2 (SEQ ID NO: 12) is mutated to glutamic acid, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 1 nM and approximately 5 nM.In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 6 of CDR1 (SEQ ID NO: 11) is mutated to lysine, amino acid positions 7 and 15 of CDR2 (SEQ ID NO: 12) are mutated to alanine and leucine, respectively, and amino acid position 6 of CDR3 (SEQ ID NO: 13) is mutated to lysine, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 6 of CDR1 (SEQ ID NO: 11) is mutated to lysine, and amino acid position 7 of CDR2 (SEQ ID NO: 12) is mutated to threonine, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 6 of CDR1 (SEQ ID NO: 11) is mutated to arginine, amino acid position 6 of CDR3 (SEQ ID NO: 12) is mutated to lysine, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 5 nM and approximately 12 nM.In some embodiments, the single-domain serum albumin-binding protein comprises an amino acid sequence in which amino acid position 6 of CDR1 (SEQ ID NO: 11) is mutated to lysine, amino acid position 7 of CDR2 (SEQ ID NO: 12) is mutated to arginine, amino acid position 6 of CDR3 (SEQ ID NO: 13) is mutated to valine, and the hKd and cKd of the single-domain serum albumin-binding protein are between approximately 1 nM and approximately 5 nM.

[0011] In another embodiment, a polynucleotide encoding a single-domain serum albumin-binding protein according to the Disclosure is provided herein. Further embodiments describe a vector comprising a polynucleotide as disclosed herein. Another embodiment describes a host cell transformed with a vector according to the Disclosure. In one embodiment, a pharmaceutical composition is provided comprising (i) a single-domain serum albumin-binding protein according to the Disclosure, a polynucleotide according to the Disclosure, a vector according to the Disclosure, or a host cell according to the Disclosure, and (ii) a pharmaceutically acceptable carrier.

[0012] In another embodiment, a process for producing a single-domain serum albumin-binding protein according to the Disclosure is described herein, which includes culturing a host transformed or transfected with a vector containing a nucleic acid sequence encoding the single-domain serum albumin-binding protein described herein under conditions that enable the expression of the single-domain serum albumin-binding protein, and recovering and purifying the protein produced from the culture.

[0013] The present disclosure further describes methods for treating or improving proliferative disorders, neoplastic disorders, inflammatory disorders, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases, including administration of a single-domain serum albumin-binding protein to a subject. In some embodiments, the subject is human. In some embodiments, the method further includes administration of a drug in combination with the single-domain serum albumin-binding protein according to the present disclosure.

[0014] In another embodiment, a polyspecific binding protein comprising the single-domain serum albumin-binding protein according to the disclosure is described. In yet another embodiment, an antibody comprising the single-domain serum albumin-binding protein according to the disclosure is described.

[0015] Further embodiments describe polyspecific antibodies, bispecific antibodies, sdAbs, variable heavy chain domains, peptides, or ligands comprising the single-domain serum albumin-binding protein according to the Disclosure. In one embodiment, an antibody comprising the single-domain serum albumin-binding protein according to the Disclosure is provided, wherein the antibody is a single-domain antibody. In some embodiments, the single-domain antibody is derived from the heavy chain variable region of IgG.

[0016] One embodiment describes a polyspecific binding protein or antibody comprising a single-domain serum albumin-binding protein and a CD3-binding domain as described herein. One embodiment describes a method for treating or improving proliferative disorders, neoplastic disorders, inflammatory disorders, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases, including administration of a polyspecific antibody as described herein to a subject. [Brief explanation of the drawing]

[0017] The novel features of the present invention are described, among other things, in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which describes embodiments in which the principles of the present invention are used, and the following appended drawings. [Figure 1] Illustrate the specific binding of parental anti-HSA phage as determined by ELISA titration to HSA antigen and CD3 antigen. [Figure 2] Illustrate the cross-reactivity of anti-HSA phage to human, cynomolgus monkey, and mouse serum albumin as determined by ELISA titration. [Figure 3] Provide the binding affinity profiles of nine clones selected for more accurate Kd determination using purified sdAbs. [Figure 4] Illustrate the temperature of hydrophobic exposure (Th°C) for multiple anti-HSA sdAb variants. [Figure 5] Illustrate the tendency of multiple anti-HSA sdAb variants to form dimers versus monomers at low pH.

Mode for Carrying Out the Invention

[0018] Preferred embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many changes, variations and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may also be utilized in the practice of the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of this claim and its equivalents be covered thereby.

[0019] Specific Definitions The terms used in this specification are for the purpose of describing only specific cases and are not intended to limit the present invention. As used in this specification, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Further, to the extent that the terms "including", "includes", "having", "has", "with", or variations thereof are used in any form of the embodiments for carrying out the invention and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0020] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measuring system. For example, "about" can mean within one or more standard deviations of any value for practice. When a particular value is recited in the present application and claims, unless otherwise specified, the term "about" is assumed to mean within an acceptable error range of the particular value.

[0021] The terms "individual", "patient", or "subject" are used interchangeably. None of these terms require (and are not limited to) a situation characterized by the supervision (e.g., constant or intermittent) of a healthcare provider (e.g., a doctor, a registered nurse, a clinical nurse, a physician assistant, a nursing assistant, or a hospice staff).

[0022] The term "framework" or "FR" residue (or region) refers to variable domain residues other than those of the CDR or hypervariable region as defined herein. "Human consensus framework" is a framework that represents the amino acid residues that most commonly occur upon selection of a human immunoglobulin VL or VH framework sequence.

[0023] As used herein, “variable region” or “variable domain” refers to the fact that a particular portion of a variable domain differs significantly in its sequence within an antibody, and is used for the binding and specificity of each particular antibody to that particular antigen. However, variability is not uniformly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions within the variable domains of both the light and heavy chains. The more highly conserved portion of the variable domain is called the framework (FR). The natural heavy and light chain variable domains each contain four FR regions that employ a β-sheet structure, connected by three CDRs that form loops connecting the β-sheet structure, and in some cases forming part of the β-sheet structure. The CDRs of each chain are held together in very close proximity by the FR region and, together with the CDRs from other chains, contribute to the formation of the antibody’s antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in antibody binding to the antigen, but exhibits various effector functions, such as the antibody's involvement in antibody-dependent cytotoxicity. “Numbering of variable domain residues as in Kabat” or “Numbering of amino acid positions as in Kabat,” and its modified forms, refer to the numbering scheme used for heavy-chain or light-chain variable domains in antibody edits in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering scheme, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of FR or CDR in the variable domain.For example, the heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues may be determined for any antibody by alignment in homologous regions of the sequence of an antibody having a “standard” Kabat-numbered sequence. The CDRs in this disclosure are not intended to necessarily correspond to any Kabat numbering convention.

[0024] As used herein, the term “percent (%) amino acid sequence identity” for a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular sequence, after the sequences have been aligned, gaps introduced, and, if necessary, the maximum percentage sequence identity has been achieved, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0025] As used herein, “elimination half-life” is used in its usual sense, as described in Goodman and Gillman's *The Pharmaceutical Basis of Therapeutics* 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). In short, the term is intended to encompass a quantitative measure of the temporal course of drug excretion. Since the drug concentration does not usually approach the concentration required for saturation of the elimination process, the elimination of most pharmaceuticals is exponential (i.e., follows first-order kinetics). The rate of an exponential process can be expressed by its rate constant k, which represents a small change per unit time, or by its half-life t¹ / ², which is the time required for 50% completion of the process. The units of these two constants are time⁻¹ and time, respectively. The first-order rate constant and half-life of a reaction are simply related (k × t¹ / ² = 0.693) and can be interchanged accordingly. Because primary elimination dynamics instructs the drug to be lost at a constant rate per unit time, the logarithmic plot of drug concentration versus time is always linear after the initial distribution phase (i.e., after drug absorption and distribution are complete). The half-life for drug excretion can be accurately determined from such graphs.

[0026] As used herein, the term “binding affinity” refers to the affinity of the proteins described herein to their binding targets and is expressed numerically using the “Kd” value. When two or more proteins are shown to have equivalent binding affinities to their binding targets, the Kd values ​​for the binding of each protein to those binding targets are within ±2 of each other. When two or more proteins are shown to have equivalent binding affinities to a single binding target, the Kd values ​​for the binding of each protein to the above single binding target are within ±2 of each other. When a protein is shown to bind to two or more targets with equivalent binding affinities, the Kd values ​​for the binding of the above protein to two or more targets are within ±2 of each other. Generally, a high Kd value corresponds to a weak binding. In some embodiments, “Kd” is measured by radiolabeled antigen-binding assay (RIA) or surface plasmon resonance assay using BIAcore®-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ). In some embodiments, "on-rate" or "rate of association or association rate" or "kon," and "off-rate" or "rate of dissociation or dissociation rate" or "koff" are also determined by surface plasmon resonance technology using BIAcore®-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ). In additional embodiments, "Kd," "kon," and "koff" are measured using Octet® Systems (Pall Life Sciences).

[0027] Single-domain serum albumin-binding proteins, pharmaceutical compositions, as well as nucleic acids, recombinant expression vectors, and host cells for producing such single-domain serum albumin-binding proteins are described herein. Furthermore, methods for using the disclosed single-domain serum albumin-binding proteins in the prevention and / or treatment of diseases, illnesses, and disorders are also provided. Single-domain serum albumin-binding proteins can specifically bind to serum albumin. In some embodiments, single-domain serum albumin-binding proteins also include additional domains, such as a CD3-binding domain, as well as binding domains for other target antigens.

[0028] <Single-domain serum albumin-binding protein> A single-domain serum albumin-binding protein is intended herein. Serum albumin is produced by the liver and dissolved in plasma, and is the most abundant blood protein in mammals. Albumin is essential for maintaining colloid osmotic pressure, which is necessary for the proper distribution of body fluids between blood vessels and body tissues; without albumin, high pressure in blood vessels would not be able to deliver more fluid to the tissues. It also acts as a plasma carrier by nonspecifically binding to several hydrophobic steroid hormones, and as a transport protein for hemin and fatty acids. Human serum albumin (HSA) (molecular weight ~67 kDa) is the most abundant protein in plasma, present at about 50 mg / ml (600 μM), and has a half-life of about 20 days in humans. HSA plays a role in maintaining plasma pH, contributes to colloidal blood pressure, functions as a carrier for many metabolites and fatty acids, and serves as a major drug transport protein in plasma. In some embodiments, a single-domain serum albumin-binding protein binds to HSA. In some embodiments, the single-domain serum albumin-binding protein binds to serum albumin protein from cynomolgus monkeys. In some embodiments, the single-domain serum albumin-binding protein binds to HSA and serum albumin protein from cynomolgus monkeys. In some embodiments, the single-domain serum albumin-binding protein further binds to mouse serum albumin protein. In some embodiments, the binding affinity to mouse serum albumin is about 1.5 to about 20 times weaker than the affinity to human or cynomolgus monkey serum albumin.

[0029] Non-covalent association with albumin extends the elimination half-life of short-lived proteins. For example, recombinant fusion of the albumin-binding domain to an FAb fragment resulted in a 25-fold and 58-fold decrease in in vitro clearance and a 26-fold and 37-fold extension of half-life when administered intravenously to mice and rabbits, respectively, compared to administration of the FAb fragment alone. In another example, when insulin was acylated with a fatty acid to promote association with albumin, long-term effects were observed when administered subcutaneously to rabbits or pigs. Together, these studies demonstrate a correlation between albumin binding and sustained action / serum half-life.

[0030] In some embodiments, the single-domain serum albumin-binding proteins described herein are single-domain antibodies, peptides, ligands, or small molecule entities, such as the heavy chain variable domain (VH), variable domain (VHH), or other domains of sdAb derived from camelids, that are specific to serum albumin. In some embodiments, the single-domain serum albumin-binding proteins described herein are single-domain antibodies, peptides, ligands, or small molecule entities, such as the heavy chain variable domain (VH), variable domain (VHH), or other domains of sdAb derived from camelids, that are specific to HSA. In some embodiments, the serum albumin-binding domain of the single-domain serum albumin-binding proteins described herein is any domain that binds to serum albumin, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, or humanized antibodies. In some embodiments, the serum albumin-binding domain is a single-domain antibody. In other embodiments, the serum albumin-binding domain is a peptide. In further embodiments, the serum albumin-binding domain is a small molecule. Single-domain serum albumin-binding proteins are quite small, and in some embodiments, they are intended to be at most 25 kD, 20 kD, 15 kD, or even 10 kD. In some cases, the single-domain serum albumin-binding protein binding is less than 5 kD if it is a peptide or small molecule entity.

[0031] In some embodiments, the single-domain serum albumin-binding proteins described herein are half-life-extending domains that result in modified pharmacokinetics and pharmacokinetics of the single-domain serum albumin-binding protein itself. As described above, the half-life-extending domain extends the elimination half-life. The half-life-extending domain further modifies the pharmacodynamic properties of the single-domain serum albumin-binding protein, including alteration of tissue distribution, penetration, and diffusion. In some embodiments, the half-life-extending domain results in improved tissue (including tumor) targeting, tissue distribution, tissue penetration, diffusion within tissue, and enhanced efficacy compared to proteins without the half-life-extending domain. In one embodiment, a therapeutic method effectively and efficiently utilizes a reduced amount of single-domain serum albumin-binding protein, resulting in reduced side effects such as decreased cytotoxicity to non-tumor cells.

[0032] Furthermore, the binding affinity of a single-domain serum albumin-binding protein to its binding target can be selected to target a specific elimination half-life in a particular single-domain serum albumin-binding protein. Therefore, in some embodiments, a single-domain serum albumin-binding protein has a high binding affinity to its binding target. In other embodiments, a single-domain serum albumin-binding protein has a moderate binding affinity to its binding target. In yet another embodiment, a single-domain serum albumin-binding protein has a low or moderate binding affinity to its binding target. Typical binding affinities include Kd values ​​of ≤10 nM (high), between 10 nM and 100 nM (moderate), and above 100 nM (low). As described above, the binding affinity of a single-domain serum albumin-binding protein to its binding target is determined by known methods such as surface plasmon resonance (SPR).

[0033] In some embodiments, the single-domain serum albumin-binding protein disclosed herein binds to HSA having human Kd (hKd). In some embodiments, the single-domain serum albumin-binding protein disclosed herein binds to cynomolgus monkey serum albumin having cynomolgus monkey Kd (cKd). In some embodiments, the single-domain serum albumin-binding protein disclosed herein binds to cynomolgus monkey serum albumin having cynomolgus monkey Kd (cKd) and to HSA having human Kd (hKd). In some embodiments, hKd ranges from 1 nM to 100 nM. In some embodiments, hKd ranges from 1 nM to 10 nM. In some embodiments, cKd ranges from 1 nM to 100 nM. In some embodiments, the range of hKd and cKd is between about 1 nM and about 5 nM, or between about 5 nM and 10 nM. In some embodiments, the single-domain serum albumin-binding protein binds to serum albumin selected from human serum albumin, cynomolgus monkey serum albumin, and mouse serum albumin. In some embodiments, the single-domain serum albumin-binding protein binds to human serum albumin, cynomolgus monkey serum albumin, and mouse serum albumin having equivalent binding affinity (Kd). In some embodiments, the single-domain serum albumin-binding protein binds to human serum albumin having a human Kd (hKd) between about 1 nM and about 10 nM, and to cynomolgus monkey serum albumin having a cynomolgus monkey Kd (cKd) between about 1 nM and about 10 nM. In some embodiments, the single-domain serum albumin-binding protein binds to mouse serum albumin having a mouse Kd (mKd) between about 10 nM and about 50 nM.

[0034] In some embodiments, hKd is approximately 1.5 nM, approximately 1.6 nM, approximately 1.7 nM, approximately 1.8 nM, approximately 1.9 nM, approximately 2 nM, approximately 2.1 nM, approximately 2.2 nM, approximately 2.3 nM, approximately 2.4 nM, approximately 2.5 nM, approximately 2.6 nM, approximately 2.7 nM, approximately 2.8 nM, approximately 2.9 nM, approximately 3 nM, approximately 3.1 nM, These values ​​are approximately 3.2nM, 3.3nM, 3.4nM, 3.5nM, 3.6nM, 3.7nM, 3.8nM, 3.9nM, 4nM, 4.5nM, 5nM, 6nM, 6.5nM, 7nM, 7.5nM, 8nM, 8.5nM, 9.0nM, 9.5nM, or 10nM.

[0035] In some embodiments, cKd is approximately 1.5 nM, approximately 1.6 nM, approximately 1.7 nM, approximately 1.8 nM, approximately 1.9 nM, approximately 2 nM, approximately 2.1 nM, approximately 2.2 nM, approximately 2.3 nM, approximately 2.4 nM, approximately 2.5 nM, approximately 2.6 nM, approximately 2.7 nM, approximately 2.8 nM, approximately 2.9 nM, approximately 3 nM, approximately 3.1 nM, These values ​​are approximately 3.2nM, 3.3nM, 3.4nM, 3.5nM, 3.6nM, 3.7nM, 3.8nM, 3.9nM, 4nM, 4.5nM, 5nM, 6nM, 6.5nM, 7nM, 7.5nM, 8nM, 8.5nM, 9.0nM, 9.5nM, or 10nM.

[0036] In some embodiments, mKd is approximately 10 nM, approximately 11 nM, approximately 12 nM, approximately 13 nM, approximately 14 nM, approximately 15 nM, approximately 16 nM, approximately 17 nM, approximately 18 nM, approximately 19 nM, approximately 20 nM, approximately 21 nM, approximately 22 nM, approximately 23 nM, approximately 24 nM, approximately 25 nM, approximately 26 nM, approximately 27 nM, and approximately 28 nM. These values ​​are approximately 29 nM, 30 nM, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, or 50 nM.

[0037] In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence selected from SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.25, SEQ ID NO.26, and SEQ ID NO.27.

[0038] In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 4, with hKd and cKd between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 4, with hKd approximately 2.3 nM and cKd approximately 2.4 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 25, with hKd and cKd between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 25, with hKd approximately 2.1 nM and cKd approximately 2.2 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 5, with hKd and cKd between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 5, with an hKd of approximately 1.9 nM and a cKd of approximately 1.7 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 6, with hKd and cKd between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 6, with an hKd of approximately 3.2 nM and a cKd of approximately 3.6 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 7, with hKd and cKd between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 7, with an hKd of approximately 2.7 nM and a cKd of approximately 2.6 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 26, with hKd and cKd between approximately 1 nM and approximately 5 nM.In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 26, with an hKd of approximately 2.1 nM and a cKd of approximately 2 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 8, with hKd and cKd between approximately 5 nM and approximately 10 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 8, with an hKd of approximately 6 nM and a cKd of approximately 7.5 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 9, where hKd and cKd are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 9, where hKd is approximately 2.2 nM and cKd is approximately 2.3 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 27, where the hKd and cKd are between approximately 1 nM and approximately 5 nM. In some embodiments, the single-domain serum albumin-binding protein has an amino acid sequence described as SEQ ID NO. 27, where the hKd is approximately 1.6 nM and the cKd is approximately 1.6 nM.

[0039] In some embodiments, the single-domain serum albumin-binding protein has the amino acid sequence set forth as SEQ ID NO.4 and has an mKd of about 17 nM. In some embodiments, the single-domain serum albumin-binding protein has the amino acid sequence set forth as SEQ ID NO.5 and has an mKd of about 12 nM. In some embodiments, the single-domain serum albumin-binding protein has the amino acid sequence set forth as SEQ ID NO.6 and has an mKd of about 33 nM. In some embodiments, the single-domain serum albumin-binding protein has the amino acid sequence set forth as SEQ ID NO.7 and has an mKd of about 14 nM. In some embodiments, the single-domain serum albumin-binding protein has the amino acid sequence set forth as SEQ ID NO.9 and has an mKd of about 16 nM. In some embodiments, the single-domain serum albumin-binding protein has the amino acid sequence set forth as SEQ ID NO.25 and has an mKd of about 17 nM. In some embodiments, the single-domain serum albumin-binding protein has the amino acid sequence set forth as SEQ ID NO.26 and has an mKd of about 17 nM. In some embodiments, the single-domain serum albumin-binding protein has the amino acid sequence set forth as SEQ ID NO.27 and has an mKd of about 16 nM.

[0040] In some embodiments, the ratio between hKd and cKd (hKd:cKd) ranges from about 20:1 to about 1:2.

[0041] In some embodiments, the single-domain serum albumin-binding protein has a half-life of disappearance of at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 20 hours, at least 25 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, or at least 100 hours.

[0042] <CD3 binding domain> The specificity of the T cell response is mediated by the recognition of antigens (major histocompatibility complex, represented in the context of MHC) by the T cell receptor complex. As part of the T cell receptor complex, CD3 is a protein complex present on the cell surface that contains CD3γ (gamma) chains, CD3δ (delta) chains, and two CD3ε (epsilon) chains. Due to its involvement with the T cell receptor complex, CD3, as well as CD3ζ (zeta), associates with the α (alpha) and β (beta) chains of the T cell receptor complex. Clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodies, induces T cell activation that resembles T cell receptor binding but is independent of the clonal specificity.

[0043] In one embodiment, a polyspecific protein comprising a single-domain serum albumin-binding protein in accordance with this disclosure is described herein. In some embodiments, the polyspecific protein further comprises a domain that specifically binds to CD3. In some embodiments, the polyspecific protein further comprises a domain that specifically binds to human CD3. In some embodiments, the polyspecific protein further comprises a domain that specifically binds to CD3γ. In some embodiments, the polyspecific protein further comprises a domain that specifically binds to CD3δ. In some embodiments, the polyspecific protein further comprises a domain that specifically binds to CD3ε.

[0044] In further embodiments, the multispecific protein further comprises a domain that specifically binds to the T cell receptor (TCR). In some embodiments, the multispecific protein further comprises a domain that specifically binds to the α chain of the TCR. In some embodiments, the multispecific protein further comprises a domain that specifically binds to the β chain of the TCR.

[0045] In some embodiments, the CD3-binding domain of a multispecific protein, including the single-domain serum albumin-binding protein described herein, not only exhibits strong CD3-binding affinity to human CD3 but also shows excellent cross-reactivity with each cynomolgus monkey CD3 protein. In some cases, the CD3-binding domain of the multispecific protein cross-reacts with CD3 from cynomolgus monkeys. In some cases, the ratio of human KD to cynomolgus monkey KD (hKd:cKd) in CD3 binding is between 20:1 and 1:2.

[0046] In some embodiments, the CD3-binding domain of a polyspecific protein, including the single-domain serum albumin-binding protein described herein, may be any domain that binds to CD3, including, but not limited to, domains from antigen-binding fragments of CD3-binding antibodies such as monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, or single-domain antibodies (sdAb), Fab, Fab′, F(ab)2, and Fv fragments, fragments composed of one or more CDRs, single-chain antibodies (e.g., single-chain Fv fragment (scFv)), disulfide-stabilized (dsFv) Fv fragments, hetero-conjugated antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain. In some examples, it is beneficial for the CD3-binding domain that the polyspecific protein, including the single-domain serum albumin-binding protein described herein, ultimately originates from the same species in which it is used. For example, for use in humans, it may be beneficial for the CD3-binding domain of a multispecific protein, including the single-domain serum albumin-binding protein described herein, to include human or humanized residues from the antigen-binding domain of the antibody or antibody fragment.

[0047] Therefore, in one embodiment, the antigen-binding domain includes a humanized antibody or a human antibody or antibody fragment, or a mouse antibody or antibody fragment. In one embodiment, the humanized anti-CD3 binding domain or human anti-CD3 binding domain comprises one or more (e.g., all three) light chain complementarity determination regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the humanized anti-CD3 binding domain or human anti-CD3 binding domain described herein, and / or one or more (e.g., all three) heavy chain complementarity determination regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the humanized anti-CD3 binding domain or human anti-CD3 binding domain described herein, which comprises, for example, one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HC CDRs.

[0048] In some embodiments, the humanized or human anti-CD3 binding domain includes a CD3-specific humanized or human light chain variable region, the CD3-specific light chain variable region includes human or non-human light chain CDRs within the human light chain framework region. In one example, the light chain framework region is a lambda (λ) light chain framework. In another example, the light chain framework region is a kappa (κ) light chain framework.

[0049] In some embodiments, the humanized or human anti-CD3 binding domain includes a CD3-specific humanized or human heavy chain variable region, and the CD3-specific heavy chain variable region includes human or non-human heavy chain CDRs within the human heavy chain framework region.

[0050] In some cases, the heavy chain and / or light chain complementarity determining regions are, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4), teprizumab (MGA031), vizilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5 These are derived from known anti-CD3 antibodies such as F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, and WT-31.

[0051] The affinity for CD3 can be determined by the ability of the multispecific protein itself, or its CD3-binding domain, to bind to CD3, for example, coated on an assay plate; displayed on the surface of microbial cells; or in solution. The binding ability of the multispecific protein itself, or its CD3-binding domain, to CD3, according to this disclosure, can be assayed by immobilizing a ligand (e.g., CD3), or the multispecific protein itself or its CD3-binding domain, on beads, substrates, cells, etc. The agent can be added to a binding partner with a suitable buffer incubated at a predetermined temperature for a set period of time. After washing to remove unbound material, the binding protein can be released, for example, in SDS, a high pH buffer, etc., and analyzed, for example, by surface plasmon resonance (SPR).

[0052] <Target antigen binding domain> In addition to the serum albumin-binding and CD3 domains described herein, the polyspecific binding proteins, including single-domain serum albumin-binding proteins, also include, in some embodiments, a domain that binds to a target antigen. The target antigen is associated with or involved in a disease, disorder, or illness. In particular, the target antigen is associated with proliferative disorders, neoplastic diseases, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases. In some embodiments, the target antigen is a tumor antigen expressed on tumor cells. Alternatively, in some embodiments, the target antigen is associated with a pathogen such as a virus or bacteria.

[0053] In some embodiments, the target antigen is a cell surface molecule such as a protein, lipid, or polysaccharide. In some embodiments, the target antigen is a tumor cell, a virus-infected cell, a bacterial-infected cell, a damaged red blood cell, an arterial plaque cell, or a fibrous tissue cell.

[0054] The design of a polyspecific binding protein, including a single-domain serum albumin-binding protein, in accordance with this disclosure, makes the binding domain flexible to the target antigen, in that the binding domain to the target antigen can be any type of binding domain, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the binding domain to the target antigen is a single-domain antibody such as a single-chain variable fragment (scFv), a heavy-chain variable domain (VH), a light-chain variable domain (VL), and a variable domain (VHH) of a camelid-derived sdAb. In other embodiments, the binding domain to the target antigen is a non-Ig binding domain, i.e., an antibody mimetic such as antikalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Knitz domain peptides, and monobodies. In further embodiments, the binding domain to the target antigen is a ligand or peptide that binds to or associates with the target antigen. In yet another embodiment, the binding domain to the target antigen is Nottin. In yet another embodiment, the binding domain to the target antigen is a small molecule entity.

[0055] <Modification of single-domain serum albumin-binding proteins> The single-domain serum albumin-binding proteins described herein include derivatives or analogs in which (i) an amino acid is substituted with an amino acid residue not encoded by the gene code, (ii) a mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretion sequence, or a sequence for blocking the immunogen domain and / or for protein purification.

[0056] Typical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamic acid formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfated, arginylation, and other transfer RNA-mediated addition of amino acids to proteins, as well as ubiquitination.

[0057] Modifications occur at any site on the single-domain serum albumin-binding proteins described herein, including the peptide backbone, amino acid side chains, and amino or carboxyl terminus of the tri-binding protein. Specific common peptide modifications useful for modifying single-domain serum albumin-binding proteins include glycosylation, lipid binding, sulfation, gamma-carboxylation, hydroxylation, blockade of amino or carboxyl groups in the polypeptide, or both, by covalent modification and ADP-ribosylation.

[0058] <Polynucleotide encoding a single-domain serum albumin-binding protein> In some embodiments, polynucleotide molecules encoding single-domain serum albumin-binding proteins described herein are also provided. In some embodiments, the polynucleotide molecules are provided as DNA constructs. In other embodiments, the polynucleotide molecules are provided as messenger RNA transcripts.

[0059] Polynucleotide molecules are constructed by combining genes encoding three binding domains, which are separated by a peptide linker or, in other embodiments, directly linked by peptide bonds, into a single gene construct operably linked to a suitable promoter and optionally a suitable transcriptional terminator, and by known methods such as expressing it in bacteria or other suitable expression systems such as CHO cells.

[0060] In some embodiments, polynucleotide molecules encoding a multispecific binding protein, including a single-domain serum albumin-binding protein, are also provided in accordance with this disclosure. In some embodiments, the polynucleotide encoding the multispecific binding protein also includes a coding sequence for a CD3-binding domain. In some embodiments, the polynucleotide encoding the multispecific binding protein also includes a coding sequence for a target antigen-binding domain. In some embodiments, the polynucleotide encoding the multispecific binding protein also includes coding sequences for a CD3-binding domain and a target antigen-binding domain. In some embodiments, the polynucleotide molecule is provided as a DNA construct. In other embodiments, the polynucleotide molecule is provided as a messenger RNA transcript. In embodiments where the target antigen-binding domain is a small molecule, the polynucleotide includes genes encoding the serum albumin-binding domain and the CD3-binding domain. In embodiments where the half-life extension domain is a small molecule, the polynucleotide includes a gene encoding a domain that binds to CD3 and the target antigen. Depending on the vector system and host used, translation elements comprising any number of suitable transcriptions and constitutively inducible promoters may be used. The promoters are selected to drive the expression of the polynucleotide in the respective host cells.

[0061] In some embodiments, polynucleotides are inserted into vectors representing further embodiments, preferably expression vectors. These recombinant vectors can be constructed by known methods. Specific desired vectors include plasmids, phagemids, phage derivatives, viruses (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, lentiviruses, etc.), and cosmids.

[0062] Various expression vectors / host systems may be used to express the described single-domain serum albumin-binding protein polypeptide, comprising polynucleotides. Examples of expression vectors for expression in E. coli include pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27), pcDNA5 (Invitrogen) for expression in mammalian cells, PICHIAPINK® Yeast Expression Systems (Invitrogen), and BACUVANCE® Baculovirus Expression System (GenScript).

[0063] Therefore, single-domain serum albumin-binding proteins as described herein are produced in some embodiments by introducing a vector encoding such a protein into host cells, culturing the host cells under conditions in which the protein domain is expressed, isolated, and optionally further purified.

[0064] <Production of single-domain serum albumin-binding protein> In some embodiments, processes for the production of single-domain serum albumin-binding proteins are disclosed herein. In some embodiments, the process includes culturing a host transformed or transfected with a vector containing a nucleic acid sequence encoding a single-domain serum albumin-binding protein under conditions that enable the expression of the serum albumin-binding protein, and recovering and purifying the protein produced from the culture.

[0065] In further embodiments, processes are provided that aim to improve one or more properties, such as affinity, stability, heat resistance, cross-reactivity, etc., of single-domain serum albumin-binding proteins and / or polyspecific binding proteins including the single-domain serum albumin-binding proteins described herein, compared to reference binding compounds. In some embodiments, multiple single-substitution libraries are provided, each corresponding to a different domain or amino acid segment of a single-domain serum albumin-binding protein or reference binding compound, such that each member of the single-substitution library encodes only a single amino acid change in its corresponding domain or amino acid segment. (This makes it possible to investigate all possible substitutions of a large protein or protein-binding site with a small number of small libraries.) In some embodiments, the multiple domains form or encompass the amino acid contig sequences of the single-domain serum albumin-binding protein or reference binding compound. The nucleotide sequences of the various single-substitution libraries overlap with the nucleotide sequences of at least one other single-substitution library. In some embodiments, the multiple single-substitution libraries are designed such that all members overlap with all members of each single-substitution library encoding adjacent domains.

[0066] The binding compounds expressed from these single-substitution libraries are separately selected to obtain a subset of mutants in each library, and these mutants possess properties at least as good as the reference binding compound, while the resulting library is smaller in size. (i.e., the number of nucleic acids encoding the selected set of binding compounds is less than the number of nucleic acids encoding the members of the original single-substitution library.) These properties include, but are not limited to, affinity for the target compound, and stability under various conditions such as heat, high or low pH, enzymatic degradation, and cross-reactivity with other proteins. The selected compounds from each single-substitution library are interchangeably referred to herein as "pre-candidate compounds" or "pre-candidate proteins." The nucleic acid sequences encoding the pre-candidate compounds from the separate single-substitution libraries are shuffled by PCR using PCR-based gene shuffling techniques to generate a shuffled library.

[0067] A typical workflow for the screening process is described herein. A library of pre-candidate compounds is generated from a single-substitution library and selected for binding to the target protein. The pre-candidate library is then shuffled to purify a library of nucleic acids encoding the candidate compounds, which are then cloned into a convenient expression vector, such as a phagemide expression system. Subsequently, the phage expression candidate compounds undergo one or more rounds of selection to improve desired properties, such as binding affinity to the target molecule. The target molecule may be adsorbed, or attached to the surface of a well or other reaction vessel, or the target molecule may be induced paired with a binding site such as biotin, which, after incubation with the candidate conjugate compound, may be captured with a complementary site such as streptavidin bound to beads, such as magnetic beads, for washing. In a typical selection regimen, the candidate conjugate compounds undergo a lengthy washing process so that only candidate compounds with very low dissociation rates from the target molecule are selected. A typical washing time for these embodiments is at least 8 hours; or, in other embodiments, at least 24 hours; or, in other embodiments, at least 48 hours; or, in other embodiments, at least 72 hours. The isolated clones after selection are amplified and subjected to additional selection cycles, or analyzed by, for example, sequencing and by comparative measurements of binding affinity by, for example, ELISA, surface plasmon resonance coupling, biolayer interferometry (e.g., Octet system, ForteBio, Menlo Park, CA). In some embodiments, the process is performed to identify one or more single-domain serum albumin-binding proteins, and / or polyspecific binding proteins containing single-domain serum albumin-binding proteins, which exhibit improved heat resistance and cross-reactivity to a selected set of binding targets compared to a reference serum albumin-binding protein, such as a protein having the amino acid sequence of SEQ ID NO. 10.A single substitution library is prepared by altering codons in the VH region of a reference serum albumin-binding protein, including codons in both the framework region and the CDR; in another embodiment, the codon-altered positions include the CDR of the heavy chain of the reference serum albumin-binding protein, or subsets of such CDRs such as CDR1 alone, CDR2 alone, CDR3 alone, or a combination thereof. In another embodiment, the codon-altered positions occur exclusively in the framework region. In some embodiments, the library contains only single codon changes from the framework region of the reference serum albumin-binding protein, with the VH numbered in the range of 10 to 250. In another embodiment, the codon-altered positions include CDR3 of the heavy chain of the reference serum albumin-binding protein, or subsets of such CDR3s. In another embodiment, the number of codon-altered positions in the coding region of the VH ranges from 10 to 250, with a maximum of 100 positions located in the framework region. Following the preparation of the single substitution library, the following steps are performed as outlined above: (a) expressing each member of the individual single substitution library separately as a pre-candidate protein; (b) selecting individual members of the single substitution library encoding a pre-candidate protein that may or may not be different from the original binding target [e.g., a desired cross-reactivity target]; (c) shuffling the library members selected by PCR to generate a shuffled combinatorial library; (d) expressing the shuffled library members as candidate serum albumin-binding proteins; and (e) selecting one or more members of the shuffled library for a candidate serum albumin-binding protein that binds to the original binding partner, and optionally (f) selecting further candidate proteins to bind to a desired cross-reactivity target, thereby providing a nucleic acid-encoded serum albumin-binding protein with enhanced cross-reactivity of one or more substances to a reference serum albumin-binding protein without loss of affinity to the original ligand.In further embodiments, the method may be carried out to obtain a serum albumin-binding protein with reduced reactivity to a selected cross-reacting substance(s) or compound(s) or epitope(s): by replacing step (f) with the following step: depleting a subset of candidate serum albumin-binding proteins that bind to undesirable cross-reacting compounds one or more times.

[0068] Recent studies have reported that therapeutic antibodies are at risk of degradation via numerous pathways during manufacturing, storage, and in vivo use. The most frequently occurring chemical degradation reactions in proteins include the deamide of asparagine (N) and isomerization of aspartic acid (D) residues. In particular, it has been hypothesized that if N and D residues are involved in antigen recognition, their chemical alteration can lead to a significant loss of potency. Asparagine and aspartic acid residues are known to share a degradation pathway that proceeds via the formation of cyclic succinimide intermediates. The formation of succinimide intermediates and their hydrolysis products (aspartic acid and isoaspartic acid) at the aspartic acid moiety of antibodies presents stability issues. When isomerization occurs, the chemical structure of the antibody changes, which can lead to poor stability, as indicated by aggregation and a shorter shelf life, for example. Accordingly, in some embodiments of this disclosure, a single-domain serum albumin-binding protein is provided, in which one or more aspartic acid residues are mutated, thereby reducing the possibility of isomerization of the single-domain serum albumin-binding protein. In some embodiments, the aspartic acid residue is located in CDR2 of the single-domain serum albumin-binding protein, and the aspartic acid residue is mutated to glutamic acid. In some embodiments, the aspartic acid residue at position 62 of the protein, as defined by SEQ ID NO. 10, is mutated to glutamic acid (D62E). In some embodiments, the serum albumin-binding affinity of the single-domain serum albumin protein containing the D62E mutation is not affected by the mutation. In some embodiments, single-domain serum albumin-binding proteins containing the D62E mutation and those not containing the mutation have equivalent binding affinity to serum albumin.

[0069] <Pharmaceutical composition> In some embodiments, pharmaceutical compositions are also provided comprising a single-domain serum albumin-binding protein as described herein, a vector comprising a polynucleotide encoding a polypeptide of the single-domain serum albumin-binding protein, or a host cell transformed by the vector, and at least one pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” includes, but is not limited to, any carrier that does not interfere with the efficacy of the biological activity of the components and is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Such carriers can be formulated by conventional methods and can be administered to a subject in an appropriate dose. Preferably, the composition is sterile. These compositions may further contain adjuvants such as preservatives, emulsifiers, and dispersants. Prevention of microbial action can be ensured by the inclusion of various antimicrobial and antifungal agents.

[0070] In some embodiments of the pharmaceutical composition, the single-domain serum albumin-binding protein described herein is encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposomes, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical composition, the single-domain serum albumin-binding protein is bound to liposomes. In some examples, the single-domain serum albumin-binding protein is conjugated to the surface of the liposome. In some examples, the single-domain serum albumin-binding protein is encapsulated within the shell of the liposome. In some examples, the liposome is a cationic liposome.

[0071] The single-domain serum albumin-binding proteins described herein are intended for use as drugs. Administration is achieved by various methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of treatment and the type of compound contained in the pharmaceutical composition. The administration regimen is determined by the attending physician and other clinical factors. The dosage for any one patient depends on many factors, including the patient's build, body surface area, age, sex, the specific compound being administered, the time and route of administration, the type of treatment, health status, and other medications being administered concurrently. “Effective dose” refers to the amount of active ingredient sufficient to affect the course and severity of the disease, resulting in a reduction or remission of such conditions, and may be determined using known methods.

[0072] <Treatment Method> In some embodiments, methods and uses are also provided for stimulating the immune system of an individual in need, including the administration of a single-domain serum albumin-binding protein or a multispecific binding protein, including a single-domain albumin-binding protein described herein. In some examples, the administration of a single-domain serum albumin-binding protein described herein causes and / or sustains cytotoxicity to cells expressing a target antigen. In some examples, the cells are cancer cells, virus-infected cells, bacterial-infected cells, autoreactive T or B cells, damaged red blood cells, arterial plaque, or fibrotic tissue.

[0073]

[0078] Furthermore, methods and uses for treating diseases, disorders, or illnesses associated with a target antigen are also provided herein, comprising administering to an individual a single-domain serum albumin-binding protein, or a multispecific binding protein including a single-domain albumin-binding protein described herein. Diseases, disorders, or illnesses associated with a target antigen include, but are not limited to, viral infections, bacterial infections, autoimmune diseases, transplant rejection, atherosclerosis, or fibrosis. In other embodiments, diseases, disorders, or illnesses associated with a target antigen are proliferative disorders, neoplastic diseases, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases. In one embodiment, the disease, disorder, or illness associated with a target antigen is cancer. In one example, the cancer is a blood cancer. In another example, the cancer is a solid tumor cancer.

[0074] As used herein, in some embodiments, “treatment,” “to treat,” or “treated” means a therapeutic treatment aimed at delaying (reducing) an undesirable physiological illness, disorder, or disease, or obtaining a beneficial or desirable clinical outcome. Beneficial or desirable clinical outcomes for the purposes described herein include, but are not limited to, symptom relief; reduction of the severity of illness, disorder, or disease; stabilization (i.e., no worsening) of the illness, disorder, or disease state; delaying the onset or progression of illness, disorder, or disease; improvement of the illness, disorder, or disease state; and remission (partial or whole), whether detectable or undetectable, or whether the illness, disorder, or disease is exacerbated or improved. Treatment includes inducing a clinically significant response without excessive levels of side effects. Treatment further includes extending survival time compared to the expected survival time without treatment. In other embodiments, “treatment,” “treating,” or “treated” refers to a preventative treatment whose purpose is to delay the onset or reduce the severity of an undesirable physiological illness, disorder, or disease, such as in a person predisposed to a disease (e.g., an individual marked with a genetic marker for a disease such as breast cancer).

[0075] In some embodiments of the methods described herein, single-domain serum albumin-binding proteins, or multispecific binding proteins containing single-domain serum albumin-binding proteins as described herein, are administered in combination with agents for treating specific diseases, disorders, or illnesses. These agents include, but are not limited to, antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiotherapy (gamma rays, X-rays, and / or directed delivery of radioisotopes, microwaves, UV radiation, etc.), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapies. In some embodiments, single-domain serum albumin-binding proteins, or multispecific binding proteins containing single-domain serum albumin-binding proteins as described herein, are administered in combination with antidiarrheal agents, antiemetics, analgesics, opioids, and / or nonsteroidal anti-inflammatory drugs. In some embodiments, single-domain serum albumin-binding proteins, or multispecific binding proteins containing single-domain serum albumin-binding proteins as described herein, are administered before, during, or after surgery. [Examples]

[0076] Example 1: Generation of an anti-HSA single-domain antibody mutant having binding properties equivalent to those of the parental anti-HSA single-domain antibody. <Characterization of parental anti-HSA phages> The specific binding of parental anti-HSA phages to HSA antigens was measured using CD3 as a negative control (Figure 1), and the cross-reactivity of anti-HSA phages to human, cynomolgus monkey, and mouse serum albumin was measured (Figure 2).

[0077] <Single-substitution HSA sdAb phage library> A single substitution library was provided for each of the three CDR domains. The single substitution libraries were conjugated to HSA and then washed with buffers containing various levels of HSA. Conjugated phages were rescued and counted at 0 and 24 hours. Phages selected by washing with 2.5 mg / ml HSA in buffer for 24 hours were used to construct two independent combinatorial phage libraries.

[0078] <Combinatorial Anti-HSA Library> In the first round, MSA was used as the selective target. Wells were washed for 24 hours after combinatorial phage binding from two independent libraries. In the second round, HSA was used as the selective target. Wells were washed for 24 hours in 1 mg / ml HSA after binding from both libraries. Inserts PCRed from the selections in the second round were subcloned into the ME10 His6 expression vector (disclosed as SEQ ID NO: 38 (6XHis sequence)). 96 clones were selected, DNA purified, sequenced, and transfected into Expi293 cells.

[0079] <Measurement of binding affinity> The supernatant was used to evaluate Kd against HSA and CSA using the Octet platform. Nine clones were selected for further characterization based on their binding affinity compared to the parental sdAb, as well as their robust production, aggregation, and stability profiles (Figure 3).

[0080] Example 2: Pharmacokinetics of a triplicate antibody containing a single-domain anti-HSA antibody A triplicate antibody was prepared using the single-domain anti-HSA antibody from Example 1, and evaluated to eliminate the half-life in animal experiments.

[0081] The triplicate antibody is administered to cynomolgus monkeys as a 0.5 mg / kg bolus intramuscular injection. Other groups of cynomolgus monkeys receive proteins that are equivalent in size to CD3 and CD20 but lack HSA binding, possessing binding domains. Groups 3 and 4 receive antibodies with CD3 and HSA binding domains, and proteins with CD20 and HSA binding domains, both of which are equivalent in size to the triplicate antibody. Each test group consists of five monkeys. Serum samples are taken at the indicated time points, sequentially diluted, and the protein concentrations are measured against CD3 and / or CD20 using a binding ELISA.

[0082] Pharmacokinetic analysis is performed using the plasma concentration of the test substance. The group-mean plasma data for each test substance, when plotted against time after administration, conform to a multiple exponential profile. The data are fitted using a standard two-compartment model with bolus injection and first-order rate constants for the distribution and efflux phases. A general formula for best fitting data for IV administration is: c(t)=Ae -αt +Be -βt The equation is given by A=D / V(α-k21) / (α-β) and B=D / V(β-k21) / (α-β), where α and β (between α and β) are the apparent first-order rate constants of the distribution and efflux phases, respectively. The α-phase is the phase constant of clearance, reflecting the distribution of the protein into all extracellular fluid of the animal, while the second phase portion of the decay curve, or the β-phase portion, represents true plasma clearance. Methods for fitting such equations are known in the art. For example, A=D / V(α-k21) / (α-β) and B=D / V(β-k21) / (α-β), where α and β (between α>β) are given by a quadratic equation: r 2 The formula is the root of +(k12+k21+k10)r+k21k10=0, and uses the following estimation parameters: V=distribution volume, k10=discharge rate, k12=transfer rate from compartment 1 to compartment 2, k21=transfer rate from compartment 2 to compartment 1, and D=dose.

[0083] Data analysis: Graphs of concentration vs. time profiles are created using KaleidaGraph (KaleidaGraph (trademark) V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than the reportable limit (LTR) are not included in the PK analysis and are not shown on the graph. Pharmacokinetic parameters are measured by compartmental analysis using WinNonlin software (WinNonlin (registered trademark) Professional V. 3.1 WinNonlin (trademark) Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters are calculated as described in Ritschel W A and Kearns G L, 1999, IN: Handbook Of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, D.C.

[0084] It is expected that the trispecific antibody containing the anti-HSA single domain antibody of Example 1 improves pharmacokinetic parameters such as an increase in the elimination half-life compared to the protein lacking the HSA binding domain.

[0085] Example 3: Heat resistance of variants of anti-HSA single domain antibodies The temperature (T h ) corresponding to the derivative of the inflection point of the peak dye fluorescence is known to correlate with the melting temperature (T m ), which is a measure of protein stability. The purpose of this study was to evaluate the T h of several variants of anti-HAS single domain antibodies.

[0086] <Protein production> The sequence of the anti-huALB single-domain antibody was cloned into pcDNA3.4 (Invitrogen) with the leader sequence preceding and a 6x histidine tag (SEQ ID NO:38) following. Expi293F cells (Life Technologies A14527) were maintained in suspensions of Optimum Growth Flasks (Thomson) at concentrations of 0.2–8x1e6 cells / mL in Expi 293 medium. The purified plasmid DNA was transfected into Expi293F cells according to the Expi293 expression system kit (Life Technologies, A14635) protocol and maintained for 4–6 days post-transfection. The prepared media were partially purified by affinity and desalting chromatography. The anti-huCD3escFv protein was concentrated using an Amicon Ultra centrifugal filtration unit (EMD Millipore), applied to Superdex 200 size exclusion medium (GE Healthcare), and degraded in a neutral buffer containing excipients. Fractional storage and final purity were evaluated by SDS-PAGE and analytical size exclusion chromatography (SEC). The absorbance of the purified protein solution was determined at 280 nm using SpectraMax M2 (Molecular Devices), and the UV-transmitting 96-well plate (Corning 3635) and its concentration were calculated from the molar extinction coefficient.

[0087] <Differential Scanning Fluorescence Quantification> Purified anti-HSA single-domain antibody protein was diluted to 0.2–0.25 mg / mL in a neutral buffer containing excipients with a final concentration of 0.15% DMSO and 5xSYPRO orange dye (Life Technologies S6651), and placed in MicroAmp EnduraPlate optical microplates and adhesive films (Applied Biosystems 4483485 and 4311971). The plates containing the diluted protein and dye mixture were loaded into an ABI 7500 Fast real-time PCR instrument (Applied Biosystems) and subjected to a multi-step temperature gradient from 25°C to 95°C. The temperature gradient consisted of 2-minute holds at each of the 25°C levels, using excitation at 500 nm, and the radiation was collected using a ROX filter. h This is presented for the purified anti-HSA single-domain antibody protein shown in Figure 4.

[0088] Example 4: Relative trend of dimerization of variants of anti-HSA single-domain antibody when exposed to pH reduction. Anti-HSA single-domain antibody proteins were expressed in Expi293-F cells as described above. Prepared media for each mutant were applied to a column packed with protein A agarose (GE Healthcare, 17519901), thoroughly washed with TRIS-buffered saline, eluted with 0.05% (vol / vol) acetic acid at pH 3, held at room temperature for up to 10 minutes before partial neutralization to pH 5, and then desalted in a neutral buffer containing excipients using a Sephadex G25 column (GE Healthcare 17058401).

[0089] As described in Example 3, the concentrations of the purified anti-HSA single-domain antibody variants were measured by absorbance at 280 nm. The purified proteins were evaluated by SDS-PAGE and analytical SEC using a Yarra 2000 SEC column (Phenomenex 00H-4512-E0) and analyzed on a 1200 LC in solvent-containing phosphate buffer using Chemstation software (Agilent). Peaks corresponding to dimers and monomers were manually combined, and the values ​​are shown in Figure 5.

[0090] [Table 1]

[0091] [Table 2]

Claims

1. A single-domain serum albumin-binding protein, Here, the single-domain serum albumin-binding protein comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO:

7. A single-domain serum albumin-binding protein.

2. The single-domain serum albumin-binding protein according to claim 1, wherein the single-domain serum albumin-binding protein has an elimination half-life of at least 12 hours.

3. The single-domain serum albumin-binding protein according to claim 1, wherein the single-domain serum albumin-binding protein has an elimination half-life of at least 20 hours.

4. The single-domain serum albumin-binding protein according to claim 1, wherein the single-domain serum albumin-binding protein has an elimination half-life of at least 30 hours.

5. The single-domain serum albumin-binding protein according to claim 1, wherein the single-domain serum albumin-binding protein has an elimination half-life of at least 40 hours.

6. A single-domain serum albumin-binding protein according to any one of claims 1 to 5, further comprising a linker.

7. The linker is (GS) ｎ , (GGS) ｎ , (GGGS) ｎ , (GGSG) ｎ , (GGSGG) ｎ , (GGGGG) ｎ , (GGG) ｎ , (GGGGGS) ｎ , (GGGGGS) ３ , or (GGGGGS) ４ The single domain serum albumin binding protein according to claim 6, comprising

8. A single-domain serum albumin-binding protein according to any one of claims 1 to 7, for use in the treatment or improvement of proliferative disorders, neoplastic disorders, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases in a subject requiring treatment.

9. A single-domain serum albumin-binding protein according to claim 8, for use in the treatment or improvement of the aforementioned neoplastic disease.

10. A multispecific binding protein comprising a single-domain serum albumin-binding protein according to any one of claims 1 to 7.

11. A multispecific binding protein according to claim 10 for use in the treatment or improvement of proliferative disorders, neoplastic disorders, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases in a subject requiring treatment.

12. The multispecific binding protein according to claim 11, for use in the treatment or improvement of the aforementioned neoplastic disease.

13. An antibody comprising a single-domain serum albumin-binding protein according to any one of claims 1 to 7, wherein the antibody is a single-domain antibody.

14. The antibody according to claim 13, for use in treating or improving proliferative disorders, neoplastic disorders, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases in a subject requiring treatment.

15. A single-domain serum albumin-binding protein according to any one of claims 1 to 7, a polynucleotide encoding a multispecific binding protein according to claim 10, or an antibody according to claim 13.

16. The polynucleotide according to claim 15 for use in treating or improving proliferative disorders, neoplastic disorders, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases in a subject requiring treatment.

17. A vector comprising the polynucleotide of claim 15.

18. The vector according to claim 17, for use in treating or improving proliferative disorders, neoplastic disorders, inflammatory disorders, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases in a subject requiring treatment.

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