Multispecific binding proteins and methods of use thereof

Abstract

To provide new and useful multi-specific binding proteins for treating cancer that have adequate therapeutic efficacy, a format straightforward to manufacture, and favorable pharmacokinetic properties such as a longer half-life.SOLUTION: The invention relates to multi-specific binding proteins that bind CD19, CD3, and serum albumin. The invention also relates to pharmaceutical compositions comprising these multi-specific binding proteins, expression vectors and host cells for making these multi-specific binding proteins, and methods of use of these multi-specific binding proteins in treating hematologic cancers.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and claims priority to U.S. Provisional Patent Application No. 62 / 681,784, filed June 7, 2018. The entire disclosure of U.S. Provisional Patent Application No. 62 / 681,784 is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to multispecific binding proteins that bind to CD19, CD3, and serum albumin. The present invention also relates to pharmaceutical compositions comprising these multispecific binding proteins, expression vectors and host cells for making these multispecific binding proteins, and methods of using these multispecific binding proteins in the treatment of hematological cancers. [Background technology]

[0003] background Bispecific molecules, such as BiTE® (bispecific T-cell engager) constructs, are recombinant protein constructs made of two operatively linked antibody-derived binding domains. One binding domain of the BiTE® construct is specific for a selected tumor-associated surface antigen on a target cell, and the other binding domain is specific for CD3, a subunit of the T cell receptor complex on a T cell. This design allows the BiTE® construct to transiently connect the T cell and the target cell while simultaneously potently activating the intrinsic cytolytic capacity of the T cell against the target cell.

[0004] The CD3 receptor complex is a protein complex composed of four polypeptide chains. In mammals, this complex contains one CD3γ (gamma) chain, one CD3δ (delta) chain, and two CD3ε (epsilon) chains. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chains are highly related cell surface proteins of the immunoglobulin superfamily that contain one extracellular immunoglobulin domain. These chains bind to the T cell receptor (TCR) to form the TCR-CD3 complex, which generates an activation signal in T lymphocytes upon antigen binding. Approximately 95% of T cells express the αβTCR, which contains one α (alpha) chain and one β (beta) chain. Two TCRξ (zeta) chains are also present in the TCR-CD3 complex. The αβTCR is responsible for the recognition of antigens presented by the major histocompatibility complex (MHC). Upon binding of the TCR with an antigenic peptide and the MHC complex, the T lymphocyte is activated by a series of biochemical events mediated by associated enzymes, coreceptors, specialized adaptor molecules, and activated or released transcription factors.

[0005] Previous BiTE® constructs bind to conformational epitopes of CD3 and are typically species specific (see PCT Publication No. WO2008119567A2). Improved BiTE® constructs, such as blinatumomab (also referred to as AMG103; see PCT Publication No. WO1999054440A1) and solitomab (also referred to as AMG110; see PCT Publication No. WO2005040220A1), bind to a context-independent epitope at the N-terminus of the CD3ε chain (e.g., amino acid residues 1-27 of the human CD3ε extracellular domain) and display cross-species specificity for human, marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus Oedipus), and squirrel monkey (Saimiri sciureus) CD3ε chains (see ibid.). These constructs do not nonspecifically activate T cells to the same extent as observed with previous BiTE® constructs and are therefore believed to pose a low risk of side effects (see Brischwein et al. (2007) J. Immunother., 30(8): 798-807).

[0006] BiTE® constructs are believed to have the disadvantage of rapid clearance from the body. Thus, although BiTE® constructs can rapidly penetrate many body regions, are readily produced, and are easy to handle, their in vivo applications may be limited by their short persistence in vivo. Due to their short in vivo half-lives, chronic administration by continuous intravenous infusion may be necessary to achieve the therapeutic effects of blinatumomab and solitomab. However, such continuous intravenous infusions may be inconvenient for patients and may increase the cost of treatment.

[0007] Although great progress has been made in constructing multispecific binding proteins, there remains a need for new and useful multispecific binding proteins for treating cancer that have sufficient therapeutic potency, a format that is easy to manufacture, and favorable pharmacokinetic properties, such as long half-lives. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2008119567A2 [Patent Document 2] WO1999054440A1 [Patent Document 3] WO2005040220A1 [Non-patent literature]

[0009] [Non-Patent Document 1] Brischwein et al. (2007) J. Immunother., 30(8): 798-807 Summary of the Invention

[0010] The multispecific binding proteins disclosed herein include a first domain that binds to CD19 (e.g., human CD19), a second domain that binds to CD3 (e.g., human and / or macaque CD3), and a third domain that binds to serum albumin (e.g., human serum albumin (HSA)). These domains are linked in a certain manner for favorable therapeutic efficacy and in vivo half-life. The multispecific binding proteins can be used to stimulate an immune response against CD19-expressing cells. As a result, the multispecific binding proteins can be used to treat diseases or disorders associated with abnormal cells expressing CD19, such as certain B-cell hematological cancers.

[0011] In one aspect, the disclosure provides a multispecific binding protein comprising: (a) a first antigen-binding site that binds to human CD19; (b) a second antigen-binding site that binds to human CD3; and (c) a third antigen-binding site that binds to human serum albumin (HSA).

[0012] In certain embodiments, the multispecific binding protein comprises one polypeptide chain, in which the third antigen-binding site is not disposed between the first and second antigen-binding sites.

[0013] In certain embodiments, the polypeptide chain has a third antigen-binding site disposed N-terminal to both the first and second antigen-binding sites. In certain embodiments, the polypeptide chain has a third antigen-binding site disposed N-terminal to the first antigen-binding site, and the first antigen-binding site disposed N-terminal to the second antigen-binding site. In certain embodiments, the polypeptide chain has a third antigen-binding site disposed N-terminal to the second antigen-binding site, and the second antigen-binding site disposed N-terminal to the first antigen-binding site.

[0014] In certain embodiments, the polypeptide chain has a third antigen-binding site disposed C-terminal to both the first and second antigen-binding sites. In certain embodiments, the polypeptide chain has a first antigen-binding site disposed N-terminal to the second antigen-binding site, and a second antigen-binding site disposed N-terminal to the third antigen-binding site. In certain embodiments, the polypeptide chain has a second antigen-binding site disposed N-terminal to the first antigen-binding site, and a first antigen-binding site disposed N-terminal to the third antigen-binding site.

[0015] In certain embodiments, the polypeptide chain has a first antigen-binding site disposed N-terminal to the third antigen-binding site, and the third antigen-binding site disposed N-terminal to the second antigen-binding site. In certain embodiments, the polypeptide chain has a second antigen-binding site disposed N-terminal to the third antigen-binding site, and the third antigen-binding site disposed N-terminal to the binding protein first antigen-binding site.

[0016] In certain embodiments, the first antigen-binding site comprises a single chain variable fragment (scFv) or a single domain antibody (sdAb). In certain embodiments, the first antigen-binding site comprises a single chain variable fragment (scFv). In certain embodiments, the first antigen-binding site binds to human CD19 with a dissociation constant (K D ) (i.e., binds equal to or stronger than 20 nM). In certain embodiments, the first antigen-binding site has a melting temperature of at least 60°C.

[0017] In certain embodiments, the first antigen-binding site comprises a heavy chain variable domain (VH) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable domain (VL) comprising complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein (i) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:3, 4, 5, 6, 7, and 8, respectively, and / or (ii) the VH comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:1, and the VL comprises an amino acid sequence set forth in SEQ ID NOs:2, 2, 3, 4, 5, 6, 7, and 8, respectively. In one particular embodiment, the VH comprises an amino acid sequence set forth in SEQ ID NO:1 and the VL comprises an amino acid sequence set forth in SEQ ID NO:2 that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:2.

[0018] In certain embodiments, the first antigen-binding site comprises a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and a VL comprising complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein (i) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:12, 13, 14, 15, 16, and 17, respectively, and / or (ii) the VH comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:10, and the VL comprises a complementarity determining region selected from the group consisting of: In one particular embodiment, the VH comprises an amino acid sequence set forth in SEQ ID NO:10 and the VL comprises an amino acid sequence set forth in SEQ ID NO:11 that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:11.

[0019] In certain embodiments, the second antigen-binding site comprises an scFv or an sdAb. In certain embodiments, the second antigen-binding site comprises an scFv. In certain embodiments, the second antigen-binding site binds to human CD3ε. In certain embodiments, the second antigen-binding site binds to human CD3 with a K equal to or lower than 10 nM. D In certain embodiments, the second antigen-binding site binds to human CD3ε with a K equal to or lower than 10 nM. D In certain embodiments, the second antigen-binding site has a melting temperature of at least 60°C.

[0020] In certain embodiments, the second antigen-binding site comprises a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and a VL comprising complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein (i) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:99, 100, 101, 102, 103, and 104, respectively, and / or (ii) the VH comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:97, and the VL comprises a complementarity determining region selected from the group consisting of: In certain embodiments, the VH comprises an amino acid sequence set forth in SEQ ID NO:97 and the VL comprises an amino acid sequence set forth in SEQ ID NO:98 that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:98. In certain embodiments, the VH comprises an amino acid sequence set forth in SEQ ID NO:97 and the VL comprises an amino acid sequence set forth in SEQ ID NO:98.

[0021] In certain embodiments, the third antigen-binding site comprises an scFv or an sdAb. In certain embodiments, the third antigen-binding site comprises an sdAb. In certain embodiments, the third antigen-binding site binds to HSA with a K equal to or lower than 20 nM. D (i.e., binds equal to or stronger than 20 nM). In certain embodiments, the third antigen-binding site has a melting temperature of at least 60°C.

[0022] In certain embodiments, the third antigen-binding site comprises a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, where (i) HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 122 or 123, 124 or 125, and 126, respectively, and / or (ii) the VH comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 121. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 121.

[0023] In certain embodiments, at least two adjacent antigen binding sites are connected by a peptide linker. In certain embodiments, in a multispecific binding protein, each of the adjacent antigen binding sites is connected by a peptide linker. In certain embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 298, 299, or 302. In certain embodiments, the peptide linker consists of the amino acid sequence of SEQ ID NO: 298, 299, or 302.

[0024] In certain embodiments, the multispecific binding protein does not comprise an antibody Fc region.

[0025] In certain embodiments, the molecular weight of the multispecific binding protein is at least 65 kD. In certain embodiments, the molecular weight of the multispecific binding protein is in the range of 50-90 kD, 50-80 kD, 50-70 kD, 50-60 kD, 60-90 kD, 60-80 kD, 60-70 kD, 65-90 kD, 65-80 kD, 65-70 kD, 70-90 kD, or 70-80 kD.

[0026] In certain embodiments, the serum half-life of the multispecific binding protein is at least 24 hours, 36 hours, 48 ​​hours, or 60 hours.

[0027] In another aspect, the disclosure provides a pharmaceutical composition comprising a multispecific binding protein disclosed herein and a pharma- ceutically acceptable carrier.

[0028] In another aspect, the present disclosure provides an isolated polynucleotide encoding a multispecific binding protein disclosed herein. In another aspect, the present disclosure provides a vector comprising said polynucleotide. In another aspect, the present disclosure provides a recombinant host cell comprising said polynucleotide or said vector.

[0029] In another aspect, the disclosure provides a method of producing a multispecific binding protein, comprising culturing a host cell disclosed herein under suitable conditions that allow expression of the multispecific binding protein. In certain aspects, the method further comprises isolating the multispecific binding protein. In certain aspects, the method further comprises formulating the isolated multispecific binding protein with a pharma- ceutically acceptable carrier.

[0030] In another aspect, the present disclosure provides a method of stimulating an immune response against cells expressing CD19, comprising exposing said cells and T lymphocytes to a multispecific binding protein or pharmaceutical composition disclosed herein.

[0031] In another aspect, the disclosure provides a method of treating a hematological cancer in a subject in need thereof, comprising administering to the subject an effective amount of a multispecific binding protein or pharmaceutical composition disclosed herein. In certain embodiments, the hematological cancer is a B-cell hematological malignancy.

[0032] In another aspect, the present disclosure provides a complex comprising a T cell expressing CD3, a B cell expressing CD19, and a multispecific binding protein disclosed herein, wherein the multispecific binding protein simultaneously binds to the T cell and the B cell. In certain embodiments, the complex further comprises HSA. [The present invention 1001] (a) a first antigen-binding site that binds to human CD19; (b) a second antigen-binding site that binds to human CD3; and (c) A third antigen-binding site that binds to human serum albumin (HSA). 2. A multispecific binding protein comprising: [The present invention 1002] Any of the multispecific binding proteins of the invention comprising a single polypeptide chain. [The present invention 1003] The multispecific binding protein of the present invention 1002, wherein in said polypeptide chains, the third antigen-binding site is not located between the first and second antigen-binding sites. [The present invention 1004] The multispecific binding protein of the present invention 1003, wherein in said polypeptide chain, a third antigen-binding site is located N-terminal to both the first antigen-binding site and the second antigen-binding site. [The present invention 1005] The multispecific binding protein of the present invention 1004, wherein in the polypeptide chain, the third antigen-binding site is located N-terminal to the first antigen-binding site, and the first antigen-binding site is located N-terminal to the second antigen-binding site. [The present invention 1006] The multispecific binding protein of the present invention 1004, wherein in the polypeptide chain, a third antigen-binding site is located N-terminal to the second antigen-binding site, and the second antigen-binding site is located N-terminal to the first antigen-binding site. [The present invention 1007] The multispecific binding protein of the present invention 1003, wherein in said polypeptide chain, a third antigen-binding site is located C-terminal to both the first antigen-binding site and the second antigen-binding site. [The present invention 1008] The multispecific binding protein of the present invention 1007, wherein in the polypeptide chain, a first antigen-binding site is located N-terminal to a second antigen-binding site, and the second antigen-binding site is located N-terminal to a third antigen-binding site. [The present invention 1009] The multispecific binding protein of the present invention, wherein in the polypeptide chain, the second antigen-binding site is located N-terminal to the first antigen-binding site, and the first antigen-binding site is located N-terminal to the third antigen-binding site. [The present invention 1010] The multispecific binding protein of the present invention 1002, wherein in the polypeptide chain, a first antigen-binding site is located N-terminal to a third antigen-binding site, and a third antigen-binding site is located N-terminal to a second antigen-binding site. [The present invention 1011] The multispecific binding protein of the present invention 1002, wherein in said polypeptide chain, the second antigen-binding site is located N-terminal to the third antigen-binding site, and the third antigen-binding site is located N-terminal to the binding protein first antigen-binding site. [The present invention 1012] Any of the preceding multispecific binding proteins of the invention, wherein the first antigen-binding site comprises a single chain variable fragment (scFv) or a single domain antibody (sdAb). [The present invention 1013] 1012. A multispecific binding protein of the invention, wherein the first antigen-binding site comprises a single-chain variable fragment (scFv). [The present invention 1014] The first antigen-binding site has a dissociation constant (K D ) any of the multispecific binding proteins of the invention that bind to human CD19. [The present invention 1015] Any of the multispecific binding proteins of the invention, wherein the first antigen-binding site has a melting temperature of at least 60°C. [The present invention 1016] the first antigen-binding site comprises a heavy chain variable domain (VH) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable domain (VL) comprising complementarity determining regions LCDR1, LCDR2, and LCDR3; (a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:3, 4, 5, 6, 7, and 8, respectively; and / or (b) the VH comprises an amino acid sequence at least 85% identical to SEQ ID NO:1, and the VL comprises an amino acid sequence at least 85% identical to SEQ ID NO:2; A multispecific binding protein according to any one of claims 1001 to 1015. [The present invention 1017] The multispecific binding protein of the present invention, wherein VH comprises the amino acid sequence shown in SEQ ID NO:1 and VL comprises the amino acid sequence shown in SEQ ID NO:2. [The present invention 1018] the first antigen-binding site comprises a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and a VL comprising complementarity determining regions LCDR1, LCDR2, and LCDR3; (a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 12, 13, 14, 15, 16, and 17, respectively; and / or (b) the VH comprises an amino acid sequence at least 85% identical to SEQ ID NO:10, and the VL comprises an amino acid sequence at least 85% identical to SEQ ID NO:11; A multispecific binding protein according to any one of claims 1001 to 1015. [The present invention 1019] The multispecific binding protein of the present invention, wherein VH comprises the amino acid sequence shown in SEQ ID NO:10 and VL comprises the amino acid sequence shown in SEQ ID NO:11. [The present invention 1020] Any of the preceding multispecific binding proteins of the invention, wherein the second antigen binding site comprises an scFv or an sdAb. [The present invention 1021] The multispecific binding protein of the present invention 1020, wherein the second antigen-binding site comprises an scFv. [The present invention 1022] Any of the multispecific binding proteins of the invention, wherein the second antigen-binding site binds to human CD3ε. [The present invention 1023] The second antigen-binding site has a K equal to or lower than 10 nM. D 5. Any of the multispecific binding proteins of the invention which bind to human CD3 at [The present invention 1024] The second antigen-binding site has a K equal to or lower than 10 nM. D Any of the multispecific binding proteins of the invention which bind to human CD3ε at [The present invention 1025] Any of the multispecific binding proteins of the invention wherein the second antigen-binding site has a melting temperature of at least 60°C. [The present invention 1026] the second antigen-binding site comprises a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and a VL comprising complementarity determining regions LCDR1, LCDR2, and LCDR3; (a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs:99, 100, 101, 102, 103, and 104, respectively; and / or (b) the VH comprises an amino acid sequence at least 85% identical to SEQ ID NO:97, and the VL comprises an amino acid sequence at least 85% identical to SEQ ID NO:98; A multispecific binding protein according to any one of claims 1001 to 1025 of the present invention. [The present invention 1027] The multispecific binding protein of the present invention 1026, wherein VH comprises the amino acid sequence shown in SEQ ID NO:97 and VL comprises the amino acid sequence shown in SEQ ID NO:98. [The present invention 1028] Any of the preceding multispecific binding proteins of the invention, wherein the third antigen-binding site comprises an scFv or an sdAb. [The present invention 1029] 1028. A multispecific binding protein of the invention, wherein the third antigen binding site comprises an sdAb. [The present invention 1030] The third antigen-binding site has a K equal to or less than 20 nM. D 5. Any of the multispecific binding proteins of the invention which bind to HSA at [The present invention 1031] Any of the multispecific binding proteins of the invention wherein the third antigen-binding site has a melting temperature of at least 60°C. [The present invention 1032] the third antigen-binding site comprises a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3; (a) HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 122 or 123, 124 or 125, and 126, respectively; and / or (b) VH comprises an amino acid sequence at least 85% identical to SEQ ID NO:121; A multispecific binding protein according to any one of claims 1001 to 1031. [The present invention 1033] A multispecific binding protein of the present invention, wherein VH comprises the amino acid sequence shown in SEQ ID NO:121. [The present invention 1034] The multispecific binding protein of any of claims 1001 to 1033, wherein at least two adjacent antigen-binding sites are connected by a peptide linker. [The present invention 1035] A multispecific binding protein according to the invention, wherein each of the adjacent antigen binding sites is connected by a peptide linker. [The present invention 1036] The multispecific binding protein of the present invention 1034 or 1035, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 298, 299, or 302. [The present invention 1037] The multispecific binding protein of the present invention 1034 or 1035, wherein the peptide linker consists of the amino acid sequence of SEQ ID NO: 298, 299, or 302. [The present invention 1038] Any of the multispecific binding proteins of the invention which do not comprise an antibody Fc region. [The present invention 1039] The multispecific binding protein of any of claims 1001 to 1038, wherein the molecular weight of the multispecific binding protein is at least 65 kD. [The present invention 1040] The multispecific binding protein of any of claims 1001 to 1038, wherein the molecular weight of the multispecific binding protein is in the range of 50 to 90 kD, 50 to 80 kD, 50 to 70 kD, 50 to 60 kD, 60 to 90 kD, 60 to 80 kD, 60 to 70 kD, 65 to 90 kD, 65 to 80 kD, 65 to 70 kD, 70 to 90 kD, or 70 to 80 kD. [The present invention 1041] Any of the multispecific binding proteins of the invention, wherein the serum half-life of the multispecific binding protein is at least 24 hours, 36 hours, 48 ​​hours, or 60 hours. [The present invention 1042] A pharmaceutical composition comprising any one of the multispecific binding proteins of the present inventions 1001 to 1041 and a pharma- ceutically acceptable carrier. [The present invention 1043] An isolated polynucleotide encoding any one of the multispecific binding proteins of the present inventions 1001 to 1041. [The present invention 1044] A vector comprising the polynucleotide of the present invention. [The present invention 1045] A recombinant host cell comprising a polynucleotide of the invention 1043 or a vector of the invention 1044. [The present invention 1046] A method for producing a multispecific binding protein, comprising culturing a host cell of the invention 1045 under suitable conditions allowing expression of said multispecific binding protein. [The present invention 1047] The method of claim 1046 further comprising the step of isolating the multispecific binding protein. [The present invention 1048] The method of claim 1047 further comprising the step of formulating the isolated multispecific binding protein with a pharma- ceutically acceptable carrier. [The present invention 1049] A method for stimulating an immune response against cells expressing CD19, comprising exposing said cells and T lymphocytes to a multispecific binding protein of any of claims 1001 to 1041 or a pharmaceutical composition of claim 1042. [The present invention 1050] A method for treating hematological cancer in a subject in need thereof, comprising administering to said subject an effective amount of a multispecific binding protein of any of claims 1001 to 1041 or a pharmaceutical composition of claim 1042. [The present invention 1051] The method of claim 1050, wherein the hematological cancer is a B-cell hematological malignancy. [The present invention 1052] A complex comprising a T cell expressing CD3, a B cell expressing CD19, and a multispecific binding protein according to any one of claims 1001 to 1041, wherein the multispecific binding protein simultaneously binds to both the T cell and the B cell. [The present invention 1053] A conjugate of 1052 of the present invention, further comprising HSA. [Brief description of the drawings]

[0033] [Figure 1]Schematic diagram of the six-domain arrangement of single-chain multispecific binding proteins. The CD19-binding domain in the form of a single domain antibody (sdAb), the CD3-binding domain in the form of a single chain variable fragment (scFv), and the HSA-binding domain in the form of an sdAb are linked in various orientations. The top of each construct represents the N-terminus of a particular polypeptide chain, and the bottom of each construct represents the C-terminus. [Diagram 2] Schematic diagram of the six-domain configuration of single-chain multispecific binding proteins, in which the CD19-binding domain in the form of an scFv, the CD3-binding domain in the form of an scFv, and the HSA-binding domain in the form of an sdAb are linked in various orientations. The top of each construct represents the N-terminus of a particular polypeptide chain, and the bottom of each construct represents the C-terminus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Detailed Description The multispecific binding protein disclosed herein comprises a first domain that binds to CD19 (e.g., human CD19), a second domain that binds to CD3 (e.g., human CD3), and a third domain that binds to serum albumin (e.g., HSA). These domains are linked in a certain manner for favorable therapeutic efficacy and in vivo half-life. Pharmaceutical compositions comprising the multispecific binding protein, methods of treating diseases or disorders with the multispecific binding protein or pharmaceutical composition, and methods of producing the multispecific binding protein are also provided. Various aspects of the invention are described in the following sections. However, aspects of the invention described in a particular section should not be limited to any particular section.

[0035] To facilitate the understanding of this invention, a number of terms and phrases are defined below.

[0036] The term "multispecific binding protein" refers to a multispecific molecule whose structure and / or function is based on the structure and / or function of an antibody, e.g., a full-length or entire immunoglobulin molecule, or is based on the heavy chain variable domain (VH) and / or the light chain variable domain (VL) of an antibody and / or single chain variants thereof. Thus, the multispecific binding protein is capable of binding to its specific target or antigen. Furthermore, any one of the binding domains of the multispecific binding protein according to the invention comprises the structural minimum of an antibody, allowing target binding. This minimum may be defined, for example, by the presence of at least three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH domain) and / or three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL domain). Another approach to define the structural minimum of an antibody is to define the epitope of the specific target to which the antibody binds, or to refer to known antibodies that compete for the antibody to bind to the same epitope to which the known antibody binds. Antibodies on which the constructs according to the invention are based include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies.

[0037] Any one of the binding domains of the multispecific binding protein according to the invention may comprise the above-mentioned group of CDRs. These CDRs may be contained within the framework of VH and / or VL. Fd fragments, for example, have two VH domains and often retain some antigen-binding function of the intact antigen-binding domain. Further examples of formats of antibody fragments, antibody variants, or binding domains include: (1) Fab fragments, which are monovalent fragments with VL, VH, CL, and CH1 domains; (2) F(ab') fragments, which are bivalent fragments in which two Fab fragments are linked by a disulfide bridge at the hinge region; 2(3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having the VL and VH domains of a single arm of an antibody; (5) dAb fragments (Ward et al., (1989) Nature 341:544-546). dAb fragments have a VH domain; (6) isolated complementarity determining regions (CDRs); and (7) single chain Fvs (scFvs). Single chain Fvs (scFvs) may be derived, for example, from an scFv library. Exemplary formats of multispecific binding proteins in accordance with the invention are described, for example, in WO2000006605A2, WO2005040220A1, WO2008119567A2, WO2010037838A2, WO2013026837A1, WO2013026833A1, US20140308285A1, US20140302037A1, WO2014144722A2, WO2014151910A1, and WO2015048272A1.

[0038] Multispecific binding proteins according to the invention also include antibody variants such as di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab 2 , Fab 3 , diabodies, single chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, "multibodies" such as triabodies or tetrabodies, or single domain antibodies, such as nanobodies, or engineered antibody fragments also referred to as single variable domain antibodies, comprising a single variable domain, which may be a VH (also referred to as VHH in the context of sdAbs) or VL, that specifically binds to an antigen or epitope independent of other V regions or domains.

[0039] As used herein, the terms "single-chain Fv", "single-chain antibody" and "scFv" refer to a single polypeptide chain antibody fragment that contains the variable regions from the heavy and light chains but lacks the constant region. Generally, single-chain antibodies further contain a peptide linker connecting the VH and VL domains, which allows them to form the desired structure and bind to the antigen. Single-chain antibodies are discussed in detail in Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994). Various methods of making single chain antibodies are known, including those described in U.S. Patent Nos. 4,694,778 and 5,260,203; International Patent Application Publication No. WO 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041. In certain embodiments, single chain antibodies may also be bispecific, multispecific, human, humanized, and / or synthetic.

[0040] Furthermore, the "multispecific binding proteins" described herein may be monovalent, bivalent, or polyvalent / multivalent constructs. Furthermore, the "multispecific binding proteins" described herein may comprise molecules consisting of only one polypeptide chain or may comprise molecules consisting of multiple polypeptide chains, which may be identical (homodimers, homotrimers, or homooligomers) or different (heterodimers, heterotrimers, or heterooligomers). Examples of the above identified antibodies and variants or derivatives thereof are described, for example, in Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988); Using Antibodies: a laboratory manual, CSHL Press (1999); Kontermann and Dibel, Antibody Engineering, Springer, 2nd ed. 2010; and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0041] The domains of the multispecific binding protein of the present invention may be connected via one or more peptide bonds and / or peptide linkers. The term "peptide linker" according to the present invention includes an amino acid sequence that links two domains. Peptide linkers can also be used to fuse a third domain with another domain of the multispecific binding protein of the present invention. An essential technical feature of such peptide linkers is that they do not contain any polymerization activity. Suitable peptide linkers include those described in US Pat. Nos. 4,751,180 and 4,935,233 or WO198809344A1.

[0042] The multispecific binding protein of the present invention may be an in vitro generated multispecific binding protein. The term "in vitro generated multispecific binding protein" refers to a multispecific binding protein according to the above definition, in which all or part of the variable region (e.g., at least one CDR) is generated by non-immune cell selection, such as in vitro phage display, protein chip, or any other method that can test candidate sequences for their ability to bind to antigen. The multispecific binding protein of the present invention may also be generated by genome rearrangement in immune cells in animals. A "recombinant antibody" is an antibody made by the use of recombinant DNA technology or genetic engineering.

[0043] The multispecific binding proteins of the present invention may be monoclonal. As used herein, the term "monoclonal" means that the proteins obtained from the population are substantially homogeneous, i.e., the individual proteins within the population are identical except for natural mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present. In terms of antibodies, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (or epitopes), monoclonal antibodies are highly specific and directed against one antigenic side or determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by a particular method.

[0044] The multispecific binding protein of the present invention or one or more antigen-binding sites thereof may be affinity matured. In immunology, affinity maturation is the process by which B cells produce antibodies with high affinity to an antigen during an immune response. Repeated exposure to the same antigen causes the host to produce antibodies with successively greater affinities. Unlike natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. After two or three rounds of mutation and selection using a display method such as phage display, antibody fragments with affinities in the low nanomolar range can be obtained.

[0045] Amino acid substitution changes can be introduced into a multispecific binding protein (e.g., a humanized or human antibody) by substituting one or more hypervariable region residues of a parent antibody. Typically, the resulting variants selected for further development have improved biological properties compared to the parent antibody from which they were derived. A convenient approach to generate such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region flanks (e.g., 6-7 flanks) are mutated to generate all possible amino acid substitutions in each flank. The antibody variants thus generated are monovalently displayed from filamentous phage particles as fusions of the M13 gene III product contained within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region flanks for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the binding domains. Such contact residues and adjacent residues are candidates for substitution according to the technique described in detail herein.Once such variants are made, the variant panel is subjected to screening as described herein.The antibody that has superior properties in one or more related assays may be selected for further development.

[0046] The multispecific binding proteins of the present invention may in particular comprise "chimeric" antibodies (immunoglobulins) or fragments thereof in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, so long as it exhibits the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 6851-55). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) or human constant region sequences. Various approaches to making chimeric antibodies have been described. See, e.g., Morrison et al. (1985) Proc. Natl. Acad. Sci. USA, 81:6851; Takeda et al. (1985) Nature, 314: 452; U.S. Pat. No. 4,816,567; U.S. Pat. No. 4,816,397; European Patent No. EP 0171496; European Patent Application Publication No. EP 0173494; and British Patent No. GB ​​2177096.

[0047] The term "binding domain" or "domain that binds to (antigen)" in the context of the present invention characterizes a domain that (specifically) binds to or (specifically) interacts with a certain target epitope or a certain target side on a target molecule (antigen), e.g., CD19, serum albumin, and CD3. The structure and function of the first binding domain, the second binding domain, and / or the third binding domain may be based on the structure and / or function of an antibody, e.g., a full-length or full immunoglobulin molecule. The binding domain may be derived from the VH and / or VL or VHH domain of an antibody or a fragment thereof. For example, the binding domain may comprise three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL domain) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH domain). The binding domain may also comprise VHH CDRs (i.e., CDR1, CDR2, and CDR3 of the VHH region).

[0048] The terms "variable domain" and "variable region" are used interchangeably and refer to the portions of an antibody or immunoglobulin domain that exhibit variability in sequence and are responsible for determining the specificity and binding affinity of a particular antibody. The variability is not uniformly distributed throughout the variable domain of an antibody; it is concentrated in subdomains of the heavy and light chain variable regions, respectively. These subdomains are called "hypervariable regions" or "complementarity determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called "framework" regions (FRMs or FRs), which provide a scaffold for the six CDRs to form an antigen-binding surface in three-dimensional space.

[0049] In the present invention, any one of the binding domains of the multispecific binding protein may comprise a single domain antibody (sdAb). Single domain antibodies comprise one monomeric antibody variable domain that can selectively bind to a specific antigen, regardless of other variable regions or domains. The first single domain antibodies were engineered from heavy chain antibodies found in camelids. These are called VHH fragments. Cartilaginous fish also have heavy chain antibodies (IgNAR), and VHH fragments can be derived from heavy chain antibodies (IgNAR). NAR Single domain antibodies, called fragments, can be obtained. An alternative approach is to split the dimeric variable domain of a common immunoglobulin, e.g., human or rodent, into monomers, thus obtaining VH or VL as single domain antibodies. Currently, most studies of single domain antibodies are based on heavy chain variable domains, but nanobodies derived from light chains have also been shown to specifically bind target epitopes. Examples of single domain antibodies include nanobodies and single variable domain antibodies.

[0050] The term "antigen-binding site" as used herein refers to the portion of an immunoglobulin molecule, or a derivative or variant thereof, that is involved in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent sections within the V regions of the heavy and light chains are called "hypervariable regions" and are inserted between more conserved adjacent sections known as "framework regions" or "FRs". Thus, the term "FR" refers to the amino acid sequences naturally found in immunoglobulins between and adjacent to the hypervariable regions. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs". In certain animals, such as camelids and cartilaginous fish, the antigen-binding site is formed by a single antibody chain providing a "single domain antibody." The antigen-binding site may be present in an intact antibody, in an antigen-binding fragment of an antibody that retains the antigen-binding surface, or in a recombinant polypeptide, e.g., an scFv, with a peptide linker connecting the heavy chain variable domain to the light chain variable domain in a single polypeptide.

[0051] As used herein, the terms "a" and "an" mean "one or more" and include pluralities unless the context is inappropriate.

[0052] As used herein, the terms "subject" and "patient" refer to an organism that is treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably include humans.

[0053] The term "effective amount" as used herein refers to an amount of a compound (e.g., a compound of the present invention) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. The term "treat" as used herein includes any effect that results in improvement of or alleviates the symptoms of a condition, disease, disorder, etc., such as, for example, relieving, alleviating, modulating, ameliorating, or eliminating.

[0054] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier, which makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic applications.

[0055] The term "pharmaceutical acceptable carrier" as used herein refers to any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0056] Throughout the description, when compositions are described as having, including, or comprising certain components, or processes and methods are described as having, including, or comprising certain steps, it is intended that there are also compositions of the invention that consist essentially of or consist of the recited components, and processes and methods according to the invention that consist essentially of or consist of the recited processing steps.

[0057] As a general matter, compositions specifying percentages are percent by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, the previous definition of the variable takes precedence.

[0058] I. Multispecific Binding Proteins In one aspect, the disclosure provides multispecific binding proteins that include a first domain that binds CD19 (e.g., human CD19), a second domain that binds CD3 (e.g., human and / or macaque CD3), e.g., CD3ε (epsilon), CD3δ (delta), and / or CD3γ (gamma), and a third domain that binds serum albumin (e.g., HSA).

[0059] In certain embodiments, the first domain is a first antigen-binding site that binds to CD19. In certain embodiments, the first antigen-binding site comprises an antibody heavy chain variable domain (VH). In certain embodiments, the first antigen-binding site comprises an antibody light chain variable domain (VL). In certain embodiments, the first antigen-binding site comprises a VH and a VL. In certain embodiments, the first antigen-binding site comprises an sdAb. In certain embodiments, the first antigen-binding site comprises an scFv.

[0060] In certain embodiments, the second domain is a second antigen-binding site that binds to CD3. In certain embodiments, the second antigen-binding site comprises a VH. In certain embodiments, the second antigen-binding site comprises a VL. In certain embodiments, the second antigen-binding site comprises a VH and a VL. In certain embodiments, the second antigen-binding site comprises an sdAb. In certain embodiments, the second antigen-binding site comprises an scFv.

[0061] In certain embodiments, the third domain is a third antigen-binding site that binds to serum albumin.In certain embodiments, the third antigen-binding site comprises VH.In certain embodiments, the third antigen-binding site comprises VL region.In certain embodiments, the third antigen-binding site comprises VH region and VL region.In certain embodiments, the third antigen-binding site comprises sdAb.In certain embodiments, the third antigen-binding site comprises scFv.

[0062] Alternatively, it is contemplated that one or more of the binding domains may not comprise an antigen-binding site. For example, U.S. Patent Application Publication No. US20130316952A1 discloses: Disclosed is a polypeptide that binds to serum albumin and has the amino acid sequence of TIFF0007661446000001.tif4144.Additional exemplary polypeptides that bind to HSA are described in Dennis et al. (2002) J. Biol. Chem., 277: 35035-43; Jacobs et al. (2015) Protein Eng. Des. Sel., 28: 385-93; and Zorzi et al. (2017) Nat. Commun., 8: 16092.

[0063] In certain embodiments, the multispecific binding protein further comprises an antibody Fc region. The presence of the Fc region may increase the serum half-life of the multispecific binding protein. Depending on the particular Fc subtype and variant used, the Fc region may also alter the activity (e.g., cytotoxic activity) of the multispecific binding protein.

[0064] In other embodiments, the multispecific binding protein does not contain an antibody Fc region. The absence of Fc contributes to the compact size of the multispecific binding protein, which can exhibit improved tissue penetration and pharmacokinetic properties. In certain embodiments, the multispecific binding protein consists of or essentially consists of a first antigen-binding site, a second antigen-binding site, and a third antigen-binding site, and a linker therebetween. In certain embodiments, the multispecific binding protein consists essentially of a first antigen-binding site, a second antigen-binding site, and a third antigen-binding site.

[0065] In certain embodiments, the multispecific binding protein binds monovalently to CD19, CD3, and / or serum albumin. Omission of additional binding domains reduces the risk of non-specific immune cell activation and reduces the size of the multispecific binding protein.

[0066] A. First antigen-binding site The first antigen-binding site of the multispecific binding protein binds to CD19 (e.g., human CD19).

[0067] CD19, also known as B cell surface antigen B4 or Leu-12, is a transmembrane protein expressed on B lymphocytes and follicular dendritic cells. CD19 is a coreceptor of the B cell antigen receptor complex on B lymphocytes (see Carter et al. (2002) Science, 256: 105-07; van Zelm et al. (2006) N. Eng. J. Med., 354: 1901-12). CD19 binds to CD21, CD81, and Leu-13 and enhances B cell receptor (BCR) signaling. Together with the BCR, CD19 regulates intrinsic and antigen receptor-induced signaling thresholds that are important for B cell clonal expansion and humoral immunity. Upon activation, the cytoplasmic tail of CD19 phosphorylates, leading to binding of Src-family kinases and recruitment of PI-3 kinase.

[0068] CD19 is a human B cell surface marker that is expressed from early stages of pre-B cell development until terminal differentiation towards plasma cells. CD19 is also expressed on many non-Hodgkin's lymphoma (NHL) cells and certain leukemias. Antibodies that bind to CD19 have been developed and tested in clinical studies for cancers of lymphatic origin, such as B cell malignancies (see, for example, Hekman et al. (1991) Cancer Immunol. Immunother., 32: 364-72; Vlasfeld et al. (1995) Cancer Immunol. Immunother., 40: 37-47; Corny et al. (1995) J. Immunother. Emphasis Tumor Immunol., 18: 231-41; and Manzke et al. (2001) Int. J. Cancer, 91: 516-22). Additionally, a BiTE® construct called Blinatumomab is being developed for clinical use.

[0069] The first antigen-binding site that binds to CD19 can be any of the antibodies described herein, for example, MT-103 (a single-chain bispecific CD19 / CD3 antibody; see Hoffman et al. (2005) Int. J. Cancer, 115: 98-104; Schlereth et al. (2006) Cancer Immunol. Immunother. 55: 503-14), CD19 / CD16 diabodies (see Schlenzka et al. (2004) Anti-cancer Drugs 15: 915-19; Kipriyanov et al. (2002) J. Immunol. 169: 137-44), BU12-saporin (see Flavell et al. (1995) Br. J. Cancer 72: 1373-79), and anti-CD19-idarubicin (Rowland et al. (1993) Cancer Immunol. Immunother. 55: 503-14). Immunol. Immunother. 55: 503-14). Further exemplary antigen binding sites that bind to CD19 from which the first antigen binding site of the present invention can be derived are disclosed in U.S. Patent Application Publication Nos. US20170174786A1, US20090042291A1, US20160046730A1, US20070154473A1, US20090142349A1, US20180142018A1, US20090136526A1, US20060257398A1, and US20180230225A1, and PCT Publication No. WO2019057100A1.

[0070] The first antigen-binding site that binds to CD19 can comprise a VH that comprises three complementarity regions (HCDR1, HCDR2, and HCDR3) and / or a VL that comprises three complementarity regions (LCDR1, LCDR2, and LCDR3). Table 1 summarizes, for each variable region, the CDRs of the variable region and the scFv constructs based on the indicated heavy and light chain variable regions. The first antigen-binding site can be obtained from the exemplary variable domain and CDR sequences as listed in Table 1.

[0071] Table 1: Exemplary first antigen binding site sequences TIFF0007661446000002.tif132150TIFF0007661446000003.tif230150TIFF0007661446000004.tif232150TIFF0007661446000005.tif232150TIFF000 7661446000006.tif232150TIFF0007661446000007.tif232150TIFF0007661 446000008.tif227150TIFF0007661446000009.tif230150TIFF00076614460 00010.tif227150TIFF0007661446000011.tif227150TIFF0007661446000012.tif230150TIFF0007661446000013.tif227150TIFF0007661446000014.tif229150TIFF0007661446000015.tif229150TIFF0007661446000016.tif119150When the VL and LCDR sequences are shown as "N / A", the antigen binding site is an sdAb having only a VH (e.g., a VHH).

[0072] In certain embodiments, the first antigen-binding site comprises a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and a VL comprising complementarity determining regions LCDR1, LCDR2, and LCDR3. In certain embodiments, the VH comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VH of an antibody shown in Table 1, and the VL comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VL of an antibody shown in Table 1. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences of an antibody shown in Table 1, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences of an antibody shown in Table 1. In certain embodiments, the VH comprises the amino acid sequence of the VH of an antibody shown in Table 1, and the VL comprises the amino acid sequence of the VL of an antibody shown in Table 1.

[0073] In certain embodiments, the VH of the first antigen-binding site comprises an amino acid sequence that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:1, and the VL of the first antigen-binding site comprises an amino acid sequence that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:2. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:3, 4, and 5, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:6, 7, and 8, respectively. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:1, and the VL comprises the amino acid sequence set forth in SEQ ID NO:2.

[0074] In certain embodiments, the VH of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:10, and the VL of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:11. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:12, 13, and 14, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:15, 16, and 17, respectively. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:10, and the VL comprises the amino acid sequence set forth in SEQ ID NO:11.

[0075] In certain embodiments, the VH of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:19, and the VL of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:20. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:21, 22, and 23, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:24, 25, and 26, respectively. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:19, and the VL comprises the amino acid sequence set forth in SEQ ID NO:20.

[0076] In certain embodiments, the VH of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:27, and the VL of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:28. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:29, 30, and 31, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:32, 33, and 34, respectively. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:27, and the VL comprises the amino acid sequence set forth in SEQ ID NO:28.

[0077] In certain embodiments, the VH of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:35, and the VL of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:36. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:29, 30, and 31, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:32, 33, and 34, respectively. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:35, and the VL comprises the amino acid sequence set forth in SEQ ID NO:36.

[0078] In certain embodiments, the VH of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:37, and the VL of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:38. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:39, 40, and 41, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:42, 43, and 44, respectively. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:37, and the VL comprises the amino acid sequence set forth in SEQ ID NO:38.

[0079] In certain embodiments, the VH of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:45, and the VL of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:46. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:47, 48, and 49, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:50, 51, and 52, respectively. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:45, and the VL comprises the amino acid sequence set forth in SEQ ID NO:46.

[0080] In certain embodiments, the VH of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:86, and the VL of the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:87. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:88, 89, and 90, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:91, 92, and 93, respectively. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:86, and the VL comprises the amino acid sequence set forth in SEQ ID NO:87.

[0081] Such antigen-binding sites may be in the form of an scFv. In certain embodiments, the first antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to an scFv sequence shown in Table 1. In certain embodiments, the first antigen binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:9, 18, 96, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, or 257.

[0082] In other embodiments, the first antigen binding site comprises an sdAb comprising a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. In certain embodiments, the VH comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VH of an sdAb antibody shown in Table 1. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequence of an antibody shown in Table 1. In certain embodiments, the VH comprises the amino acid sequence of the VH of an sdAb shown in Table 1.

[0083] In certain embodiments, the first antigen-binding site competes for binding to CD19 (e.g., human CD19) with an antibody or antigen-binding fragment thereof comprising a VH, VL, and / or scFv sequence shown in Table 1.

[0084] In certain embodiments, the first antigen-binding site of the multispecific binding protein binds to CD19 (e.g., human CD19) with a dissociation constant (K D ) to join. K D can be measured by methods known in the art. In certain embodiments, K D is measured by SPR against CD19 or an extracellular fragment thereof immobilized on a chip. D is measured, for example, by flow cytometry for cell surface expressed CD19 according to the method described in Example 5 below.

[0085] In certain embodiments, the first antigen-binding site has a K for CD19 that is less than or equal to 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. D For example, in certain embodiments, the first antigen-binding site binds to CD19 at about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about .1nM to about 7nM, about 0.1nM to about 6nM, about 0.1nM to about 5nM, about 0.1nM to about 4nM, about 0.1nM to about 3nM, about 0.1nM to about 2nM, about 0.1nM to about 1nM, about 0.1nM to about 0.5nM, about 0.5nM to about 10 nM, about 1nM to about 10nM, about 2nM to about 10nM, about 3nM to about 10nM, about 4nM to about 10nM, about 5nM to about 10nM, about 6nM to about 10nM, about 7nM to about 10nM, about 8nM to about 10nM, or about 9nM to about 10nM. D Combine with.

[0086] In certain embodiments, the first antigen-binding site binds to CD19 with a K greater than or equal to 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM. D In certain embodiments, the first antigen-binding site binds to CD19 at a concentration of about 10 nM to about 1000 nM, about 10 nM to about 900 nM, about 10 nM to about 800 nM, about 10 nM to about 700 nM, about 10 nM to about 600 nM, about 10 nM to about 500 nM, about 10 nM to about 400 nM, about 10 nM to about 300 nM, about 10 nM to about 200 nM, about 10 nM to about 100 nM, about 10 K of about 50 nM to about 1000 nM, about 100 nM to about 1000 nM, about 200 nM to about 1000 nM, about 300 nM to about 1000 nM, about 400 nM to about 1000 nM, about 500 nM to about 1000 nM, about 600 nM to about 1000 nM, about 700 nM to about 1000 nM, about 800 nM to about 1000 nM, or about 900 nM to about 1000 nM. D Combine with.

[0087] It is understood that the binding affinity of the first antigen-binding site alone to CD19 may be different from that of the same antigen-binding site in the context of the multispecific binding protein disclosed herein.This is likely due to conformational constraints from other domains.Context-dependent binding affinities are described in subsection IG entitled "Binding Affinity".

[0088] The melting temperature represents the thermostability of the antigen-binding site, and can be measured, for example, by differential scanning fluorimetry, as described in Durowoju et al. (2017) J. Vis. Exp. (121): 55262. The thermostability of an antibody or fragment thereof can be enhanced by linking CDRs to a stable framework, by introducing non-classical disulfide bonds, and by other mutagenesis, as described in McConnell et al. (2014) MAbs, 6(5): 1274-82; and Goldman et al. (2017) Front. Immunol., 8: 865.

[0089] In certain embodiments, the first antigen binding site has a melting temperature of at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C, at least 64°C, at least 65°C, at least 70°C, at least 75°C, or at least 80°C. In certain embodiments, the first antigen-binding site has a melting temperature in the range of 50-80°C, 50-70°C, 50-65°C, 50-60°C, 50-55°C, 55-70°C, 55-65°C, 55-60°C, 56-65°C, 56-60°C, 57-65°C, 57-60°C, 58-65°C, 58-60°C, 59-65°C, 59-60°C, 60-80°C, 60-75°C, 60-70°C, 60-65°C, 65-80°C, 65-75°C, 65-70°C, 70-80°C, or 70-75°C.

[0090] B. Secondary antigen-binding site The second antigen-binding site of the multispecific binding protein binds to CD3 (e.g., human CD3 and / or macaque CD3). In certain embodiments, the second antigen-binding site binds to CD3ε (epsilon). In certain embodiments, the second antigen-binding site binds to CD3δ (delta). In certain embodiments, the second antigen-binding site binds to CD3γ (gamma).

[0091] In certain embodiments, the second antigen-binding site of the multispecific binding protein binds to an epitope at the N-terminus of the CD3ε chain. In certain embodiments, the second antigen-binding site binds to an epitope located at amino acid residues 1-27 of the human CD3ε extracellular domain. This epitope or a homologous variant thereof is also present in certain non-human primates. Thus, in certain embodiments, the second antigen-binding site binds to CD3 in various primates, such as humans, New World primates (e.g., marmosets, cotton-top tamarins, or squirrel monkeys), Old World primates (e.g., baboons and macaques), gibbons, and non-human ape. Marmosets and cotton-top tamarins are New World primates belonging to the Callitrichidae family. In contrast, squirrel monkeys are New World primates belonging to the Cebidae family. In certain embodiments, the second antigen-binding site binds to human CD3ε and / or macaque CD3ε, hi certain embodiments, the second antigen-binding site further binds to marmoset, cotton-top tamarin, and / or squirrel monkey CD3ε.

[0092] The second antigen binding site that binds to an extracellular epitope of human and / or macaque CD3 is, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, Fl11- 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, WT-31, and the antibodies described in WO2008119567A2. For example, the second binding domain optionally comprises (a) a CDR-L1 as depicted in SEQ ID NO:27 of WO2008119567A2, a CDR-L2 as depicted in SEQ ID NO:28 of WO2008119567A2, and a CDR-L3 as depicted in SEQ ID NO:29 of WO2008119567A2; (b) a CDR-L1 as depicted in SEQ ID NO:117 of WO2008119567A2, a CDR-L2 as depicted in SEQ ID NO:118 of WO2008119567A2, and a CDR-L3 as depicted in SEQ ID NO:119 of WO2008119567A2; and (c) a CDR-L1 as depicted in SEQ ID NO:117 of WO2008119567A2, a CDR-L2 as depicted in SEQ ID NO:118 of WO2008119567A2, and a CDR-L3 as depicted in SEQ ID NO:119 of WO2008119567A2. The second binding domain may comprise a VL domain comprising a CDR-L1, CDR-L2, and CDR-L3 selected from CDR-L1 depicted in SEQ ID NO:153 of WO2008119567A2, CDR-L2 depicted in SEQ ID NO:154 of WO2008119567A2, and CDR-L3 depicted in SEQ ID NO:155 of WO2008119567A2. Alternatively, one or more amino acid mutations may be introduced into group (a), (b), or (c) of CDR-L1, CDR-L2, and CDR-L3, and the second binding domain may comprise any group of mutations of CDR-L1, CDR-L2, and CDR-L3.

[0093] For example, the second binding domain may be selected from the group consisting of: (a) CDR-H1 as depicted in SEQ ID NO:12 of WO2008119567A2, CDR-H2 as depicted in SEQ ID NO:13 of WO2008119567A2, and CDR-H3 as depicted in SEQ ID NO:14 of WO2008119567A2; (b) CDR-H1 as depicted in SEQ ID NO:30 of WO2008119567A2, CDR-H2 as depicted in SEQ ID NO:31 of WO2008119567A2, and CDR-H3 as depicted in SEQ ID NO:32 of WO2008119567A2; (c) CDR-H1 as depicted in SEQ ID NO:33 of WO2008119567A2, and CDR-H3 as depicted in SEQ ID NO:34 of WO2008119567A2; (d) CDR-H1 as depicted in SEQ ID NO:66 of WO2008119567A2, CDR-H2 as depicted in SEQ ID NO:67 of WO2008119567A2, and CDR-H3 as depicted in SEQ ID NO:68 of WO2008119567A2; (e) CDR-H1 as depicted in SEQ ID NO:84 of WO2008119567A2, CDR-H2 as depicted in SEQ ID NO:86 of WO2008119567A2, and CDR-H3 as depicted in SEQ ID NO:90 of WO2008119567A2. NO:85 and CDR-H3 as depicted in SEQ ID NO:86 of WO2008119567A2; (f) CDR-H1 as depicted in SEQ ID NO:102 of WO2008119567A2, CDR-H2 as depicted in SEQ ID NO:103 of WO2008119567A2 and CDR-H3 as depicted in SEQ ID NO:104 of WO2008119567A2; (g) CDR-H1 as depicted in SEQ ID NO:120 of WO2008119567A2, CDR-H2 as depicted in SEQ ID NO:121 of WO2008119567A2 and CDR-H3 as depicted in SEQ ID NO:122 of WO2008119567A2;(h) CDR-H1 as depicted in SEQ ID NO:138 of WO2008119567A2, CDR-H2 as depicted in SEQ ID NO:139 of WO2008119567A2, and CDR-H3 as depicted in SEQ ID NO:140 of WO2008119567A2; (i) CDR-H1 as depicted in SEQ ID NO:156 of WO2008119567A2, CDR-H2 as depicted in SEQ ID NO:157 of WO2008119567A2, and CDR-H3 as depicted in SEQ ID NO:158 of WO2008119567A2; and (j) CDR-H1 as depicted in SEQ ID NO:156 of WO2008119567A2, CDR-H2 as depicted in SEQ ID NO:157 of WO2008119567A2, and CDR-H3 as depicted in SEQ ID NO:158 of WO2008119567A2. The second binding domain may comprise a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 selected from CDR-H1 depicted in SEQ ID NO:174 of WO2008119567A2, CDR-H2 depicted in SEQ ID NO:175 of WO2008119567A2, and CDR-H3 depicted in SEQ ID NO:176 of WO2008119567A2. Alternatively, one or more amino acid mutations may be introduced into groups (a), (b), (c), (d), (e), (f), (g), (h), (i), and (j) of CDR-H1, CDR-H2, and CDR-H3, and the second binding domain may comprise any group of mutations of CDR-H1, CDR-H2, and CDR-H3. The sequences mentioned in WO2008119567A2 are incorporated herein by reference.;

[0094] Alternatively, the second domain may be a CD3 antibody, such as muromonab-CD3 (OKT3) as illustrated in WO2008101154, otelixizumab (TRX4) as illustrated in WO2007145941A2, teplizumab (MGA031) as illustrated in WO2013040164A1, visilizumab (Nuvion) ​​as illustrated in WO2004052397A1, SP34 as illustrated in WO2015181098A1, X35 illustrated in WO2015006749A2, VIT3 illustrated in WO2015006749A2, BMA030 illustrated in WO2015006749A2 (BW264 / 56), CLB-T3 / 3 illustrated in WO2004106381A1, CRIS7 illustrated in WO2004106381A1, YTH12.5 illustrated in WO2004106383A1, Fl illustrated in WO2012084895A2 11-409, CLB-T3.4.2 illustrated in WO2004106381A1, TR-66 illustrated in WO2013158856A2, WT32 illustrated in WO2004106381A1, SPv-T3b illustrated in WO2004106383A1, 11D8 illustrated in WO2004106381A1, XIII-141 illustrated in WO2004106381A1, XIII-46 illustrated in WO2004106381A1, XIII-87 illustrated in WO2004106381A1, 12F6 as shown, T3 / RW2-8C8 as shown in WO2004106381A1, T3 / RW2-4B6 as shown in WO2004106381A1, OKT3D as shown in WO2004106381A1, M-T301 as shown in WO2004106381A1, SMC2 as shown in WO2004106381A1, F101.01 as shown in WO2004106381A1, UCHT-1 as shown in WO2000041474A2, and WT-31 as shown in WO2016085889A1.The above-referenced sequences disclosed in WO2008101154, WO2007145941A2, WO2013040164A1, WO2004052397A1, WO2015181098A1, WO2015006749A2, WO2004106381A1, WO2004106383A1, WO2012084895A2, WO2013158856A2, WO2000041474A2, and WO2016085889A1 are incorporated by reference herein.

[0095] The second antigen-binding site that binds to CD3 can comprise a VH comprising three complementarity regions (HCDR1, HCDR2, and HCDR3) and / or a VL comprising three complementarity regions (LCDR1, LCDR2, and LCDR3). Table 2 summarizes, for each variable region, the CDRs of the variable region and scFv constructs based on the indicated heavy and light chain variable regions. The second antigen-binding site can be obtained from exemplary variable domain and CDR sequences as listed in Table 2.

[0096] Table 2: Exemplary second antigen binding site sequences TIFF0007661446000017.tif99150TIFF0007661446000018.tif230150TIFF0007661446000019.ti f229150TIFF0007661446000020.tif230150TIFF0007661446000021.tif230150TIFF00076614460 00022.tif233150TIFF0007661446000023.tif230150TIFF0007661446000024.tif233150TIFF0007661446000025.tif136150When the VL and LCDR sequences are shown as "N / A", the antigen binding site is an sdAb having only VH (e.g., VHH).

[0097] In certain embodiments, the second antigen-binding site comprises a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and a VL comprising complementarity determining regions LCDR1, LCDR2, and LCDR3. In certain embodiments, the VH comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VH of an antibody shown in Table 2, and the VL comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VL of an antibody shown in Table 2. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences of an antibody shown in Table 2, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences of an antibody shown in Table 2. In certain embodiments, the VH comprises the amino acid sequence of the VH of an antibody shown in Table 2, and the VL comprises the amino acid sequence of the VL of an antibody shown in Table 2.

[0098] In certain embodiments, the VH of the second antigen binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:97, and the VL of the second antigen binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:98. In certain embodiments, the VH comprises the HCDR1, HCDR2, HCDR3 sequences set forth in SEQ ID NOs:99, 100, and 101, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:102, 103, and 104, respectively. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:97, and the VL comprises the amino acid sequence set forth in SEQ ID NO:98.

[0099] In certain embodiments, the VH of the second antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:106, and the VL of the second antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:107. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:108, 109, and 110, respectively, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:111, 112, and 113, respectively. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:106, and the VL comprises the amino acid sequence set forth in SEQ ID NO:107.

[0100] Such antigen binding sites may be in the form of scFv. In certain embodiments, the second antigen binding site comprises an amino acid sequence that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to an scFv sequence shown in Table 2. In certain embodiments, the second antigen binding site comprises an amino acid sequence that is at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:105 or 114.

[0101] In other embodiments, the second antigen binding site comprises an sdAb comprising a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. In certain embodiments, the VH comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VH of an sdAb antibody shown in Table 2. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequence of an antibody shown in Table 2. In certain embodiments, the VH comprises the amino acid sequence of the VH of an sdAb shown in Table 2.

[0102] In certain embodiments, the second antigen-binding site competes for binding to CD3 (e.g., human CD3 and / or macaque CD3) with an antibody or antigen-binding fragment thereof comprising a VH, VL, and / or scFv sequence shown in Table 2.

[0103] In certain embodiments, the second antigen-binding site of the multispecific binding protein binds to CD3 (e.g., human CD3 and / or macaque CD3) with a dissociation constant (K D ) to join. K D can be measured by methods known in the art. D is measured by SPR against CD3 or an extracellular fragment thereof immobilized on a chip. D is measured, for example, by flow cytometry for cell surface expressed CD3 according to the methods described in Example 5 below.

[0104] In certain embodiments, the second antigen-binding site has a K for CD3 that is less than or equal to 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. D For example, in certain embodiments, the first antigen-binding site binds to CD3 at about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0. 1nM to about 7nM, about 0.1nM to about 6nM, about 0.1nM to about 5nM, about 0.1nM to about 4nM, about 0.1nM to about 3nM, about 0.1nM to about 2nM, about 0.1nM to about 1nM, about 0.1nM to about 0.5nM, about 0.5nM to about 10 nM, about 1nM to about 10nM, about 2nM to about 10nM, about 3nM to about 10nM, about 4nM to about 10nM, about 5nM to about 10nM, about 6nM to about 10nM, about 7nM to about 10nM, about 8nM to about 10nM, or about 9nM to about 10nM. D Combine with.

[0105] In certain embodiments, the second antigen-binding site has a K for CD3 that is greater than or equal to 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM. DIn certain embodiments, the second antigen-binding site binds to CD3 at about 10 nM to about 1000 nM, about 10 nM to about 900 nM, about 10 nM to about 800 nM, about 10 nM to about 700 nM, about 10 nM to about 600 nM, about 10 nM to about 500 nM, about 10 nM to about 400 nM, about 10 nM to about 300 nM, about 10 nM to about 200 nM, about 10 nM to about 100 nM, about 10 nM to about 10 nM, about 10 nM to about 2 ... K of about 50 nM to about 1000 nM, about 100 nM to about 1000 nM, about 200 nM to about 1000 nM, about 300 nM to about 1000 nM, about 400 nM to about 1000 nM, about 500 nM to about 1000 nM, about 600 nM to about 1000 nM, about 700 nM to about 1000 nM, about 800 nM to about 1000 nM, or about 900 nM to about 1000 nM D Combine with.

[0106] It is understood that the binding affinity to CD3 of the second antigen-binding site alone may be different from that of the same antigen-binding site in the context of the multispecific binding protein disclosed herein. This is likely due to conformational constraints from other domains. Context-dependent binding affinities are described in subsection IG entitled "Binding Affinity".

[0107] In certain embodiments, the second antigen binding site has a melting temperature of at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C, at least 64°C, at least 65°C, at least 70°C, at least 75°C, or at least 80°C. In certain embodiments, the second antigen-binding site has a melting temperature in the range of 50-80°C, 50-70°C, 50-65°C, 50-60°C, 50-55°C, 55-70°C, 55-65°C, 55-60°C, 56-65°C, 56-60°C, 57-65°C, 57-60°C, 58-65°C, 58-60°C, 59-65°C, 59-60°C, 60-80°C, 60-75°C, 60-70°C, 60-65°C, 65-80°C, 65-75°C, 65-70°C, 70-80°C, or 70-75°C.

[0108] C. The third antigen-binding site The third antigen-binding site of the multispecific binding protein binds to serum albumin (e.g., HSA). In certain embodiments, the binding affinity of the third antigen-binding site to human serum albumin is greater than the binding affinity of the third antigen-binding site to mouse serum albumin. In certain embodiments, the third antigen-binding site does not bind to the D-III domain of HSA. In certain embodiments, the third antigen-binding site extends the serum half-life of the multispecific binding protein.

[0109] The third antigen-binding site that binds serum albumin can be derived, for example, from the antigen-binding sites disclosed in U.S. Pat. No. 8,188,223, and PCT Publication Nos. WO2017085172, and WO2018050833.

[0110] The third antigen-binding site that binds to HSA can comprise VH that comprises three complementary regions (HCDR1, HCDR2, and HCDR3) and / or VL that comprises three complementary regions (LCDR1, LCDR2, and LCDR3).Table 3 summarizes, for each variable region, the CDRs of the variable region and the scFv constructs based on the heavy chain variable region and light chain variable region shown.The third antigen-binding site can be obtained from the exemplary variable domain and CDR sequences as listed in Table 3.

[0111] Table 3: Exemplary third antigen-binding site sequences TIFF0007661446000026.tif177150TIFF0007661446000027.tif232150TIFF0007661446000028.tif226150TIFF0007661446000029.t if232150TIFF0007661446000030.tif232150TIFF0007661446000031.tif226150TIFF0007661446000032.tif232150TIFF0007661446 000033.tif232150TIFF0007661446000034.tif226150TIFF0007661446000035.tif232155TIFF0007661446000036.tif232150TIFF0007661446000037.tif227150TIFF0007661446000038.tif35150When the VL and LCDR sequences are indicated as "N / A", the antigen binding site is an sdAb having only a VH (e.g., VHH).When the VH and HCDR sequences are indicated as "N / A", the antigen binding site is an sdAb having only a VL.

[0112] In certain embodiments, the third antigen-binding site comprises a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and a VL comprising complementarity determining regions LCDR1, LCDR2, and LCDR3. In certain embodiments, the VH comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VH of an antibody shown in Table 3, and the VL comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VL of an antibody shown in Table 3. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences of an antibody shown in Table 3, and the VL comprises the LCDR1, LCDR2, and LCDR3 sequences of an antibody shown in Table 3. In certain embodiments, the VH comprises the amino acid sequence of the VH of an antibody shown in Table 3, and the VL comprises the amino acid sequence of the VL of an antibody shown in Table 3.

[0113] In other embodiments, the third antigen binding site comprises an sdAb comprising a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. In certain embodiments, the VH comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VH of an sdAb antibody shown in Table 3. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequence of an antibody shown in Table 3. In certain embodiments, the VH comprises the amino acid sequence of the VH of an sdAb shown in Table 3.

[0114] In certain embodiments, the VH of the third antigen-binding site comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:121. In certain embodiments, the VH comprises an HCDR1 sequence set forth in SEQ ID NO:122 or 123, an HCDR2 sequence set forth in SEQ ID NO:124 or 125, and an HCDR3 sequence set forth in SEQ ID NO:126. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NO:122, 125, and 126, respectively. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 123, 124, 126, respectively. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:121.

[0115] In certain embodiments, the third antigen-binding site competes for binding to serum albumin (e.g., HSA) with an antibody or antigen-binding fragment thereof comprising a VH, VL, and / or scFv sequence shown in Table 3.

[0116] In certain embodiments, the third antigen-binding site of the multispecific binding protein has a dissociation constant (K) of about 0.1 nM to about 100 μM for serum albumin (e.g., HSA). D ) to join. K D can be measured by methods known in the art. D is measured by SPR against serum albumin or a fragment thereof immobilized on the chip.

[0117] In certain embodiments, the third antigen-binding site has a K of less than or equal to 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM to serum albumin. DFor example, in certain embodiments, the third antigen-binding site binds to serum albumin at about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1nM to about 8nM, about 0.1nM to about 7nM, about 0.1nM to about 6nM, about 0.1nM to about 5nM, about 0.1nM to about 4nM, about 0.1nM to about 3nM, about 0.1nM to about 2nM, about 0.1nM to about 1nM, about 0.1n M ~ about 0.5nM, about 0.5nM to about 10nM, about 1nM to about 10nM, about 2nM to about 10nM, about 3nM to about 10nM, about 4nM to about 10nM, about 5nM to about 10nM, about 6nM to about 10nM, about 7nM to about 10nM, about 8 nM to about 10nM, about 9nM to about 10nM, about 1nM to about 15nM, about 2nM to about 15nM, about 3nM to about 15nM, about 4nM to about 15nM, about 5nM to about 15nM, about 6nM to about 15nM, about 7nM to about 15nM, about 8nM M ~ about 15nM, about 9nM - about 15nM, about 10nM - about 15nM, about 11nM - about 15nM, about 12nM - about 15nM, about 13nM - about 15nM, about 14nM - about 15nM, about 1nM - about 20nM, about 2nM - about 20nM, Approximately 3nM to approximately 20nM, approximately 4nM to approximately 20nM, approximately 5nM to approximately 20nM, approximately 6nM to approximately 20nM, approximately 7nM to approximately 20nM, approximately 8nM to approximately 20nM, approximately 9nM to approximately 20nM, approximately 10nM to approximately 20nM, approximately 11nM to approximately 20nM , about 12nM to about 20nM, about 13nM to about 20nM, about 14nM to about 20nM, about 15nM to about 20nM, about 16nM to about 20nM, about 17nM to about 20nM, about 18nM to about 20nM, or about 19nM to about 20nM. D Combine with.

[0118] In certain embodiments, the third antigen-binding site has a K for serum albumin that is greater than or equal to 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM. DIn certain embodiments, the third antigen-binding site binds to serum albumin at a concentration of about 10 nM to about 1000 nM, about 10 nM to about 900 nM, about 10 nM to about 800 nM, about 10 nM to about 700 nM, about 10 nM to about 600 nM, about 10 nM to about 500 nM, about 10 nM to about 400 nM, about 10 nM to about 300 nM, about 10 nM to about 200 nM, about 10 nM to about 100 nM, about K of 0 nM to about 50 nM, about 50 nM to about 1000 nM, about 100 nM to about 1000 nM, about 200 nM to about 1000 nM, about 300 nM to about 1000 nM, about 400 nM to about 1000 nM, about 500 nM to about 1000 nM, about 600 nM to about 1000 nM, about 700 nM to about 1000 nM, about 800 nM to about 1000 nM, or about 900 nM to about 1000 nM. D Combine with.

[0119] It is understood that the binding affinity of the third antigen-binding site alone to serum albumin may be different from that of the same antigen-binding site in the context of the multispecific binding protein disclosed herein.This is probably due to conformational constraints from other domains.The binding affinity that depends on the context is described in subsection IG entitled "binding affinity".

[0120] In certain embodiments, the third antigen binding site has a melting temperature of at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C, at least 64°C, at least 65°C, at least 70°C, at least 75°C, or at least 80°C. In certain embodiments, the third antigen-binding site has a melting temperature in the range of 50-80°C, 50-70°C, 50-65°C, 50-60°C, 50-55°C, 55-70°C, 55-65°C, 55-60°C, 56-65°C, 56-60°C, 57-65°C, 57-60°C, 58-65°C, 58-60°C, 59-65°C, 59-60°C, 60-80°C, 60-75°C, 60-70°C, 60-65°C, 65-80°C, 65-75°C, 65-70°C, 70-80°C, or 70-75°C.

[0121] D. Humanization and Deimmunization To optimize the immunogenicity and binding properties of the multispecific binding protein, and thereby improve the therapeutic index of the multispecific binding protein, the first antigen-binding site, the second antigen-binding site, and / or the third antigen-binding site of the multispecific binding protein disclosed herein may be humanized, e.g., from one or more of the antigen-binding sites disclosed above.

[0122] A "humanized" antibody or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, sdAb, scFv, or other antigen-binding subsequence of an antibody) contains mostly human sequences, but also (a) contains minimal sequence derived from a non-human immunoglobulin. Humanized antibodies are mostly human immunoglobulins (recipient antibody) in which residues from one or more hypervariable regions (CDRs) of the recipient have been replaced by residues from one or more hypervariable regions of a non-human species (donor antibody) such as a rodent (e.g., mouse, rat, or hamster), rabbit, or camelid (e.g., llama) having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin have been replaced by corresponding non-human residues. Furthermore, as used herein, "humanized antibodies" may also include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine and optimize antibody performance. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. (1986) Nature, 321: 522-25; Reichmann et al. (1988) Nature, 332: 323-29; and Presta (1992) Curr. Op. Struct. Biol., 2: 593-96.

[0123] Humanized antibodies may be produced using transgenic animals such as mice that express human heavy and light chain genes but cannot express endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR grafting method that can be used to prepare the humanized antibodies described herein (see U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only a portion of the CDRs may be replaced with a non-human CDR. All that is required is to replace as many CDRs as necessary to allow the humanized antibody to bind to a predetermined antigen.

[0124] Humanized antibodies can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or back mutations. Such altered immunoglobulin molecules can be generated by any of several techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. USA, 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth. Enzymol, 92: 3-16, 1982, and EP 239 400).

[0125] Antibodies or fragments thereof may also be modified by specifically deleting human T cell epitopes in a process called "deimmunization". Deimmunization methods are disclosed, for example, in WO1998052976A1 or WO2000034317A2. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC class II. These peptides are potential T cell epitopes. To detect potential T cell epitopes, a computer modeling approach called "peptide threading" can be applied, in addition to searching a database of human MHC class II binding peptides for motifs present in the VH and VL sequences. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes. Detected potential T cell epitopes can be eliminated by substituting a small number of amino acid residues in the variable domain, or preferably by single amino acid substitutions. Typically, conservative substitutions are made. In many cases, but not exclusively, amino acids commonly found at positions in human germline antibody sequences may be used. Human germline sequences are disclosed, for example, in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798; Cook, GP et al. (1995) Immunol. Today Vol. 16 (5): 237-242; and Tomlinson et al. (1995) EMBO J. 14: 4628-38. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, L A. et al. MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, e.g., framework regions and CDRs. Consensus human framework regions can also be used, for example, as described in U.S. Patent No. 6,300,064.

[0126] E. Construct Format The first antigen-binding site, the second antigen-binding site, and the third antigen-binding site may take various forms. In certain embodiments, the first antigen-binding site, the second antigen-binding site, and / or the third antigen-binding site comprise two antibody variable domains (e.g., VH and VL). VH and VL can be mutated to introduce a disulfide bond (e.g., between H44 and L100) that stabilizes the antigen-binding site (see Zhao et al. (2010) Int. J. Mol. Sci., 12(1):1-11). In certain embodiments, the first antigen-binding site, the second antigen-binding site, and / or the third antigen-binding site comprise one antibody variable domain (e.g., sdAb).

[0127] In an antigen-binding site containing a VH and a VL, the VH and VL can be linked to form an scFv. The VH can be located N-terminal or C-terminal to the VL. The VH and VL are typically linked via a linker, such as a peptide linker. Exemplary sequences of peptide linkers are provided in subsection IF entitled "Linkers." In certain embodiments, the VH of the antigen-binding domain is connected to the VL of the antigen-binding domain via a peptide linker having an amino acid sequence listed in Table 4. In certain embodiments, the VH of the antigen-binding domain is connected to the VL of the antigen-binding domain via a peptide linker having an amino acid sequence of SEQ ID NO: 298, 299, or 302, and the VH is located N-terminal to the VL. In other certain embodiments, the VH of the antigen-binding domain is connected to the VL of the antigen-binding domain via a peptide linker having an amino acid sequence of SEQ ID NO: 298, 299, or 302, and the VH is located C-terminal to the VL.

[0128] Alternatively, the VH and VL may be present on separate polypeptide chains and the formation of the VH-VL complex may be facilitated by additional domains, such as the antibody constant regions CH1 and CL. Thus, in certain embodiments, the multispecific binding protein comprises a Fab comprising a VH and a VL as disclosed herein.

[0129] In certain embodiments, the multispecific binding proteins of the invention comprise a first antigen-binding site comprising one antibody variable domain, a second antigen-binding site comprising one antibody variable domain, and a third antigen-binding site comprising one antibody variable domain. In certain embodiments, the multispecific binding proteins comprise a first antigen-binding site in an sdAb format, a second antigen-binding site in an sdAb format, and a third antigen-binding site in an sdAb format.

[0130] In certain embodiments, the multispecific binding proteins of the invention comprise a first antigen-binding site comprising one antibody variable domain, a second antigen-binding site comprising one antibody variable domain, and a third antigen-binding site comprising two antibody variable domains. In certain embodiments, the multispecific binding proteins comprise a first antigen-binding site in an sdAb format, a second antigen-binding site in an sdAb format, and a third antigen-binding site in an scFv format.

[0131] In certain embodiments, the multispecific binding proteins of the invention comprise a first antigen-binding site comprising one antibody variable domain, a second antigen-binding site comprising two antibody variable domains, and a third antigen-binding site comprising one antibody variable domain, hi certain embodiments, the multispecific binding proteins comprise a first antigen-binding site in sdAb format, a second antigen-binding site in scFv format, and a third antigen-binding site in sdAb format.

[0132] In certain embodiments, the multispecific binding proteins of the invention comprise a first antigen-binding site comprising one antibody variable domain, a second antigen-binding site comprising two antibody variable domains, and a third antigen-binding site comprising two antibody variable domains, hi certain embodiments, the multispecific binding proteins comprise a first antigen-binding site in sdAb format, a second antigen-binding site in scFv format, and a third antigen-binding site in scFv format.

[0133] In certain embodiments, the multispecific binding proteins of the invention comprise a first antigen-binding site comprising two antibody variable domains, a second antigen-binding site comprising one antibody variable domain, and a third antigen-binding site comprising one antibody variable domain, hi certain embodiments, the multispecific binding proteins comprise a first antigen-binding site in scFv format, a second antigen-binding site in sdAb format, and a third antigen-binding site in sdAb format.

[0134] In certain embodiments, the multispecific binding proteins of the invention comprise a first antigen-binding site comprising two antibody variable domains, a second antigen-binding site comprising one antibody variable domain, and a third antigen-binding site comprising two antibody variable domains, hi certain embodiments, the multispecific binding proteins comprise a first antigen-binding site in scFv format, a second antigen-binding site in sdAb format, and a third antigen-binding site in scFv format.

[0135] In certain embodiments, the multispecific binding proteins of the invention comprise a first antigen-binding site comprising two antibody variable domains, a second antigen-binding site comprising two antibody variable domains, and a third antigen-binding site comprising one antibody variable domain, hi certain embodiments, the multispecific binding proteins comprise a first antigen-binding site in scFv format, a second antigen-binding site in scFv format, and a third antigen-binding site in sdAb format.

[0136] In certain embodiments, the multispecific binding proteins of the invention comprise a first antigen-binding site comprising two antibody variable domains, a second antigen-binding site comprising two antibody variable domains, and a third antigen-binding site comprising two antibody variable domains, hi certain embodiments, the multispecific binding proteins comprise a first antigen-binding site in scFv format, a second antigen-binding site in scFv format, and a third antigen-binding site in scFv format.

[0137] The three antigen-binding sites of the multispecific binding protein are oriented from amino to carboxyl as follows: (i) a first antigen-binding site (CD19-binding domain)-a second antigen-binding site (CD3-binding domain)-a third antigen-binding site (serum albumin-binding domain); (ii) first antigen-binding site (CD19-binding domain)-third antigen-binding site (serum albumin-binding domain)-second antigen-binding site (CD3-binding domain); (iii) second antigen-binding site (CD3-binding domain)-first antigen-binding site (CD19-binding domain)-third antigen-binding site (serum albumin-binding domain); (iv) second antigen-binding site (CD3-binding domain)-third antigen-binding site (serum albumin-binding domain)-first antigen-binding site (CD19-binding domain); (v) a third antigen-binding site (serum albumin-binding domain)-a first antigen-binding site (CD19-binding domain)-a second antigen-binding site (CD3-binding domain); and (vi) third antigen-binding site (serum albumin-binding domain)-second antigen-binding site (CD3-binding domain)-first antigen-binding site (CD19-binding domain) where the dashes above represent a peptide bond and / or a linker (e.g., a peptide linker).

[0138] In certain embodiments, the third antigen-binding site is not located between the first and second antigen-binding sites. In certain embodiments, the third antigen-binding site is located N-terminally to both the first and second antigen-binding sites, or C-terminally to both the first and second antigen-binding sites. In certain embodiments, the third antigen-binding site is located N-terminally to both the first and second antigen-binding sites. In certain embodiments, the third antigen-binding site is located C-terminally to both the first and second antigen-binding sites.

[0139] The location (N-terminus or C-terminus) of an antigen-binding site relative to another antigen-binding site is determined by the definition of "N-terminus" and "C-terminus" as known in the art, if one polypeptide chain contains both antigen-binding sites. It is understood that if an antigen-binding site contains two separate polypeptide chains, the location (N-terminus or C-terminus) of an antigen-binding site relative to another antigen-binding site (having one polypeptide chain or two polypeptide chains) can be determined in the same manner, if one polypeptide chain contains at least one of the former polypeptide chain and at least one of the latter polypeptide chain. Furthermore, it is understood that if antigen-binding site A is N-terminal to antigen-binding site B, and antigen-binding site B is N-terminal to antigen-binding site C, antigen-binding site A is considered to be located N-terminal to antigen-binding site C, even if antigen-binding sites A and C are not present in one common polypeptide chain. More complex structures of multispecific binding proteins are also contemplated, some of which may have positions that are difficult to characterize using the above-mentioned "N-terminal" and "C-terminal" terms, for example, because of differences in the relative positions of two antigen binding sites on one polypeptide chain versus another, or because of the presence of loop structures.

[0140] According to the present invention, the multispecific binding proteins and their constituent binding domains are in the form of one or more polypeptides. Such polypeptides may comprise protein and non-protein moieties (e.g., chemical linkers or chemical cross-linking agents, e.g., glutaraldehyde). In certain embodiments, the multispecific binding proteins of the present invention comprise a first antigen-binding site, a second antigen-binding site, and a third antigen-binding site, all of which are linked together to form a single polypeptide chain. In certain embodiments, the first antigen-binding site, the second antigen-binding site, and the third antigen-binding site are in the form of scFvs and / or sdAbs, e.g., in the above combinations, to form a single polypeptide chain.

[0141] F. Linker As described above, the antigen-binding sites of the multispecific binding proteins of the present invention can be linked via peptide bonds or linkers (e.g., peptide linkers). In certain embodiments, at least two adjacent antigen-binding sites are connected by a linker (e.g., a peptide linker). In certain embodiments, two adjacent antigen-binding sites are each connected by a linker (e.g., a peptide linker).

[0142] In certain embodiments, the three antigen binding sites of the multispecific binding protein are oriented from amino to carboxyl as follows: (i) the first antigen-binding site (CD19-binding domain)—L 1 - Second antigen-binding site (CD3-binding domain) - L 2 - a third antigen-binding site (serum albumin-binding domain); (ii) the first antigen-binding site (CD19-binding domain)—L 1 -Third antigen-binding site (serum albumin-binding domain)-L 2 - a second antigen-binding site (CD3-binding domain); (iii) the second antigen-binding site (CD3-binding domain)—L 1 -First antigen-binding site (CD19-binding domain)-L 2- a third antigen-binding site (serum albumin-binding domain); (iv) the second antigen-binding site (CD3-binding domain)—L 1 -Third antigen-binding site (serum albumin-binding domain)-L 2 - the first antigen-binding site (CD19-binding domain); (v) the third antigen-binding site (serum albumin-binding domain)—L 1 -First antigen-binding site (CD19-binding domain)-L 2 - a second antigen-binding site (the CD3-binding domain); and (vi) the third antigen-binding site (serum albumin-binding domain)—L 1 - Second antigen-binding site (CD3-binding domain) - L 2 -First antigen-binding site (CD19-binding domain) Either one of the following, L 1 and L 2 In certain constructs, L 1 or L 2 It is understood that may be replaced with a peptide bond.

[0143] If one polypeptide chain comprises two adjacent antigen-binding sites, the peptide linker connecting the two antigen-binding sites is understood to be an amino acid sequence between the two antigen-binding sites. If the antigen-binding sites comprise two separate polypeptide chains, one of the polypeptide chains is present in one common polypeptide as an adjacent antigen-binding site or as a polypeptide chain, and the peptide linker connecting the two antigen-binding sites is an amino acid sequence between the two antigen-binding sites in one common polypeptide.

[0144] In certain embodiments, the linker L 1 and L 2 is a peptide linker. 1 and L 2 The appropriate length of L can be independently selected. For example, in certain embodiments, L 1 and / or L 2is about 50 amino acid residues or less in length. 1 In certain embodiments, L consists of about 50 amino acid residues or less. 1 In certain embodiments, L consists of about 20 amino acid residues or less. 2 In certain embodiments, L consists of about 50 amino acid residues or less. 2 In certain embodiments, L consists of about 20 amino acid residues or less. 1 and L 2 and L independently consist of about 50 or less amino acid residues. 1 and L 2 are independently comprised of about 20 or less amino acid residues.

[0145] In some embodiments, the peptide linker L 1 and L 2 has an optimized length and / or amino acid composition. 1 and L 2 are the same length and have the same amino acid composition. 1 and L 2 In certain embodiments, L 1 and / or L 2 is "short", i.e., consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain embodiments, the linker consists of about 12 amino acid residues or less. In certain embodiments, L 1 and / or L 2 is "long", e.g., consisting of 15, 20, or 25 amino acid residues. 1 and / or L 2 consists of about 3 to about 15, for example 8, 9, or 10, consecutive amino acid residues.

[0146] L 1 and L 2With respect to the amino acid composition of the peptide, the peptide is selected to have the properties of conferring flexibility to the multispecific binding protein of the invention, not interfering with the binding domain, and being resistant to cleavage from proteases. For example, glycine and serine residues generally confer protease resistance. Examples of linkers suitable for linking domains in multispecific binding proteins include (GS) n , (GGS) n , (GGGS) n , (GGSG) n , (GGSGG) n , and (GGGGS) n In some embodiments, L is an aryl group, including, but not limited to, L, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. 1 and / or L 2 is independently selected from the peptide sequences listed in Table 4. In some embodiments, L 1 and / or L 2 is independently selected from SEQ ID NOs: 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, or 302. 1 and / or L 2 is independently selected from SEQ ID NOs: 298, 299, and 302. 1 and / or L 2 In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 298, 299, or 302. 1 and / or L 2 consists of the amino acid sequence of SEQ ID NO: 298, 299, or 302. 1 and L 2 In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 298, 299, or 302, respectively. 1 and L 2 consists of the amino acid sequence of SEQ ID NO:298, 299, or 302, respectively.

[0147] Table 4: Exemplary peptide linker sequences TIFF0007661446000039.tif143150

[0148] Linkers disclosed herein, e.g., peptide linkers, can also be used to connect the VH and VL of an scFv, as mentioned in subsection IE entitled "Construct Formats."

[0149] G. Binding affinity In certain embodiments, the multispecific binding protein has a K of about 0.1 nM to about 100 μM for CD19 (e.g., human CD19), CD3 (e.g., human CD3 and / or macaque CD3), and / or serum albumin (e.g., HSA). D Join with K D can be measured by methods known in the art, for example, by SPR or flow cytometry as described in Example 5 below.

[0150] In certain embodiments, the multispecific binding protein has a K for CD19, CD3, and / or serum albumin that is less than or equal to 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. DFor example, in certain embodiments, the multispecific binding protein binds to CD19, CD3, and / or serum albumin at about 10 pM to about 1 nM, about 10 pM to about 0.9 nM, about 10 pM to about 0.8 nM, about 10 pM to about 0.7 nM, about 10 pM to about 0.6 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.4 nM, about 10 pM to about 0.3 nM, about 10 pM to about 0.2 nM, about 10 pM to about 0.1 nM, about 10 pM to about 50 pM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0. 1nM to about 8nM, about 0.1nM to about 7nM, about 0.1nM to about 6nM, about 0.1nM to about 5nM, about 0.1nM to about 4nM, about 0.1nM to about 3nM, about 0.1nM to about 2nM, about 0.1nM to about 1nM, about 0.1nM to about 0.5nM, about 0.5 K of nM to about 10nM, about 1nM to about 10nM, about 2nM to about 10nM, about 3nM to about 10nM, about 4nM to about 10nM, about 5nM to about 10nM, about 6nM to about 10nM, about 7nM to about 10nM, about 8nM to about 10nM, or about 9nM to about 10nM D Combine with.

[0151] In certain embodiments, the multispecific binding protein has a K for CD19, CD3, and / or serum albumin that is greater than or equal to 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM. D In certain embodiments, the multispecific binding protein binds to CD19, CD3, and / or serum albumin at about 10 nM to about 1000 nM, about 10 nM to about 900 nM, about 10 nM to about 800 nM, about 10 nM to about 700 nM, about 10 nM to about 600 nM, about 10 nM to about 500 nM, about 10 nM to about 400 nM, about 10 nM to about 300 nM, about 10 nM to about 200 nM, about 10 nM K of about 100 nM, about 10 nM to about 50 nM, about 50 nM to about 1000 nM, about 100 nM to about 1000 nM, about 200 nM to about 1000 nM, about 300 nM to about 1000 nM, about 400 nM to about 1000 nM, about 500 nM to about 1000 nM, about 600 nM to about 1000 nM, about 700 nM to about 1000 nM, about 800 nM to about 1000 nM, or about 900 nM to about 1000 nM.D Combine with.

[0152] In certain embodiments, the K of binding to CD19 or CD3 D is measured in the presence of serum albumin (e.g., HSA). In certain embodiments, the K D is measured in the substantial absence of serum albumin (e.g., HSA). In certain embodiments, the K D is measured in the presence of serum albumin (e.g., HSA), for example, in the presence of about 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL serum albumin (e.g., HSA).

[0153] In certain embodiments, the multispecific binding proteins of the disclosure bind to CD19, CD3, and / or serum albumin with Ks similar to those of the respective antigen binding sites alone or to monoclonal antibodies with the same antigen binding sites. D In certain embodiments, the multispecific binding proteins for CD19, CD3, and / or serum albumin bind at a K value. D The values ​​are an increase of no more than 1.5-fold, no more than 2-fold, no more than 3-fold, no more than 4-fold, no more than 5-fold, no more than 6-fold, no more than 7-fold, no more than 8-fold, no more than 9-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 35-fold, no more than 40-fold, no more than 45-fold, or no more than 50-fold compared to the respective antigen-binding site alone or to a monoclonal antibody having the same antigen-binding site.

[0154] In certain embodiments, in the presence of serum albumin, the multispecific binding proteins of the disclosure have a K D In certain embodiments, the K of the multispecific binding protein for binding to CD19 and / or CD3 in the presence of serum albumin is DThe values ​​are an increase of no more than 1.5-fold, no more than 2-fold, no more than 3-fold, no more than 4-fold, no more than 5-fold, no more than 6-fold, no more than 7-fold, no more than 8-fold, no more than 9-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 35-fold, no more than 40-fold, no more than 45-fold, or no more than 50-fold compared to the absence or significant absence of serum albumin.

[0155] H. Therapeutic Activity of the Constructs The multispecific binding protein disclosed herein is designed to bind to B cells and T cells simultaneously. Once T cells are recruited, B cell lysis is promoted with cytolytic synapse formation and delivery of perforin and granzymes. The bound T cells can continuously lyse the target cells and are not subject to immune evasion mechanisms that interfere with peptide antigen processing and presentation or clonal T cell differentiation. See, for example, WO2007042261A2. Thus, the binding of the multispecific binding protein to the target B cells destroys the target cells and / or reduces the progression of B cell-related diseases.

[0156] The cytotoxicity mediated by the multispecific binding proteins of the invention can be measured in vitro in a variety of ways. Effector cells can be, for example, stimulated enriched (human) CD8 positive T cells or unstimulated (human) peripheral blood mononuclear cells (PBMCs). If the target cells are derived from macaques or express or are transfected with a macaque target cell surface antigen to which the first domain is bound, the effector cells must also be derived from macaques, such as a macaque T cell line, e.g., 4119LnPx. The target cells must express CD19, e.g., human or macaque CD19. The target cells can be a cell line (e.g., CHO) stably or transiently transfected with CD19. Alternatively, the target cells can be a cell line that naturally expresses CD19, such as B lymphocytes. Usually, the effector:target cell (E:T) ratio is about 10:1, but this can also vary. The killing of target cells can be 51It may be measured in a Cr release assay (incubation time of about 18 hours) or in a FACS-based cytotoxicity assay (incubation time of about 48 hours). Other methods for measuring cell death are well known to those skilled in the art, such as MTT or MTS assay, ATP-based assay including bioluminescence assay, sulforhodamine B (SRB) assay, WST assay, in vitro tumor cell susceptibility test (clonogenic assay), and ECIS technology.

[0157] In certain embodiments, the cytotoxic activity mediated by the multispecific binding proteins disclosed herein is measured in the cell-based cytotoxicity assay described above, which is expressed as an EC50 value corresponding to the median effective concentration (the concentration of the multispecific binding protein that induces a cytotoxic response halfway between the baseline and maximum). 50 In certain embodiments, the EC of the multispecific binding protein is expressed as 50 The value is ≦5000pM, e.g., ≦4000pM, ≦3000pM, ≦2000pM, ≦1000pM, ≦500pM, ≦400pM, ≦300pM, ≦200pM, ≦100pM, ≦50pM, ≦20pM, ≦10pM, ≦5pM, ≦4pM, ≦3pM, ≦2pM, or ≦1pM.

[0158] EC 50 Values ​​are generally based on stimulated / enriched CD8 + When T cells were used as effector cells, it was found to be lower than that of unstimulated PBMCs. 50 It is understood that the value is generally low when the target cells express high levels of the target cell surface antigen compared to low levels of the target antigen. For example, stimulated / enriched human CD8 + When T cells were used as effector cells (and either cells transfected with the target cell surface antigen, e.g., CHO cells, or human cell lines positive for the target cell surface antigen, were used as target cells), the EC 50The value is ≦1000 pM, e.g., ≦500 pM, ≦250 pM, ≦100 pM, 50 pM, ≦10 pM, or ≦5 pM. The EC 50 The EC value is ≦5000 pM, e.g., ≦4000 pM, ≦2000 pM, ≦1000 pM, ≦500 pM, ≦200 pM, ≦150 pM, ≦100 pM, ≦50 pM, ≦10 pM, or ≦5 pM. The EC value of the multispecific binding protein is ≦5000 pM, e.g., ≦4000 pM, ≦2000 pM, ≦1000 pM, ≦500 pM, ≦200 pM, ≦150 pM, ≦100 pM, ≦50 pM, ≦10 pM, or ≦5 pM. 50 The value is ≦2000 pM, for example ≦1500 pM, ≦1000 pM, ≦500 pM, ≦300 pM, ≦250 pM, ≦100 pM, ≦50 pM, ≦10 pM, or ≦5 pM.

[0159] Thus, in certain embodiments, EC 50 Values ​​are based on stimulated / enriched human CD8 + In certain embodiments, EC 50 Values ​​are measured using human PBMCs as effector cells. In certain embodiments, EC 50 Values ​​are measured using a macaque T cell line such as LnPx4119 as effector cells and cells engineered to express macaque CD19 (eg, CHO cells) as target cells.

[0160] In certain embodiments, the multispecific binding proteins of the invention do not induce or mediate lysis of cells that do not express CD 19. The term "does not induce lysis" or "does not mediate lysis" or grammatical equivalents mean that the multispecific binding protein, at a concentration of up to 500 nM, does not induce or mediate lysis of more than 30% of cells that do not express CD19, e.g., 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less, thereby setting the lysis of CD19 expressing cells to 100%.

[0161] In certain embodiments, the multispecific binding proteins disclosed herein are more effective at killing CD19-expressing cells (e.g., cancer cells) than the corresponding respective anti-CD19 monoclonal antibodies or anti-CD3 monoclonal antibodies at the same molar concentration. In certain embodiments, the multispecific binding proteins are more effective at killing CD19-expressing cells (e.g., cancer cells) than the corresponding respective anti-CD19 monoclonal antibodies and anti-CD3 monoclonal antibodies in combination, each at the same molar concentration.

[0162] The cytotoxic activity of the multispecific binding protein can be measured in the presence or absence of serum albumin (e.g., HSA). In certain embodiments, the cytotoxic activity disclosed above is measured in the absence of serum albumin (e.g., HSA). In certain embodiments, the cytotoxic activity disclosed above is measured in the substantial absence of serum albumin (e.g., HSA). In certain embodiments, the cytotoxic activity disclosed above is measured in the presence of serum albumin (e.g., HSA), for example, in the presence of about 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL serum albumin (e.g., HSA).

[0163] In certain aspects, the multispecific binding proteins of the disclosure in the presence of serum albumin inhibit EC in the absence or significant absence of serum albumin. 50 Values ​​similar to EC 50 In certain embodiments, the EC value of the multispecific binding protein for killing CD19-expressing cells in the presence of serum albumin is 50The EC value is an increase of no more than 1.5-fold, no more than 2-fold, no more than 3-fold, no more than 4-fold, no more than 5-fold, no more than 6-fold, no more than 7-fold, no more than 8-fold, no more than 9-fold, no more than 10-fold, no more than 15-fold, no more than 20-fold, no more than 25-fold, no more than 30-fold, no more than 35-fold, no more than 40-fold, no more than 45-fold, or no more than 50-fold compared to the absence or significant absence of serum albumin. The presence of serum albumin (e.g., about 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL serum albumin) also increases the EC value of the multispecific binding protein. 50 It is also understood that non-specific effects may alter the EC values ​​non-specifically. Non-specific effects are measured using the EC values ​​of a control protein that does not contain a serum albumin binding domain in the presence or absence of serum albumin. 50 In certain embodiments, the fold change is offset by the non-specific effect of serum albumin on a control protein, e.g., a bispecific protein that binds CD19 and CD3.

[0164] I. Structure Size In certain embodiments, the molecular weight of the multispecific binding protein is about 40 kD to about 100 kD. In certain embodiments, the molecular weight of the multispecific binding protein is at least 60 kD, at least 65 kD, at least 70 kD, at least 75 kD, at least 80 kD, at least 85 kD, at least 90 kD, or at least 95 kD. It is understood that, although small size generally contributes to rapid diffusion and tissue penetration, reduced size may not be critical for purposes of treating indications where there is a significant presence of target cells (e.g., cancer cells) in the blood.

[0165] In certain embodiments, the molecular weight of the multispecific binding protein is about 40 kD to about 90 kD, about 40 kD to about 80 kD, about 40 kD to about 70 kD, about 40 kD to about 60 kD, about 40 kD to about 50 kD, about 50 kD to about 100 kD, about 50 kD to about 90 kD, about 50 kD to about 80 kD, about 50 kD to about 70 kD, about 50 kD to about 60 kD, about 60 kD to about 100 kD, about 60 kD to about 90 kD, about 60 kD to about 80 kD, about 60 kD to about 70 kD, about 65 kD to about 100 kD, about 65 kD to about 90 kD, about 65 kD to about 80 kD, about 65 kD to about 70 kD, about 70 kD to about 100 kD, about 70 kD to about 90 kD, about 70 kD to about 80 kD, about 80 kD to about 100 kD, about 80 kD to about 90 kD, or about 90 kD to about 100 kD. In certain embodiments, the multispecific binding protein is smaller than 40 kD. In certain embodiments, the multispecific binding protein is about 50 kD to about 90 kD, about 50 kD to about 80 kD, about 50 kD to about 70 kD, about 50 kD to about 60 kD, about 60 kD to about 90 kD, about 60 kD to about 80 kD, about 60 kD to about 70 kD, about 65 kD to about 90 kD, about 65 kD to about 80 kD, about 65 kD to about 70 kD, about 70 kD to about 90 kD, or about 70 kD to about 80 kD.

[0166] J. Serum Half-Life Fusion proteins have been developed to extend the in vivo half-life of small proteins, particularly antibody fragments.For example, fusion with heterodimeric antibody Fc region, for example, Fc with one or more mutations that extend in vivo half-life, is described in US Patent Application Publication No. US20140302037A1, US20140308285A1, and PCT Publication No. WO2014144722A2, WO2014151910A1, and WO2015048272A1.An alternative strategy is fusion with human serum albumin (HSA) or HSA-binding peptide (see, for example, PCT Publication No. WO2013128027A1 and WO2014140358A1). The fetal Fc receptor (FcRn) appears to be involved in extending the life of albumin in the circulation (see Chaudhury et al. (2003) J. Exp. Med., 3: 315-22). Albumin and IgG bind non-cooperatively to distinct sites on FcRn, forming tri-moleculars (see ibid.). The binding of human FcRn to HSA and to human IgG is pH-dependent, being strong at acidic pH and weak at neutral or physiological pH (see ibid.). This observation suggests that proteins and protein complexes containing albumin, as well as those containing IgG (especially Fc), are protected from degradation by pH-sensitive interactions with FcRn (see ibid.). Using surface plasmon resonance (SPR) to measure the ability of individual HSA domains to bind to immobilized soluble human FcRn, it has been shown that FcRn and albumin interact in a pH-dependent manner through the D-III domain of albumin at a site that is distinct from the IgG binding site (see Chaudhury et al. (2006) Biochemistry 45:4983-90 and PCT Publication No. WO2008068280A1).

[0167] The present disclosure provides multispecific binding proteins with extended half-life. In certain embodiments, the serum half-life of the multispecific binding protein is at least 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, or 96 hours. In certain embodiments, the serum half-life of the multispecific binding protein is at least about 50 hours. In certain embodiments, the serum half-life of the multispecific binding protein is at least about 100 hours. Methods for measuring serum half-life are known in the art, and an exemplary method is described in Example 4. In certain embodiments, the serum half-life is measured in non-human primates. In certain embodiments, the serum half-life is measured in humans.

[0168] In certain embodiments, the serum concentration of the multispecific binding protein 50 hours after intravenous administration to a subject is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the serum concentration of the multispecific binding protein 1 hour after administration to said subject.

[0169] In certain embodiments, the serum half-life of the multispecific binding protein is at least 20% longer than a control multispecific binding protein that comprises a first domain identical to a first antigen-binding site of the multispecific binding protein, a second domain identical to a second antigen-binding site of the multispecific binding protein, but does not comprise a third domain identical or substantially identical to a third antigen-binding site of the multispecific binding protein. In certain embodiments, the control multispecific binding protein is identical to the multispecific binding protein except for the absence of the third antigen-binding site. In certain embodiments, the serum half-life of the multispecific binding protein is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% longer than the serum half-life of the control multispecific binding protein. In certain embodiments, the serum half-life of a multispecific binding protein is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold longer than the serum half-life of a control multispecific binding protein.

[0170] II. Preparation method Said multispecific binding protein can be produced using recombinant DNA technology well known to those skilled in the art.For example, one or more isolated polynucleotides encoding the multispecific binding protein can be linked with other suitable nucleotide sequences, including, for example, constant region coding sequences and expression control sequences, to generate a conventional gene expression construct (i.e., expression vector) that encodes the desired multispecific binding protein.The generation of a defined gene construct is within the scope of routine skill in the art.

[0171] The nucleic acid encoding the desired multispecific binding protein can be incorporated (ligated) into an expression vector and introduced into a host cell by conventional transfection or transformation methods. Exemplary host cells are Escherichia coli (E. coli) cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), and myeloma cells that do not produce IgG protein. The transformed host cells can be grown under conditions that allow the host cells to express the gene encoding the multispecific binding protein.

[0172] Specific expression and purification conditions vary depending on the expression system used.For example, when expressing a gene in E. coli, first, the engineered gene is cloned into an expression vector by placing it downstream of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence.The expressed protein may be secreted.The expressed protein may accumulate in refractile or inclusion bodies, which can be collected after disrupting cells by French press or sonication.The refractile bodies are then solubilized, and the protein may be refolded and / or cleaved by methods known in the art.

[0173] When the engineered gene is expressed in eukaryotic host cells, such as CHO cells, it is first inserted into an expression vector that contains a suitable eukaryotic promoter, secretion signal, polyA sequence and stop codon.Optionally, the vector or gene construct may contain enhancer and intron.In an embodiment involving a fusion protein that contains an antibody or a portion thereof, the expression vector may optionally contain a sequence that codes for all or part of a constant region, thereby allowing all or part of a heavy or light chain to be expressed.The gene construct can be introduced into eukaryotic host cells using conventional techniques.

[0174] The multispecific binding proteins disclosed herein may comprise one polypeptide chain. In this case, one vector expressing the polypeptide (e.g., containing an expression control sequence operably linked to a nucleotide sequence encoding the polypeptide) can be transfected into a host cell. Alternatively, the multispecific binding proteins disclosed herein may comprise two or more polypeptides. In this case, multiple expression vectors, e.g., one expression vector expressing each polypeptide, can be co-transfected into a host cell. Also, one expression vector expressing two or more polypeptides can be transfected into a host cell. For example, the coding sequences of two or more polypeptides can be operably linked to different expression control sequences (e.g., promoters, enhancers, and / or internal ribosome entry sites (IRES)). The coding sequences of two or more polypeptides can be separated by a ribosomal skip sequence or a self-cleaving sequence, e.g., a 2A peptide.

[0175] In certain embodiments, an N-terminal signal sequence is included in the protein construct to express the multispecific binding protein. Exemplary N-terminal signal sequences include those derived from interleukin-2, CD-5, IgG kappa light chain, trypsinogen, serum albumin, and prolactin.

[0176] After transfection, single clones can be isolated for generating cell banks using methods known in the art such as limiting dilution, ELISA, FACS, microscopy, or Clonepix. Clones can be cultured under conditions suitable for bioreactor scale-up and sustained expression of the multispecific binding proteins.

[0177] Multispecific binding proteins can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0178] III. Pharmaceutical Compositions The present disclosure also features a pharmaceutical composition comprising a therapeutically effective amount of the multispecific binding protein described herein. The composition can be formulated for use in various drug delivery systems. For suitable formulation, one or more physiologically acceptable excipients or carriers can also be included in the composition. Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of drug delivery methods, see, for example, Langer (Science 249:1527-1533, 1990).

[0179] In certain embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeability of the composition.In such embodiments, suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (e.g., glycine, glutamine, asparagine, arginine, or lysine); agents) (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, proline, terpenes ... These include, but are not limited to, parabens, chlorhexidine, sorbic acid, or hydrogen peroxide; solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients, and / or pharmaceutical adjuvants (see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990)).

[0180] In certain embodiments, the pharmaceutical composition may contain nanoparticles, such as polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED. 1: 10-29).

[0181] In certain embodiments, the pharmaceutical composition may contain sustained or controlled delivery formulations. Techniques for formulating sustained or controlled delivery means, such as liposome carriers, bio-erodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. Sustained release formulations may include, for example, porous polymeric microparticles, or semipermeable polymer matrices in the form of molded articles, such as films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactic acid, copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, poly(2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions may include liposomes, which can be prepared by any of several methods known in the art.

[0182] The pharmaceutical composition containing the multispecific binding protein disclosed herein can be provided in dosage unit form and can be prepared by any suitable method. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal administration. In certain embodiments, the recombinant human sialidase, recombinant human sialidase fusion protein, or antibody conjugate disclosed herein is administered by IV infusion. In certain embodiments, the recombinant human sialidase, recombinant human sialidase fusion protein, or antibody conjugate disclosed herein is administered by intratumoral injection. Useful formulations can be prepared by methods known in the pharmaceutical arts. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as EDTA; buffers, such as acetate, citrate, or phosphate; and agents for adjusting tonicity, such as sodium chloride or dextrose.

[0183] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The carrier must be stable under the conditions of manufacture and storage and must be preserved against microorganisms. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0184] The intravenous drug delivery formulation may be contained in a syringe, pen, or bag. In certain embodiments, the bag may be connected to a conduit with a tube and / or needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and contained in about 12 to 60 vials. In certain embodiments, the formulation may be freeze-dried and 45 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, about 40 mg to about 100 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, freeze-dried formulations from 12, 27, or 45 vials are combined to obtain a therapeutic amount of protein in the intravenous drug formulation. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 250 mg / vial to about 1,000 mg / vial. In certain embodiments, the formulation may be a liquid formulation and may be stored at about 600 mg / vial. In certain embodiments, the formulation is a liquid formulation and may be stored at about 250 mg / vial.

[0185] These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged as is for use or lyophilized and packaged, with the lyophilized preparation being mixed with a sterile aqueous carrier prior to administration. The pH of the preparation is typically 3-11, more preferably 5-9 or 6-8, and most preferably 7-8, e.g., 7-7.5. The resulting compositions in solid form may be packaged as a plurality of single dose units, each unit containing a fixed amount of the above-mentioned agent. The compositions in solid form may also be packaged in containers for flexible amounts.

[0186] In certain aspects, the present disclosure provides an extended shelf life formulation comprising a combination of a protein of the present disclosure and mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0187] In certain embodiments, an aqueous formulation is prepared comprising the protein of the present disclosure in a pH buffer solution. The pH of the buffer of the present invention may be about 4 to about 8, for example, about 4.5 to about 6.0, or about 4.8 to about 5.5, and the pH of the buffer of the present invention may be about 5.0 to about 5.2. Intermediate ranges of the pH recited above are also intended to be part of the present disclosure. For example, ranges of values ​​using a combination of any of the recited values ​​above as upper and / or lower limits are intended to be included. Examples of buffers that control the pH within this range include acetate (e.g., sodium acetate), succinate (e.g., sodium succinate), gluconate, histidine, citrate, and other organic acid buffers.

[0188] In certain embodiments, the formulation includes a buffer system containing citrate and phosphate to maintain a pH in the range of about 4 to about 8. In certain embodiments, the pH range can be about 4.5 to about 6.0, or about pH 4.8 to about 5.5, or the pH range can be about 5.0 to about 5.2. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3 mg / ml citric acid (e.g., 1.305 mg / ml), about 0.3 mg / ml sodium citrate (e.g., 0.305 mg / ml), about 1.5 mg / ml disodium hydrogen phosphate dihydrate (e.g., 1.53 mg / ml), about 0.9 mg / ml sodium dihydrogen phosphate dihydrate (e.g., 0.86), and about 6.2 mg / ml sodium chloride (e.g., 6.165 mg / ml). In certain embodiments, the buffer system comprises 1-1.5 mg / ml citric acid, 0.25-0.5 mg / ml sodium citrate, 1.25-1.75 mg / ml disodium hydrogen phosphate dihydrate, 0.7-1.1 mg / ml sodium dihydrogen phosphate dihydrate, and 6.0-6.4 mg / ml sodium chloride. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.

[0189] Polyols may act as tonicifiers and stabilize the multispecific binding protein and may also be included in the formulation. Polyols are added to the formulation in amounts that may vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also vary with respect to the polyol molecular weight. For example, a small amount of a monosaccharide (e.g., mannitol) may be added compared to a disaccharide (e.g., trehalose). In certain embodiments, a polyol that may be used in the formulation as a tonicity agent is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg / ml. In certain embodiments, the mannitol concentration may be about 7.5 to 15 mg / ml. In certain embodiments, the mannitol concentration may be about 10 to 14 mg / ml. In certain embodiments, the mannitol concentration may be about 12 mg / ml. In certain embodiments, the polyol sorbitol may be included in the formulation.

[0190] A surfactant or a surfactant may also be added to the formulation. Exemplary surfactants include non-ionic surfactants, such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of surfactant added is such that it reduces the aggregation of the formulated antibody and / or minimizes the formation of particles in the formulation and / or reduces adsorption. In certain embodiments, the formulation may include a surfactant that is a polysorbate. In certain embodiments, the formulation may contain the surfactant polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edi., 1996). In certain embodiments, the formulation may contain about 0.1 mg / mL to about 10 mg / mL, or about 0.5 mg / mL to about 5 mg / mL, of polysorbate 80. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

[0191] In an embodiment, the protein product of the present disclosure is formulated as a liquid formulation. The liquid formulation may be provided in a USP / Ph Eur Type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure at a concentration of 10 mg / mL. The stopper may be made of an elastomer that complies with USP and Ph Eur. In a particular embodiment, the liquid formulation may be diluted with 0.9% saline.

[0192] In certain embodiments, the liquid formulation of the present disclosure may be prepared as a solution of 10mg / mL concentration in combination with stabilizing level of sugar.In certain embodiments, the liquid formulation may be prepared by dissolving in an aqueous carrier.In certain embodiments, the stabilizer may be added in an amount that does not exceed the amount that may cause undesirable or inappropriate viscosity for intravenous administration.In certain embodiments, the sugar may be a disaccharide, for example, sucrose.In certain embodiments, the liquid formulation may also include one or more of a buffer, a surfactant, and a preservative.

[0193] In certain embodiments, the pH of the liquid formulation may be adjusted by adding a pharma- ceutically acceptable acid and / or base. In certain embodiments, the pharma-ceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

[0194] The aqueous carrier of interest herein is pharma- ceutically acceptable (safe and non-toxic for human administration) and is useful for preparing liquid formulations.Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0195] Preservatives may optionally be added to the formulations herein to reduce bacterial activity. Addition of a preservative may, for example, facilitate the production of multi-use (multiple dose) formulations.

[0196] The multispecific binding protein may be lyophilized to produce a lyophilized formulation comprising the protein and a lyoprotectant. The lyoprotectant may be a sugar, e.g., a disaccharide. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0197] The amount of sucrose or maltose useful for stabilizing the lyophilized formulation may be at least a 1:2 protein:sucrose or maltose weight ratio. In certain embodiments, the protein:sucrose or maltose weight ratio may be 1:2 to 1:5. In certain embodiments, the pH of the formulation may be adjusted by adding a pharma- ceutically acceptable acid and / or base prior to lyophilization. In certain embodiments, the pharma- ceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharma- ceutically acceptable base may be sodium hydroxide. Prior to lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted to 6-8. In certain embodiments, the pH range of the lyophilized formulation may be 7-8.

[0198] Actual dosage levels of the active ingredients in pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without toxicity to the patient.

[0199] The specific dose may be a uniform dose for each patient, for example, 50-5,000 mg of protein. Alternatively, the patient's dose may be tailored to the patient's approximate weight or surface area. Other factors in determining the appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex, and medical condition of the patient. Further refinement of the calculations required to determine the dosage appropriate for treatment is routinely performed by those skilled in the art, especially in light of the dosage information and assays disclosed herein. Dosages can also be determined by the use of known assays for determining dosages used with appropriate dose-response data. As disease progression is monitored, dosages for individual patients can be adjusted. Blood concentrations of the targetable construct or complex in the patient can be measured to see if dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics may be used to ascertain which targetable constructs and / or complexes and dosages are most likely to be effective for a particular individual (Schmitz et al., Clinica Chimica Acta 308: 43-53, 2001; Steimer et al., Clinica Chimica Acta 308: 33-41, 2001).

[0200] In general, the dosage based on body weight is about 0.01 μg to about 100 mg / kg body weight, e.g., about 0.01 μg to about 100 mg / kg body weight, about 0.01 μg to about 50 mg / kg body weight, about 0.01 μg to about 10 mg / kg body weight, about 0.01 μg to about 1 mg / kg body weight, about 0.01 μg to about 100 μg / kg body weight, about 0.01 μg to about 50 μg / kg body weight, about 0.01 μg to about 10 μg / kg body weight, about 0.01 μg to about 1 μg / kg body weight, about 0.01 μg to about 0.1 μg / kg body weight, about 0.1 μg to about 100 mg / kg body weight, about 0.1 μg to about 50 mg / kg body weight, about 0.1 μg to about 10 mg / kg body weight, about 0.1 μg to about 1 mg / kg body weight, about 0.1 μg to about 100 μg / kg body weight, about 0. 1 μg to about 10 μg / kg body weight, about 0.1 μg to about 1 μg / kg body weight, about 1 μg to about 100 mg / kg body weight, about 1 μg to about 50 mg / kg body weight, about 1 μg to about 10 mg / kg body weight, about 1 μg to about 1 mg / kg body weight, about 1 μg to about 100 μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg about 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, about 50 mg to about 100 mg / kg body weight.

[0201] Doses may be given daily, weekly, monthly, or annually or more, or even once every 2-20 years. One skilled in the art can easily assess the repetition rate for dosing based on the measured residence time and concentration of the targetable construct or complex in bodily fluids or tissues. Administration of the present invention may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion via a catheter, or by direct intralesional injection. It may be administered once or more daily, once or more weekly, once or more monthly, or once or more annually.

[0202] IV. Therapeutic uses It is contemplated that the multispecific binding proteins may be used alone or in combination with other therapeutic agents.

[0203] A. Indications The present disclosure provides a method 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 in a subject in need thereof comprising administration of a multispecific binding protein as described herein.

[0204] In certain embodiments, the cancer to be treated is non-Hodgkin's lymphoma, for example, B-cell lymphoma.In certain embodiments, the non-Hodgkin's lymphoma is B-cell lymphoma, for example, diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, or primary central nervous system lymphoma.In certain other embodiments, the cancer to be treated is multiple myeloma.In certain other embodiments, the cancer to be treated is acute lymphoblastic leukemia (ALL).In certain embodiments, the ALL is relapsed / refractory adult ALL and childhood ALL.

[0205] B. Combination Therapy The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. The term "administered in combination" as used herein is understood to mean that two (or more) different treatments are delivered to a subject so that the effects of the treatments on the patient overlap at some point while the subject is suffering from the disorder. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is an overlap in the administration period. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments in either case, the effect of the treatments is enhanced because of the combined administration. For example, the second treatment is more effective. For example, the second treatment does not have an equivalent effect to that seen when the second treatment is administered in the absence of the first treatment, or the second treatment reduces symptoms to a greater extent than that seen when the second treatment is administered in the absence of the first treatment, or a similar situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction of symptoms or other parameters associated with the disorder is greater than that observed with one treatment delivered in the absence of the other treatment.The effect of the two treatments may be partially additive, may be totally additive, or may be greater than additive.Delivery may be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0206] In one aspect, the disclosure provides methods of treating a subject by administering a second therapeutic agent in combination with one or more multispecific binding proteins described herein.

[0207] Exemplary therapeutic agents that may be used as part of a combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, fumarate, furantoin ... lutamide, drogenil, butocin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epithiostanol, formestane, interferon-alpha, interferon-2alpha, interferon-beta, interferon-gamma, colony stimulating factor-1, colony stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone releasing factor, and variations of the aforementioned agents that exhibit specific binding to their cognate receptors and may exhibit increased or decreased serum half-lives.

[0208] Another class of drugs that may be used as part of combination therapy in the treatment of cancer is immune checkpoint inhibitors.Checkpoint inhibitors can be selected from, for example, PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, adenosine A2A receptor antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, KIR antagonists, LAG3 antagonists, TIM-3 antagonists, VISTA antagonists or TIGIT antagonists.

[0209] In certain embodiments, the checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. PD-1 is a receptor present on the surface of T cells that serves as an immune system checkpoint that inhibits or otherwise regulates T cell activity when appropriate to prevent an overly active immune response. However, cancer cells can exploit this checkpoint by expressing a ligand, e.g., PD-L1, that interacts with PD-1 on the surface of T cells to shut down or regulate T cell activity. Exemplary PD-1 / PD-L1-based immune checkpoint inhibitors include antibody-based therapeutic agents. Exemplary therapeutic methods using PD-1 / PD-L1-based immune checkpoint inhibition are described in U.S. Pat. Nos. 8,728,474 and 9,073,994 and European Patent No. 1537878 B1, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, e.g., in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149.Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (Tecentriq®, Genentech), duvalumab (AstraZeneca), MEDI4736, avelumab, and BMS936559 (Bristol Myers Squibb Co.).

[0210] In certain embodiments, the methods or compositions described herein are administered in combination with CTLA-4 inhibitors.In the CTLA-4 pathway, T cells are inhibited when CTLA-4 on T cells interacts with its ligands (e.g., CD80, also known as B7-1, and CD86) on the surface of antigen-presenting cells (not cancer cells).Exemplary CTLA-4-based immune checkpoint inhibition methods are described in U.S. Patent Nos. 5,811,097, 5,855,887, and 6,051,227. Exemplary anti-CTLA-4 antibodies are described in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910; 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, and 8,143,379. Nos. 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984, International (PCT) Publication Nos. WO 98 / 42752, WO 00 / 37504, and WO 01 / 14424, and European Patent No. EP 1212422 B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0211] In certain embodiments, the methods or compositions described herein are administered in combination with (i) a PD-1 or PD-L1 inhibitor, such as a PD-1 or PD-L1 inhibitor disclosed herein, and (ii) a CTLA-4 inhibitor, such as a CTLA-4 inhibitor disclosed herein.

[0212] In certain embodiments, the methods or compositions described herein are administered in combination with an IDO inhibitor.Exemplary IDO inhibitors include 1-methyl-D-tryptophan (known as indoximod), epacadostat (INCB24360), navoximod (GDC-0919), and BMS-986205.

[0213] Still other agents that may be used as part of a combination therapy in the treatment of cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0214] Further categories of anticancer drugs include, for example, (i) ALK inhibitors, ATR inhibitors, A2A antagonists, Base Excision Repair inhibitors, Inhibitors selected from: Inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors + 2-chloro-deoxyadenosine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, MEK inhibitors, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositide 3-kinase inhibitors, inhibitors of both PARP1 and DHODH, proteasome inhibitors, topoisomerase-II inhibitors, tyrosine kinase inhibitors, VEGFR inhibitors, and WEE1 inhibitors; (ii) agonists of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) cytokines selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0215] It is understood that the multispecific antibodies disclosed herein are designed to activate T lymphocytes and may cause side effects such as neurotoxicity. Thus, in certain embodiments, the second therapeutic agent that can be used in combination with the multispecific binding protein includes an agent that alleviates the side effects of the multispecific binding protein, e.g., reduces neurotoxicity. In certain embodiments, the second therapeutic agent inhibits T cell trafficking, e.g., reduces or inhibits immune cells from crossing the blood-brain barrier. Non-limiting examples of such therapeutic agents include antagonists (e.g., antagonist antibodies) of adhesion molecules (e.g., α4 integrin) on the surface of immune cells, e.g., natalizumab. In certain embodiments, the second therapeutic agent, e.g., fingolimod or ozanimod, increases the internalization of sphingosine-1-phosphate (SIP) receptors (e.g., S1PR1 or S1PR5). In certain embodiments, the second therapeutic agent is a nitric oxide synthase (NOS) inhibitor, such as ronopterin, cindunistat, A-84643, ONO-1714, L-NOARG, NCX-456, VAS-2381, GW-273629, NXN-462, CKD-712, KD-7040, or guanidinoethyldisulfide. In certain embodiments, the second therapeutic agent is a CSF1 or CSF1R antagonist, such as pexidartinib, emactuzumab, cabiralizumab, LY-3022855, JNJ-40346527, or MCS110. Further non-limiting examples of second therapeutic agents include pentosan polysulfate, minocycline, anti-ICAM-1 antibodies, anti-P-selectin antibodies, anti-CD11a antibodies, anti-CD162 antibodies, and anti-IL-6R antibodies (eg, tocilizumab).

[0216] The amounts and relative timing of administration of the multispecific binding protein and additional therapeutic agents may be selected to achieve a desired combined therapeutic effect. For example, when a combination therapy is administered to a patient in need of such administration, the combined therapeutic agents, or pharmaceutical compositions containing such therapeutic agents, can be administered in any order, e.g., sequentially, concurrently, together, simultaneously, etc. Furthermore, for example, the multispecific binding protein may be administered while the additional therapeutic agent exerts a prophylactic or therapeutic effect, or vice versa.

[0217] Throughout the description, when compositions are described as having, including, or comprising certain components, or processes and methods are described as having, including, or comprising certain steps, it is intended that there are also compositions of the invention that consist essentially of or consist of the recited components, and processes and methods according to the invention that consist essentially of or consist of the recited processing steps.

[0218] In this application, when an element or component is said to be included in and / or selected from a recited list of elements or components, it should be understood that the element or component may be any one of the recited elements or components, or the element or component may be selected from the group consisting of two or more of the recited elements or components.

[0219] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether express or implied, can be combined in various ways without departing from the spirit and scope of the present invention.For example, when a specific compound is mentioned, unless otherwise understood from the context, this compound can be used in various embodiments of the compositions of the present invention and / or the methods of the present invention.In other words, although embodiments are described and illustrated in this application in a manner that allows a clear and concise application to be written and drawn, it is intended and understood that these embodiments can be combined or separated in various ways without departing from the disclosure and the present invention.For example, it is understood that all features described and illustrated in this specification are applicable to all aspects of the invention described and illustrated in this specification.

[0220] The phrase "at least one of" shall be understood to include each of the listed objects following the phrase individually and to include combinations of two or more of the listed objects, unless otherwise understood from context and usage. The phrase "and / or" in the context of more than two listed objects shall be understood to have the same meaning, unless otherwise understood from context.

[0221] Unless otherwise specified or understood from the context, use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, should be understood as generally open-ended and open-ended, without excluding, for example, additional, unrecited elements or steps.

[0222] When the term "about" is used before a quantitative value, the present invention also includes the specific quantitative value itself, unless otherwise specified. As used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise specifically indicated or inferred.

[0223] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0224] The use of any and all examples or exemplary language herein, such as "such as" or "including," is merely for the purpose of better illustrating the invention and does not pose a limitation on the scope of the invention unless recited in the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0225] The above description sets forth several aspects and embodiments of the present invention. This patent application specifically contemplates all combinations and permutations of these aspects and embodiments. EXAMPLES

[0226] Having now generally described the invention, the present invention will be readily understood by reference to the following examples, which are included solely to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

[0227] Example 1. Production of multispecific binding proteins This example describes the production and purification of multispecific binding proteins.

[0228] The nucleic acid encoding the single chain polyspecific binding protein (see Table 5) was constructed, codon-optimized for expression in human cells, and cloned into a mammalian expression vector according to standard procedures. After sequence verification, the expression vector in the form of a plasmid was prepared in sufficient amount for transfection using Plasmid Plus purification kits (Qiagen). Human embryonic kidney 293 (HEK293) cells were passaged to an appropriate density for transient transfection. The cells were transiently transfected with the expression vector and cultured for 6 days.

[0229] The amino acid sequences of the various multispecific binding proteins are summarized in Table 5. Constructs tAb0027-tAb0032 each contained an anti-CD19 scFv having the amino acid sequence shown in SEQ ID NO:9, an anti-CD3 scFv having the amino acid sequence shown in SEQ ID NO:105, and an anti-HSA sdAb having the amino acid sequence shown in SEQ ID NO:121. Constructs tAb0033-tAb0038 each contained an anti-CD19 scFv having the amino acid sequence shown in SEQ ID NO:18, an anti-CD3 scFv having the amino acid sequence shown in SEQ ID NO:105, and an anti-HSA sdAb having the amino acid sequence shown in SEQ ID NO:121.

[0230] Table 5. Exemplary multispecific binding proteins TIFF0007661446000040.tif142150TIFF0007661446000041.tif228150TIFF0007661446000042.tif22815 0TIFF0007661446000043.tif228150TIFF0007661446000044.tif228150TIFF0007661446000045.tif93150

[0231] The culture was harvested by centrifugation at 4000 rpm and the supernatant was filtered through a 0.22 mm filter. The multispecific binding protein with a C-terminal 10xHis tag was purified in two steps. The first step was nickel affinity chromatography with elution with PBS containing 400 mM imidazole. The second step was size-exclusion chromatography with elution in PBS (phosphate-buffered saline) pH 7.2. The multispecific binding protein concentration was determined by UV spectroscopy and protein samples were concentrated when necessary. The purity of the protein was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). Specifically, HPLC was performed on an Agilent 1100 series instrument using a MabPac size-exclusion column with PBS flow at 0.2 mL / min. The fraction with elution time of approximately 225-240 min was collected for further characterization.

[0232] As described above, the generated constructs contained anti-CD19 scFvs having the amino acid sequences shown in SEQ ID NO:9 or 18. The binding affinity of the two CD19 binding domains to CD19 was measured by SPR using monomeric and dimeric CD19 extracellular domains fused to human IgG1 Fc. Binding kinetic parameters were generally measured using a ForteBio instrument as previously described (see Estep et al. (2013) MAbs, 5(2): 270-78). The K of the CD19 binding domain having the sequence of SEQ ID NO:9 when measured using monomeric CD19 protein was 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.40, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.60, 0.70, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, 0.99, 0.99, 0.99, D The K value of the CD19-binding domain having the sequence of SEQ ID NO:18 is 7 nM. D The K value of the CD19 binding domain having the sequence of SEQ ID NO:9 was 11 nM when measured using a dimeric CD19 protein. D The K value is 5 nM, and the K value of the CD19 binding domain having the sequence of SEQ ID NO:18 D The value was 15 nM.

[0233] Example 2. Multispecific binding protein is CD19 + Induce T cell cytotoxicity against target cells This example illustrates the cytotoxic activity of multispecific binding proteins.

[0234] The T cell redirection activity of multispecific binding proteins and BiTE proteins was evaluated using the KILR Raji Cell Model. Briefly, panT cells were isolated from primary human PBMCs derived from one healthy donor by negative selection using a commercially available kit (e.g., Easy Sep Human T Cell Enrichment Kit, StemCell Technologies). To expand T cells, they were maintained in RPMI1640 medium supplemented with 10% serum and 300 IU / mL IL-2. The collected T cells were washed twice to remove serum.

[0235] KILR Raji cells express CD19 on their surface and were used as target cells. To opsonize target cells, each multispecific binding protein or BiTE (see Table 5) was incubated with target cells for 30 minutes at 37°C in RPMI1640 supplemented with 5% heat-inactivated low IgG fetal bovine serum and penicillin-streptomycin-glutamine. The proteins were added in serial dilutions at 10 different doses, with each dose repeated in duplicate. Human serum albumin was added to the medium of some samples at a final concentration of 15mg / mL. Selected proteins were also evaluated using KILR SKOV3 cells, which are CD19 negative, as a negative control.

[0236] Following opsonization, target cells were incubated with panT cells at an effector:target (E:T) ratio of 10:1 for 6 hours at 37°C. Upon killing of KILR Raji cells, these cells released labeled housekeeping proteins into the medium. Labeled housekeeping proteins were quantified by adding KILR detection reagent (DiscoverX). Luminescent signals from all wells were read on an Envision plate reader. Spontaneous release and total lysis controls were included on each plate to calculate percent killing.

[0237] The following formula: % Killed = (value from test protein sample - mean value from spontaneous release control) / (mean value from total lysis control - mean value from spontaneous release control) x 100 Percent killing was calculated from the luminescence signal values ​​using GraphPad Prism software. EC50 values ​​were calculated from the percent killing by fitting dose-response curves.

[0238] Table 6 lists the EC50 values ​​for T cell redirected killing of exemplary multispecific binding proteins and control anti-CD19 BiTE proteins in the absence and presence of human serum albumin. No significant killing was observed with CD19-negative KILR SKOV3 cells.

[0239] Table 6. Cytotoxic activity of multispecific binding proteins TIFF0007661446000046.tif103155

[0240] As shown in Table 6, regardless of construct format, CD3 binding domain, HSA binding domain, and the presence or absence of HSA in the assay medium, multispecific binding proteins containing anti-CD19 scFv having the amino acid sequence of SEQ ID NO:9 exhibited stronger cytotoxic activity than those containing anti-CD19 scFv having the amino acid sequence of SEQ ID NO:18. This data suggests that constructs containing this anti-CD19 scFv, which has a high binding affinity to CD19, will exhibit stronger therapeutic activity than constructs containing other anti-CD19 scFvs with lower binding affinity.

[0241] Furthermore, all of the multispecific binding proteins tested had a lower EC in the absence of HSA than in the presence of HSA. 50 EC values ​​in the presence of HSA were significantly higher (i.e., greater ability to induce cytotoxicity). Without wishing to be bound by theory, it is believed that the presence of HSA alters the protein complex. This was specific to the multispecific binding protein containing the HSA-binding domain, rather than a nonspecific effect as observed with blinatumomab. 50 Values ​​and EC in the absence of HSA 50 The ratio of values, also referred to herein as "fold change," was used to assess the effect of HSA on the potential therapeutic activity of multispecific binding proteins. As shown in Table 6, construct formats with an HSA-binding domain N-terminal to both the CD19-binding domain and the CD3-binding domain (i.e., tAb0031, tAb0032, tAb0037, and tAb0038) showed less fold change than other construct formats, regardless of which CD19-binding domain was used in the construct.

[0242] Furthermore, among constructs with the same CD19-, CD3-, and HSA-binding domains, constructs in the CD19:CD3:HSA format (i.e., the CD19-binding domain is located N-terminal to the CD3-binding domain, and the CD3-binding domain is located N-terminal to the HSA-binding domain), i.e., tAb0027 and tAb0033, had the lowest EC in the absence and presence of HSA. 50 value or the second lowest EC 50 The values ​​were shown.

[0243] Example 3.CD19 + Cytotoxicity of multispecific binding proteins against target cells This example provides an alternative method for determining the cytotoxic activity of multispecific binding proteins.

[0244] The multispecific binding proteins disclosed herein can be evaluated in in vitro assays for mediating T cell dependent cytotoxicity against B cell antigen positive target cells. For example, the CD19 binding multispecific binding proteins disclosed herein can be used to mediate T cell dependent cytotoxicity against B cell antigen positive target cells. + The antibodies are assessed in an in vitro assay for mediation of T cell dependent cytotoxicity against target cells.

[0245] Fluorescently labeled CD19 + MEC-1 cells (CD19 + Human chronic B-cell leukemia cell line) are incubated with isolated PBMCs of random donors or CB15 T cells (standardized T-cell line) as effector cells in the presence of CD19-binding multispecific binding protein. After 4 hours of incubation at 37°C in a humidified incubator, fluorescent dye release from target cells into the supernatant is determined by spectrofluorometer. Target cells incubated without CD19-binding multispecific binding protein serve as negative control, and target cells completely lysed by adding saponin at the end of incubation serve as positive control. Based on the measurement of remaining viable target cells, the following formula: [1-(viable target (試料) Number of / Surviving targets(自然) The percentage of specific cell lysis can be calculated according to the following: [number of cells per 100] × 100%. Sigmoidal dose-response curves and EC 50 Values ​​are calculated by nonlinear regression / 4-parameter logistic fit using GraphPad software. The lysis values ​​obtained for a given multispecific binding protein concentration are used to calculate a sigmoidal dose-response curve by 4-parameter logistic fit analysis using Prism software. The percentage of target cell lysis induced by CD19-binding multispecific binding proteins is expected to be higher than that induced by similar constructs lacking the CD19-binding or CD3-binding domains.

[0246] Alternatively, a human T-cell dependent cytotoxicity (TDCC) assay is used to measure the ability of multispecific binding proteins to induce T cells to kill tumor cells (Nazarian et al. 2015, J. Biomol. Screen, 20:519-27). In this assay, T cells and target cancer cell line cells are mixed together in a 10:1 ratio in a 384-well plate, and various amounts of multispecific binding proteins are added. After 48 hours, the T cells are washed away, leaving the plate with target cells that were not killed by the T cells attached. To quantify the remaining viable cells, a CellTiter-Glo® Luminescent Cell Viability Assay (Promega) is used. It is believed that the killing rate of cancer cells expressing B cell antigens induced by CD19-binding multispecific binding proteins is higher than the killing rate induced by similar constructs lacking either the CD19-binding domain or the CD3-binding domain and / or other negative control molecules.

[0247] Example 4. Pharmacokinetics of multispecific binding proteins with HSA-binding domains This example is designed to determine the pharmacokinetics of a multispecific binding protein.

[0248] A multispecific binding protein containing a domain that binds CD19, a domain that binds CD3, and a domain that binds serum albumin is tested in cynomolgus monkeys in terms of pharmacokinetic (PK) studies to assess the serum excretion time of the multispecific binding protein.

[0249] The multispecific binding proteins are administered as an intravenous bolus or intravenous infusion. The multispecific binding proteins are administered in a linear pharmacokinetically relevant range of doses: 0.5 μg / kg to 3 μg / kg, 6 μg / kg, 12 μg / kg, and 15 μg / kg. For comparative purposes, serum concentrations of the multispecific binding proteins are normalized to dose and normalized to molecular weight (expressed in nmol).

[0250] Groups of at least 2-3 animals are used for each polyspecific binding protein. Blood samples are collected and serum is prepared to determine serum concentrations of the polyspecific binding protein. The polyspecific binding protein levels in serum are measured using an immunoassay, which is performed by capturing the polyspecific binding protein via the CD19 binding domain, against which an antibody directed against the CD3 binding domain of the polyspecific binding protein is used for detection. The serum concentration-time profiles are used to determine PK parameters using known analytical methods, e.g., methods and software described in Ritschel WA and Kearns GL, 1999, IN: Handbook Of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, DC, e.g., WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.).

[0251] Alternatively, the serum half-lives of various multispecific binding proteins containing a serum albumin binding domain are compared to the serum half-lives of a control construct capable of binding to CD19 and CD3 but lacking the serum albumin binding domain by including in the experiment a separate group of cynomolgus monkeys receiving the control construct. Additional domains can be included so that the control construct is similar in size to the multispecific binding protein.

[0252] The serum half-life of the multispecific binding protein that binds CD19 is expected to be significantly longer compared to similar constructs and / or other negative control molecules that can bind CD19 and CD3 but lack the serum albumin binding domain.

[0253] Example 5. Determination of antigen affinity by flow cytometry This example is designed to determine the affinity of a multispecific binding protein for an antigen.

[0254] Various of the multispecific binding proteins disclosed herein are synthesized using human CD3 + cells and corresponding B cell surface antigen positive cells, e.g., human CD19 + The multispecific binding proteins are tested for binding affinity to cynomolgus monkey CD3 cells. + Cells and corresponding B cell surface antigen positive cells, e.g., cynomolgus monkey CD19 + Binding affinity to cells is also tested.

[0255] CD3 + Cells and CD19 +The cells are incubated with 100 μL of serial dilutions of the polyspecific binding protein. After washing three times with FACS buffer, the cells are incubated with 0.1 mL of 10 μg / mL mouse monoclonal antibody idiotypic antibody dissolved in the same buffer for 45 min on ice. After the second washing cycle, the cells are incubated with 0.1 mL of 15 μg / mL FITC-conjugated goat anti-mouse IgG antibody under the same conditions as before. As a control, the cells are incubated with anti-His IgG followed by FITC-conjugated goat anti-mouse IgG antibody without the polyspecific binding protein. The cells are then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg / mL propidium iodide (PI) to exclude dead cells. 1 × 10 4 The fluorescence of living cells is measured using commercially available flow cytometers and software. The mean fluorescence intensity of the cell sample is calculated using software such as CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany). The K D Values ​​can be calculated using normalized fluorescence intensity values ​​and known formulas, for example, those provided in GraphPad Prism software (GraphPad Software, La Jolla Calif. USA). CD3 binding affinity and cross-reactivity can be calculated using CD3 + Jurkat cells and cynomolgus CD3 + The CD19 binding and cross-reactivity are evaluated in titration and flow cytometry experiments against the HSC-F cell line. + The cross-reactivity is evaluated in tumor cell lines. D Ratios were determined for CHO cell lines expressing either recombinant human or recombinant cynomolgus antigens. D The values ​​can be used to calculate:

[0256] Example 6. Cytokine production induced by multispecific binding proteins This example is designed to determine the ability of multispecific binding proteins to induce cytokine production from immune cells.

[0257] CD19 + To obtain evidence that T cells are activated by the multispecific binding proteins of the present invention, for example, multispecific binding proteins that bind to CD19, in the presence of target cells such as B cells, AlphaLISA assays (Perkin Elmer) targeting TNFα and interferon-γ are used. For this assay, primary human T cells and human tumor cells expressing B cell surface antigens are incubated in the presence of multispecific binding proteins that bind to CD19, as described under the name of cytotoxicity assay. After 48 hours of incubation, 2 microliter aliquots of the assay supernatant are analyzed according to the manufacturer's instructions. The levels of TNFα or interferon-γ induced by multispecific binding proteins that bind to CD19 are considered to be higher than those induced by similar constructs lacking the CD19 or CD3 binding domains and / or other negative control molecules.

[0258] Example 7. Identification of scFv variants that bind to human CD3ε This example is designed to identify variants of the antigen binding site disclosed herein that bind to human CD3ε.

[0259] Characterize the binding properties of the parent CD3ε binding constructs for biotin-CD3ε and biotin-HSA. Prepare single substitution libraries of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 domains to construct anti-CD3ε scFv phage libraries. Change one residue at a time by mutagenesis. Bind single substitution libraries to biotinylated human CD3ε, wash, elute, and count to select clones and confirm binding affinity. Use biotinylated cynomolgus CD3ε as the first round selection target, wash 4 hours after combinatorial phage binding from two independent libraries (~2× selection). Use biotinylated human CD3ε as the second round selection target, wash 3 hours after binding of both libraries (<2× selection). Subclone PCRed inserts from the second round selection into pcDNA3.4 His6 expression vector. 180 clones were picked, DNA purified, sequenced, and introduced into Expi293 by transfection. D ), dissociation rate (k d or k off ), and association rate (k a or k on To more precisely determine parameters such as affinity, a panel of 16 clones with a range of affinities for human CD3ε is selected.

[0260] INCORPORATION BY REFERENCE All publications and patents cited throughout the text of this specification, whether supra or infra, including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, and the like, are hereby incorporated by reference in their entirety for all purposes. To the extent that the material incorporated by reference contradicts or is inconsistent with this specification, the present specification takes precedence over such material.

[0261] Equivalent The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore considered in all respects to be illustrative and not limiting of the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are intended to be within the scope of the invention.

[0262] Sequence information SEQUENCE LISTING <110> CULLINAN ONCOLOGY, INC. <120> MULTI-SPECIFIC BINDING PROTEINS AND METHODS OF USE THEREOF <150> US 62 / 681,784 <151> 2018-06-07 <160> 407 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polyp <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 2 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Glu Thr Ser 20 25 30 Thr Gly Thr Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Leu 85 90 95 Leu Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 3 Asp Tyr Ile Met His 1 5 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 4 Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe Gln 1 5 10 15 Gly <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 5 Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp Tyr 1 5 10 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 6 Lys Ser Ser Gln Ser Leu Glu Thr Ser Thr Gly Thr Thr Tyr Leu Asn 1 5 10 15 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 7 Arg Val Ser Lys Arg Phe Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 Leu Gln Leu Leu Glu Asp Pro Tyr Thr 1 5 <210> 9 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Thr Pro 130 135 140 Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Lys 145 150 155 160 Ser Ser Gln Ser Leu Glu Thr Ser Thr Gly Thr Thr Tyr Leu Asn Trp 165 170 175 Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Val 180 185 190 Ser Lys Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser 195 200 205 Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val 210 215 220 Gly Val Tyr Tyr Cys Leu Gln Leu Leu Glu Asp Pro Tyr Thr Phe Gly 225 230 235 240 Gln Gly Thr Lys Leu Glu Ile Lys 245 <210> 10 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 11 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 12 Thr Ser Gly Met Gly Val Gly 1 5 <210> 13 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr 1 5 10 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 16 Asp Thr Ser Lys Leu Ala Ser 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Phe Gln Gly Ser Val Tyr Pro Phe Thr 1 5 <210> 18 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 18 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala 130 135 140 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala 145 150 155 160 Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln 165 170 175 Ala Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile 180 185 190 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln 210 215 220 Gly Ser Val Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 225 230 235 240 Lys Arg <210> 19 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Ser Ser Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Tyr Tyr Tyr Gly Thr Arg Val Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 20 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Lys Ser Leu Gln Asn Val 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Gln Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Asn Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Ile Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 21 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 21 Ser Tyr Val Met His 1 5 <210> 22 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr 1 5 10 <210> 23 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Gly Thr Tyr Tyr Tyr Gly Thr Arg Val Phe Asp Tyr 1 5 10 <210> 24 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 24 Arg Ser Ser Lys Ser Leu Gln Asn Val Asn Gly Asn Thr Tyr Leu Tyr 1 5 10 15 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 25 Arg Met Ser Asn Leu Asn Ser 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 26 Met Gln His Leu Glu Tyr Pro Ile Thr 1 5 <210> 27 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 27 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 28 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 28 Asp Ile Leu Leu Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 29 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 29 Ser Tyr Trp Met Asn 1 5 <210> 30 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 30 Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 1 5 10 15 Gly <210> 31 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 31 Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr 1 5 10 15 <210> 32 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 32 Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn 1 5 10 15 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 33 Asp Ala Ser Asn Leu Val Ser 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 34 Gln Gln Ser Thr Glu Asp Pro Trp Thr 1 5 <210> 35 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 35 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Ser Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 36 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 36 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg 100 105 110 <210> 37 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 37 Gln Val Gln Leu Glu Gln Pro Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30 Trp Met His Trp Val Lys Gln Thr Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Asp Gly Lys Ala Lys Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Val Ser Asp Leu Thr Ala Glu Asp Ser Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 38 <211> 104 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 38 Gln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Ala Thr Met Thr Cys Ser Ala Ser Ser Gly Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Asp Lys Thr Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Gly Ser Tyr Thr Phe Gly 85 90 95 Gly Gly Thr Lys Leu Glu Ile Lys 100 <210> 39 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 39 Ser Asn Trp Met His 1 5 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 40 Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn 1 5 10 <210> 41 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 41 Gly Ser Asn Pro Tyr Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 42 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 42 Ser Ala Ser Ser Gly Val Asn Tyr Met His 1 5 10 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 43 Asp Thr Asp Lys Thr Ala Ser 1 5 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 44 His Gln Arg Gly Ser Tyr Thr 1 5 <210> 45 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 45 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Ser Ser 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Arg Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Val Thr Met Ile Trp Gly Val Ile Ile Asp Phe Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 46 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 46 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 47 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 47 Ser Ser Trp Ile Gly 1 5 <210> 48 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 48 Ile Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln 1 5 10 15 Gly <210> 49 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 49 His Val Thr Met Ile Trp Gly Val Ile Ile Asp Phe 1 5 10 <210> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 50 Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala 1 5 10 <210> 51 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 51 Asp Ala Ser Ser Leu Glu Ser 1 5 <210> 52 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 52 Gln Gln Phe Asn Ser Tyr Pro Phe Thr 1 5 <210> 53 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 53 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 54 Gln Gln Phe Asn Ser Tyr Pro Tyr Thr 1 5 <210> 55 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 55 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Asp Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Gln Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ala Val Ala Ala Asp Trp Leu Asp Pro Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 56 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 56 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Arg 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 57 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 57 Ser Tyr Ala Ile Ser 1 5 <210> 58 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 58 Gly Ile Ile Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Gln Phe Gln 1 5 10 15 Gly <210> 59 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 59 Glu Ala Val Ala Ala Asp Trp Leu Asp Pro 1 5 10 <210> 60 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 60 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 61 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 61 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 62 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 62 Gln Gln Tyr Gly Ser Ser Arg Phe Thr 1 5 <210> 63 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 63 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Tyr Ser Ser Gly Trp Asp Ser Tyr Tyr Gly Met Gly 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 64 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 64 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 65 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 65 Ser Tyr Trp Ile Ala 1 5 <210> 66 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 66 Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln 1 5 10 15 Gly <210> 67 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 67 Gln Gly Tyr Ser Ser Gly Trp Asp Ser Tyr Tyr Gly Met Gly Val 1 5 10 15 <210> 68 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 68 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Ile Pro Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ser Gly Gly Ser Ala Asp Tyr Ser Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Ala Val Thr Val Ser Ser 115 120 <210> 69 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 69 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Lys Arg Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 70 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 70 Ser Tyr Thr Ile Asn 1 5 <210> 71 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 71 Gly Ile Ile Pro Ile Phe Gly Ile Pro Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 72 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 72 Ala Ser Gly Gly Ser Ala Asp Tyr Ser Tyr Gly Met Asp Val 1 5 10 <210> 73 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 73 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 74 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 74 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 75 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 75 Gln Gln Tyr Lys Arg Tyr Pro Tyr Thr 1 5 <210> 76 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 76 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Asn Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Asn Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Val Ser Met Ile Trp Gly Val Ile Met Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 77 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 77 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 78 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 78 Ser Tyr Trp Ile Gly 1 5 <210> 79 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 79 Gly Val Ser Met Ile Trp Gly Val Ile Met Asp Val 1 5 10 <210> 80 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 80 Gln Gln Phe Asn Ser Tyr Pro Trp Thr 1 5 <210> 81 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 81 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Gln Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Gly Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Tyr Ser Ser Gly Trp Arg Ser Tyr Tyr Gly Met Gly 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 82 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 82 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro His 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 83 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 83 Asn Tyr Trp Ile Ala 1 5 <210> 84 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 84 Gln Gly Tyr Ser Ser Gly Trp Arg Ser Tyr Tyr Gly Met Gly Val 1 5 10 15 <210> 85 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 85 Gln Gln Phe Asn Ser Tyr Pro His Thr 1 5 <210> 86 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 86 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Val Ser Gly Ile Tyr Asn Leu His Gly Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 87 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 87 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Arg Phe Gly Ser Pro 85 90 95 Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 88 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 88 Gly Tyr Ser Phe Thr Ser Tyr Trp 1 5 <210> 89 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 89 Ile Tyr Pro Gly Asp Ser Asp Thr 1 5 <210> 90 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 90 Ala Arg Gly Val Ser Gly Ile Tyr Asn Leu His Gly Phe Asp Ile 1 5 10 15 <210> 91 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 91 Gln Ser Ile Ser Ser Tyr 1 5 <210> 92 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 92 Ala Ala Ser 1 <210> 93 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 93 Gln Gln Gly Arg Phe Gly Ser Pro Phe Thr 1 5 10 <210> 94 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 94 Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Tyr Ser Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 95 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 95 Gln Thr Val Val Thr Gln Glu Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala Gly Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Ser 100 105 110 Arg <210> 96 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 96 Gln Thr Val Val Thr Gln Glu Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala Gly Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly Ser 100 105 110 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Leu Glu Met Ala Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 145 150 155 160 Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly 165 170 175 Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr 180 185 190 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 195 200 205 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 210 215 220 Val Tyr Tyr Cys Ala Arg Tyr Tyr Tyr Ser Arg Leu Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser 245 <210> 97 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 97 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Leu Glu Asn Ala Asn Thr Ile Tyr Asp Ala Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Tyr Gly Arg Tyr Phe Tyr Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 98 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 98 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ala 20 25 30 Arg Thr Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Ser Arg Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 99 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 99 Phe Asn Ile Lys Asp Tyr Tyr Met His 1 5 <210> 100 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 100 Trp Ile Asp Leu Glu Asn Ala Asn Thr Ile Tyr Asp Ala Lys Phe Gln 1 5 10 15 Gly <210> 101 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 101 Ala Arg Asp Ala Tyr Gly Arg Tyr Phe Tyr Asp Val 1 5 10 <210> 102 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 102 Lys Ser Ser Gln Ser Leu Leu Asn Ala Arg Thr Gly Lys Asn Tyr Leu 1 5 10 15 Ala <210> 103 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 103 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 104 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 104 Lys Gln Ser Tyr Ser Arg Arg Thr 1 5 <210> 105 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 105 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ala 20 25 30 Arg Thr Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Ser Arg Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 130 135 140 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn 145 150 155 160 Ile Lys Asp Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg 165 170 175 Leu Glu Trp Met Gly Trp Ile Asp Leu Glu Asn Ala Asn Thr Ile Tyr 180 185 190 Asp Ala Lys Phe Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala 195 200 205 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala 210 215 220 Val Tyr Tyr Cys Ala Arg Asp Ala Tyr Gly Arg Tyr Phe Tyr Asp Val 225 230 235 240 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250 <210> 106 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 106 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 107 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 107 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Lys Phe Leu Val Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 108 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 108 Gly Phe Thr Phe Asn Lys Tyr Ala Ile Asn 1 5 10 <210> 109 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 109 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Gln 1 5 10 15 Val Lys <210> 110 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 110 His Ala Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr 1 5 10 <210> 111 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 111 Ala Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn 1 5 10 <210> 112 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 112 Gly Thr Lys Phe Leu Val Pro 1 5 <210> 113 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 113 Thr Leu Trp Tyr Ser Asn Arg Trp Val 1 5 <210> 114 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 114 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Gln Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Ala Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Thr Val Val 130 135 140 Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu 145 150 155 160 Thr Cys Ala Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn 165 170 175 Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly 180 185 190 Thr Lys Phe Leu Val Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu 195 200 205 Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp 210 215 220 Glu Ala Glu Tyr Tyr Cys Thr Leu Trp Tyr Ser Asn Arg Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 115 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 115 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Leu Glu Asn Ala Asn Thr Ile Tyr Asp Ala Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Tyr Gly Arg Tyr Phe Tyr Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 116 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 116 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Ser Arg Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 117 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 117 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Leu Glu Asn Ala Asn Thr Ile Tyr Asp Ala Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Val Tyr Gly Arg Tyr Phe Tyr Asp Leu Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 118 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 118 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Glu Ser 20 25 30 Arg Thr Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Ser Arg Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 119 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 119 Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Val Glu 115 120 <210> 120 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 120 Val Asp Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser 1 5 10 15 Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser 20 25 30 Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp 35 40 45 Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 121 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 121 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 122 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 122 Ser Phe Gly Met Ser 1 5 <210> 123 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 123 Gly Phe Thr Phe Ser Ser Phe Gly Met Ser 1 5 10 <210> 124 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 124 Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu 1 5 10 <210> 125 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 125 Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 126 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 126 Gly Gly Ser Leu Ser Arg 1 5 <210> 127 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 127 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 128 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 128 Gly Phe Thr Phe Arg Ser Phe Gly Met Ser 1 5 10 <210> 129 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 129 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 130 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 130 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 131 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 131 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 132 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 132 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 133 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 133 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 134 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 134 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 135 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 135 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 136 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 136 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 137 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 137 Ala Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Gly Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Tyr Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Asn Gly Arg Gly Asp Asp Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Glu Tyr Tyr Cys 85 90 95 Thr Ile Gly Arg Ser Val Ser Arg Ser Arg Thr Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 138 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 138 Gly Phe Ser Phe Gly Ser Phe Gly Met Ser 1 5 10 <210> 139 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 139 Ser Ile Asn Gly Arg Gly Asp Asp Thr Arg 1 5 10 <210> 140 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 140 Ser Ile Asn Gly Arg Gly Asp Asp Thr Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 141 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 141 Gly Arg Ser Val Ser Arg Ser 1 5 <210> 142 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 142 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Asp Gln Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asp Ser Ser Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Pro Gly Thr Gln Val Thr Val 100 105 110 Ser Ser <210> 143 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 143 Ala Ile Ser Ala Asp Ser Ser Thr Lys Asn 1 5 10 <210> 144 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 144 Ala Ile Ser Ala Asp Ser Ser Thr Lys Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 145 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 145 Gly Arg Gly Ser Pro 1 5 <210> 146 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 146 Gln Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Gly Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asp Ser Ser Asp Lys Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ala Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser <210> 147 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 147 Gly Phe Thr Phe Gly Ser Phe Gly Met Ser 1 5 10 <210> 148 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 148 Ala Ile Ser Ala Asp Ser Ser Asp Lys Arg 1 5 10 <210> 149 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 149 Ala Ile Ser Ala Asp Ser Ser Asp Lys Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 150 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 150 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Tyr Trp Val Arg Val Ala Pro Gly Lys Gly Leu Glu Arg Ile 35 40 45 Ser Arg Asp Ile Ser Thr Gly Gly Gly Tyr Ser Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Lys Asp Arg Glu Ala Gln Val Asp Thr Leu Asp Phe Asp Tyr 100 105 110 Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 151 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 151 Asn Tyr Trp Met Tyr 1 5 <210> 152 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 152 Gly Phe Thr Phe Ser Asn Tyr Trp Met Tyr 1 5 10 <210> 153 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 153 Arg Asp Ile Ser Thr Gly Gly Gly Tyr Ser Tyr 1 5 10 <210> 154 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 154 Arg Asp Ile Ser Thr Gly Gly Gly Tyr Ser Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly <210> 155 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 155 Asp Arg Glu Ala Gln Val Asp Thr Leu Asp Phe Asp Tyr 1 5 10 <210> 156 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 156 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Gly Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Glu Arg Ile Phe Asp Leu Asn 20 25 30 Leu Met Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Arg Glu Leu Val 35 40 45 Ala Thr Cys Ile Thr Val Gly Asp Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Met Asp Tyr Thr Lys Gln Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Leu Tyr Tyr Cys 85 90 95 Lys Ile Arg Arg Thr Trp His Ser Glu Leu Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 157 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 157 Leu Asn Leu Met Gly 1 5 <210> 158 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 158 Ser Glu Arg Ile Phe Asp Leu Asn Leu Met Gly 1 5 10 <210> 159 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 159 Thr Cys Ile Thr Val Gly Asp Ser Thr Asn 1 5 10 <210> 160 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 160 Thr Cys Ile Thr Val Gly Asp Ser Thr Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 161 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 161 Arg Arg Thr Trp His Ser Glu Leu 1 5 <210> 162 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 162 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Glu Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Glu Arg Ile Trp Asp Ile Asn 20 25 30 Leu Leu Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr Val Gly Asp Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Tyr Asp Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Leu Tyr Tyr Cys Lys 85 90 95 Ile Arg Arg Thr Trp His Ser Glu Leu Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 163 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 163 Ile Asn Leu Leu Gly 1 5 <210> 164 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 164 Ser Glu Arg Ile Trp Asp Ile Asn Leu Leu Gly 1 5 10 <210> 165 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 165 Thr Ile Thr Val Gly Asp Ser Thr Ser 1 5 <210> 166 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 166 Thr Ile Thr Val Gly Asp Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 167 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 167 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 <210> 168 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 168 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 <210> 169 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 169 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Lys 100 105 110 Val Ser Ser Ala 115 <210> 170 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 170 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 171 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 171 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 <210> 172 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 172 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ala 115 <210> 173 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 173 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ala Ala 115 <210> 174 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 174 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly 115 <210> 175 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 175 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly 115 <210> 176 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 176 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly 115 <210> 177 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 177 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Val Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 178 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 178 Gly Phe Thr Phe Ser Lys Phe Gly Met Ser 1 5 10 <210> 179 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 179 Ser Ile Ser Gly Ser Gly Arg Asp Thr Leu Tyr Ala Glu Ser Val Lys 1 5 10 15 <210> 180 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 180 Gly Gly Ser Leu Ser Val 1 5 <210> 181 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 181 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 182 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 182 Gly Phe Thr Phe Ser Arg Phe Gly Met Ser 1 5 10 <210> 183 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 183 Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Glu Ser Val Lys 1 5 10 15 <210> 184 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 184 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Thr Asp Thr Leu Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 185 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 185 Ser Ile Ser Gly Ser Gly Thr Asp Thr Leu Tyr Ala Glu Ser Val Lys 1 5 10 15 <210> 186 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 186 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Leu Glu Asn Ala Asn Thr Ile Tyr Asp Ala Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Tyr Gly Gly Tyr Phe Tyr Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 187 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 187 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Ser Arg Arg Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 188 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 188 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Leu Glu Asn Gly Asn Thr Ile Tyr Asp Ala Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Tyr Gly Arg Tyr Phe Tyr Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 189 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 189 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Ser Arg Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 190 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 190 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Leu Glu Asn Ala Asn Thr Ile Tyr Asp Ala Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Val Tyr Gly Arg Tyr Leu Tyr Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 191 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 191 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Asn 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Ser Arg Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 192 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 192 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Leu Glu Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Tyr Gly Arg Tyr Phe Tyr Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 193 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 193 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Ser Arg Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 194 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 194 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Leu Glu Glu Gly Asn Thr Ile Tyr Asp Ala Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Tyr Gly Arg Tyr Phe Tyr Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 195 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 195 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Gly 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Thr Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Ser Arg Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 196 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 196 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asp Leu Glu Asn Ala Asn Thr Ile Tyr Asp Ala Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Asn Tyr Gly Gly Tyr Phe Tyr Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 197 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 197 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Pro Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro L...

Claims

1. (a) a first antigen-binding site that binds to human CD19 with a dissociation constant (KD) of about 10 pM to about 1 μM; (b) a second antigen-binding site that binds to human CD3; and (c) A third antigen-binding site that binds to human serum albumin (HSA).

2. A multispecific binding protein comprising:

2. The multispecific binding protein of claim 1, wherein the first antigen binding site has a melting temperature of 50°C to 80°C.

3. (a) a first antigen-binding site that binds to human CD19; (b) a second antigen-binding site that binds to human CD3, CD3ε, CD3δ, or CD3γ, and that binds to human CD3 with a dissociation constant (KD) of about 0.1 nM to about 1 μM; and (c) A third antigen-binding site that binds to human serum albumin (HSA).

2. A multispecific binding protein comprising:

4. The multispecific binding protein of claim 3, wherein the second antigen binding site has a melting temperature of 50°C to 80°C.

5. (a) a first antigen-binding site that binds to human CD19; (b) a second antigen-binding site that binds to human CD3; and (c) a third antigen-binding site that binds to human serum albumin (HSA) with a dissociation constant (KD) of about 0.1 nM to about 100 μM; 2. A multispecific binding protein comprising:

6. The multispecific binding protein of claim 5, wherein the third antigen binding site has a melting temperature of 50°C to 80°C.

7. The multispecific binding protein of claim 1, wherein the molecular weight of the multispecific binding protein is in the range of 40 to 100 kD.

8. A pharmaceutical composition comprising the multispecific binding protein of any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, which mediates a cytotoxic activity level (EC 50 value) of ≦5000 pM to ≦1 pM.

10. The pharmaceutical composition of claim 8, wherein the multispecific binding protein does not induce or mediate lysis of more than 30% of cells that do not express CD19.

11. The pharmaceutical composition of claim 8, wherein the serum concentration of the multispecific binding protein 50 hours after intravenous administration to a subject is at least 10% of the serum concentration of the multispecific binding protein 1 hour after administration to the subject.

Images