Single-domain serum albumin-binding proteins

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 benefit of U.S. Provisional Application No. 62 / 339,682, filed May 20, 2016, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on May 18, 2017, has the filename 47517-703_601_SEQ.txt and is 22,218 bytes in size.

[0003] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, and in its entirety. [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 a variety of ways to increase the circulating half-life of therapeutic molecules. The present disclosure provides single-domain albumin-binding proteins that can be used to extend the half-life of therapeutic molecules. Summary of the Invention

[0005] In one embodiment, provided herein is a single domain serum albumin binding protein comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in GFX1X2X3X4FGMS (SEQ ID NO. 1), wherein 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 as set forth in SISGSGX5X6TLYAX7SX8K (SEQ ID NO. No. 2), wherein 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 (SEQ ID NO. 3), wherein X9 is serine, arginine, threonine, or lysine; 10 is arginine, lysine, valine, proline, or asparagine, where X1, X2, X3, X4, X5, X6, X7, X8, X9, and X 10are 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 framework residues selected such that the protein is at least 80 percent identical to the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the single domain serum albumin binding protein comprises the 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, wherein 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, wherein 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, wherein r1 comprises SEQ ID NO. 14. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein 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, wherein r1 comprises SEQ ID NO. 16 and r3 comprises SEQ ID NO. 23. In some embodiments, the single domain serum albumin binding protein comprises amino acid sequences, wherein r1 comprises SEQ ID NO.15 and r2 comprises SEQ ID NO.18.In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence, wherein r1 comprises SEQ ID NO. 14 and r3 comprises SEQ ID NO. 23. In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence, wherein 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 the amino acid sequence, wherein r1 comprises SEQ ID NO. 14 and r2 comprises SEQ ID NO. 20. In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence, wherein r1 comprises SEQ ID NO. 15 and r2 comprises SEQ ID NO. 21. In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence, wherein 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 comprises the amino acid sequence set forth as SEQ ID NO.4. In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.7. In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.9. In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.26. In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.27.In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 5. In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 6. In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 8. In some embodiments, the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 25.

[0006] In some embodiments, the single domain serum albumin binding protein binds to a 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 with comparable binding affinities (Kd). In some embodiments, the single domain serum albumin binding protein binds to mouse serum albumin with a binding affinity (Kd) that is about 1.5- to about 20-fold weaker than the binding affinity (Kd) of the protein for human and cynomolgus monkey serum albumin. In some embodiments, the single domain serum albumin binding protein binds to human serum albumin with a human Kd (hKd) of between about 1 nM and about 100 nM and binds to cynomolgus monkey serum albumin with a cynomolgus monkey Kd (cKd) of between 1 nM and 100 nM. In some embodiments, the hKd and cKd of the single domain serum albumin binding protein are between 1 nM and about 5 nM, or between about 5 nM and about 10 nM. In some embodiments, the hKd and cKd of the 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 between the hKd and cKd of the single domain serum albumin binding protein (hKd:cKd) ranges from about 20:1 to about 1:2.

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

[0008] In some embodiments, the single domain serum albumin binding protein comprises 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, the sequence of which is set forth 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, wherein amino acid position 28 is substituted with arginine, lysine, serine, or proline, amino acid position 29 is substituted with tyrosine, and amino acid position 30 is substituted with arginine or is substituted with lysine, amino acid position 31 is substituted with lysine, arginine, or alanine, 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 protein comprises 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 comprises a substitution at position 29. In some embodiments, the single domain serum albumin binding protein comprises a substitution at position 31. In some embodiments, the single domain serum albumin binding protein comprises a substitution at position 56. In some embodiments, the single domain serum albumin binding protein comprises a substitution at position 62. In some embodiments, the single domain serum albumin binding protein comprises a substitution at position 64. In some embodiments, the single domain serum albumin binding protein comprises a substitution at position 104. In some embodiments, the single domain serum albumin binding protein comprises substitutions at amino acid positions 31 and 62.In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence in which position 31 is substituted with arginine. In some embodiments, the single domain serum albumin binding protein comprises 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 comprises substitutions at amino acid positions 31, 56, 64, and 104. In some embodiments, the single domain serum albumin binding protein comprises 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 comprises substitutions at amino acid positions 29 and 104. 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 and amino acid position 104 is substituted with lysine. In some embodiments, the single domain serum albumin binding protein comprises substitutions at amino acid positions 31 and 56. 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 and amino acid position 56 is substituted with threonine. In some embodiments, the single domain serum albumin binding protein comprises substitutions at amino acid positions 31, 56, and 62. 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 amino acid position 62 is substituted with glutamic acid. In some embodiments, the single domain serum albumin binding protein comprises substitutions at amino acid positions 31 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 arginine and amino acid position 104 is substituted with lysine. In some embodiments, the single domain serum albumin binding protein comprises 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 about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein amino acid position 31 is substituted with arginine and amino acid position 62 is substituted with glutamic acid, and the hKd and cKd of the single domain serum albumin binding protein are between about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein amino acid 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, and the hKd and cKd of the single domain serum albumin binding protein are between about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein amino acid position 29 is substituted with tyrosine and amino acid position 104 is substituted with lysine, and the hKd and cKd are between about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein amino acid position 31 is substituted with lysine and amino acid position 56 is substituted with threonine, and the hKd and cKd of the single domain serum albumin binding protein are between about 1 nM and about 5 nM.In some embodiments, the single domain serum albumin comprises an amino acid sequence, wherein amino acid position 31 is substituted with lysine, amino acid position 56 is substituted with threonine, and amino acid position 62 is substituted with glutamic acid, and the hKd and cKd are between about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein amino acid position 31 is substituted with arginine, and amino acid position 104 is substituted with lysine, and the hKd and cKd of the single domain serum albumin binding protein are between about 5 nM and about 10 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein 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, and the hKd and cKd of the single domain serum albumin binding protein are between about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein 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, and the hKd and cKd of the single domain serum albumin binding protein are between about 1 nM and about 5 nM.

[0010] In another embodiment, provided herein is a single domain serum albumin binding protein comprising at least one mutation in CDR1, CDR2, or CDR3, wherein CDR1 comprises the sequence as set forth in SEQ ID NO:11, CDR2 comprises the sequence as set forth in SEQ ID NO:12, and CDR3 comprises the sequence as set forth in SEQ ID NO:13, wherein the at least one mutation is not at amino acid position 1, 2, 7, 8, 9, or 10 of SEQ ID NO:11, position 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 comprises at least one mutation at an amino acid position 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 comprises 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 comprises a mutation at amino acid position 6 of CDR1 (SEQ ID NO:11). In some embodiments, the single domain serum albumin binding protein comprises a mutation 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 comprises a mutation 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 comprises a mutation 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 comprises a mutation 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 comprises a mutation 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 comprises a mutation 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 comprises a mutation 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 comprises a mutation 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 wherein 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 about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein 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 about 1 nM and about 5 nM.In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein 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 about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein amino acid position 4 of CDR1 (SEQ ID NO:11) is mutated to tyrosine, 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 about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein 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 about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein amino acid position 6 of CDR1 (SEQ ID NO:11) is mutated to lysine and amino acid positions 7 and 13 of CDR2 (SEQ ID NO:12) are mutated to threonine and glutamic acid, respectively, and the hKd and cKd of the single domain serum albumin binding protein are between about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein amino acid position 6 of CDR1 (SEQ ID NO:11) is mutated to arginine and 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 about 5 nM and about 12 nM.In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein 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, and 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 about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein comprises an amino acid sequence, wherein amino acid position 6 of CDR1 (SEQ ID NO:11) is mutated to lysine, amino acid positions 7 and 13 of CDR2 (SEQ ID NO:12) are mutated to arginine and glutamic acid, respectively, and 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 about 1 nM and about 5 nM.

[0011] In another embodiment, provided herein is a polynucleotide encoding a single domain serum albumin binding protein according to the present disclosure. A further embodiment describes a vector comprising such a polynucleotide. Another embodiment describes a host cell transformed with a vector according to the present disclosure. In one embodiment, a pharmaceutical composition is provided comprising (i) a single domain serum albumin binding protein according to the present disclosure, a polynucleotide according to the present disclosure, a vector according to the present disclosure, or a host cell according to the present disclosure, and (ii) a pharmaceutically acceptable carrier.

[0012] In another embodiment, described herein is a process for the production of a single domain serum albumin binding protein according to the present disclosure, said process comprising culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding a single domain serum albumin binding protein described herein under conditions allowing expression of the single domain serum albumin binding protein, and recovering and purifying the protein produced from the culture.

[0013] Methods for treating or ameliorating a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease, or a host-versus-graft disease are further described, comprising administering to a subject a single domain serum albumin binding protein according to the present disclosure. In some embodiments, the subject is a human. In some embodiments, the method further comprises administering a drug in combination with a single domain serum albumin binding protein according to the present disclosure.

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

[0015] Further embodiments describe multispecific antibodies, bispecific antibodies, sdAbs, variable heavy chain domains, peptides, or ligands comprising the single domain serum albumin binding proteins of the present disclosure. In one embodiment, an antibody comprising the single domain serum albumin binding protein of the present 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 an IgG.

[0016] One embodiment describes a multispecific binding protein or antibody comprising a single-domain serum albumin-binding protein and a CD3-binding domain according to the present disclosure. In one embodiment, a method is described for treating or ameliorating a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease, or a host-versus-graft disease, comprising administering to a subject a multispecific antibody according to the present disclosure. [Brief explanation of the drawings]

[0017] The novel features of the invention are set forth with particularity 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 that sets forth embodiments in which the principles of the invention are utilized, and the accompanying drawings in which: [Figure 1] 1 illustrates the specific binding of the parental anti-HSA phage to HSA and CD3 antigens as determined by ELISA titration. [Figure 2] 1 illustrates the cross-reactivity of anti-HSA phage to human, cynomolgus monkey, and mouse serum albumin as determined by ELISA titration. [Figure 3] Purified sdAbs are used to provide binding affinity profiles of nine clones selected for more accurate Kd determination. [Figure 4] 1 illustrates the temperature of hydrophobic exposure (Th°C) for several anti-HSA sdAb variants. [Figure 5] 1 illustrates the tendency of multiple anti-HSA sdAb mutants to form dimers versus monomers at low pH. DETAILED DESCRIPTION OF THE INVENTION

[0018] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0019] Specific Definitions The terms used herein are intended to describe particular cases only and are not intended to limit the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0020] The terms "about" or "approximately" mean within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean 1 or more than 1 standard deviation per practice for any value. When particular values ​​are described in this application and in the claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable range of error for the particular value.

[0021] The terms "individual," "patient," or "subject" are used interchangeably. None of the terms require or are limited to situations characterized by the supervision (e.g., full-time or intermittent) of a medical professional (e.g., a physician, registered nurse, bedside nurse, physician assistant, nursing assistant, or hospice worker).

[0022] The term "framework" or "FR" residues (or regions) refers to variable domain residues other than the CDR or hypervariable region residues as defined herein. A "human consensus framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences.

[0023] As used herein, the terms "variable region" or "variable domain" refer to the fact that certain portions of the variable domain differ significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Naturally occurring heavy- and light-chain variable domains each contain four FR regions adopting a β-sheet structure, connected by three CDRs that form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. "Kabat-like variable domain residue numbering" or "Kabat-like amino acid position numbering," and variations thereof, refer to the numbering system used for the heavy chain variable domains or light chain variable domains of the compilation of antibodies 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 system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.For example, a heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and inserted residues 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 of the homologous regions of the antibody's sequence with the "standard" Kabat numbered sequence. It is not intended that the CDRs of this disclosure necessarily correspond to the Kabat numbering convention.

[0024] As used herein, the term "percent (%) amino acid sequence identity" with respect to 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 aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of 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 alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0025] As used herein, "elimination half-life" is used in its conventional 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). Briefly, the term is intended to encompass a quantitative measure of the time course of drug elimination. The elimination of most pharmaceuticals is exponential (i.e., follows first-order kinetics) because drug concentrations usually do not approach those required for saturation of the elimination process. The rate of an exponential process may be expressed by its rate constant, k, which expresses the fraction of change per unit time, or by its half-time, t, which is the time required for 50% completion of the process. The units of these two constants are time and time, respectively. The first-order rate constant and half-time of a reaction are simply related (k × t = 0.693) and may be interchanged accordingly. First-order elimination kinetics dictates that a constant rate of drug is lost per unit time, so a plot of the logarithm of drug concentration versus time is always linear after the initial distribution phase (i.e., after drug absorption and distribution are complete). The half-time for drug elimination can be accurately determined from such a graph.

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

[0027] Described herein are 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. Also provided are methods of using the disclosed single-domain serum albumin-binding proteins in the prevention and / or treatment of diseases, illnesses, and disorders. The single-domain serum albumin-binding proteins can specifically bind to serum albumin. In some embodiments, the single-domain serum albumin-binding proteins also contain additional domains, such as a CD3-binding domain, as well as binding domains for other target antigens.

[0028] <Single domain serum albumin binding protein> Single-domain serum albumin-binding proteins are contemplated herein. Serum albumin is produced by the liver, occurs dissolved in plasma, and is the most abundant blood protein in mammals. Albumin is essential for maintaining the oncotic pressure required for proper distribution of fluid between blood vessels and body tissues; without albumin, high pressure in blood vessels would drive more fluid into tissues. It also acts as a plasma carrier by nonspecifically binding 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 approximately 50 mg / ml (600 μM), and has a half-life of approximately 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 the major drug transport protein in plasma. In some embodiments, the 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] Noncovalent association with albumin extends the elimination half-life of short-lived proteins. For example, recombinant fusion of an albumin-binding domain to a FAb fragment resulted in a 25-fold and 58-fold reduction in in vitro clearance and a 26-fold and 37-fold increase in half-life when administered intravenously to mice and rabbits, respectively, compared with administration of the FAb fragment alone. In another example, when insulin was acylated with fatty acids to promote albumin association, long-lasting effects were observed when injected subcutaneously into rabbits or pigs. Together, these studies demonstrate the link 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, such as the heavy chain variable domain (VH) or variable domain (VHH) of a camelid-derived sdAb specific for serum albumin, peptides, ligands, or small molecular entities. In some embodiments, the single-domain serum albumin binding proteins described herein are single-domain antibodies, such as the heavy chain variable domain (VH) or variable domain (VHH) of a camelid-derived sdAb specific for HSA, peptides, ligands, or small molecular entities. 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, and humanized antibodies. In certain 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. It is contemplated that single domain serum albumin binding proteins are fairly small, in some embodiments no more than 25 kD, no more than 20 kD, no more than 15 kD, or no more than 10 kD. In some instances, single domain serum albumin binding proteins bind no more than 5 kD, if they are peptides or small molecule entities.

[0031] In some embodiments, the single domain serum albumin binding proteins described herein comprise a half-life prolonging domain, which results in altered pharmacokinetics and pharmacodynamics of the single domain serum albumin binding protein itself. As described above, the half-life prolonging domain extends the elimination half-life. The half-life prolonging domain further alters the pharmacodynamic properties of the single domain serum albumin binding protein, including altered tissue distribution, penetration, and diffusion. In some embodiments, the half-life prolonging domain results in improved tissue (including tumor) targeting, tissue distribution, tissue penetration, diffusion within tissues, and enhanced efficacy compared to proteins without the half-life prolonging domain. In one embodiment, a treatment method effectively and efficiently utilizes reduced amounts of the single domain serum albumin binding protein, resulting in reduced side effects, such as reduced cytotoxicity to non-tumor cells.

[0032] Furthermore, the binding affinity of a single domain serum albumin binding protein for its binding target can be selected to target a specific elimination half-life in a particular single domain serum albumin binding protein. Thus, in some embodiments, the single domain serum albumin binding protein has a high binding affinity for its binding target. In other embodiments, the single domain serum albumin binding protein has a moderate binding affinity for its binding target. In still other embodiments, the single domain serum albumin binding protein has a low or moderate binding affinity for its binding target. Typical binding affinities include Kds of 10 nM or less (high), between 10 nM and 100 nM (moderate), and above 100 nM (low). As noted above, the binding affinity of a single domain serum albumin binding protein for a binding target can be determined by known methods, such as surface plasmon resonance (SPR).

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

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

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

[0036] In some embodiments, the mKd is about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, about 20 nM, about 21 nM, about 22 nM, about 23 nM, about 24 nM, about 25 nM, about 26 nM, about 27 nM, about 28 nM , about 29 nM, about 30 nM, about 31 nM, about 32 nM, about 33 nM, about 34 nM, about 35 nM, about 36 nM, about 37 nM, about 38 nM, about 39 nM, about 40 nM, about 41 nM, about 42 nM, about 43 nM, about 44 nM, about 45 nM, about 46 nM, about 47 nM, about 48 nM, or about 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 the amino acid sequence set forth as SEQ ID NO.4, and the hKd and cKd are between about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein has the amino acid sequence set forth as SEQ ID NO.4, and the hKd is about 2.3 nM and the cKd is about 2.4 nM. In some embodiments, the single domain serum albumin binding protein has the amino acid sequence set forth as SEQ ID NO.25, and the hKd and cKd are between about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein has the amino acid sequence set forth as SEQ ID NO.25, and the hKd is about 2.1 nM and the cKd is about 2.2 nM. In some embodiments, the single domain serum albumin binding protein has the amino acid sequence set forth as SEQ ID NO.5, and the hKd and cKd are between about 1 nM and about 5 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 hKd of about 1.9 nM and a cKd of about 1.7 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 hKd and cKd of between about 1 nM and about 5 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 hKd of about 3.2 nM and a cKd of about 3.6 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 hKd and cKd of between about 1 nM and about 5 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 hKd of about 2.7 nM and a cKd of about 2.6 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 hKd and cKd between about 1 nM and about 5 nM.In some embodiments, the single domain serum albumin binding protein has the amino acid sequence set forth as SEQ ID NO.26, wherein the hKd is about 2.1 nM and the cKd is about 2 nM. In some embodiments, the single domain serum albumin binding protein has the amino acid sequence set forth as SEQ ID NO.8, wherein the hKd and cKd are between about 5 nM and about 10 nM. In some embodiments, the single domain serum albumin binding protein has the amino acid sequence set forth as SEQ ID NO.8, wherein the hKd is about 6 nM and the cKd is about 7.5 nM. In some embodiments, the single domain serum albumin binding protein has the amino acid sequence set forth as SEQ ID NO.9, wherein the hKd and cKd are between about 1 nM and about 5 nM. In some embodiments, the single domain serum albumin binding protein has the amino acid sequence set forth as SEQ ID NO.9, wherein the hKd is about 2.2 nM and the cKd is about 2.3 nM. In some embodiments, the single domain serum albumin binding protein has the amino acid sequence set forth as SEQ ID NO.27, wherein the hKd and cKd are between about 1 nM and about 5 nM, hi some embodiments, the single domain serum albumin binding protein has the amino acid sequence set forth as SEQ ID NO.27, wherein the hKd is about 1.6 nM and the cKd is about 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 T cell responses is mediated by the recognition of antigens (represented in the context of the major histocompatibility complex, 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 includes the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. To comprise the T cell receptor complex, CD3 associates with the α (alpha) and β (beta) chains of the T cell receptor complex, as well as CD3ζ (zeta). Clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodies, triggers T cell activation, similar to T cell receptor engagement but independent of the specificity typical of that clone.

[0043] In one aspect, described herein are multispecific proteins comprising single-domain serum albumin-binding proteins in accordance with the present disclosure. In some embodiments, the multispecific protein further comprises a domain that specifically binds to CD3. In some embodiments, the multispecific protein further comprises a domain that specifically binds to human CD3. In some embodiments, the multispecific protein further comprises a domain that specifically binds to CD3γ. In some embodiments, the multispecific protein further comprises a domain that specifically binds to CD3δ. In some embodiments, the multispecific 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 a T cell receptor (TCR). In some embodiments, the multispecific protein further comprises a domain that specifically binds to the alpha chain of the TCR. In some embodiments, the multispecific protein further comprises a domain that specifically binds to the beta chain of the TCR.

[0045] In certain embodiments, the CD3-binding domain of a multispecific protein comprising a single-domain serum albumin-binding protein described herein not only exhibits potent CD3-binding affinity with human CD3, but also exhibits excellent cross-reactivity with the respective cynomolgus monkey CD3 protein. In some examples, the CD3-binding domain of the multispecific protein cross-reacts with CD3 from cynomolgus monkeys. In some examples, the ratio of human KD:cynomolgus monkey KD (hKd:cKd) for CD3 binding is between 20:1 and 1:2.

[0046] In some embodiments, the CD3-binding domain of a multispecific protein comprising a single-domain serum albumin-binding protein described herein can be any domain that binds to CD3, including, but not limited to, a domain from an antigen-binding fragment of a CD3-binding antibody, such as a monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody, or single-domain antibody (sdAb), Fab, Fab', F(ab)2, and Fv fragment, a fragment composed of one or more CDRs, a single-chain antibody (e.g., single-chain Fv fragment (scFv)), a disulfide-stabilized (dsFv) Fv fragment, a heteroconjugate antibody (e.g., a bispecific antibody), a pFv fragment, a heavy chain monomer or dimer, a light chain monomer or dimer, and a dimer consisting of one heavy chain and one light chain. In some instances, it is beneficial for the CD3-binding domain to be derived from the same species as the multispecific protein comprising a single-domain serum albumin-binding protein described herein will ultimately be used. For example, for human use, it may be beneficial for the CD3 binding domain of a multispecific protein, including the single domain serum albumin binding proteins described herein, to include human or humanized residues from the antigen binding domain of an antibody or antibody fragment.

[0047] Thus, in one embodiment, the antigen binding domain comprises a humanized or human antibody or antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-CD3 binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a humanized or human anti-CD3 binding domain described herein, and / or one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a humanized or human anti-CD3 binding domain described herein, including, for example, a humanized or human anti-CD3 binding domain comprising 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 comprises a humanized or human light chain variable region specific for CD3, the light chain variable region comprising human or non-human light chain CDRs within a human light chain framework region. In some examples, the light chain framework region is a λ (lambda) light chain framework. In other examples, the light chain framework region is a κ (kappa) light chain framework.

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

[0050] In some examples, the complementarity determining regions of the heavy and / or light chains are selected from the group consisting of, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, and YTH12.5. These antibodies 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 binding to CD3 can be determined by the ability of the multispecific protein comprising a single-domain serum albumin-binding protein itself or its CD3-binding domain to bind to CD3, for example, coated on an assay plate; displayed on the surface of a microbial cell; in solution, etc. The binding ability of the multispecific protein comprising a single-domain serum albumin-binding protein according to the present disclosure itself or its CD3-binding domain can be assayed by immobilizing a ligand (e.g., CD3) or the multispecific protein itself or its CD3-binding domain to beads, a substrate, cells, etc. The agent can be added to the binding partner in an appropriate buffer, incubated for a period of time at a predetermined temperature. After washing to remove unbound material, the binding protein can be released, for example, with 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 multispecific binding proteins, including single-domain serum albumin-binding proteins, also include, in certain embodiments, a domain that binds to a target antigen. The target antigen is involved in, and / or associated with, a disease, disorder, or condition. In particular, the target antigen is associated with a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic response, a parasitic response, a graft-versus-host disease, or a host-versus-graft disease. 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 a bacterium.

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

[0054] The design of multispecific binding proteins, including single-domain serum albumin-binding proteins, in accordance with the present disclosure allows for flexibility in the binding domain 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, polyclonal, recombinant, human, 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), or the heavy chain variable domain (VH), light chain variable domain (VL), and 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 anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies. In further embodiments, the binding domain for the target antigen is a ligand or peptide that binds to or associates with the target antigen. In still further embodiments, the binding domain for the target antigen is a knottin. In still further embodiments, the binding domain for 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 encompass derivatives or analogs in which (i) amino acids are substituted with amino acid residues not encoded by the genetic code, (ii) the mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as leader or secretory sequences or sequences for blocking the immunogenic domain and / or for purification of the protein.

[0056] Exemplary modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0057] Modifications may be made anywhere in the single domain serum albumin binding proteins described herein, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Particular common peptide modifications useful for modifying single domain serum albumin binding proteins include glycosylation of glutamic acid residues, lipid attachment, sulfation, gamma-carboxylation, hydroxylation, blocking 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 are also provided that encode the single domain serum albumin binding proteins described herein.In some embodiments, the polynucleotide molecules are provided as DNA constructs.In other embodiments, the polynucleotide molecules are provided as messenger RNA transcripts.

[0059] The polynucleotide molecule is constructed by known methods, such as by combining the genes encoding the three binding domains, separated by peptide linkers or, in other embodiments, directly linked by peptide bonds, into a single gene construct operably linked to a suitable promoter and, optionally, a suitable transcription terminator, and expressing it in bacteria or other suitable expression systems, such as, for example, CHO cells.

[0060] In some embodiments, polynucleotide molecules encoding multispecific binding proteins, including single-domain serum albumin-binding proteins, are also provided in accordance with the present 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 a coding sequence 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-extending domain is a small molecule, the polynucleotide includes genes encoding domains that bind to CD3 and the target antigen. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. Promoters are selected to drive expression of the polynucleotide in the respective host cells.

[0061] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, representing a further embodiment. The recombinant vector can be constructed by known methods. Particularly desirable vectors include plasmids, phagemids, phage derivatives, viruses (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc.), and cosmids.

[0062] A variety of expression vector / host systems may be utilized to contain and express polynucleotides encoding the described single domain serum albumin binding protein polypeptides. Examples of expression vectors for expression in E. coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27), and for expression in mammalian cells are pcDNA5 (Invitrogen), PICHIAPINK™ Yeast Expression Systems (Invitrogen), and BACUVANCE™ Baculovirus Expression System (GenScript).

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

[0064] <Production of single-domain serum albumin-binding proteins> In some embodiments, disclosed herein is a process for producing a single domain serum albumin binding protein, in some embodiments, the process comprises culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the single domain serum albumin binding protein under conditions that allow 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 are directed to improving one or more properties, such as affinity, stability, thermostability, cross-reactivity, etc., of single-domain serum albumin binding proteins and / or multispecific binding proteins, including the single-domain serum albumin binding proteins described herein, relative to a reference binding compound. In some embodiments, multiple single-substitution libraries are provided, each corresponding to a different domain or amino acid segment of the 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 allows all possible substitutions of a large protein or protein-binding site to be explored with a few small libraries.) In some embodiments, the multiple domains form a contiguous sequence or encompass the amino acids 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 an adjacent domain.

[0066] The binding compounds expressed from these single-substitution libraries are separately selected to obtain a subset of variants in each library, with properties at least as good as those of the reference binding compound, and the resulting libraries are reduced in size (i.e., the number of nucleic acids encoding the selected set of binding compounds is fewer 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, stability against 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 "proto-candidate compounds" or "proto-candidate proteins." The nucleic acid sequences encoding the proto-candidate compounds from the separate single-substitution libraries are PCR-shuffled using PCR-based gene shuffling techniques to generate shuffled libraries.

[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 produce a library of nucleic acids encoding the candidate compounds, which are then cloned into a convenient expression vector, such as a phagemid expression system. The phage-expressed candidate compounds then 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 otherwise attached to the surface of a well or other reaction vessel, or it may be derivatized with a binding moiety, such as biotin, which, after incubation with the candidate binding compound, may be captured with a complementary moiety, such as streptavidin, attached to beads, such as magnetic beads, for washing. In a typical selection regimen, the candidate binding compounds undergo an extended washing step to select only candidate compounds with very low dissociation rates from the target molecule. Typical wash times for such embodiments are 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. Isolated clones following selection are amplified and subjected to additional cycles of selection or analyzed, for example, by sequencing and comparative measurements of binding affinity, for example, by ELISA, surface plasmon resonance binding, biolayer interferometry (e.g., Octet system, ForteBio, Menlo Park, CA), etc. In some embodiments, the process is performed to identify one or more single domain serum albumin binding proteins and / or multispecific binding proteins comprising single domain serum albumin binding proteins, with improved thermostability, cross-reactivity to a selected set of binding targets, compared to a reference serum albumin binding protein, such as the protein having the amino acid sequence of SEQ ID NO. 10.Single substitution libraries are prepared by altering codons in the VH region of a reference serum albumin binding protein, including codons in both framework regions and CDRs; in another embodiment, the positions at which the codons are altered comprise the CDRs of the heavy chain of the reference serum albumin binding protein, or a subset of such CDRs, such as solely CDR1, solely CDR2, solely CDR3, or any combination thereof. In another embodiment, the positions at which the codons are altered occur exclusively in the framework regions. In some embodiments, the library contains only single codon changes from the reference serum albumin binding protein in only the framework regions of the VH, numbered in the range of 10 to 250. In another embodiment, the positions at which the codons are altered comprise the CDR3 of the heavy chain of the reference serum albumin binding protein, or a subset of such CDR3. In another embodiment, the number of positions at which the codons are altered in the VH coding region ranges from 10 to 250, with a maximum of 100 positions being in the framework regions. After preparation of the single-permutation libraries, the following steps are performed, as outlined above: (a) separately expressing each member of the individual single-permutation libraries as pre-candidate proteins; (b) selecting individual single-permutation library members that encode pre-candidate proteins that bind to a binding partner that may or may not be different from the original binding target (e.g., a desired cross-reactive target); (c) shuffling the selected library members by PCR to generate a shuffled combinatorial library; (d) expressing the shuffled library members as candidate serum albumin-binding proteins; and (e) selecting the shuffled library members one or more times for candidate serum albumin-binding proteins that bind to the original binding partner, and optionally (f) selecting additional candidate proteins for binding to the desired cross-reactive target, thereby providing nucleic acid-encoded serum albumin-binding proteins that have enhanced cross-reactivity of one or more substances relative to a reference serum albumin-binding protein without losing affinity for the original ligand.In a further embodiment, the method may be carried out to obtain serum albumin binding proteins with reduced reactivity towards the selected cross-reacting substance(s) or compound(s) or epitope(s) by replacing step (f) with the following step: depleting candidate binding compounds one or more times from the subset of candidate serum albumin binding proteins that bind to the undesired cross-reacting compounds.

[0068] Recent studies have demonstrated that therapeutic antibodies are at risk of degradation via multiple pathways during production, storage, and in vivo use. The most frequent chemical degradation reactions in proteins include deamidation of asparagine (N) and isomerization of aspartic acid (D) residues. It has been hypothesized that chemical alteration of N and D residues, particularly those involved in antigen recognition, could result in 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 aspartic acid sites in antibodies presents stability issues. When isomerization occurs, the chemical structure of the antibody is altered, potentially resulting in poor stability, as manifested, for example, by aggregation and a shorter shelf life. Accordingly, some embodiments of the present disclosure provide single-domain serum albumin binding proteins, in which one or more aspartic acid residues are mutated, thereby reducing the likelihood of isomerization of the single-domain serum albumin binding protein. In some embodiments, the aspartic acid residue is in CDR2 of the single-domain serum albumin binding protein, and the aspartic acid residue is mutated to glutamic acid. In certain embodiments, the aspartic acid residue at position 62 of the protein 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, the single-domain serum albumin binding proteins containing and not containing the D62E mutation have equivalent binding affinity to serum albumin.

[0069] <Pharmaceutical Composition> In some embodiments, pharmaceutical compositions are provided comprising a single-domain serum albumin-binding protein described herein, a vector containing a polynucleotide encoding a single-domain serum albumin-binding protein polypeptide, or a host cell transformed with this 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 effectiveness of the biological activity of the component 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 administered to a subject in appropriate dosages. Preferably, the compositions are sterile. These compositions may further contain adjuvants such as preservatives, emulsifiers, and dispersing agents. Prevention of microbial attack can be ensured by the inclusion of various antibacterial and antifungal agents.

[0070] In some embodiments of the pharmaceutical composition, the single domain serum albumin binding protein described herein is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a fullerene, a liquid crystal, a liposome, a quantum dot, a superparamagnetic nanoparticle, a dendrimer, or a nanorod. In other embodiments of the pharmaceutical composition, the single domain serum albumin binding protein is bound to a liposome. 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 can be 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 included in the pharmaceutical composition. The dosing regimen will be determined by the attending physician and other clinical factors. The dosage for any one patient will depend on many factors, including the patient's size, 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 concomitantly. An "effective amount" refers to an amount of active ingredient sufficient to affect the course and severity of a disease, resulting in a reduction or remission of such symptoms, and may be determined using known methods.

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

[0073]

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

[0074] As used herein, in some embodiments, "treatment" or "treating" or "treated" refers to therapeutic treatment aimed at delaying (reducing) an undesirable physiological disease, disorder, or condition, or to obtain a beneficial or desired clinical result. For purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reduction in the severity of the disease, disorder, or condition; stabilization of the disease, disorder, or condition (i.e., not worsening); delay in the onset or slowing of progression of the disease, disorder, or condition; improvement of the disease, disorder, or disease state; and remission (whether partial or total), whether detectable or undetectable, or whether progression or improvement of the disease, disorder, or condition. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment further includes prolonging survival compared to expected survival in the absence of treatment. In another embodiment, "treatment" or "treating" or "treated" refers to prophylactic treatment, the purpose of which is to delay the onset of or reduce the severity of an unwanted physiological disease, disorder, or condition, for example, in an individual predisposed to the condition (e.g., an individual bearing a genetic marker for a condition such as breast cancer).

[0075] In some embodiments of the methods described herein, the single-domain serum albumin binding protein or multispecific binding protein comprising the single-domain serum albumin binding protein described herein is administered in combination with an agent for treating a particular disease, disorder, or condition. The agent includes, but is not limited to, antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiation therapy (directed delivery of gamma rays, X-rays, and / or radioisotopes, microwaves, UV radiation, etc.), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapeutic treatments. In some embodiments, the single-domain serum albumin binding protein or multispecific binding protein comprising the single-domain serum albumin binding protein described herein is administered in combination with an antidiarrheal, antiemetic, analgesic, opioid, and / or nonsteroidal anti-inflammatory drug. In some embodiments, the single-domain serum albumin binding protein or multispecific binding protein comprising the single-domain serum albumin binding protein described herein is administered before, during, or after surgery. [Example]

[0076] Example 1: Generation of anti-HSA single domain antibody variants with binding properties comparable to those of the parent anti-HSA single domain antibody Characterization of the parent anti-HSA phage The specific binding of the parental anti-HSA phage to the HSA antigen was measured using CD3 as a negative control (Fig. 1), and the cross-reactivity of the anti-HSA phage to human, cynomolgus monkey, and mouse serum albumin was measured (Fig. 2).

[0077] <Single substitution HSA sdAb phage library> A single-substitution library was provided for each of the three CDR domains. The single-substitution library was bound to HSA and then washed with buffer containing various levels of HSA. Bound phages at 0 and 24 hours were rescued and enumerated. Selected phages washed with 2.5 mg / ml HSA in buffer for 24 hours were used to generate two independent combinatorial phage libraries.

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

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

[0080] Example 2: Pharmacokinetics of trispecific antibodies containing anti-HSA single domain antibodies The anti-HSA single domain antibody of Example 1 will be used to prepare a trispecific antibody, which will be evaluated to eliminate half-time in animal studies.

[0081] The trispecific antibody is administered to cynomolgus monkeys as a 0.5 mg / kg intramuscular bolus injection. Another group of cynomolgus monkeys receives a protein with binding domains comparable in size to CD3 and CD20 but lacking HSA binding. The third and fourth groups receive an antibody with CD3 and HSA binding domains, and a protein with CD20 and HSA binding domains, respectively, both of which are comparable in size to the trispecific antibody. Each test group consists of five monkeys. Serum samples are taken at the indicated time points, serially diluted, and the protein concentrations are measured using binding ELISA for CD3 and / or CD20.

[0082] Pharmacokinetic analysis is performed using the plasma concentrations of the test article. Group mean plasma data for each test article fit a multi-exponential profile when plotted against time post-dose. Data are fit by a standard two-compartment model with bolus administration and first-order rate constants for the distribution and elimination phases. The general equation for best fit of data for iv administration is: c(t) = Ae -αt +Be -βt where c(t) is the plasma concentration at time t, A and B are the intercepts on the Y-axis, and α and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The α-phase is the phase constant of clearance and reflects the distribution of the protein into all extracellular fluids of the animal, while the second or β-phase portion of the decay curve represents the true plasma clearance. Methods for fitting such equations are known in the art. For example, A = D / V(α-k21) / (α-β), B = D / V(β-k21) / (α-β), where α and β (between α and β) are determined by the quadratic equation: r 2 +(k12+k21+k10)r+k21k10=0, with estimated parameters of V=volume of distribution, k10=excretion rate, k12=transfer rate from compartment 1 to compartment 2, and k21=transfer rate from compartment 2 to compartment 1, and D=dose.

[0083] Data analysis: Concentration versus time profile graphs are generated using KaleidaGraph (KaleidaGraph™ V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values ​​reported as less than reportable (LTR) are not included in the PK analysis and are not shown on the graph. Pharmacokinetic parameters are determined by compartmental analysis using WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters are calculated as described in Ritschel WA and Kearns GL, 1999, IN: Handbook of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, DC.

[0084] It is expected that trispecific antibodies comprising the anti-HSA single domain antibody of Example 1 will have improved pharmacokinetic parameters, such as increased elimination half-life, compared to proteins lacking the HSA binding domain.

[0085] Example 3: Heat resistance of anti-HSA single domain antibody mutants The temperature of hydrophobic exposure of proteins (T h ) corresponds to the derivative of the inflection point of the peak dye fluorescence and is the melting temperature (T m The purpose of this study is to compare the T of several anti-HAS single domain antibody mutants. h The purpose was to evaluate the following.

[0086] <Protein production> The anti-huALB single-domain antibody sequence was cloned into pcDNA3.4 (Invitrogen) preceded by a leader sequence and followed by a 6x histidine tag (SEQ ID NO:38). Expi293F cells (Life Technologies A14527) were maintained in suspension in Optimum Growth Flasks (Thomson) at 0.2–8x1e6 cells / mL in Expi293 medium. 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. Conditioned medium was partially purified by affinity and desalting chromatography. The anti-huCD3escFv protein was concentrated using an Amicon Ultra centrifugal filter unit (EMD Millipore), applied to Superdex 200 size-exclusion medium (GE Healthcare), and digested in a neutral buffer containing excipients. Fraction pooling and final purity were assessed by SDS-PAGE and analytical size-exclusion chromatography (SEC). The absorbance of the purified protein solution was determined at 280 nm using a SpectraMax M2 (Molecular Devices) and plated onto a UV-transparent 96-well plate (Corning 3635), and the concentration was calculated from the molar extinction coefficient.

[0087] <Differential scanning fluorimetry> 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 5x SYPRO orange dye (Life Technologies S6651) and placed in a MicroAmp EnduraPlate optical microplate with adhesive film (Applied Biosystems 4483485 and 4311971). The plate containing the diluted protein and dye mixture was 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 a 2-minute hold at each step, with excitation at 500 nm and emission collected with a ROX filter. T in degrees Celsius h is presented for the purified anti-HSA single domain antibody protein in Figure 4.

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

[0089] The concentrations of purified anti-HSA single domain antibody variants were measured by absorbance at 280 nm as described in Example 3. The purified proteins were assessed by SDS-PAGE and analytical SEC using a Yarra 2000 SEC column (Phenomenex 00H-4512-E0) and analyzed in phosphate buffer containing solvent on a 1200LC 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 comprising complementarity determining regions CDR1, CDR2, and CDR3, where: (a) The amino acid sequence of CDR1 is GFX １ X ２ X ３ X ４ FGMS (SEQ ID NO. 1), １ is threonine, arginine, lysine, serine, or proline, and X ２ is phenylalanine or tyrosine, and X ３ is serine, arginine, or lysine, and X ４ is serine, lysine, arginine, or alanine; (b) The amino acid sequence of CDR2 is SISGSGX ５ X ６ TLYAX ７ SX ８ K (SEQ ID NO. 2), ５ is serine, arginine, threonine, or alanine, and X ６ is aspartic acid, histidine, valine, or threonine, and X ７ is aspartic acid, histidine, arginine, or serine, and X ８ is valine or leucine; and (c) the amino acid sequence of CDR3 is GGSLX ９ X １０ (SEQ ID NO. 3), ９ is serine, arginine, threonine, or lysine; X １０ is arginine, lysine, valine, proline, or asparagine, where X １ , X ２ , X ３ , X ４ , X ５ , X ６ , X ７ , X ８ , X ９ , and X １０ are single-domain serum albumin-binding proteins that are not simultaneously threonine, phenylalanine, serine, serine, serine, aspartic acid, aspartic acid, valine, serine, and arginine, respectively.

2. The single domain serum albumin binding protein has the following formula: including f1-r1-f2-r2-f3-r3-f4, wherein r1 is SEQ ID NO. 1, r2 is SEQ ID NO. 2, and r3 is SEQ ID NO. 3, where f １ , f ２ , f ３ , and f ４ are framework residues selected such that the single domain serum albumin binding protein is at least 80 percent identical to the amino acid sequence set forth in SEQ ID NO:

10.

3. 3. The single domain serum albumin binding protein of claim 2, wherein r1 comprises SEQ ID NO. 14, SEQ ID NO. 15, or SEQ ID NO.

16.

4. 3. The single domain serum albumin binding protein of claim 2, wherein 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.

5. 3. The single domain serum albumin binding protein of claim 2, wherein r3 comprises SEQ ID NO. 23 or SEQ ID NO.

24.

6. 6. The single domain serum albumin binding protein of any one of claims 2-5, wherein r1 comprises SEQ ID NO.

14.

7. 6. The single domain serum albumin binding protein of any one of claims 2-5, wherein r1 comprises SEQ ID NO. 15, r2 is SEQ ID NO. 17, and r3 is SEQ ID NO.

23.

8. 6. The single domain serum albumin binding protein of claim 2, wherein r1 comprises SEQ ID NO. 16 and r3 comprises SEQ ID NO.

23.

9. 6. The single domain serum albumin binding protein of claim 2, wherein r1 comprises SEQ ID NO. 15 and r2 comprises SEQ ID NO.

18.

10. 6. The single domain serum albumin binding protein of claim 2, wherein r1 comprises SEQ ID NO. 14 and r3 comprises SEQ ID NO.

23.

11. 6. The single domain serum albumin binding protein of claim 2, wherein r1 comprises SEQ ID NO. 15, r2 comprises SEQ ID NO. 19, and r3 comprises SEQ ID NO.

24.

12. 6. The single domain serum albumin binding protein of claim 2, wherein r1 comprises SEQ ID NO. 14 and r2 comprises SEQ ID NO.

20.

13. 6. The single domain serum albumin binding protein of claim 2, wherein r1 comprises SEQ ID NO. 15 and r2 comprises SEQ ID NO.

21.

14. 6. The single domain serum albumin binding protein of any one of claims 2-5, wherein r1 comprises SEQ ID NO. 15, r2 comprises SEQ ID NO. 22, and r3 comprises SEQ ID NO.

24.

15. 2. The single domain serum albumin binding protein of claim 1, wherein 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.

16. 16. A single domain serum albumin binding protein according to any one of claims 1 to 15, wherein the single domain serum albumin binding protein binds to a serum albumin selected from human serum albumin, cynomolgus monkey serum albumin, and mouse serum albumin.

17. 17. A single domain serum albumin binding protein according to any one of claims 1 to 16, wherein the single domain serum albumin binding protein binds to human serum albumin and cynomolgus serum albumin with comparable binding affinities (Kd).

18. 18. The single domain serum albumin binding protein of any one of claims 1 to 17, wherein the single domain serum albumin binding protein binds to mouse serum albumin with a binding affinity (Kd) that is about 1.5 to about 20 times weaker than the binding affinity (Kd) of the single domain serum albumin binding protein for human and cynomolgus monkey serum albumin.

19. 19. The single domain serum albumin binding protein of any one of claims 1 to 18, wherein the single domain serum albumin binding protein binds to human serum albumin with a human Kd (hKd) of between about 1 nM and about 100 nM and binds to cynomolgus monkey serum albumin with a cynomolgus monkey Kd (cKd) of between about 1 nM and about 100 nM.

20. 20. The single domain serum albumin binding protein of claim 19, wherein the hKd and cKd are between 1 nM and about 5 nM, or between about 5 nM and about 10 nM.

21. 21. The single domain serum albumin binding protein of claim 20, wherein the hKd and cKd 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.

22. 22. The single domain serum albumin binding protein of any one of claims 16-21, wherein the ratio between hKd and cKd (hKd:cKd) ranges from about 20:1 to about 1:

2.

23. 23. The single domain serum albumin binding protein of any one of claims 1-22, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 4, wherein the hKd and cKd are between about 1 nM and about 5 nM.

24. 23. The single domain serum albumin binding protein of any one of claims 1-22, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 5, wherein the hKd and cKd are between about 1 nM and about 5 nM.

25. 23. The single domain serum albumin binding protein of any one of claims 1-22, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 6, wherein the hKd and cKd are between about 1 nM and about 5 nM.

26. 23. The single domain serum albumin binding protein of any one of claims 1-22, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 7, wherein the hKd and cKd are between about 1 nM and about 5 nM.

27. 23. The single domain serum albumin binding protein of any one of claims 1-22, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 8, wherein hKd and cKd are between about 5 nM and about 10 nM.

28. 23. The single domain serum albumin binding protein of any one of claims 1-22, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 9, wherein the hKd and cKd are between about 1 nM and about 5 nM.

29. 23. The single domain serum albumin binding protein of any one of claims 1-22, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 25, wherein the hKd and cKd are between about 1 nM and about 5 nM.

30. 23. The single domain serum albumin binding protein of any one of claims 1-22, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 26, wherein the hKd and cKd are between about 1 nM and about 5 nM.

31. 23. The single domain serum albumin binding protein of any one of claims 1-22, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 27, wherein the hKd and cKd are between about 1 nM and about 5 nM.

32. 32. The single domain serum albumin binding protein of any one of claims 1-31, wherein 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.

33. A single domain serum albumin binding protein comprising CDR1, CDR2, and CDR3, comprising the sequence set forth as SEQ ID NO. 10 (wt anti-HSA), 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 lysine; amino acid position 31 is substituted with lysine, arginine, or alanine; 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; A single domain serum albumin binding protein, wherein amino acid position 104 is substituted with lysine, valine, proline, or asparagine.

34. 34. The single domain serum albumin binding protein of claim 33, comprising one or more additional substitutions at amino acid positions other than positions 28, 29, 30, 31, 56, 57, 62, 64, 103, and 104.

35. 35. A single domain serum albumin binding protein according to claim 33 or 34, comprising a substitution at position 29.

36. 35. A single domain serum albumin binding protein according to claim 33 or 34, comprising a substitution at position 31.

37. 35. A single domain serum albumin binding protein according to claim 33 or 34, comprising a substitution at position 56.

38. 35. A single domain serum albumin binding protein according to claim 33 or 34, comprising a substitution at position 62.

39. 35. A single domain serum albumin binding protein according to claim 33 or 34, comprising a substitution at position 64.

40. 35. A single domain serum albumin binding protein according to claim 33 or 34, comprising a substitution at position 104.

41. 35. A single domain serum albumin binding protein according to claim 33 or 34, comprising substitutions at amino acid positions 31 and 62.

42. 37. The single domain serum albumin binding protein of claim 36, wherein amino acid position 31 is substituted with arginine.

43. 42. The single domain serum albumin binding protein of claim 41, wherein amino acid position 31 is substituted with arginine and amino acid position 62 is substituted with glutamic acid.

44. 35. A single domain serum albumin binding protein according to claim 33 or 34, comprising substitutions at amino acid positions 31, 56, 64 and 104.

45. 45. The single domain serum albumin binding protein of claim 44, wherein amino acid 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.

46. 35. A single domain serum albumin binding protein according to claim 33 or 34, wherein amino acid positions 29 and 104 are substituted.

47. 47. The single domain serum albumin binding protein of claim 46, wherein amino acid position 29 is substituted with tyrosine and amino acid position 104 is substituted with lysine.

48. 35. A single domain serum albumin binding protein according to claim 33 or 34, wherein amino acid positions 31 and 56 are substituted.

49. 49. The single domain serum albumin binding protein of claim 48, wherein amino acid position 31 is substituted with lysine and amino acid position 56 is substituted with threonine.

50. 35. A single domain serum albumin binding protein according to claim 33 or 34, wherein amino acid positions 31, 56 and 62 are substituted.

51. 51. The single domain serum albumin binding protein of claim 50, wherein amino acid position 31 is substituted with lysine, amino acid position 56 is substituted with threonine, and amino acid position 62 is substituted with glutamic acid.

52. 35. A single domain serum albumin binding protein according to claim 33 or 34, wherein amino acid positions 31 and 104 are substituted.

53. 52. The single domain serum albumin binding protein of claim 51, wherein amino acid position 31 is substituted with arginine and amino acid position 104 is substituted with lysine.

54. 35. A single domain serum albumin binding protein according to claim 33 or 34, wherein amino acid positions 31, 56 and 104 are substituted.

55. 55. The single domain serum albumin binding protein of claim 54, wherein 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.

56. 35. A single domain serum albumin binding protein according to claim 33 or 34, wherein amino acid positions 31, 56, 62 and 104 are substituted.

57. 57. The single domain serum albumin binding protein of claim 56, wherein 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.

58. 58. A single domain serum albumin binding protein according to any one of claims 33-57, wherein the single domain serum albumin binding protein binds to human serum albumin and cynomolgus serum albumin with comparable binding affinities (Kd).

59. 59. The single domain serum albumin binding protein of any one of claims 33-58, wherein the single domain serum albumin binding protein binds to mouse serum albumin with a binding affinity (Kd) that is about 1.5 to about 20 times weaker than the binding affinity (Kd) of the single domain serum albumin binding protein for human and cynomolgus monkey serum albumin.

60. 60. The single domain serum albumin binding protein of any one of claims 33-59, wherein the single domain serum albumin binding protein binds to human serum albumin with a human Kd (hKd) of between about 1 nM and about 100 nM and binds to cynomolgus monkey serum albumin with a cynomolgus monkey Kd (cKd) of between 1 nM and 100 nM.

61. 61. The single domain serum albumin binding protein of any one of claims 33-60, wherein the hKd and cKd are between 1 nM and about 5 nM, or between about 5 nM and about 10 nM.

62. 62. The single domain serum albumin binding protein of any one of claims 33-61, wherein the hKd and cKd 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.

63. 63. The single domain serum albumin binding protein of any one of claims 33-62, wherein the single domain serum albumin binding protein binds to human serum albumin with a human Kd (hKd) and binds to cynomolgus monkey serum albumin with a cynomolgus monkey Kd (cKd), wherein the ratio between hKd and cKd (hKd:cKd) is from about 20:1 to about 1:

2.

64. 64. A single domain serum albumin binding protein according to any one of claims 33-63, wherein amino acid position 31 is substituted with arginine and the hKd and cKd are between about 1 nM and about 5 nM.

65. 64. A single domain serum albumin binding protein according to any one of claims 33-63, wherein amino acid position 31 is substituted with arginine and amino acid position 62 is substituted with glutamic acid, and the hKd and cKd are between about 1 nM and about 5 nM.

66. 64. The single domain serum albumin binding protein of any one of claims 33-63, wherein 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 are between about 1 nM and about 5 nM.

67. 64. A single domain serum albumin binding protein according to any one of claims 33-63, wherein amino acid position 29 is substituted with tyrosine, amino acid position 104 is substituted with lysine, and the hKd and cKd are between about 1 nM and about 5 nM.

68. 64. A single domain serum albumin binding protein according to any one of claims 33-63, wherein amino acid position 31 is substituted with lysine, amino acid position 56 is substituted with threonine, and the hKd and cKd are between about 1 nM and about 5 nM.

69. 64. The single domain serum albumin binding protein of any one of claims 33-63, wherein 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 about 1 nM and about 5 nM.

70. 64. A single domain serum albumin binding protein according to any one of claims 33-63, wherein amino acid position 31 is substituted with arginine, amino acid position 104 is substituted with lysine, and the hKd and cKd are between about 5 nM and about 10 nM.

71. 64. The single domain serum albumin binding protein of any one of claims 33-63, wherein 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 hKd and cKd are between about 1 nM and about 5 nM.

72. 64. The single domain serum albumin binding protein of any one of claims 33-63, wherein 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 are between about 1 nM and about 5 nM.

73. 73. The single domain serum albumin binding protein of any one of claims 33-72, wherein 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.

74. 1. A single domain serum albumin binding protein comprising at least one mutation in CDR1, CDR2, or CDR3, wherein CDR1 has the sequence as set forth in SEQ ID NO: 11, CDR2 has the sequence as set forth in SEQ ID NO: 12, and CDR3 has the sequence as set forth in SEQ ID NO: 13; wherein at least one mutation is not at amino acid position 1, 2, 7, 8, 9, or 10 of SEQ ID NO: 11, position 1, 3, 6, 10, or 11 of SEQ ID NO: 12, or position 1 or 2 of SEQ ID NO:

13.

75. 75. The single domain serum albumin binding protein of claim 74, comprising at least one mutation at an amino acid position 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).

76. 75. The single domain serum albumin binding protein of claim 74, comprising 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).

77. 77. A single domain serum albumin binding protein according to any one of claims 74 to 76, comprising a mutation at amino acid position 6 of CDR1 (SEQ ID NO: 11).

78. 77. A single domain serum albumin binding protein according to any one of claims 74-76, comprising a mutation at amino acid position 6 of CDR1 (SEQ ID NO: 11) and at amino acid position 13 of CDR2 (SEQ ID NO: 12).

79. 77. The single domain serum albumin binding protein of any one of claims 74-76, comprising 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).

80. 77. A single domain serum albumin binding protein according to any one of claims 74 to 76, comprising a mutation at amino acid position 4 of CDR1 (SEQ ID NO: 11) and at amino acid position 6 of CDR3 (SEQ ID NO: 13).

81. 77. A single domain serum albumin binding protein according to any one of claims 74-76, comprising a mutation at amino acid position 6 of CDR1 (SEQ ID NO: 11) and at amino acid position 7 of CDR2 (SEQ ID NO: 12).

82. 77. A single domain serum albumin binding protein according to any one of claims 74-76, comprising a mutation at amino acid position 6 of CDR1 (SEQ ID NO: 11) and at amino acid positions 7 and 13 of CDR2 (SEQ ID NO: 12).

83. 77. A single domain serum albumin binding protein according to any one of claims 74-76, comprising a mutation at amino acid position 6 of CDR1 (SEQ ID NO: 11) and at amino acid position 6 of CDR3 (SEQ ID NO: 13).

84. 77. The single domain serum albumin binding protein of any one of claims 74-76, comprising a mutation 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).

85. 77. The single domain serum albumin binding protein of any one of claims 74-76, comprising 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).

86. 86. A single domain serum albumin binding protein according to any one of claims 74-85, wherein the single domain serum albumin binding protein binds to human serum albumin and cynomolgus serum albumin with comparable binding affinities (Kd).

87. 87. The single domain serum albumin binding protein of any one of claims 74-86, wherein the single domain serum albumin binding protein binds to mouse serum albumin with a binding affinity (Kd) that is about 1.5 to about 20 times weaker than the binding affinity (Kd) of the single domain serum albumin binding protein for human and cynomolgus monkey serum albumin.

88. 88. The single domain serum albumin binding protein of any one of claims 74-87, wherein the single domain serum albumin binding protein binds to human serum albumin with a human Kd (hKd) of between about 1 nM and about 100 nM and binds to cynomolgus monkey serum albumin with a cynomolgus monkey Kd (cKd) of between about 1 nM and about 100 nM.

89. 89. The single domain serum albumin binding protein of any one of claims 74-88, wherein the hKd and cKd are between 1 nM and about 5 nM, or between about 5 nM and about 10 nM.

90. 90. The single domain serum albumin binding protein of any one of claims 74-89, wherein the hKd and cKd 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.

91. 86. The single domain serum albumin binding protein of any one of claims 74-85, wherein the single domain serum albumin binding protein binds to human serum albumin with a human Kd (hKd) and binds to cynomolgus monkey serum albumin with a cynomolgus monkey Kd (cKd), and the ratio between hKd and cKd (hKd:cKd) is from about 20:1 to about 1:

2.

92. 92. The single domain serum albumin binding protein of any one of claims 74-91, wherein amino acid position 6 of CDR1 (SEQ ID NO: 11) is mutated to arginine, and the hKd and cKd are between about 1 nM and about 5 nM.

93. 92. The single domain serum albumin binding protein of any one of claims 74-91, wherein 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 wherein the hKd and cKd of the single domain serum albumin binding protein are between about 1 nM and about 5 nM.

94. 92. The single domain serum albumin binding protein of any one of claims 74-91, wherein 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 wherein hKd and cKd are between about 1 nM and about 5 nM.

95. 92. The single domain serum albumin binding protein of any one of claims 74-91, wherein amino acid position 4 of CDR1 (SEQ ID NO: 11) is mutated to tyrosine and amino acid position 6 of CDR3 (SEQ ID NO: 13) is mutated to lysine, and wherein hKd and cKd are between about 1 nM and about 5 nM.

96. 92. The single domain serum albumin binding protein of any one of claims 74-91, wherein 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 wherein hKd and cKd are between about 1 nM and about 5 nM.

97. 92. The single domain serum albumin binding protein of any one of claims 74-91, wherein amino acid position 6 of CDR1 (SEQ ID NO: 11) is mutated to lysine, and amino acid positions 7 and 13 of CDR2 (SEQ ID NO: 12) are mutated to threonine and glutamic acid, respectively, and wherein hKd and cKd are between about 1 nM and about 5 nM.

98. 92. The single domain serum albumin binding protein of any one of claims 74-91, wherein amino acid position 6 of CDR1 (SEQ ID NO: 11) is mutated to arginine, and amino acid position 6 of CDR3 (SEQ ID NO: 12) is mutated to lysine, and wherein hKd and cKd are between about 5 nM and about 12 nM.

99. 92. The single domain serum albumin binding protein of any one of claims 74-91, wherein 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, and amino acid position 6 of CDR3 (SEQ ID NO: 13) is mutated to valine, and wherein hKd and cKd are between about 1 nM and about 5 nM.

100. 92. The single domain serum albumin binding protein of any one of claims 74-91, wherein amino acid position 6 of CDR1 (SEQ ID NO: 11) is mutated to lysine, amino acid positions 7 and 13 of CDR2 (SEQ ID NO: 12) are mutated to arginine and glutamic acid, respectively, and amino acid position 6 of CDR3 (SEQ ID NO: 13) is mutated to valine, and wherein hKd and cKd are between about 1 nM and about 5 nM.

101. 101. The single domain serum albumin binding protein of any one of claims 74-100, wherein 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.

102. A polynucleotide encoding the single domain serum albumin binding protein of any one of claims 1-101.

103. A vector comprising the polynucleotide of claim 102.

104. A host cell transformed with the vector of claim 103.

105. 104. A pharmaceutical composition comprising: (i) the single domain serum albumin binding protein of any one of claims 1-101, the polynucleotide of claim 102, the vector of claim 103, or the host cell of claim 104; and (ii) a pharmaceutically acceptable carrier.

106. A process for the production of a single domain serum albumin binding protein according to any one of claims 1-101, comprising: The process comprises culturing a host transformed or transfected with a vector comprising a nucleic acid sequence encoding the single domain serum albumin binding protein of any one of claims 1-101 under conditions allowing expression of the single domain serum albumin binding protein, and recovering and purifying the protein produced from the culture.

107. 102. A method for the treatment or amelioration of a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease, or a host-versus-graft disease, comprising administering to a subject the single domain serum albumin binding protein of any one of claims 1-101.

108. 108. The method of claim 107, wherein the subject is a human.

109. 109. The method of claim 108, wherein said method further comprises administering a drug in combination with the single domain serum albumin binding protein of any one of claims 1-100.

110. 103. A multispecific binding protein comprising the single domain serum albumin binding protein of any one of claims 1, 33, or 74.

111. 80. An antibody comprising the single domain serum albumin binding protein of claim 1, 33, or 74.

112. 80. A multispecific antibody, bispecific antibody, sdAb, variable heavy domain, peptide, or ligand comprising the single domain serum albumin binding protein of claim 1, 33, or 74.

113. 100. An antibody comprising the single domain serum albumin binding protein of claim 1, 33, or 74, wherein the antibody is a single domain antibody.

114. The antibody of claim 113, wherein the antibody is derived from the heavy chain variable region of an IgG.

115. 100. A multispecific binding protein or antibody comprising the single domain serum albumin binding protein of any one of claims 1, 33, or 74 and a CD3 binding domain.

116. 116. A method for the treatment or amelioration of a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease, or a host-versus-graft disease, comprising administering to a subject the multispecific antibody of any one of claims 111-115.

117. 102. The single domain serum albumin binding protein of any one of claims 1-101, wherein said single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.

4.

118. 102. The single domain serum albumin binding protein of any one of claims 1-101, wherein said single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.

7.

119. 102. The single domain serum albumin binding protein of any one of claims 1-101, wherein said single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.

9.

120. 102. The single domain serum albumin binding protein of any one of claims 1-101, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.

26.

121. 102. The single domain serum albumin binding protein of any one of claims 1-101, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.

27.

122. 102. The single domain serum albumin binding protein of any one of claims 1-101, wherein said single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.

5.

123. 102. The single domain serum albumin binding protein of any one of claims 1-101, wherein said single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.

6.

124. 102. The single domain serum albumin binding protein of any one of claims 1-101, wherein said single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO.

8.

125. 102. The single domain serum albumin binding protein of any one of claims 1-101, wherein the single domain serum albumin binding protein comprises the amino acid sequence set forth as SEQ ID NO. 25.

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