Anti-CD19 antibodies and multispecific binding proteins

Abstract

Provided herein are anti-CD19 antibodies and multispecific binding proteins that bind to CD19, CD3, and serum albumin. Also provided are pharmaceutical compositions comprising these antibodies or multispecific binding proteins, expression vectors and host cells for producing these antibodies or multispecific binding proteins, and methods of using these antibodies or multispecific binding proteins in the treatment of hematological cancers. TIFF2023506017000065.tif76153

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 / 946,931, filed December 11, 2019, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] FIELD OF THE INVENTION The present invention relates to anti-CD19 antibodies and multispecific binding proteins that bind to CD19, CD3, and optionally serum albumin. The invention also relates to pharmaceutical compositions comprising these antibodies or multispecific binding proteins, expression vectors and host cells for producing these antibodies or multispecific binding proteins, and methods of using these antibodies or 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 from two operably linked antibody-derived binding domains. One binding domain of the BiTE® construct is specific for a selected tumor-associated surface antigen on the target cell, and the other binding domain is specific for CD3, a subunit of the T-cell receptor complex on the 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 a single 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 engagement. Approximately 95% of T cells express the αβ TCR, which contains one α (alpha) chain and one β (beta) chain. Two TCRξ (zeta) chains are also present in the TCR-CD3 complex. The αβ TCR is responsible for recognizing antigens presented by the major histocompatibility complex (MHC). Upon engagement of the TCR with an antigenic peptide and 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] 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 (Non-Patent Document 1); van Zelm et al. (2006) N. Eng. J. Med., 354: 1901-12 (Non-Patent Document 2)). Together with the B cell receptor (BCR), CD19 regulates the intrinsic and antigen receptor-induced signaling thresholds that are important for B cell clonal proliferation and humoral immunity. CD19 is a human B cell surface marker expressed from early stages of pre-B cell development until terminal differentiation into 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 lymphoid origin, such as B-cell malignancies (see, for example, Hekman et al. (1991) Cancer Immunol. Immunother., 32: 364-72 (Non-Patent Document 3); Vlasfeld et al. (1995) Cancer Immunol. Immunother., 40: 37-47 (Non-Patent Document 4); Corny et al. (1995) J. Immunother. Emphasis Tumor Immunol., 18: 231-41 (Non-Patent Document 5); and Manzke et al. (2001) Int. J. Cancer, 91: 516-22 (Non-Patent Document 6)). Additionally, a BiTE® construct called blinatumomab has been developed for clinical use.

[0006] BiTE® constructs are believed to have the disadvantage of rapid clearance from the body. Thus, although BiTE® constructs can rapidly penetrate many areas of the body, 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, achieving the therapeutic effects of blinatumomab and solitomab may require long-term administration by continuous intravenous infusion. However, such continuous intravenous infusion can be inconvenient for patients and increase the cost of treatment.

[0007] Although significant developments have been made in the construction of anti-CD19 antibodies and multispecific binding proteins, there remains a need for new and useful proteins for treating cancer that have sufficient therapeutic efficacy, formats that are easy to manufacture, and favorable pharmacokinetic properties, such as longer half-lives. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Carter et al. (2002) Science, 256: 105-07 [Non-patent document 2] van Zelm et al. (2006) N. Eng. J. Med., 354: 1901-12 [Non-patent document 3] Hekman et al. (1991) Cancer Immunol. Immunother., 32: 364-72 [Non-patent document 4] Vlasfeld et al. (1995) Cancer Immunol. Immunother., 40: 37-47 [Non-Patent Document 5] Corny et al. (1995) J. Immunother. Emphasis Tumor Immunol., 18: 231-41 [Non-patent document 6] Manzke et al. (2001) Int. J. Cancer, 91: 516-22 Summary of the Invention

[0009] The present invention is based, in part, on the development of new antibodies that bind to CD19. Also provided are multispecific binding proteins comprising a first domain that binds to CD19 (e.g., human CD19), a second domain that binds to CD3 (e.g., human CD3), and optionally a half-life-extending domain, which can be a third domain that binds to serum albumin (e.g., human serum albumin), where the first domain is derived from these new antibodies. These domains are linked in a specific manner for favorable therapeutic efficacy and in vivo half-life. Anti-CD19 antibodies and multispecific binding proteins are useful for treating diseases and disorders associated with abnormal cells that express CD19, such as certain B-cell hematological malignancies.

[0010] Thus, in one aspect, the present disclosure provides a method for treating a pulmonary artery disease (PA) comprising administering to a subject a therapeutically effective amount of a pulmonary artery disease (PA) having a K in the range of 5 pM to 1 nM. D (b) a first antigen-binding site that binds to human CD19 with a K in the range of 0.5 nM to 20 nM; and D The present invention provides a multispecific binding protein comprising a second antigen-binding site that binds to human CD3 at a specific site.

[0011] In certain embodiments, the multispecific binding protein is a fusion protein comprising a first antigen-binding site and a second antigen-binding site. In certain embodiments, the multispecific binding protein further comprises a half-life prolonging domain. In certain embodiments, the half-life prolonging domain comprises a third antigen-binding site that binds to human serum albumin. In certain embodiments, the half-life prolonging domain is not located between the first antigen-binding site and the second antigen-binding site in the polypeptide chain.

[0012] In certain embodiments, each K Dis measured by surface plasmon resonance. In certain embodiments, the first antigen-binding site has a K in the range of 5 pM to 0.1 nM. D and the second antigen-binding site binds to human CD19 with a K in the range of 0.5 nM to 10 nM. D In certain embodiments, the second antigen-binding site binds to human CD3 at K D vs. K, whose first antigen-binding site binds to human CD19 D The ratio is in the range of 10:1 to 1,000:1.

[0013] In certain embodiments, each K D is measured by biolayer interferometry. In certain embodiments, the first antigen-binding site has a K in the range of 50 pM to 1 nM. D and the second antigen-binding site binds to human CD19 with a K in the range of 1 nM to 20 nM. D In certain embodiments, the second antigen-binding site binds to human CD3 at K D vs. K, whose first antigen-binding site binds to human CD19 D The ratio is in the range of 10:1 to 100:1.

[0014] In another aspect, the present disclosure provides an antigen-binding site that binds to human CD19, wherein the antibody 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 110, 111, 113, 114, 115, and 71, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 93, 87, 68, 80, 70, and 71, respectively.

[0015] In certain embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 110, 117, 119, 114, 120, and 71, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 93, 87, 68, 80, 70, and 71, respectively. In certain embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 65, 66, 68, 69, 70, and 71, respectively. In certain embodiments, the VH comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:62, and the VL comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:63. In certain embodiments, the antigen-binding site comprises the amino acid sequence of SEQ ID NO:72. In certain embodiments, the antigen-binding site has a K D It binds to human CD19.

[0016] In another aspect, the present disclosure provides an antigen-binding site that binds to human CD19, wherein the antibody comprises a VH comprising complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a VL comprising complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs:53, 54, 56, 57, 58, and 59, respectively, but do not comprise the amino acid sequences of SEQ ID NOs:4, 23, 17, 18, 19, and 10, respectively. In certain embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 53, 54, 56, 57, 60, and 59, respectively, but do not include the amino acid sequences of SEQ ID NOs: 4, 23, 17, 18, 19, and 10, respectively. In certain embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 53, 61, 56, 57, 60, and 59, respectively, but do not include the amino acid sequences of SEQ ID NOs: 4, 23, 17, 18, 19, and 10, respectively. In certain embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, 7, 8, 9, and 10, respectively. In certain embodiments, the VH comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:1, and the VL comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:2. In certain embodiments, the antigen-binding site comprises the amino acid sequence of SEQ ID NO:11. In certain embodiments, the antigen-binding site has a K D It binds to human CD19.

[0017] In another aspect, the present disclosure provides an antibody comprising an antigen-binding site that binds to CD19 as disclosed herein.

[0018] In another aspect, the present disclosure provides a multispecific binding protein comprising: (a) a first antigen-binding site that binds to human CD19 as disclosed herein; and (b) a second antigen-binding site that binds to human CD3.

[0019] In certain embodiments, the multispecific binding protein further comprises a half-life prolonging domain, hi certain embodiments, the half-life prolonging domain comprises a third antigen-binding site that binds to human serum albumin.

[0020] In certain embodiments, the third antigen-binding site comprises a VH comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 184, 409, and 411, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 129, 133, and 135, respectively.

[0021] In certain embodiments, HCDR1, HCDR2, and HCDR3 of the third antigen-binding site comprise the amino acid sequences of SEQ ID NOs: 184, 185, and 187, respectively, but do not include the amino acid sequences of SEQ ID NOs: 129, 133, and 135, respectively. In certain embodiments, HCDR1, HCDR2, and HCDR3 of the third antigen-binding site comprise the amino acid sequences of SEQ ID NOs: 189, 190, and 192, respectively, but do not include the amino acid sequences of SEQ ID NOs: 129, 133, and 135, respectively. In certain embodiments, HCDR1, HCDR2, and HCDR3 of the third antigen-binding site comprise the amino acid sequences of SEQ ID NOs: 189, 193, and 195, respectively, but do not include the amino acid sequences of SEQ ID NOs: 129, 133, and 135, respectively. In certain embodiments, HCDR1, HCDR2, and HCDR3 of the third antigen-binding site comprise the amino acid sequences of SEQ ID NOs: 123, 124, and 126, respectively. In certain embodiments, the VH of the third antigen-binding site comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 121. In certain embodiments, the third antigen-binding site has a K D In certain embodiments, the third antigen-binding site binds to human serum albumin with a K of less than or equal to 2 nM. D In certain embodiments, the third antigen-binding site has a melting temperature greater than or equal to 60°C.

[0022] In certain embodiments, the multispecific binding protein comprises a single polypeptide chain, in which the third antigen-binding site is not located between the first and second antigen-binding sites.

[0023] In certain embodiments, the polypeptide chain has a third antigen-binding site located N-terminal to both the first and second antigen-binding sites. In certain embodiments, the polypeptide chain has a third antigen-binding site located N-terminal to the first antigen-binding site, and the first antigen-binding site located N-terminal to the second antigen-binding site. In certain embodiments, the polypeptide chain has a third antigen-binding site located N-terminal to the second antigen-binding site, and the second antigen-binding site located N-terminal to the first antigen-binding site.

[0024] In certain embodiments, the polypeptide chain has a third antigen-binding site located C-terminal to both the first and second antigen-binding sites. In certain embodiments, the polypeptide chain has a first antigen-binding site located N-terminal to the second antigen-binding site, and a second antigen-binding site located N-terminal to the third antigen-binding site. In certain embodiments, the polypeptide chain has a second antigen-binding site located N-terminal to the first antigen-binding site, and a first antigen-binding site located N-terminal to the third antigen-binding site.

[0025] In certain embodiments, the polypeptide chains have a first antigen-binding site located N-terminal to the third antigen-binding site, and the third antigen-binding site located N-terminal to the second antigen-binding site, while in other embodiments, the polypeptide chains have a second antigen-binding site located N-terminal to the third antigen-binding site, and the third antigen-binding site located N-terminal to the binding protein first antigen-binding site.

[0026] In certain embodiments, the first antigen-binding site comprises a single-chain variable fragment (scFv). In certain embodiments, the third antigen-binding site comprises a single-domain antibody (sdAb). In certain embodiments, the second antigen-binding site comprises an scFv.

[0027] In certain embodiments, the second antigen-binding site binds to human CD3ε. In certain embodiments, the second antigen-binding site has a K in the range of 1 to 100 nM. D It binds to human CD3ε.

[0028] 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 415, 416, 418, 419, 420, and 421, respectively. In certain embodiments, the VH comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 412, and the VL comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 413. In certain embodiments, the antigen-binding site comprises the amino acid sequence of SEQ ID NO: 422 or 423.

[0029] 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 415, 416, 426, 419, 420, and 421, respectively. In certain embodiments, the VH comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 424, and the VL comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 413. In certain embodiments, the antigen-binding site comprises the amino acid sequence of SEQ ID NO: 427 or 428.

[0030] 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 415, 431, 418, 419, 420, and 432, respectively. In certain embodiments, the VH comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 429, and the VL comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 430. In certain embodiments, the antigen-binding site comprises the amino acid sequence of SEQ ID NO: 433 or 434.

[0031] In certain embodiments, at least two adjacent antigen-binding sites are connected by a peptide linker. In certain embodiments, 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.

[0032] In certain embodiments, the multispecific binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:694-710.

[0033] In certain embodiments, the multispecific binding protein does not comprise an antibody Fc region. In certain embodiments, the molecular weight of the multispecific binding protein is at least 65 kD. In certain embodiments, the serum half-life of the multispecific binding protein is at least 24, 36, 48, or 60 hours.

[0034] In another aspect, the present disclosure provides a pharmaceutical composition comprising: (a) a multispecific binding protein or antibody disclosed herein; and (b) a pharmaceutically acceptable carrier.

[0035] The present disclosure also provides isolated polynucleotides encoding the multispecific binding proteins or antibodies disclosed herein. In addition, the present disclosure provides vectors comprising the polynucleotides disclosed herein, and recombinant host cells comprising the polynucleotides or vectors disclosed herein.

[0036] The present disclosure also provides methods of producing a multispecific binding protein or antibody, comprising culturing a host cell disclosed herein under suitable conditions that allow for expression of the multispecific binding protein or antibody. In certain aspects, the method further comprises isolating the multispecific binding protein or antibody. In certain aspects, the method further comprises formulating the isolated multispecific binding protein or antibody with a pharmaceutically acceptable carrier.

[0037] Additionally, the present disclosure provides a method of stimulating an immune response against cells expressing CD19, comprising exposing the cells and T lymphocytes to a multispecific binding protein, antibody, or pharmaceutical composition disclosed herein.

[0038] The present disclosure also 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, antibody, or pharmaceutical composition disclosed herein. In certain embodiments, the hematological cancer is a B-cell hematological malignancy.

[0039] Additionally, 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 both the T cell and the B cell. In certain embodiments, the complex further comprises serum albumin. [The present invention 1001] (a) K in the range of 5 pM to 1 nM D a first antigen-binding site that binds to human CD19 at (b) K in the range of 0.5 nM to 20 nM D A second antigen-binding site that binds to human CD3 in 1. A multispecific binding protein comprising: [The present invention 1002] 1001. A multispecific binding protein of the present invention, which is a fusion protein comprising a first antigen-binding site and a second antigen-binding site. [The present invention 1003] The multispecific binding protein of the present invention 1001 or 1002, further comprising a half-life prolonging domain. [The present invention 1004] 1003. The multispecific binding protein of the present invention, wherein said half-life prolonging domain comprises a third antigen binding site that binds to human serum albumin. [The present invention 1005] The multispecific binding protein of any of claims 1002 to 1004, wherein the half-life prolonging domain is not located between the first antigen-binding site and the second antigen-binding site in the polypeptide chain. [The present invention 1006] Each K D The multispecific binding protein of any of 1001 to 1005, wherein the binding activity is measured by surface plasmon resonance. [The present invention 1007] The first antigen-binding site has a K in the range of 5 pM to 0.1 nM. D and the second antigen-binding site binds to human CD19 with a K in the range of 0.5 nM to 10 nM. D 1006. A multispecific binding protein of the present invention that binds to human CD3 at [The present invention 1008] The second antigen-binding site binds to human CD3. D vs. K, whose first antigen-binding site binds to human CD19 D The multispecific binding protein of the present invention 1006 or 1007, wherein the ratio of [The present invention 1009] Each K D The multispecific binding protein of any one of 1001 to 1005, wherein the binding activity is measured by biolayer interferometry. [The present invention 1010] The first antigen-binding site has a K in the range of 50 pM to 1 nM. D and the second antigen-binding site binds to human CD19 with a K in the range of 1 nM to 20 nM. D 1009. A multispecific binding protein of the present invention that binds to human CD3 at [The present invention 1011] The second antigen-binding site binds to human CD3. D vs. K, whose first antigen-binding site binds to human CD19 D The multispecific binding protein of the present invention 1009 or 1010, wherein the ratio of [The present invention 1012] An antigen-binding site that binds to human CD19, wherein the antibody 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 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 110, 111, 113, 114, 115, and 71, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 93, 87, 68, 80, 70, and 71, respectively. [The present invention 1013] 1012. An antigen-binding site of the present invention, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 110, 117, 119, 114, 120, and 71, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 93, 87, 68, 80, 70, and 71, respectively. [The present invention 1014] 1013. An antigen-binding site of the present invention, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 65, 66, 68, 69, 70, and 71, respectively. [The present invention 1015] An antigen-binding site according to any one of claims 1012 to 1014, wherein VH comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:62, and VL comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:63. [The present invention 1016] An antigen-binding site according to any one of claims 1012 to 1015 of the present invention, comprising the amino acid sequence of SEQ ID NO:72. [The present invention 1017] K less than or equal to 1 nM D Any of the antigen-binding sites of 1012 to 1016 of the present invention, which bind to human CD19 at the [The present invention 1018] An antigen-binding site that binds to human CD19, wherein the antibody comprises a VH having complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a VL having complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 53, 54, 56, 57, 58, and 59, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 4, 23, 17, 18, 19, and 10, respectively. [The present invention 1019] An antigen-binding site of the present invention 1018, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 53, 54, 56, 57, 60, and 59, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 4, 23, 17, 18, 19, and 10, respectively. [The present invention 1020] An antigen-binding site of the present invention 1019, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 53, 61, 56, 57, 60, and 59, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 4, 23, 17, 18, 19, and 10, respectively. [The present invention 1021] 10. The antigen-binding site of the present invention 1020, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 4, 5, 7, 8, 9, and 10, respectively. [The present invention 1022] An antigen-binding site of any of claims 1018 to 1021, wherein VH comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:1, and VL comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:2. [The present invention 1023] The antigen-binding site of the present invention 1022, comprising the amino acid sequence of SEQ ID NO:11. [The present invention 1024] K less than or equal to 0.3 nM D The antigen-binding site of any one of 1018 to 1023 of the present invention, which binds to human CD19. [The present invention 1025] An antibody comprising an antigen-binding site of any one of 1012 to 1024 of the present invention. [The present invention 1026] (a) a first antigen-binding site that binds to human CD19, which is any one of the antigen-binding sites of the present invention 1012 to 1024; and (b) A second antigen-binding site that binds to human CD3 1. A multispecific binding protein comprising: [The present invention 1027] 1026. A multispecific binding protein of the invention further comprising a half-life prolonging domain. [The present invention 1028] 1027. The multispecific binding protein of the present invention, wherein said half-life prolonging domain comprises a third antigen-binding site that binds to human serum albumin. [The present invention 1029] 1028. A multispecific binding protein of the present invention, wherein the third antigen-binding site comprises a VH comprising complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 184, 409, and 411, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 129, 133, and 135, respectively. [The present invention 1030] 1029. The multispecific binding protein of the present invention, wherein HCDR1, HCDR2, and HCDR3 of the third antigen-binding site comprise the amino acid sequences of SEQ ID NOs: 184, 185, and 187, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 129, 133, and 135, respectively. [The present invention 1031] 1029. The multispecific binding protein of the present invention, wherein HCDR1, HCDR2, and HCDR3 of the third antigen-binding site comprise the amino acid sequences of SEQ ID NOs: 189, 190, and 192, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 129, 133, and 135, respectively. [The present invention 1032] 1032. The multispecific binding protein of any of claims 1029 to 1031, wherein HCDR1, HCDR2, and HCDR3 of the third antigen-binding site comprise the amino acid sequences of SEQ ID NOs: 189, 193, and 195, respectively, but do not comprise the amino acid sequences of SEQ ID NOs: 129, 133, and 135, respectively. [The present invention 1033] 1032. The multispecific binding protein of the present invention, wherein HCDR1, HCDR2, and HCDR3 of the third antigen-binding site comprise the amino acid sequences of SEQ ID NOs: 123, 124, and 126, respectively. [The present invention 1034] 1034. The multispecific binding protein of any of claims 1029 to 1033, wherein the VH of the third antigen-binding site comprises an amino acid sequence at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:121. [This invention 1035] 1035. The multispecific binding protein of any of claims 1029 to 1034, wherein the VH of the third antigen-binding site comprises the amino acid sequence of SEQ ID NO:121. [The present invention 1036] The third antigen-binding site has a K less than or equal to 10 nM. D 1036. The multispecific binding protein of any one of claims 1029 to 1035, which binds to human serum albumin at [This invention 1037] The third antigen-binding site has a K of less than or equal to 2 nM. D 1036. The multispecific binding protein of any one of claims 1029 to 1036, which binds to Protein A at [The present invention 1038] 1038. The multispecific binding protein of any of claims 1029 to 1037, wherein the third antigen-binding site has a melting temperature greater than or equal to 60°C. [This invention 1039] A multispecific binding protein of any of claims 1001 to 1011 and 1026 to 1038, comprising a single polypeptide chain. [The present invention 1040] 1039. The multispecific binding protein of the present invention, wherein in said polypeptide chains, the third antigen-binding site is not located between the first and second antigen-binding sites. [This invention 1041] The multispecific binding protein of the present invention 1040, wherein in the 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 1042] 1041. The multispecific binding protein of the present invention, 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. [This invention 1043] 1041. The multispecific binding protein of the present invention, wherein, in the polypeptide chain, the 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. [This invention 1044] The multispecific binding protein of the present invention 1040, wherein in the polypeptide chain, the third antigen-binding site is located C-terminal to both the first antigen-binding site and the second antigen-binding site. [This invention 1045] The multispecific binding protein of the present invention 1044, wherein, in the polypeptide chains, the first 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 third antigen-binding site. [The present invention 1046] The multispecific binding protein of the present invention 1044, 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. [This invention 1047] 1039. The multispecific binding protein of the present invention, wherein, in the polypeptide chains, the first 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 second antigen-binding site. [This invention 1048] 1039. The multispecific binding protein of the present invention, wherein, in the 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 first antigen-binding site of the binding protein. [This invention 1049] The multispecific binding protein of any of claims 1001 to 1011 and 1026 to 1048, wherein the first antigen-binding site comprises a single-chain variable fragment (scFv). [The present invention 1050] 1028-1049. The multispecific binding protein of any of claims 1004-1011 and 1028-1049, wherein the third antigen-binding site comprises a single domain antibody (sdAb). [This invention 1051] The multispecific binding protein of any of claims 1001 to 1011 and 1026 to 1050, wherein the second antigen-binding site comprises an scFv. [This invention 1052] The multispecific binding protein of any of claims 1001 to 1011 and 1026 to 1051, wherein the second antigen-binding site binds to human CD3ε. [This invention 1053] The second antigen-binding site has a K in the range of 1 to 100 nM. D 1052. A multispecific binding protein of the present invention that binds to human CD3ε at [This invention 1054] The multispecific binding protein of any of claims 1001 to 1011 and 1026 to 1053, wherein 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, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 415, 416, 418, 419, 420, and 421, respectively. [This invention 1055] 1054. A multispecific binding protein of the present invention, wherein VH comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:412, and VL comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:413. [The present invention 1056] The multispecific binding protein of the invention 1054 or 1055, wherein the antigen-binding site comprises the amino acid sequence of SEQ ID NO: 422 or 423. [This invention 1057] The multispecific binding protein of any of claims 1001 to 1011 and 1026 to 1053, wherein 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, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 415, 416, 426, 419, 420, and 421, respectively. [This invention 1058] 1057. A multispecific binding protein of the invention, wherein VH comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:424, and VL comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:413. [This invention 1059] The multispecific binding protein of the invention 1057 or 1058, wherein the antigen-binding site comprises the amino acid sequence of SEQ ID NO: 427 or 428. [The present invention 1060] The multispecific binding protein of any of claims 1001 to 1011 and 1026 to 1053, wherein 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, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 415, 431, 418, 419, 420, and 432, respectively. [This invention 1061] 1060. A multispecific binding protein of the present invention, wherein VH comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:429, and VL comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:430. [This invention 1062] 1060 or 1061, a multispecific binding protein of the invention, wherein the antigen-binding site comprises the amino acid sequence of SEQ ID NO: 433 or 434. [This invention 1063] The multispecific binding protein of any of claims 1001 to 1011 and 1026 to 1062, wherein at least two adjacent antigen-binding sites are connected by a peptide linker. [This invention 1064] 1063. A multispecific binding protein of the invention, wherein each of the adjacent antigen-binding sites is connected by a peptide linker. [This invention 1065] The multispecific binding protein of the present invention 1063 or 1064, wherein said peptide linker comprises the amino acid sequence of SEQ ID NO: 298, 299, or 302. [The present invention 1066] The multispecific binding protein of the present invention 1063 or 1064, wherein the peptide linker consists of the amino acid sequence of SEQ ID NO: 298, 299, or 302. [This invention 1067] A multispecific binding protein of any one of claims 1001 to 1011 and 1026 to 1066, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 694 to 710. [The present invention 1068] The multispecific binding protein of any one of 1001 to 1011 and 1026 to 1067, which does not comprise an antibody Fc region. [The present invention 1069] The multispecific binding protein of any one of claims 1001 to 1011 and 1026 to 1068, wherein the molecular weight of the multispecific binding protein is at least 65 kD. [The present invention 1070] The multispecific binding protein of any of claims 1001 to 1011 and 1026 to 1069, wherein the serum half-life of the multispecific binding protein is at least 24, 36, 48, or 60 hours. [This invention 1071] (a) any one of the multispecific binding proteins 1001 to 1011 and 1026 to 1070 of the present invention or the antibody 1025 of the present invention; and (b) a pharmaceutically acceptable carrier 10. A pharmaceutical composition comprising: [This invention 1072] An isolated polynucleotide encoding any one of the multispecific binding proteins of the present invention 1001 to 1011 and 1026 to 1070 or the antibody of the present invention 1025. [This invention 1073] A vector comprising the polynucleotide of the present invention. [This invention 1074] A recombinant host cell comprising a polynucleotide of the present invention 1072 or a vector of the present invention 1073. [This invention 1075] A method for producing a multispecific binding protein or antibody, comprising culturing a host cell of the invention 1074 under suitable conditions that allow expression of the multispecific binding protein or antibody. [This invention 1076] The method of claim 1075, further comprising the step of isolating the multispecific binding protein or antibody. [This invention 1077] The method of claim 1076, further comprising the step of formulating the isolated multispecific binding protein or antibody with a pharmaceutically acceptable carrier. [This invention 1078] A method for stimulating an immune response against cells expressing CD19, comprising the step of exposing said cells and T lymphocytes to any of the multispecific binding proteins of the present inventions 1001 to 1011 and 1026 to 1070, the antibody of the present invention 1025, or the pharmaceutical composition of the present invention 1071. [This invention 1079] A method of treating hematological cancer in a subject in need thereof, comprising the step of administering to said subject an effective amount of a multispecific binding protein of any of 1001 to 1011 and 1026 to 1070 of the present invention, an antibody of 1025 of the present invention, or a pharmaceutical composition of 1071 of the present invention. [The present invention 1080] 1079. The method of claim 1079, wherein said hematological cancer is a B-cell hematological malignancy. [This invention 1081] A complex comprising a T cell expressing CD3, a B cell expressing CD19, and any one of the multispecific binding proteins of the present inventions 1001 to 1011 and 1026 to 1070, wherein the multispecific binding protein simultaneously binds to both the T cell and the B cell. [This invention 1082] A conjugate of the present invention 1081, further comprising serum albumin. [Brief explanation of the drawings]

[0040] [Figure 1] Schematic diagram of the six-domain arrangement of single-chain multispecific binding proteins. 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 given polypeptide chain, and the bottom of each construct represents the C-terminus. [Figure 2] 2A-2B are bar graphs depicting EC50 values ​​of the indicated multispecific binding proteins for inducing T cell cytotoxicity against KILR-Raji cells. [Figure 3] Figure 3A is a graph showing the EC50 values ​​of the indicated multispecific binding proteins to induce production of the cytokines IL2, IFNγ, and TNFα compared to the EC50 values ​​of the same multispecific binding proteins to induce T cell cytotoxicity ("% survival") against Raji cells when T cells and Raji cells are cultured at a ratio of 5:1. Figure 3B is a bar graph showing the percentage T cell cytotoxicity mediated by the multispecific binding proteins tAb0050, tAb0063, and the control blinatumomab when T cells and Raji cells are cultured at a ratio of 0.5:1. [Figure 4-1] 4A-4D illustrate the results of in vivo experiments to assess tumor regression following treatment with a multispecific binding protein. In one experiment, mice received high-dose treatment (0.1 mg / kg / dose) with the test multispecific binding protein, and tumor volume (FIG. 4A) and percent survival (FIG. 4B) were assessed. In another experiment, mice received low-dose treatment (0.01 mg / kg / dose) with the test multispecific binding protein, and tumor volume (FIG. 4C) and percent survival (FIG. 4D) were assessed. [Figure 4-2] See description of Figure 4-1. [Figure 5A]Figures 5A-5D illustrate the results of an in vivo dose-efficacy study in mice treated with the multispecific binding protein tAb0050 or blinatumomab. Tumor growth data is shown in Figure 5A. Percent survival for each treatment is shown for 0.01 mg / kg / dose (Figure 5B), 0.03 mg / kg / dose (Figure 5C), and 0.1 mg / kg / dose (Figure 5D). [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description The present invention is based, in part, on the development of new antibodies that bind to CD19. Also provided are multispecific binding proteins comprising a first domain that binds to CD19 (e.g., human CD19), a second domain that binds to CD3 (e.g., human CD3), and optionally a half-life-extending domain, which can be a third domain that binds to serum albumin (e.g., human serum albumin), where the first domain is derived from these new antibodies. These domains are linked in a specific manner for favorable therapeutic efficacy and in vivo half-life. Anti-CD19 antibodies and multispecific binding proteins are useful for treating diseases and disorders associated with abnormal cells that express CD19, such as certain B-cell hematological malignancies.

[0042] To facilitate the understanding of this invention, a number of terms and phrases are defined below.

[0043] The term "multispecific binding protein" refers to a protein or protein conjugate capable of binding to two or more different targets (e.g., two or more different antigens or two or more different epitopes of the same antigen). For example, a multispecific binding protein can bind to two or more different targets through two or more different binding domains. The structure and / or function of a multispecific binding protein can be based on the structure and / or function of an antibody, e.g., a full-length or entire immunoglobulin molecule, the heavy chain variable domain (VH) and / or the light chain variable domain (VL) of an antibody, and / or a single-chain antibody. In one example, each one of the binding domains of a multispecific binding protein in accordance with the invention comprises the minimum structural requirements of an antibody, enabling target binding. This minimum requirement can 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 defining the minimum structural requirements of an antibody is to define the epitope of a specific target to which the antibody binds, or to refer to known antibodies that compete with the antibody for binding to the same epitope as the known antibody. Antibodies that can serve as the basis for constructs according to the present invention include, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies.

[0044] Any one of the binding domains of the multispecific binding proteins according to the invention may comprise the above-mentioned group of CDRs. These CDRs may be contained within a VH and / or VL framework. Fd fragments, for example, have two VH domains and often retain some antigen-binding function of the intact antigen-binding domain. Further examples of antibody fragment, antibody variant, or binding domain formats include: (1) Fab fragments, which are monovalent fragments containing VL, VH, CL, and CH1 domains; (2) F(ab')2 fragments, which are bivalent fragments in which two Fab fragments are linked by a disulfide bridge at the hinge region; (3) Fd fragments, which contain two VH and CH1 domains; (4) Fv fragments, which contain the VL and VH domains of a single antibody arm; and (5) dAb fragments (Ward et al., (1989) Nature 341:544-546). dAb fragments include VH domains; (6) isolated complementarity determining regions (CDRs); and (7) single-chain Fvs (scFvs). Single-chain Fvs (scFvs) can be derived, for example, from scFv libraries. Exemplary formats of multispecific binding proteins according to the present invention are described, for example, in WO2000006605A2, WO2005040220A1, WO2008119567A2, WO2010037838A2, WO2013026837A1, WO2013026833A1, US20140308285A1, US20140302037A1, WO2014144722A2, WO2014151910A1, and WO2015048272A1.

[0045] Multispecific binding proteins according to the invention may also include antibody variants such as di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, 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.

[0046] As used herein, the terms "single-chain Fv," "single-chain antibody," and "scFv" refer to a single polypeptide chain antibody fragment that contains variable regions derived from the heavy and light chains but lacks a constant region. Typically, single-chain antibodies further contain a peptide linker connecting the VH and VL domains, enabling them to form the desired structure and bind to an 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 for 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.

[0047] Furthermore, the "multispecific binding proteins" described herein can be monovalent, bivalent, or polyvalent / multivalent constructs. Furthermore, the "multispecific binding proteins" described herein can include molecules consisting of only one polypeptide chain or molecules consisting of multiple polypeptide chains, which can 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.

[0048] The domains of the multispecific binding proteins 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 connects two domains. Peptide linkers can also be used to fuse a third domain to 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 U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO198809344A1.

[0049] The multispecific binding proteins of the present invention may be multispecific binding proteins generated in vitro. The term "in vitro generated multispecific binding protein" refers to a multispecific binding protein according to the above definition in which all or a portion of the variable region (e.g., at least one CDR) has been generated by non-immune cell selection, such as in vitro phage display, protein chips, or any other method that can test candidate sequences for their ability to bind to an antigen. The multispecific binding proteins of the present invention may also be generated by genome rearrangement in immune cells in animals. A "recombinant antibody" is an antibody produced by the use of recombinant DNA technology or genetic engineering.

[0050] The multispecific binding proteins of the invention may be monoclonal. As used herein, the term "monoclonal" means that the proteins obtained from a 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, which typically include different antibodies directed against different determinants (or epitopes), monoclonal antibodies are highly specific, being directed against a single antigenic side or determinant on an 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 any particular method.

[0051] The multispecific binding proteins 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 antigens during an immune response. Repeated exposure to the same antigen causes the host to produce antibodies with successively higher 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 display methods such as phage display, antibody fragments with affinities in the low nanomolar range can be obtained.

[0052] Amino acid substitution changes can be introduced into multispecific binding proteins (e.g., humanized or human antibodies) by substituting one or more hypervariable region residues of a parent antibody. Generally, 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 generating 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 to the M13 gene III product packaged 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 additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the binding domains. These contact residues and adjacent residues are candidates for substitution according to the method described in detail herein.After making such variants, the variant panel is subjected to screening as described herein.The antibody that has excellent properties in one or more relevant assays may be selected for further development.

[0053] The multispecific binding proteins of the present invention may specifically comprise "chimeric" antibodies (immunoglobulins) or fragments thereof in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, so long as the desired biological activity is exhibited (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 containing 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 producing chimeric antibodies have been described. See, for example, Morrison et al. (1985) Proc. Natl. Acad. Sci. USA, 81:6851; Takeda et al. (1985) Nature, 314:452; U.S. Patent No. 4,816,567; U.S. Patent No. 4,816,397; European Patent No. EP 0171496; European Patent Application Publication No. EP 0173494; and British Patent No. GB ​​2177096.

[0054] The terms "binding domain" or "domain that binds to (an antigen)" in the context of the present invention characterize a domain that (specifically) binds to or (specifically) interacts with a certain target epitope or a certain target side on a target molecule (antigen), such as CD19, serum albumin, and CD3. The structure and function of the first binding domain, second binding domain, and / or third binding domain may be based on the structure and / or function of an antibody, e.g., a full-length or entire immunoglobulin molecule. The binding domain can be obtained 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 domain).

[0055] 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 evenly distributed throughout the variable domain of an antibody; rather, the variability 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 the "framework" regions (FRMs or FRs), which provide a scaffold for the six CDRs to form the antigen-binding surface in three-dimensional space.

[0056] 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 possess 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, such as a human or rodent, into monomers, thus obtaining a VH or VL as a single-domain antibody. Currently, most research on single-domain antibodies is based on heavy chain variable domains, but nanobodies derived from light chains have also been shown to specifically bind to target epitopes. Examples of single-domain antibodies include nanobodies and single variable domain antibodies.

[0057] As used herein, the term "antigen-binding site" 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 from 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 adjacent, more conserved sections known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions in immunoglobulins. 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 a 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, using a peptide linker to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide.

[0058] As used herein, the term "antibody" refers to a protein or protein conjugate that contains an antigen-binding site. Antibodies can be monospecific or multispecific (e.g., bispecific).

[0059] As used herein, the terms "a" and "an" mean "one or more" and include pluralities unless the context is inappropriate.

[0060] As used herein, the terms "subject" and "patient" refer to an organism treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, etc.), and more preferably include humans.

[0061] As used herein, the term "effective amount" 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. As used herein, the term "treat" includes any effect that results in improvement of or alleviates the symptoms of a condition, disease, disorder, etc., such as alleviating, mitigating, modulating, ameliorating, or eliminating the effect.

[0062] 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 diagnostic or therapeutic use in vivo or ex vivo.

[0063] As used herein, the term "pharmaceutically acceptable carrier" 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).

[0064] Throughout the description, when compositions are described as having, including, or comprising particular ingredients, or processes and methods are described as having, including, or comprising particular steps, it is intended that there are also compositions of the invention that consist essentially of or consist of the recited ingredients, and processes and methods according to the invention that consist essentially of or consist of the recited processing steps.

[0065] 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.

[0066] I. Anti-CD19 antibody In one aspect, the present disclosure provides antigen-binding sites that bind to CD19 (e.g., human CD19) derived from the antibodies listed in Table 116. The present disclosure also provides antibodies comprising the antigen-binding sites. CDR sequences are identified under the Kabat numbering scheme unless indicated by an asterisk (*).

[0067] Table 1. Exemplary antibody sequences that bind to CD19 TIFF0007780434000001.tif161150TIFF0007780434000002.tif231150TIFF0007780434 000003.tif233150TIFF0007780434000004.tif232150TIFF0007780434000005.tif51150

[0068] In certain embodiments, an antigen-binding site that binds to CD19 of the present invention comprises an antibody heavy chain variable domain (VH) comprising 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%, at least 99%, or 100%) identical to the VH of an antibody disclosed in Table 1, and an antibody light chain variable domain (VL) comprising 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%, at least 99%, or 100%) identical to the VL of the same antibody disclosed in Table 1. In certain embodiments, the antigen-binding site comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the VH and VL sequences of the antibodies disclosed in Table 1, as determined by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262: 732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibody disclosed in Table 1. In certain embodiments, the antigen-binding site comprises the VH and VL sequences of an antibody disclosed in Table 1.

[0069] Series 1 Constructs In certain embodiments, the antigen-binding site that binds to CD19 comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:52, 54, and 55, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:57, 58, and 59, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:22, 23, 16, 18, 19, and 10, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs:3, 14, 22, and 29; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs:5, 15, 23, 34, 39, 42, and 48; the HCDR3 sequence is selected from the group consisting of SEQ ID NOs:6 and 16; the LCDR1 sequence is selected from the group consisting of SEQ ID NOs:8, 18, 24, 31, 35, 45, and 49; the LCDR2 sequence is selected from the group consisting of SEQ ID NOs:9, 19, 25, and 51; and / or the LCDR3 sequence is selected from the group consisting of SEQ ID NOs:10, 26, and 36.

[0070] In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:53, 54, and 56, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:57, 58, and 59, respectively, wherein the antigen-binding site does not comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:4, 23, 17, 18, 19, and 10, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs:4 and 30; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs:5, 15, 23, 34, 39, 42, and 48; the HCDR3 sequence is selected from the group consisting of SEQ ID NOs:7 and 17; the LCDR1 sequence is selected from the group consisting of SEQ ID NOs:8, 18, 24, 31, 35, 45, and 49; the LCDR2 sequence is selected from the group consisting of SEQ ID NOs:9, 19, 25, and 51; and / or the LCDR3 sequence is selected from the group consisting of SEQ ID NOs:10, 26, and 36.

[0071] In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:1, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:2.

[0072] In certain embodiments, the antigen-binding site has a higher binding affinity to human and / or cynomolgus CD19 compared to the antigen-binding site having the VH and VL sequences shown in SEQ ID NOs: 20 and 13, respectively.

[0073] Series 2 Constructs In certain embodiments, the antigen-binding site that binds to CD19 comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:52, 54, and 55, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:57, 60, and 59, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:22, 23, 16, 18, 19, and 10, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs:3, 14, 22, and 29; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs:5, 15, 23, 34, 39, 42, and 48; the HCDR3 sequence is selected from the group consisting of SEQ ID NOs:6 and 16; the LCDR1 sequence is selected from the group consisting of SEQ ID NOs:8, 18, 24, 31, 35, 45, and 49; the LCDR2 sequence is selected from the group consisting of SEQ ID NOs:9, 19, and 25; and / or the LCDR3 sequence is selected from the group consisting of SEQ ID NOs:10, 26, and 36.

[0074] In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:53, 54, and 56, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:57, 60, and 59, respectively, wherein the antigen-binding site does not comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:4, 23, 17, 18, 19, and 10, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs:4 and 30; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs:5, 15, 23, 34, 39, 42, and 48; the HCDR3 sequence is selected from the group consisting of SEQ ID NOs:7 and 17; the LCDR1 sequence is selected from the group consisting of SEQ ID NOs:8, 18, 24, 31, 35, 45, and 49; the LCDR2 sequence is selected from the group consisting of SEQ ID NOs:9, 19, and 25; and / or the LCDR3 sequence is selected from the group consisting of SEQ ID NOs:10, 26, and 36.

[0075] In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:1, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:2.

[0076] In certain embodiments, the antigen-binding site has a higher binding affinity for human CD19 compared to an antigen-binding site having the VH and VL sequences set forth in SEQ ID NOs: 20 and 13, respectively. In certain embodiments, the antigen-binding site has a K of less than or equal to 0.3 nM as measured by surface plasmon resonance (SPR) when the antigen-binding site exists as a monomer. D In certain embodiments, the antigen-binding site has a K in the range of 0.05 to 0.3 nM or in the range of 0.1 to 0.3 nM when the antigen-binding site exists as a monomer, as measured by SPR. D and binds to human CD19 or its extracellular fragments.

[0077] Series 3 Constructs In certain embodiments, the antigen-binding site that binds to CD19 comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:52, 61, and 55, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:57, 60, and 59, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:22, 23, 16, 18, 19, and 10, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs:3, 14, 22, and 29; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs:5, 23, 34, 42, and 48; the HCDR3 sequence is selected from the group consisting of SEQ ID NOs:6 and 16; the LCDR1 sequence is selected from the group consisting of SEQ ID NOs:8, 18, 24, 31, 35, 45, and 49; the LCDR2 sequence is selected from the group consisting of SEQ ID NOs:9, 19, and 25; and / or the LCDR3 sequence is selected from the group consisting of SEQ ID NOs:10, 26, and 36.

[0078] In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:53, 61, and 56, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:57, 60, and 59, respectively, wherein the antigen-binding site does not comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:4, 23, 17, 18, 19, and 10, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs:4 and 30; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs:5, 23, 34, 42, and 48; the HCDR3 sequence is selected from the group consisting of SEQ ID NOs:7 and 17; the LCDR1 sequence is selected from the group consisting of SEQ ID NOs:8, 18, 24, 31, 35, 45, and 49; the LCDR2 sequence is selected from the group consisting of SEQ ID NOs:9, 19, and 25; and / or the LCDR3 sequence is selected from the group consisting of SEQ ID NOs:10, 26, and 36.

[0079] In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:1, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:2.

[0080] In certain embodiments, the antigen-binding site has a higher binding affinity for human CD19 compared to the antigen-binding site having the VH and VL sequences set forth in SEQ ID NOs: 20 and 13, respectively. In certain embodiments, the antigen-binding site has a K of less than or equal to 0.2 nM as measured by SPR when the antigen-binding site exists as a monomer. D In certain embodiments, the antigen-binding site has a K in the range of 0.05 to 0.2 nM or in the range of 0.1 to 0.2 nM when the antigen-binding site exists as a monomer, as measured by SPR. D and binds to human CD19 or its extracellular fragments.

[0081] Individual constructs In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-101. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:3, 5, and 6, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:8, 9, and 10, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:4, 5, and 7, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:8, 9, and 10, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:1, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:2. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:1 and 2, respectively.

[0082] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-102. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 18, 19, and 10, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 4, 15, and 17, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 18, 19, and 10, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 12, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 13. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 12 and 13, respectively.

[0083] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-103. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:22, 23, and 16, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:24, 25, and 26, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:4, 23, and 17, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:24, 25, and 26, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:20, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:21. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:20 and 21, respectively.

[0084] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-104. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 29, 23, and 6, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 31, 25, and 10, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 30, 23, and 7, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 31, 25, and 10, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:27, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:28. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:27 and 28, respectively.

[0085] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-105. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 14, 34, and 6, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 35, 25, and 36, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 4, 34, and 7, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 35, 25, and 36, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 32, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 33. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 32 and 33, respectively.

[0086] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-106. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:3, 39, and 16, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:24, 25, and 10, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:4, 39, and 17, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:24, 25, and 10, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 37, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 38. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 37 and 38, respectively.

[0087] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-107. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 3, 42, and 16, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 18, 25, and 10, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 4, 42, and 17, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 18, 25, and 10, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 40, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 41. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 40 and 41, respectively.

[0088] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-108. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:29, 23, and 16, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:45, 19, and 36, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:30, 23, and 17, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:45, 19, and 36, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:43, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:44. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:43 and 44, respectively.

[0089] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-109. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 29, 48, and 16, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 49, 9, and 26, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 30, 48, and 17, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 49, 9, and 26, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 46, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 47. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 46 and 47, respectively.

[0090] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-110. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 22, 23, and 16, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 18, 51, and 36, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 4, 23, and 17, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 18, 51, and 36, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:20, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:50. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:20 and 50, respectively.

[0091] In certain embodiments, the antigen-binding site derived from CNG-CD19-101, CNG-CD19-102, CNG-CD19-103, CNG-CD19-110, CNG-CD19-104, CNG-CD19-105, CNG-CD19-106, CNG-CD19-107, CNG-CD19-108, or CNG-CD19-109 has a higher binding affinity for human and / or cynomolgus CD19 compared to the antigen-binding site having the VH and VL sequences set forth in SEQ ID NOs:20 and 13, respectively.

[0092] In certain embodiments, an antigen-binding site derived from CNG-CD19-101, CNG-CD19-102, CNG-CD19-103, CNG-CD19-104, CNG-CD19-105, CNG-CD19-106, CNG-CD19-107, CNG-CD19-108, or CNG-CD19-109 has a K of less than or equal to 0.3 nM as measured by SPR when the antigen-binding site exists as a monomer. D In certain embodiments, the antigen-binding site derived from CNG-CD19-101, CNG-CD19-102, CNG-CD19-103, CNG-CD19-104, CNG-CD19-105, CNG-CD19-106, CNG-CD19-107, CNG-CD19-108, or CNG-CD19-109 has a K in the range of 0.05 to 0.3 nM or in the range of 0.1 to 0.3 nM as measured by SPR when the antigen-binding site exists as a monomer. D and binds to human CD19 or its extracellular fragments.

[0093] In certain embodiments, an antigen-binding site derived from CNG-CD19-101, CNG-CD19-103, CNG-CD19-104, CNG-CD19-105, CNG-CD19-107, CNG-CD19-108, or CNG-CD19-109 has a K of less than or equal to 0.2 nM as measured by SPR when the antigen-binding site exists as a monomer. DIn certain embodiments, the antigen-binding site derived from CNG-CD19-101, CNG-CD19-103, CNG-CD19-104, CNG-CD19-105, CNG-CD19-107, CNG-CD19-108, or CNG-CD19-109 has a K in the range of 0.05 to 0.2 nM or in the range of 0.1 to 0.2 nM when the antigen-binding site exists as a monomer, as measured by SPR. D and binds to human CD19 or its extracellular fragments.

[0094] In certain embodiments, an antigen-binding site derived from CNG-CD19-103, CNG-CD19-110, CNG-CD19-104, CNG-CD19-105, CNG-CD19-106, CNG-CD19-107, CNG-CD19-108, or CNG-CD19-109 has a K of less than or equal to 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM as measured by SPR when the antigen-binding site exists as a monomer. D In certain embodiments, the antigen-binding site derived from CNG-CD19-103, CNG-CD19-110, CNG-CD19-104, CNG-CD19-105, CNG-CD19-106, CNG-CD19-107, CNG-CD19-108, or CNG-CD19-109 has a K in the range of 1 to 9 nM, 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 1 to 4 nM, or 1 to 3 nM, as measured by SPR when the antigen-binding site exists as a monomer. D and binds to cynomolgus monkey CD19.

[0095] The present disclosure also provides antigen-binding sites that compete with antibodies or antigen-binding sites comprising the VH, VL, and / or scFv sequences provided in Table 1 for binding to CD19 (e.g., human CD19).

[0096] In certain embodiments, the antigen-binding site that binds to CD19 is in the form of an scFv. In certain embodiments, the VH is located C-terminal to the VL. In certain embodiments, the VH is located N-terminal to the VL. In certain embodiments, the VH and VL are linked by a peptide linker, e.g., a linker disclosed in subsection E below entitled "Linker." To stabilize the scFv, the amino acid residues at positions 44 of the VH and 100 of the VL (under Kabat numbering) can be substituted with Cys, thereby facilitating disulfide bond formation between the VH and VL. Thus, in certain embodiments, the VH and VL contain Cys at positions 100 and 44, respectively. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:11.

[0097] In another aspect, the present disclosure provides antigen-binding sites that bind to CD19 (e.g., human CD19) from the antibodies listed in Table 2. The present disclosure also provides antibodies comprising the antigen-binding sites. CDR sequences are identified under the Kabat numbering scheme unless indicated by an asterisk (*).

[0098] Table 2: Exemplary antibody sequences that bind to CD19 TIFF0007780434000006.tif110150TIFF0007780434000007.tif231150TIFF0007780434 000008.tif232150TIFF0007780434000009.tif232150TIFF0007780434000010.tif41150

[0099] In certain embodiments, an antigen-binding site that binds to CD19 of the present invention comprises a VH that 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%, at least 99%, or 100%) identical to the VH of an antibody disclosed in Table 2, and a VL that 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%, at least 99%, or 100%) identical to the VL of the same antibody disclosed in Table 2. In certain embodiments, the antigen-binding site comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the VH and VL sequences of the antibodies disclosed in Table 2, as determined by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262: 732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibody disclosed in Table 2. In certain embodiments, the antigen-binding site comprises the VH and VL sequences of an antibody disclosed in Table 2.

[0100] Series 1 Constructs In certain embodiments, the antigen-binding site that binds to CD19 comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 109, 111, and 112, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 114, 115, and 71, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 92, 87, 67, 80, 70, and 71, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs:64, 75, 85, 92, 96, and 105; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs:66, 77, 87, and 107; the HCDR3 sequence is selected from the group consisting of SEQ ID NOs:67, 78, 82, and 88; the LCDR1 sequence is selected from the group consisting of SEQ ID NOs:69, 80, and 108; the LCDR2 sequence is selected from the group consisting of SEQ ID NOs:70, 94, 100, and 102; and / or the LCDR3 sequence is SEQ ID NO:71.

[0101] In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:110, 111, and 113, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:114, 115, and 71, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:93, 87, 68, 80, 70, and 71, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs:65, 76, 86, 93, 97, and 106; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs:66, 77, 87, and 107; the HCDR3 sequence is selected from the group consisting of SEQ ID NOs:68, 79, 83, and 89; the LCDR1 sequence is selected from the group consisting of SEQ ID NOs:69, 80, and 108; the LCDR2 sequence is selected from the group consisting of SEQ ID NOs:70, 94, 100, and 102; and / or the LCDR3 sequence is SEQ ID NO:71.

[0102] In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:62, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:63.

[0103] In certain embodiments, the antigen-binding site has a higher binding affinity for human CD19 compared to an antigen-binding site having the VH and VL sequences set forth in SEQ ID NOs:90 and 74, respectively. In certain embodiments, the antigen-binding site has a K of less than or equal to 2 nM, 1 nM, or 0.5 nM as measured by surface plasmon resonance (SPR) when the antigen-binding site exists as a monomer. D In certain embodiments, the antigen-binding site has a K in the range of 0.05 to 2 nM, in the range of 0.05 to 1 nM, or in the range of 0.05 to 0.5 nM, as measured by SPR when the antigen-binding site exists as a monomer. D and binds to human CD19 or its extracellular fragments.

[0104] Series 2 Constructs In certain embodiments, the antigen-binding site that binds to CD19 comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:116, 117, and 118, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:114, 120, and 71, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:92, 87, 67, 80, 70, and 71, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs:64, 96, and 105; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs:66, 87, and 107; the HCDR3 sequence is selected from the group consisting of SEQ ID NOs:67 and 78; the LCDR1 sequence is selected from the group consisting of SEQ ID NOs:69 and 108; the LCDR2 sequence is selected from the group consisting of SEQ ID NOs:70, 100, and 102; and / or the LCDR3 sequence is SEQ ID NO:71.

[0105] In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:110, 117, and 119, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:114, 120, and 71, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:93, 87, 68, 80, 70, and 71, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs:65, 97, and 106; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs:66, 87, and 107; the HCDR3 sequence is selected from the group consisting of SEQ ID NOs:68 and 79; the LCDR1 sequence is selected from the group consisting of SEQ ID NOs:69 and 108; the LCDR2 sequence is selected from the group consisting of SEQ ID NOs:70, 100, and 102; and / or the LCDR3 sequence is SEQ ID NO:71.

[0106] In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:62, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:63.

[0107] In certain embodiments, the antigen-binding site has a higher binding affinity for human CD19 compared to an antigen-binding site having the VH and VL sequences set forth in SEQ ID NOs:90 and 74, respectively. In certain embodiments, the antigen-binding site has a K of less than or equal to 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM as measured by SPR when the antigen-binding site exists as a monomer. D In certain embodiments, the antigen-binding site has a K in the range of 0.05-0.4 nM, 0.05-0.3 nM, 0.05-0.2 nM, or 0.05-0.1 nM, as measured by SPR when the antigen-binding site exists as a monomer. D and binds to human CD19 or its extracellular fragments.

[0108] Individual constructs In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-701. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:64, 66, and 67, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:65, 66, and 68, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:62, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:63. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:62 and 63, respectively.

[0109] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-702. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:75, 77, and 78, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:80, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:76, 77, and 79, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:80, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:73, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:74. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:73 and 74, respectively.

[0110] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-703. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:64, 66, and 82, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:80, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:65, 66, and 83, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:80, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:81, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:74. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:81 and 74, respectively.

[0111] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-704. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:85, 87, and 88, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:80, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:86, 87, and 89, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:80, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:84, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:74. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:84 and 74, respectively.

[0112] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-705. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:92, 87, and 67, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 94, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:93, 87, and 68, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 94, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:90, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:91. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:90 and 91, respectively.

[0113] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-706. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:96, 87, and 78, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:97, 87, and 79, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:95, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:63. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:95 and 63, respectively.

[0114] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-707. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:64, 66, and 78, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:65, 66, and 79, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:98, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:63. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:98 and 63, respectively.

[0115] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-708. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:64, 66, and 78, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 100, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:65, 66, and 79, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 100, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:98, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:99. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:98 and 99, respectively.

[0116] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-709. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:64, 66, and 78, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 102, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:65, 66, and 79, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:69, 102, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:98, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:101. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs:98 and 101, respectively.

[0117] In certain embodiments, the antigen-binding site that binds to CD19 is derived from CNG-CD19-710. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 105, 107, and 78, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 108, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 106, 107, and 79, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs: 108, 70, and 71, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 103, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 104. In certain embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 103 and 104, respectively.

[0118] In certain embodiments, the antigen-binding site derived from CNG-CD19-701, CNG-CD19-702, CNG-CD19-703, CNG-CD19-704, CNG-CD19-705, CNG-CD19-706, CNG-CD19-707, CNG-CD19-708, CNG-CD19-709, or CNG-CD19-710 has a higher binding affinity for human CD19 compared to the antigen-binding site having the VH and VL sequences set forth in SEQ ID NOs:90 and 74, respectively.

[0119] In certain embodiments, an antigen-binding site derived from CNG-CD19-701, CNG-CD19-702, CNG-CD19-703, CNG-CD19-704, CNG-CD19-705, CNG-CD19-706, CNG-CD19-707, CNG-CD19-708, CNG-CD19-709, or CNG-CD19-710 has a K of less than or equal to 2 nM, 1 nM, or 0.5 nM as measured by surface plasmon resonance (SPR) when the antigen-binding site exists as a monomer. D and binds to human CD19 or an extracellular fragment thereof. In certain embodiments, the antigen-binding site derived from CNG-CD19-701, CNG-CD19-702, CNG-CD19-703, CNG-CD19-704, CNG-CD19-705, CNG-CD19-706, CNG-CD19-707, CNG-CD19-708, CNG-CD19-709, or CNG-CD19-710 has a K in the range of 0.05 to 2 nM, in the range of 0.05 to 1 nM, or in the range of 0.05 to 0.5 nM, as measured by SPR when the antigen-binding site exists as a monomer. D and binds to human CD19 or its extracellular fragments.

[0120] In certain embodiments, an antigen-binding site derived from CNG-CD19-701, CNG-CD19-706, CNG-CD19-707, CNG-CD19-708, CNG-CD19-709, or CNG-CD19-710 has a K of less than or equal to 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM as measured by SPR when the antigen-binding site exists as a monomer. D In certain embodiments, the antigen-binding site derived from CNG-CD19-701, CNG-CD19-706, CNG-CD19-707, CNG-CD19-708, CNG-CD19-709, or CNG-CD19-710 has a K in the range of 0.05-0.4 nM, 0.05-0.3 nM, 0.05-0.2 nM, or 0.05-0.1 nM, as measured by SPR when the antigen-binding site exists as a monomer. D and binds to human CD19 or its extracellular fragments.

[0121] In certain embodiments, an antigen-binding site derived from CNG-CD19-701, CNG-CD19-702, CNG-CD19-703, CNG-CD19-704, CNG-CD19-705, CNG-CD19-706, CNG-CD19-707, CNG-CD19-708, CNG-CD19-709, or CNG-CD19-710 has a K of less than or equal to 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM as measured by SPR when the antigen-binding site exists as a monomer. D In certain embodiments, the antigen-binding site derived from CNG-CD19-701, CNG-CD19-702, CNG-CD19-703, CNG-CD19-704, CNG-CD19-705, CNG-CD19-706, CNG-CD19-707, CNG-CD19-708, CNG-CD19-709, or CNG-CD19-710 has a K in the range of 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 1 to 4 nM, or 1 to 3 nM, as measured by SPR when the antigen-binding site exists as a monomer. D and binds to cynomolgus monkey CD19.

[0122] The present disclosure also provides antigen-binding sites that compete with antibodies or antigen-binding sites comprising the VH, VL, and / or scFv sequences provided in Table 2 for binding to CD19 (e.g., human CD19).

[0123] In certain embodiments, the antigen-binding site that binds to CD19 is in the form of an scFv. In certain embodiments, the VH is located C-terminal to the VL. In certain embodiments, the VH is located N-terminal to the VL. In certain embodiments, the VH and VL are linked by a peptide linker, such as the linkers disclosed in subsection E below entitled "Linkers." To stabilize the scFv, the amino acid residues at positions 44 of the VH and 100 of the VL (according to the Kabat numbering) can be substituted with Cys, thereby facilitating disulfide bond formation between the VH and VL. Thus, in certain embodiments, the VH and VL contain Cys at positions 100 and 44, respectively.

[0124] However, it has been found that CNG-CD19-701 to CNG-CD19-710 exhibit high stability, with melting temperatures in the range of 80-85°C, when present in the form of Fab. Consequently, the antigen-binding sites derived from these antibodies are believed to be stable in the absence of such Cys substitutions. Given that a multispecific binding protein may contain another antigen-binding site stabilized by a disulfide bridge formed between the two introduced Cys residues, the absence of Cys substitutions is further believed to be advantageous in the context of a multispecific binding protein. Without intending to be bound by theory, the presence of two pairs of Cys residues may increase the risk of misfolding as a result of the formation of undesired disulfide bridges. Therefore, the use of an antigen-binding site that is stable in the absence of intramolecular disulfide bridges may provide flexibility in selecting another antigen-binding site when constructing a multispecific binding protein. Accordingly, in certain embodiments, the VH and VL do not contain Cys at positions 100 and 44, respectively. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:72.

[0125] II. Multispecific Binding Proteins In one aspect, the present disclosure provides a multispecific binding protein comprising a first domain (e.g., a first antigen-binding site) that binds to CD19 (e.g., human CD19); a second domain (e.g., a second antigen-binding site) that binds to CD3 (e.g., human and / or macaque CD3), e.g., CD3ε (epsilon), CD3δ (delta), and / or CD3γ (gamma); and, optionally, a half-life-extending domain. The multispecific binding protein is configured to bring CD19-expressing cells, such as B cells, into spatial proximity with CD3-expressing cells, such as T cells, to enhance the cytotoxicity of the CD3-expressing cells against the CD19-expressing cells. This target-specific cytotoxicity is desirable for treating diseases associated with CD19-expressing cells. Activation of CD3-expressing cells can also result in cytokine production. Such cytokine production, when in appropriate amounts, can be beneficial for T cell activation and proliferation, resulting in optimal and sustained T cell-redirected target killing. However, excessive cytokine production can result in undesirable systemic toxicity, such as cytokine release syndrome. High affinity for CD19 (K > 5 pM and < 1 nM as measured by SPR) was required to achieve target-specific cytotoxicity without eliciting excessive cytokine production. D ) and varying affinity for CD3 (K ranging from below 10 nM to above 50 nM as measured by SPR) D Multispecific binding proteins have been designed with binding affinities of 1 nM, 0.1 nM, 50 pM, or 20 pM (e.g., K values ​​of 1 nM, 0.1 nM, 50 pM, or 20 pM as measured by SPR). D K lower than 10 nM D Specifically, we demonstrate the benefit of using a CD3 binding domain with a K in the range of 0.5 nM to 10 nM as measured by SPR. D The CD3-binding domain binds to CD3 at a K greater than 10 nM as measured by SPR. DCompared to the CD3-binding domain, which binds to CD3 at 1 kJ / s, the improved cytotoxic activity of the multispecific binding protein is at least as robust as its increased ability to induce cytokine production. Thus, a K in the range of 5 pM to 1 nM as measured by SPR is achieved. D affinity for CD19, and a K in the range of 0.5 nM to 10 nM as measured by SPR D Multispecific binding proteins having an affinity for CD3 of 1 pM to 1 nM can be used, for example, at low doses to achieve therapeutic efficacy while minimizing potential safety issues. Thus, in certain embodiments of the multispecific binding proteins disclosed herein, the first domain has a K in the range of 5 pM to 1 nM. D and the second domain binds to CD19 (e.g., human CD19) with a K in the range of 0.5 nM to 10 nM. D binds to CD3 (e.g., human CD3) at each K D is measured by SPR.

[0126] In certain embodiments, each K D is measured by surface plasmon resonance (SPR). In certain embodiments, the first antigen-binding site has a K measured by SPR within the range of 5 pM to 1 nM, 5 pM to 0.5 nM, 5 pM to 0.2 nM, 5 pM to 0.1 nM, 5 pM to 50 pM, 5 pM to 20 pM, 5 pM to 10 pM, 10 pM to 1 nM, 10 pM to 0.5 nM, 10 pM to 0.2 nM, 10 pM to 0.1 nM, 10 pM to 50 pM, or 10 pM to 20 pM. D In certain embodiments, the second antigen-binding site has a K within the range of 0.5 nM to 10 nM, 0.5 nM to 5 nM, 0.5 nM to 2 nM, 0.5 nM to 1 nM, 1 nM to 10 nM, 1 nM to 5 nM, or 1 nM to 2 nM, as measured by SPR. D In certain embodiments, the first antigen-binding site has a K in the range of 5 pM to 0.1 nM, 5 pM to 50 pM, 5 pM to 20 pM, 10 pM to 1 nM, 10 pM to 0.1 nM, 10 pM to 50 pM, or 10 pM to 20 pM, as measured by SPR. Dand binds to CD19 (e.g., human CD19), and the second antigen-binding site has a K in the range of 0.5 nM to 10 nM as measured by SPR. D In certain embodiments, the first antigen-binding site has a K in the range of 5 pM to 0.1 nM, 5 pM to 50 pM, 5 pM to 20 pM, 10 pM to 1 nM, 10 pM to 0.1 nM, 10 pM to 50 pM, or 10 pM to 20 pM, as measured by SPR. D and binds to CD19 (e.g., human CD19), and the second antigen-binding site has a K in the range of 1 nM to 10 nM as measured by SPR. D and binds to CD3 (e.g., human CD3).

[0127] In certain embodiments, each K D is measured by biolayer interferometry (BLI). In certain embodiments, the first antigen-binding site has a K within the range of 50 pM to 1 nM, 50 pM to 0.5 nM, 50 pM to 0.2 nM, 50 pM to 0.1 nM, 0.1 nM to 1 nM, 0.1 nM to 0.5 nM, or 0.1 nM to 0.2 nM, as measured by BLI. D In certain embodiments, the second antigen-binding site has a K in the range of 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 100 nM, 10 nM to 50 nM, or 10 nM to 20 nM, as measured by BLI. D In certain embodiments, the first antigen-binding site has a K in the range of 50 pM to 1 nM, 50 pM to 0.5 nM, 50 pM to 0.2 nM, 0.1 nM to 10 nM, 0.1 nM to 0.5 nM, or 0.1 nM to 0.2 nM, as measured by BLI. D and binds to CD19 (e.g., human CD19), and the second antigen-binding site has a K in the range of 5 nM to 100 nM as measured by BLI. DIn certain embodiments, the first antigen-binding site has a K in the range of 50 pM to 1 nM, 50 pM to 0.5 nM, 50 pM to 0.2 nM, 0.1 nM to 10 nM, 0.1 nM to 0.5 nM, or 0.1 nM to 0.2 nM, as measured by BLI. D and binds to CD19 (e.g., human CD19), and the second antigen-binding site has a K in the range of 10 nM to 100 nM as measured by BLI. D and binds to CD3 (e.g., human CD3).

[0128] In certain embodiments, the second antigen-binding site is a K that binds to CD3 (e.g., human CD3). D vs. K, where the first antigen-binding site binds to CD19 (e.g., human CD19). D is within the range of 10:1 to 1,000:1, 10:1 to 500:1, 10:1 to 200:1, 10:1 to 150:1, 20:1 to 1,000:1, 20:1 to 500:1, 20:1 to 200:1, 20:1 to 150:1, 50:1 to 1,000:1, 50:1 to 500:1, 50:1 to 200:1, 50:1 to 150:1, 100:1 to 1,000:1, 100:1 to 500:1, 100:1 to 200:1, or 100:1 to 150:1. In certain embodiments, the first antigen-binding site is a K that binds to CD19. D and K, whose second antigen-binding site binds to CD3. D are measured by the same method (e.g., both by SPR or both by BLI).

[0129] An optional half-life extending domain can be a third domain (e.g., a third antigen-binding site) that binds serum albumin (e.g., HSA). In certain embodiments of the multispecific binding proteins disclosed herein, the first domain comprises an antigen-binding site disclosed in Section I above, entitled "Anti-CD19 Antibodies." In certain embodiments, the first, second, and third domains comprise a first antigen-binding site, a second antigen-binding site, and a third antigen-binding site, respectively. In certain embodiments, the first antigen-binding site of the multispecific binding protein is an antigen-binding site that binds CD19, disclosed in Section I above, entitled "Anti-CD19 Antibodies."

[0130] Each of the antigen-binding sites of the multispecific binding protein can take a variety of forms, such as a single-chain variable fragment (scFv), a Fab fragment, or a single-domain antibody (sdAb). In certain embodiments, the first antigen-binding site comprises an scFv. In certain embodiments, the second antigen-binding site comprises an scFv. In certain embodiments, the third antigen-binding site comprises an sdAb.

[0131] 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: The present invention discloses a serum albumin binding polypeptide having the amino acid sequence of TIFF0007780434000011.tif4144.Other exemplary HSA binding polypeptides 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.

[0132] 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.

[0133] In other embodiments, the multispecific binding protein does not contain an antibody Fc region. The absence of Fc contributes to the reduced 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, with 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.

[0134] 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.

[0135] A. Antigen-binding site that binds to CD19 The first antigen-binding site of the multispecific binding protein binds to CD19 (e.g., human CD19). In certain embodiments, the first antigen-binding site is an antigen-binding site that binds to CD19, as disclosed above in Section I entitled "Anti-CD19 Antibodies."

[0136] Alternatively, the first antigen-binding site that binds to CD19 can be selected from antibodies such as, 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). Additional 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. For example, in certain embodiments, the first antigen-binding site that binds to CD19 is derived from an antibody listed in Table 3.

[0137] Table 3: Exemplary antibody sequences that bind to CD19 TIFF0007780434000012.tif105150TIFF0007780434000013.tif233150TIFF00077804340 00014.tif231150TIFF0007780434000015.tif231150TIFF0007780434000016.tif229150 TIFF0007780434000017.tif230150TIFF0007780434000018.tif229150TIFF0007780434000019.tif168150When the VL and LCDR sequences are shown as "N / A", the antigen binding site is an sdAb having only VH (e.g., VHH).

[0138] In certain embodiments, the first antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to the VH of an antibody disclosed in Table 3, and a VL comprising 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%, at least 99%, or 100%) identical to the VL of the same antibody disclosed in Table 3. In certain embodiments, the antigen-binding site comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the VH and / or VL sequences of the antibodies disclosed in Table 3, as determined by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262: 732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibody disclosed in Table 3. In certain embodiments, the antigen-binding site comprises the VH and VL sequences of an antibody disclosed in Table 3.

[0139] Such antigen-binding sites may take the form of scFvs. In certain embodiments, the VH is positioned C-terminal to the VL. In certain embodiments, the VH is positioned N-terminal to the VL. In certain embodiments, the VH and VL are linked by a peptide linker, e.g., a linker disclosed in subsection E below entitled "Linkers." To stabilize the scFv, the amino acid residues at positions 44 of the VH and 100 of the VL (under Kabat numbering) can be substituted with Cys, thereby facilitating disulfide bond formation between the VH and VL. Thus, in certain embodiments, the VH and VL contain Cys at positions 100 and 44, respectively.

[0140] 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 provided in Table 3. In certain embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 of the VH sequences of the antibodies disclosed in Table 3, as determined by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262: 732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences of an antibody provided in Table 3. In certain embodiments, the VH comprises the amino acid sequence of the VH of an sdAb provided in Table 3.

[0141] In certain embodiments, the first 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. DFor example, in certain embodiments, the first antigen-binding site binds to CD19 at a concentration of about 5 pM to about 1 nM, about 5 pM to about 0.9 nM, about 5 pM to about 0.8 nM, about 5 pM to about 0.7 nM, about 5 pM to about 0.6 nM, about 5 pM to about 0.5 nM, about 5 pM to about 0.4 nM, about 5 pM to about 0.3 nM, about 5 pM to about 0.2 nM, about 5 pM to about 0.1 nM, about 5 pM to about 50 pM, about 5 pM to about 20 pM, or about 5 pM to about 10 pM. , about 10pM to about 1nM, about 10pM to about 0.9nM, about 10pM to about 0.8nM, about 10pM to about 0.7nM, about 10pM to about 0.6nM, about 10pM to about 0.5nM, about 10pM to about 0.4nM , 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, about 10 pM to about 20 pM, about 50 pM to about 1 nM, about 50 pM to about 0.9 nM, about 50pM to about 0.8nM, about 50pM to about 0.7nM, about 50pM to about 0.6nM, about 50pM to about 0.5nM, about 50pM to about 0.4nM, about 50pM to about 0.3nM, about 50pM to about 0.2 nM, about 50pM to about 0.1nM, about 0.1nM to about 10nM, about 0.1nM to about 9nM, about 0.1nM to about 8nM, about 0.1nM to about 7nM, about 0.1nM to about 6nM, about 0.1nM to about 5nM, Approximately 0.1nM to approximately 4nM, approximately 0.1nM to approximately 3nM, approximately 0.1nM to approximately 2nM, approximately 0.1nM to approximately 1nM, approximately 0.1nM to approximately 0.5nM, approximately 0.5nM to approximately 10nM, approximately 1nM to approximately 10nM, approximately 2n K of M ~ 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 In certain embodiments, K D The K value is measured by surface plasmon resonance (SPR). D The values ​​are measured by biolayer interferometry (BLI).

[0142] It is understood that the binding affinity for CD19 of a first antigen-binding site alone may differ from the binding affinity of the same antigen-binding site in the context of a multispecific binding protein disclosed herein, possibly due to conformational constraints from other domains. Context-dependent binding affinities are described below in subsection G entitled "Binding Affinity."

[0143] In certain embodiments, the first antigen-binding site, when present in the form of Fab, has a melting temperature of at least 60°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, when present in the form of Fab, has a melting temperature in the range of 60-85°C, 60-80°C, 60-75°C, 60-70°C, 60-65°C, 65-85°C, 65-80°C, 65-75°C, 65-70°C, 70-85°C, 70-80°C, 70-75°C, 75-85°C, 75-80°C, or 80-85°C.

[0144] B. Antigen-binding site that binds to CD3 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).

[0145] 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 known as AMG103; see PCT Publication No. WO1999054440A1) and solitomab (also known 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 exhibit cross-species specificity for the CD3ε chain of humans, common marmosets (Callithrix jacchus), cotton-top tamarins (Saguinus Oedipus), and squirrel monkeys (Saimiri sciureus) (see ibid.). These constructs are believed to not nonspecifically activate T cells to the same extent as observed with previous BiTE® constructs and therefore have a lower risk of side effects (see Brischwein et al. (2007) J. Immunother., 30(8): 798-807).

[0146] In certain embodiments, the second antigen-binding site of the multispecific binding protein binds to an epitope located 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., common marmosets, cotton-top tamarins, or squirrel monkeys), Old World primates (e.g., baboons and macaques), gibbons, and non-human homininae. Common marmosets and cotton-top tamarins are New World primates belonging to the Callitrichidae family, while 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 common marmoset, cotton-top tamarin, and / or squirrel monkey CD3ε.

[0147] The second antigen-binding site that binds to an extracellular epitope of human and / or macaque CD3 can be, for example, muromonab-CD3 (OKT3) as described in WO2008101154; otelixizumab (TRX4) as described in WO2007145941; teplizumab (MGA031) as described in WO2013040164; visilizumab (Nuvion) ​​as described in WO2004052397; SP34 as described in WO2015181098; X35, VIT3, or BMA030 (BW264 / 56), as described in WO2004106381; CLB-T3 / 3, CRIS7, CLB-T3.4.2, WT32, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, or F101.01, as described in WO2004106383; YTH12.5 or SPv-T3b, as described in WO2012084895; 11-409; TR-66 as described in WO2013158856; UCHT-1 as described in WO2000041474; WT-31 as illustrated in WO2016085889, or an antibody described in WO2008119567. For example, in certain embodiments, the second antigen-binding site that binds to CD3 is derived from an antibody listed in Table 4.

[0148] Table 4: Exemplary antibody sequences that bind to CD3 TIFF0007780434000020.tif162150TIFF0007780434000021.tif231150TIFF0007780434000022.tif233150TIFF0007780434000023.tif232150TIFF0007780434000024.tif232150TIFF0007780434000025.tif227150TIFF0007780434000026.tif111150When the VL and LCDR sequences are shown as "N / A", the antigen binding site is an sdAb having only VH (e.g., VHH).

[0149] In certain embodiments, the second antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to the VH of an antibody disclosed in Table 4, and a VL comprising 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%, at least 99%, or 100%) identical to the VL of the same antibody disclosed in Table 4. In certain embodiments, the antigen-binding site comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the VH and / or VL sequences of the antibodies disclosed in Table 4, as determined by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262: 732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibody disclosed in Table 4. In certain embodiments, the antigen-binding site comprises the VH and VL sequences of an antibody disclosed in Table 4.

[0150] In certain embodiments, the second antigen-binding site that binds to CD3 is derived from CNG-CD3-1. In certain embodiments, the second antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:414, 416, and 417, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:419, 420, and 421, respectively. In certain embodiments, the second antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:415, 416, and 418, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:419, 420, and 421, respectively. In certain embodiments, the second antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 412, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 413. In certain embodiments, the VH and VL of the second antigen-binding site comprise the amino acid sequences of SEQ ID NOs: 412 and 413, respectively.

[0151] In certain embodiments, the second antigen-binding site that binds to CD3 is derived from CNG-CD3-2. In certain embodiments, the second antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:414, 416, and 425, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:419, 420, and 421, respectively. In certain embodiments, the second antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:415, 416, and 426, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:419, 420, and 421, respectively. In certain embodiments, the second antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:424, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:413. In certain embodiments, the VH and VL of the second antigen-binding site comprise the amino acid sequences of SEQ ID NOs:424 and 413, respectively.

[0152] In certain embodiments, the second antigen-binding site that binds to CD3 is derived from CNG-CD3-3. In certain embodiments, the second antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:414, 431, and 417, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:419, 420, and 432, respectively. In certain embodiments, the second antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:415, 431, and 418, respectively, and a VL comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NOs:419, 420, and 432, respectively. In certain embodiments, the second antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 429, and a VL comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 430. In certain embodiments, the VH and VL of the second antigen-binding site comprise the amino acid sequences of SEQ ID NOs: 429 and 430, respectively.

[0153] Such antigen-binding sites may take the form of scFvs. In certain embodiments, the VH is positioned C-terminal to the VL. In certain embodiments, the VH is positioned N-terminal to the VL. In certain embodiments, the VH and VL are linked by a peptide linker, e.g., a linker disclosed in subsection E below entitled "Linkers." In certain embodiments, the second antigen-binding site comprises the amino acid sequence of SEQ ID NO: 422, 427, or 433. To stabilize the scFv, the amino acid residues at positions 44 of the VH and 100 of the VL (according to Kabat numbering) can be substituted with Cys, thereby facilitating disulfide bond formation between the VH and VL. Thus, in certain embodiments, the VH and VL comprise Cys at positions 100 and 44, respectively. In certain embodiments, the second antigen-binding site comprises the amino acid sequence of SEQ ID NO: 423, 428, or 434.

[0154] 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 provided in Table 4. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequence of an antibody provided in Table 4. In certain embodiments, the VH comprises the amino acid sequence of the VH of an sdAb provided in Table 4.

[0155] 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 provided in Table 4.

[0156] 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 ) and connect them. D can be measured by methods known in the art. D is measured by SPR against CD3 or its extracellular fragments immobilized on a chip. D is measured by flow cytometry for CD3 expressed on the surface of cells, for example, according to the method described in Example 7 below.

[0157] In certain embodiments, the second 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 as measured by SPR. DFor example, in certain embodiments, the first antigen-binding site has an affinity for CD3 of 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 0.5 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 0.5 nM, about 10 pM to about 0.5 nM, about 10 pM to about 0.6 ... pM ~ about 0.1nM, about 10pM - about 50pM, 0.1nM - about 10nM, about 0.1nM - about 9nM, about 0.1nM - about 8nM, about 0.1nM - about 7nM, about 0.1nM - Approximately 6nM, approximately 0.1nM to approximately 5nM, approximately 0.1nM to approximately 4nM, approximately 0.1nM to approximately 3nM, approximately 0.1nM to approximately 2nM, approximately 0.1nM to approximately 1nM, approximately 0.1nM to approximately 0.5nM , about 0.5nM to about 10nM, about 0.5nM to about 9nM, about 0.5nM to about 8nM, about 0.5nM to about 7nM, about 0.5nM to about 6nM, about 0.5nM to about 5nM, about 0. 5nM to about 4nM, about 0.5nM to about 3nM, about 0.5nM to about 2nM, about 0.5nM to about 1nM, about 1nM to about 10nM, about 1nM to about 9nM, about 1nM to about 8nM, about 1nM to about 7nM, about 1nM to about 6nM, about 1nM to about 5nM, about 1nM to about 4nM, about 1nM to about 3nM, about 1nM to about 2nM, about 2nM to about 10nM, about 3nM to about 1 K in the range of 0nM, 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 and binds to CD3.

[0158] In certain embodiments, the second antigen-binding site has a K of less than or equal to 50 nM, 40 nM, 30 nM, 20 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, or 0.1 nM as measured by BLI. DFor example, in certain embodiments, the first antigen-binding site has a specific binding affinity, as measured by BLI, of approximately about 0.1 nM to about 20 nM, about 0.1 nM to about 19 nM, about 0.1 nM to about 18 nM, about 0.1 nM to about 17 nM, about 0.1 nM to about 16 nM, about 0.1 nM to about 15 nM, about 0.1 nM to about 14 nM, about 0.1 nM to about 13 nM, about 0.1 nM to about 12 nM, about 0.1 nM to about 11 nM, 0.1 nM to about 10 nM, about 0.1 nM to about 9 nM, about 0.1 nM to about 8 nM, about 0.1 nM to about 7 nM, about 0.1 nM to about 6 nM, about 0.1 nM to about 1 Approximately 5nM, approximately 0.1nM to approximately 4nM, approximately 0.1nM to approximately 3nM, approximately 0.1nM to approximately 2nM, approximately 0.1nM to approximately 1nM, approximately 0.1nM ~about 0.5nM, about 1nM to about 50nM, about 1nM to about 40nM, about 1nM to about 30nM, about 1nM to about 20nM, about 1nM to about 19 K in the range of nM, about 1nM to about 18nM, about 1nM to about 17nM, about 1nM to about 16nM, about 1nM to about 15nM, about 1nM to about 14nM, about 1nM to about 13nM, about 1nM to about 12nM, about 1nM to about 11nM, about 1nM to about 10nM, or about 1nM to about 5nM D and binds to CD3.

[0159] In certain embodiments, the second antigen-binding site has a K greater than or equal to 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM as measured by SPR. D and binds to CD3 (e.g., human CD3, e.g., human CD3ε). In certain embodiments, the second antigen-binding site has a K within the range of about 1 nM to about 100 nM, about 1 nM to about 90 nM, about 1 nM to about 80 nM, about 1 nM to about 70 nM, about 1 nM to about 60 nM, about 1 nM to about 50 nM, about 1 nM to about 40 nM, about 1 nM to about 30 nM, about 1 nM to about 20 nM, about 1 nM to about 10 nM, about 10 nM to about 100 nM, about 10 nM to about 90 nM, about 10 nM to about 80 nM, about 10 nM to about 70 nM, about 10 nM to about 60 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, or about 10 nM to about 20 nM, as measured by SPR.D In certain embodiments, the second antigen-binding site has 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, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1 μM as measured by BLI. D and binds to CD3 (e.g., human CD3, e.g., human CD3ε). In certain embodiments, the second antigen-binding site has an affinity for at least one of about 10 nM to about 1 μM, 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, or about 10 nM to about 200 nM as measured by BLI. , about 10 nM to about 100 nM, about 100 nM to about 1 μM, about 100 nM to about 900 nM, about 100 nM to about 800 nM, about 100 nM to about 700 nM, about 100 nM to about 600 nM, about 100 nM to about 500 nM, about 100 nM to about 400 nM, about 100 nM to about 300 nM, or about 100 nM to about 200 nM D and binds to CD3.

[0160] It is understood that the binding affinity of the second antigen-binding site alone to CD3 may differ from the binding affinity of the same antigen-binding site in the context of a multispecific binding protein disclosed herein, possibly due to conformational constraints from other domains. Context-dependent binding affinities are described below in subsection G entitled "Binding Affinity."

[0161] In certain embodiments, the second antigen-binding site, when present in the form of Fab, has a melting temperature of at least 60°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, when present in the form of Fab, has a melting temperature in the range of 60-85°C, 60-80°C, 60-75°C, 60-70°C, 60-65°C, 65-85°C, 65-80°C, 65-75°C, 65-70°C, 70-85°C, 70-80°C, 70-75°C, 75-85°C, 75-80°C, or 80-85°C.

[0162] C. Half-life extending domain In certain embodiments, the multispecific binding protein comprises a half-life prolonging domain. As used herein, the term "half-life prolonging domain" refers to a protein domain that prolongs the half-life of a protein to which it is fused within a subject (e.g., the subject's blood). Exemplary half-life prolonging domains include an Fc domain, a serum albumin domain, and a protein domain that binds to serum albumin. In certain embodiments, the half-life prolonging domain in the multispecific binding protein comprises a third antigen-binding site that binds to serum albumin (e.g., HSA). It is believed that the serum albumin-binding domain may promote the recycling of the multispecific binding protein through binding to the fetal Fc receptor (FcRn), thereby prolonging the serum half-life of the multispecific binding protein. Thus, in certain embodiments, the third antigen-binding site does not bind to the D-III domain of HSA (the domain that mediates the interaction between HSA and FcRn). In certain embodiments, the third antigen-binding site prolongs the serum half-life of the multispecific binding protein.

[0163] In certain embodiments, the third antigen-binding site is an antigen-binding site that binds serum albumin (e.g., human serum albumin (HSA)) from a single domain antibody listed in Table 5. CDR sequences are identified under the Kabat numbering scheme unless indicated by an asterisk (*).

[0164] Table 5. Exemplary antibody sequences that bind serum albumin TIFF0007780434000027.tif223155TIFF0007780434000028.tif229155TIFF00077804340 00029.tif229155TIFF0007780434000030.tif229155TIFF0007780434000031.tif195155

[0165] In certain embodiments, the antigen-binding site that binds to serum albumin comprises a VH comprising 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%, at least 99%, or 100%) identical to the VH of an antibody disclosed in Table 5. In certain embodiments, the antigen-binding site comprises HCDR1, HCDR2, and HCDR3 of the VH sequences of the antibodies disclosed in Table 5, as determined by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262: 732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In certain embodiments, the antigen binding site comprises the HCDR1, HCDR2, and HCDR3 sequences of an antibody disclosed in Table 5. In certain embodiments, the antigen binding site comprises the VH sequence of an antibody disclosed in Table 5.

[0166] Series 1 Constructs In certain embodiments, the antigen-binding site that binds to HSA comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:408, 409, and 410, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs:128, 133, and 134, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs: 122, 128, 132, 137, 145, 157, and 169; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs: 124, 133, 146, 151, 153, 161, 165, 171, 179, and 181; and / or the HCDR3 sequence is selected from the group consisting of SEQ ID NOs: 125, 134, 142, 147, 154, 158, 162, 166, 172, and 176.

[0167] In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 184, 409, and 411, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and 135, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs: 123, 129, and 170; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs: 124, 133, 146, 151, 153, 161, 165, 171, 179, and 181; and / or the HCDR3 sequence is selected from the group consisting of SEQ ID NOs: 126, 135, 143, 148, 155, 159, 163, 167, 173, and 177.

[0168] In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:121.

[0169] In certain embodiments, the antigen-binding site has a higher binding affinity for human serum albumin, cynomolgus monkey serum albumin, mouse serum albumin, and / or protein A compared to an antigen-binding site having the VH sequence set forth in SEQ ID NO:196.

[0170] Series 2 Constructs In certain embodiments, the antigen-binding site that binds to HSA comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 183, 185, and 186, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 133, and 134, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs: 122, 128, 132, 145, 157, and 169; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs: 124, 133, 146, 151, 153, 171, 179, and 181; and / or the HCDR3 sequence is selected from the group consisting of SEQ ID NOs: 125, 134, 147, 154, 158, 172, and 176.

[0171] In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 184, 185, and 187, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and 135, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs: 123, 129, and 170; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs: 124, 133, 146, 151, 153, 171, 179, and 181; and / or the HCDR3 sequence is selected from the group consisting of SEQ ID NOs: 126, 135, 148, 155, 159, 173, and 177.

[0172] In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:121.

[0173] In certain embodiments, the antigen-binding site has a higher binding affinity for human serum albumin compared to an antigen-binding site having the VH sequence set forth in SEQ ID NO: 196. In certain embodiments, the antigen-binding site has a K of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM as measured by SPR when the antigen-binding site exists as a monomer. D In certain embodiments, the antigen-binding site has a K in the range of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM, as measured by SPR when the antigen-binding site exists as a monomer. D It binds to human serum albumin.

[0174] Series 3 Constructs In certain embodiments, the antigen-binding site that binds to HSA comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 188, 190, and 191, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 133, and 134, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs: 122, 128, 132, and 145; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs: 124, 133, and 161; and / or the HCDR3 sequence is selected from the group consisting of SEQ ID NOs: 125, 134, 162, 147, and 176.

[0175] In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 189, 190, and 192, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and 135, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs: 123 and 129; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs: 124, 133, and 161; and / or the HCDR3 sequence is selected from the group consisting of SEQ ID NOs: 126, 135, 163, 148, and 177.

[0176] In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:121.

[0177] In certain embodiments, the antigen-binding site has a higher binding affinity for Protein A compared to an antigen-binding site having the VH sequence set forth in SEQ ID NO: 196. In certain embodiments, the antigen-binding site has a K of less than or equal to 2.5 nM or 2 nM as measured by SPR when the antigen-binding site exists as a monomer. D In certain embodiments, the antigen-binding site has a K in the range of 1-2.5 nM or 1-2 nM as measured by SPR when the antigen-binding site exists as a monomer. D It binds to protein A.

[0178] Series 4 Constructs In certain embodiments, the antigen-binding site that binds to HSA comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 188, 193, and 194, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 133, and 134, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs: 122, 128, 132, and 145; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs: 124 and 133; and / or the HCDR3 sequence is selected from the group consisting of SEQ ID NOs: 125, 134, 147, and 176.

[0179] In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 189, 193, and 195, respectively, and the antigen-binding site does not comprise the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and 135, respectively. In certain embodiments, the HCDR1 sequence is selected from the group consisting of SEQ ID NOs: 123 and 129; the HCDR2 sequence is selected from the group consisting of SEQ ID NOs: 124 and 133; and / or the HCDR3 sequence is selected from the group consisting of SEQ ID NOs: 126, 135, 148, and 177.

[0180] In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO:121.

[0181] In certain embodiments, the antigen-binding site has a higher binding affinity for human serum albumin and a higher affinity for Protein A than an antigen-binding site having the VH sequence set forth in SEQ ID NO: 196. In certain embodiments, the antigen-binding site has a K of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM, as measured by SPR when the antigen-binding site exists as a monomer. D binds to human serum albumin and has a K less than or equal to 2.5 nM or 2 nM D In certain embodiments, the antigen-binding site has a K in the range of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM, as measured by SPR when the antigen-binding site exists as a monomer. D binds to human serum albumin and has a K in the range of 1–2.5 nM or 1–2 nM D It binds to protein A.

[0182] Individual constructs In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-101. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 122, 124, and 125, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 123, 124, and 126, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 121. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:121.

[0183] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-102. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 124, and 125, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 124, and 126, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 127. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:127.

[0184] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-103. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 122, 124, and 125, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 123, 124, and 126, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 130. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:130.

[0185] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-104. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 132, 133, and 134, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and 135, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 131. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:131.

[0186] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-105. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 137, 133, and 134, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and 135, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 136. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:136.

[0187] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-106. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 124, and 139, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 124, and 140, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 138. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:138.

[0188] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-107. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 124, and 142, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 124, and 143, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 141. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:141.

[0189] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-108. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 145, 146, and 147, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 146, and 148, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 144. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:144.

[0190] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-109. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 145, 133, and 134, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and 135, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 149. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:149.

[0191] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-110. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 151, and 134, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 151, and 135, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 150. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:150.

[0192] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-111. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 153, and 154, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 153, and 155, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 152. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:152.

[0193] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-112. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 157, 133, and 158, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and 159, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 156. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:156.

[0194] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-113. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 161, and 162, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 161, and 163, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 160. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:160.

[0195] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-114. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 165, and 166, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 165, and 167, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 164. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:164.

[0196] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-115. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 169, 171, and 172, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 170, 171, and 173, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 168. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:168.

[0197] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-116. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 133, and 147, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and 148, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 174. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:174.

[0198] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-117. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 133, and 176, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and 177, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 175. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:175.

[0199] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-118. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 179, and 147, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 179, and 148, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 178. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:178.

[0200] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-119. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 181, and 125, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 181, and 126, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 180. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:180.

[0201] In certain embodiments, the antigen-binding site that binds serum albumin is derived from CNG-HSA-120. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 128, 133, and 154, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NOs: 129, 133, and 155, respectively. In certain embodiments, the antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to SEQ ID NO: 180. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO:180.

[0202] In certain embodiments, the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has a higher binding affinity for human, cynomolgus monkey, and / or mouse serum albumin compared to the antigen-binding site having the VH sequence set forth in SEQ ID NO:196.

[0203] In certain embodiments, an antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has a K of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM as measured by SPR when the antigen-binding site exists as a monomer. D In certain embodiments, an antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has a K in the range of 1 to 10 nM, 1 to 9 nM, 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 1 to 4 nM, or 1 to 3 nM as measured by SPR when the antigen-binding site exists as a monomer. D It binds to human serum albumin.

[0204] In certain embodiments, an antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has a K of less than or equal to 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM as measured by SPR when the antigen-binding site exists as a monomer. D In certain embodiments, an antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has a K in the range of 1 to 9 nM, 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 1 to 4 nM, or 1 to 3 nM as measured by SPR when the antigen-binding site exists as a monomer. D and binds to cynomolgus monkey serum albumin.

[0205] In certain embodiments, an antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, or CNG-HSA-120 has a K of less than or equal to 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM as measured by SPR when the antigen-binding site is present as a monomer. Dand binds to mouse serum albumin. In certain embodiments, CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, CNG-HSA-200, CNG-HSA-201, CNG-HSA-202, CNG-HSA-203, CNG-HSA-204, CNG-HSA-205, CNG-HSA-206, CNG-HSA-207, CNG-HSA-208, CNG-HSA-209, CNG-HSA-310, CNG-HSA-311, CNG-HSA-312, CNG-HSA-313, CNG-HSA-314, CNG-HSA-315, CNG-HSA-316, CNG-HSA-317, CNG-HSA-318, CNG-HSA-319, CNG-HSA-320, CNG-HSA-321, CNG-HSA-322, CNG-HSA-323, CNG-HSA-324, CNG-HSA-325, CNG-HSA-326, CNG-HSA-327, CNG-HSA-328, CNG-HSA-329, CNG-HSA-330, CNG-HSA-331, CNG-HSA-332, CNG-HSA-333, CNG-HSA-334, CNG-HSA-33 - the antigen-binding site derived from HSA-118, CNG-HSA-119, or CNG-HSA-120 has a K in the range of 1-100 nM, 1-90 nM, 1-80 nM, 1-70 nM, 1-60 nM, 1-50 nM, 1-40 nM, 1-30 nM, 1-20 nM, or 1-10 nM, as measured by SPR when the antigen-binding site exists as a monomer. D and binds to mouse serum albumin.

[0206] In certain embodiments, the antigen-binding site derived from CNG-HSA-101, CNG-HSA-103, CNG-HSA-106, CNG-HSA-107, CNG-HSA-108, CNG-HSA-109, CNG-HSA-111, CNG-HSA-113, CNG-HSA-114, CNG-HSA-115, CNG-HSA-116, CNG-HSA-118, or CNG-HSA-120 is selected from the group consisting of a first K D binds to human serum albumin at a second K D binds to mouse serum albumin at the second K D Against the first King D is in the range of 0.5 to 10, 0.5 to 9, 0.5 to 8, 0.5 to 7, 0.5 to 6, 0.5 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.9 to 10, 0.9 to 9, 0.9 to 8, 0.9 to 7, 0.9 to 6, 0.9 to 5, 0.9 to 4, 0.9 to 3, 0.9 to 2, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. It is understood that an antigen-binding site having a ratio closer to 1 has an affinity for mouse serum albumin that is more similar to its affinity for human serum albumin, which enables more accurate evaluation of the pharmacokinetics of an antigen-binding site or a protein containing the same using a mouse model.

[0207] In certain embodiments, the antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, or CNG-HSA-117 has a higher binding affinity for Protein A compared to the antigen-binding site having the VH sequence set forth in SEQ ID NO: 196. It is understood that the increased affinity for Protein A allows for the purification of the antigen-binding site, or proteins comprising the antigen-binding site but not the antibody Fc region, by Protein A chromatography. In certain embodiments, an antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, or CNG-HSA-117 has a K of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, or 3 nM as measured by SPR when the antigen-binding site exists as a monomer. D binds to human serum albumin and has a K less than or equal to 2.5 nM or 2 nM D In certain embodiments, the antigen-binding site from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, or CNG-HSA-117 has a K in the range of 1-10 nM, 1-9 nM, 1-8 nM, 1-7 nM, 1-6 nM, 1-5 nM, 1-4 nM, or 1-3 nM, as measured by SPR when the antigen-binding site exists as a monomer. D binds to human serum albumin and has a K in the range of 1–2.5 nM or 1–2 nM D It binds to protein A.

[0208] In certain embodiments, an antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-116, or CNG-HSA-117 has a higher binding affinity for human serum albumin and a higher affinity for protein A compared to an antigen-binding site having the VH sequence set forth in SEQ ID NO: 196. In certain embodiments, an antigen-binding site derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-116, or CNG-HSA-117 has a K of less than or equal to 2.5 nM or 2 nM as measured by SPR when the antigen-binding site exists as a monomer. D In certain embodiments, the antigen-binding site from CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-116, or CNG-HSA-117 has a K in the range of 1-2.5 nM or 1-2 nM as measured by SPR when the antigen-binding site exists as a monomer. D It binds to protein A.

[0209] The melting temperature represents the thermostability of the antigen-binding site and can be measured by differential scanning fluorimetry, for example, as described in Durowoju et al. (2017) J. Vis. Exp. (121): 55262. The thermostability of an antibody or a fragment thereof can be enhanced by grafting CDRs onto a stable framework, introducing non-classical disulfide bonds, and other mutagenesis, as described in McConnell et al. (2014) MAbs, 6(5): 1274-82 and Goldman et al. (2017) Front. Immunol., 8: 865. In certain embodiments, antigen-binding sites derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-105, CNG-HSA-106, CNG-HSA-108, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, CNG-HSA-117, or CNG-HSA-120 have a melting temperature greater than or equal to 60° C. as measured by differential scanning fluorimetry. In certain embodiments, antigen-binding sites derived from CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-106, or CNG-HSA-120 have a melting temperature greater than or equal to 65° C. as measured by differential scanning fluorimetry.

[0210] In certain embodiments, the third antigen-binding site competes for binding to serum (e.g., human serum albumin) and / or for binding to Protein A with an antibody or antigen-binding site comprising a VH sequence provided in Table 5.

[0211] Alternatively, the third antigen-binding site that binds to serum albumin can be derived from the antigen-binding site disclosed in, for example, U.S. Patent No. 8,188,223 and PCT Publication Nos. WO2017085172 and WO2018050833. For example, in certain embodiments, the third antigen-binding site that binds to serum albumin is derived from an antibody listed in Table 6.

[0212] Table 6: Exemplary antigen-binding site sequences that bind to serum albumin TIFF0007780434000032.tif78150TIFF0007780434000033.tif230150TIFF0007780434000034.ti f232150TIFF0007780434000035.tif228150TIFF0007780434000036.tif232150TIFF00077804340 00037.tif228150TIFF0007780434000038.tif232150TIFF0007780434000039.tif232150TIFF0007780434000040.tif54150When the VL and LCDR sequences are shown as "N / A", the antigen-binding site is an sdAb having only a VH (e.g., VHH).When the VH and HCDR sequences are shown as "N / A", the antigen-binding site is an sdAb having only a VL.

[0213] In certain embodiments, the third antigen-binding site comprises a VH comprising 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%, at least 99%, or 100%) identical to the VH of an antibody disclosed in Table 6, and a VL comprising 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%, at least 99%, or 100%) identical to the VL of the same antibody disclosed in Table 6. In certain embodiments, the antigen-binding site comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the VH and / or VL sequences of the antibodies disclosed in Table 6, as determined by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262: 732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibody disclosed in Table 6. In certain embodiments, the antigen-binding site comprises the VH and VL sequences of an antibody disclosed in Table 6.

[0214] 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 provided in Table 6. In certain embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 of the VH sequences of the antibodies disclosed in Table 6 as determined by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J Mol Biol 262: 732-745), IMGT (see Lefranc, (1999) The Immunologist, 7, 132-136), or any other CDR determination method known in the art. In certain embodiments, the VH comprises the HCDR1, HCDR2, and HCDR3 sequences of an antibody provided in Table 6. In certain embodiments, the VH comprises the amino acid sequence of the VH of an sdAb provided in Table 6.

[0215] In certain embodiments, the third antigen-binding site has a K of less than or equal to 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 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, or 0.1 nM. DFor example, in certain embodiments, the third antigen-binding site binds to serum albumin (e.g., HSA) with a K of about 0.1 nM to about 100 nM, about 0.1 nM to about 50 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 1 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 1 nM to about 10 nM, about 10 nM to about 100 nM, or about 10 nM to about 50 nM. D The third antigen-binding site binds to serum albumin at a high affinity. Given the high abundance of serum albumin in blood, it is understood that a very high affinity of the third antigen-binding site for serum albumin may not be required for effective extension of the serum half-life of the multispecific binding protein. Therefore, an antigen-binding site with a lower affinity for serum albumin is also contemplated.

[0216] It is understood that the binding affinity of the third antigen-binding site alone to serum albumin may differ from the binding affinity of the same antigen-binding site in the context of a multispecific binding protein disclosed herein, possibly due to conformational constraints from other domains. Context-dependent binding affinities are described below in subsection G entitled "Binding Affinity."

[0217] 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.

[0218] D. 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 comprises two antibody variable domains (e.g., VH and VL). The 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 constitute a single antibody variable domain (e.g., sdAb).

[0219] 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 shown in subsection E below under the heading "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 7. 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 the 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 the amino acid sequence of SEQ ID NO: 298, 299, or 302, and the VH is located C-terminal to the VL.

[0220] Alternatively, the VH and VL may be present on separate polypeptide chains, and formation of the VH-VL complex may be facilitated by additional domains, such as antibody constant regions CH1 and CL. Thus, in certain embodiments, the multispecific binding protein comprises a Fab comprising a VH and a VL disclosed herein.

[0221] In certain embodiments, a multispecific binding protein of the invention comprises 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, hi certain embodiments, a multispecific binding protein comprises a first antigen-binding site in sdAb format, a second antigen-binding site in sdAb format, and a third antigen-binding site in sdAb format.

[0222] In certain embodiments, a multispecific binding protein of the invention comprises 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, hi certain embodiments, a multispecific binding protein comprises a first antigen-binding site in sdAb format, a second antigen-binding site in sdAb format, and a third antigen-binding site in scFv format.

[0223] In certain embodiments, a multispecific binding protein of the invention comprises 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, a multispecific binding protein comprises 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.

[0224] In certain embodiments, a multispecific binding protein of the invention comprises 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, a multispecific binding protein comprises 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.

[0225] In certain embodiments, a multispecific binding protein of the invention comprises 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, a multispecific binding protein comprises 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.

[0226] In certain embodiments, a multispecific binding protein of the invention comprises 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 protein comprises 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.

[0227] In certain embodiments, a multispecific binding protein of the invention comprises 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, a multispecific binding protein comprises 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.

[0228] 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.

[0229] The three antigen-binding sites of the multispecific binding protein are oriented from amino to carboxyl as follows: (i) first antigen-binding site (CD19-binding domain) - second antigen-binding site (CD3-binding domain) - 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) wherein the dashes above represent peptide bonds and / or linkers (e.g., peptide linkers).

[0230] In certain embodiments, the third antigen-binding site is not located between the first antigen-binding site and the second antigen-binding site. Constructs with such a format are intended to have favorable therapeutic efficacy and in vivo half-life. In certain embodiments, the third antigen-binding site is located at the N-terminal side of both the first antigen-binding site and the second antigen-binding site, or at the C-terminal side of both the first antigen-binding site and the second antigen-binding site. In certain embodiments, the third antigen-binding site is located at the N-terminal side of both the first antigen-binding site and the second antigen-binding site. In certain embodiments, the third antigen-binding site is located at the C-terminal side of both the first antigen-binding site and the second antigen-binding site.

[0231] The location (N- or C-terminal) of one antigen-binding site relative to another antigen-binding site is determined by defining "N- or C-terminal" as known in the art, provided that a single polypeptide chain contains both antigen-binding sites. When an antigen-binding site comprises two separate polypeptide chains, the location (N- or C-terminal) of an antigen-binding site relative to another antigen-binding site (having one or two polypeptide chains) can be similarly determined, provided that a single polypeptide chain contains at least one of the former polypeptide chain and at least one of the latter polypeptide chains. Furthermore, if antigen-binding site A is N-terminal to antigen-binding site B, which is N-terminal to antigen-binding site C, it is understood that 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 on a single common polypeptide chain. More complex structures of multispecific binding proteins are also contemplated, some of which may have locations that are difficult to characterize using the aforementioned terms "N-terminal" and "C-terminal," e.g., due to differences in the relative positions of two antigen-binding sites on one polypeptide chain compared to another, or due to the presence of loop structures.

[0232] According to the present invention, multispecific binding proteins and their component binding domains are in the form of one or more polypeptides. Such polypeptides may comprise proteinaceous and non-proteinaceous portions (e.g., chemical linkers or chemical cross-linking agents, e.g., glutaraldehyde). In certain embodiments, 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 aforementioned combinations, to form a single polypeptide chain.

[0233] E. Linker As described above, the antigen-binding sites of the multispecific binding proteins of the 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).

[0234] In certain embodiments, the three antigen binding sites of the multispecific binding protein are oriented from amino to carboxyl as follows: (i) first antigen-binding site (CD19-binding domain)-L1-second antigen-binding site (CD3-binding domain)-L2-third antigen-binding site (serum albumin-binding domain); (ii) the first antigen-binding site (CD19-binding domain)-L1-the third antigen-binding site (serum albumin-binding domain)-L2-the second antigen-binding site (CD3-binding domain); (iii) second antigen-binding site (CD3-binding domain)-L1-first antigen-binding site (CD19-binding domain)-L2-third antigen-binding site (serum albumin-binding domain); (iv) second antigen-binding site (CD3-binding domain)-L1-third antigen-binding site (serum albumin-binding domain)-L2-first antigen-binding site (CD19-binding domain); (v) a third antigen-binding site (serum albumin-binding domain)-L1-a first antigen-binding site (CD19-binding domain)-L2-a second antigen-binding site (CD3-binding domain); and (vi) third antigen-binding site (serum albumin-binding domain) - L1 - second antigen-binding site (CD3-binding domain) - L2 - first antigen-binding site (CD19-binding domain) and can be linked by linkers (e.g., peptide linkers) designated L1 and L2. It is understood that in certain constructs, L1, L2, or both L1 and L2 can be replaced with peptide bonds.

[0235] If one polypeptide chain comprises two adjacent antigen-binding sites, the peptide linker connecting the two antigen-binding sites is understood to be the amino acid sequence located between the two antigen-binding sites. If the antigen-binding sites comprise two separate polypeptide chains, one of the polypeptide chains is present in a common polypeptide as an adjacent antigen-binding site or polypeptide chain, and the peptide linker connecting the two antigen-binding sites is the amino acid sequence located between the two antigen-binding sites in a common polypeptide.

[0236] In certain embodiments, linkers L1 and L2 are peptide linkers. The appropriate lengths of L1 and L2 can be independently selected. For example, in certain embodiments, L1 and / or L2 are about 50 amino acid residues or less in length. In certain embodiments, L1 consists of about 50 amino acid residues or less. In certain embodiments, L1 consists of about 20 amino acid residues or less. In certain embodiments, L2 consists of about 50 amino acid residues or less. In certain embodiments, L2 consists of about 20 amino acid residues or less. In certain embodiments, L1 and L2 independently consist of about 50 amino acid residues or less. In certain embodiments, L1 and L2 independently consist of about 20 amino acid residues or less.

[0237] In some embodiments, the peptide linkers L1 and L2 have optimized lengths and / or amino acid compositions. In some embodiments, L1 and L2 are the same length and have the same amino acid composition. In other embodiments, L1 and L2 are different. In certain embodiments, L1 and / or L2 are "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 or fewer amino acid residues. In certain embodiments, L1 and / or L2 are "long," e.g., consisting of 15, 20, or 25 amino acid residues. In some embodiments, L1 and / or L2 consist of about 3 to about 15, e.g., 8, 9, or 10, consecutive amino acid residues.

[0238] Regarding the amino acid composition of L1 and L2, peptides are selected that have the properties of imparting flexibility to the multispecific binding proteins of the present invention, not interfering with the binding domain, and being resistant to cleavage by 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 where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, L1 and / or L2 are independently selected from the peptide sequences listed in Table 7. In some embodiments, L1 and / or L2 are independently selected from SEQ ID NOs: 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, or 302. In some embodiments, L1 and / or L2 are independently selected from SEQ ID NOs: 298, 299, and 302. In some embodiments, L1 and / or L2 comprise the amino acid sequence of SEQ ID NOs: 298, 299, or 302. In some embodiments, L1 and / or L2 consist of the amino acid sequence of SEQ ID NO: 298, 299, or 302. In some embodiments, L1 and L2 each comprise the amino acid sequence of SEQ ID NO: 298, 299, or 302. In some embodiments, L1 and L2 each consist of the amino acid sequence of SEQ ID NO: 298, 299, or 302.

[0239] Table 7: Exemplary peptide linker sequences TIFF0007780434000041.tif143150

[0240] The linkers disclosed herein, e.g., peptide linkers, can also be used to connect the VH and VL of an scFv, as mentioned above in subsection D entitled "Construct Formats."

[0241] F. Exemplary Multispecific Binding Proteins Examples of multispecific binding proteins comprising an scFv that binds CD19, an scFv that binds CD3, and an sdAb that binds serum albumin are listed in Table 8 below.

[0242] Table 8. Exemplary multispecific binding proteins TIFF0007780434000042.tif149161 1 The multispecific binding proteins in this table exist as a single polypeptide, and the format shows the CD19-binding scFv, CD3-binding scFv, and HSA-binding sdAb in the order listed from N-terminus to C-terminus. 2 The CD3-binding scFv sequences can contain or lack Cys substitutions at VH position 44 and VL position 100. As a result, two sequences are provided for the scFvs derived from each of the antibodies CNG-CD3-1, CNG-CD3-2, and CNG-CD3-3 (see also Table 4).

[0243] TIFF0007780434000043.tif195151TIFF0007780434000044.tif215151TIFF0007780434000045.tif230151TIFF0007780434000046.tif231151

[0244] The antigen binding sites listed in a particular row of Table 8 can be linked at the positions designated in the row through a peptide linker, as described above in subsection E. In certain embodiments, at least two adjacent antigen binding sites are linked by a peptide linker having the amino acid sequence of SEQ ID NO: 302. In certain embodiments, each of two adjacent antigen binding sites is linked by a peptide linker having the amino acid sequence of SEQ ID NO: 302, thereby forming a multispecific binding protein present in a single polypeptide.

[0245] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 121, 72, and 422. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO: 694.

[0246] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 121, 422, and 72. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO: 695.

[0247] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 72, 121, and 422. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO: 696.

[0248] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 422, 121, and 72. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO: 697.

[0249] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 72, 422, and 121. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO: 698.

[0250] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 422, 72, and 121. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO: 699.

[0251] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 121, 11, and 422. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO:700.

[0252] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 121, 422, and 11. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO:701.

[0253] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 422, 121, and 11. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO: 702.

[0254] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 11, 422, and 121. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO: 703.

[0255] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 121, 72, and 423. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO:704.

[0256] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 121, 72, and 427. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO:705.

[0257] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 121, 72, and 428. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO:706.

[0258] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 121, 72, and 433. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO:707.

[0259] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 121, 72, and 434. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO:708.

[0260] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 121, 11, and 427. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO:709.

[0261] In certain embodiments, the multispecific binding protein comprises, from N-terminus to C-terminus, SEQ ID NOs: 121, 11, and 433. In certain embodiments, the multispecific binding protein comprises the amino acid sequence of SEQ ID NO: 710.

[0262] In certain embodiments, the multispecific binding protein comprises an antigen-binding site that binds to CD19, an antigen-binding site that binds to CD3, and a half-life extending domain comprising an antibody Fc region as disclosed herein. The multispecific binding protein can take a variety of formats for combining the antigen-binding sites and the Fc region.

[0263] In certain embodiments, the multispecific binding protein comprises an anti-CD19 antibody in an IgG antibody format fused to a CD3-binding scFv at the C-terminus of the IgG Fc region. In certain embodiments, the multispecific binding protein comprises a first polypeptide chain comprising, from N-terminus to C-terminus, a VH of an antigen-binding site that binds to CD19, the CH1 domain, hinge, CH2 domain, and CH3 domain of an IgG antibody (e.g., human IgG1, IgG2, IgG3, or IgG4), and an scFv that binds to CD3, and a second polypeptide chain comprising, from N-terminus to C-terminus, a VL of an antigen-binding site that binds to CD19, and the light chain constant (CL) domain of an IgG antibody. In certain embodiments, the scFv that binds to CD3 comprises a VL domain located N-terminal to the VH domain. In certain embodiments, the IgG antibody is a human IgG1 antibody. In certain embodiments, the multispecific binding protein comprises two first polypeptide chains and two second polypeptide chains, thereby forming a dimeric antibody Fc region.

[0264] In certain embodiments, the multispecific binding protein comprises CNG-CD19-701 as the CD19-binding domain and CNG-CD3-1 as the CD3-binding domain. In certain embodiments, the multispecific binding protein comprises a polypeptide chain having the following two amino acid sequences: TIFF0007780434000047.tif105151

[0265] In certain embodiments, the multispecific binding protein comprises CNG-CD19-701 as the CD19-binding domain and CNG-CD3-2 as the CD3-binding domain. In certain embodiments, the multispecific binding protein comprises a polypeptide chain having the following two amino acid sequences: TIFF0007780434000048.tif105151

[0266] In certain embodiments, the multispecific binding protein comprises CNG-CD19-701 as the CD19-binding domain and CNG-CD3-3 as the CD3-binding domain. In certain embodiments, the multispecific binding protein comprises a polypeptide chain having the following two amino acid sequences: TIFF0007780434000049.tif104151

[0267] In certain embodiments, the multispecific binding protein comprises an anti-CD19 antibody in an IgG antibody format fused to a CD3-binding scFv at the C-terminus of the IgG light chain constant region. In certain embodiments, the multispecific binding protein comprises a first polypeptide chain comprising, from N-terminus to C-terminus, a VH of an antigen-binding site that binds to CD19, a CH1 domain, hinge, CH2 domain, and CH3 domain of an IgG antibody (e.g., human IgG1, IgG2, IgG3, or IgG4), and a second polypeptide chain comprising, from N-terminus to C-terminus, a VL of an antigen-binding site that binds to CD19, a light chain constant (CL) domain of an IgG antibody, and an scFv that binds to CD3. In certain embodiments, the scFv that binds to CD3 comprises a VL domain located N-terminal to the VH domain. In certain embodiments, the IgG antibody is a human IgG1 antibody. In certain embodiments, the multispecific binding protein comprises two first polypeptide chains and two second polypeptide chains, thereby forming a dimeric antibody Fc region.

[0268] In certain embodiments, the multispecific binding protein comprises CNG-CD19-701 as the CD19-binding domain and CNG-CD3-1 as the CD3-binding domain. In certain embodiments, the multispecific binding protein comprises a polypeptide chain having the following two amino acid sequences: TIFF0007780434000050.tif109151

[0269] In certain embodiments, the multispecific binding protein comprises CNG-CD19-701 as the CD19-binding domain and CNG-CD3-2 as the CD3-binding domain. In certain embodiments, the multispecific binding protein comprises a polypeptide chain having the following two amino acid sequences: TIFF0007780434000051.tif109151

[0270] In certain embodiments, the multispecific binding protein comprises CNG-CD19-701 as the CD19-binding domain and CNG-CD3-3 as the CD3-binding domain. In certain embodiments, the multispecific binding protein comprises a polypeptide chain having the following two amino acid sequences: TIFF0007780434000052.tif109151

[0271] In certain embodiments, the multispecific binding proteins disclosed herein further comprise a tag peptide, such as a Flag tag, a 6xHis tag, or a 10xHis tag (HHHHHHHHHH, SEQ ID NO:711). Such tag peptides are useful for purifying the multispecific binding protein. In certain embodiments, the tag peptide (e.g., a 10xHis tag) is located at the C-terminus of the multispecific binding protein. In certain embodiments, the tag peptide (e.g., a 10xHis tag) is located at the N-terminus of the multispecific binding protein.

[0272] G. Binding affinity In certain embodiments, the multispecific binding protein has a K in the range of about 5 pM to about 100 μM. D and binds to CD19 (e.g., human CD19), CD3 (e.g., human CD3 and / or macaque CD3), and / or serum albumin (e.g., HSA). D can be measured by methods known in the art, for example, by SPR, BLI, or flow cytometry as described in Examples 1, 2, or 7 below.

[0273] In certain embodiments, the multispecific binding protein 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. D For example, in certain embodiments, the multispecific binding protein binds to CD19, CD3, and / or serum albumin with a binding affinity of 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, or about 0.1 nM. ~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 10nM, K in the range of 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 and binds to CD19, CD3, and / or serum albumin.

[0274] 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. DIn certain embodiments, the multispecific binding protein binds with an affinity 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 nM to about 150 nM, or about 10 nM to about 200 nM. K in the range of 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 and binds to CD19, CD3, and / or serum albumin.

[0275] 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).

[0276] In certain embodiments, the multispecific binding proteins of the present disclosure bind to CD19, CD3, and / or serum albumin with K s that are 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 K of the multispecific binding protein to CD19, CD3, and / or serum albumin is DThe value is an increase of 1.5-fold or less, 2-fold or less, 3-fold or less, 4-fold or less, 5-fold or less, 6-fold or less, 7-fold or less, 8-fold or less, 9-fold or less, 10-fold or less, 15-fold or less, 20-fold or less, 25-fold or less, 30-fold or less, 35-fold or less, 40-fold or less, 45-fold or less, or 50-fold or less compared to that of the respective antigen-binding site alone or a monoclonal antibody having the same antigen-binding site.

[0277] 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 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 absence or significant absence of serum albumin.

[0278] H. Therapeutic activity The multispecific binding proteins disclosed herein are designed to simultaneously bind to B cells and T cells. Once T cells are recruited, B cell lysis is promoted, accompanied by cytolytic synapse formation and delivery of perforin and granzymes. Engaged T cells can continuously lyse target cells and are not subject to immune evasion mechanisms that interfere with peptide antigen processing and presentation or clonal T cell differentiation. See, e.g., WO2007042261A2. Thus, binding of the multispecific binding protein to target B cells results in target cell destruction and / or attenuates the progression of B cell-related diseases.

[0279] The cytotoxicity mediated by the multispecific binding proteins of the present 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. Typically, the effector:target cell (E:T) ratio is about 10:1, but this can also vary. Target cell killing is 51 Cell death 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, such as MTT or MTS assays, ATP-based assays including bioluminescence assays, sulforhodamine B (SRB) assays, WST assays, clonogenic assays, and ECIS techniques, are well known to those skilled in the art.

[0280] 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 provides an EC corresponding to the median effective concentration (the concentration of the multispecific binding protein that induces a cytotoxic response halfway between baseline and maximum). 50 In certain embodiments, the EC of the multispecific binding protein is expressed as a 50 The value is ≦5000 pM, e.g., ≦4000 pM, ≦3000 pM, ≦2000 pM, ≦1000 pM, ≦500 pM, ≦400 pM, ≦300 pM, ≦200 pM, ≦100 pM, ≦50 pM, ≦20 pM, ≦10 pM, ≦5 pM, ≦4 pM, ≦3 pM, ≦2 pM, or ≦1 pM.

[0281] EC 50 Values ​​are generally based on stimulated / enriched CD8 + When T cells are used as effector cells, it is understood that the activity is lower than that of unstimulated PBMCs. 50 It is generally understood that the value is 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 are 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 are used as target cells), the EC of the multispecific binding protein 50 The EC value of the multispecific binding protein when human PBMCs are used as effector cells is ≦1000 pM, e.g., ≦500 pM, ≦250 pM, ≦100 pM, 50 pM, ≦10 pM, or ≦5 pM. 50 The EC20 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 EC20 value of the multispecific binding protein ... 50 The value is ≦2000 pM, e.g., ≦1500 pM, ≦1000 pM, ≦500 pM, ≦300 pM, ≦250 pM, ≦100 pM, ≦50 pM, ≦10 pM, or ≦5 pM.

[0282] 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. 50Values ​​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.

[0283] 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 terms "do not induce lysis" or "do not mediate lysis" or grammatical equivalents mean that the multispecific binding protein, at concentrations 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%.

[0284] 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, each at the same molar concentration.

[0285] The cytotoxic activity of a 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 significant 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).

[0286] In certain embodiments, the multispecific binding proteins of the disclosure in the presence of serum albumin inhibit EC2 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 50 The 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 nonspecific effects may alter the EC values ​​nonspecifically. Nonspecific effects are measured by the EC values ​​of a control protein that does not contain a serum albumin binding domain in the presence or absence of serum albumin. 50In 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.

[0287] I. Construct 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. While small size generally contributes to rapid diffusion and tissue penetration, it is understood that 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.

[0288] In certain embodiments, the molecular weight of the multispecific binding protein is approximately between about 40 kD and about 90 kD, about 40 kD and about 80 kD, about 40 kD and about 70 kD, about 40 kD and about 60 kD, about 40 kD and about 50 kD, about 50 kD and about 100 kD, about 50 kD and about 90 kD, about 50 kD and about 80 kD, about 50 kD and about 70 kD, about 50 kD and about 60 kD, or about 60 kD and 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 between about 50 kD and about 90 kD, between about 50 kD and about 80 kD, between about 50 kD and about 70 kD, between about 50 kD and about 60 kD, between about 60 kD and about 90 kD, between about 60 kD and about 80 kD, between about 60 kD and about 70 kD, between about 65 kD and about 90 kD, between about 65 kD and about 80 kD, between about 65 kD and about 70 kD, between about 70 kD and about 90 kD, or between about 70 kD and about 80 kD.

[0289] 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 a tri-molecular (see ibid.). The binding of human FcRn to HSA and 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 distinct from the IgG binding site (see Chaudhury et al. (2006) Biochemistry 45:4983-90 and PCT Publication No. WO2008068280A1).

[0290] The present disclosure provides multispecific binding proteins with extended half-lives. 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 6. In certain embodiments, the serum half-life is measured in non-human primates. In certain embodiments, the serum half-life is measured in humans.

[0291] 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.

[0292] In certain embodiments, the serum half-life of the multispecific binding protein is at least 20% longer than that of a control multispecific binding protein, where the control multispecific binding protein comprises a first domain identical to the first antigen-binding site of the multispecific binding protein, a second domain identical to the second antigen-binding site of the multispecific binding protein, but does not comprise a third domain identical or substantially identical to the 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 half-life extending domain. 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 the 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.

[0293] III. Preparation method The antibodies and multispecific binding proteins described above can be produced using recombinant DNA technology well known to those skilled in the art. For example, one or more isolated polynucleotides encoding an antibody or multispecific binding protein can be linked to other appropriate nucleotide sequences, including, for example, constant region coding sequences and expression control sequences, to generate a conventional gene expression construct (i.e., expression vector) encoding the desired antibody or multispecific binding protein. The production of a defined gene construct is within the routine skill of those in the art.

[0294] Nucleic acids encoding the desired antibodies or multispecific binding proteins can be incorporated (ligated) into expression vectors and introduced into host cells 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. Transformed host cells can be grown under conditions that allow the host cells to express the genes encoding the antibodies or multispecific binding proteins.

[0295] Specific expression and purification conditions vary depending on the expression system used.For example, when expressing a gene in E. coli, first clone the engineered gene into an expression vector by placing it downstream of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence.Expressed protein may be secreted.Expressed protein may accumulate in refractile or inclusion bodies, which can be collected after cell disruption 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.

[0296] When the engineered gene is expressed in eukaryotic host cells, such as CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, secretion signal, polyA sequence, and stop codon. Optionally, the vector or gene construct may contain an enhancer and intron. In embodiments involving fusion proteins containing antibodies or portions thereof, the expression vector optionally contains a sequence encoding all or part of the constant region, thereby enabling all or part of the heavy or light chain to be expressed. Conventional techniques can be used to introduce this gene construct into eukaryotic host cells.

[0297] The antibodies or multispecific binding proteins disclosed herein may comprise a single polypeptide chain. In this case, a single 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 antibodies or 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. Alternatively, a single expression vector expressing two or more polypeptides can be transfected into a host cell. For example, the coding sequences for 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 for two or more polypeptides can also be separated by a ribosomal skip sequence or a self-cleaving sequence, e.g., a 2A peptide.

[0298] In certain embodiments, an N-terminal signal sequence is included in the protein construct for expression of an antibody or multispecific binding protein. Exemplary N-terminal signal sequences include those derived from interleukin-2, CD5, IgG kappa light chain, trypsinogen, serum albumin, and prolactin.

[0299] After transfection, single clones can be isolated for generation of 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 antibody or multispecific binding protein.

[0300] Antibodies or 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.

[0301] IV. Pharmaceutical Compositions The present disclosure also features pharmaceutical compositions containing a therapeutically effective amount of an antibody or multispecific binding protein described herein. The compositions can be formulated for use in various drug delivery systems. For appropriate 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).

[0302] 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, absorption 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., PEG-10 ... 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, flavoring agents, 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, solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronic, 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 chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients, and / or pharmaceutical adjuvants (see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990)).

[0303] 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).

[0304] In certain embodiments, the pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bioerodible 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 shaped 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.

[0305] Pharmaceutical compositions containing the antibodies or multispecific binding proteins disclosed herein can be provided in dosage unit form and can be prepared by any suitable method. Pharmaceutical compositions should be formulated to be compatible with their intended route of administration. Examples of routes of administration include intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal administration. In certain embodiments, recombinant human sialidase, recombinant human sialidase fusion protein, or antibody conjugate disclosed herein is administered by IV infusion. In certain embodiments, 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; an antibacterial agent, such as benzyl alcohol or methyl parabens; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as EDTA; a buffer, such as acetate, citrate, or phosphate; and an agent for adjusting tonicity, such as sodium chloride or dextrose.

[0306] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (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 a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0307] Intravenous drug delivery formulations may be contained in a syringe, pen, or bag. In certain embodiments, the bag may be connected to a conduit equipped with tubing and / or a 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 an intravenous drug formulation. In certain embodiments, the formulation is a liquid formulation and may be stored at about 250 mg to about 1,000 mg per vial. In certain embodiments, the formulation is a liquid formulation and may be stored at about 600 mg per vial. In certain embodiments, the formulation is a liquid formulation and may be stored at about 250 mg per vial.

[0308] 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 combined with a sterile aqueous carrier prior to administration. The pH of the preparation is typically 3 to 11, more preferably 5 to 9 or 6 to 8, and most preferably 7 to 8, e.g., 7 to 7.5. The resulting solid-form composition may be packaged as a plurality of single-dose units, each unit containing a predetermined amount of the aforementioned drug. The solid-form composition may also be packaged in containers for flexible amounts.

[0309] In certain aspects, the present disclosure provides an extended shelf life formulation comprising a protein of the present disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0310] In certain embodiments, aqueous formulations are prepared containing proteins of the present disclosure in a pH buffer solution. The pH of the buffers of the present invention may be about 4 to about 8, e.g., about 4.5 to about 6.0, or about 4.8 to about 5.5. The pH of the buffers of the present invention may be about 5.0 to about 5.2. Intermediate pH ranges within the above-listed ranges are also intended to be part of the present disclosure. For example, ranges of values ​​using any combination of the above-listed values ​​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.

[0311] 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 mg / ml), 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.

[0312] Polyols may act as tonicifiers, stabilizing the antibody or 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 relative to a disaccharide (e.g., trehalose). In certain embodiments, the 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.

[0313] A surfactant or surfactant may also be added to the formulation. Exemplary surfactants include nonionic 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 aggregation of the formulated antibody and / or minimizes particle formation 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 refer to 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.

[0314] In some embodiments, the protein product of the present disclosure is formulated as a liquid formulation. The liquid formulation may be provided at a concentration of 10 mg / mL in a USP / Ph Eur Type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made of an elastomer that complies with USP and Ph Eur. In certain embodiments, the liquid formulation may be diluted with 0.9% saline.

[0315] In certain embodiments, the liquid formulation of the present disclosure may be prepared as a solution of 10 mg / mL concentration by combining with a 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.

[0316] In certain embodiments, the pH of the liquid formulation may be adjusted by adding a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

[0317] The aqueous carrier of interest herein is pharmaceutically 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.

[0318] Preservatives may optionally be added to the formulations herein to reduce bacterial activity, which may, for example, facilitate the production of multi-use (multiple dose) formulations.

[0319] The antibody or 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.

[0320] 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 pharmaceutically acceptable acid and / or base prior to lyophilization. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharmaceutically 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.

[0321] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied to provide 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.

[0322] A specific dose may be a uniform dose for each patient, e.g., 50-5,000 mg of protein. Alternatively, a patient's dose can be tailored to the patient's approximate body weight or surface area. Other factors in determining the appropriate dosage may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and medical condition. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely performed by those skilled in the art, particularly in light of the dosage information and assays disclosed herein. Dosages can also be determined by the use of known assays for determining dosages, along with appropriate dose-response data. Individual patient dosages can be adjusted as disease progression is monitored. Blood concentrations of the targetable construct or complex in the patient can be measured to determine whether dosage adjustments are necessary to reach or maintain effective concentrations. Pharmacogenomics can be used to determine which targetable constructs and / or complexes and their 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).

[0323] Generally, 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, and about 50 mg to about 100 mg / kg body weight.

[0324] Doses may be given daily, weekly, monthly, or yearly or more frequently, or even once every 2 to 20 years. Those skilled in the art can readily assess repetition rates 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, or intracavity, 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.

[0325] V. Therapeutic uses It is contemplated that the antibodies or multispecific binding proteins can be used alone or in combination with other therapeutic agents.

[0326] A. Indications The present disclosure provides methods for treating or ameliorating a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease, or a host-versus-graft disease in a subject in need thereof, comprising administration of a multispecific binding protein or an antibody that binds CD19 disclosed herein.

[0327] In certain embodiments, the cancer to be treated is non-Hodgkin's lymphoma, for example, B-cell lymphoma.In certain embodiments, 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, ALL is relapsed / refractory adult ALL and pediatric ALL.

[0328] B. Combination Therapy The methods and compositions described herein can be used alone or in combination with other therapeutic agents and / or modalities. As used herein, the term "administered in combination" 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 a disorder. In certain embodiments, delivery of one treatment is still occurring when 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, delivery of one treatment ends before delivery of the other treatment begins. In certain embodiments in either case, the combined administration enhances the effectiveness of the treatments. For example, the second treatment is more effective. For example, the second treatment may not have an effect equivalent to that seen when the second treatment is administered in the absence of the first treatment, or the second treatment may reduce 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 may occur with the first treatment. In certain embodiments, the delivery is such that the reduction in 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 effects of the two treatments may be partially additive, wholly additive, or greater than additive. The delivery may be such that the effect of the first treatment delivered remains detectable when the second treatment is delivered.

[0329] In one aspect, the present disclosure provides methods of treating a subject by administering a second therapeutic agent in combination with one or more multispecific binding proteins and / or antibodies that bind CD19 disclosed herein.

[0330] 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, furunculosis ... These include thrombin, thrombin time, thrombin time, thrombin time (time), ...

[0331] Another class of drugs that may be used as part of combined therapy in cancer treatment 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.

[0332] 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, inhibiting or otherwise regulating 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. Patent Nos. 8,728,474 and 9,073,994 and European Patent No. 1537878B1, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, for example, 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.).

[0333] In certain embodiments, the methods or compositions described herein are administered in combination with CTLA-4 inhibitor.In the CTLA-4 pathway, T cell is inhibited when CTLA-4 on T cell interacts with its ligand (for example, CD80, also known as B7-1, and CD86) on the surface of antigen-presenting cell (not cancer cell).Exemplary CTLA-4-based immune checkpoint inhibition methods are described in US Patent No. 5,811,097, US Patent No. 5,855,887 and US Patent No. 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 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.

[0334] In certain embodiments, the methods or compositions described herein are administered in combination with (i) a PD-1 or PD-L1 inhibitor, e.g., a PD-1 or PD-L1 inhibitor disclosed herein, and (ii) a CTLA-4 inhibitor, e.g., a CTLA-4 inhibitor disclosed herein.

[0335] 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.

[0336] 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).

[0337] Further categories of anti-cancer 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 plus 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.

[0338] It is understood that the antibodies or multispecific binding proteins disclosed herein are designed to activate T lymphocytes and may cause side effects such as neurotoxicity. Accordingly, in certain embodiments, a second therapeutic agent that can be used in combination with an antibody or multispecific binding protein includes an agent that alleviates the side effects of the antibody or 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 lonopterin, 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 (e.g., tocilizumab).

[0339] The amounts and relative timing of administration of the antibody or multispecific binding protein and additional therapeutic agent 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 antibody or multispecific binding protein may be administered while the additional therapeutic agent exerts a prophylactic or therapeutic effect, or vice versa.

[0340] Throughout the description, when compositions are described as having, including, or comprising particular ingredients, or processes and methods are described as having, including, or comprising particular steps, it is intended that there are also compositions of the invention that consist essentially of or consist of the recited ingredients, and processes and methods according to the invention that consist essentially of or consist of the recited processing steps.

[0341] 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 that the element or component may be selected from the group consisting of two or more of the recited elements or components.

[0342] 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 particular compound is referred to, unless otherwise understood from the context, this compound can be used in various embodiments of the compositions of the present invention and / or in 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 present 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.

[0343] 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 connection with more than two listed objects shall be understood to have the same meaning, unless otherwise understood from context.

[0344] Unless otherwise stated or understood from the context, use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, is to be understood as generally open-ended and open-ended, without excluding, for example, additional, unrecited elements or steps.

[0345] 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.

[0346] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Further, two or more steps or actions may be conducted simultaneously.

[0347] The use of any and all examples or exemplary language herein, such as "such as" or "including," is merely intended to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise 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.

[0348] 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. [Example]

[0349] Having now generally described the invention, the same will be more 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.

[0350] Example 1. Characterization of new anti-CD19 antibodies This example describes new anti-CD19 antibodies, CNG-CD19-101 to CNG-CD19-110 and CNG-CD19-701 to CNG-CD19-710. The amino acid sequences of CNG-CD19-101 to CNG-CD19-110 are provided in Table 1 above, and the amino acid sequences of CNG-CD19-701 to CNG-CD19-710 are provided in Table 2 above.

[0351] CNG-CD19-101 to CNG-CD19-110 and CNG-CD19-701 to CNG-CD19-710 were optimized from the parent antibodies CNG-CD19-1 and CNG-CD19-7, respectively, by introducing diversity into the VH and / or VL and shuffling fragments of the VH and VL sequences. Antibody clones were selected for improved binding affinity to biotinylated human CD19 compared to their respective parent antibodies. Selected antibodies were then produced from yeast cells and purified using a Protein A column.

[0352] The binding affinity of the antibody to isolated CD19 was measured by surface plasmon resonance using a ForteBio Octet HTX system as previously described (see, e.g., Estep et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs 5(2), 270-278 (2013)). Briefly, ForteBio affinity measurements were performed by online loading of IgG onto an anti-hIgG Fc Capture (AHC) sensor. The sensor was equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. The IgG-loaded sensor was exposed to 100 nM human serum albumin for 3 minutes and then transferred to assay buffer for 3 minutes for off-rate measurements. All reaction kinetics were analyzed using a 1:1 binding model.

[0353] The binding affinity of the antibody to CD19 expressed on the cell surface was also measured. Briefly, approximately 100,000 Chinese hamster ovary (CHO) cells overexpressing the antigen were washed with wash buffer and incubated with 100 μl of 100 nM IgG for 15 minutes at room temperature. The cells were then washed twice with wash buffer and incubated with 100 μl of 1:100 Human-PE for 15 minutes on ice. The cells were then washed twice more with wash buffer and analyzed using a FACS Canto II analyzer (BD Biosciences).

[0354] Table 9. Binding of anti-CD19 antibodies to CD19 protein and CD19-expressing cells TIFF0007780434000053.tif106155

[0355] As shown in Table 9, CNG-CD19-101 to CNG-CD19-110 showed higher binding affinity to human CD19 and / or cynomolgus monkey CD19 than CNG-CD19-1. In particular, CNG-CD19-101 to CNG-CD19-109 showed lower K binding affinity to human CD19 than CNG-CD19-1. D CNG-CD19-103 to CNG-CD19-110 bind to cynomolgus monkey CD19 with a lower K value than CNG-CD19-1. D CNG-CD19-101 and CNG-CD19-103 to CNG-CD19-110 showed increased binding to CHO cells expressing human CD19 compared to CNG-CD19-1.

[0356] Table 10. Binding of anti-CD19 antibodies to CD19 protein and CD19-expressing cells TIFF0007780434000054.tif106155

[0357] As shown in Table 10, CNG-CD19-701 to CNG-CD19-710 showed higher binding affinity to human CD19 and cynomolgus monkey CD19 than CNG-CD19-7. In particular, CNG-CD19-701 to CNG-CD19-710 showed lower K values ​​to human CD19 and cynomolgus monkey CD19 than CNG-CD19-7. D CNG-CD19-701 to CNG-CD19-710 also showed increased binding to CHO cells expressing human CD19 compared to CNG-CD19-7.

[0358] Additional kinetic analysis of anti-CD19 VL-VH scFvs with or without disulfide bonds introduced by Cys substitutions at positions 44 in VH and 100 in VL was performed by surface plasmon resonance (SPR). The scFv proteins were serially diluted and assayed at 25°C in an HBS-EP+ running buffer system (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20) using a Biacore 8K optical biosensor (Global Life Sciences Solutions USA, Marlborough, MA). The sample compartment was maintained at 10°C for the duration of each experiment.

[0359] Each experimental cycle began with the injection of a 1:20 solution of biotin CAPture reagent (Global Life Sciences Solutions USA) in running buffer across flow cells 1 and 2 (2 μL / min for 500 s). This was followed by the injection of biotinylated CD-19 (40 nM) into flow cell 2 (4.0 μL / min for 300 s). Upon capture of CD19 onto the sensor surface, a test scFv (16.2–0.067 nM, 3-fold diluted in running buffer) was injected across flow cells 1 and 2 (30 μL / min for 300 s). Dissociation of the scFv was monitored over a duration of 2564 s. Finally, the sensor surface was prepared for another cycle with the injection of regeneration solution (6 M guanidine-HCl in 0.25 M NaOH) across flow cells 1 and 2 (10 μL / min for 90 s).

[0360] The resulting data were processed and fitted as follows: the sensorgrams were trimmed to include only the association and dissociation phases of the scFv. The trimmed data were then aligned, double-referenced, and fitted to a 1:1 binding model using nonlinear least-squares fitting with Biacore Insight Evaluation software version 3.0.11.15423 (Myszka D. (1999) J. Mol. Recognit. 12(5):279-284).

[0361] The resulting K for each tested scFv D and k off The values ​​are summarized in Table 11.

[0362] Table 11. Binding of anti-CD19 antibodies to human CD19 protein TIFF0007780434000055.tif60152 1 In each scFv construct, disulfide bonds were introduced by Cys substitutions at positions 44 in VH and 100 in VL.

[0363] Example 2. Characterization of new anti-serum albumin antibodies This example describes new anti-serum albumin antibodies, CNG-HSA-101 through CNG-HSA-120, whose amino acid sequences are provided in Table 5 above.

[0364] CNG-HSA-101 to CNG-HSA-120 were optimized from the parent single-domain antibody CNG-HSA-1 by introducing diversity into the heavy chain variable region, generating random mutations by error-prone PCR, and shuffling the VH fragment. Antibody clones were selected for improved binding affinity to biotinylated human serum albumin compared to the parent antibody. Additionally, thermal selection pressure was applied for the VH shuffling optimization cycles. Thermal selection pressure was applied by incubating the library at various temperatures and then selecting antibodies that retained antigen binding after thermal incubation. The selected antibodies were then produced from yeast cells and purified using a protein A column.

[0365] The binding affinity of the antibody to isolated serum albumin was measured by surface plasmon resonance using a ForteBio Octet HTX system as previously described (see, e.g., Estep et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs 5(2), 270-278 (2013)). Briefly, ForteBio affinity measurements were performed by online loading of heavy chain antibodies (HCAbs) onto AHC sensors. The sensors were equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. The HCAb-loaded sensors were exposed to 100 nM human serum albumin for 3 minutes and then transferred to assay buffer for 3 minutes for off-rate measurements. All reaction kinetics were analyzed using a 1:1 binding model.

[0366] The melting temperatures (Tm) of VHH fragments were measured by dynamic scanning fluorimetry (DSF). Briefly, 10 μL of 20× Sypro Orange dye was added to 20 μL of 0.2–1 mg / mL HCAb. Using a BioRad CFX96 RT PCR machine, the sample plate temperature was increased from 40°C to 90°C in 0.5°C increments, with a 2-minute equilibration at each temperature. Tm was extracted using the negative first derivative from the raw data.

[0367] Table 12. Binding of anti-serum albumin antibodies to serum albumin and protein A TIFF0007780434000056.tif207155 1 NB means that no binding was detected under the conditions of the assay. 2 ND means not determined.

[0368] As shown in Table 12, CNG-HSA-101 to CNG-HSA-120 exhibited higher binding affinity than CNG-HSA-1 for human serum albumin, cynomolgus monkey serum albumin, mouse serum albumin, and / or protein A. Notably, all of these antibodies exhibited lower K values ​​for mouse serum albumin than CNG-HSA-1. D CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-108, CNG-HSA-109, CNG-HSA-110, CNG-HSA-111, CNG-HSA-112, CNG-HSA-115, CNG-HSA-116, CNG-HSA-117, CNG-HSA-118, CNG-HSA-119, and CNG-HSA-120 bound to human serum albumin and cynomolgus monkey serum albumin with lower K values ​​than CNG-HSA-1. DCNG-HSA-101, CNG-HSA-103, CNG-HSA-106, CNG-HSA-107, CNG-HSA-108, CNG-HSA-109, CNG-HSA-111, CNG-HSA-113, CNG-HSA-114, CNG-HSA-115, CNG-HSA-116, CNG-HSA-118, and CNG-HSA-120 bound to mouse serum albumin at K values ​​similar to those observed for the same antibodies bound to human serum albumin. D K less than 4 times higher than D CNG-HSA-101, CNG-HSA-102, CNG-HSA-104, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, and CNG-HSA-117 bound to Protein A with lower K values ​​than CNG-HSA-1. D CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-104, CNG-HSA-105, CNG-HSA-106, CNG-HSA-108, CNG-HSA-109, CNG-HSA-113, CNG-HSA-116, CNG-HSA-117, and CNG-HSA-120 exhibited melting temperatures greater than or equal to 60°C, and among them, CNG-HSA-101, CNG-HSA-102, CNG-HSA-103, CNG-HSA-106, and CNG-HSA-120 exhibited melting temperatures greater than or equal to 65°C.

[0369] Example 3. Production of multispecific binding proteins This example describes the production and purification of a multispecific binding protein.

[0370] Nucleic acids encoding single-chain multispecific binding proteins (see Table 13) were constructed, codon-optimized for expression in human cells, and cloned into mammalian expression vectors according to standard procedures. After sequence verification, the expression vectors in the form of plasmids were prepared in sufficient quantities 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 vectors and cultured for 6 days.

[0371] The amino acid sequences of the various multispecific binding proteins are summarized in Table 13. Constructs tAb0027 to 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 to 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.

[0372] Table 13. Exemplary multispecific binding proteins TIFF0007780434000057.tif203150TIFF0007780434000058.tif228150TIFF00077804340 00059.tif228150TIFF0007780434000060.tif228150TIFF0007780434000061.tif252150

[0373] 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 in 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 the protein sample was concentrated as needed. The protein purity 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. Fractions with elution times of approximately 225–240 min were collected for further characterization.

[0374] As described above, some of the generated constructs contained anti-CD19 scFvs having the amino acid sequences shown in SEQ ID NO:9 or 18. The binding affinities of the two CD19 binding domains to CD19 were 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 the monomeric CD19 protein was 0.01. 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.

[0375] Some of the multispecific binding proteins contained anti-CD3 scFv derived from CNG-CD3-1, CNG-CD3-2, or CNG-CD3-3, having the amino acid sequences of SEQ ID NOs: 422, 427, or 433, respectively, in Table 4. The binding af...

Claims

1. (a) a first antigen-binding site that binds to human CD19, comprising a heavy chain complementarity-determining region (HCDR) and a light chain complementarity-determining region (LCDR), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:65, HCDR2 comprises the amino acid sequence of SEQ ID NO:66, HCDR3 comprises the amino acid sequence of SEQ ID NO:68, LCDR1 comprises the amino acid sequence of SEQ ID NO:69, LCDR2 comprises the amino acid sequence of SEQ ID NO:70, and LCDR3 comprises the amino acid sequence of SEQ ID NO:71; (b) a second antigen-binding site that binds to human CD3, comprising a heavy chain complementarity-determining region (HCDR) and a light chain complementarity-determining region (LCDR), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:415, HCDR2 comprises the amino acid sequence of SEQ ID NO:416, HCDR3 comprises the amino acid sequence of SEQ ID NO:418, LCDR1 comprises the amino acid sequence of SEQ ID NO:419, LCDR2 comprises the amino acid sequence of SEQ ID NO:420, and LCDR3 comprises the amino acid sequence of SEQ ID NO:421; (c) a third antigen-binding site that binds to human serum albumin, comprising heavy chain complementarity-determining regions (HCDRs), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 123, HCDR2 comprises the amino acid sequence of SEQ ID NO: 124, and HCDR3 comprises the amino acid sequence of SEQ ID NO:

125. wherein the first antigen-binding site and the second antigen-binding site comprise single-chain variable fragments (scFvs), and the third antigen-binding site comprises a single-domain antibody (sdAb).

2. 10. The multispecific binding protein of claim 1, wherein the multispecific binding protein comprises one polypeptide chain.

3. In the polypeptide chain, the third antigen-binding site is located on the N-terminal side of both the first antigen-binding site and the second antigen-binding site. 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; or 3. The multispecific binding protein of claim 2, wherein in the polypeptide chains, the 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.

4. In the polypeptide chain, the third antigen-binding site is located C-terminally to both the first antigen-binding site and the second antigen-binding site. In the polypeptide chain, a first antigen-binding site is located N-terminal to a second antigen-binding site, and a second antigen-binding site is located N-terminal to a third antigen-binding site; or 3. The multispecific binding protein of claim 2, wherein in the polypeptide chains, 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.

5. in the polypeptide chain, the first 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 second antigen-binding site; or 3. The multispecific binding protein of claim 2, wherein in the polypeptide chains, 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 first antigen-binding site.

6. (a) the multispecific binding protein of any one of claims 1 to 5; and (b) a pharmaceutically acceptable carrier 10. A pharmaceutical composition comprising:

7. 6. An isolated polynucleotide encoding the multispecific binding protein of any one of claims 1 to 5.

8. A vector comprising the polynucleotide of claim 7.

9. A recombinant host cell comprising the polynucleotide of claim 7 or the vector of claim 8.

10. 6. A pharmaceutical composition comprising the multispecific binding protein of any one of claims 1 to 5, for stimulating an immune response against cells expressing CD19 by exposing said cells and T lymphocytes to said multispecific binding protein.

11. 10. A pharmaceutical composition for treating hematological cancer in a subject in need thereof, comprising an effective amount of the multispecific binding protein of any one of claims 1 to 5.

12. 12. The pharmaceutical composition of claim 11, wherein the hematological cancer is a B-cell hematological malignancy.

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