Multispecific antigen binding proteins capable of binding CD19 and CD3, and use thereof

Abstract

To provide multispecific antigen binding proteins capable of binding CD19 and CD3, and use thereof.SOLUTION: Provided are multispecific antigen binding proteins (MSAPs) that specifically bind to CD3 and CD19. Further provided are uses of the MSAPs for the preparation of pharmaceutical compositions, methods of treating cancer, and kits comprising the MSAPs. Some antigens are overexpressed, mutagenized or selectively mutagenized in tumor tissues. Therefore, antibodies targeting specific antigens on the surface of cancer cells can be used as cancer therapeutics.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to International Patent Application No. PCT / CN2018 / 123108, filed December 24, 2018, the entire contents of which are incorporated herein by reference.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of Sequence Listing (Filename: 720622001641SEQLIST.TXT, Recorded: December 11, 2019, Size: 54KB).

[0003] The present invention relates to a multispecific antigen binding protein (MSAP) that specifically binds to CD3 and CD 19. Further provided herein are pharmaceutical compositions comprising MSAP, methods of treating cancer using MSAP, and kits comprising MSAP. [Background technology]

[0004] Some antigens are overexpressed, mutated, or selectively mutated in tumor tissue. Therefore, antibodies targeting specific antigens on the surface of cancer cells can be used as cancer therapeutics. B-lymphocyte antigen CD19 is also known as CD19 molecule (cluster of differentiation 19), B-lymphocyte surface antigen B4, T-cell surface antigen Leu-12, and CVID3. CD19 is expressed on both normal and malignant B-lymphocytes and is considered a B-cell tumor-associated antigen. It can be used as a biomarker for B-lymphocyte differentiation, a diagnosis of lymphoma, and a target for leukemia immunotherapy.

[0005] CD3 is an antigen expressed by T cells that contains three distinct polypeptide chains (ε, δ and γ chains). The three CD3 polypeptide chains associate with the T cell receptor (TCR) and ζ chain to form the TCR complex, which functions to activate the signaling cascade in T cells. Currently, many therapeutic strategies target TCR signaling and use anti-human CD3 monoclonal antibodies to treat diseases. The CD3-specific antibody OKT3 was the first monoclonal antibody approved for therapeutic use in humans and is used in the clinic as an immunomodulatory agent for the treatment of allograft rejection.

[0006] Although bispecific antibodies have been shown to be capable of effectively killing cancer cells, the severe adverse effects of these molecules, including systemic immune activation, immunogenicity (anti-drug antibody effect), and generally poor manufacturability, have significantly limited the broad application of this class of drugs.For example, one drawback of the CD19xCD3 bispecific scFv-scFv (single-chain variable fragment) fusion protein (blinatumomab) is that the drug has a short half-life and is incompatible with subcutaneous administration, so it must be administered intravenously (iv) every day, and still neurological effects such as disorientation, confusion, speech and language disorders, tremors or convulsions still occur during clinical trials (Bargou et al. Science 321(5891):974-797,2008). The shortcomings of current bispecific antibody formats remain a major challenge for their widespread use in treating cancer patients with good efficacy and safety.Therefore, there is an urgent need in the art to develop new bispecific antibodies or treatment regimens with improved efficacy, stability, safety and manufacturability. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Bargou et al.Science 321(5891):974-797,2008 Summary of the Invention [Means for solving the problem]

[0008] The present invention provides multispecific antigen binding proteins ("MSAP," bispecific antigen binding proteins, "BSAP," etc.) that specifically bind to CD3 and CD19, pharmaceutical compositions comprising MSAP, and methods of treating cancer using MSAP.

[0009] In one aspect of the invention, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an immunoglobulin (Ig) heavy chain variable region (VH) and an Ig heavy chain constant region (CH1), and (b) an Ig light chain variable region (VL) and an Ig light chain constant region (CL), optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19; and III. optionally, a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the MSAP (such as a BSAP) comprises a first anti-CD19 antigen-binding fragment (e.g., an scFv) and a second anti-CD19 antigen-binding fragment (e.g., an scFv), where the first anti-CD19 antigen-binding fragment is fused to the N-terminus of the VH of an anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.

[0010] In some embodiments according to any of the MSAPs (such as BSAPs) described above, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε, such as an epitope within amino acids 1-27 of CD3ε. In some embodiments, the VH of the anti-CD3 Fab fragment comprises a heavy chain hypervariable region H1 (HVR-H1) comprising the amino acid sequence of SEQ ID NO:9, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:11, and / or the VL of the anti-CD3 Fab fragment comprises a light chain hypervariable region L1 (HVR-L1) comprising the amino acid sequence of SEQ ID NO:12, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds, such as about 2 disulfide bonds. In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18 and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond.

[0011] In some embodiments according to any of the MSAPs (such as BSAPs) described above, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or a VL comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, HVR-L2 comprising the amino acid sequence of SEQ ID NO:48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:6 or 49. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH, wherein the VH of the anti-CD19 antigen-binding fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VL, wherein the VL of the anti-CD19 antigen-binding fragment comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, HVR-L2 comprising the amino acid sequence of SEQ ID NO:48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:49. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50. In some embodiments, the VH of the anti-CD19 antigen-binding fragment (e.g., scFv) comprises the amino acid sequence of SEQ ID NO:7, and / or the VL of the anti-CD19 antigen-binding fragment (e.g., scFv) comprises the amino acid sequence of SEQ ID NO:50.

[0012] In some embodiments according to any of the MSAPs (such as BSAPs) described above, the anti-CD19 antigen-binding fragment is a single chain variable fragment (scFv). In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the MSAP (such as BSAP) comprises a first anti-CD19 scFv and a second anti-CD19 scFv. In some embodiments, the first and second anti-CD19 scFv have the same amino acid sequence. In some embodiments, the first and second anti-CD19 scFv have different amino acid sequences.

[0013] In some embodiments according to any of the MSAPs (e.g., BSAPs) described above, the linker comprises about 2 to about 30 amino acid residues (e.g., about 2 to about 15 amino acid residues) selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44, such as SEQ ID NO: 44.

[0014] In some embodiments according to any of the MSAPs (such as BSAPs) described above, the MSAP comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:53 or 60, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:53, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:60, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:61.

[0015] In some embodiments according to any of the MSAPs (such as BSAPs) described above, the MSAP comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:53 or 60, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:58. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:53, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:58. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:60, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:58.

[0016] In some embodiments according to any of the MSAPs (such as BSAPs) described above, the MSAP (such as BSAPs) comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:57 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:57 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:57 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:61.

[0017] Further provided are isolated nucleic acid molecules encoding any of the above-described MSAPs (such as BSAPs), expression vectors carrying the isolated nucleic acid molecules, isolated host cells comprising the expression vectors, and methods of producing any of the above-described MSAPs comprising culturing the isolated host cells and recovering the MSAP from the cell culture.

[0018] Also provided herein are uses, compositions (such as pharmaceutical compositions), kits and articles of manufacture comprising any of the MSAPs (such as BSAPs) described above. In some embodiments, provided are compositions (such as pharmaceutical compositions) comprising any of the MSAPs (such as BSAPs) described above, and optionally a pharma- ceutically acceptable carrier.

[0019] Further provided herein is the use of any of the MSAPs (such as BSAPs) described above in the preparation of a medicament for treating cancer. In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual in need thereof is provided, the method comprising administering to the individual an effective amount of any of the MSAPs (such as BSAPs) described above or a composition (such as a pharmaceutical composition) thereof. In some embodiments, the MSAP (such as BSAP) or composition (such as a pharmaceutical composition) is administered intravenously. In some embodiments, the individual is a human. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), including blast crisis of CML and CML including the Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), including Richter's syndrome or Richter's transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), including NHL including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma. In some embodiments, the cancer is DLBCL. In some embodiments, the cancer is ALL.

[0020] These and other aspects and advantages of the present invention will be apparent from the following detailed description and the appended claims. It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention.

[0021] The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entirety. [Brief description of the drawings]

[0022] [Figure 1] A illustrates the structure of an exemplary CD19xCD3 MSAP ITAB2009. B illustrates the structure of an exemplary CD19xCD3 MSAP ITAB2007. [Diagram 2] Figure 1 shows CD19xCD3 MSAP (ITAB2009 or ITAB2007) mediated human PBMC cytotoxicity against autologous B cells. B cells mixed with human PBMC only without the addition of CD19xCD3 MSAP served as control. [Diagram 3] Figure 1 shows CD19xCD3 MSAP (ITAB2009 or ITAB2007) mediated human PBMC cytotoxicity against Daudi tumor cells. Daudi tumor cells mixed with human PBMC only without the addition of CD19xCD3 MSAP served as control. [Figure 4] The CD19 expression levels of different cells as reflected by anti-CD19 staining signals are shown. [Figure 5-1] CD19xCD3 MSAP (ITAB2009 or ITAB2007)-mediated T cell activation in the presence of Pfeiffer cells (FIG. 5A). T cells mixed with Pfeiffer cells without the addition of CD19xCD3 MSAP served as a control. [Figure 5-2] CD19xCD3 MSAP (ITAB2009 or ITAB2007)-mediated T cell activation in the presence of Daudi cells (FIG. 5B). T cells mixed with Daudi cells without the addition of CD19xCD3 MSAP served as a control. [Figure 6] 1 shows CD19×CD3 MSAP (ITAB2009 or ITAB2007)-mediated human Pri-T cell cytotoxicity against Reh cells. [Figure 7]Figure 1 shows the growth inhibitory effect of ITAB2007 CD19xCD3 MSAP on subcutaneous Raji xenograft tumors in immune reconstituted NOG mice inoculated with human PBMCs. Vehicle injection (PBST) served as a control. The results showed that ITAB2007 inhibited the growth of Raji xenograft tumors in huPBMC-implanted mice in a concentration-dependent manner. [Figure 8] Figure 2 shows the survival rate of Reh-vaccinated mice with immune systems reconstituted with human primary T cells upon treatment with CD19xCD3 MSAP or vehicle control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention provides multispecific antigen binding proteins (MSAPs) comprising an anti-CD3 Fab fragment that specifically binds to CD3 and an anti-CD19 antigen binding fragment (e.g., scFv) that specifically binds to CD19 (hereinafter referred to as "CD3xCD19 MSAPs"). In some embodiments, the CD3xCD19 MSAP (such as a BSAP) comprises a first anti-CD19 scFv and a second anti-CD19 scFv, wherein the first anti-CD19 scFv binds an anti-CD3 A first anti-CD19 scFv is fused to the N-terminus of the VH of the Fab fragment and a second anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the two anti-CD19 scFvs are the same. The invention also provides methods of treating cancer, particularly hematological malignancies, using the CD3xCD19 MSAPs (such as BSAPs) described herein.

[0024] Current anti-cancer bispecific antibodies suffer from several drawbacks, such as poor manufacturability, aggregation, short half-life, severe adverse effects (systemic immune activation, immunogenicity (anti-drug antibody response), etc.), long infusion time, and inability to be retained in tumor tissue, which poses a great challenge to the broad application of these anti-cancer bispecific antibodies in cancer treatment with good efficacy and safety. For example, blinatumomab (BLINCYTO®, anti-CD3 / anti-CD19 bispecific scFv-scFv) was approved in the United States in 2014 as a second-line treatment for Philadelphia chromosome-negative relapsed or refractory acute lymphoblastic leukemia (ALL). However, due to its short half-life and incompatibility with subcutaneous administration, blinatumomab must be administered intravenously (iv) daily, and neurological effects, such as disorientation, confusion, speech and language disorders, tremors or convulsions, still occurred during clinical trials (Bargou et al. Science 321(5891):974-797, 2008).

[0025] After extensive research, the inventors of the present application unexpectedly discovered the CD3×CD19 MSAP (such as BSAP) format, which fuses an anti-CD19 antigen-binding fragment (such as scFv) to the N-terminus of an anti-CD3 Fab fragment. The CD3×CD19 MSAP (such as BSAP) described herein has been found to have several advantages over other multispecific proteins known in the art. First, the CD3×CD19 MSAP (such as BSAP) described herein has enhanced cytotoxic activity against cancer cells, particularly against cancer cells of CD19-low expressing tumors, such as B-cell lymphomas and acute lymphoblastic leukemias (ALL). Second, the CD3×CD19 MSAP (such as BSAP) described herein cross-reacts with non-human primates, such as cynomolgus monkeys, which may be useful for toxicity studies on non-human primates (such as cynomolgus monkeys) for the benefit of predicting human clinical trials. Third, the CD3×CD19 MSAPs (such as BSAPs) described herein have extended half-lives, potentially allowing for less frequent dosing and shorter infusion times, providing greater convenience for patients.

[0026] Thus, in one aspect, the invention provides an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an immunoglobulin (Ig) heavy chain variable region (VH) and an Ig heavy chain constant region (CH1), and (b) an Ig light chain variable region (VL) and an Ig light chain constant region (CL); II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19; and III. optionally, a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv). In another aspect, the invention provides an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an immunoglobulin (Ig) heavy chain variable region (VH) and an Ig heavy chain constant region (CH1), and (b) an Ig light chain variable region (VL) and an Ig light chain constant region (CL), wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds CD19; and III. optionally, a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv).

[0027] Pharmaceutical compositions and kits comprising the CD3xCD19 MSAPs (such as BSAPs) described herein, and methods of their use to treat cancer are also provided.

[0028] I. Definition The practice of the present invention will employ, unless specifically indicated to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology within the skill of those in the art, many of which are described below for illustrative purposes. Such techniques are explained fully in the literature; see, for example, Current Protocols in Molecular Biology, 1999, 1999, 1999, 1999, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1999, 1998, 1999, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2020, 2021, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2050, 2051, 2052, 2060, 2061, 2070, 2071, 2072 Biology or Current Protocols in Immunology, John Wiley&Sons, New York, NY (2009), Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley&Sons, 1995, Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001), Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), DNA Cloning: A Practical Approach, vol. I&II (D. Glover, ed.), Oligonucleotide Synthesis (N. Gait, ed., 1984), Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985), Transcription and Translation (B. Hames & S. Higgins, eds., 1984), Animal Cell Culture (R. Freshney, ed., 1986), Perbal, A Practical See Guide to Molecular Cloning (1984) and other similar references.

[0029] As used herein, the term "treatment" refers to a clinical intervention designed to change the natural history of the individual or cells being treated in the course of clinical pathology. The desired effects of treatment include slowing the rate of disease progression, improving or alleviating the disease state, and remission or improving prognosis. For example, an individual is successfully "treated" when one or more symptoms associated with cancer are alleviated or eliminated, including, but not limited to, reducing (or destroying) the proliferation of cancerous cells, reducing symptoms caused by the disease, improving the quality of life of patients suffering from the disease, reducing the dose of other drugs required to treat the disease, and / or prolonging the survival of the individual.

[0030] As used herein, "effective amount" refers to an amount of an agent or drug that is effective to treat a disease or disorder of interest. In the case of cancer, an effective amount of an agent may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. As will be understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be obtained in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in relation to the administration of one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount when a desired result may or is achieved in combination with one or more other agents.

[0031] As used herein, an "individual" or "subject" refers to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0032] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and fragments thereof, so long as the fragment exhibits the desired biological activity or function. As used herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0033] The terms "native antibody", "full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein and refer to an antibody in its substantially intact form, not to an antibody fragment as defined below. The term specifically refers to an antibody having a heavy chain containing an Fc region. Naturally occurring antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ) followed by multiple constant domains. Each light chain has a variable domain (V L ) and a constant domain at its other end, with the light chain constant domain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.

[0034] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain is the C H 1. C H 2 and C H It contains 3 domains (collectively CH) as well as the CHL (or CL) domain of the light chain.

[0035] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0036] The term "variable" refers to the fact that certain portions of the variable domains vary widely in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs, also called CDRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions, which are primarily in a beta-sheet configuration with three HVRs connecting the beta-sheet structure to form loops that in some cases form part of the beta-sheet structure. The HVRs within each chain are held in close proximity to each other by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0037] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0038] The term IgG "isotype" or "subclass" as used herein means any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions.

[0039] Depending on the amino acid sequence in the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and generally described in, for example, Abbas et al. al. Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or noncovalent association of the antibody with one or more other proteins or peptides.

[0040] An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen-binding region thereof. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(AB')2, and Fv fragments (such as single-chain variable fragments, scFv), diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0041] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and the remainder is an "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0042] An "Fv" is the smallest antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight non-covalent association. In a single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to that of a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site.

[0043] A Fab fragment has two polypeptide chains containing heavy and light chain variable domains (VH, VL) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). Herein, we refer to "Fd" as the heavy chain polypeptide portion of the Fab fragment containing the VH and CH1 domains. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical bonds of antibody fragments are also known.

[0044] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a general review of scFvs, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies. Springer Berlin Heidelberg, 1994. 269-315.

[0045] The "Fc" fragment contains the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of the antibody are determined by the sequence of the Fc region, which is also the region recognized by the Fc receptors (FcR) found on certain cell types.

[0046] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., individual antibodies in the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the characteristic of the antibody that is not a mixture of separate antibodies. In some embodiments, such monoclonal antibodies typically include antibodies that include a polypeptide sequence that binds to a target, where the target-binding polypeptide sequence was obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a pool of multiple clones, e.g., hybridoma clones, phage clones, or recombinant DNA clones, etc. The selected target-binding sequence can be further modified, e.g., to improve affinity for the target, humanize the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, create a multispecific antibody, etc., and it should be understood that an antibody that includes an altered target-binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0047] The modifier "monoclonal" indicates the character of the antibody as obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced using a variety of techniques, including the hybridoma method (e.g., Kohler and Milstein, Nature 256:495-97 (1975); Hongo et al., Hybridoma 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (see, e.g., Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellowes, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Mol. Biol. 103(34):12467-12472 (2004)). al., J. Immunol. Methods 284(1-2):119-132 (2004)), as well as techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893, WO 1996 / 34096, WO 1996 / 33735, WO 1991 / 10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993), Jakobovits et al., Nature 362:255-258 (1993), Bruggemann et al., Year in Immunol. 7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992), Lonberg et al., Nature 368:856-859 (1994), Morrison, Nature 368:812-813 (1994), Fishwild et al., Nature Biotechnol. 14:845-851 (1996), Neuberger, Nature Biotechnol. 14:826 (1996), and Lonberg and Huszar,Intern.Rev.Immunol. 13:65-93 (1995)).

[0048] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as the fragments exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATTZED® antibodies, whose antigen-binding regions are derived from antibodies produced, for example, by immunizing macaque monkeys with the antigen of interest.

[0049] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some cases, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are FRs of human immunoglobulin sequences. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani, See also, Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994), and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0050] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or an antibody produced using any of the techniques for producing human antibodies as disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991). Methods available for the preparation of human monoclonal antibodies are also described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 77 (1985); Boerner et al., J. Immunol. 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous loci have been inactivated, e.g., immunized xenomice (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See also Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006), for human antibodies generated by human B cell hybridoma technology.

[0051] The term "hypervariable region", "HVR", or "HV" as used herein refers to a region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Generally, antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In natural antibodies, H3 and L3 show the highest diversity of the six HVRs, and H3 in particular is thought to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable even in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). HVRs are also referred to as "CDRs" or "complementarity determining regions."

[0052] The structure and location of immunoglobulin variable regions may be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and the latest edition thereof currently available on the Internet (immuno.bme.nwu.edu).

[0053] "Framework" or "FR" residues are those variable domain residues other than HVR residues as herein defined.

[0054] The term "covalently linked" as used herein refers to a direct linkage through one or more chemical bonds or an indirect linkage through one or more linkers. Any suitable chemical bond can be used to create a direct linkage, including, but not limited to, a covalent bond, such as a peptide bond and a disulfide bond, or a non-covalent bond, such as a hydrogen bond, a hydrophobic bond, an ionic bond, or a van der Waals bond.

[0055] "Covalent bond" as used herein refers to a stable bond between two atoms that share one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, "peptide bond" refers to a covalent bond formed between a carboxyl group of an amino acid and an amine group of an adjacent amino acid. "Disulfide bond" as used herein refers to a covalent bond formed between two sulfur atoms, such as the combination of a heavy chain fragment CH1 and a light chain fragment CL by one or more disulfide bonds. One or more disulfide bonds may be formed between two fragments by linking thiol groups of the two fragments. In some embodiments, one or more disulfide bonds may be formed between one or more cysteines of the heavy chain fragment and the light chain fragment, respectively. Disulfide bonds may be formed by oxidation of two thiol groups. In some embodiments, the covalent linkage is directly linked by a covalent bond. In some embodiments, the covalent linkage is directly linked by a peptide bond or a disulfide bond.

[0056] As used herein, the terms "bind", "specifically bind to" or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for a longer period of time than the antibody binds to other targets. In one embodiment, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a dissociation constant (K) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. d In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can, but need not, include exclusive binding.

[0057] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0058] Amino acid substitutions may include, but are not limited to, the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table A. Amino acid substitutions may be introduced into the antibody of interest and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. Table A [Table A]

[0059] Amino acids may be grouped by common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) acidic: Asp, Glu, (4) basic: His, Lys, Arg, (5) residues that affect chain orientation: Gly, Pro, (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would involve exchanging a member of one of these classes for another class.

[0060] As used herein, a "multispecific antigen-binding protein" (MSAP) refers to a protein having a Fab fragment covalently linked to one or more antigen-binding fragments with different properties compared to the Fab fragment. The properties may be biological properties, such as in vitro or in vivo activity. The properties may also be simple chemical or physical properties, such as binding to a target molecule, catalytic reaction, etc. The Fab fragment and the one or more antigen-binding fragments may be directly linked by a single peptide bond or linked to each other in an in-frame manner, but via a peptide linker. The terms "multispecific antigen-binding protein" and "MSAP" are used interchangeably herein and refer to an antigen-binding protein with polyepitopic specificity.

[0061] The term "multispecific" when used in reference to an antibody or antigen binding protein (such as a multispecific antigen binding protein, MSAP), refers to an antibody or antigen binding protein that has polyepitopic specificity (i.e., can specifically bind to two, three, or more different epitopes on one biomolecule or can specifically bind to epitopes on two, three, or more different biomolecules). Unless otherwise indicated, the antigens bound by a multispecific antibody or MSAP can be listed in any order in the multispecific antibody or MSAP name. That is, the terms "anti-CD3 / CD19", "anti-CD19 / CD3", "CD19xCD3" and "CD3xCD19" may be used interchangeably and refer to a multispecific antibody (such as MSAP) that specifically binds to both CD3 and CD19 (e.g., different CD19 epitopes). In some embodiments, the MSAP is a BSAP.

[0062] The term "bispecific" when used in reference to an antibody or antigen-binding protein (such as a bispecific antigen-binding protein, BSAP), refers to an antibody or antigen-binding protein that can specifically bind to two different epitopes on one biomolecule or that can specifically bind to epitopes on two different biomolecules. Unless otherwise indicated, the antigens bound by a bispecific antibody or BSAP may be listed in any order in the bispecific antibody or BSAP name. That is, the terms "anti-CD3 / CD19", "anti-CD19 / CD3", "CD19xCD3" and "CD3xCD19" may be used interchangeably and refer to a bispecific antibody (such as a BSAP) that specifically binds both the CD3 and CD19 epitopes. In some embodiments, the BSAP is bivalent. In some embodiments, the BSAP is multivalent (e.g., trivalent).

[0063] As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide which donates its amine group to form a peptide bond with the carboxyl group of the adjacent amino acid residue. The "N-terminus" of a polypeptide, as used herein, refers to the first amino acid of the polypeptide which donates its carboxyl group to form a peptide bond with the amine group of the adjacent amino acid residue.

[0064] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell to which the vector is introduced. Certain vectors are capable of inducing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0065] The term "cell" includes the primary subject cell and its progeny.

[0066] The term "cytokine storm", also known as "cytokine cascade" or "hypercytokinemia", can lead to a potentially fatal immune response that typically consists of a positive feedback loop between cytokines and immune cells, resulting in highly elevated levels of various cytokines (e.g., INF-γ, IL-10, IL-6, CCL2, etc.).

[0067] It is understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0068] Reference herein to "about" a value or parameter includes (and describes) variations that are directed to the value or parameter itself. For example, a reference to "about X" includes the description of "X."

[0069] As used herein, a reference to "not being" a value or parameter generally means and describes "other than" a value or parameter. For example, a method is not used to treat cancer type X means that the method is used to treat cancer types other than X.

[0070] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0071] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the content clearly dictates otherwise.

[0072] II. CD3×CD19 Multispecific Antigen Binding Protein (CD3×CD19 MSAP) The present invention provides a multispecific antigen binding protein (MSAP) comprising an anti-CD3 Fab fragment that specifically binds CD3 (hereinafter referred to as "anti-CD3 Fab fragment") and an anti-CD19 antigen binding fragment (e.g., scFv) that specifically binds CD19 (hereinafter referred to as "anti-CD19 antigen binding fragment", e.g., anti-CD19 scFv), fused to each other by an optional linker, hereinafter referred to as "CD3xCD19 MSAP". In some embodiments, the anti-CD3 Fab fragment comprises (a) an immunoglobulin (Ig) heavy chain variable region (VH) and an Ig heavy chain constant region (CH1), and an Ig light chain variable region (VL) and an Ig light chain constant region (CL). In some embodiments, the anti-CD3 Fab fragment comprises (a) an immunoglobulin (Ig) heavy chain variable region (VH) and an Ig heavy chain constant region (CH1), and an Ig light chain variable region (VL) and an Ig light chain constant region (CL), wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, a CD3xCD19 MSAP (such as a BSAP) comprises a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv), where the first anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of an anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the CD3xCD19 MSAP is trivalent and trispecific, i.e., the two anti-CD19 antigen-binding fragments (e.g., scFvs) specifically bind to different epitopes of CD19, hereafter referred to as a "CD3xCD19 trispecific antigen-binding protein" or "CD3xCD19 TSAP". In some embodiments, the CD3xCD19 MSAP is trivalent and bispecific, i.e., two anti-CD19 antigen-binding fragments (e.g., scFvs) specifically bind to the same CD19 epitope, hereafter referred to as a "trivalent CD3xCD19 BSAP."In some embodiments, the CD3xCD19 MSAP is bivalent and bispecific, hereafter referred to as a "bivalent CD3xCD19 BSAP." An exemplary CD3xCD19 MSAP is shown in Figure 1B.

[0073] The CD3×CD19 MSAPs (e.g., BSAPs) of the invention have important advantages, including, but not limited to, 1) demonstrated enhanced cancer cell killing efficacy, 2) demonstrated superior in vivo therapeutic efficacy against Burkitt's lymphoma and precursor B-cell (preB) acute lymphoblastic leukemia (ALL) in animal models, and 3) cross-reactivity with non-human primates, such as cynomolgus monkeys, which may be useful for toxicity studies in non-human primates (e.g., cynomolgus monkeys) for benefits predictive of human clinical trials.

[0074] Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, II. an anti-CD19 antigen-binding fragment (e.g., an scFv) that specifically binds to CD19, and III. optionally, a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., the scFv). In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond. Thus, in some embodiments, there is provided an MSAP (such as a BSAP) that includes: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, the CH1 and CL of the anti-CD3 Fab fragment being linked by a disulfide bond, II. an anti-CD19 antigen-binding fragment (e.g., an scFv) that specifically binds to CD19, and III. optionally, a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., the scFv). In some embodiments, the anti-CD19 antigen-binding fragment (e.g., the scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., the scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the MSAP (such as a BSAP) comprises a first anti-CD19 antigen binding fragment (e.g., scFv) and a second anti-CD19 antigen binding fragment (e.g., scFv), where the first anti-CD19 antigen binding fragment (e.g., scFv) is fused to the N-terminus of the VH of an anti-CD3 Fab fragment and the second anti-CD19 antigen binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε).In some embodiments, the VH of the anti-CD3 Fab fragment comprises a heavy chain hypervariable region H1 (HVR-H1) comprising the amino acid sequence of SEQ ID NO: 9, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprises a light chain hypervariable region L1 (HVR-L1) comprising the amino acid sequence of SEQ ID NO: 12, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46), which can form an intermolecular disulfide bond. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or a VL comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, HVR-L2 comprising the amino acid sequence of SEQ ID NO:48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:49.In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7 and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7 and / or a VL comprising the amino acid sequence of SEQ ID NO:50. In some embodiments, the anti-CD19 antigen-binding fragment is an scFv. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51 or 59. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51. In some embodiments, the MSAP (such as a BSAP) comprises two anti-CD19 scFvs having the same amino acid sequence. In some embodiments, the MSAP comprises two anti-CD19 scFvs having different amino acid sequences. In some embodiments, the linker comprises about 2 to about 30 amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker comprises about 2 to about 15 amino acid residues. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide and a second polypeptide, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 53 or 60, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 54 or 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide and a second polypeptide, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 53, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide and a second polypeptide, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 60, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58.In some embodiments, an MSAP (e.g., a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:58. In some embodiments, an MSAP (e.g., a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, an MSAP (e.g., a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61.

[0075] In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) is an anti-CD3 It is fused to the N-terminus of the VH of the Fab fragment.

[0076] Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 antigen-binding fragment (e.g., an scFv) that specifically binds CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., the scFv), wherein the anti-CD19 antigen-binding fragment (e.g., the scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, there is provided an MSAP (such as a BSAP) that includes: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 scFv that specifically binds to CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 scFv, wherein the anti-CD19 scFv is an anti-CD3 A linker fused to the N-terminus of the VH of the Fab fragment. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:9, HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:11, and / or the VL of the anti-CD3 Fab fragment comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18 and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:6 or 49. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50. In some embodiments, an anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51 or 59.In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58.

[0077] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. Fab fragments, II. anti-CD19 antigen-binding fragments (e.g., scFvs) that specifically bind to CD19, wherein the anti-CD19 antigen-binding fragments (e.g., scFvs) comprise a VH, and the VH of the anti-CD19 antigen-binding fragments (e.g., scFvs) comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the anti-CD19 antigen-binding fragments (e.g., scFvs) comprise a VL, and the VL of the anti-CD19 antigen-binding fragments (e.g., scFvs) comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49, and III. A linker connecting a Fab fragment and an anti-CD19 antigen-binding fragment (e.g., scFv), in which the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of an anti-CD3 Fab fragment.In some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 scFv comprising a VH and an anti-CD19 an anti-CD19 scFv, wherein the VH of the scFv comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or the anti-CD19 scFv comprises a VL, wherein the VL of the anti-CD19 scFv comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, HVR-L2 comprising the amino acid sequence of SEQ ID NO:48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:6 or 49, and III. optionally, a linker linking an anti-CD3 Fab fragment and an anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15 and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds).In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and / or a VL comprising the amino acid sequence of SEQ ID NO: 8 or 50. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59. Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 III. an anti-CD19 scFv comprising the amino acid sequence of SEQ ID NO: 51 or 59, and III. optionally a linker linking an anti-CD3 Fab fragment and an anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NO: 20 to 22, 29, and 31 to 44 (e.g., SEQ ID NO: 44).In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:58. In some embodiments, an MSAP (e.g., a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:58.

[0078] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19, wherein the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH, and the VH of the anti-CD19 antigen-binding fragment (e.g., scFv) comprises the amino acid sequence of SEQ ID NO: 7, and / or the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VL, and the VL of the anti-CD19 antigen-binding fragment (e.g., scFv) comprises the amino acid sequence of SEQ ID NO: 8 or 50; and, optionally, a linker that links an anti-CD3 Fab fragment and an anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment.In some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 scFv comprising a VH and an anti-CD19 an anti-CD19 scFv, wherein the VH of the scFv comprises the amino acid sequence of SEQ ID NO: 7, and / or the VL of the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 8 or 50; and III. optionally, a linker linking an anti-CD3 Fab fragment and an anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:18 and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:52.In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59. Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 III. An anti-CD19 scFv, the scFv comprising the amino acid sequence of SEQ ID NO: 51 or 59, and III. optionally, a linker linking an anti-CD3 Fab fragment and an anti-CD19 scFv, the anti-CD19 scFv being fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NO: 20 to 22, 29, and 31 to 44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58.

[0079] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16. Fab fragments, II. anti-CD19 antigen-binding fragments (e.g., scFvs) that specifically bind to CD19, wherein the anti-CD19 antigen-binding fragments (e.g., scFvs) comprise a VH, and the VH of the anti-CD19 antigen-binding fragments (e.g., scFvs) comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the anti-CD19 antigen-binding fragments (e.g., scFvs) comprise a VL, and the VL of the anti-CD19 antigen-binding fragments (e.g., scFvs) comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49, and III. A linker connecting a Fab fragment and an anti-CD19 antigen-binding fragment (e.g., scFv), in which the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of an anti-CD3 Fab fragment.In some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, optionally wherein the anti-CD3 Fab fragment comprises (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16; II. an anti-CD19 scFv that specifically binds to CD19, wherein the anti-CD19 scFv comprises a VH, and wherein the VH of the anti-CD19 scFv comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or wherein the anti-CD19 scFv comprises a VL, and wherein the anti-CD19 III. an anti-CD19 scFv, the VL of the scFv comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49; and III. optionally, a linker linking an anti-CD3 Fab fragment and an anti-CD19 scFv, the anti-CD19 scFv being fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond.In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50. In some embodiments, an anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51 or 59. Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16; II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 scFv comprising the amino acid sequence of SEQ ID NO: 51 or 59; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58.

[0080] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19, wherein the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH, and the VH of the anti-CD19 antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 7, and / or the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VL, and the VL of the anti-CD19 antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 8 or 50; and, optionally, a linker that links an anti-CD3 Fab fragment and an anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or An anti-CD3 Fab fragment, the VL of the Fab fragment comprising the amino acid sequence of SEQ ID NO: 16; II. an anti-CD19 scFv that specifically binds to CD19, the VH of the anti-CD19 scFv comprising the amino acid sequence of SEQ ID NO: 7 and / or the VL of the anti-CD19 scFv comprising the amino acid sequence of SEQ ID NO: 8 or 50; and III. optionally, a linker linking the anti-CD3 Fab fragment and the anti-CD19 scFv, the anti-CD19 scFv being fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18 and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NO: 20 to 22, 29, and 31 to 44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58.

[0081] In some embodiments, a MSAP (such as a BSAP) is provided that comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:53 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:58. In some embodiments, a MSAP (such as a BSAP) is provided that comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:60 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:58.

[0082] In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) is an anti-CD3 It is fused to the N-terminus of the VL of the Fab fragment.

[0083] Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 antigen-binding fragment (e.g., an scFv) that specifically binds CD19; and III. optionally, a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., the scFv), wherein the anti-CD19 antigen-binding fragment (e.g., the scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, there is provided an MSAP (such as a BSAP) that includes: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 scFv that specifically binds to CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 scFv, wherein the anti-CD19 scFv is an anti-CD3 A linker fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:9, HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:11, and / or the VL of the anti-CD3 Fab fragment comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18 and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:6 or 49. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50. In some embodiments, an anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51 or 59.In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 61.

[0084] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. Fab fragments, II. anti-CD19 antigen-binding fragments (e.g., scFvs) that specifically bind to CD19, wherein the anti-CD19 antigen-binding fragments (e.g., scFvs) comprise a VH, and the VH of the anti-CD19 antigen-binding fragments (e.g., scFvs) comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the anti-CD19 antigen-binding fragments (e.g., scFvs) comprise a VL, and the VL of the anti-CD19 antigen-binding fragments (e.g., scFvs) comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49, and III. A linker connecting a Fab fragment and an anti-CD19 antigen-binding fragment (e.g., scFv), in which the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.In some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, optionally wherein the anti-CD3 Fab fragment comprises (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. an anti-CD19 scFv that specifically binds to CD19, optionally wherein the anti-CD19 scFv comprises a VH and an anti-CD19 an anti-CD19 scFv, wherein the VH of the scFv comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or the anti-CD19 scFv comprises a VL, wherein the VL of the anti-CD19 scFv comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, HVR-L2 comprising the amino acid sequence of SEQ ID NO:48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:6 or 49, and III. optionally, a linker linking an anti-CD3 Fab fragment and an anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15 and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds).In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and / or a VL comprising the amino acid sequence of SEQ ID NO: 8 or 50. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59. Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 III. an anti-CD19 scFv comprising the amino acid sequence of SEQ ID NO: 51 or 59, and III. optionally a linker linking an anti-CD3 Fab fragment and an anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NO: 20 to 22, 29, and 31 to 44 (e.g., SEQ ID NO: 44).In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, an MSAP (e.g., a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61.

[0085] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19, wherein the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH, and the VH of the anti-CD19 antigen-binding fragment (e.g., scFv) comprises the amino acid sequence of SEQ ID NO: 7, and / or the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VL, and the VL of the anti-CD19 antigen-binding fragment (e.g., scFv) comprises the amino acid sequence of SEQ ID NO: 8 or 50; and, optionally, a linker that links an anti-CD3 Fab fragment and an anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.In some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 scFv comprising a VH and an anti-CD19 an anti-CD19 scFv, wherein the VH of the scFv comprises the amino acid sequence of SEQ ID NO: 7, and / or the VL of the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 8 or 50; and III. optionally, a linker linking an anti-CD3 Fab fragment and an anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:18 and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:52.In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59. Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 III. an anti-CD19 scFv comprising the amino acid sequence of SEQ ID NO: 51 or 59, and III. optionally a linker linking an anti-CD3 Fab fragment and an anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20 to 22, 29, and 31 to 44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 61.

[0086] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, optionally wherein the anti-CD3 Fab fragment comprises (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16. Fab fragments, II. anti-CD19 antigen-binding fragments (e.g., scFvs) that specifically bind to CD19, wherein the anti-CD19 antigen-binding fragments (e.g., scFvs) comprise a VH, and the VH of the anti-CD19 antigen-binding fragments (e.g., scFvs) comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the anti-CD19 antigen-binding fragments (e.g., scFvs) comprise a VL, and the VL of the anti-CD19 antigen-binding fragments (e.g., scFvs) comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49, and III. A linker connecting a Fab fragment and an anti-CD19 antigen-binding fragment (e.g., scFv), in which the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.In some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16; II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 scFv comprising a VH, the VH of the anti-CD19 scFv comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or wherein the anti-CD19 scFv comprises a VL, the anti-CD19 III. an anti-CD19 scFv, the VL of the scFv comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49; and III. optionally, a linker linking an anti-CD3 Fab fragment and an anti-CD19 scFv, the anti-CD19 scFv being fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond.In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50. In some embodiments, an anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51 or 59. Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16; II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 scFv comprising the amino acid sequence of SEQ ID NO: 51 or 59; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 61.

[0087] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19, wherein the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH, wherein the VH of the anti-CD19 antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 7, and / or the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VL, wherein the VL of the anti-CD19 antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 8 or 50; and, optionally, III. a linker that links an anti-CD3 Fab fragment and an anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or An anti-CD3 Fab fragment, the VL of the Fab fragment comprising the amino acid sequence of SEQ ID NO: 16; II. an anti-CD19 scFv that specifically binds to CD19, the VH of the anti-CD19 scFv comprising the amino acid sequence of SEQ ID NO: 7 and / or the VL of the anti-CD19 scFv comprising the amino acid sequence of SEQ ID NO: 8 or 50; and III. optionally, a linker linking the anti-CD3 Fab fragment and the anti-CD19 scFv, the anti-CD19 scFv being fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18 and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NO: 20 to 22, 29, and 31 to 44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 61.

[0088] In some embodiments, a MSAP (such as a BSAP) is provided that comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:57 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:54. In some embodiments, a MSAP (such as a BSAP) is provided that comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:57 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:61.

[0089] In some embodiments, the MSAP (such as a BSAP) comprises a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv), where the first anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of an anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.

[0090] Thus, in some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19, the first anti-CD19 antigen-binding fragment (e.g., scFv) being fused to the N-terminus of the VH of the anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv) being fused to the N-terminus of the VH of the anti-CD3 Fab fragment. III. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) fused to the N-terminus of the VL of the Fab fragment, and optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 antigen-binding fragment (e.g., scFv), and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv). In some embodiments, there is provided an MSAP (such as a BSAP) that includes: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. a first anti-CD19 scFv and a second anti-CD19 scFv that specifically bind to CD19, the first anti-CD19 scFv being fused to the N-terminus of the VH of the anti-CD3 Fab fragment and the second anti-CD19 scFv being fused to the N-terminus of the VL of the anti-CD3 Fab fragment; and III. A second linker that links the scFvs.In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:9, HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:11, and / or the VL of the anti-CD3 Fab fragment comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46), which can form an intermolecular disulfide bond. In some embodiments, the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49. In some embodiments, the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50. In some embodiments, the first and / or second anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51 or 59.In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) are identical in sequence. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) have different sequences. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to different CD19 epitopes. In some embodiments, the first and / or second linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the first and / or second linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44 (e.g., SEQ ID NO: 44). In some embodiments, the first and second linker are identical in sequence. In some embodiments, the first and second linkers have different sequences. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61.

[0091] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. II. A first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) that specifically bind to CD19, wherein the first anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of an anti-CD3 Fab fragment, and the second anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of an anti-CD3 Fab fragment. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) comprising a VH fused to the N-terminus of the VL of the Fab fragment, wherein the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH, wherein the VH of the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VL, wherein the VL of the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49; and III.Optionally, a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 antigen-binding fragment (e.g., scFv), and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv). In some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. a first anti-CD19 scFv and a second anti-CD19 that specifically bind to CD19. a first anti-CD19 scFv and a second anti-CD19 scFv, wherein the first anti-CD19 scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment, and the second anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment, and the VH of the first and / or second anti-CD19 scFv comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or the VL of the first and / or second anti-CD19 scFv comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49; and III. a first linker that optionally links the VH of the anti-CD3 Fab fragment and the first anti-CD19 scFv, and an anti-CD3 A second linker linking the VL of the Fab fragment and the second anti-CD19 scFv. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO:45) or CPPCS (SEQ ID NO:46) capable of forming an intermolecular disulfide bond. In some embodiments, the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50. In some embodiments, the first and / or second anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51 or 59.Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. a first anti-CD19 scFv and a second anti-CD19 scFv that specifically bind to CD19, the first anti-CD19 III. a first anti-CD19 scFv and a second anti-CD19 scFv, wherein the first and / or second anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59, and III. optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 scFv, and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 scFv. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) are identical in sequence. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) have different sequences. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to different CD19 epitopes. In some embodiments, the first and / or second linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine.In some embodiments, the first and / or second linker is selected from the group consisting of SEQ ID NOs:20-22, 29, and 31-44 (e.g., SEQ ID NO:44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61.

[0092] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. II. A first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) that specifically bind to CD19, wherein the first anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of an anti-CD3 Fab fragment, and the second anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of an anti-CD3 Fab fragment. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) fused to the N-terminus of the VL of the Fab fragment, wherein the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH, and the VH of the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises the amino acid sequence of SEQ ID NO:7, and / or the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VL, and the VL of the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises the amino acid sequence of SEQ ID NO:8 or 50; and III. optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 antigen-binding fragment (e.g., scFv), and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv).In some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. An anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO:9, HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:14. a Fab fragment; II. a first anti-CD19 scFv and a second anti-CD19 scFv that specifically bind to CD19, wherein the first anti-CD19 scFv is fused to the N-terminus of the VH of an anti-CD3 Fab fragment and the second anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment, and wherein the VH of the first and / or the second anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 7 and / or the VL of the first and / or the second anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 8 or 50; and III. optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 scFv, and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 scFv. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO:45) or CPPCS (SEQ ID NO:46) capable of forming an intermolecular disulfide bond. In some embodiments, the first and / or second anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51 or 59.Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. a first anti-CD19 scFv and a second anti-CD19 scFv that specifically bind to CD19, the first anti-CD19 III. a first anti-CD19 scFv and a second anti-CD19 scFv, wherein the first and / or second anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59, and III. optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 scFv, and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 scFv. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) are identical in sequence. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) have different sequences. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to different CD19 epitopes. In some embodiments, the first and / or second linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine.In some embodiments, the first and / or second linker is selected from the group consisting of SEQ ID NOs:20-22, 29, and 31-44 (e.g., SEQ ID NO:44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61.

[0093] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16; II. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19, the first anti-CD19 antigen-binding fragment (e.g., scFv) being fused to the N-terminus of the VH of the anti-CD3 Fab fragment, and the second anti-CD19 antigen-binding fragment (e.g., scFv) being fused to the N-terminus of the VH of the anti-CD3 Fab fragment. and / or the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH, wherein the VH of the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or the first and / or second anti-CD19 antigen-binding fragment (e.g., a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) comprising a VL, and the VL of the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49, and III. optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 antigen-binding fragment (e.g., scFv), and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv).In some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprising the amino acid sequence of SEQ ID NO: 16; II. a first anti-CD19 scFv and a second anti-CD19 scFv that specifically bind to CD19, the first anti-CD19 scFv being fused to the N-terminus of the VH of the anti-CD3 Fab fragment and the second anti-CD19 scFv being fused to the N-terminus of the VL of the anti-CD3 Fab fragment, and a first anti-CD19 scFv and a second anti-CD19 scFv, wherein the VH of the scFv comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or the VL of the first and / or second anti-CD19 scFv comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, HVR-L2 comprising the amino acid sequence of SEQ ID NO:48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:6 or 49, and III. optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 scFv, and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 scFv. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:18 and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:52.In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO:45) or CPPCS (SEQ ID NO:46) capable of forming an intermolecular disulfide bond. In some embodiments, the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50. In some embodiments, the first and / or second anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51 or 59. Thus, in some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. An anti-CD3 Fab fragment that specifically binds to CD3, wherein the anti-CD3 Fab fragment is selected from the group consisting of: (a) an Ig. an anti-CD3 Fab fragment comprising (a) a VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16; II. a first anti-CD19 scFv and a second anti-CD19 scFv that specifically bind to CD19, wherein the first anti-CD19 scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment, and the second anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment, and wherein the first and / or the second anti-CD19 scFv comprise the amino acid sequence of SEQ ID NO: 51 or 59; and III. A first linker that links the VH of the Fab fragment and the first anti-CD19 scFv, and a second linker that links the VL of the anti-CD3 Fab fragment and the second anti-CD19 scFv. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) are identical in sequence. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) have different sequences. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to different CD19 epitopes. In some embodiments, the first and / or second linkers comprise about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the first and / or second linker is selected from the group consisting of SEQ ID NOs:20-22, 29, and 31-44 (e.g., SEQ ID NO:44). In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61.In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61.

[0094] In some embodiments, there is provided an MSAP (such as a BSAP) that comprises: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16; II. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19, the first anti-CD19 antigen-binding fragment (e.g., scFv) being fused to the N-terminus of the VH of the anti-CD3 Fab fragment, and the second anti-CD19 antigen-binding fragment (e.g., scFv) being fused to the N-terminus of the VH of the anti-CD3 Fab fragment. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) fused to the N-terminus of the VL of the Fab fragment, wherein the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH, and the VH of the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises the amino acid sequence of SEQ ID NO:7, and / or the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VL, and the VL of the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises the amino acid sequence of SEQ ID NO:8 or 50; and III. optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 antigen-binding fragment (e.g., scFv), and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv).In some embodiments, there is provided an MSAP (such as a BSAP) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, and wherein the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 15 and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 16; II. a first anti-CD19 scFv and a second anti-CD19 scFv that specifically bind to CD19, the first anti-CD19 scFv being anti-CD3. III. a first anti-CD19 scFv and a second anti-CD19 scFv, wherein the first and / or second anti-CD19 scFv are fused to the N-terminus of the VH of the Fab fragment, and the second anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment, and the VH of the first and / or second anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 7, and / or the VL of the first and / or second anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 8 or 50, and III. optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 scFv, and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 scFv. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO:45) or CPPCS (SEQ ID NO:46) capable of forming an intermolecular disulfide bond. In some embodiments, the first and / or second anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51 or 59. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) are identical in sequence. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) have different sequences. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to different CD19 epitopes. In some embodiments, the first and / or second linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the first and / or second linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44 (e.g., SEQ ID NO: 44).In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61.

[0095] In some embodiments, MSAP is provided that comprises a first polypeptide and a second polypeptide, such as a BSAP, where the first polypeptide comprises the amino acid sequence of SEQ ID NO:53 or 60 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, MSAP is provided that comprises a first polypeptide and a second polypeptide, such as a BSAP, where the first polypeptide comprises the amino acid sequence of SEQ ID NO:53 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:54. In some embodiments, MSAP is provided that comprises a first polypeptide and a second polypeptide, such as a BSAP, where the first polypeptide comprises the amino acid sequence of SEQ ID NO:60 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:61.

[0096] The present invention further provides a fusion protein comprising any of the CD3xCD19 MSAPs (such as BSAPs) described herein, a CD3xCD19 MSAP complex (e.g. a small molecule drug conjugate), or an isolated cell expressing any of the CD3xCD19 MSAPs described herein.

[0097] Anti-CD3 Fab fragment The anti-CD3 Fab fragment of the CD3xCD19 MSAP (such as BSAP) described herein specifically binds to CD3, such as human CD3. "CD3" is known in the art as a six-chain multiprotein complex (see Abbas and Lichtman, 2003; Janeway et al., p172 and 178, 1999). In mammals, the T cell receptor (TCR) complex comprises a homodimer of the CD3 gamma chain, the CD3 delta chain, two CD3 epsilon chains, and the CD3 zeta chain. The CD3 gamma chain, the CD3 delta chain, and the CD3 epsilon chain are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3 gamma chain, the CD3 delta chain, and the CD3 epsilon chain are negatively charged, a property that allows these chains to associate with the positively charged TCR chain. The intracellular tails of the CD3 gamma, delta, and epsilon chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3 zeta chain has three. Without being bound by theory, ITAMs are believed to be important for the signaling ability of the TCR complex. CD3, as used herein, may be derived from a variety of animal species, including, for example, humans, primates, mice, rats, or other mammals.

[0098] In some embodiments, the CD3xCD19 MSAPs (such as BSAPs) described herein have an increased in vivo half-life compared to the anti-CD3 Fab fragments alone, hi some embodiments, the CD3xCD19 MSAPs (such as BSAPs) have a half-life that is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times that of the individual anti-CD3 Fab fragments.

[0099] In some embodiments, the anti-CD3 Fab fragment of the CD3×CD19 MSAP (such as a BSAP) has an equilibrium binding constant (K d), e.g., ≦100 nM, preferably ≦10 nM, more preferably ≦1 nM, etc. d In some embodiments, the anti-CD3 Fab fragment is at a concentration of about 0.1×10 -9 M ~ approx. 1×10 -7 M, for example, about 5.99 x 10 -9 K such as M d In some embodiments, the anti-CD3 Fab fragment binds to human CD3 at about 0.1 x 10 -9 M ~ approx. 1×10 -7 M, for example, about 1.88 x 10 -8 K such as M d binds to monkey (e.g., cynomolgus monkey) CD3.

[0100] In some embodiments, the anti-CD3 Fab fragment of a CD3xCD19 MSAP (such as a BSAP) specifically binds to an individual CD3 chain, such as the CD3 gamma chain, the CD3 delta chain, or the CD3 epsilon chain. In some embodiments, the anti-CD3 Fab fragment specifically binds to a complex formed from two or more individual CD3 chains (e.g., a complex of more than one CD3 epsilon chain, a complex of the CD3 gamma chain and the CD3 epsilon chain, a complex of the CD3 delta chain and the CD3 epsilon chain). In some embodiments, the anti-CD3 The Fab fragment specifically binds to the CD3 epsilon chain. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3 epsilon. In some embodiments, the anti-CD3 Fab fragment specifically binds to an epitope within amino acids 1-27 of CD3 epsilon.

[0101] Anti-CD3 Fab fragments can be produced by various methods known in the art (see, e.g., U.S. Pat. Nos. 6,291,161 and 6,291,158). Sources of anti-CD3 Fab include those from various species, e.g., humans, camelids (camel, dromedary, or llama, Hamers-Casterman et al. (Roux et al. (1993) Nature, 363:446 and Nguyen et al. (1998) J. Mol. Biol., 275:413), shark (Roux et al. (1998) Proc. Nat'l. Acad. Sci. (USA) 95:11804), fish (Nguyen et al. (2002) Immunogenetics, 54:39), rodent, bird, or sheep. In some embodiments, the anti-CD3 Fab fragment is derived from a human antibody or a humanized antibody. In some embodiments, the anti-CD3 Fab fragment is chimeric. In some embodiments, the anti-CD3 Fab fragment is derived from a fully human antibody developed using, for example, phage display, yeast display, or transgenic mice with human Ig genes.

[0102] In some embodiments, the anti-CD3 Fab fragment specifically binds to both human and non-human primate (such as cynomolgus monkey) CD3. Exemplary anti-human CD3 antibodies that are cross-reactive with monkey CD3 include, but are not limited to, the SP34 mouse monoclonal antibody (see, e.g., Pressano, S. The EMBO J. 4:337-344, 1985; Alarcon, B. EMBO J. 10:903-912, 1991; Salmeron A. et al., J. Immunol. 147:3047-52, 1991; Yoshino N. et al., Exp. Anim 49:97-110, 2000; Conrad M L. et al., Cytometry 71A:925-33, 2007; and Yang et al., J. Immunol. 137:1097-1100:1986). CD3xCD19 MSAPs (such as BSAPs) with anti-CD3 Fab fragments that are cross-reactive to monkey CD3 may facilitate toxicity testing in non-human primates, which may provide a more appropriate safety assessment for human clinical trial candidates without the need to perform toxicity testing in chimpanzees or with surrogate molecules.

[0103] In some embodiments, the anti-CD3 Fab fragment is derived from an anti-CD3 antibody that has no cross-reactivity to non-human primates. Exemplary anti-CD3 antibodies include the Cris-7 monoclonal antibody (Reinherz, EL et al. (eds.), Leukocyte typing II, Springer Verlag, New York, (1986)), the BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med. 172:1691), OKT3 (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) and its derivatives such as OKT3 ala-ala (Herold et al. (2003) J. Clin. Invest. 11:409), visilizumab (Carpenter et al. (2002) Blood 99:2712), and the 145-2C11 monoclonal antibody (Hirsch et al. (1988) J. Immunol. 140:3766). Further CD3 binding molecules contemplated herein include UCHT-1 (Beverley, PC and Callard, RE (1981) Eur. J. Immunol. 11:329-334) and the CD3 binding molecules described in WO2004 / 106380, WO2010 / 037838, WO2008 / 119567, WO2007 / 042261, WO2010 / 0150918.

[0104] In some embodiments, the anti-CD3 Fab fragment comprises one constant (CH1) region and one variable (VH) region of an immunoglobulin (Ig) heavy chain, and one constant (CL) region and one variable (VL) region of an Ig light chain. In some embodiments, the CH1 and VH heterodimerize with the VL and CL and are covalently linked by a disulfide bond between the heavy and light chain constant regions. In some embodiments, the anti-CD3 Fab fragment has the basic structure NH2-VL-CL-SS-CH1-VH-NH2. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by one or more disulfide bonds. In some embodiments, the number of disulfide bonds between the CH1 and CL of the anti-CD3 Fab fragment is at least one, such as two, three, four, or more. In some embodiments, cysteine ​​residues are engineered in the anti-CD3 Fab fragment (such as in the CH1 and CL regions) to introduce disulfide bonds.

[0105] In some embodiments, the anti-CD3 Fab fragment of a CD3xCD19 MSAP (such as a BSAP) does not contain a disulfide bond. For example, the heavy and light chains may be engineered in such a way that they stably interact without the need for disulfide bonds. In some embodiments, the heavy or light chains may be engineered to remove cysteine ​​residues, but the heavy and light chains still stably interact and function as a Fab. In some embodiments, mutations are made to promote stable interactions between the heavy and light chains. For example, a "knobs-into-holes" engineering strategy may be used to promote dimerization between the heavy and light chains of a Fab (see, e.g., 1996 Protein Engineering, 9:617-621). Variant Fab fragments designed for specific purposes, such as amino acid changes in the constant domains of CH1 and / or CL, as well as disulfide bond removal or addition of tags for purification, are also contemplated for use herein.

[0106] In some embodiments, the arrangement of the variable and constant regions in an anti-CD3 Fab fragment may differ from that found in a native anti-CD3 Fab, hi some embodiments, the orientation of the variable and constant regions may be VH-CL in one chain and VL-CH1 in another chain (see, e.g., Shaefer et al. (2011), PNAS, 108:111870-92).

[0107] In some embodiments, the anti-CD3 Fab fragment of the CD3xCD19 MSAP (such as a BSAP) is derived from a monoclonal antibody. Suitable monoclonal antibodies may be of any type, including IgA, IgM, IgD, IgG, IgE and subtypes thereof, such as IgG1, IgG2, IgG3, and IgG4. The light chain domain may be derived from a kappa or lambda chain. In some embodiments, the anti-CD3 Fab fragment is recombinantly engineered.

[0108] In some embodiments, the anti-CD3 Fab fragment comprises a human immunoglobulin CH1. In some embodiments, the human immunoglobulin CH1 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-CD3 Fab fragment comprises a human lambda light chain constant region. In one embodiment, the human lambda light chain constant region comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 (such as 1, 2, 3, 4, or 5) disulfide bonds. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 2 disulfide bonds. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. SEQ ID NO: 18 (human CH1) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS SEQ ID NO:52 (human lambda CL) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE

[0109] The anti-CD3 Fab fragment of a CD3xCD19 MSAP (such as a BSAP) specifically binds to CD3 via an antigen-binding site formed between the heavy chain variable region (VH) and the light chain variable region (VL). The antigen-binding site comprises at least one (such as one, two or three) HVR of an immunoglobulin heavy chain and / or at least one (such as one, two or three) HVR of an immunoglobulin light chain. In some embodiments, the CD3xCD19 An MSAP (such as a BSAP) comprises one, two, three, four, five, or all six HVRs of the VH and VL sequences of a full-length antibody that specifically binds CD3.

[0110] In some embodiments, the anti-CD3 Fab fragment is derived from SP34. In some embodiments, the anti-CD3 Fab fragment is a CD3 Fab fragment described in U.S. Pat. No. 8,846,042. In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising one, two or three HVRs (or CDRs) from SEQ ID NO: 15, and / or a VL comprising one, two or three HVRs (or CDRs) from SEQ ID NO: 16. In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising three HVRs from SEQ ID NO: 15, and / or a VL comprising three HVRs from SEQ ID NO: 16. In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising one, two or three HVRs selected from SEQ ID NO: 9-11, and / or a VL comprising one, two or three HVRs selected from SEQ ID NO: 12-14. In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:9, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:11, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:12, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO:15. In some embodiments, the anti-CD3 Fab fragment comprises a VL comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO:16.In some embodiments, a VH or VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but an anti-CD3 Fab fragment comprising the sequence retains the ability to bind to CD3. In some embodiments, one or two amino acids are substituted, inserted, and / or deleted in any one or more of the HVRs. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the framework regions, "FR"). In some embodiments, the anti-CD3 Fab fragment comprises a VH comprising the amino acid sequence of SEQ ID NO:15, and / or a VL comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the anti-CD3 Fab fragment comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:57, and / or a light chain polypeptide comprising the amino acid sequence of SEQ ID NO:58. SEQ ID NO:9 (Anti-CD3 HVR-H1) TYAMN SEQ ID NO: 10 (Anti-CD3 HVR-H2) RIRSKYNNYATYYADSVKD SEQ ID NO: 11 (anti-CD3 HVR-H3) HGNFGNSYVSWFAY SEQ ID NO: 12 (Anti-CD3 HVR-L1) RSSTGAVTTSNYAN SEQ ID NO: 13 (Anti-CD3 HVR-L2) GTNKRAP SEQ ID NO: 14 (Anti-CD3 HVR-L3) ALWYSNLWV SEQ ID NO: 15 (anti-CD3 VH, HVR is underlined) [ka] SEQ ID NO: 16 (anti-CD3 VL, HVR is underlined) [ka] SEQ ID NO: 57 (Fd polypeptide of anti-CD3 Fab fragment, HVR is underlined) [ka] SEQ ID NO:58 (light chain polypeptide of anti-CD3 Fab fragment, HVR is underlined) [ka]

[0111] In some embodiments, a particular VH and / or VL of an anti-CD3 Fab fragment may be used to screen a library of complementary variable regions to identify a VH / VL with desirable properties, such as high affinity for CD3. Such methods are described, for example, in Portolano et al., J. Immunol. (1993) 150:880-887; Clarkson et al., Nature (1991) 352:624-628; and Klimka et al., British Journal of Cancer (2000) 83:252-260; Beiboer et al. al., J. Mol. Biol. (2000) 296:833-849, and Rader et al., PNAS (1998) 95:8910-8915.

[0112] Anti-CD19 antigen binding fragment The CD3xCD19 MSAP (such as a BSAP) described herein comprises one or two anti-CD19 antigen-binding fragments (e.g., scFvs) that specifically bind to CD 19. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFvs) is fused at the N-terminus of an anti-CD3 Fab fragment, e.g., at the N-terminus of the VH of the anti-CD3 Fab fragment and / or at the N-terminus of the VL of the anti-CD3 Fab fragment.

[0113] B-lymphocyte antigen CD19 is also known as CD19 molecule (cluster of differentiation 19), B-lymphocyte surface antigen B4, T-cell surface antigen Leu-12, and CVID3. In humans, CD19 is expressed in all B-lineage cells except plasma cells, and in follicular dendritic cells. CD19 has two major roles: 1) functioning as an adaptor protein that recruits cytoplasmic signaling proteins to the membrane, and 2) functioning within the CD19 / CD21 complex to reduce the threshold of the B-cell receptor signaling pathway. CD19 is expressed on both normal and malignant B-lymphocytes and is considered a B-cell tumor-associated antigen. For example, CD19 can function as a biomarker for B-lymphocyte differentiation, a cancer diagnostic marker, or a target for immunotherapy, such as B-cell lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

[0114] In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) can specifically bind to CD19 on the cell surface. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is in a solid tumor. In some embodiments, the cancer cell is a metastatic cancer cell, such as a hematological cancer, such as ALL, CLL, MCL, B-cell lymphoma, etc.

[0115] In some embodiments, the CD3xCD19 MSAPs (such as BSAPs) described herein have an increased in vivo half-life compared to the anti-CD19 antigen-binding fragments (e.g., scFvs) alone, hi some embodiments, the CD3xCD19 MSAPs (such as BSAPs) have a half-life that is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times that of the individual anti-CD19 antigen-binding fragments (e.g., scFvs).

[0116] In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) of the CD3xCD19 MSAP (such as a BSAP) has an equilibrium binding constant (K d), e.g., ≦100 nM, preferably ≦10 nM, more preferably ≦1 nM, etc. For example, the K d In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) has a concentration of about 0.01×10 -9 M ~ approx. 1×10 -7 M, for example, about 8.91 x 10 -10 K such as M d In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) binds to human CD19 at a concentration of about 0.1 x 10 -9 M ~ approx. 1×10 -7 M, for example, about 8.91 x 10 -9 K such as M d binds to monkey (e.g., cynomolgus) CD19.

[0117] The anti-CD19 antigen-binding fragments (e.g., scFv) in the CD3xCD19 MSAP (e.g., BSAP) not only provide additional binding specificity and enhanced properties (e.g., increased serum half-life or activation of the immune activation cascade), but also create steric hindrance by fusion to the N-terminus of the VH and / or VL chain that significantly reduces the binding affinity of the anti-CD3 Fab fragments to CD3. This is in stark contrast to other Fab fusion proteins, such as TRIBODY™, which fuse an additional binding domain to the C-terminus of the Fab fragment (see, e.g., Journal of Immunology, 2000, 165:7050-7057). The anti-CD19 antigen-binding fragments (e.g., scFv) are not intended to dimerize, unlike other known fusion proteins, such as those described in WO2008 / 024188 and WO2009 / 149185. A further distinguishing feature of CD3xCD19 MSAPs (such as BSAPs) is that when the MSAP (such as BSAP) is not bound to CD19 on tumor cells, the anti-CD19 antigen-binding fragment (e.g., scFv) reduces the binding affinity of the anti-CD3 Fab fragment for CD3.

[0118] The anti-CD19 antigen-binding fragments described herein can be of any format and derived from any suitable anti-CD19 antibody. For example, the anti-CD19 antigen-binding fragment can be selected from scFv, VH, VL, scFv-scFv, Fv, Fab, Fab', (Fab')2, minibody, diabody, domain antibody variant (dAb), single domain antibody (sdAb), camelid antibody (VHH), fibronectin 3 domain variant, ankyrin repeat variant, and other antigen-specific binding domains derived from other protein scaffolds. In some embodiments, the anti-CD19 antigen-binding fragment is an scFv. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) is humanized. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) is chimeric. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) is derived from a mouse, rat, monkey, or rabbit monoclonal antibody. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) is derived from any monoclonal antibody selected from B43, MEDI-551, CLB-CD19, 4G7, SJ25-C1LT19, Leu-12, HD37, or any other known anti-CD19 antibody. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) is derived from a fully human antibody developed using, for example, phage display, yeast display, or transgenic mice with human Ig genes.

[0119] In some embodiments, the anti-CD19 antigen binding fragment (e.g., scFv) specifically binds to both human and non-human primate (e.g., cynomolgus monkey) CD19. CD3xCD19 MSAPs (such as BSAPs) with anti-CD19 antigen binding fragments (e.g., scFvs) that are cross-reactive to monkey CD19 may facilitate toxicity testing in non-human primates, which may provide a more appropriate safety assessment for human clinical trial candidates without the need to perform toxicity testing in chimpanzees or with surrogate molecules.

[0120] An anti-CD19 antigen-binding fragment (e.g., scFv) of the invention may fully or partially modulate, block, inhibit, reduce, antagonize, neutralize, or interfere with the functional activity of CD19. An anti-CD19 antigen-binding fragment (e.g., scFv) is considered to be able to fully modulate, block, inhibit, reduce, antagonize, neutralize, or interfere with the functional activity of CD19 if the functional activity of CD19 is reduced by at least 95% (such as 96%, 97%, 98%, 99% or 100%) in the presence of the anti-CD19 antigen-binding fragment (e.g., scFv) compared to when the anti-CD19 antigen-binding fragment (e.g., scFv) does not bind. An anti-CD19 antigen-binding fragment (e.g., scFv) is considered to be capable of significantly modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or interfering with the functional activity of CD19 if the functional activity of CD19 is reduced by at least 50% (e.g., 55%, 60%, 75%, 80%, 85%, or 90%) in the presence of the anti-CD19 antigen-binding fragment (e.g., scFv) compared to when the anti-CD19 antigen-binding fragment (e.g., scFv) is not bound. An anti-CD19 antigen-binding fragment (e.g., scFv) is considered to be able to partially modulate, block, inhibit, reduce, antagonize, neutralize, or interfere with the functional activity of CD19 if the functional activity of CD19 is reduced by less than 95% (e.g., reduced by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%) in the presence of the anti-CD19 antigen-binding fragment (e.g., scFv) compared to when the anti-CD19 antigen-binding fragment (e.g., scFv) does not bind.

[0121] In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises specific sequences or certain variants of these sequences. In some embodiments, amino acid substitutions in the variant sequences do not substantially reduce the ability of the anti-CD19 antigen-binding fragment (e.g., scFv) to bind to CD19. Modifications that substantially improve the binding affinity or other properties of the anti-CD19 antigen-binding fragment (e.g., scFv) for CD19, such as specificity, immunogenicity, antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), and / or cross-reactivity with CD19 variants, are also contemplated.

[0122] An anti-CD19 antigen-binding fragment (e.g., scFv) may comprise at least one (such as one, two, or three) HVR of an immunoglobulin heavy chain and / or at least one (such as one, two, or three) HVR of an immunoglobulin light chain of a full-length antibody that specifically binds to CD19. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises one, two, three, four, five, or all six HVRs of the VH and VL sequences of a full-length antibody that specifically binds to CD19.

[0123] In some embodiments, the anti-CD19 antigen-binding fragment is an scFv that specifically binds to CD19 (also referred to herein as "anti-CD19 scFv"). In some embodiments, the VH and VL of the anti-CD19 scFv are linked to each other by a peptide linker, such as a flexible linker that includes glycine and / or serine. Any of the peptide linkers in the "Linkers" subsection below can be used as a linking peptide between the VH and VL of the anti-CD19 scFv. In some embodiments, the VH and VL of the anti-CD19 scFv are directly linked to each other. In some embodiments, the anti-CD19 scFv comprises an N'-VH-L-VL-C' fusion polypeptide, where L is any peptide linker. In some embodiments, the anti-CD19 scFv comprises an N'-VL-L-VH-C' fusion polypeptide, where L is any peptide linker.

[0124] In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH and / or a VL. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH that comprises one, two or three HVRs (or CDRs) from SEQ ID NO:7, and / or a VL that comprises one, two or three HVRs (or CDRs) from SEQ ID NO:8 or 50. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH that comprises one, two or three HVRs (or CDRs) from SEQ ID NO:7, and / or a VL that comprises one, two or three HVRs (or CDRs) from SEQ ID NO:50. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH that comprises three HVRs from SEQ ID NO:7, and / or a VL that comprises three HVRs from SEQ ID NO:8 or 50. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising three HVRs from SEQ ID NO:7, and / or a VL comprising three HVRs from SEQ ID NO:50. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:6 or 49. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or a VL comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, HVR-L2 comprising the amino acid sequence of SEQ ID NO:48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:49.In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:7, and / or a VL comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:8 or 50 (e.g., SEQ ID NO:50). In some embodiments, an anti-CD19 antigen-binding fragment (e.g., an scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., an scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:50. In some embodiments, an anti-CD19 antigen-binding fragment is an scFv. In some embodiments, an anti-CD19. The scFv comprises the amino acid sequence of SEQ ID NO:51 or 59. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51. SEQ ID NO:1 (Anti-CD19 HVR-H1) SYVMH SEQ ID NO:2 (Anti-CD19 HVR-H2) WIGYINPYNDGTKY SEQ ID NO: 3 (Anti-CD19 HVR-H3) GTYYYGSRVFDY SEQ ID NO: 47 (Anti-CD19 HVR-L1-1.3) RSSKSLQNVNGNTYLY SEQ ID NO: 48 (Anti-CD19 HVR-L2-1.3) RMSNLNS SEQ ID NO: 49 (Anti-CD19 HVR-L3-1.3) MQHLEYPLT SEQ ID NO:4 (Anti-CD19 HVR-L1-1.2) RSSKSLQNVNGNTYLY SEQ ID NO:5 (Anti-CD19 HVR-L2-1.2) RMSNLNS SEQ ID NO:6 (Anti-CD19 HVR-L3-1.2) MQHLEYPIT SEQ ID NO:7 (anti-CD19 VH, HVR is underlined) [ka] SEQ ID NO:50 (anti-CD19 VL1.3, HVR is underlined) [ka] SEQ ID NO:8 (anti-CD19 VL1.2, HVR is underlined) [ka] SEQ ID NO:51 (Anti-CD19 scFv(VL-VH)1.3), HVR is underlined, peptide linker is in bold) [ka] SEQ ID NO:59 (Anti-CD19 scFv(VL-VH)1.2), HVR is underlined, peptide linker is in bold) [ka]

[0125] Linker The CD3xCD19 MSAP (such as a BSAP) described herein may comprise a linker (such as a peptide linker) linking the VH or VL of the anti-CD3 Fab fragment and the anti-CD19 antigen binding fragment (e.g., scFv). In some embodiments, the CD3xCD19 MSAP (such as a BSAP) comprises two anti-CD19 antigen binding fragments (e.g., scFv). In some embodiments, the linker between the VH of the anti-CD3 Fab fragment and the first anti-CD19 antigen binding fragment (e.g., scFv) is the same as the linker between the VL of the anti-CD3 Fab fragment and the second anti-CD19 antigen binding fragment (e.g., scFv). In some embodiments, the linker between the VH of the anti-CD3 Fab fragment and the first anti-CD19 antigen binding fragment (e.g., scFv) is different from the linker between the VL of the anti-CD3 Fab fragment and the second anti-CD19 antigen binding fragment (e.g., scFv). In some embodiments, the anti-CD19 antigen binding fragment is a scFv. In some embodiments, the anti-CD19 scFv comprises a linker (such as a peptide linker) connecting the VH and VL of the anti-CD19 scFv, which may be the same as or different from any of the linkers between the VH and VL of the anti-CD3 Fab fragment and the anti-CD19 scFv.

[0126] The linker can be a peptide linker of any length. In some embodiments, the peptide linker can be about 1 to about 10 amino acids in length, about 2 to about 15 amino acids in length, about 3 to about 12 amino acids in length, about 4 to about 10 amino acids in length, about 5 to about 9 amino acids in length, about 6 to about 8 amino acids in length, about 1 to about 20 amino acids in length, about 21 to about 30 amino acids in length, about 1 to about 30 amino acids in length, about 2 to about 20 amino acids in length, about 10 to about 30 amino acids in length, about 2 to about 19 amino acids in length, about 2 to about 18 amino acids in length, or about 3 to about 4 amino acids in length. The peptide linker may be about 2 to about 17 amino acids long, about 2 to about 16 amino acids long, about 2 to about 10 amino acids long, about 2 to about 14 amino acids long, about 2 to about 13 amino acids long, about 2 to about 12 amino acids long, about 2 to about 11 amino acids long, about 2 to about 9 amino acids long, about 2 to about 8 amino acids long, about 2 to about 7 amino acids long, about 2 to about 6 amino acids long, about 2 to about 5 amino acids long, about 2 to about 4 amino acids long, or about 2 to about 3 amino acids long. In some embodiments, the peptide linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long. In some embodiments, the peptide linker is 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. For example, in some embodiments, the peptide linker is about 5 amino acids in length. In some embodiments, the N-terminus of the peptide linker is covalently linked to the C-terminus of an anti-CD19 antigen-binding fragment (e.g., scFv) and the C-terminus of the peptide linker is covalently linked to the N-terminus of the VH or VL of an anti-CD3 Fab fragment.

[0127] The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, for example, WO 1996 / 34103. In some embodiments, the peptide linker is a human IgG1 or IgG4 hinge. In some embodiments, the peptide linker is a mutated human IgG1 or IgG4 hinge. In some embodiments, the linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G n (SEQ ID NO: 31), glycine-serine polymers (e.g., (GS) n (SEQ ID NO: 32), (GSGGS) n (SEQ ID NO: 33), (GGGS) n (SEQ ID NO: 34), or (GGGGS) n(SEQ ID NO:35), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and may therefore be able to function as neutral tethers between building blocks. Glycine utilizes the phi-psi space to a much greater extent than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Exemplary flexible linkers include Gly-Gly (SEQ ID NO: 36), Gly-Gly-Ser-Gly (SEQ ID NO: 37), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 38), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 39), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 40), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 41), Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 42), Gly-Gly Examples of the linker include, but are not limited to, y-Ser-Gly-Gly-Gly-Ser (SEQ ID NO: 20), Ser-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 21), Gly-Arg-Ala-Gly-Gly-Gly-Gly-Ala-Gly-Gly-Gly-Gly (SEQ ID NO: 22), Gly-Arg-Ala-Gly-Gly-Gly (SEQ ID NO: 29), GGGSGGGGSGGGGGS (SEQ ID NO: 43), GGGGS (SEQ ID NO: 44), and the like. In some embodiments, the linker between the VH of the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv) is GGGGS (SEQ ID NO: 44). In some embodiments, the linker between the VL of the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv) is GGGGS (SEQ ID NO: 44). In some embodiments, the linker connecting the VH and VL of the anti-CD19 scFv is GGGGSGGGSGGGGS (SEQ ID NO: 43).The skilled artisan will recognize that the design of a CD3xCD19 MSAP (such as a BSAP) may comprise a linker that is wholly or partially flexible, such that the linker may comprise, in addition to a flexible linker portion, one or more portions that confer a less flexible structure to provide the desired CD3xCD19 MSAP (such as a BSAP) structure.

[0128] In some embodiments, the linker between the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (eg, scFv) is a stable linker (not cleavable by proteases, particularly MMPs).

[0129] In some embodiments, the linker is a cleavable linker. In some embodiments, the linker between the VH or VL of the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a protease substrate cleavage sequence, for example, an MMP substrate cleavage sequence. Substrate sequences that can be cleaved by MMPs have been widely studied. For example, the sequence of PLGLAG (SEQ ID NO: 30) can be cleaved by most MMPs. In some embodiments, the protease cleavage site is recognized by MMP-2, MMP-9, or a combination thereof.

[0130] In some embodiments, the CD3×CD19 MSAP (such as a BSAP) described herein has the structure: N′-anti-CD19 VH-L1-anti-CD19 VL-L2-anti-CD3 VH-CH1-C', and a second polypeptide having the structure: N'-anti-CD3 VL-CL-C'. In some embodiments, a CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide having the structure: N'-anti-CD19 VL-L1-anti-CD19 VH-L2-anti-CD3 VH-CH1-C', and a second polypeptide having the structure: N'-anti-CD3 VL-CL-C'. In some embodiments, a CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide having the structure: N'-anti-CD3 VH-CH1-C', and a second polypeptide having the structure: N'-anti-CD19 VH-L1-anti-CD19 VL-L2-anti-CD3 VL-CL-C'. In some embodiments, a CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the structure: N'-anti-CD3 VH-CH1-C', and a second polypeptide comprising the structure: N'-anti-CD19 VL-L1-anti-CD19 VH-L2-anti-CD3 VL-CL-C'. In some embodiments, a CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the structure: N'-anti-CD19 VH-L1-anti-CD19 VL-L2-anti-CD3 VH-CH1-C', and a second polypeptide comprising the structure: N'-anti-CD19 VH-L3-anti-CD19 VL-L4-anti-CD3 VL-CL-C'. In some embodiments, a CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the structure: N'-anti-CD19 VH-L1-anti-CD19 VL-L2-anti-CD3 VH-CH1-C', and a second polypeptide comprising the structure: N'-anti-CD19 VL-L3-anti-CD19 VH-L4-anti-CD3 VL-CL-C'. In some embodiments, a CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the structure: N'-anti-CD19 VL-L1-anti-CD19 VH-L2-anti-CD3 VH-CH1-C', and a second polypeptide comprising the structure: N'-anti-CD19 VL-L3-anti-CD19 VH-L4-anti-CD3 VL-CL-C'. L1, L2, L3, and L4 are any possible linkers (such as peptide liners) described herein. L1-L4 can be the same or different.

[0131] In some embodiments, the CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53, or an amino acid sequence that is at least about 85% (such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) identical to the sequence of SEQ ID NO:53. In some embodiments, the CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60, or an amino acid sequence that is at least about 85% identical to the sequence of SEQ ID NO:60 (such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%). In some embodiments, the CD3xCD19 MSAP (such as a BSAP) comprises the amino acid sequence of SEQ ID NO:54, or a second polypeptide comprising an amino acid sequence at least about 85% (such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) identical to the sequence of SEQ ID NO:54. In some embodiments, the CD3xCD19 MSAP (such as a BSAP) comprises the amino acid sequence of SEQ ID NO:61, or a second polypeptide comprising an amino acid sequence at least about 85% (such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) identical to the sequence of SEQ ID NO:61.In some embodiments, the CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, the first polypeptide is encoded by a nucleic acid comprising the sequence of SEQ ID NO:55. In some embodiments, the second polypeptide is encoded by a nucleic acid comprising the sequence of SEQ ID NO:56. In some embodiments, the CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61. In some embodiments, the CD3xCD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:58. In some embodiments, the CD3×CD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:58. In some embodiments, the CD3×CD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, the CD3×CD19 MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61. Further provided are CD3×CD19 MSAP (such as a BSAP) and compositions thereof (such as pharmaceutical compositions) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. Further provided are CD3×CD19 MSAP (such as a BSAP) and compositions thereof (such as pharmaceutical compositions) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:60 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61.Further provided is a CD3xCD19 MSAP (such as a BSAP) and compositions (such as pharmaceutical compositions) thereof, comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:58. Further provided is a CD3xCD19 MSAP (such as a BSAP) and compositions (such as pharmaceutical compositions) thereof, comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61.

[0132] In some embodiments, the C-terminus of the first and / or second polypeptide of the CD3xCD19 MSAP (such as a BSAP) comprises the covalent bond region CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. For example, in some embodiments, the covalent bond region capable of forming an intermolecular disulfide bond is located at the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment. In some embodiments, the N-terminus or C-terminus of the first and / or second polypeptide may comprise a histidine tag (HIS tag) for protein purification. For example, in some embodiments, the N-terminus of the anti-CD19 antigen-binding fragment (e.g., scFv) is further fused to a histidine tag. In some embodiments, the C-terminus of the CH1 and / or CL of the anti-CD3 Fab fragment is further fused to a histidine tag. In some embodiments, the N-terminus of the first and / or second polypeptide of the CD3xCD19 MSAP (such as a BSAP) is further fused to a signal peptide for good expression, such as the signal peptide sequence shown in SEQ ID NO: 25 or the signal peptide encoded by the nucleic acid sequence of SEQ ID NO: 26. SEQ ID NO: 53 (amino acid sequence of the first polypeptide anti-CD19 VL-anti-CD19 VH-GGGGS linker-anti-CD3 VH-CH1-CPPCS, linker is in bold and HVR is underlined) [ka] SEQ ID NO: 54 (amino acid sequence of the second polypeptide anti-CD19 VL-anti-CD19 VH-GGGGS linker-anti-CD3 VL-CL-CPPCS, linker is in bold, HVR is underlined) [ka] SEQ ID NO: 55 (nucleic acid sequence encoding the first polypeptide anti-CD19 VL-anti-CD19 VH-GGGGS linker-anti-CD3 VH-CH1-CPPCS) SEQ ID NO: 56 (Nucleic acid sequence encoding the second polypeptide anti-CD19 VL-anti-CD19 VH-GGGGS linker-anti-CD3 VL-CL-CPPCS) SEQ ID NO: 60 (amino acid sequence of the first polypeptide anti-CD19 VL-anti-CD19 VH-GGGGS linker-anti-CD3 VH-CH1-CPPCS, the linker is in bold and the HVR is underlined) [ka] SEQ ID NO: 61 (amino acid sequence of the second polypeptide anti-CD19 VL-anti-CD19 VH-GGGGS linker-anti-CD3 VL-CL-CPPCS, the linker is in bold and the HVR is underlined) [ka]

[0133] III. Preparation method The CD3xCD19 MSAP (such as a BSAP) described herein may be prepared by any of the protein expression and purification methods known in the art. See, for example, Example 1. The DNA sequence encoding the CD3xCD19 MSAP (such as a BSAP) may be completely synthetic. Once such a sequence is obtained, it is cloned into a suitable expression vector and then transfected into a suitable host cell. The transfected host cells are cultured and the supernatant is harvested and purified to obtain the CD3xCD19 MSAP (such as a BSAP) of the invention.

[0134] In some embodiments, the application provides an isolated nucleic acid encoding one or more of the polypeptides of any one of the CD3xCD19 MSAPs (such as BSAPs) described herein. In some embodiments, the isolated nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 55 or 56. The isolated nucleic acid may be DNA or RNA.

[0135] In some embodiments, the isolated nucleic acid is inserted into a vector, such as an expression vector, a viral vector, or a cloning vector. For nucleic acid expression, the vector may be introduced into a host cell to allow expression of the nucleic acid in the host cell. The expression vector may contain various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be appropriately selected by those skilled in the art. For example, the promoter sequence may be selected to promote transcription of the polynucleotide in the vector. Suitable promoter sequences include, but are not limited to, T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. The enhancer sequence may be selected to enhance transcription of the nucleic acid. The selectable marker may be selected to allow selection of host cells into which the vector has been inserted from host cells into which it has not been inserted, for example, the selectable marker may be a gene that confers antibiotic resistance. The signal sequence may be selected to allow the expressed polypeptide to be transported outside the host cell. In some embodiments, the isolated nucleic acid further comprises a nucleic acid sequence encoding a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the nucleic acid sequence encoding the signal peptide comprises the nucleic acid sequence of SEQ ID NO: 26. SEQ ID NO: 25 (amino acid sequence of signal peptide) MEWSWVFLFFLSVTTGVHS SEQ ID NO: 26 (nucleic acid encoding a signal peptide) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcc

[0136] In some embodiments, an isolated host cell containing the vector described above is provided. The host cell containing the vector may be useful for expressing or cloning the isolated nucleic acid. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells. The expression of antibodies and antigen-binding fragments in prokaryotic cells, such as E. coli, is well established in the art. For a review, see, for example, Pluckthun, A. BioTechnology 9:545-551 (1991). Expression in cultured eukaryotic cells is also available to those skilled in the art as an option for producing antibodies or antigen-binding fragments thereof, see recent reviews, for example, Ref, ME (1993) Curr. Opinion Biotech. 4:573-576, Trill JJ et al. (1995) Curr. Opinion Biotech 6:553-560. Higher eukaryotic cells, particularly those derived from multicellular organisms, can be used for the expression of glycosylated polypeptides. Suitable higher eukaryotic cells include, but are not limited to, invertebrate and insect cells, as well as vertebrate cells.

[0137] The vector can be introduced into the host cell using any suitable method known in the art, including but not limited to DEAE-dextran mediated delivery, calcium phosphate precipitation, cationic lipid mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor mediated gene delivery, polylysine, histone, chitosan, and peptide mediated delivery. Standard methods for transfection and transformation of cells to express a vector of interest are well known in the art. In some embodiments, the host cell comprises a first vector encoding a first polypeptide and a second vector encoding a second polypeptide. In some embodiments, the host cell comprises a single vector comprising isolated nucleic acids encoding the first and second polypeptides.

[0138] In some embodiments, the present application provides a method of expressing any of the CD3xCD19 MSAPs (such as BSAPs) described herein, the method comprising culturing isolated host cells containing the vector and recovering the CD3xCD19 MSAPs (such as BSAPs) from the cell culture. The isolated host cells are cultured under conditions that allow expression of the isolated nucleic acid inserted in the vector. Suitable conditions for expression of a polynucleotide may include, but are not limited to, a suitable medium, a suitable density of host cells in the culture medium, the presence of necessary nutrients, the presence of cofactors, suitable temperature and humidity, and the absence of microbial contaminants. The skilled person is able to select suitable conditions for expression as appropriate.

[0139] In some embodiments, the polypeptides expressed in the host cell may form dimers and thus produce the CD3xCD19 MSAP (such as a BSAP) described herein. In some embodiments, the polypeptides expressed in the host cell may form a polypeptide complex that is a homodimer. In some embodiments, the host cell expresses a first polynucleotide and a second polynucleotide, and the first polynucleotide and the second polynucleotide may form a polypeptide complex that is a heterodimer.

[0140] In some embodiments, the polypeptide complex (such as a CD3×CD19 MSAP or BSAP) may be formed within the host cell. For example, a dimer may be formed within the host cell with the aid of appropriate enzymes and / or cofactors. In some embodiments, the polypeptide complex may be secreted extracellularly. In some embodiments, the first and second polypeptides may be secreted extracellularly and form a dimer (such as a CD3×CD19 MSAP or BSAP) outside the host cell.

[0141] In some embodiments, the first and second polypeptides may be expressed separately and allowed to dimerize under suitable conditions to form a CD3xCD19 MSAP (such as a BSAP). For example, the first and second polypeptides may be combined in a suitable buffer to allow the first and second protein monomers to dimerize by suitable interactions, such as hydrophobic interactions. In some embodiments, the first and second polypeptides may be combined in a suitable buffer containing enzymes and / or cofactors that may promote dimerization of the first and second polypeptides. In some embodiments, the first and second polypeptides may be combined in a suitable vehicle to allow them to react with each other in the presence of suitable reagents and / or catalysts.

[0142] The expressed polypeptide(s) and / or polypeptide complexes can be collected using any suitable method. The polypeptide(s) and / or polypeptide complexes can be expressed in the periplasmic space within the cell or secreted into the medium outside the cell. If the polypeptide and / or polypeptide complexes are expressed intracellularly, the host cells containing the polypeptide and / or polypeptide complexes may be lysed, and the polypeptide and / or polypeptide complexes may be isolated from the lysate by removing undesirable debris by centrifugation or ultrafiltration. If the polypeptide and / or polypeptide complexes are secreted into the periplasmic space of E. coli, the cell paste may be thawed for about 30 minutes in the presence of agents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF), and the cell debris may be removed by centrifugation (Carter et al., BioTechnology 10:163-167 (1992)). If the polypeptide and / or polypeptide complex is secreted into the medium, the cell culture supernatant may be harvested and concentrated using a commercially available protein concentration filter, e.g., an Amincon or Millipore Pellicon ultrafiltration unit. Protease inhibitors and / or antibiotics may be included during the harvest and concentration steps to inhibit proteolysis and / or the growth of contaminating microorganisms.

[0143] The expressed polypeptide(s) and / or polypeptide complexes may be further purified by suitable methods, such as, but not limited to, affinity chromatography, hydroxyapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation (for reviews see Bonner, PL, Protein purification, published by Taylor & Francis., 2007; Janson, JC, et al, Protein purification: principles, high resolution methods and applications, published by Wiley-VCH, 1998).

[0144] In some embodiments, polypeptides and / or polypeptide dimer complexes can be purified by affinity chromatography. In some embodiments, Protein A chromatography or Protein A / G (a fusion protein of Protein A and Protein G) chromatography can be useful for purifying polypeptides and / or polypeptide complexes that contain components derived from antibody CH2 and / or CH3 domains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)); Zettlit, KA, Antibody Engineering, Part V, 531-535, 2010). In some embodiments, Protein G chromatography can be useful for purifying polypeptides and / or polypeptide complexes that contain IgG gamma 3 heavy chains (Guss et al., EMBO J. 5:1567 1575 (1986)). In some embodiments, protein L chromatography may be useful for the purification of polypeptides and / or polypeptide complexes that contain kappa light chains (Sudhir, P., Antigen engineering protocols, Chapter 26, published by Humana Press, 1995; Nilson, BH Kat al, J. Biol. Chem., 267, 2234-2239 (1992)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices, such as porous glass powder or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. When the CD3xCD19 MSAP (such as the BSAP) contains an additional CH3 domain, Bakerbond ABX resin (JT Baker, Phillipsburg, NJ) is useful for purification.

[0145] IV. PHARMACEUTICAL COMPOSITIONS, UNIT DOSE DOSE, ARTICLE ARTICLE, AND KITS Further provided by the present application is a pharmaceutical composition comprising any one of the CD3xCD19 MSAPs (such as BSAPs) described herein, and optionally a pharma- ceutically acceptable carrier.

[0146] The pharmaceutical composition may be suitable for a variety of administration methods described herein, including, for example, systemic or local administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for local administration to a tumor site. In some embodiments, the pharmaceutical composition is formulated for intratumoral injection.

[0147] "Carrier" as used herein includes pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. In many cases, a physiologically acceptable carrier is an aqueous pH buffered solution. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers, such as phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin. , gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0148] In some embodiments, the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 9.0, including any one of the following pH ranges: about 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0. In some embodiments, the pharmaceutical composition may also be made isotonic with blood by the addition of a suitable tonicity adjusting agent, such as glycerol.

[0149] Pharmaceutical compositions used for in vivo administration are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Sterility is easily achieved by filtration through a sterile filtration membrane. In some embodiments, the composition is pathogen-free. For injection, the pharmaceutical composition can be in the form of a solution, for example, in a physiologically compatible buffer, such as Hank's solution or Ringer's solution. In addition, the pharmaceutical composition can be in solid form and redissolved or suspended immediately before use. Lyophilized compositions are also included.

[0150] In some embodiments, the pharmaceutical composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous, intraperitoneal, or intravitreal injection. Typically, the composition for injection is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. In general, the ingredients are supplied either separately or mixed together in unit dosage form, for example as a dry lyophilized powder or water-free concentrate in a sealed, sealed container, such as an ampoule or sachet indicating the amount of active agent. When the composition is administered by injection, it can be prepared using an infusion bottle containing sterile water or saline of pharmaceutical grade. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0151] In some embodiments, the pharmaceutical composition is suitable for administration to humans. In some embodiments, the pharmaceutical composition is suitable for administration to rodents (e.g., mice, rats) or non-human primates (e.g., cynomolgus monkeys). In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is stored frozen.

[0152] Also provided are unit dosage forms of the CD3xCD19 MSAP (such as a BSAP) described herein, or compositions thereof (such as pharmaceutical compositions). The term "unit dosage form" refers to physically discrete units suitable as single administration to an individual, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient. These unit dosage forms may be stored in suitable packaging in single or multiple unit doses, and may further be sterilized and sealed.

[0153] The present application further provides an article of manufacture comprising the compositions (such as pharmaceutical compositions) described herein in suitable packaging. Suitable packaging for the compositions (such as pharmaceutical compositions) described herein is known in the art and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (such as sealed Mylar or plastic bags), and the like. These articles may be further sterilized and / or sealed.

[0154] The application also provides kits that include the compositions (such as pharmaceutical compositions) described herein, and may further include instruction(s) regarding how to use the compositions, such as the uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for practicing any of the methods described herein.

[0155] V. Cancer Treatment Methods One aspect of the present application provides a method of treating cancer (e.g. DLBCL or ALL) in an individual (such as a human), the method comprising administering to the individual an effective amount of any of the CD3xCD19 MSAPs (such as BSAPs) described herein, or compositions (such as pharmaceutical compositions) thereof. In some embodiments, the CD3xCD19 MSAP (such as BSAP) or compositions (such as pharmaceutical compositions) thereof are administered intravenously. In some embodiments, the method does not induce a cytokine storm.

[0156] In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds CD19, and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv). In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment.In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. An anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond. a Fab fragment; II. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) that specifically bind to CD19, where the first anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment; and III. A first linker links the VH of the Fab fragment and the first anti-CD19 antigen-binding fragment (e.g., scFv), and a second linker links the VL of the anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv). In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) have the same amino acid sequence. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) have different sequences. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) bind to different CD19 epitopes. In some embodiments, the first and second linkers are identical in sequence. In some embodiments, the first and second linkers have different sequences. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:9, HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:11, and / or the VL of the anti-CD3 Fab fragment comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:18 and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:52.In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and / or a VL comprising the amino acid sequence of SEQ ID NO: 8 or 50. In some embodiments, the anti-CD19 antigen-binding fragment is a scFv. In some embodiments, the anti-CD19 antigen-binding fragment is an scFv comprising the amino acid sequence of SEQ ID NO: 51 or 59. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54 or 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:58.In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the MSAP (such as a BSAP) or a composition thereof (such as a pharmaceutical composition) is administered intravenously. In some embodiments, the method does not induce a cytokine storm. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), e.g., blast crisis of CML and CML including the Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), e.g., CLL including Richter's syndrome or Richter's transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), e.g., NHL including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma. In some embodiments, the methods of treating cancer described herein have one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting the proliferation of cancer cells, (3) inducing a redistribution of peripheral T cells (e.g., recruiting T cells to tissues or tumors expressing CD19), (4) reducing tumor size, (5) alleviating one or more symptoms in an individual with cancer, (6) inhibiting tumor metastasis (e.g., metastasis to lymph nodes), (7) increasing survival time, (8) increasing the time to cancer progression, and (9) preventing, inhibiting, or reducing the likelihood of cancer recurrence. In some embodiments, the methods of killing cancer cells mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.In some embodiments, a method of reducing tumor size mediated by a CD3xCD19 MSAP (such as a BSAP) or a pharmaceutical composition thereof described herein may reduce tumor size by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, a method of inhibiting tumor metastasis (e.g. metastasis to lymph nodes) mediated by a CD3xCD19 MSAP (such as a BSAP) or a pharmaceutical composition thereof described herein may inhibit metastasis by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the method of extending the survival time of an individual (such as a human) mediated by a CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may extend the survival time of the individual by at least any of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, or 24 months, or more. In some embodiments, the method of extending the time to cancer progression mediated by a CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may extend the time to cancer progression by at least any of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks ... The pharmaceutical composition may increase, enhance or stimulate a subject's immune response or function by activating effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells). In some embodiments, the individual's CD4 and / or CD8 T cells have increased or enhanced priming, activation, proliferation, cytokine release and / or cytolytic activity compared to before administration of a CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein.

[0157] In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; Fab fragment, II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19, wherein the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49, and III. optionally a linker that links an anti-CD3 Fab fragment and an anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment.In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; Fab fragment, II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19, wherein the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49, and III. optionally a linker that links an anti-CD3 Fab fragment and an anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; II. A first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) that specifically bind to CD19, wherein the first anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of an anti-CD3 Fab fragment, and the second anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of an anti-CD3 Fab fragment. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) fused to the N-terminus of the VL of the Fab fragment, wherein the first and / or second anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49; and III. optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 antigen-binding fragment (e.g., scFv), and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv).In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) have the same amino acid sequence. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) have different sequences. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFvs) bind to different CD19 epitopes. In some embodiments, the first and second linkers are identical in sequence. In some embodiments, the first and second linkers have different sequences. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15 and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and / or a VL comprising the amino acid sequence of SEQ ID NO: 8 or 50. In some embodiments, the anti-CD19 antigen-binding fragment is an scFv comprising the amino acid sequence of SEQ ID NO: 51 or 59. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20 to 22, 29, and 31 to 44 (e.g., SEQ ID NO: 44).In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:58. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the MSAP (such as a BSAP) or a composition thereof (such as a pharmaceutical composition) is administered intravenously. In some embodiments, the method does not induce a cytokine storm. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), e.g., blast crisis of CML and CML including the Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), e.g., CLL including Richter's syndrome or Richter's transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), e.g., NHL including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma.In some embodiments, the methods of treating cancer described herein have one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting the proliferation of cancer cells, (3) inducing a redistribution of peripheral T cells (e.g., recruiting T cells to tissues or tumors expressing CD19), (4) reducing tumor size, (5) alleviating one or more symptoms in an individual with cancer, (6) inhibiting tumor metastasis (e.g., metastasis to lymph nodes), (7) increasing survival time, (8) increasing the time to cancer progression, and (9) preventing, inhibiting, or reducing the likelihood of cancer recurrence. In some embodiments, the methods of killing cancer cells mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the methods of reducing tumor size mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may reduce tumor size by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the methods of inhibiting tumor metastasis (e.g. metastasis to lymph nodes) mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may reduce metastasis by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the method of extending the survival time of an individual (such as a human) mediated by a CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may extend the survival time of the individual by at least any of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, or 24 months, or more. In some embodiments, the method of extending the time to cancer progression mediated by a CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may extend the time to cancer progression by at least any of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, or more. In some embodiments, the CD3×CD19 MSAP (such as a BSAP) described herein or a pharmaceutical composition thereof may increase, enhance or stimulate the immune response or function of a subject by activating effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells). In some embodiments, the CD4 and / or CD8 T cells of the individual have increased or enhanced priming, activation, proliferation, cytokine release and / or cytolytic activity compared to before administration of the CD3×CD19 MSAP (such as a BSAP) described herein or a pharmaceutical composition thereof.

[0158] In some embodiments, the MSAP specifically binds to more than two (such as three or more) epitopes. For example, in some embodiments, the MSAP comprises a first anti-CD19 antigen-binding fragment (e.g., scFv) fused to the N-terminus of the VH of an anti-CD3 Fab fragment, and a second anti-CD19 antigen-binding fragment (e.g., scFv) fused to the N-terminus of the VL of an anti-CD3 Fab fragment, where the first and second anti-CD19 antigen-binding fragments (e.g., scFv) bind different CD19 epitopes. In some embodiments, the MSAP is trispecific. In some embodiments, the MSAP is bispecific (e.g., BSAP) and bivalent. For example, in some embodiments, a bivalent and bispecific MSAP (or BSAP) comprises one anti-CD19 antigen-binding fragment (e.g., scFv) fused to the N-terminus of either the VH or VL of an anti-CD3 Fab fragment. In some embodiments, the MSAP is bispecific (e.g., BSAP) and trivalent. For example, in some embodiments, the MSAP comprises a first anti-CD19 antigen-binding fragment (e.g., scFv) fused to the N-terminus of the VH of the anti-CD3 Fab fragment, and a second anti-CD19 antigen-binding fragment (e.g., scFv) fused to the N-terminus of the VL of the anti-CD3 Fab fragment, wherein the first and second anti-CD19 antigen-binding fragments (e.g., scFv) bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFv) have different amino acid sequences and / or structures but bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragments (e.g., scFv) have the same amino acid sequence. In some embodiments, the anti-CD19 antigen-binding fragment is an scFv. In some embodiments, the anti-CD19 antigen-binding fragment is an scFv comprising the amino acid sequence of SEQ ID NO:51 or 59.

[0159] Thus, in some embodiments, there is provided a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human), the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 scFv that specifically binds to CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 scFv that specifically binds to CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising: (a) an Ig VH and an Ig and (b) an anti-CD3 Fab fragment comprising a CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. a first anti-CD19 scFv and a second anti-CD19 scFv that specifically bind to CD19, wherein the first anti-CD19 scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment and the second anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment; and III. optionally, a first linker that links the VH of the anti-CD3 Fab fragment and the first anti-CD19 scFv, and a second linker that links the VL of the anti-CD3 Fab fragment and the second anti-CD19 scFv. In some embodiments, the first and second anti-CD19 scFv have the same amino acid sequence, such as SEQ ID NO: 51 or 59. In some embodiments, the first and second anti-CD19 scFvs have different sequences. In some embodiments, the first and second anti-CD19 scFvs bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 scFvs bind to different CD19 epitopes. In some embodiments, the first and second linkers are identical in sequence. In some embodiments, the first and second linkers have different sequences. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the anti-CD3 The VH of the Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:9, HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:11, and / or the VL of the anti-CD3 Fab fragment comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or the VL of the anti-CD3 The VL of the Fab fragment comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18 and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, the anti-CD19 scFv comprises a VH comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49. In some embodiments, the anti-CD19 scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and / or a VL comprising the amino acid sequence of SEQ ID NO: 8 or 50. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs:20-22, 29, and 31-44 (e.g., SEQ ID NO:44). In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:53 or 60, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:53, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:54.In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide and a second polypeptide, the first polypeptide comprising the amino acid sequence of SEQ ID NO:60 and the second polypeptide comprising the amino acid sequence of SEQ ID NO:61. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide and a second polypeptide, the first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60 and the second polypeptide comprising the amino acid sequence of SEQ ID NO:58. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide and a second polypeptide, the first polypeptide comprising the amino acid sequence of SEQ ID NO:57 and the second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the MSAP (such as a BSAP) or a composition thereof (such as a pharmaceutical composition) is administered intravenously. In some embodiments, the method does not induce a cytokine storm. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), e.g., blast crisis of CML and CML including the Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), e.g., CLL including Richter's syndrome or Richter's transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), e.g., NHL including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma.In some embodiments, the methods of treating cancer described herein have one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting the proliferation of cancer cells, (3) inducing a redistribution of peripheral T cells (e.g., recruiting T cells to tissues or tumors expressing CD19), (4) reducing tumor size, (5) alleviating one or more symptoms in an individual with cancer, (6) inhibiting tumor metastasis (e.g., metastasis to lymph nodes), (7) increasing survival time, (8) increasing the time to cancer progression, and (9) preventing, inhibiting, or reducing the likelihood of cancer recurrence. In some embodiments, the methods of killing cancer cells mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, a method of reducing tumor size mediated by a CD3xCD19 MSAP (such as a BSAP) or a pharmaceutical composition thereof described herein may reduce tumor size by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, a method of inhibiting tumor metastasis (e.g. metastasis to lymph nodes) mediated by a CD3xCD19 MSAP (such as a BSAP) or a pharmaceutical composition thereof described herein may inhibit metastasis by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the method of extending survival of an individual (such as a human) mediated by a CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may extend survival of the individual by at least any of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months or 24 months or more.In some embodiments, the method of extending the time to cancer progression mediated by the CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may extend the time to cancer progression by at least any of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more. In some embodiments, the CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may increase, enhance, or stimulate the immune response or function of the subject by activating effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells). In some embodiments, the CD4 and / or CD8 T cells of the individual have increased or enhanced priming, activation, proliferation, cytokine release and / or cytolytic activity compared to before administration of the CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein.

[0160] In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; Fab fragment, II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 scFv comprising a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49, and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment.In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; Fab fragment, II. an anti-CD19 scFv that specifically binds to CD19, the anti-CD19 scFv comprising a VH comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49, and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 scFv, wherein the anti-CD19 scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; a first anti-CD19 scFv and a second anti-CD19 scFv that specifically bind to CD19, wherein the first anti-CD19 scFv is fused to the N-terminus of the VH of an anti-CD3 Fab fragment, and the second anti-CD19 scFv is fused to the N-terminus of the VL of an anti-CD3 Fab fragment, and the first and / or second anti-CD19 scFv comprises a VH comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49; and III. a first linker linking the VL of the anti-CD3 Fab fragment and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 scFv. In some embodiments, the first and second anti-CD19 scFv have the same amino acid sequence. In some embodiments, the first and second anti-CD19 scFv have different sequences. In some embodiments, the first and second anti-CD19 scFv bind to the same CD19 epitope.In some embodiments, the first and second anti-CD19 scFvs bind different CD19 epitopes. In some embodiments, the first and second linkers are identical in sequence. In some embodiments, the first and second linkers have different sequences. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or an anti-CD3. The VL of the Fab fragment comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, the anti-CD19 scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and / or a VL comprising the amino acid sequence of SEQ ID NO: 8 or 50. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54 or 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61.In some embodiments, MSAP (such as BSAP) or a composition thereof (such as a pharmaceutical composition) is administered intravenously. In some embodiments, the method does not induce a cytokine storm. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), e.g., blast crisis of CML and CML including the Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), e.g., CLL including Richter's syndrome or Richter's transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), e.g., NHL including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma. In some embodiments, the methods of treating cancer described herein have one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting the proliferation of cancer cells, (3) inducing a redistribution of peripheral T cells (e.g., recruiting T cells to tissues or tumors expressing CD19), (4) reducing tumor size, (5) alleviating one or more symptoms in an individual with cancer, (6) inhibiting tumor metastasis (e.g., metastasis to lymph nodes), (7) increasing survival time, (8) increasing the time to cancer progression, and (9) preventing, inhibiting, or reducing the likelihood of cancer recurrence. In some embodiments, the methods of killing cancer cells mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of reducing tumor size mediated by a CD3×CD19 MSAP (such as a BSAP) or a pharmaceutical composition thereof described herein may reduce tumor size by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%).In some embodiments, the method of inhibiting tumor metastasis (e.g. metastasis to lymph nodes) mediated by the CD3×CD19 MSAP (such as BSAP) or pharmaceutical composition thereof described herein may inhibit metastasis by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the method of extending survival of an individual (such as a human) mediated by the CD3×CD19 MSAP (such as BSAP) or pharmaceutical composition thereof described herein may extend survival of the individual by at least any of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, or 24 months or more. In some embodiments, the time to cancer progression mediated by the CD3×CD19 MSAP (such as BSAP) or pharmaceutical composition thereof described herein is extended. The method may extend the time to cancer progression by at least any of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more. In some embodiments, the CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may increase, enhance, or stimulate the immune response or function of the subject by activating effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells). In some embodiments, the CD4 and / or CD8 T cells of the individual have increased or enhanced priming, activation, proliferation, cytokine release and / or cytolytic activity compared to before administration of the CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein.

[0161] In some embodiments, the MSAP (such as a BSAP) comprises two anti-CD19 scFvs, hi some embodiments, the two anti-CD19 scFvs both comprise the amino acid sequence of SEQ ID NO:51 or 59.

[0162] Thus, in some embodiments, there is provided a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human), the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond, the VH of the anti-CD3 Fab fragment comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and / or the VL of the anti-CD3 Fab fragment comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; Fab fragment, II. a first anti-CD19 scFv and a second anti-CD19 scFv that specifically bind to CD19, the first anti-CD19 scFv being fused to the N-terminus of the VH of an anti-CD3 Fab fragment, and the second anti-CD19 scFv being fused to the N-terminus of the VL of an anti-CD3 Fab fragment, both of the first and second anti-CD19 scFv comprising the amino acid sequence of SEQ ID NO: 51 or 59, and III. optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 scFv, and a second linker linking the VL of the anti-CD3 Fab fragment and the second anti-CD19 scFv. In some embodiments, the first and second linkers are identical in sequence. In some embodiments, the first and second linkers have different sequences. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15 and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16.In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20 to 22, 29, and 31 to 44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54. In some embodiments, the MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:61. In some embodiments, the MSAP (such as a BSAP) or a composition thereof (such as a pharmaceutical composition) is administered intravenously. In some embodiments, the method does not induce a cytokine storm.In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), e.g., blast crisis of CML and CML including the Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), e.g., CLL including Richter's syndrome or Richter's transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), e.g., NHL including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma. In some embodiments, the methods of treating cancer described herein have one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting the proliferation of cancer cells, (3) inducing a redistribution of peripheral T cells (e.g., recruiting T cells to tissues or tumors expressing CD19), (4) reducing tumor size, (5) alleviating one or more symptoms in an individual with cancer, (6) inhibiting tumor metastasis (e.g., metastasis to lymph nodes), (7) increasing survival time, (8) increasing the time to cancer progression, and (9) preventing, inhibiting, or reducing the likelihood of cancer recurrence. In some embodiments, the methods of killing cancer cells mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of reducing tumor size mediated by a CD3×CD19 MSAP (such as a BSAP) or a pharmaceutical composition thereof described herein may reduce tumor size by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%).In some embodiments, a method of inhibiting tumor metastasis (e.g. metastasis to lymph nodes) mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may inhibit metastasis by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, a method of extending survival time of an individual (such as a human) mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may extend survival time of the individual by at least any of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, or 24 months or more. In some embodiments, the method of extending the time to cancer progression mediated by the CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may extend the time to cancer progression by at least any of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more. In some embodiments, the CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may increase, enhance, or stimulate the immune response or function of the subject by activating effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells). In some embodiments, the CD4 and / or CD8 T cells of the individual have increased or enhanced priming, activation, proliferation, cytokine release and / or cytolytic activity compared to before administration of the CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein.

[0163] In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (e.g., BSAP, or a pharmaceutical composition thereof) comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 53 or 60 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 54 or 61. In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (e.g., BSAP, or a pharmaceutical composition thereof) comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 53 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (e.g., BSAP, or a pharmaceutical composition thereof) comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 60 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 61. In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as BSAP, or a pharmaceutical composition thereof) comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 53 or 60, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, a method of treating cancer (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as BSAP, or a pharmaceutical composition thereof) comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 57, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 54 or 61. In some embodiments, the MSAP (such as BSAP) or a composition thereof (such as a pharmaceutical composition) is administered intravenously. In some embodiments, the method does not induce a cytokine storm.In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), e.g., blast crisis of CML and CML including the Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), e.g., CLL including Richter's syndrome or Richter's transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), e.g., NHL including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma. In some embodiments, the methods of treating cancer described herein have one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting the proliferation of cancer cells, (3) inducing a redistribution of peripheral T cells (e.g., recruiting T cells to tissues or tumors expressing CD19), (4) reducing tumor size, (5) alleviating one or more symptoms in an individual with cancer, (6) inhibiting tumor metastasis (e.g., metastasis to lymph nodes), (7) increasing survival time, (8) increasing the time to cancer progression, and (9) preventing, inhibiting, or reducing the likelihood of cancer recurrence. In some embodiments, the methods of killing cancer cells mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may achieve a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method of reducing tumor size mediated by a CD3×CD19 MSAP (such as a BSAP) or a pharmaceutical composition thereof described herein may reduce tumor size by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%).In some embodiments, a method of inhibiting tumor metastasis (e.g. metastasis to lymph nodes) mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may inhibit metastasis by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, a method of extending survival time of an individual (such as a human) mediated by a CD3xCD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may extend survival time of the individual by at least any of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, or 24 months or more. In some embodiments, the method of extending the time to cancer progression mediated by the CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may extend the time to cancer progression by at least any of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or more. In some embodiments, the CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein may increase, enhance, or stimulate the immune response or function of the subject by activating effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells). In some embodiments, the CD4 and / or CD8 T cells of the individual have increased or enhanced priming, activation, proliferation, cytokine release and / or cytolytic activity compared to before administration of the CD3×CD19 MSAP (such as a BSAP) or pharmaceutical composition thereof described herein.

[0164] In some embodiments, a method of killing cancer cells (e.g., DLBCL or ALL) in an individual (such as a human) is provided, the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, a method of killing cancer cells (e.g., DLBCL or ALL) in an individual (e.g., a human) is provided, the method comprising administering to the individual an effective amount of MSAP (e.g., BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, a method is provided for killing cancer cells (e.g., DLBCL or ALL) in an individual (such as a human), the method comprising administering to the individual an effective amount of MSAP (such as BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 antibody that specifically binds to CD3; an anti-CD3 Fab fragment, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) that specifically bind to CD19, the first anti-CD19 antigen-binding fragment (e.g., scFv) being fused to the N-terminus of the VH of the anti-CD3 Fab fragment, and the second anti-CD19 antigen-binding fragment (e.g., scFv) being fused to the N-terminus of the VL of the anti-CD3 Fab fragment; and III. A first linker links the VH of the Fab fragment and the first anti-CD19 antigen-binding fragment (e.g., scFv), and a second linker links the VL of the anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv). In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) have the same amino acid sequence. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) have different amino acid sequences. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) bind to different CD19 epitopes. In some embodiments, the first and second linkers have the same amino acid sequence. In some embodiments, the first and second linkers have different amino acid sequences. In some embodiments, the tumor cell death rate mediated by MSAP (such as BSAP) is at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the MSAP (such as BSAP, or a pharmaceutical composition thereof) is administered intravenously. In some embodiments, the method does not induce a cytokine storm.In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:9, HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:11, and / or the VL of the anti-CD3 Fab fragment comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46), which can form an intermolecular disulfide bond. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50. In some embodiments, an anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO:51 or 59.In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54 or 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:57 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), e.g., blast crisis of CML and CML including the Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), e.g., CLL including Richter's syndrome or Richter's transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), e.g., NHL including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma.

[0165] In some embodiments, a method is provided for inhibiting the proliferation of cancer cells (e.g., DLBCL or ALL) in an individual (e.g., a human), the method comprising administering to the individual an effective amount of MSAP (e.g., BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; a Fab fragment, II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19, and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, a method of inhibiting the proliferation of cancer cells (e.g., DLBCL or ALL) in an individual (e.g., a human) is provided, the method comprising administering to the individual an effective amount of MSAP (e.g., BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.In some embodiments, a method of inhibiting the proliferation of cancer cells (e.g., DLBCL or ALL) in an individual (e.g., a human) is provided, the method comprising administering to the individual an effective amount of MSAP (e.g., BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. a first anti-CD19 antigen binding fragment (e.g., scFv) and a second anti-CD19 antigen binding fragment (e.g., scFv) that specifically bind to CD19, the first anti-CD19 antigen binding fragment (e.g., scFv) being fused to the N-terminus of the VH of the anti-CD3 Fab fragment and the second anti-CD19 antigen binding fragment (e.g., scFv) being fused to the N-terminus of the VH of the anti-CD3 Fab fragment; III. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) fused to the N-terminus of the VL of the Fab fragment, and optionally a first linker linking the VH of the anti-CD3 Fab fragment and the first anti-CD19 antigen-binding fragment (e.g., scFv), and a second linker linking the VH of the anti-CD3 Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv). In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) have the same amino acid sequence. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) have different amino acid sequences. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) bind to different CD19 epitopes. In some embodiments, the first and second linkers have the same amino acid sequence. In some embodiments, the first and second linkers have different amino acid sequences. In some embodiments, the cancer cell proliferation rate is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more in the presence of MSAP (such as BSAP, or a pharmaceutical composition thereof).In some embodiments, MSAP (such as BSAP, or a pharmaceutical composition thereof) is administered intravenously. In some embodiments, the method does not induce a cytokine storm. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (e.g., an epitope within amino acids 1-27 of CD3ε). In some embodiments, the VH of the anti-CD3 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:9, HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:11, and / or the VL of the anti-CD3 Fab fragment comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18 and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalently linked sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46) capable of forming an intermolecular disulfide bond. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:6 or 49. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and / or a VL comprising the amino acid sequence of SEQ ID NO:8 or 50.In some embodiments, anti-CD19. The scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20 to 22, 29, and 31 to 44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54 or 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:58. In some embodiments, an MSAP (such as a BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:54 or 61. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), e.g., blast crisis of CML and CML including the Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), e.g., CLL including Richter's syndrome or Richter's transformation of CLL, hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), e.g., NHL including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma.

[0166] In some embodiments, a method is provided for inducing peripheral T cell redistribution in an individual (such as a human) (e.g., recruiting T cells to tissues or tumors expressing CD19), the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising: (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig an anti-CD3 Fab fragment comprising a CL, and optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 antigen-binding fragment (e.g., an scFv) that specifically binds to CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., an scFv), wherein the anti-CD19 antigen-binding fragment (e.g., the scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment. In some embodiments, there is a method of inducing peripheral T cell redistribution in an individual (such as a human) (e.g., recruiting T cells to tissues or tumors expressing CD19), the method comprising administering to the individual an effective amount of MSAP (such as a BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising (a) an Ig VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. an anti-CD19 antigen-binding fragment (e.g., scFv) that specifically binds to CD19; and III. optionally a linker linking the anti-CD3 Fab fragment and the anti-CD19 antigen-binding fragment (e.g., scFv), wherein the anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment. In some embodiments, a method is provided for inducing a redistribution of peripheral T cells in an individual (such as a human) (e.g., recruiting T cells to tissues or tumors expressing CD19), the method comprising administering to the individual an effective amount of MSAP (such as BSAP, or a pharmaceutical composition thereof) comprising: I. an anti-CD3 Fab fragment that specifically binds to CD3, the anti-CD3 Fab fragment comprising: (a) an Ig an anti-CD3 Fab fragment comprising (a) a VH and an Ig CH1, and (b) an Ig VL and an Ig CL, optionally wherein the CH1 and CL of the anti-CD3 Fab fragment are linked by a disulfide bond; II. a first anti-CD19 antigen-binding fragment (e.g., scFv) and a second anti-CD19 antigen-binding fragment (e.g., scFv) that specifically bind to CD19, wherein the first anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VH of the anti-CD3 Fab fragment, and the second anti-CD19 antigen-binding fragment (e.g., scFv) is fused to the N-terminus of the VL of the anti-CD3 Fab fragment; and III. A second linker links the VH of the Fab fragment and the second anti-CD19 antigen-binding fragment (e.g., scFv). In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) have the same amino acid sequence. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) have different amino acid sequences. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) bind to the same CD19 epitope. In some embodiments, the first and second anti-CD19 antigen-binding fragment (e.g., scFv) bind to different CD19 epitopes. In some embodiments, the first and second linkers have the same amino acid sequence. In some embodiments, the first and second linkers have different amino acid sequences. In some embodiments, the MSAP (such as BSAP, or a pharmaceutical composition thereof) is administered intravenously. In some embodiments, the method does not induce a cytokine storm. In some embodiments, the anti-CD3 Fab fragment specifically binds to the N-terminus of CD3ε (eg, an epitope within amino acids 1-27 of CD3ε).In some embodiments, the VH of the anti-CD3 Fab fragment comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:9, HVR-H2 comprising the amino acid sequence of SEQ ID NO:10, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:11, and / or the VL of the anti-CD3 Fab fragment comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:12, HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the VH of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:15, and / or the VL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the CH1 and CL of the anti-CD3 Fab fragment are linked by about 1 to about 5 disulfide bonds (e.g., 2 disulfide bonds). In some embodiments, the CH1 of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 18, and / or the CL of the anti-CD3 Fab fragment comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the C-terminus of the CH1 and CL of the anti-CD3 Fab fragment each further comprises a covalent sequence of CPPC (SEQ ID NO: 45) or CPPCS (SEQ ID NO: 46), which can form an intermolecular disulfide bond. In some embodiments, an anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and / or a VL comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 or 49. In some embodiments, the anti-CD19 antigen-binding fragment (e.g., scFv) comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and / or a VL comprising the amino acid sequence of SEQ ID NO: 8 or 50. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 51 or 59. In some embodiments, the linker comprises about 2 to about 30 (e.g., about 2 to about 15) amino acid residues selected from the group consisting of glycine, serine, arginine, and alanine.In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 20-22, 29, and 31-44 (e.g., SEQ ID NO: 44). In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54 or 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 53 or 60, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the MSAP (e.g., BSAP) comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 54 or 61. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), e.g., blast crisis of CML and CML including the Abelson oncogene associated with CML (Bcr-ABL translocation), myelodysplastic syndrome (MDS), acute B-lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), e.g., CLL i...

Claims

[Claim 1] The invention described in the specification.

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