CD19 binding molecules and uses thereof

Abstract

The present disclosure provides CD19 binding molecules that specifically bind to CD19, including monospecific, bispecific, and trispecific binding molecules, conjugates comprising the CD19 binding molecules, and pharmaceutical compositions comprising the CD19 binding molecules and conjugates. The present disclosure further provides methods of using the CD19 binding molecules to treat diseases and disorders associated with CD19 expression. The present disclosure still further provides recombinant host cells engineered to express the CD19 binding molecules and methods of producing the CD19 binding molecules by culturing the host cells under conditions in which the CD19 binding molecules are expressed.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 110,501, filed November 6, 2020, U.S. Provisional Patent Application No. 63 / 114,371, filed November 16, 2020, U.S. Provisional Patent Application No. 63 / 147,501, filed February 9, 2021, and U.S. Provisional Patent Application No. 63 / 110,490, filed November 6, 2020, the contents of each of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on November 3, 2021, is named NOV-014WO_SL.txt, and is 768,034 bytes in size.

[0003] The present disclosure relates generally to CD19 binding molecules, including monospecific, bispecific and trispecific binding molecules, that specifically bind to CD19 and their use to treat diseases and disorders associated with expression of CD19. [Background technology]

[0004] B cells express a wide range of cell surface molecules during their differentiation and proliferation. CD19 is a pan-B cell membrane glycoprotein expressed from early stages of pre-B cell development through terminal differentiation and regulates the development and function of B lymphocytes. CD19 expression has been identified in many cancers of lymphoid origin, the majority of non-Hodgkin's lymphomas (NHL), and leukemias, including chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), and Waldenstrom's macroglobulinemia (WM).

[0005] Blinatumomab, a CD19-CD3 bispecific T-cell aggregate, has been approved for the treatment of ALL. However, treatment with blinatumomab is characterized by a lack of durable responses and a high relapse rate. (Von Stackelberg et al., 2016, Journal of Clinical Oncology 34(36):4381-4389) Furthermore, blinatumomab has a short half-life, thus requiring continuous exposure to the drug for adequate efficacy with manageable toxicity. (Porter et al., 2013, Clin Pharmacol. 5(Suppl 1):5-11)

[0006] Despite significant improvements in cancer therapy, B-cell malignancies, such as the B-cell subtype of non-Hodgkin's lymphoma and chronic lymphocytic leukemia, are significant causes of cancer-related deaths. Thus, there remains a need for additional therapeutic agents for the treatment of B-cell malignancies. Summary of the Invention

[0007] The present disclosure provides CD19 binding molecules that specifically bind to human CD19, eg, antibodies, antigen-binding fragments thereof, and multispecific molecules that specifically bind to human CD19. CD19 binding molecules of the disclosure typically comprise an Fc domain comprising a first variant human IgG1 Fc region and a second variant human IgG1 Fc region having L234A, L235A, and G237A ("LALAGA") substitutions, L234A, L235A, S267K, and P329A ("LALASKPA") substitutions, D265A, P329A, and S267K ("DAPASK") substitutions, G237A, D265A, and P329A ("GADAPA") substitutions, G237A, D265A, P329A, and S267K ("GADAPASK") substitutions, L234A, L235A, and P329G ("LALAPG") substitutions, or L234A, L235A, and P329A ("LALAPA") substitutions.

[0008] In one aspect, the present disclosure provides monospecific CD19 binding molecules (e.g., antibodies and antigen-binding fragments thereof) comprising a CD19 antigen-binding domain or antigen-binding module ("ABM"). Exemplary CD19 binding molecules that may be monospecific are described in Section 7.2, below, and in specific embodiments 1-15.

[0009] In another embodiment, the present disclosure provides a multispecific binding molecule ("MBM") comprising a CD19 ABM of the present disclosure.

[0010] In certain embodiments, the MBM is a bispecific binding molecule ("BBM"). A BBM of the present disclosure includes a first ABM ("ABM1" or "CD19 ABM") that specifically binds human CD19 and a second ABM ("ABM2") that specifically binds a second antigen, such as human CD3 or another component of the T cell receptor (TCR) complex (sometimes referred to herein as a "TCR ABM"). The terms ABM1, ABM2, CD19 ABM, and TCR ABM are used for convenience only and are not intended to convey any particular form of BBM. In some embodiments, the TCR ABM binds CD3 (referred to herein as a "CD3 ABM" or the like). Thus, the disclosure regarding ABM2 and TCR ABMs is also applicable to CD3 ABMs. Such multispecific molecules can be used to target CD3+ effector T cells to CD19+ sites, thereby enabling the CD3+ effector T cells to attack and lyse CD19+ cells and tumors. Exemplary MBM features are described below in Sections 7.5-7.6 and in specific embodiments 16-783.

[0011] The present disclosure also relates to the principle of redirected targeted T cell lysis (RTCC) by providing a trispecific binding molecule ("TBM") that associates with either CD19, CD3, or other components of the TCR complex on T cells and CD2 or a human tumor-associated antigen ("TAA"), e.g., a B cell antigen other than CD19. The TBM of the present disclosure comprises at least three antigen binding modules ("ABM") capable of binding to (i) CD19 (ABM1), (ii) a component of the TCR complex (ABM2), and (iii) either CD2 or a TAA (ABM3). A TBM that binds (1) human CD19, (2) CD3 or other components of the TCR complex, and (3) CD2 is conveniently referred to herein as a "Type 1 TBM." A TBM that binds (1) human CD19, (2) CD3 or other components of the TCR complex, and (3) a TAA is conveniently referred to herein as a "Type 2 TBM."

[0012] Without being bound by theory, the inventors believe that the combined engagement of the CD2 and TCR complex in type 1 TBM can stimulate both a primary signaling pathway that promotes T cell-mediated tumor cell lysis (e.g., by TCR clustering) and a secondary costimulatory pathway that induces T cell proliferation, potentially overcoming anergy. Without being bound by theory, it is also believed that the engagement of a TAA in addition to CD19 and components of the TCR complex in type 2 TBM may improve clinical outcomes of RTCC therapy of cancer, e.g., B cell malignancies, by targeting more cancerous B cells than with bispecific associations that target only CD19 and TCR complex components.

[0013] Thus, in one aspect, the disclosure provides a type 1 TBM that binds to (1) human CD19, (2) CD3 or other components of the TCR complex, and (3) CD2.

[0014] In another aspect, the present disclosure provides a type 2 TBM that binds (1) human CD19, (2) CD3 or other components of the TCR complex, and (3) a TAA.

[0015] Unless expressly stated otherwise or the context dictates otherwise, references to TBM in this disclosure apply to both Type 1 and Type 2 TBM.

[0016] In some embodiments, each antigen binding module of an MBM of the present disclosure is capable of binding to its respective target simultaneously with each of the one or more additional antigen binding modules. ABM1 is immunoglobulin-based, while ABM2 and, if present, ABM3, can be immunoglobulin-based or non-immunoglobulin-based. Thus, an MBM can include immunoglobulin-based ABMs or any combination of immunoglobulin-based and non-immunoglobulin-based ABMs. Immunoglobulin-based ABMs that may be used in an MBM are described below in Section 7.3.1 and in specific embodiments 17-21 and 24-29. Non-immunoglobulin-based ABMs that may be used in an MBM are described below in Section 7.3.2 and in specific embodiments 22-23. Further characteristics of an exemplary ABM that binds to human CD19 are described below in Section 7.2 and in specific embodiments 17-21. Further characteristics of an exemplary ABM that binds to a component of the TCR complex are described below in Section 7.7 and in specific embodiments 30-223. Further features of exemplary ABMs that bind to CD2 are described below in Section 7.8 and in specific embodiments 325-374. Further features of exemplary ABMs that bind to TAAs are described below in Section 7.9 and in specific embodiments 375-493.

[0017] The ABMs of an MBM (or portion thereof) can be linked to one another by, for example, a short peptide linker or an Fc domain. Methods and components for linking ABMs to form an MBM are described in Section 7.4 below and in specific embodiments 497-770.

[0018] A BBM has at least two ABMs (e.g., the BBM is at least bivalent), and a TBM has at least three ABMs (e.g., the TBM is at least trivalent), although they can have greater valencies. For example, a BBM can have three, four, or more ABMs (i.e., trivalent, tetravalent, or greater than tetravalent). Exemplary bivalent, trivalent, and tetravalent BBM forms are shown in Figure 1 and described below in Section 7.5 and in specific embodiments 226-286.

[0019] A TBM can have four ABMs (i.e., tetravalent), five ABMs (i.e., pentavalent), or six ABMs (i.e., hexavalent), provided that the TBM has at least one ABM capable of binding to CD19, at least one ABM capable of binding to a component of the TCR complex, and at least one ABM capable of binding to either CD2 or a TAA. Exemplary trivalent, tetravalent, pentavalent, and hexavalent TBM formats are shown in Figure 2 and described below in Section 7.6 and in specific embodiments 289-323.

[0020] The present disclosure further provides nucleic acids encoding CD19 binding molecules (either in a single nucleic acid or multiple nucleic acids) and recombinant host cells and cell lines engineered to express the nucleic acids and CD19 binding molecules of the disclosure. Exemplary nucleic acids, host cells, and cell lines are described in Section 7.10, below, and in specific embodiments 885-892.

[0021] The present disclosure further provides drug conjugates comprising the CD19 binding molecules of the present disclosure. For convenience, such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs," even though part of the ABM may be a non-immunoglobulin domain. Examples of ADCs are described in Section 7.12, below, and in specific embodiments 784-822. Pharmaceutical compositions comprising the CD19 binding molecules are also provided. Examples of pharmaceutical compositions are described in Section 7.15, below, and in specific embodiment 823.

[0022] Further provided herein are methods of using the CD19 binding molecules, ADCs, and pharmaceutical compositions of the present disclosure, for example, to treat proliferative diseases in which CD19 is expressed (e.g., cancer), to treat autoimmune diseases, and to treat other diseases and conditions associated with CD19 expression. In some embodiments, the cancer is a B-cell malignancy (e.g., NHL, such as DLBCL or MCL). In some embodiments, the CD19 binding molecules, ADCs, and pharmaceutical compositions of the present disclosure are administered to a subject with NHL, e.g., DLBCL or MCL, who (i) has failed at least one prior (and optionally up to five prior) standard of care therapy, e.g., anti-CD20 therapy such as rituximab, and / or (ii) is intolerant to or ineligible for one or more other approved therapies, e.g., autologous stem cell transplantation (ASCT), and / or (iii) is a non-responder to chimeric antigen receptor (CAR) T-cell therapy. The NHL may be relapsed and / or refractory. Further exemplary methods are described below in Section 7.16 and in specific embodiments 824-881.

[0023] The present disclosure further provides methods of using the CD19 binding molecules, ADCs, and pharmaceutical compositions in combination with other agents and therapies. Exemplary agents, therapies, and methods of combination treatment are described in Section 7.17, below, and in specific embodiment 881.

[0024] a first mutant human IgG1 Fc region and a second mutant human IgG1 Fc region that specifically bind to human CD19 and that form an Fc domain; and (a) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; and Provided herein are CD19 binding molecules comprising an Fc region, wherein the first and second variant Fc regions comprise L234A, L235A, and G237A ("LALAGA") substitutions, L234A, L235A, S267K, and P329A ("LALASKPA") substitutions, D265A, P329A, and S267K ("DAPASK") substitutions, G237A, D265A, and P329A ("GADAPA") substitutions, G237A, D265A, P329A, and S267K ("GADAPASK") substitutions, L234A, L235A, and P329G ("LALAPG") substitutions, or L234A, L235A, and P329A ("LALAPA") substitutions, wherein the amino acid residues are numbered according to the EU numbering system.

[0025] a first mutant human IgG1 Fc region and a second mutant human IgG1 Fc region that specifically bind to human CD19 and that form an Fc domain; and (a) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; and Provided herein are CD19 binding molecules comprising an Fc region, wherein the first and second variant Fc regions comprise L234A, L235A, and G237A ("LALAGA") substitutions, L234A, L235A, S267K, and P329A ("LALASKPA") substitutions, D265A, P329A, and S267K ("DAPASK") substitutions, G237A, D265A, and P329A ("GADAPA") substitutions, G237A, D265A, P329A, and S267K ("GADAPASK") substitutions, L234A, L235A, and P329G ("LALAPG") substitutions, or L234A, L235A, and P329A ("LALAPA") substitutions, wherein the amino acid residues are numbered according to the EU numbering system.

[0026] a first mutant human IgG1 Fc region and a second mutant human IgG1 Fc region that specifically bind to human CD19 and that form an Fc domain; and (a) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; and Provided herein are CD19 binding molecules comprising an Fc region, wherein the first and second variant Fc regions comprise L234A, L235A, and G237A ("LALAGA") substitutions, L234A, L235A, S267K, and P329A ("LALASKPA") substitutions, D265A, P329A, and S267K ("DAPASK") substitutions, G237A, D265A, and P329A ("GADAPA") substitutions, G237A, D265A, P329A, and S267K ("GADAPASK") substitutions, L234A, L235A, and P329G ("LALAPG") substitutions, or L234A, L235A, and P329A ("LALAPA") substitutions, wherein the amino acid residues are numbered according to the EU numbering system.

[0027] a first mutant human IgG1 Fc region and a second mutant human IgG1 Fc region that specifically bind to human CD19 and that form an Fc domain; and (a) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25. Provided herein are CD19 binding molecules comprising an Fc region, wherein the first and second variant Fc regions comprise L234A, L235A, and G237A ("LALAGA") substitutions, L234A, L235A, S267K, and P329A ("LALASKPA") substitutions, D265A, P329A, and S267K ("DAPASK") substitutions, G237A, D265A, and P329A ("GADAPA") substitutions, G237A, D265A, P329A, and S267K ("GADAPASK") substitutions, L234A, L235A, and P329G ("LALAPG") substitutions, or L234A, L235A, and P329A ("LALAPA") substitutions, wherein the amino acid residues are numbered according to the EU numbering system.

[0028] In some embodiments, a CD19 binding molecule may comprise a VH having the amino acid sequence of SEQ ID NO: 13. A CD19 binding molecule may also comprise a VL having the amino acid sequence of SEQ ID NO:26.

[0029] In some embodiments, the CD19 binding molecule is a multispecific binding molecule (MBM) that includes (a) an antigen binding module 1 (ABM1) that specifically binds to CD19; and (b) an antigen binding module 2 (ABM2) that specifically binds to a different target molecule. In some embodiments, the target molecule is a component of the human T-cell receptor (TCR) complex.

[0030] In some embodiments, the CD19 binding molecule has an ABM1 that is an antibody, antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab')2, single domain antibody (SDAB), VH or VL domain, or camelid VHH domain. For example, in some embodiments, the ABM1 is a Fab. In other embodiments, the ABM1 is an anti-CD19 antibody or an antigen-binding domain thereof.

[0031] In some embodiments, the CD19-binding molecule has an ABM2 that is an immunoglobulin scaffold-based ABM. For example, the ABM2 can be an antibody, antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab')2, single-domain antibody (SDAB), VH or VL domain, or camelid VHH domain. As a specific example, the ABM2 is an scFv. In some embodiments, the ABM2 can specifically bind to a component of the human T cell receptor (TCR) complex. For example, the component of the TCR complex can be CD3. In some embodiments, the ABM2 is an anti-CD3 antibody or an antigen-binding domain thereof. When the CD19-binding molecule binds to CD3, the ABM2 can comprise the CDR sequence of any one of CD3-1 to CD3-130. More specifically, the ABM2 can comprise the CDR sequence of CD3-21.

[0032] In some embodiments, the CDR sequences of the CD3 binding portions of the binding molecules described herein can be defined by Kabat numbering, as shown in Table 12B. The CDR sequences of the CD3 binding portions of the binding molecules described herein can also be defined by Chothia numbering, as shown in Table 12C. The CDR sequences of the CD3 binding portions of the binding molecules described herein can also be defined by a combination of Kabat numbering and Chothia numbering, as shown in Table 12D.

[0033] In some embodiments, the CD19 binding molecule may have an ABM2 comprising the heavy and light chain variable sequences of CD3-21, as shown in Table 12A.

[0034] More specifically, the CD19 binding molecule can be a trispecific binding molecule (TBM) that includes an antigen binding module 3 (ABM3) that specifically binds to a target molecule other than CD19.

[0035] In some embodiments, the CD19 binding molecule may have an ABM2 that specifically binds to a component of the human T cell receptor (TCR) complex and an ABM3 that specifically binds to human CD2. In some embodiments, the CD19 binding molecule is trivalent. The CD19 binding molecule may have any one of the forms shown in Figures 2A-2P. For example, the CD19 binding molecule has the form shown in Figure 2I. In some embodiments, the CD19 binding molecule has the form referred to as T2 in Section 7.6.1.

[0036] In certain embodiments, the CD19 binding molecule has an ABM3 that specifically binds to human CD2. In some embodiments, the ABM3 is a non-immunoglobulin scaffold-based ABM. When the ABM3 binds to human CD2, the ABM3 can include a receptor binding domain of a CD2 ligand. For example, the ABM3 can be a CD58 portion. In some embodiments, the CD58 portion includes the amino acid sequence of CD58-6 shown in Table 15.

[0037] The CD19 binding molecule may have modifications in its Fc region. For example, the first and second variant Fc regions may comprise L234A, L235A, and G237A ("LALAGA") substitutions, with the amino acid residues numbered according to the EU numbering system. In another embodiment, the first and second variant Fc regions may comprise L234A, L235A, S267K, and P329A ("LALASKPA") substitutions, with the amino acid residues numbered according to the EU numbering system. In yet another embodiment, the first and second variant Fc regions may comprise D265A, P329A, and S267K ("DAPASK") substitutions, with the amino acid residues numbered according to the EU numbering system. In other embodiments, the first and second variant Fc regions may comprise G237A, D265A, and P329A ("GADAPA") substitutions, with the amino acid residues numbered according to the EU numbering system. In another embodiment, the first and second variant Fc regions may comprise G237A, D265A, P329A, and S267K ("GADAPASK") substitutions, with the amino acid residues numbered according to the EU numbering system. In another embodiment, the first and second variant Fc regions may comprise L234A, L235A, and P329G ("LALAPG") substitutions, with the amino acid residues numbered according to the EU numbering system. In another embodiment, the first variant Fc region and the second variant Fc region may comprise L234A, L235A and P329A ("LALAPA") substitutions, wherein the amino acid residues are numbered according to the EU numbering system.

[0038] The Fc region of the CD19 binding molecule can have a first variant Fc region and a second variant Fc region, which together form an Fc heterodimer. For example, the Fc heterodimer can include a knobs-in-holes ("KIH") modification. In some embodiments, the first and second variant Fc regions can include the amino acid substitutions T366W:T366S / L368A / Y407V.

[0039] In a particular example, the CD19 binding molecule comprises: (a) an antigen binding module 1 (ABM1) that specifically binds to CD19 and includes CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; (b) an antigen binding module 2 (ABM2) that specifically binds to a component of the human T cell receptor (TCR) complex; (c) an antigen binding module 3 (ABM3) that specifically binds to human CD2; and (d) a first mutant human IgG1 Fc region and a second mutant human IgG1 Fc region that form an Fc domain. and a trispecific binding molecule (TBM) comprising an Fc region, wherein the first and second variant Fc regions comprise L234A, L235A, and G237A ("LALAGA") substitutions, L234A, L235A, S267K, and P329A ("LALASKPA") substitutions, D265A, P329A, and S267K ("DAPASK") substitutions, G237A, D265A, and P329A ("GADAPA") substitutions, G237A, D265A, P329A, and S267K ("GADAPASK") substitutions, L234A, L235A, and P329G ("LALAPG") substitutions, or L234A, L235A, and P329A ("LALAPA") substitutions, wherein amino acid residues are numbered according to the EU numbering system. In this particular example, the CD19 binding molecule is trivalent. Additionally, ABM1 is a Fab. In this particular example, the Fab comprises a VH having the amino acid sequence of SEQ ID NO: 13 and a VL having the amino acid sequence of SEQ ID NO: 26. Furthermore, the CD19 binding molecule can bind to a component of the TCR complex, which is CD3. In this example, ABM2 is an anti-CD3 antibody or its antigen-binding domain. The sequence of ABM2 comprises the CDR sequences of CD3-21. For example, ABM2 comprises the heavy and light chain variable sequences of CD3-21, as shown in Table 12A. The anti-CD3 antibody or its antigen-binding domain is in the form of an scFv in this example. Furthermore, ABM2 comprises the amino acid sequence of the scFv shown as CD3-21 in Table 12A. Regarding the ABM3 arm in this example, ABM3 is a CD58 moiety. More specifically, ABM3 comprises the amino acid sequence of CD58-6, as shown in Table 15.

[0040] In another specific example, the CD19 binding molecule comprises: (a) antigen binding module 1 (ABM1) that specifically binds to CD19 and is a Fab comprising CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; (b) antigen binding module 2 (ABM2) that specifically binds to CD3 and comprises the amino acid sequence of the scFv shown as CD3-21 in Table 12A; (c) antigen binding module 3 (ABM3) that specifically binds to human CD2 and comprises the amino acid sequence of CD58-6 shown in Table 15; and (d) a first mutant human IgG1 Fc region and a second mutant human IgG1 Fc region that form an Fc domain. and a trispecific binding molecule (TBM) comprising an Fc region, wherein the first and second variant Fc regions comprise L234A, L235A, and G237A ("LALAGA") substitutions, L234A, L235A, S267K, and P329A ("LALASKPA") substitutions, D265A, P329A, and S267K ("DAPASK") substitutions, G237A, D265A, and P329A ("GADAPA") substitutions, G237A, D265A, P329A, and S267K ("GADAPASK") substitutions, L234A, L235A, and P329G ("LALAPG") substitutions, or L234A, L235A, and P329A ("LALAPA") substitutions, wherein amino acid residues are numbered according to the EU numbering system. In this example, ABM1 comprises a VH having the amino acid sequence of SEQ ID NO: 13 and a VL having the amino acid sequence of SEQ ID NO: 26. Additionally, the CD19 binding molecule has the form shown in Figure 2I and referred to as T2 in Section 7.6.1.

[0041] The CD19 binding molecules described throughout the specification can be made into pharmaceutical compositions. For example, the pharmaceutical composition includes (a) any one of the CD19 binding molecules or conjugates described throughout the specification, and (b) an excipient. Furthermore, the CD19 binding molecules or conjugates described throughout the specification can be used in combination therapy. For example, combinations including the CD19 binding molecules described throughout the specification and at least one additional therapeutic agent are described. The one or more additional therapeutic agents can include an immunomodulatory imid drug (IMiD). For example, the IMiD can be lenalidomide, thalidomide, pomalidomide, or iveldomide. In a specific example, the IMiD is lenalidomide.

[0042] The CD19 binding molecules (or compositions or combinations) described throughout this specification can be used as pharmaceuticals. The CD19 binding molecules (or compositions or combinations) described throughout this specification can also be used to treat a subject in need thereof. For example, the CD19 binding molecules (or compositions or combinations) can be used in a method for treating a subject having a CD19-related disease or disorder, the method comprising administering to the subject an effective amount of the CD19 binding molecules described throughout this specification. The conjugates described throughout this specification can also be used to treat a subject in need thereof. In some embodiments, the pharmaceutical compositions described throughout this specification can be used in a method for treating a subject having a CD19-related disease or disorder, the method comprising administering to the subject an effective amount of the pharmaceutical composition (or combination) described throughout this specification. The CD19-related disease or disorder can be diffuse large B-cell lymphoma (DLBCL). More specifically, the DLBCL can be relapsed or refractory DLBCL. The CD19-related disease or disorder can be acute lymphoblastic leukemia (ALL). More specifically, the ALL can be relapsed or refractory ALL.

[0043] The CD19 binding molecules described throughout this specification can be encoded by one or more nucleic acids.

[0044] Cells can be engineered to express the CD19-binding molecules described throughout the present specification. These cells can be used in methods for producing CD19-binding molecules. For example, methods for producing CD19-binding molecules (described throughout the present specification) are described, which include (a) culturing cells engineered to express the CD19-binding molecules described throughout the present specification under conditions in which the CD19-binding molecules are expressed; and (b) recovering the CD19-binding molecules from the cell culture. [Brief explanation of the drawings]

[0045] [Figure 1-1] Exemplary BBM Configurations. Figures 1A-1AH show components of the exemplary BBM configurations shown in Figures 1B-1AH. Not all regions connecting the different domains of each chain are shown (e.g., the linker connecting the VH and VL domains of an scFv, the hinge connecting the CH2 and CH3 domains of an Fc domain, etc. are omitted). Figures 1B-1F show a bivalent BBM; Figures 1G-1Z show a trivalent BBM; and Figures 1AA-1AH show a tetravalent BBM. [Figure 1-2] (As mentioned above.) [Figure 1-3] (As mentioned above.) [Figure 1-4] (As mentioned above.) [Figure 1-5] (As mentioned above.) [Figure 1-6] (As mentioned above.) [Figure 1-7] (As mentioned above.) [Figure 1-8] (As mentioned above.) [Figure 1-9] (As mentioned above.) [Figure 1-10] (As mentioned above.) [Figure 1-11] (As mentioned above.) [Figure 1-12] (As mentioned above.) [Figure 1-13] (As mentioned above.) [Figure 1-14](As mentioned above.) [Figure 1-15] (As mentioned above.) [Figure 1-16] (As mentioned above.) [Figure 1-17] (As mentioned above.) [Figure 1-18] (As mentioned above.) [Figure 1-19] (As mentioned above.) [Figure 1-20] (As mentioned above.) [Figure 1-21] (As mentioned above.) [Figure 1-22] (As mentioned above.) [Figure 1-23] (As mentioned above.) [Figure 1-24] (As mentioned above.) [Figure 1-25] (As mentioned above.) [Figure 1-26] (As mentioned above.) [Figure 1-27] (As mentioned above.) [Figure 1-28] (As mentioned above.) [Figure 1-29] (As mentioned above.) [Figure 1-30] (As mentioned above.) [Figure 1-31] (As mentioned above.) [Figure 1-32] (As mentioned above.) [Figure 1-33] (As mentioned above.) [Figure 1-34] (As mentioned above.) [Figure 2-1] Exemplary TBM Configurations. Figure 2A shows components of the exemplary TBM configurations shown in Figures 2B-2V. Not all regions connecting the different domains of each chain are shown (e.g., the linker connecting the VH and VL domains of an scFv, the hinge connecting the CH2 and CH3 domains of an Fc, etc. are omitted). Figures 2B-2P show a trivalent TBM; Figures 2Q-2S show a tetravalent TBM; Figure 2T show a pentavalent TBM, and Figures 2U-2V show a hexavalent TBM. [Figure 2-2] (As mentioned above.) [Figure 2-3] (As mentioned above.) [Figure 2-4] (As mentioned above.) [Figure 2-5] (As mentioned above.) [Figure 2-6] (As mentioned above.) [Figure 2-7] (As mentioned above.) [Figure 2-8] (As mentioned above.) [Figure 2-9] (As mentioned above.) [Figure 2-10] (As mentioned above.) [Figure 2-11] (As mentioned above.) [Figure 2-12] (As mentioned above.) [Figure 2-13] (As mentioned above.) [Figure 2-14] (As mentioned above.) [Figure 2-15] (As mentioned above.) [Figure 2-16] (As mentioned above.) [Figure 2-17] (As mentioned above.) [Figure 2-18] (As mentioned above.) [Figure 2-19] (As mentioned above.) [Figure 2-20] (As mentioned above.) [Figure 2-21] (As mentioned above.) [Figure 2-22] (As mentioned above.) [Figure 3-1] Schematic diagram of the bispecific (FIGS. 3A and 3C) and trispecific (FIG. 3B) constructs of Example 1. [Figure 3-2] (As mentioned above.) [Figure 3-3] (As mentioned above.) [Figure 4-1]Ability of CD19 BBM to induce redirected T cytotoxicity (RTCC) against CD19+ target cells. Both NEG258- and NEG218-based BBMs mediated RTCC activity against CD19+ target cell lines. Nalm6-luc (Figure 4A) and Karpas422-luc (Figure 4B) cells were cocultured with expanded T cells at a 3:1 effector:target (E:T) ratio in the presence of serially diluted BBM. After 24 hours of incubation, luminescence signals were measured. [Figure 4-2] (As mentioned above.) [Figure 5-1] Ability of CD19 BBM to induce T cell proliferation. Both NEG258- and NEG218-based BBMs induced T cell proliferation. Karpas422-luc (Figure 5A) and Nalm6-luc (Figure 5B) cells were co-cultured with expanded T cells at a 1:1 E:T ratio in the presence of serially diluted BBM. After 96 hours of incubation, luminescence signals were measured. [Figure 5-2] (As mentioned above.) [Figure 6-1] Ability of CD19 TBM to induce CD2-dependent T cell activation. Knockout of CD2 attenuated the benefit of the trispecific construct. Figures 6A-6B show representative flow cytometry analysis of CD2 expression on JNL CD2 WT (Figure 6A) and KO (Figure 6B) cells. Staining with anti-CD2 mAb (histograms with dotted shading) is overlaid with staining with mIgG1 isotype control (hatched histograms) or unstained (open histograms). Figures 6C-6F show data from JNL CD2+ (Figures 6C-6D) and CD2- (Figures 6E-6F) cells cocultured with CD19+ target cells at a 3:1 E:T ratio in the presence of serially diluted BBM and TBM. Luminescence signals were measured after 24 h of incubation. [Figure 6-2] (As mentioned above.) [Figure 6-3] (As mentioned above.) [Figure 6-4] (As mentioned above.) [Figure 6-5](As mentioned above.) [Figure 6-6] (As mentioned above.) [Figure 7-1] Binding of CD19 TBM to cyno B cells. Figure 7A shows data for TBM with a NEG218-based CD19-binding arm, and Figure 7B shows data for TBM with a NEG258-based CD19-binding arm. [Figure 7-2] (As mentioned above.) [Figure 8-1] Ability of CD19 TBM to induce T cell activation upon cyno B cell depletion in PBMCs. In Figure 8A, PBMCs were isolated from cynomolgus monkey whole blood using Ficoll density gradient centrifugation and incubated overnight with bispecific or trispecific constructs. Samples were collected and simultaneously stained for CD3 and CD20 to identify B and T cells in the PBMC population. The B cell depletion rate was calculated as described in Section 8.6.1. Figures 8B-8H show the results of FACS analysis of CD69 and CD25 expression on CD3+ T cells to determine single-positive cells (CD69+CD25- or CD69-CD25+) or double-positive cells (CD69+CD25+). Figure 8B: Untreated (medium only); Figures 8C-8E: CD3hi TSP1L; Figures 8F-8H: CD3hi TSP1. [Figure 8-2] (As mentioned above.) [Figure 8-3] (As mentioned above.) [Figure 8-4] (As mentioned above.) [Figure 8-5] (As mentioned above.) [Figure 8-6] (As mentioned above.) [Figure 8-7] (As mentioned above.) [Figure 8-8] (As mentioned above.) [Figure 9-1] Ability of NEG258- and NEG218-based TBMs to induce redirected T cytotoxicity by human donor cells against Nalm6 (Figures 9A-9H) and Karpas422 (Figures 9I-9P) target cells. [Figure 9-2] (As mentioned above.) [Figure 9-3] (As mentioned above.) [Figure 9-4] (As mentioned above.) [Figure 9-5] (As mentioned above.) [Figure 9-6] (As mentioned above.) [Figure 9-7] (As mentioned above.) [Figure 9-8] (As mentioned above.) [Figure 9-9] (As mentioned above.) [Figure 9-10] (As mentioned above.) [Figure 9-11] (As mentioned above.) [Figure 9-12] (As mentioned above.) [Figure 9-13] (As mentioned above.) [Figure 9-14] (As mentioned above.) [Figure 9-15] (As mentioned above.) [Figure 9-16] (As mentioned above.) [Figure 10-1] Ability of NEG258- and NEG218-based TBMs with different CD3 affinities to induce redirected T cytotoxicity by human donor cells against Nalm6 (Figures 10A-H) and Karpas422 (Figures 10I-P) target cells. [Figure 10-2] (As mentioned above.) [Figure 10-3] (As mentioned above.) [Figure 10-4] (As mentioned above.) [Figure 10-5] (As mentioned above.) [Figure 10-6] (As mentioned above.) [Figure 10-7] (As mentioned above.) [Figure 10-8] (As mentioned above.) [Figure 10-9] (As mentioned above.) [Figure 10-10] (As mentioned above.) [Figure 10-11] (As mentioned above.) [Figure 10-12] (As mentioned above.) [Figure 10-13] (As mentioned above.) [Figure 10-14] (As mentioned above.) [Figure 10-15] (As mentioned above.) [Figure 10-16] (As mentioned above.) [Figure 11-1] Ability of NEG258-based TBMs with CD2-binding arms and control lysozyme-binding arms to induce redirected T cytotoxicity by human donor cells against Nalm6 (Figures 11A-11H) and Karpas422 (Figures 11I-11L) target cells. [Figure 11-2] (As mentioned above.) [Figure 11-3] (As mentioned above.) [Figure 11-4] (As mentioned above.) [Figure 11-5] (As mentioned above.) [Figure 11-6] (As mentioned above.) [Figure 11-7] (As mentioned above.) [Figure 11-8] (As mentioned above.) [Figure 11-9] (As mentioned above.) [Figure 11-10] (As mentioned above.) [Figure 11-11] (As mentioned above.) [Figure 11-12] (As mentioned above.) [Figure 12-1] Induction of T cell cytokine release by NEG258- and NEG218-based TBM. Figure 12A: IFN-γ; Figure 12B: TNF-α; Figure 12C: IL2. [Figure 12-2] (As mentioned above.) [Figure 12-3] (As mentioned above.) [Figure 13-1] Binding of NEG258- and NEG218-based TBMs to the murine 300.19 cell line overexpressing human CD19 (Figure 13A) or cyno CD19 (Figure 13B). The TBMs show only negligible binding to the wild-type 300.19 cell line (Figure 13C). [Figure 13-2] (As mentioned above.) [Figure 13-3] (As mentioned above.) [Figure 14] Schematic representation of CD58. [Figure 15] Redirected T-cytotoxicity by TBM containing CD58 mutant sequences. [Figure 16] Antigen-independent T cell activation by TBM containing CD58 mutant sequences. Data are expressed as relative luminescence units (RLU). [Figure 17-1] CD19 and CD58 expression in various cell lines: Figures 17A-17B: CD19 and CD58 expression, respectively, in OCI-LY-19 cells; Figures 17C-17D: CD19 and CD58 expression, respectively, in Karpas-422 cells; Figures 17E-17F: CD19 and CD58 expression, respectively, in Toledo cells; Figures 17G-17H: CD19 and CD58 expression, respectively, in Nalm-6 cells. [Figure 17-2] (As mentioned above.) [Figure 17-3] (As mentioned above.) [Figure 17-4] (As mentioned above.) [Figure 17-5] (As mentioned above.) [Figure 17-6] (As mentioned above.) [Figure 17-7] (As mentioned above.) [Figure 17-8] (As mentioned above.) [Figure 18-1] Ability of NEG258-based TBM and BBM to induce redirected T cytotoxicity by human donor cells against Karpas422 target cells. Figures 18A and 18B show data using T cells from two different donors. [Figure 18-2] (As mentioned above.) [Figure 19-1]Induction of T cell cytokine release by NEG258-based TBM and BBM. Figures 19A-19B: IFN-γ (donor 1 and donor 2, respectively); Figures 19C-19D: IL-2 (donor 1 and donor 2, respectively); Figures 19E-19F: TNF-α (donor 1 and donor 2, respectively). Triangles on the x-axis indicate decreasing concentrations of constructs from left to right in the figure. [Figure 19-2] (As mentioned above.) [Figure 19-3] (As mentioned above.) [Figure 19-4] (As mentioned above.) [Figure 19-5] (As mentioned above.) [Figure 19-6] (As mentioned above.) [Figure 20] Binding of NEG258-based TBM and BBM to T cells. [Figure 21-1] NEG258-based TBM- and BBM-mediated T cell proliferation. Figure 21A: T cell proliferation in OC-LY-19 co-culture; Figure 21B: T cell proliferation in Karpas422 co-culture; Figure 21C: T cell proliferation in Toledo co-culture. [Figure 21-2] (As mentioned above.) [Figure 21-3] (As mentioned above.) [Figure 22-1] Ability of NEG258-based TBM and BBM to induce redirected T cytotoxicity by human donor cells against Karpas422 target cells. Figures 22A and 22B show data using T cells from two different donors. [Figure 22-2] (As mentioned above.) [Figure 23-1]Ability of NEG258-based TBM and BBM to induce redirected T cytotoxicity by human donor cells against various target cells: Figures 23A-23B: OC-LY-19 (donor 1 and donor 2, respectively); Figures 23C-23D: Toledo (donor 1 and donor 2, respectively); Figures 23E-23F: Nalm6 (donor 1 and donor 2, respectively); Figures 23G-23H: Nalm6 KO (donor 1 and donor 2, respectively); Figures 23I-23J: K562 (donor 1 and donor 2, respectively). [Figure 23-2] (As mentioned above.) [Figure 23-3] (As mentioned above.) [Figure 23-4] (As mentioned above.) [Figure 23-5] (As mentioned above.) [Figure 23-6] (As mentioned above.) [Figure 23-7] (As mentioned above.) [Figure 23-8] (As mentioned above.) [Figure 23-9] (As mentioned above.) [Figure 23-10] (As mentioned above.) [Figure 24-1] Induction of T cell cytokine release by NEG258-based TBM and BBM in various target cells. Figures 24A-24B: TNF-α from OC-LY-19 (donor 1 and donor 2, respectively); Figures 24C-24D: TNF-α from Toledo (donor 1 and donor 2, respectively); Figures 24E-24F: TNF-α from Nalm6 (donor 1 and donor 2, respectively); Figures 24G-24H: TNF-α from Nalm6 KO (donor 1 and donor 2, respectively); Figures 24I-24J: TNF-α from K562 (donor 1 and donor 2, respectively). [Figure 24-2] (As mentioned above.) [Figure 24-3] (As mentioned above.) [Figure 24-4] (As mentioned above.) [Figure 24-5] (As mentioned above.) [Figure 24-6] (As mentioned above.) [Figure 24-7] (As mentioned above.) [Figure 24-8] (As mentioned above.) [Figure 24-9] (As mentioned above.) [Figure 24-10] (As mentioned above.) [Figure 25-1] Re-challenge RTCC assay with Karpas 422 and OCI-LY-19 cell lines. Figure 25A: Assay setup. Figures 25B-25D: Karpas 422 (after the first, second, and third challenges, respectively); Figures 25E-25H: OCI-LY-19 (after the first, second, third, and fourth challenges, respectively). [Figure 25-2] (As mentioned above.) [Figure 25-3] (As mentioned above.) [Figure 25-4] (As mentioned above.) [Figure 25-5] (As mentioned above.) [Figure 25-6] (As mentioned above.) [Figure 25-7] (As mentioned above.) [Figure 25-8] (As mentioned above.) [Figure 26-1] Rechallenge T cell phenotyping with Karpas 422 and OCI-LY-19 cell lines. Figures 26A-H: Karpas 422 phenotyping; Figures 26I-P: OCI-LY-19 phenotyping. Figures 26A and 26I: % IL-2+ CD4 T cells; Figures 26B and 26J: % IFNγ+ CD4 T cells; Figures 26C and 26K: % IL-2+ CD8 T cells; Figures 26D and 26L: % IFNγ+ CD8 T cells; Figures 26E and 26M: CD3 young; Figures 26F and 26N: CD4 old; Figures 26G and 26O: CD8 young; Figures 26H and 26P: CD8 old. Lines in the figures represent different T cell donors. [Figure 26-2] (As mentioned above.) [Figure 26-3] (As mentioned above.) [Figure 26-4] (As mentioned above.) [Figure 26-5] (As mentioned above.) [Figure 26-6] (As mentioned above.) [Figure 26-7] (As mentioned above.) [Figure 26-8] (As mentioned above.) [Figure 26-9] (As mentioned above.) [Figure 26-10] (As mentioned above.) [Figure 26-11] (As mentioned above.) [Figure 26-12] (As mentioned above.) [Figure 26-13] (As mentioned above.) [Figure 26-14] (As mentioned above.) [Figure 26-15] (As mentioned above.) [Figure 26-16] (As mentioned above.) [Figure 27-1] Ability of CD3hi TSP1 to induce T cell proliferation in the presence of CD19+ target cells compared with CD3hi BSP1. Nalm6-luc cells were cocultured with sorted CD28+ or CD28- CD8 T cells at an E:T ratio of 1:3 for 72 hours in the presence of 1 nM (Figures 27A-B) or 0.1 nM (Figures 27C-D) CD3hi TSP1 or CD3hi BSP1 and in the presence (Figures 27A and 27C) or absence (Figures 27B and 27D) of irradiated autologous PBMCs (T cell depleted). Proliferation was measured as the percentage of CFSE-diluted cells among viable cells. [Figure 27-2] (As mentioned above.) [Figure 27-3] (As mentioned above.) [Figure 27-4] (As mentioned above.) [Figure 28-1]Ability of CD3hi TSP1 and CD3hi BSP1 to induce T cell cytokine production in the presence of Nalm6 CD19+ target cells (E:T 1:3). Figures 28A-B: Median fluorescence intensity (MFI) of CD28- and CD28+ CD8 T cells producing GzB (Figure 28A) and IFN-γ (Figure 28B) when co-cultured with 1 nM CD3hi TSP1 or 1 nM CD3hi BSP1 in the presence of irradiated PBMCs. Figures 28C-D: MFI of CD28- and CD28+ CD8 T cells producing GzB (Figure 28C) and IFN-γ (Figure 28D) when co-cultured with 1 nM CD3hi TSP1 or 1 nM CD3hi BSP1 in the absence of irradiated PBMCs. Figures 28E-F: MFI of CD28- and CD28+ CD8 T cells producing GzB (Figure 28E) and IFN-γ (Figure 28F) when co-cultured with 0.1 nM CD3hi TSP1 or 0.1 nM CD3hi BSP1 in the presence of irradiated PBMCs. Figures 28G-H: MFI of CD28- and CD28+ CD8 T cells producing GzB (Figure 28G) and IFN-γ (Figure 28H) when co-cultured with 0.1 nM CD3hi TSP1 or 0.1 nM CD3hi BSP1 in the absence of irradiated PBMCs. Figures 28I-L: Percentage of viable T cells when co-cultured with 1 nM (Figures 28I and 28J) or 0.1 nM (Figures 28K and 28L) CD3hi TSP1 or CD3hi BSP1 in the presence (Figures 28I and 28K) or absence (Figures 28J and 28L) of irradiated PBMCs. [Figure 28-2] (As mentioned above.) [Figure 28-3] (As mentioned above.) [Figure 28-4] (As mentioned above.) [Figure 28-5] (As mentioned above.) [Figure 28-6] (As mentioned above.) [Figure 28-7] (As mentioned above.) [Figure 28-8] (As mentioned above.) [Figure 28-9] (As mentioned above.) [Figure 28-10] (As mentioned above.) [Figure 28-11] (As mentioned above.) [Figure 28-12] (As mentioned above.) [Figure 29-1] Ability of CD3hi TSP1 to induce changes in T cell phenotype compared to CD3hi BSP1. Figure 29A: Representative examples of CD28- and CD28+ T cells sorted for CCR7 and CD45RO expression. Figures 29B-29I: Distribution of different T cell populations defined based on the combined expression of the two surface markers CD45RO and CCR7 (naive, CD45RO-CCR7+; central memory (CM), CD45RO+CCR7+; effector memory (EM), CD45RO+CCR7-; and terminally differentiated (TEMRA), CD45RO-CCR7-) after 72 hours of co-culture (E:T 1:3) in the presence (Figures 29B-29E) or absence (Figures 29F-29I) of PBMCs and in the presence of 1 nM (Figures 29B-29C and Figures 29F-29G) or 0.1 nM (Figures 29D-29E and Figures 29H-29I) of CD3hi TSP1 or CD3hi BSP1. Data for proliferating cells (CFSE-) are shown in Figures 29B, 29D, 29F, and 29H. Data for non-proliferating cells (CSFE+) are shown in Figures 29C, 29E, 29G, and 291. Data for CD28- cells are shown on the left side of each figure, and data for CD28+ cells are shown on the right side of the figure. [Figure 29-2] (As mentioned above.) [Figure 29-3] (As mentioned above.) [Figure 29-4] (As mentioned above.) [Figure 29-5] (As mentioned above.) [Figure 29-6] (As mentioned above.) [Figure 29-7] (As mentioned above.) [Figure 29-8] (As mentioned above.) [Figure 29-9] (As mentioned above.) [Figure 30-1]Ability of CD3hi TSP1 to induce redirected T cytotoxicity (RTCC) against CD19+ target cells compared with CD3hi BSP1. RTCC results from Nalm6-luc cells cocultured with sorted CD28+ or CD28- CD8 T cells at an E:T ratio of 1:3 for 72 hours in the presence of 1 nM (Figures 30A and 30C) or 0.1 nM (Figures 30B and 30D) CD3hi BSP1, CD3hi TSP1, or CD3hi TSP1C, and in the presence (Figures 30A and 30B) or absence (Figures 30C and 30D) of irradiated autologous PBMCs (T cell depleted). (n=3) Luminescence signal was measured at the end of the coculture incubation. Results are expressed as fold increase over the untreated condition, where no antibody was added to assess background signal caused by the control antibody. [Figure 30-2] (As mentioned above.) [Figure 30-3] (As mentioned above.) [Figure 30-4] (As mentioned above.) [Figure 31-1] Antitumor activity of CD3hi TSP1 (Figure 31A) and CD3med TSP1 (Figure 31B) in a human PBMC adoptive transfer application in the OCI-LY-19 subcutaneous tumor model. [Figure 31-2] (As mentioned above.) [Figure 32-1] Body weight changes following treatment with CD3hi TSP1 (FIG. 32A) and CD3med TSP1 (FIG. 32B) in a human PBMC adoptive transfer application in the OCI-LY-19 subcutaneous tumor model. [Figure 32-2] (As mentioned above.) [Figure 33] Schematic representation of the NSG mouse humanization process. [Figure 34-1] Antitumor activity of CD3 TSP1, CD3hi BSP1, and CD3med TSP1 in a DLBCL subcutaneous tumor model in huCD34+ NSG mice (Figure 34A) and body weight change after treatment with CD3 TSP1, CD3hi BSP1, and CD3med TSP1 in a DLBCL subcutaneous tumor model in huCD34+ NSG mice (Figure 34B). [Figure 34-2] (As mentioned above.) [Figure 35-1] Antitumor activity (Figures 35A and 35C) and body weight response (Figures 35B and 35D) following antibody treatment with CD3hi TSP1 (Figures 35A and 35B) and CD3med TSP1 (Figures 35C and 35D) in the OCI-LY-19 DLBCL subcutaneous tumor model in huCD34+ NSG mice. [Figure 35-2] (As mentioned above.) [Figure 35-3] (As mentioned above.) [Figure 35-4] (As mentioned above.) [Figure 36-1] Antitumor activity of CD3hi BSP1 (Figure 36A), CD3hi TSP1 (Figure 36B) and CD3med TSP1 (Figure 36C) in a human PBMC adoptive transfer application in the Daudi-Luc subcutaneous tumor model. [Figure 36-2] (As mentioned above.) [Figure 36-3] (As mentioned above.) [Figure 37-1] Body weight changes following antibody treatment with CD3hi BSP1 (Figure 37A), CD3hi TSP1 (Figure 37B) or CD3med TSP1 (Figure 37C) in a human PBMC adoptive transfer application of the Daudi-Luc subcutaneous tumor model. [Figure 37-2] (As mentioned above.) [Figure 37-3] (As mentioned above.) [Figure 38-1] 1 shows a schematic diagram of the Biacore measurement cycle. [Figure 38-2] (As mentioned above.) [Figure 39-1]Representative sensorgrams and plots of response and concentration are shown. Figures 39A.1 to 39A.11 (collectively "Figure 39A") show representative sensorgrams and response plots of WT IgG1, LALAPA-IgG1, LALAGA-IgG1, LALAPG-IgG1, DAPA-IgG1, LALASKPA-IgG1, DAPASK-IgG1, GADAPA-IgG1, GADAPASK-IgG1, and DANAPA-IgG1 (concentration range: 0.2 nM to 100 nM for human FcγR1A); Figures 39B.1 to 39B.11 (collectively "Figure 39B") show representative sensorgrams and response plots of WT, LALAPA-IgG1, LALAGA-IgG1, LALAPG-IgG1, DAPA-IgG1, LALASKPA-IgG1, DAPASK-IgG1, GADAPA-IgG1, GADAPASK-IgG1, and DANAPA-IgG1 (concentration range: 0.2 nM to 100 nM for human FcγR3A) against FcγR3A V158. Figures 39C.1 to 39C.11 (collectively, Figure 39C) show the sensorgrams and binding kinetics of WT, LALAPA-IgG1, LALAGA-IgG1, LALAPG-IgG1, DAPA-IgG1, LALASKPA-IgG1, DAPASK-IgG1, GADAPA-IgG1, GADAPASK-IgG1, and DANAPA-IgG1 to C1q (concentration range: 0.49 nM to 250 nM for human C1q). [Figure 39-2] (As mentioned above.) [Figure 39-3] (As mentioned above.) [Figure 39-4] (As mentioned above.) [Figure 39-5] (As mentioned above.) [Figure 39-6] (As mentioned above.) [Figure 39-7] (As mentioned above.) [Figure 39-8] (As mentioned above.) [Figure 39-9] (As mentioned above.) [Figure 39-10] (As mentioned above.) [Figure 39-11] (As mentioned above.) [Figure 39-12] (As mentioned above.) [Figure 39-13] (As mentioned above.) [Figure 39-14] (As mentioned above.) [Figure 39-15] (As mentioned above.) [Figure 39-16] (As mentioned above.) [Figure 39-17] (As mentioned above.) [Figure 39-18] (As mentioned above.) [Figure 39-19] (As mentioned above.) [Figure 39-20] (As mentioned above.) [Figure 39-21] (As mentioned above.) [Figure 39-22] (As mentioned above.) [Figure 39-23] (As mentioned above.) [Figure 39-24] (As mentioned above.) [Figure 39-25] (As mentioned above.) [Figure 39-26] (As mentioned above.) [Figure 39-27] (As mentioned above.) [Figure 39-28] (As mentioned above.) [Figure 39-29] (As mentioned above.) [Figure 39-30] (As mentioned above.) [Figure 39-31] (As mentioned above.) [Figure 39-32] (As mentioned above.) [Figure 39-33] (As mentioned above.) [Figure 40-1] Figure 40: Figure 40A shows the nuclear factor of activated T cells (NFAT) pathway activity of wild-type and mutant antibodies. Figure 40B shows the NFAT pathway activity of cells primed by the addition of wild-type and mutant antibodies and INFγ. [Figure 40-2] (As mentioned above.) [Figure 41-1]Representative sensorgrams and response plots are shown for WT, DANAPA, GADAPASK, LALA, and LALASKPA mutants (concentration range: 0.2 nM to 25 nM for human FcγR1A). [Figure 41-2] (As mentioned above.) [Figure 41-3] (As mentioned above.) [Figure 41-4] (As mentioned above.) [Figure 41-5] (As mentioned above.) [Figure 42] Nuclear factor of activated T cells (NFAT) pathway activity of wild-type and mutant antibodies is shown. DETAILED DESCRIPTION OF THE INVENTION

[0046] 7.1.Definition As used herein, the following terms are intended to have the following meanings:

[0047] ABM chain: An individual ABM can exist as a single polypeptide chain (e.g., in the case of an scFv) or can be formed by the association of two or more polypeptide chains (e.g., in the case of a Fab). As used herein, the term "ABM chain" refers to all or a portion of the ABM present on a single polypeptide chain. The use of the term "ABM chain" is intended for convenience and descriptive purposes only and does not imply a particular form or method of production.

[0048] ADCC: As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibody on target cells and subsequently cause lysis of the target cells. ADCC correlates with binding to FcγRIIIa; increased binding to FcγRIIIa results in increased ADCC activity.

[0049] ADCP: As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis refers to a cell-mediated reaction in which nonspecific phagocytes expressing FcγRs recognize bound antibody on target cells and subsequently cause phagocytosis of the target cells.

[0050] Additional Agents: For convenience, agents used in combination with the antigen-binding molecules of the present disclosure are referred to herein as "additional" agents.

[0051] Antibody: As used herein, the term "antibody" refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that is capable of non-covalently, reversibly, and specifically binding to an antigen. For example, a naturally occurring "antibody" of the IgG type is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the present disclosure).

[0052] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light (CL) and heavy chains (CH1, CH2, or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. In wild-type antibodies, the N-terminus is the variable region and the C-terminus is the constant region; the CH3 and CL domains actually comprise the carboxy-termini of the heavy and light chains, respectively.

[0053] Antibody fragment: As used herein, the term "antibody fragment" of an antibody refers to one or more portions of an antibody. In certain embodiments, these portions are portions of the contact domain of an antibody. In certain other embodiments, these portions are antigen-binding fragments that retain the ability to non-covalently, reversibly, and specifically bind to antigen, sometimes referred to herein as "antigen-binding fragments," "antigen-binding fragments thereof," "antigen-binding portions," etc. Examples of binding fragments include, but are not limited to, single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of the VH domain (Ward et al., 1989, Nature 341:544-546); and isolated complementarity-determining regions (CDRs). Thus, the term "antibody fragment" encompasses proteolytic fragments of antibodies (eg, Fab and F(ab)2 fragments) as well as modified proteins comprising one or more portions of an antibody (eg, scFv).

[0054] Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23:1126-1136). Antibody fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).

[0055] Antibody fragments can be incorporated into single-chain molecules comprising a pair of tandem Fv segments (e.g., VH-CH1-VH-CH1), which, together with complementary light chain polypeptides (e.g., VL-VC-VL-VC), form a pair of antigen-binding regions (Zapata et al., 1995, Protein Eng. 8:1057-1062; and U.S. Pat. No. 5,641,870).

[0056] Antibody numbering system: As used herein, references to numbered amino acid residues in antibody domains are based on the EU numbering system (e.g., in Table 1), unless otherwise specified. This system was first devised by Edelman et al., 1969, Proc. Nat'l Acad. Sci. USA 63:78-85, and is described in detail in Kabat et al., 1991, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.

[0057] Antigen-binding module: The term "antigen-binding module" or "ABM," as used herein, refers to a portion of an MBM that has the ability to non-covalently, reversibly, and specifically bind to an antigen. An ABM can be immunoglobulin-based or non-immunoglobulin-based. As used herein, the terms "ABM1" and "CD19 ABM" (and the like) refer to ABMs that specifically bind to CD19, the terms "ABM2" and "TCR ABM" (and the like) refer to ABMs that specifically bind to components of the TCR complex, the term "ABM3" refers to ABMs that specifically bind to CD2 or TAA (depending on the context), the term "CD2 ABM" (and the like) refers to ABMs that specifically bind to CD2, and the term "TAA ABM" (and the like) refers to ABMs that specifically bind to TAA. The terms ABM1, ABM2, and ABM3 are used for convenience only and are not intended to convey any particular form of MBM. In some embodiments, ABM2 binds to CD3 (referred to herein as "CD3 ABM"), and therefore, disclosure relating to one or more ABM2s is also applicable to CD3 ABMs.

[0058] Antigen-binding fragment: The term "antigen-binding fragment" of an antibody refers to a portion of an antibody that retains the ability to non-covalently, reversibly, and specifically bind to an antigen.

[0059] Antigen-binding molecule: The term "antigen-binding molecule" refers to a molecule, such as an antibody, comprising one or more antigen-binding domains. An antigen-binding molecule may comprise one or more polypeptide chains, for example, one, two, three, four, or more polypeptide chains. The polypeptide chains in an antigen-binding molecule may be directly or indirectly associated with each other (e.g., a first polypeptide chain may be associated with a second polypeptide chain, which may then be associated with a third polypeptide chain to form an antigen-binding molecule in which the first and second polypeptide chains are directly associated with each other, or the second and third polypeptide chains are directly associated with each other, and the first and third polypeptide chains are indirectly associated with each other via the second polypeptide chain).

[0060] Associated: The term "associated," in reference to an antigen-binding molecule, refers to a functional relationship between two or more polypeptide chains and / or two or more portions of a single polypeptide chain. Specifically, the term "associated" means that two or more polypeptides (or portions of a single polypeptide) associate with each other, e.g., noncovalently through molecular interactions and / or covalently through one or more disulfide or chemical bridges, thereby generating a functional antigen-binding molecule, e.g., a BBM or TBM, in which the antigen-binding domains therein are capable of binding to their respective targets. Examples of associations that may be present in an MBM include (but are not limited to) associations between Fc regions of Fc domains (homodimers or heterodimers as described in Section 7.4.1.5), associations between the VH and VL regions of a Fab or Fv, and associations between the CH1 and CL of a Fab.

[0061] B cell: As used herein, the term "B cell" refers to a cell of the B cell lineage, which is a type of white blood cell of the lymphocyte subtype. Examples of B cells include plasmablasts, plasma cells, lymphoplasmacytoid cells, memory B cells, follicular B cells, marginal zone B cells, B-1 cells, B-2 cells, and regulatory B cells.

[0062] B cell malignancies: As used herein, B cell malignancies refer to the uncontrolled proliferation of B cells. Examples of B cell malignancies include non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, leukemia, and myeloma. For example, the B-cell malignancy can be, but is not limited to, multiple myeloma, chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), follicular lymphoma, mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma (Waldenstrom's macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, primary mediastinal large B-cell lymphoma, mediastinal gray zone lymphoma (MGZL), splenic marginal zone B-cell lymphoma, MALT-type extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, and primary effusion lymphoma, as well as plasmacytoid dendritic cell neoplasm. For example, DLBCL includes relapsed or refractory DLBCL (RR DLBCL or R / R DLBCL).

[0063] Binding Sequence: With reference to Table 1, 12, 13, 14, 16 or 17 (including subportions thereof), the term "binding sequence" means an ABM having a set of CDRs, VH-VL pair or scFv set out in the appropriate table.

[0064] Bispecific Binding Molecule: The term "bispecific binding molecule" or "BBM" refers to a molecule that specifically binds two antigens and comprises two or more ABMs. A BBM of the present disclosure comprises at least one antigen-binding domain specific for CD19 and at least one antigen-binding domain specific for a different antigen, e.g., a component of a TCR complex. Representative BBMs are shown in Figures 1B-1AH. A BBM can comprise one, two, three, four, or even more polypeptide chains.

[0065] Bivalent: The term "bivalent" as used herein in reference to an antigen-binding molecule refers to an antigen-binding molecule having two antigen-binding domains. The domains can be the same or different. Thus, a bivalent antigen-binding molecule can be monospecific or bispecific. A bivalent BBM can include an ABM that specifically binds to CD19 and another ABM that binds to another antigen, such as a component of a TCR complex.

[0066] Cancer: The term "cancer" refers to a disease characterized by uncontrolled (often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers are described herein and include, but are not limited to, leukemia, multiple myeloma, asymptomatic myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma, including any CD19-positive cancer of any of the above types. The term "cancerous B cell" refers to a B cell that is undergoing or has undergone uncontrolled proliferation.

[0067] CD3: The term "CD3" or "cluster of differentiation 3" refers to the cluster of three coreceptors of the T cell receptor. CD3 promotes activation of both cytotoxic T cells (e.g., naive CD8+ T cells) and T helper cells (e.g., naive CD4+ T cells) and is composed of four distinct chains: one CD3γ chain (e.g., GenBank accession numbers NM_000073 and MP_000064 (human)), one CD3δ chain (e.g., GenBank accession numbers NM_000732, NM_001040651, NP_00732, and NP_001035741 (human)), and two CD3ε chains (e.g., GenBank accession numbers NM_000733 and NP_00724 (human)). The CD3 chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The CD3 molecule associates with the T cell receptor (TCR) and the ζ-chain to form the T cell receptor (TCR) complex, which functions in generating activation signals in T lymphocytes. Unless explicitly indicated, reference to CD3 in this application may refer to the CD3 co-receptor, the CD3 co-receptor complex, or any polypeptide chain of the CD3 co-receptor complex.

[0068] CD19: The term "CD19" or "cluster of differentiation 19" refers to the cluster of differentiation 19 protein, an antigenic determinant detectable on leukemia progenitor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found under UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098. CD19 is expressed in most cancers of B-cell lineage, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Other cells with CD19 expression are provided below in the definition of "diseases associated with CD19 expression." It is also an early marker of B-cell progenitor cells. See, e.g., Nicholson et al., 1997, Mol. Immun. 34(16-17):1157-1165.

[0069] Chimeric antibody: The term "chimeric antibody" (or antigen-binding fragment thereof) refers to an antibody molecule (or antigen-binding fragment thereof) in which (a) the constant region or portion thereof has been altered, substituted, or exchanged so that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or to an entirely different molecule, such as an enzyme, toxin, hormone, growth factor, drug, etc., that confers new properties to the chimeric antibody; or (b) the variable region or portion thereof has been altered, substituted, or exchanged with a variable region having a different or altered antigen specificity. For example, a murine antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. The replacement with a human constant region allows the chimeric antibody to retain its specificity in recognizing the antigen, while having reduced antigenicity in humans compared to the original murine antibody.

[0070] Chimeric Antigen Receptor: The term "chimeric antigen receptor" or alternatively "CAR" refers to a set of polypeptides, typically two in the simplest embodiment, which, when present in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule, as defined below. The set of polypeptides may or may not be contiguous with each other. If the polypeptides are not contiguous with each other, the set of polypeptides comprises a dimerization switch that, upon the presence of a dimerization molecule, allows the polypeptides to link to each other, e.g., link the antigen-binding domain to the intracellular signaling domain. CAR molecules are typically administered to a subject by administration of immune effector cells (e.g., T cells, preferably autologous to the subject) engineered to express the CAR molecule.

[0071] In combination: As used herein, administered "in combination" means that two (or more) different therapies are delivered to a subject during the subject's illness, e.g., after the subject is diagnosed with a disorder and before the disorder is cured or eliminated, or before the treatments are discontinued for other reasons. The terms "combination" and "in combination" are not limited to the precise simultaneous administration of two or more therapies, but rather refer to administering to a subject a pharmaceutical composition comprising an agent (e.g., an anti-CD19 agent) in an order and time intervals that allow the agents to act together with the additional therapy to provide a greater benefit than if administered otherwise.

[0072] Complementarity-determining region: As used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. For example, there are generally three CDRs in each heavy chain variable region (e.g., CDR-H1, CDR-H2, and CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, and CDR-L3). The precise amino acid sequence boundaries of a given CDR can be determined using the "Kabat" numbering scheme, Kabat et al., 1991, "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., 1997, JMB 273, 927-948 ("Chothia" numbering scheme), and ImMunoGenTics (IMGT) numbering scheme (Lefranc, 1999, The Immunologist 7:132-136; Lefranc, et al. The CDR numbering scheme can be determined using any of several well-known schemes, including those described by Kabat et al., 2003, Dev. Comp. Immunol. 27:55-77 ("IMGT" numbering scheme). For example, for the classical format, Kabat numbers the CDR amino acid residues in the heavy chain variable domain (VH) as 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3); The CDR amino acid residues in VH are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). According to Chothia, the CDR amino acids in VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3); and the amino acid residues in VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3).Combining the Kabat and Chothia CDR definitions, a CDR consists of amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in a human VH and amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in a human VL. According to IMGT, the CDR amino acid residues in a VH are numbered approximately 26-35 (CDR-H1), 51-57 (CDR-H2), and 93-102 (CDR-H3), and the CDR amino acid residues in a VL are numbered approximately 27-32 (CDR-L1), 50-52 (CDR-L2), and 89-97 (CDR-L3) ("Kabat" numbering). With IMGT, the CDR regions of an antibody can be determined using the program IMGT / Domain Gap Align.

[0073] Concurrently: The term "concurrently" is not limited to administration of therapeutic agents (e.g., prophylactic or therapeutic agents) at exactly the same time, but means that a pharmaceutical composition comprising an antigen-binding molecule of the present disclosure is administered to a subject in an order and within a time interval such that it can act together with additional therapeutic agents to provide an increased benefit over when they are administered in other ways.

[0074] Conservative sequence modifications: The term "conservative sequence modifications" refers to amino acid modifications that do not substantially affect or alter the binding characteristics of a CD19 binding molecule or a component thereof (e.g., a CD19 binding domain or Fc region). Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into a binding molecule by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a binding molecule can be substituted with other amino acid residues from the same side chain family, and the modified binding molecules can be tested, for example, for binding to a target molecule and / or effective heterodimerization and / or effector function.

[0075] Bispecific antibody: As used herein, the term "bispecific antibody" refers to a small antibody fragment having two antigen-binding sites, typically formed by pairing of scFv chains. Each scFv comprises a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL, where VH is either N-terminal or C-terminal to VL). Unlike typical scFvs, in which the VH and VL are separated by a linker that allows pairing of the VH and VL on the same polypeptide chain to form the antigen-binding domain, bispecific antibodies typically contain a linker that is too short to allow pairing between the VH and VL domains on the same chain, thereby allowing pairing of the VH and VL domains with complementary domains on another chain to form two antigen-binding sites. Bispecific antibodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0076] dsFv: The term "dsFv" refers to a disulfide-stabilized Fv fragment. In dsFv, the VH and VL are linked by an interdomain disulfide bond. To generate such molecules, one amino acid in each of the framework regions of the VH and VL is mutated to cysteine, which then forms a stable interchain disulfide bond. Typically, positions 44 in the VH and 100 in the VL are mutated to cysteine. See Brinkmann, 2010, Antibody Engineering 181-189, DOI: 10.1007 / 978-3-642-01147-4_14. The term dsFv encompasses both what are known as dsFv (molecules in which the VH and VL are linked by an interchain disulfide bond rather than a linker peptide) and scdsFv (molecules in which the VH and VL are linked by a linker and an interchain disulfide bond).

[0077] Effector function: The term "effector function" refers to an activity of an antibody molecule mediated by binding through a domain of the antibody other than the antigen-binding domain, typically mediated by the binding of an effector molecule. Effector function includes, for example, complement-mediated effector function, which is mediated by the binding of the C1 component of complement to an antibody. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. Effector function also includes Fc receptor (FcR)-mediated effector function, which can be triggered upon binding of the constant domain of an antibody to an Fc receptor (FcR). Binding of an antibody to an Fc receptor on the cell surface triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental passage, and regulation of immunoglobulin production. The effector function of an antibody can be modified by altering, e.g., increasing or decreasing, the affinity of the antibody for an effector molecule, such as an Fc receptor or a complement component. Binding affinity is generally altered by modifying the effector molecule binding site; in this case, it is appropriate to locate the site of interest and modify at least a portion of this site in a suitable manner. It is also contemplated that modification of the binding site on an antibody for an effector molecule need not substantially alter the overall binding affinity, but may alter the geometry of the interaction so as to render the effector mechanism ineffective, such as in non-productive binding. It is further contemplated that effector function can be altered by modifying sites not directly involved in effector molecule binding but otherwise involved in the performance of the effector function.

[0078] Epitope: An epitope or antigenic determinant is the portion of an antigen that is recognized by an antibody or other antigen-binding moiety as described herein. Epitopes can be linear or conformational.

[0079] Fab: As used herein, "Fab" or "Fab region" refers to a polypeptide region comprising the VH, CH1, VL, and CL immunoglobulin domains. These terms can refer to this region alone or in the context of an antigen-binding molecule of the present disclosure.

[0080] The Fab domain is formed by the association of a CH1 domain linked to a VH domain with a CL domain linked to a VL domain. The VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the link module. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0081] Fab regions can be generated by proteolytic cleavage of immunoglobulin molecules (e.g., with an enzyme such as papain) or by recombinant expression. In native immunoglobulin molecules, Fab is formed by the association of two different polypeptide chains (e.g., VH-CH1 on one chain associated with VL-CL on the other chain). Fab regions are typically expressed on two polypeptide chains, typically recombinantly, although single-chain Fabs are also contemplated herein.

[0082] Fc domain: The term "Fc domain" refers to a pair of associated Fc regions. These two Fc regions dimerize to create an Fc domain. The two Fc regions within an Fc domain can be the same (such an Fc domain is referred to herein as an "Fc homodimer") or different from each other (such an Fc domain is referred to herein as an "Fc heterodimer").

[0083] Fc region: As used herein, the term "Fc region" or "Fc chain" refers to a polypeptide comprising the CH2-CH3 domains of an IgG molecule, and in some cases, the hinge. In EU numbering for human IgG1, the CH2-CH3 domain comprises amino acids 231-447, and the hinge is 216-230. Thus, the definition of "Fc region" includes both (CH2-CH3) or (hinge-CH2-CH3) or fragments thereof. An "Fc fragment" in this context may contain fewer amino acids from either or both the N-terminus and C-terminus, but generally still retains the ability to form a dimer with another Fc region, as can be detected using standard size-based methods (e.g., non-denaturing chromatography, size exclusion chromatography).

[0084] Fv: The term "Fv" refers to the minimum antibody fragment derivable from an immunoglobulin that contains a complete target recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Often, these six CDRs confer target binding specificity to the antibody. However, in some instances, even a single variable domain (or half of an Fv comprising only three target-specific CDRs) may be capable of recognizing and binding to a target. The reference herein to a VH-VL dimer is not intended to convey any particular configuration. By way of example and without limitation, a VH and a VL may combine to form a half-antibody in any of the configurations described herein, or each may be present in separate half-antibodies that combine to form an antigen-binding domain when the separate half-antibodies associate, e.g., to form a TBM of the present disclosure. When present on a single polypeptide chain (eg, scFv), it is N-terminal or C-terminal to the VH and VL.

[0085] Half antibody: The term "half antibody" refers to a molecule that contains at least one ABM or ABM chain and can associate with another molecule that contains an ABM or ABM chain, for example, by disulfide bridges or molecular interactions (e.g., knob-in-hole interactions between Fc heterodimers). A half antibody can be composed of one polypeptide chain or two or more polypeptide chains (e.g., two polypeptide chains of a Fab). In one embodiment, a half antibody comprises an Fc region.

[0086] An example of a half antibody is a molecule comprising the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a half antibody is a molecule comprising a first polypeptide comprising a VL domain and a CL domain, and a second polypeptide comprising a VH domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain, where the VL and VH domains form an ABM. Yet another example of a half antibody is a polypeptide comprising an scFv domain, a CH2 domain, and a CH3 domain.

[0087] A half antibody can comprise two or more ABMs, for example, a half antibody comprises (in order from N-terminus to C-terminus) an scFv domain, a CH2 domain, a CH3 domain and another scFv domain.

[0088] A half antibody may also include an ABM chain that forms a complete ABM when associated with another ABM chain in another half antibody.

[0089] Thus, an MBM may comprise one, more typically two or even three or more half antibodies, and a half antibody may comprise one or more ABMs or ABM chains.

[0090] In some MBMs, a first half antibody associates with, e.g., heterodimerizes with, a second half antibody. In other MBMs, a first half antibody is covalently linked to a second half antibody, e.g., by disulfide bridges or chemical crosslinking. In still other MBMs, a first half antibody associates with a second half antibody through both covalent and non-covalent interactions, e.g., disulfide bridges and knob-in-hole interactions.

[0091] The term "half antibody" is intended for descriptive purposes only and does not imply a particular form or method of production. Descriptions of half antibodies as a "first" half antibody, a "second" half antibody, a "left" half antibody, a "right" half antibody, etc. are for convenience and descriptive purposes only.

[0092] Hexavalent: The term "hexavalent," as used herein in reference to an antigen-binding molecule (e.g., a TBM), refers to an antigen-binding molecule having six antigen-binding domains. A hexavalent TBM of the present disclosure generally has three pairs of antigen-binding domains, each binding to the same antigen, although different configurations (e.g., three antigen-binding domains that bind to CD19, two antigen-binding domains that bind to a component of the TCR complex and one antigen-binding domain that binds to CD2 or a TAA, or three antigen-binding domains that bind to CD19, two antigen-binding domains that bind to CD2 or a TAA, and one antigen-binding domain that binds to a component of the TCR complex) are within the scope of the present disclosure. Examples of hexavalent TBMs are shown schematically in Figures 1U-1V.

[0093] Hole: In reference to knob-into-hole, "hole" refers to at least one amino acid side chain that is recessed from the interface of a first Fc chain and thus available for positioning in a complementary "knob" at the adjacent interface of a second Fc chain, so as to stabilize the Fc heterodimer and thereby, for example, favor Fc heterodimer formation over Fc homodimer formation.

[0094] Host cell or recombinant host cell: The terms "host cell" or "recombinant host cell" refer to cells that have been genetically modified, for example, by the introduction of heterologous nucleic acid. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may exist in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Host cells may harbor heterologous nucleic acid transiently, for example, on an extrachromosomal heterologous expression vector, or stably, for example, by integration of the heterologous nucleic acid into the host cell genome. For the purpose of expressing antigen-binding molecules, host cells can be mammalian-derived or mammalian-like cell lines, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives and / or engineered variants thereof. Engineered variants include, for example, those with modified glycan profiles and / or site-specific integration sites.

[0095] Human antibody: As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region can also be derived from such human sequences, e.g., human germline sequences or mutant human germline sequences, or antibody-containing consensus framework sequences derived from human framework sequence analysis, e.g., as described in Knappik et al., 2000, J Mol Biol 296, 57-86. The structure and location of immunoglobulin variable domains, e.g., CDRs, can be defined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g., Lazikani et al., 1997, J. Mol. Biol. 273:927-948; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 USDepartment of Health and Human Services; Chothia et al., 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883).

[0096] Human antibodies may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo, or conservative substitutions to facilitate stability or manufacturing). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0097] Humanized: The term "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. To a large extent, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (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 the donor antibody. Such modifications are made to further refine antibody performance. In general, humanized antibodies 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 those of a human immunoglobulin sequence. 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 Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596. See also the following review articles and references cited therein: Vaswani and Hamilton, 1998, Ann. Allergy, Asthma & Immunol. 1:105-115; Harris, 1995, Biochem. Soc. Transactions 23:1035-1038; Hurle and Gross, 1994, Curr. Op. Biotech. 5:428-433.

[0098] Knob: In reference to knob-into-hole, "knob" refers to at least one amino acid side chain that protrudes from the interface of a first Fc chain and is therefore capable of positioning in a complementary "hole" at the interface with a second Fc chain, so as to stabilize the Fc heterodimer, thereby, for example, favoring Fc heterodimer formation over Fc homodimer formation.

[0099] Knobs and holes (or knobs-into-holes): One mechanism of Fc heterodimerization is commonly referred to in the art as "knobs and holes," or "knobs-in-holes," or "knobs-into-holes." These terms refer to amino acid mutations that create steric effects that favor the formation of Fc heterodimers over Fc homodimers, as described, for example, in Ridgway et al., 1996, Protein Engineering 9(7):617; Atwell et al., 1997, J. Mol. Biol. 270:26; and U.S. Patent No. 8,216,805. Knobs-in-hole mutations can be combined with other approaches to improve heterodimerization, as described, for example, in Section 7.4.1.6.

[0100] Monoclonal antibody: As used herein, the term "monoclonal antibody" refers to polypeptides, including antibodies, antibody fragments, molecules (including MBM), etc., that are derived from the same genetic source.

[0101] Monovalent: The term "monovalent" as used herein in reference to an antigen-binding molecule refers to an antigen-binding molecule that has a single antigen-binding domain.

[0102] Multispecific binding molecule: The term "multispecific binding molecule" or "MBM" refers to a molecule that specifically binds to at least two antigens and comprises two or more antigen-binding domains, each of which can independently be an antibody fragment (e.g., scFv, Fab, nanobody), a ligand, or a non-antibody-derived binder (e.g., fibronectin, Fynomer, DARPin).

[0103] Mutation or modification: In reference to the primary amino acid sequence of a polypeptide, the terms "modification" and "mutation" refer to amino acid substitutions, insertions, and / or deletions in a polypeptide sequence compared to a reference polypeptide. Furthermore, the term "modification" further encompasses alterations to amino acid residues, for example, by chemical conjugation (e.g., of a drug or polyethylene glycol moiety) or post-translational modification (e.g., glycosylation).

[0104] Nucleic Acid: The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids that are synthetic, natural, and unnatural, and contain known nucleotide analogs or modified backbone residues or linkages that have similar binding properties as the reference nucleic acid and are metabolized similarly to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0105] Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly set forth, but also conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences of that nucleic acid sequence. Specifically, as detailed below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; and Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).

[0106] Operably linked: The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of a fusion protein or other polypeptide, the term "operably linked" means that two or more amino acid segments are linked to produce a functional polypeptide. For example, in the context of an antigen-binding molecule, separate ABMs (or chains of ABMs) can be operably linked via a peptide linker sequence. In the context of a nucleic acid encoding a fusion protein, such as a polypeptide chain of an antigen-binding molecule, "operably linked" means that the two nucleic acids are joined such that the amino acid sequences encoded by the two nucleic acids remain in frame. In the context of transcriptional regulation, the term refers to the functional relationship of a transcriptional regulatory sequence to a transcriptional sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system.

[0107] Pentavalent: The term "pentavalent," as used herein in reference to an antigen-binding molecule (e.g., a TBM), refers to an antigen-binding molecule having five antigen-binding domains. Pentavalent TBMs of the present disclosure generally have either (a) two pairs of antigen-binding domains that each bind to the same antigen and a single antigen-binding domain that binds to a third antigen, or (b) three antigen-binding domains that bind to the same antigen and two antigen-binding domains that each bind to a separate antigen. An example of a pentavalent TBM is shown schematically in Figure 1T.

[0108] Polypeptide and Protein: The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. This term encompasses amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as natural and unnatural amino acid polymers. Furthermore, this term encompasses amino acid polymers that are derivatized, for example, by synthetic derivatization of one or more side chains or termini, glycosylation, PEGylation, circular permutation, cyclization, linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels.

[0109] Recognize: As used herein, the term "recognize" refers to an ABM finding and interacting with (eg, binding to) the epitope.

[0110] Sequence identity: Sequence identity between two similar sequences (e.g., antibody variable domains) can be determined by methods such as Smith, T.F. & Waterman, M.S. (1981) "Comparison Of Biosequences", Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, S.B. & Wunsch, C.D. (1970) "A General Method Applicable To the Search For Similarities In Amino Acid Sequence Of Two Proteins", J. Mol. Biol. 48:443 [homology alignment algorithm]; Pearson, W.R. & Lipman, D.J. (1988) "Improved Tools For Biological Sequence Comparison", Proc. Natl. Acad. Sci. (USA) 85:2444 [method of searching for similarity]; or Altschul, S.F. et al., 1990, "Basic Local Alignment Search Tool“, J. Mol. Biol. 215:403-10, “BLAST” algorithm (see blast.ncbi.nlm.nih.gov / Blast.cgi). When using any of the above algorithms, default parameters (such as window length, gap penalty, etc.) are used. In one embodiment, sequence identity is performed using the BLAST algorithm using default parameters.

[0111] Optionally, identity is determined over a region at least about 50 nucleotides (or, in the case of a peptide or polypeptide, at least about 10 amino acids) in length, or in some cases 100-500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. In certain embodiments, identity is determined over a defined domain, e.g., the VH or VL of an antibody. Unless otherwise specified, sequence identity between two sequences is determined over the entire length of the shorter of the two sequences.

[0112] Single-chain Fab or scFab: The terms "single-chain Fab" and "scFab" refer to a polypeptide comprising an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker, such that VH and VL are associated with each other and CH1 and CL are associated with each other. In certain embodiments, the antibody domains and linker are arranged in one of the following orders from N- to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. The linker can be a polypeptide of at least 30 amino acids, e.g., 32-50 amino acids. Single-chain Fabs are stabilized by a native disulfide bond between the CL and CH1 domains.

[0113] Single-chain Fv or scFv: The term "single-chain Fv" or "scFv," as used herein, refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Plueckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., 1994, Springer-Verlag, New York, pp. 269-315.

[0114] Specific (or selective) binding: The term "specifically (or selectively) binding" to an antigen or epitope refers to a binding reaction that determines the presence of the cognate antigen or epitope in a heterogeneous population of proteins and other biological substances. This binding reaction can be, but need not be, mediated by an antibody or antibody fragment, but can be mediated by any type of ABM described in Section 7.3, e.g., a ligand, a DARPin, etc. ABMs are typically 5×10 -2 Under M, 10 -2 Less than M, 5 x 10 -3 Under M, 10 -3 Less than M, 5 x 10 -4 Under M, 10 -4 Less than M, 5 x 10 -5 Under M, 10 -5 Less than M, 5 x 10 -6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Less than M or 10 -9It also has a dissociation rate constant (KD) (koff / k) less than M and binds to the target antigen with an affinity at least two-fold higher than its affinity for binding to a non-specific antigen (e.g., HSA). Binding affinity can be measured using Biacore, SPR, or BLI assays. The term "specifically binds" does not exclude cross-species cross-reactivity. For example, an antigen-binding module (e.g., an antigen-binding fragment of an antibody) that "specifically binds" to an antigen from one species may also "specifically bind" to the corresponding antigen in one or more other species. Therefore, such cross-species cross-reactivity does not itself change the classification of the antigen-binding module as a "specific" binder. In certain embodiments, an antigen binding module that specifically binds to a human antigen is cross-species compatible with one or more non-human mammalian species, such as a primate species (including, but not limited to, one or more of cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatta), and pigtailed macaques (Macaca nemestrina)) or a rodent species, such as the house mouse (Mus musculus). In other embodiments, the antigen binding module is not cross-species compatible.

[0115] Subject: The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably herein.

[0116] VH domain tandem: As used herein, the term "VH domain (or VH) tandem" refers to a series of VH domains consisting of multiple identical VH domains of an antibody. Each VH domain, except for the last one at the end of the tandem, has its C-terminus linked, with or without a linker, to the N-terminus of another VH domain. A tandem has at least two VH domains, and in certain embodiments of the antigen binding module, has 3, 4, 5, 6, 7, 8, 9, or 10 VH domains. VH tandems can be generated by combining the encoding nucleic acids for each VH domain in the desired order using recombinant methods (e.g., as described in Section 7.4.3) with or without linkers that allow them to be produced as a single polypeptide chain. The N-terminus of the first VH domain of the tandem is defined as the N-terminus of the tandem, while the C-terminus of the last VH domain of the tandem is defined as the C-terminus of the tandem.

[0117] Tandem VL domains: As used herein, the term "tandem VL domains (or VLs)" refers to a series of VL domains consisting of multiple identical VL domains of an antibody. Each VL domain, except for the last one at the end of the tandem, has its C-terminus linked to the N-terminus of another VL, with or without a linker. A tandem has at least two VL domains, and in certain embodiments of the antigen binding module, has 3, 4, 5, 6, 7, 8, 9, or 10 VL domains. A tandem of VLs can be generated by linking the encoding nucleic acids for each VL domain in the desired order using recombinant methods (e.g., as described in Section 7.4.3) with or without linkers that allow them to be produced as a single polypeptide chain. The N-terminus of the first VL domain of the tandem is defined as the N-terminus of the tandem, while the C-terminus of the last VL domain of the tandem is defined as the C-terminus of the tandem.

[0118] Target antigen: As used herein, "target antigen" refers to a molecule that is non-covalently, reversibly, and specifically bound by an antigen-binding domain.

[0119] Tetravalent: The term "tetravalent," as used herein in reference to an antigen-binding molecule (e.g., a BBM or TBM), refers to an antigen-binding molecule having four antigen-binding domains. A tetravalent TBM of the present disclosure generally has two antigen-binding domains that bind to the same antigen (e.g., CD19) and two antigen-binding domains that each bind to a distinct antigen (e.g., a component of the TCR complex and either CD2 or a TAA). Examples of tetravalent BBMs are shown schematically in Figures 1AA-1AH, and examples of tetravalent TBMs are shown schematically in Figures 2Q-2S.

[0120] Therapeutically effective amount: A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.

[0121] Treat, Treatment, Treating: As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a disease or disorder (e.g., a proliferative disease) or an improvement in one or more symptoms (e.g., one or more discernible symptoms) of the disease resulting from the administration of one or more CD19 binding molecules of the present disclosure. In certain embodiments, the terms "treat," "treatment," and "treating" refer to an improvement in at least one measurable physical parameter of the disease, such as tumor growth, not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to an inhibition of disease progression, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In some embodiments, the terms "treat," "treatment," and "treating" may refer to a reduction or stabilization of tumor size or cancerous cell number.

[0122] Trispecific binding molecule: The term "trispecific binding molecule" or "TBM" refers to a molecule that specifically binds to three antigens and comprises three or more antigen-binding domains. The TBM of the present disclosure comprises at least one antigen-binding domain specific for CD19, at least one antigen-binding domain specific for a component of the TCR complex, and at least one antigen-binding domain specific for CD2 or a TAA. Each antigen-binding domain can independently be an antibody fragment (e.g., scFv, Fab, nanobody), a ligand, or a non-antibody-derived binder (e.g., fibronectin, Fynomer, DARPin). Representative TBMs are illustrated in Figure 1. TBMs can comprise one, two, three, four, or even more polypeptide chains. For example, the TBM illustrated in Figure 1M comprises a single polypeptide chain comprising three scFvs linked by an ABM linker. The TBM illustrated in Figure 1K comprises, inter alia, two polypeptide chains comprising three scFvs linked by an Fc domain. The TBM illustrated in Figure 1J includes three polypeptide chains linked by an Fc domain, forming, inter alia, an scFv, a ligand, and a Fab. The TBM illustrated in Figure 1C includes four polypeptide chains linked by an Fc domain, forming, inter alia, three Fabs. The TBM illustrated in Figure 1U includes six polypeptide chains linked by an Fc domain, forming, inter alia, four Fabs and two scFvs.

[0123] Trivalent: The term "trivalent," as used herein in reference to an antigen-binding molecule (e.g., MBM), refers to an antigen-binding molecule having three antigen-binding domains. MBMs of the present disclosure are typically bispecific or trispecific. A bispecific BBM specifically binds to CD19 and a component of the TCR complex. A trispecific TBM specifically binds to CD19, a component of the TCR complex, and CD2 or a TAA. Thus, a trivalent BBM has three antigen-binding domains, two of which bind to CD19 and one of which binds to a component of the TCR, or vice versa. A TBM has three antigen-binding domains, each of which binds a different antigen. Examples of trivalent BBMs are shown schematically in Figures 1G-1Z, and examples of trivalent TBMs are shown schematically in Figures 2B-2V.

[0124] Tumor: The term "tumor" is used interchangeably herein with the term "cancer," e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors.

[0125] Tumor-associated antigen: The term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed on the surface of cancer cells, either whole or as a fragment (e.g., MHC / peptide), that is useful for preferentially targeting pharmacological agents to cancer cells. In some embodiments, the TAA is a marker expressed on both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, the TAA is a cell surface molecule that is overexpressed on cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more compared to normal cells. In some embodiments, the TAA is a cell surface molecule that is inappropriately synthesized on cancer cells, e.g., a molecule that contains a deletion, addition, or mutation compared to the molecule expressed on normal cells. In some embodiments, the TAA will be expressed only on the cell surface of cancer cells, either whole or as a fragment (e.g., MHC / peptide), and is not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific to cancer cells, sometimes referred to as tumor-specific antigens ("TSAs"). Although CD19 has the characteristics of a tumor-associated antigen, the terms "tumor-associated antigen" and "TAA" are used throughout this disclosure to refer to molecules other than CD19.

[0126] Variable region: As used herein, "variable region" or "variable domain" refers to a region of an immunoglobulin that includes one or more Ig domains substantially encoded by any of the Vκ, Vλ, and / or VH genes that constitute the κ, λ, and heavy chain immunoglobulin loci, respectively, and contains CDRs that confer antigen specificity. A "variable heavy domain" can pair with a "variable light domain" to form an antigen-binding domain ("ABD") or antigen-binding module ("ABM"). Each variable domain further includes three hypervariable regions ("complementarity-determining regions," "CDRs") (CDR-H1, CDR-H2, CDR-H3 for the variable heavy chain domain and CDR-L1, CDR-L2, CDR-L3 for the variable light chain domain) and four framework (FR) regions, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0127] Vector: The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby be replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As the plasmid is the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, the present disclosure is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0128] VH: The term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, dsFv or Fab.

[0129] VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.

[0130] VH-VL or VH-VL Pair: With reference to a VH-VL pair, whether on the same polypeptide chain or on different polypeptide chains, the terms "VH-VL" and "VH-VL pair" are used for convenience and are not intended to denote any particular orientation unless the context requires otherwise. Thus, an scFv comprising a "VH-VL" or "VH-VL pair" can have the VH and VL domains in any orientation, e.g., from the VH N-terminus to the VL or the VL N-terminus to the VH.

[0131] 7.2.CD19 binding molecules In one aspect, the present disclosure provides CD19 binding molecules, including monospecific and multispecific molecules that bind to human CD19. Typically, the CD19 binding molecules of the disclosure comprise an Fc domain comprising a first variant human IgG1 Fc region and a second variant human IgG1 Fc region having L234A, L235A, and G237A ("LALAGA") substitutions, L234A, L235A, S267K, and P329A ("LALASKPA") substitutions, D265A, P329A, and S267K ("DAPASK") substitutions, G237A, D265A, and P329A ("GADAPA") substitutions, G237A, D265A, P329A, and S267K ("GADAPASK") substitutions, L234A, L235A, and P329G ("LALAPG") substitutions, or L234A, L235A, and P329A ("LALAPA") substitutions.

[0132] In certain embodiments, the CD19 binding molecule is a monospecific binding molecule. For example, the monospecific binding molecule can be an antibody or an antigen-binding fragment thereof (e.g., an antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab')2, or a single domain antibody (SDAB). In other embodiments, the CD19 binding molecule is a multispecific (e.g., bispecific) CD19 binding molecule (e.g., a bispecific antibody).

[0133] In certain embodiments, the CD19 binding molecule is a chimeric or humanized monoclonal antibody. Chimeric and / or humanized antibodies can be engineered to minimize immune responses by human patients to antibodies produced in non-human subjects or obtained from the expression of non-human antibody genes. Chimeric antibodies contain non-human animal antibody variable regions and human antibody constant regions. Such antibodies retain the epitope-binding specificity of the original monoclonal antibody but may be less immunogenic when administered to humans, and are therefore more likely to be tolerated by patients. For example, one or all (e.g., one, two, or three) of the light chain variable regions and / or one or all (e.g., one, two, or three) of the heavy chain variable regions of a murine antibody (e.g., a murine monoclonal antibody) can each be linked to a human constant region, such as, but not limited to, an IgG1 human constant region. Chimeric monoclonal antibodies can be produced by known recombinant DNA techniques. For example, the genes encoding the constant regions of a non-human antibody molecule can be replaced with genes encoding human constant regions (see PCT Patent Publication PCT / US86 / 02269 to Robinson et al.; European Patent Application No. 184,187 to Akira et al.; or European Patent Application No. 171,496 to Taniguchi, M.). Furthermore, other suitable techniques that can be used to generate chimeric antibodies are described, for example, in U.S. Pat. Nos. 4,816,567; 4,978,775; 4,975,369; and 4,816,397.

[0134] Chimeric or humanized antibodies and antigen-binding fragments thereof of the present disclosure can be prepared based on the sequences of mouse monoclonal antibodies. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to generate chimeric antibodies, mouse variable regions can be linked to human constant regions using known methods (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). To generate humanized antibodies, mouse CDR regions can be inserted into a human framework using known methods. See, e.g., U.S. Pat. No. 5,225,539 to Winter and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.

[0135] Humanized antibodies can be produced by techniques such as CDR grafting (see, e.g., EP 239,400; WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (see, e.g., EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973), chain shuffling, and the like. shuffling) (see, e.g., U.S. Pat. No. 5,565,332) and, for example, U.S. Patent Application Publication No. 2005 / 0042664, U.S. Patent Application Publication No. 2005 / 0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al. al., Protein Eng., 9(10):895-904(1996), Couto et al., Cancer Res., 55(23 Supp):5973s-5977s(1995), Couto et al., Cancer Res., 55(8):1717-22(1995), Sandhu JS, Gene, 150(2):409-10(1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73(1994).In many cases, framework residues in the framework regions are substituted with corresponding residues from the CDR donor antibody to alter, e.g., improve, antigen binding. These framework substitutions, e.g., conservative substitutions, are identified by well-known methods, such as modeling the interactions between CDRs and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual frameworks at specific positions. (See, e.g., U.S. Patent No. 5,585,089 to Queen et al.; and Riechmann et al., 1988, Nature, 332:323.)

[0136] As provided herein, a humanized antibody or antibody fragment can comprise one or more CDR and framework regions from a non-human immunoglobulin molecule, wherein the amino acid residues comprising the framework are derived entirely or predominantly from germline sequences. Several techniques for humanizing antibodies or antibody fragments are known, including the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Nature, 332:323-327 (1988)). Humanization can be essentially accomplished by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR grafting (European Patent No. 239,400; PCT Publication No. WO 91 / 09967; and U.S. Patent Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; and 6,548,640). In such humanized antibodies and antibody fragments, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332).

[0137] The selection of human variable domains, both light and heavy, used to generate humanized antibodies results in reduced antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (e.g., Nicholson et al. Mol. Immun. 34(16-17):1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al. See, e.g., et al., J. Immunol., 151:2623 (1993). In certain embodiments, the framework regions, e.g., all four framework regions of the heavy chain variable region, are derived from the VH4_4-59 germline sequence. In one embodiment, the framework regions may include, e.g., one, two, three, four, or five modifications, e.g., substitutions, e.g., conservative substitutions, from an amino acid in the corresponding murine sequence. In one embodiment, the framework regions, e.g., all four framework regions of the light chain variable region, are derived from the VK3_1.25 germline sequence. In one embodiment, the framework regions may include, e.g., one, two, three, four, or five modifications, e.g., substitutions, e.g., conservative substitutions, from an amino acid in the corresponding murine sequence.

[0138] In certain embodiments, a CD19 binding molecule comprises a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and / or a light chain variable region from a particular germline light chain immunoglobulin gene. For example, such an antibody can comprise or consist of a human antibody comprising a heavy or light chain variable region that is "the product of" or "derived from" a particular germline sequence. Human antibodies that are "the product of" or "derived from" a germline immunoglobulin sequence can be identified, for example, by comparing the amino acid sequence of a human antibody to the amino acid sequence of a germline immunoglobulin and selecting the germline immunoglobulin sequence that is closest in sequence (i.e., highest percent identity) to the sequence of the human antibody (using methods outlined herein). Human antibodies that are "the product of" or "derived from" a particular germline immunoglobulin sequence can contain amino acid differences compared to the germline sequence, for example, due to natural somatic mutations or the intentional introduction of site-specific mutations. However, a humanized antibody typically has an amino acid sequence that is at least 90% identical to the amino acid sequence encoded by a germline immunoglobulin gene and includes amino acid residues that identify the antibody as derived from a human sequence when compared to germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In some cases, a humanized antibody may have an amino acid sequence that is at least 95, 96, 97, 98, or 99% identical, or at least 96, 97, 98, or 99% identical to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a humanized antibody derived from a particular germline sequence will exhibit no more than 10-20 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene (before the introduction of any skew, pI, and ablation mutations described herein; i.e., the number of mutations is generally low before the introduction of the mutations disclosed herein). In some cases, a humanized antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene (again, before the introduction of any distortion, pI, and elimination mutations herein; i.e., the number of mutations is generally low, before the introduction of the mutations of the present disclosure).

[0139] In one embodiment, the parent antibody is affinity matured. For example, structure-based methods can be used for humanization and affinity maturation, as described in U.S. Patent Application No. 11 / 004,590. Selection-based methods can be used to humanize and / or affinity mature antibody variable regions, including, but not limited to, those described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37):22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95:8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759. Other humanization methods, including but not limited to those described in U.S. Patent No. 09 / 810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084, may involve grafting only a portion of the CDRs.

[0140] In one embodiment, the CD19-binding molecule comprises an ABM that is a Fab. Fab domains can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain or by recombinant expression. Fab domains typically comprise a CH1 domain linked to a VH domain, which is paired with a CL domain linked to a VL domain. In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the link module. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0141] In some embodiments, the CD19 binding molecule comprises an ABM that is an scFab. In one embodiment, the antibody domains and linker in the scFab fragment have one of the following orders from N- to C-terminus: a) VH-CH1-linker-VL-CL, or b) VL-CL-linker-VH-CH1. In some cases, VL-CL-linker-VH-CH1 is used.

[0142] In another embodiment, the antibody domains and linker in the scFab fragment have one of the following orders from N- to C-terminus: a) VH-CL-linker-VL-CH1 or b) VL-CH1-linker-VH-CL.

[0143] Optionally, in the scFab fragment, in addition to the natural disulfide bond between the CL-domain and the CH1 domain, the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL) are also disulfide-stabilized by the introduction of a disulfide bond between the following positions: i) heavy chain variable domain position 44 and light chain variable domain position 100, ii) heavy chain variable domain position 105 and light chain variable domain position 43, or iii) heavy chain variable domain position 101 and light chain variable domain position 100 (numbering according to the EU index of Kabat).

[0144] Such further disulfide stabilization of scFab fragments is achieved by the introduction of a disulfide bond between the variable domains VH and VL of the single-chain Fab fragment. Techniques for introducing non-native disulfide bridges for the stabilization of single-chain Fvs are described, for example, in WO 94 / 029350; Rajagopal et al., 1997, Prot. Engin. 10:1453-59; Kobayashi et al., 1998, Nuclear Medicine & Biology, 25:387-393; and Schmidt et al., 1999, Oncogene 18:1711-1721. In one embodiment, the optional disulfide bond between the variable domains of the scFab fragment is located between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, the optional disulfide bond between the variable domains of the scFab fragment is between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (numbering according to EU index of Kabat).

[0145] In one embodiment, the CD19 binding molecule comprises an ABM that is an scFv. Single-chain Fv antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain and can be expressed as single-chain polypeptides, retaining the specificity of the intact antibody from which they are derived. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for linking the VH and VL chains of an scFv are any of the ABM linkers identified in Section 7.4.3, e.g., linkers designated L1-L58.

[0146] Unless specified, as used herein, an scFv can have the VL and VH variable regions in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL.

[0147] To generate an scFv-encoding nucleic acid, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a linker, such as any of the linkers described in Section 7.4.3 (such as the amino acid sequence (Gly4 to Ser)3 (SEQ ID NO:53)), such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0148] CD19 binding molecules may also comprise ABMs, which are Fvs, dsFvs, (Fab')2s, single domain antibodies (SDABs), VH or VL domains or camelid VHH domains (also called nanobodies).

[0149] CD19 binding molecules may include single domain antibodies composed of a single VH or VL domain that exhibits sufficient affinity for CD 19. In one embodiment, the single domain antibody is a camelid VHH domain (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38; WO 94 / 04678).

[0150] Tables 1A and 1B (collectively "Table 1") list exemplary CD19 binding sequences that may be included in a CD19 binding molecule. The sequences shown in Table 1A are based on the CD19 antibody NEG258.

[0151] [Table 1]

[0152] In some embodiments, the CD19 binding molecule comprises the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences of NEG258 set forth in Table 1A. The CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences can be as defined by Kabat (SEQ ID NOS: 17-19 and 4-6, respectively), Chothia (SEQ ID NOS: 20-22 and 7-9, respectively), or IMGT (SEQ ID NOS: 23-25 ​​and 10-12, respectively), or the combined CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences of Chothia and Kabat (SEQ ID NOS: 14-16 and 1-3, respectively). The CD19 binding molecule may also comprise the light chain variable sequence (SEQ ID NO: 26) and / or the heavy chain variable sequence (SEQ ID NO: 13) of the anti-CD19 antibody NEG258 described in Table 1A.

[0153] The sequences listed in Table 1B are based on the CD19 antibody NEG218.

[0154] [Table 2]

[0155] [Table 3]

[0156] In some embodiments, the CD19 binding molecule comprises the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences of NEG218 set forth in Table 1B. The CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences can be as defined by Kabat (SEQ ID NOS: 43-45 and 30-32, respectively), Chothia (SEQ ID NOS: 46-48 and 33-35, respectively), or IMGT (SEQ ID NOS: 49-51 and 36-38, respectively), or the combined CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences of Chothia and Kabat (SEQ ID NOS: 40-42 and 27-29, respectively). The CD19 binding molecule may also comprise the light chain variable sequence (SEQ ID NO: 52) and / or the heavy chain variable sequence (SEQ ID NO: 39) of the anti-CD19 antibody NEG218 described in Table 1B.

[0157] Other CD19 binding molecules are mutated but contain amino acids in the CDR regions that have at least 80, 85, 90, 95, 96, 97, 98, or 99 percent identity to the CDR sequences set forth in Table 1. In certain embodiments, such CD19 binding molecules contain mutated amino acid sequences in which no more than 1, 2, 3, 4, or 5 amino acids are mutated in the CDR regions when compared to the CDR sequences set forth in Table 1.

[0158] Other CD19 binding molecules comprise VH and / or VL domains comprising amino acid sequences having at least 80, 85, 90, 95, 96, 97, 98, or 99 percent identity to a VH and / or VL sequence set forth in Table 1. In certain embodiments, the CD19 binding molecule comprises a VH and / or VL domain in which no more than 1, 2, 3, 4, or 5 amino acids have been mutated compared to the VH and / or VL domain set forth in the sequence set forth in Table 1 while retaining substantially the same therapeutic activity.

[0159] The CD19 binding molecules can be fused or chemically conjugated (including both covalent and non-covalent conjugation) to heterologous proteins or polypeptides (or fragments thereof, e.g., polypeptides of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids). For example, the CD19 binding molecules can be fused directly or indirectly to a detectable protein, such as an enzyme or fluorescent protein. Methods for fusing or conjugating proteins, polypeptides, or peptides to antibodies or antibody fragments are known and can be used to fuse or conjugate proteins or polypeptides to the CD19 binding molecules of the present disclosure. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. 307,434 and 367,166; International Publication Nos. WO 96 / 04388 and WO 91 / 06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341.

[0160] Additional CD19 binding molecules can be generated by the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and / or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to modify the activity of the molecules of the present disclosure or fragments thereof (e.g., molecules or fragments thereof with higher affinity and lower off-rates). See generally U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313. The CD19 binding molecules or fragments thereof described herein can be modified prior to recombination by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods. Polynucleotides encoding fragments of the CD19 binding molecules described herein may be recombined with one or more components, motifs, sections, portions, domains, fragments, etc. of one or more heterologous molecules.

[0161] Additionally, CD19-binding molecules can be fused to a marker sequence, such as a peptide, to facilitate purification. In one embodiment, the marker amino acid sequence is a hexahistidine peptide (SEQ ID NO: 54), such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA 91311), among others, many of which are commercially available. For example, hexahistidine (SEQ ID NO: 54) provides convenient purification of the fusion protein, as described by Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the "flag" tag.

[0162] 7.3. Antigen-Binding Modules of Multispecific Binding Molecules Typically, one or more ABMs of an MBM comprise sequences or derivatives of immunoglobulin-based antigen-binding domains, such as antibody fragments. These antibody fragments and derivatives typically contain the CDRs of an antibody and may include larger fragments and derivatives thereof, such as Fab, scFab, Fv, and scFv.

[0163] Immunoglobulin-based ABMs can include modifications to framework residues within the VH and / or VL, e.g., to improve the properties of the MBM that contains the ABM. For example, framework modifications can be made to reduce the immunogenicity of the MBM. One approach to making such framework modifications is to "backmutate" one or more framework residues of the ABM to the corresponding germline sequence. Such residues can be identified by comparing the framework sequence to the germline sequence from which the ABM is derived. To "match" the framework region sequence to the desired germline form, residues can be "backmutated" to the corresponding germline sequence, e.g., by site-directed mutagenesis. MBMs with such "backmutated" ABMs are intended to be encompassed by the present disclosure.

[0164] Another type of framework modification involves mutating one or more residues within the framework regions or even one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of MBM. This approach is also referred to as "deimmunization" and is described in more detail in U.S. Patent Application Publication No. 20030153043 by Carr et al.

[0165] ABMs can also be modified to exhibit altered glycosylation, which can be useful, for example, to increase the affinity of the MBM for one or more of its antigens. Such carbohydrate modifications can be achieved, for example, by modifying one or more glycosylation sites within the ABM sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such deglycosylation can increase the affinity of the MBM for its antigen. Such techniques are described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0166] 7.3.1. Immunoglobulin-based ABM 7.3.1.1.Fab In certain embodiments, the ABM is a Fab domain.

[0167] In the MBMs of the present disclosure, it is advantageous to use a Fab heterodimerization approach to enable proper association of Fab domains belonging to the same ABM and minimize aberrant pairing of Fab domains belonging to different ABMs. For example, the Fab heterodimerization approach shown in Table 2 below can be used.

[0168] [Table 4]

[0169] Thus, in certain embodiments, proper association between the two polypeptides of a Fab is facilitated by swapping the VL and VH domains of the Fab with one another, or by swapping the CH1 and CL domains with one another, as described, for example, in WO 2009 / 080251.

[0170] Proper Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CL domain of the Fab, and / or one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces, such that the Fab components preferentially pair with each other over other Fab components.

[0171] In one embodiment, one or more amino acid modifications are limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains, as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0172] In one embodiment, modifications introduced into the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of various interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock-and-key, knob-into-hole, protrusion-and-cavity, donor-and-acceptor, etc., all of which suggest the nature of a structural and chemical match between two interacting surfaces.

[0173] In one embodiment, the one or more modifications introduced introduce new hydrogen bonds across the interface of the Fab component. In one embodiment, the one or more modifications introduced introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO 2014 / 150973 and WO 2014 / 082179.

[0174] In one embodiment, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduce a salt bridge between the CH1 and CL domains (see Golay et al., 2016, J Immunol 196:3199-211).

[0175] In one embodiment, the Fab domain contains 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to shuffle the hydrophobic and polar contact regions between the CH1 and CL domains (see Golay et al., 2016, J Immunol 196:3199-211).

[0176] In certain embodiments, the Fab domain can contain modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab boundaries that promote proper assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In one embodiment, a 39K, 62E modification is introduced in the VH domain, an H172A, F174G modification is introduced in the CH1 domain, a 1R, 38D, (36F) modification is introduced in the VL domain, and an L135Y, S176W modification is introduced in the CL domain. In another embodiment, a 39Y modification is introduced in the VH domain and a 38R modification is introduced in the VL domain.

[0177] Fab domains can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of Fab component pairing. For example, an engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see Mazor et al., 2015, MAbs 7:377-89).

[0178] Fab domains can also be modified by replacing the CH1 and CL domains with other domains that promote proper assembly. For example, Wu et al., 2015, MAbs 7:364-76, describe replacing the CH1 domain of the α T cell receptor with a constant domain and the CL domain of the T cell receptor with a β domain, and combining these domain replacements with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0179] An ABM can include a single-chain Fab fragment, which is a polypeptide consisting of an antibody heavy chain variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL), and a linker. In some embodiments, the antibody domains and linker have one of the following orders, from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. The linker can be a polypeptide of at least 30 amino acids, e.g., 32-50 amino acids. The single-chain Fab domain is stabilized by a native disulfide bond between the CL domain and the CH1 domain.

[0180] In some embodiments, the antibody domains and linker of a single-chain Fab fragment have one of the following orders, from N- to C-terminus: a) VH-CH1-linker-VL-CL, or b) VL-CL-linker-VH-CH1. In some cases, VL-CL-linker-VH-CH1 is used.

[0181] In another embodiment, the antibody domains and linker of the single-chain Fab fragment have one of the following orders, from N-terminal to C-terminal: a) VH-CL-linker-VL-CH1 or b) VL-CH1-linker-VH-CL.

[0182] Optionally, in the single-chain Fab fragment, in addition to the natural disulfide bond between the CL-domain and the CH1-domain, the antibody heavy chain variable domain (VH) and antibody light chain variable domain (VL) ABM are also disulfide-stabilized by the introduction of a disulfide bond between: i) heavy chain variable domain position 44 to light chain variable domain position 100, ii) heavy chain variable domain position 105 to light chain variable domain position 43, or iii) heavy chain variable domain position 101 to light chain variable domain position 100 (numbering according to EU index of Kabat).

[0183] In one embodiment, the optional disulfide bond between the variable domains of the single-chain Fab fragment is between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, the optional disulfide bond between the variable domains of the single-chain Fab fragment is between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (numbering according to EU index of Kabat).

[0184] 7.3.1.2.scFv In certain embodiments, the ABM is a single chain Fv or "scFv." Exemplary linkers connecting the VH and VL chains of an scFv are any of the ABM linkers identified in Section 7.4.3, such as those designated as L1-L54.

[0185] To generate nucleic acids encoding scFvs, the DNA fragments encoding the VH and VL are ligated to another fragment encoding a linker, such as any of the ABM linkers described in Section 7.4.3 (amino acid sequence (Gly4 ~ The fragment is operably linked to another fragment encoding a nucleotide sequence such as Ser)3 (SEQ ID NO: 53).

[0186] 7.3.1.3. Other Immunoglobulin-Based ABMs MBMs may also include ABMs with immunoglobulin formats other than Fab or scFv, such as Fv, dsFv, (Fab')2, single domain antibodies (SDAB), VH or VL domains, or camelid VHH domains (also called nanobodies).

[0187] The ABM can be a single domain antibody composed of a single VH or VL domain that exhibits sufficient affinity for the target. In embodiments, the single domain antibody is a camelid VHH domain (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38; WO 94 / 04678).

[0188] 7.3.2. Non-immunoglobulin-based ABM In certain embodiments, the MBM comprises one or more ABMs derived from non-antibody scaffold proteins (including, but not limited to, designed ankyrin repeat proteins (DARPins), avimers (short for avidity multimers), anticalins / lipocalins, centilins, Kunitz domains, adnexins, affilins, affitins (also known as nanophytins), knottins, pronectins, versabodies, duocalins, and fynomers), ligands, receptors, cytokines, or chemokines.

[0189] Non-immunoglobulin scaffolds that can be used in MBM include those listed in Tables 3 and 4 of Mintz and Crea, 2013, Bioprocess International 11(2):40-48; Figure 1, Table 1, and Figure I of Vazquez-Lombardi et al., 2015, Drug Discovery Today 20(10):1271-83; and Table 1 and Box 2 of Skrlec et al., 2015, Trends in Biotechnology 33(7):408-18. Tables 3 and 4 of Mintz and Crea, 2013, Bioprocess International 11(2):40-48; Figure 1, Table 1, and Figure I of Vazquez-Lombardi et al., 2015, Drug Discovery Today 20(10):1271-83; and the contents of Table 1 and Box 2 of Skrlec et al., 2015, Trends in Biotechnology 33(7):408-18 (collectively the "Scaffold Disclosure"). In certain embodiments, the scaffold disclosures are incorporated by reference for what they disclose with respect to adnexins. In other embodiments, the scaffold disclosures are incorporated by reference for what they disclose with respect to avimers. In other embodiments, the scaffold disclosures are incorporated by reference for what they disclose with respect to affibodies. In yet other embodiments, the scaffold disclosures are incorporated by reference for what they disclose with respect to anticalins. In yet other embodiments, the scaffold disclosures are incorporated by reference for what they disclose with respect to DARPins. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to Kunitz domains. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to knottins. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to Pronectins. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to nanophytins.In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to affilins. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to Adnectins. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to ABMs. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to Advirons. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to Affimers. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to alphabodies. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to armadillo repeat proteins. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to atrimers / tetranectins. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to obodies / OB-folds. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to sentinins. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to repebodies. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to anticalins. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to atrimers. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to bicyclic peptides. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to cys-knots. In yet another embodiment, the scaffold disclosures are incorporated by reference for what they disclose with respect to Fn3 scaffolds (including adnectins, sentinins, pronectins, and Tn3).

[0190] In one embodiment, the ABM can be a designed ankyrin repeat protein ("DARPin"). DARPins are antibody mimetic proteins that typically exhibit highly specific and high-affinity target protein binding. They are typically genetically engineered and derived from natural ankyrin proteins and consist of at least three, usually four or five, repeat motifs of these proteins. Their molecular weight is approximately 14 or 18 kDa (kilodaltons) for four or five repeat DARPins, respectively. Examples of DARPins can be found, for example, in U.S. Patent No. 7,417,130. Multispecific binding molecules comprising DARPin binding modules and immunoglobulin-based binding modules are disclosed, for example, in U.S. Patent Application Publication No. 2015 / 0030596 A1.

[0191] In another embodiment, the ABM can be an affibody, which is well known and refers to an affinity protein based on a 58 amino acid residue protein domain derived from one of the IgG-binding domains of staphylococcal protein A.

[0192] In another embodiment, the ABM can be an anticalin. Anticalins are well known and refer to another antibody mimetic technology in which the binding specificity is derived from lipocalins. Anticalins can also be formatted as dual-targeting proteins called duocalins.

[0193] In another embodiment, the ABM can be a Versabody. Versabodies are a well-known and distinct antibody mimetic technology. They are small proteins of 3-5 kDa with more than 15% cysteines, forming a high disulfide density scaffold that replaces the hydrophobic core of typical proteins.

[0194] Other non-immunoglobulin ABMs include "A" domain oligomers (also known as avimers) (see, e.g., U.S. Patent Application Publication Nos. 2005 / 0164301, 2005 / 0048512, and 2004 / 017576), Fn3-based protein scaffolds (see, e.g., U.S. Patent Application Publication No. 2003 / 0170753), VASP polypeptides, avian pancreatic polypeptide (aPP), tetranectin (based on CTLD3), affilin (based on γB-crystallin / ubiquitin), knottins, SH3 domains, PDZ domains, tendamistat, neocarzinostatin, protein A domains, lipocalin, transferrin, or Kunitz domains. In one embodiment, ABMs useful for constructing MBMs include fibronectin-based scaffolds, as exemplified in WO 2011 / 130324.

[0195] Furthermore, in certain embodiments, the ABM comprises a ligand binding domain of a receptor or a receptor binding domain of a ligand.

[0196] Connectors It is contemplated that a CD19 binding molecule may, in some cases, comprise a pair of ABMs or ABM chains (e.g., the VH-CH1 or VL-CL components of an Fab) directly linked to each other, e.g., as a fusion protein without a linker. For example, a CD19 binding molecule may comprise a connector moiety linking individual ABMs or ABM chains. The use of a connector moiety may improve target binding, for example, by increasing the flexibility of the ABMs within the CD19 binding molecule, thereby reducing steric hindrance. The ABM or ABM chains may be linked to each other, for example, via an Fc domain (each Fc domain representing a pair of associated Fc regions) and / or an ABM linker. The use of an Fc domain typically requires the use of a hinge region as a connector for the ABM or ABM chain for optimal antigen binding. Thus, the term "connector" encompasses, but is not limited to, an Fc region, an Fc domain, and a hinge region.

[0197] The connector can be selected or modified to, for example, increase or decrease the biological half-life of the CD19-binding molecule. For example, to decrease the biological half-life, one or more amino acid mutations can be introduced into the CH2-CH3 domain interface of the Fc-hinge fragment so that the CD19-binding molecule containing the fragment has reduced Staphylococcus protein A (SpA) binding compared to native Fc-hinge region SpA binding. This approach is described in more detail in U.S. Pat. No. 6,165,745 to Ward et al. Alternatively, the CD19-binding molecule can be modified to increase its biological half-life. For example, as described in U.S. Pat. No. 6,277,375 to Ward, one or more of the following mutations can be introduced: T252L, T254S, and T256F. Alternatively, to increase biological half-life, CD19 binding molecules can be modified within the CH1 or CL region to contain salvage receptor binding epitopes taken from two loops of the CH2 domain of the Fc region of IgG, as described by Presta et al. in U.S. Pat. Nos. 5,869,046 and 6,121,022.

[0198] Examples of Fc domains (formed by pairing of two Fc regions), hinge regions, and ABM linkers are described in Sections 7.4.1, 7.4.2, and 7.4.3, respectively.

[0199] Fc Domain The CD19 binding molecule may comprise an Fc domain derived from any suitable species. In one embodiment, the Fc domain is derived from a human Fc domain.

[0200] The Fc domain may be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0201] An Fc domain contains two polypeptide chains, each referred to as a heavy chain Fc region. These two heavy chain Fc regions dimerize to create an Fc domain. The two Fc regions within an Fc domain can be the same or different from each other. In natural antibodies, the Fc regions are typically identical, but for purposes of generating multispecific binding molecules of the present disclosure, the Fc regions can advantageously be different to allow heterodimerization, as described in Section 7.4.1.5 below.

[0202] Typically, each heavy chain Fc region comprises or consists of two or three heavy chain constant domains.

[0203] In natural antibodies, the heavy chain Fc regions of IgA, IgD, and IgG are composed of two heavy chain constant domains (CH2 and CH3), while the heavy chain Fc regions of IgE and IgM are composed of three heavy chain constant domains (CH2, CH3, and CH4), which dimerize to generate the Fc domain.

[0204] In the present disclosure, the heavy chain Fc region can comprise heavy chain constant domains from one or more different classes of antibodies, for example, from one, two, or three different classes.

[0205] In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG1. An exemplary sequence of a heavy chain Fc region derived from human IgG1 is provided in SEQ ID NO:1109. [ka] In some embodiments, a CD19 binding molecule of the present disclosure comprises an Fc region, the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO: 1109 modified with one or more of the substitutions described in Section 7.4.1 and subdivisions thereof.

[0206] In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG2.

[0207] In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG3.

[0208] In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG4.

[0209] In one embodiment, the heavy chain Fc region comprises a CH4 domain derived from IgM. The IgM CH4 domain is typically located C-terminal to the CH3 domain.

[0210] In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from an IgG and a CH4 domain derived from an IgM.

[0211] It will be understood that heavy chain constant domains for use in generating heavy chain Fc regions for CD19 binding molecules of the present disclosure can include variants of the native constant domains described above. Such variants can include one or more amino acid changes compared to the wild-type constant domain. In one example, the heavy chain Fc region of the present disclosure includes at least one constant domain that differs in sequence from the wild-type constant domain. It will be understood that the variant constant domain can be longer or shorter than the wild-type constant domain. For example, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In another example, the variant constant domain is at least 75% identical or similar. In another example, the variant constant domain is at least 80% identical or similar. In another example, the variant constant domain is at least 85% identical or similar. In another example, the variant constant domain is at least 90% identical or similar. In another example, the variant constant domains are at least 95% identical or similar. In another example, the variant constant domains are at least 99% identical or similar. Exemplary Fc variants are described in Sections 7.4.1.1-7.4.1.5, below.

[0212] IgM and IgA naturally occur in humans as covalently linked multimers of a common H2L2 antibody unit. IgM exists as a pentamer when incorporating a J chain or as a hexamer when lacking a J chain. IgA exists in both monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension to the C-terminal constant domain, known as the tail. The tail contains cysteine ​​residues that form disulfide bonds between heavy chains in the polymer and is thought to play an important role in polymerization. The tail also contains glycosylation sites. In certain embodiments, the CD19-binding molecules of the present disclosure do not include a tail.

[0213] The Fc domain incorporated into the CD19 binding molecules of the present disclosure may include one or more modifications that alter one or more functional properties of the protein, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Additionally, the CD19 binding molecule may be chemically modified (e.g., one or more chemical moieties may be attached to the CD19 binding molecule) or modified to alter its glycosylation, which in turn may alter one or more functional properties of the CD19 binding molecule.

[0214] Effector functions of antibody molecules include, for example, complement-mediated effector functions mediated by the binding of the C1 component of complement to antibodies. Complement activation is important in the opsonization and direct lysis of pathogens. Furthermore, it stimulates inflammatory responses by recruiting and activating phagocytes to the site of complement activation. Effector functions include Fc receptor (FcR)-mediated effector functions, which can be triggered upon binding of the constant domain of an antibody to an Fc receptor (FcR). Antigen-antibody complex-mediated cross-linking of Fc receptors on the surface of effector cells triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental passage, and regulation of immunoglobulin production.

[0215] The Fc region can be modified by substituting at least one amino acid residue with a different amino acid residue to alter effector function. For example, one or more amino acids can be substituted with a different amino acid residue so that the Fc region has altered affinity for an effector ligand. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al. Modified Fc regions can also alter C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al. Modified Fc regions can also alter the ability of the Fc region to fix complement. This approach is described, for example, in PCT Publication WO 94 / 29351 by Bodmer et al. Allotypic amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses and the constant region of the light chain of the kappa isotype, as described by Jefferis et al., 2009, MAbs, 1:332-338.

[0216] The Fc region can also be modified to "silence" effector functions, for example, to reduce or eliminate the ability of the CD19-binding molecule to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). This can be achieved, for example, by introducing mutations in the Fc region. Such mutations have been described in the art: LALA and N297A (Strohl, 2009, Curr. Opin. Biotechnol. 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. 181:6664-69; Strohl, supra). An example of a silent Fc IgG1 antibody includes the so-called LALA mutant, which contains L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgG1 antibody contains the D265A mutation. Another silent IgG1 antibody contains the so-called DAPA mutant, which contains the D265A and P329A mutations in the IgG1 Fc amino acid sequence. Another silent IgG1 antibody contains the N297A mutation, which results in an aglycosylated / non-glycosylated antibody.

[0217] The Fc region can be modified to improve the ability of a CD19-binding molecule containing the Fc region to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP), for example, by modifying one or more amino acid residues to increase the affinity of the CD19-binding molecule for activating Fcγ receptors or decrease the affinity of the CD19-binding molecule for inhibitory Fcγ receptors. Human activating Fcγ receptors include FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb, and human inhibitory Fcγ receptors include FcγRIIb. This approach is described, for example, in PCT Publication WO 00 / 42072 by Presta. Furthermore, the binding sites in human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and mutants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001). Optimization of Fc-mediated effector functions of monoclonal antibodies, such as improving ADCC / ADCP function, has been described (see Strohl, 2009, Current Opinion in Biotechnology 20:685-691). Mutations that may improve ADCC / ADCP function include one or more mutations selected from G236A, S239D, F243L, P247I, D280H, K290S, R292P, S298A, S298D, S298V, Y300L, V305I, A330L, I332E, E333A, K334A, A339D, A339Q, A339T and P396L (all positions according to EU numbering).

[0218] The Fc region can also be modified to improve the ability of the CD19-binding molecule to mediate ADCC and / or ADCP, for example, by modifying one or more amino acids to increase the affinity of the CD19-binding molecule for activating receptors that typically do not recognize the parent CD19-binding molecule, such as FcαRI. This approach is described, for example, in Borrok et al., 2015, mAbs. 7(4):743-751.

[0219] Thus, in certain embodiments, CD19-binding molecules of the present disclosure may comprise an Fc domain with an altered effector function, such as, but not limited to, binding to an Fc-receptor such as FcRn or a leukocyte receptor (e.g., as described above or in Section 7.4.1.1), complement binding (e.g., as described above or in Section 7.4.1.2), a modified disulfide bond structure (e.g., as described above or in Section 7.4.1.3), or an altered glycosylation pattern (e.g., as described above or in Section 7.4.1.4). The Fc domain may also be altered to include modifications that improve the manufacturability of asymmetric CD19-binding molecules, for example, by enabling heterodimerization, which is the preferential pairing of non-identical over identical Fc regions. Heterodimerization allows for the generation of CD19-binding molecules in which different ABMs are linked to each other by Fc domains containing Fc regions that differ in sequence. Examples of heterodimerization techniques are illustrated in Section 7.4.1.5 (and its subsections).

[0220] It will be understood that any of the modifications described in Sections 7.4.1.1-7.4.1.5 can be combined in any suitable manner to achieve the desired functional property and / or can be combined with other modifications to alter the properties of the CD19 binding molecule. In some embodiments, the CD19 binding molecule comprises an IgG1 Fc domain with mutations at 1, 2, 3, 4, 5, 6, or more than 6 of positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332 (EU numbering). For example, a CD19 binding molecule may comprise the IgG1 sequence of SEQ ID NO: 1109 with mutations at 1, 2, 3, 4, 5, 6 or more than 6 of positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331 and 332.

[0221] In some embodiments, the CD19 binding molecule comprises first and second human IgG1 Fc regions having amino acid substitutions selected from the following combinations of substitutions: substitutions L234A, L235A, and G237A ("LALAGA"); substitutions L234A, L235A, S267K, and P329A ("LALASKPA"); substitutions D265A, P329A, and S267K ("DAPASK"); substitutions G237A, D265A, and P329A ("GADAPA"); substitutions G237A, D265A, P329A, and S267K ("GADAPASK"); substitutions L234A, L235A, and P329G ("LALAPG"), and substitutions L234A, L235A, and P329A ("LALAPA"), wherein the amino acid residues are numbered according to the EU numbering system. It should be understood that the terms "LALAGA," "LALASKPA," "DAPASK," "GADAPA," "GADAPASK," "LALAPG," and "LALAPA" do not represent consecutive amino acid sequences, but rather shorthand terms that represent various combinations of the substitutions described in this paragraph.

[0222] In another embodiment, the CD19 binding molecule comprises a human IgG1 Fc region with amino acid substitutions selected from the combination of substitutions L234A, L235A, S267K, P329A ("LALASKPA") or G237A, D265A, P329A, S267K ("GADAPASK"), wherein the amino acid residues are numbered according to the EU numbering system.

[0223] In a further embodiment, the CD19 binding molecule comprises an Fc region selected from FCV1-FCV7 (see Table A below).

[0224] In still further embodiments, the CD19 binding molecule comprises an Fc region that is FCV4 or FCV7.

[0225] In some embodiments, the CD19 binding molecule has reduced or undetectable binding affinity to an Fcγ receptor or C1q compared to a polypeptide comprising a wild-type human IgG1 Fc region, optionally measured by surface plasmon resonance using a Biacore T200 instrument, wherein the Fcγ receptor is selected from the group consisting of FcγR1A, FcγRIIIaV158 mutant, and FcγRIIIaF158 mutant, and has 50%, 80%, 90%, 95%, 98%, 99% reduced or undetectable binding compared to wild-type.

[0226] In some embodiments, the CD19 binding molecule has reduced or undetectable effector function compared to a polypeptide comprising a wild-type human IgG1 Fc region.

[0227] In some embodiments, the CD19 binding molecule can bind to an antigen without causing detectable antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). In some embodiments, the effector function that is reduced or decreased is antibody-dependent cell-mediated cytotoxicity (ADCC) in the individual. In some embodiments, the effector function that is reduced or decreased is antibody-dependent cellular phagocytosis (ADCP) in the individual. In some embodiments, the effector function that is reduced or decreased is complement-dependent cytotoxicity (CDC) in the individual. In some embodiments, the first and second Fc regions of the Fc domain each comprise a nucleic acid sequence selected from the nucleic acid sequences listed in Table A below, or any sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this nucleic acid sequence.

[0228] In one embodiment, the nucleic acid encoding the Fc region comprises a nucleic acid sequence of FCV-7 (see Table A below) or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto. In one embodiment, the nucleic acid encoding the Fc region comprises a nucleic acid sequence of FCV-4 (see Table A below) or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto. In some embodiments, the Fc domain comprises a first and a second Fc region, each of which comprises an amino acid sequence selected from the amino acid sequences listed in Table A below, or any sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0229] In one embodiment, the Fc domain comprises first and second Fc regions comprising the amino acid sequence of FCV-7 (see Table A below) or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto. In one embodiment, the Fc domain comprises first and second Fc regions comprising the amino acid sequence of FCV-4 (see Table A below) or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0230] Additionally, provided herein is a vector comprising a polynucleotide encoding a CD19 binding molecule comprising an Fc region selected from FCV1 to FCV7 (see Table A below).

[0231] Also provided herein are host cells comprising vectors or polynucleotides encoding and capable of expressing CD19-binding molecules comprising an Fc region selected from FCV1 to FCV7 (see Table A below).

[0232] Table 5

[0233] Table 6

[0234] Table 7

[0235] Table 8

[0236] Table 9

[0237] Table 10

[0238] Table 11

[0239] Table 12

[0240] Table 13

[0241] Table 14

[0242] 7.4.1.1. Fc Domains with Altered FcR Binding The Fc domain of a CD19 binding molecule may exhibit altered binding to one or more Fc-receptors (FcRs) compared to the corresponding native immunoglobulin. Binding to any particular Fc-receptor may be increased or decreased. In one embodiment, the Fc domain contains one or more modifications that alter its Fc-receptor binding profile.

[0243] Human cells can express several membrane-bound FcRs, selected from FcαR, FcεR, FcγR, FcRn, and glycan receptors. Some cells can also express soluble (extracellular domain) FcRs (Fridman et al., 1993, J Leukocyte Biology 54:504-512). FcγRs can be further divided by IgG-binding affinity (high / low) and biological effect (activating / inhibiting). Human FcγRI is widely considered to be the only "high-affinity" receptor, while all others are considered to have intermediate to low affinity. FcγRIIb is the only receptor with "inhibitory" functionality due to its intracellular ITIM motif, while all others are considered to be "activating" due to ITAM motifs or pair with the common FcγR-γ chain. FcγRIIIb is active but also unique in that it associates with cells via a GPI anchor. Overall, humans express six "canonical" FcγRs: FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In addition to these sequences, there are numerous sequence or allotypic variants spread across these families. Some of these have been shown to have important functional consequences and may therefore be considered receptor subtypes in their own right. Examples include FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. H134R , FcγRIIb I190T , FcγRIIIa F158V , FcγRIIIb NA1 , FcγRIIIb NA2 and FcγRIII SHEach receptor sequence has been shown to have different affinities for the four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4 (Bruhns, 1993, Blood 113:3716-3725). Other species have somewhat different numbers and functionalities of FcγRs, with the murine system being the most studied to date and containing four FcγRs: FcγRI, FcγRIIb, FcγRIII, and FcγRIV (Bruhns, 2012, Blood 119:5640-5649). Human FcγRI on cells usually exhibits a similar affinity (approximately 10) for IgG1 / IgG3 / IgG4. -8 Due to the low affinity (approximately 10 M) and the concentration of these IgGs in serum (approximately 10 mg / ml), they are thought to be "occupied" by monomeric IgG under normal serum conditions. Therefore, cells bearing FcγRI on their surface are thought to be able to "screen" or "sample" their antigen environment vicariously by bound multispecific IgG. The lower affinity (approximately 10 M) for IgG subclasses -5 ~10 -7 Other receptors with affinity (M range) are typically considered "unoccupied." Therefore, low-affinity receptors are inherently susceptible to detection and activation by antibody-associated immune complexes. Increased Fc density in antibody immune complexes results in increased functional affinity of binding avidity to low-affinity FcγRs. This has been demonstrated in vitro using several methods (Shields et al., 2001, J Biol Chem 276(9):6591-6604; Lux et al., 2013, J Immunol 190:4315-4323). It has also been suggested to be one of the primary modes of action in the use of anti-RhD to treat ITP in humans (Crow, 2008, Transfusion Medicine Reviews 22:103-116).

[0244] Many cell types express multiple types of FcγR, and therefore, binding of IgG or antibody immune complexes to FcγR-bearing cells can have multiple and complex outcomes depending on the biological context. Most simply, cells can receive either activating, inhibitory, or mixed signals. This can result in events such as phagocytosis (e.g., macrophages and neutrophils), antigen processing (e.g., dendritic cells), reduced IgG production (e.g., B cells), or degranulation (e.g., neutrophils, mast cells). There is data supporting that inhibitory signals from FcγRIIb can dominate those of activating signals (Proulx, 2010, Clinical Immunology 135:422-429).

[0245] There are several useful Fc substitutions that can be made to alter binding to one or more FcγR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, it is known that increased binding to FcγRIIIa generally results in increased ADCC (antibody-dependent cell-mediated cytotoxicity; a cell-mediated reaction in which nonspecific cytotoxic cells that express FcγR recognize bound antibodies on target cells and subsequently cause lysis of the target cells). Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can be beneficial in certain situations as well. Amino acid substitutions utilized in this disclosure include those listed in U.S. Patent Application Publication No. 2006 / 0024298 (particularly FIG. 41), U.S. Patent Application Publication No. 2006 / 0121032, U.S. Patent Application Publication No. 2006 / 0235208, U.S. Patent Application Publication No. 2007 / 0148170, and U.S. Patent Application Publication No. 2019 / 0100587. Specific mutations that may be utilized include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 243A, 243L, 264A, 264V, 299T, 265A / 297A / 329A, 265N / 297D / 329G, and 265E / 297Q / 329S.

[0246] FcRn plays an important role in maintaining the long half-life of IgG in the serum of adults and children: the receptor binds to IgG in acidified vesicles (pH<6.5) to protect the IgG molecule from degradation, and then releases it at the higher pH of 7.4 in the blood.

[0247] FcRn differs from leukocyte Fc receptors and instead shares structural similarity with MHC class I molecules. It is a heterodimer composed of a β2-microglobulin chain noncovalently linked to a membrane-associated chain containing three extracellular domains. One of these domains, containing a carbohydrate chain, interacts with the β2-microglobulin at a site between the CH2 and CH3 domains of Fc. The interaction involves a salt bridge made to a histidine residue on IgG, which is positively charged at pH < 6.5. At higher pHs, the His residues lose their positive charge, weakening the FcRn-IgG interaction and causing the IgG to dissociate.

[0248] In one embodiment, the CD19 binding molecule comprises an Fc domain that binds to human FcRn.

[0249] In one embodiment, the Fc domain has an Fc region (e.g., one or two) that includes a histidine residue at position 310 and, in some cases, also at position 435. These histidine residues are important for human FcRn binding. In one embodiment, the histidine residues at positions 310 and 435 are natural residues, i.e., positions 310 and 435 are unmodified. Alternatively, one or both of these histidine residues may be present as a result of modification.

[0250] A CD19 binding molecule can comprise one or more Fc regions that alter Fc binding to FcRn. The altered binding can be increased or decreased binding.

[0251] In one embodiment, a CD19 binding molecule comprises an Fc domain in which at least one (and optionally both) Fc region comprises one or more modifications such that it binds to FcRn with higher affinity and avidity than the corresponding native immunoglobulin.

[0252] Fc substitutions that increase binding to the FcRn receptor and increase serum half-life are described in U.S. Patent Application Publication No. 2009 / 0163699 and include, but are not limited to, 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I or V / 434S, 436V / 428L, and 259I / 308F / 428L.

[0253] In one embodiment, the Fc region is modified by substituting the threonine residue at position 250 with a glutamine residue (T250Q).

[0254] In one embodiment, the Fc region is modified by substituting the methionine residue at position 252 with a tyrosine residue (M252Y).

[0255] In one embodiment, the Fc region is modified by substituting the serine residue at position 254 with a threonine residue (S254T).

[0256] In one embodiment, the Fc region is modified by substituting the threonine residue at position 256 with a glutamic acid residue (T256E).

[0257] In one embodiment, the Fc region is modified by substituting the threonine residue at position 307 with an alanine residue (T307A).

[0258] In one embodiment, the Fc region is modified by substituting the threonine residue at position 307 with a proline residue (T307P).

[0259] In one embodiment, the Fc region is modified by substituting the valine residue at position 308 with a cysteine ​​residue (V308C).

[0260] In one embodiment, the Fc region is modified by substituting the valine residue at position 308 with a phenylalanine residue (V308F).

[0261] In one embodiment, the Fc region is modified by substituting the valine residue at position 308 with a proline residue (V308P).

[0262] In one embodiment, the Fc region is modified by substituting the glutamine residue at position 311 with an alanine residue (Q311A).

[0263] In one embodiment, the Fc region is modified by substituting the glutamine residue at position 311 with an arginine residue (Q311R).

[0264] In one embodiment, the Fc region is modified by substituting the methionine residue at position 428 with a leucine residue (M428L).

[0265] In one embodiment, the Fc region is modified by substituting the histidine residue at position 433 with a lysine residue (H433K).

[0266] In one embodiment, the Fc region is modified by substituting the asparagine residue at position 434 with a phenylalanine residue (N434F).

[0267] In one embodiment, the Fc region is modified by substituting the asparagine residue at position 434 with a tyrosine residue (N434Y).

[0268] In one embodiment, the Fc region is modified by substituting the methionine residue at position 252 with a tyrosine residue, the serine residue at position 254 with a threonine residue, and the threonine residue at position 256 with a glutamic acid residue (M252Y / S254T / T256E).

[0269] In one embodiment, the Fc region is modified by substituting the valine residue at position 308 with a proline residue and the asparagine residue at position 434 with a tyrosine residue (V308P / N434Y).

[0270] In one embodiment, the Fc region is modified by substituting the methionine residue at position 252 with a tyrosine residue, the serine residue at position 254 with a threonine residue, the threonine residue at position 256 with a glutamic acid residue, the histidine residue at position 433 with a lysine residue, and the asparagine residue at position 434 with a phenylalanine residue (M252Y / S254T / T256E / H433K / N434F).

[0271] It will be appreciated that any of the modifications listed above may be combined to alter FcRn binding.

[0272] In one embodiment, a CD19 binding molecule comprises an Fc domain, wherein one or both Fc regions comprise one or more modifications such that the Fc domain binds to FcRn with lower affinity and avidity than the corresponding native immunoglobulin.

[0273] In one embodiment, the Fc region comprises any amino acid residue at positions 310 and / or 435 other than histidine.

[0274] The CD19 binding molecule can comprise an Fc domain in which one or both Fc regions contain one or more modifications that increase its binding to FcγRIIb, which is the only inhibitory receptor in humans and the only Fc receptor found on B cells.

[0275] In one embodiment, the Fc region is modified by substituting the proline residue at position 238 with an aspartic acid residue (P238D).

[0276] In one embodiment, the Fc region is modified by substituting the glutamic acid residue at position 258 with an alanine residue (E258A).

[0277] In one embodiment, the Fc region is modified by substituting the serine residue at position 267 with an alanine residue (S267A).

[0278] In one embodiment, the Fc region is modified by substituting the serine residue at position 267 with a glutamic acid residue (S267E).

[0279] In one embodiment, the Fc region is modified by substituting the leucine residue at position 328 with a phenylalanine residue (L328F).

[0280] In one embodiment, the Fc region is modified by substituting the glutamic acid residue at position 258 with an alanine residue and the serine residue at position 267 with an alanine residue (E258A / S267A).

[0281] In one embodiment, the Fc region is modified by substituting the serine residue at position 267 with a glutamic acid residue and the leucine residue at position 328 with a phenylalanine residue (S267E / L328F).

[0282] It will be appreciated that any of the modifications listed above may be combined to increase FcγRIIb binding.

[0283] In one embodiment, a CD19 binding molecule is provided that comprises an Fc domain that exhibits reduced binding to FcγR.

[0284] In one embodiment, the CD19 binding molecule comprises an Fc domain in which one or both Fc regions contain one or more modifications that decrease Fc binding to FcγR.

[0285] The Fc domain may be derived from IgG1.

[0286] In one embodiment, the Fc region is modified by substituting the leucine residue at position 234 with an alanine residue (L234A).

[0287] In one embodiment, the Fc region is modified by substituting the leucine residue at position 235 with an alanine residue (L235A).

[0288] In one embodiment, the Fc region is modified by substituting the glycine residue at position 236 with an arginine residue (G236R).

[0289] In one embodiment, the Fc region is modified by substituting the asparagine residue at position 297 with an alanine residue (N297A) or a glutamine residue (N297Q).

[0290] In one embodiment, the Fc region is modified by substituting the serine residue at position 298 with an alanine residue (S298A).

[0291] In one embodiment, the Fc region is modified by substituting the leucine residue at position 328 with an arginine residue (L328R).

[0292] In one embodiment, the Fc region is modified by substituting the leucine residue at position 234 with an alanine residue and the leucine residue at position 235 with an alanine residue (L234A / L235A).

[0293] In embodiments, the Fc region is modified by substituting the phenylalanine residue at position 234 with an alanine residue and the leucine residue at position 235 with an alanine residue (F234A / L235A).

[0294] In one embodiment, the Fc region is modified by substituting the glycine residue at position 236 with an arginine residue and the leucine residue at position 328 with an arginine residue (G236R / L328R).

[0295] In one embodiment, the Fc region is modified by substituting the aspartic acid residue at position 265 with an alanine residue, the asparagine residue at position 297 with an alanine residue, and the proline residue at position 329 with an alanine residue (D265A / N297A / P329A).

[0296] In one embodiment, the Fc region is modified by substituting the aspartic acid residue at position 265 with an asparagine residue, the asparagine residue at position 297 with an aspartic acid residue, and the proline residue at position 329 with a glycine residue (D265N / N297D / P329G).

[0297] In one embodiment, the Fc region is modified by substituting the aspartic acid residue at position 265 with a glutamic acid residue, the asparagine residue at position 297 with a glutamine residue, and the proline residue at position 329 with a serine residue (D265E / N297Q / P329S).

[0298] It will be appreciated that any of the modifications listed above may be combined to reduce FcγR binding.

[0299] In one embodiment, the CD19 binding molecule comprises an Fc domain in which one or both Fc regions contain one or more modifications that decrease Fc binding to FcγRIIIa without affecting Fc binding to FcγRII.

[0300] In one embodiment, the Fc region is modified by substituting the serine residue at position 239 with an alanine residue (S239A).

[0301] In one embodiment, the Fc region is modified by substituting the glutamic acid residue at position 269 with an alanine residue (E269A).

[0302] In one embodiment, the Fc region is modified by substituting the glutamic acid residue at position 293 with an alanine residue (E293A).

[0303] In one embodiment, the Fc region is modified by substituting the tyrosine residue at position 296 with a phenylalanine residue (Y296F).

[0304] In one embodiment, the Fc region is modified by substituting the valine residue at position 303 with an alanine residue (V303A).

[0305] In one embodiment, the Fc region is modified by substituting the alanine residue at position 327 with a glycine residue (A327G).

[0306] In one embodiment, the Fc region is modified by substituting the lysine residue at position 338 with an alanine residue (K338A).

[0307] In one embodiment, the Fc region is modified by substituting the aspartic acid residue at position 376 with an alanine residue (D376A).

[0308] It will be appreciated that any of the modifications listed above may be combined to reduce FcγRIIIa binding.

[0309] Fc region mutations with reduced FcR binding may be referred to as "FcγR depletion mutations," "FcγR silencing mutations," or "Fc knockout (FcKO or KO)" mutations. In certain therapeutic applications, it is desirable to reduce or eliminate normal binding of the Fc domain to one or more or all of the Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa) to avoid additional mechanisms of action. That is, for example, in many embodiments, particularly in the use of MBMs that bind monovalently to CD3, it is generally desirable to eliminate FcγRIIIa binding to eliminate or substantially reduce ADCC activity. In certain embodiments, at least one of the Fc regions of the MBMs described herein comprises one or more Fcγ receptor depletion mutations. In certain embodiments, both Fc regions comprise one or more Fcγ receptor depletion mutations. These deletion mutations are shown in Table 3, and each can be independently and optionally included or excluded. Some embodiments include G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K ... Deletion mutations are used that are selected from the group consisting of 34V / L235A / G236del / S267K / A327G, E233P / L234V / L235A / G236del, D265A / N297A / P329A, D265N / N297D / P329G, and D265E / N297Q / P329S (where "del" indicates a deletion, e.g., G236del refers to the deletion of glycine at position 236). It should be noted that the deletion mutations referred to herein eliminate FcγR binding, but generally do not eliminate FcRn binding.

[0310] [Table 15]

[0311] In some embodiments, the MBMs of the present disclosure comprise a first Fc region and a second Fc region, hi some embodiments, the first Fc region and / or the second Fc region may comprise the following mutations: E233P, L234V, L235A, G236del, and S267K.

[0312] The Fc domain of human IgG1 has the highest binding to Fcγ receptors, therefore, when the constant domain (or Fc domain) in the framework of the heterodimeric antibody is IgG1, deletion mutations can be used.

[0313] Alternatively, or in addition to deleting mutations in an IgG1 background, mutations at glycosylation position 297, e.g., substituting the asparagine residue at position 297 with an alanine residue (N297A) or glutamine residue (N297Q), can significantly eliminate binding to, for example, FcγRIIIa. Human IgG2 and IgG4 have naturally reduced binding to Fcγ receptors, and therefore these backbones can be used with or without deleting mutations.

[0314] 7.4.1.2. Fc Domain with Altered Complement Binding A CD19 binding molecule can comprise an Fc domain in which one or both Fc regions contain one or more modifications that alter Fc binding to complement. The altered complement binding can be increased or decreased binding.

[0315] In one embodiment, the Fc region comprises one or more modifications that reduce its binding to C1q. Initiation of the classical complement pathway begins with the binding of the hexameric C1q protein to the CH2 domain of antigen-bound IgG and IgM.

[0316] In one embodiment, the CD19 binding molecule comprises an Fc domain in which one or both Fc regions contain one or more modifications that reduce Fc binding to C1q.

[0317] In one embodiment, the Fc region is modified by substituting the leucine residue at position 234 with an alanine residue (L234A).

[0318] In one embodiment, the Fc region is modified by substituting the leucine residue at position 235 with an alanine residue (L235A).

[0319] In one embodiment, the Fc region is modified by substituting the leucine residue at position 235 with a glutamic acid residue (L235E).

[0320] In one embodiment, the Fc region is modified by substituting the glycine residue at position 237 with an alanine residue (G237A).

[0321] In one embodiment, the Fc region is modified by substituting the lysine residue at position 322 with an alanine residue (K322A).

[0322] In one embodiment, the Fc region is modified by substituting the proline residue at position 331 with an alanine residue (P331A).

[0323] In one embodiment, the Fc region is modified by substituting the proline residue at position 331 with a serine residue (P331S).

[0324] In one embodiment, the CD19-binding molecule comprises an Fc domain derived from IgG4. IgG4 naturally has a lower complement activation profile than IgG1, as well as weaker FcγR binding. Thus, in one embodiment, the CD19-binding molecule comprises an IgG4 Fc domain and also contains one or more modifications that increase FcγR binding.

[0325] It will be appreciated that any of the modifications listed above may be combined to reduce C1q binding.

[0326] 7.4.1.3. Fc Domains with Engineered Disulfide Structures A CD19 binding molecule may comprise an Fc domain containing one or more modifications to create and / or remove cysteine ​​residues. Cysteine ​​residues play an important role in the spontaneous assembly of Fc-based multispecific binding molecules by forming disulfide bridges between individual pairs of polypeptide monomers. Thus, by altering the number and / or location of cysteine ​​residues, it is possible to modify the structure of a CD19 binding molecule to produce a protein with improved therapeutic properties.

[0327] A CD19 binding molecule of the disclosure can comprise an Fc domain in which one or both Fc regions, e.g., both Fc regions, comprise a cysteine ​​residue at position 309. In one embodiment, the cysteine ​​residue at position 309 is generated by modification, e.g., in the case of an Fc domain derived from IgG1, a leucine residue at position 309 is replaced with a cysteine ​​residue (L309C), or in the case of an Fc domain derived from IgG2, a valine residue at position 309 is replaced with a cysteine ​​residue (V309C).

[0328] In one embodiment, the Fc region is modified by substituting the valine residue at position 308 with a cysteine ​​residue (V308C).

[0329] In one embodiment, two disulfide bonds in the hinge region are eliminated by mutating the core hinge sequence CPPC (SEQ ID NO: 55) to SPPS (SEQ ID NO: 56).

[0330] 7.4.1.4. Fc Domains with Altered Glycosylation In certain embodiments, CD19 binding molecules are provided that contain fewer glycosylation sites than the corresponding immunoglobulins, resulting in improved manufacturability. These proteins have simpler post-translational glycosylation patterns and are therefore simpler and cheaper to produce.

[0331] In one embodiment, a glycosylation site in the CH2 domain is eliminated by substituting the asparagine residue at position 297 with an alanine residue (N297A) or a glutamine residue (N297Q). In addition to improved manufacturability, these glycosyl mutants also reduce FcγR binding as described herein above.

[0332] In certain embodiments, CD19-binding molecules can be generated with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to improve the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the CD19-binding molecule in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to internally express CD19-binding molecules, thereby producing CD19-binding molecules with altered glycosylation. For example, European Patent No. 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation. PCT Publication WO 03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, and also results in hypofucosylation of antibodies expressed in the corresponding host cells (see also Shields et al., 2002, J. Biol. Chem. 277:26733-26740). Umana et al., PCT Publication WO 99 / 54342, describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines display increased bisecting GlcNac structures, which result in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0333] Fc Heterodimerization Many multispecific molecule formats, unlike native immunoglobulins, involve dimerization between two Fc regions that are operably linked to non-identical antigen-binding domains (or portions thereof, e.g., VH or VH-CH1 of a Fab). Inefficient heterodimerization of two Fc regions to form an Fc domain has always been an obstacle to increasing the yield of the desired multispecific molecules and presents a challenging purification problem. Various techniques available in the art can be used to promote dimerization of Fc regions that may be present in CD19 binding molecules (and particularly the MBMs of the present disclosure), as disclosed, for example, in European Patent Application Publication No. 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO 2009 / 089004A1.

[0334] The present disclosure provides Fc heterodimers, i.e., CD19-binding molecules comprising Fc domains comprising heterologous, non-identical Fc regions. Heterodimerization techniques are used to promote dimerization of Fc regions operably linked to different ABMs (or portions thereof, e.g., VH or VH-CH1 of Fabs) and reduce dimerization of Fc regions operably linked to the same ABM or portion thereof. Typically, each Fc region in an Fc heterodimer comprises an antibody CH3 domain. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, in some cases the IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the previous section.

[0335] Typically, MBMs contain, in addition to a CH3 domain, a CH1 domain, a CH2 domain, a hinge region, a VH domain, a VL domain, CDRs, and / or other antibody fragments, such as antigen-binding fragments described herein. In certain embodiments, the two heteropolypeptides are two heavy chains that form a bispecific or multispecific molecule. Heterodimerization of two different heavy chains at the CH3 domain generates the desired antibody or antibody-like molecule, while homodimerization of identical heavy chains reduces the yield of the desired antibody or molecule. In exemplary embodiments, the two or more heteropolypeptide chains comprise two chains that comprise a CH3 domain and form any of the multispecific molecule formats described above in this disclosure. In one embodiment, the two heteropolypeptide chains that comprise a CH3 domain contain a modification that favors heterodimeric association of the polypeptides compared to the unmodified chains. Various examples of modification approaches are provided in Table 4 below and in Sections 7.4.1.5.1-7.4.1.5.7.

[0336] [Table 16]

[0337] [Table 17]

[0338] [Table 18]

[0339] [Table 19]

[0340] [Table 20]

[0341] [Table 21]

[0342] Table 22

[0343] Table 23

[0344] Table 24

[0345] Table 25

[0346] Table 26

[0347] Table 27

[0348] Table 28

[0349] Table 29

[0350] Table 30

[0351] Table 31

[0352] [Table 32]

[0353] [Table 33]

[0354] [Table 34]

[0355] [Table 35]

[0356] [Table 36]

[0357] Exemplary pairs of non-identical heterologous Fc sequences that can be paired to form Fc heterodimers and that can be included in the CD19 binding molecules of the present disclosure include (i) SEQ ID NO: 1106 and SEQ ID NO: 1107, and (ii) SEQ ID NO: 1106 and SEQ ID NO: 1108. [ka] An Fc region having the amino acid sequence of one of SEQ ID NOs: 1106-1108 can be modified to include one or more of the substitutions described in Section 7.4.1 (including subportions thereof), e.g., to include one or more substitutions corresponding to the deletion variants described in Table 3. In some embodiments, a CD19 binding molecule comprises an Fc region having the amino acid sequence of one of SEQ ID NOs: 1106-1108 with a mutation at one, two, three, four, five, six, or more than six of positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332 (EU numbering), e.g., one or more mutations described in Section 7.4.1 (including subportions thereof). For example, the CD19 binding molecule can comprise an Fc region having the amino acid sequence of SEQ ID NO: 1106 with one, two, three, four, five, six, or more than six mutations at positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331, and 332, and / or , 329, 330, 331 and 332 and / or an Fc region having the amino acid sequence of SEQ ID NO: 1108 with mutations at 1, 2, 3, 4, 5, 6 or more of positions 233, 234, 235, 236, 237, 239, 265, 266, 267, 268, 269, 297, 299, 322, 327, 328, 329, 330, 331 and 332.

[0358] 7.4.1.5.1. Stereomutation A CD19 binding molecule may include one or more, e.g., multiple, modifications to one or more of the constant domains of the Fc domain, e.g., the CH3 domain. In one example, a CD19 binding molecule of the present disclosure includes two polypeptides each including an antibody heavy chain constant domain, e.g., the CH2 or CH3 domain. In one example, the two heavy chain constant domains, e.g., the CH2 or CH3 domain, of a CD19 binding molecule include one or more modifications that enable heterodimeric association between the two chains. In one embodiment, one or more modifications are located on the CH2 domains of the two heavy chains. In one embodiment, one or more modifications are located on the CH3 domains of at least two polypeptides of the CD19 binding molecule.

[0359] One mechanism for Fc heterodimerization is commonly referred to as "knobs and holes" or "knobs-into-holes." These terms refer to amino acid mutations that result in steric effects that favor the formation of Fc heterodimers over Fc homodimers, as described, for example, in Ridgway et al., 1996, Protein Engineering 9(7):617; Atwell et al., 1997, J. Mol. Biol. 270:26; and U.S. Patent No. 8,216,805. Knobs-in-hole mutations can be combined with other approaches to improve heterodimerization.

[0360] In one embodiment, one or more modifications to a first polypeptide of a CD19 binding molecule comprising a heavy chain constant domain can generate a "knob," and one or more modifications to a second polypeptide of the CD19 binding molecule can generate a "hole," such that heterodimerization of the polypeptides of the CD19 binding molecule comprising a heavy chain constant domain causes the "knob" to associate with the "hole" (e.g., interact, e.g., the CH2 domain of the first polypeptide interacts with the CH2 domain of the second polypeptide, or the CH3 domain of the first polypeptide interacts with the CH3 domain of the second polypeptide). The knob can be positioned in a complementary "hole" at the interface of the second polypeptide of the CD19 binding molecule comprising a heavy chain constant domain so as to protrude from the interface of the first polypeptide of the CD19 binding molecule comprising a heavy chain constant domain, thereby stabilizing the heteromultimer and thereby favoring, for example, heteromultimer formation over homomultimer formation. The knob can be present at the original interface or can be synthetically introduced (e.g., by modifying the nucleic acid encoding the interface). The import residue for forming the knob is generally a natural amino acid residue and may be selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some cases, tryptophan and tyrosine are selected. In embodiments, the original residue for forming the protrusion has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0361] The "hole" is recessed from the boundary of the second polypeptide of a CD19-binding molecule comprising a heavy chain constant domain and thus contains at least one amino acid side chain that fits into a corresponding knob on the adjacent interaction surface of the first polypeptide of a CD19-binding molecule comprising a heavy chain constant domain. The hole can be present in the original boundary or can be synthetically introduced (e.g., by modifying the nucleic acid encoding the boundary). The import residue for hole formation is typically a natural amino acid residue, and in some embodiments is selected from alanine (A), serine (S), threonine (T), and valine (V). In one embodiment, the amino acid residue is serine, alanine, or threonine. In another embodiment, the original residue for hole formation has a large side chain capacity, such as tyrosine, arginine, phenylalanine, or tryptophan.

[0362] In embodiments, the first CH3 domain is modified at residue 366, 405, or 407 to generate either a "knob" or a "hole" (as described above), and the second CH3 domain that heterodimerizes with the first CH3 domain is modified at residue 366 if residue 366 is modified in the first CH3 domain, or at residue 407 if residue 405 is modified in the first CH3 domain, or at residue 366 if residue 394 or residue 407 is modified in the first CH3 domain to generate a "hole" or a "knob" complementary to the "knob" or "hole" of the first CH3 domain.

[0363] In another embodiment, the first CH3 domain is modified at residue 366, and the second CH3 domain that heterodimerizes with the first CH3 domain is modified at residues 366, 368, and / or 407 to create a "hole" or "knob" complementary to the "knob" or "hole" of the first CH3 domain. In one embodiment, the modification to the first CH3 domain introduces a tyrosine (Y) residue at position 366. In one embodiment, the modification to the first CH3 domain is T366Y. In one embodiment, the modification to the first CH3 domain introduces a tryptophan (W) residue at position 366. In one embodiment, the modification to the first CH3 domain is T366W. In certain embodiments, modifications to a second CH3 domain that heterodimerizes with a first CH3 domain modified at position 366 (e.g., comprising the modifications T366Y or T366W, e.g., having a tyrosine (Y) or tryptophan (W) introduced at position 366) comprise a modification at position 366, a modification at position 368, and a modification at position 407. In certain embodiments, the modification at position 366 introduces a serine (S) residue, the modification at position 368 introduces an alanine (A), and the modification at position 407 introduces a valine (V). In certain embodiments, the modifications comprise T366S, L368A, and Y407V. In one embodiment, the first CH3 domain of the multispecific molecule comprises the modification T366Y and the second CH3 domain that heterodimerizes with the first CH3 domain comprises the modifications T366S, L368A, and Y407V, or vice versa. In one embodiment, the first CH3 domain of the multispecific molecule comprises the modification T366W and the second CH3 domain that heterodimerizes with the first CH3 domain comprises the modifications T366S, L368A and Y407V, or vice versa.

[0364] Additional steric or "distortion" (e.g., knobs-in-holes) modifications are described in PCT Publication No. WO 2014 / 145806 (e.g., Figures 3, 4, and 12 of WO 2014 / 145806), PCT Publication No. WO 2014 / 110601, and PCT Publication Nos. WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, and WO 2016 / 182751. An example of a KIH mutant comprises a first constant chain comprising L368D and K370S modifications paired with a second constant chain comprising S364K and E357Q modifications.

[0365] Additional knobs-into-hole modification pairs suitable for use in any of the CD19 binding molecules of the present disclosure are further described, for example, in WO 1996 / 027011 and Merchant et al., 1998, Nat. Biotechnol., 16:677-681.

[0366] In a further embodiment, the CH3 domain can be further modified to introduce a pair of cysteine ​​residues. Without being bound by theory, it is believed that the introduction of a pair of cysteine ​​residues capable of forming a disulfide bond confers stability to a heterodimerized CD19-binding molecule, e.g., MBM, comprising the paired CH3 domains. In one embodiment, the first CH3 domain comprises a cysteine ​​at position 354, and the second CH3 domain that heterodimerizes with the first CH3 domain comprises a cysteine ​​at position 349. In one embodiment, the first CH3 domain comprises a cysteine ​​at position 354 (e.g., with the modification S354C) and a tyrosine (Y) at position 366 (e.g., with the modification T366Y), and the second CH3 domain that heterodimerizes with the first CH3 domain comprises a cysteine ​​at position 349 (e.g., with the modification Y349C), a serine at position 366 (e.g., with the modification T366S), an alanine at position 368 (e.g., with the modification L368A), and a valine at position 407 (e.g., with the modification Y407V). In one embodiment, the first CH3 domain comprises a cysteine ​​at position 354 (e.g., with the modification S354C) and a tryptophan (W) at position 366 (e.g., with the modification T366W), and the second CH3 domain that heterodimerizes with the first CH3 domain comprises a cysteine ​​at position 349 (e.g., with the modification Y349C), a serine at position 366 (e.g., with the modification T366S), an alanine at position 368 (e.g., with the modification L368A), and a valine at position 407 (e.g., with the modification Y407V).

[0367] An additional mechanism utilized to generate heterodimers is sometimes referred to as "electrostatic steering," as described in Gunasekaran et al., 2010, J. Biol. Chem. 285(25):19637. This is sometimes referred to herein as "charge pairing." In this embodiment, electrostatics are used to skew formation toward heterodimerization. As one skilled in the art will appreciate, this may affect pI and therefore purification, and therefore may in some cases be considered pI mutations. However, because they were generated to promote heterodimerization and were not used as a purification tool, they are classified as "stereotypic mutations." These include, but are not limited to, D221E / P228E / L368E paired with D221R / P228R / K409R and C220E / P228E / 368E paired with C220R / E224R / P228R / K409R.

[0368] The additional mutations can be optionally and independently combined in any amount with other mutations, such as the pI mutations outlined herein or other conformational mutations shown in Figure 37 of US Patent Application Publication No. 2012 / 0149876.

[0369] In certain embodiments, the conformational mutations outlined herein can optionally and independently incorporate any pI mutations (or other mutations such as Fc mutations, FcRn mutations, etc.) in one or both Fc regions and can independently and optionally be included or excluded from the CD19 binding molecules of the present disclosure.

[0370] A list of suitable distortion mutations is found in Table 5, which shows some pairs that are particularly useful in many embodiments. Particularly useful in many embodiments are pairs of sets including, but not limited to, S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; and K370S:S364K / E357Q. In terms of nomenclature, the pair "S364K / E357Q:L368D / K370S" means that one of the Fc regions has the double mutation set S364K / E357Q and the other has the double mutation set L368D / K370S.

[0371] [Table 37]

[0372] [Table 38]

[0373] [Table 39]

[0374] [Table 40]

[0375] [Table 41]

[0376] [Table 42]

[0377] In some embodiments, the CD19 binding molecule comprises a first Fc region and a second Fc region. In some embodiments, the first Fc region comprises the following mutations: L368D and K370S, and the second Fc region comprises the following mutations: S364K and E357Q. In some embodiments, the first Fc region comprises the following mutations: S364K and E357Q, and the second Fc region comprises the following mutations: L368D and K370S.

[0378] 7.4.1.5.2. Alternative Knobs and Holes: IgG Heterodimerization Heterodimerization of polypeptide chains of CD19 binding molecules comprising paired CH3 domains can be increased by introducing one or more modifications into the CH3 domains derived from an IgG1 antibody class. In one embodiment, the modifications comprise a K409R modification to one CH3 domain paired with a F405L modification in the second CH3 domain. Additional modifications can also or alternatively be at positions 366, 368, 370, 399, 405, 407, and 409. In some cases, heterodimerization of polypeptides comprising such modifications is performed under reducing conditions, e.g., at 25-37°C, e.g., at 25°C or 37°C, for 1-10 hours, e.g., 1.5-5 hours, e.g., 5 hours, in 10-100 mM 2-MEA (e.g., 25, 50, or 100 mM 2-MEA).

[0379] The amino acid substitutions described herein can be introduced into the CH3 domain using well-known techniques (see, e.g., McPherson, ed., 1991, Directed Mutagenesis: a Practical Approach; Adelman et al., 1983, DNA, 2:183).

[0380] IgG heterodimerization techniques are further described, for example, in WO 2008 / 119353, WO 2011 / 131746 and WO 2013 / 060867.

[0381] In any of the embodiments described in this section, the CH3 domain may be further modified to introduce a pair of cysteine ​​residues, as described in Section 7.4.1.3.

[0382] 7.4.1.5.3.pI (isoelectric point) mutation In general, as will be understood by those skilled in the art, there are two general categories of pI mutations: those that increase the pI of a protein (basic changes) and those that decrease the pI of a protein (acidic changes). As described herein, all combinations of these mutations can be made: one Fc region can be wild-type or a variant that does not exhibit a pI that is significantly different from wild-type, and the other can be either more basic or more acidic. Alternatively, each Fc region can be altered, one more basic and one more acidic.

[0383] Exemplary combinations of pI mutations are shown in Table 6. Although these changes are shown relative to IgG1 as outlined herein and shown in Table 6, all isotypes can be modified in this manner, as can isotype hybrids. R133E and R133Q can also be used when the heavy chain constant domain is derived from IgG2-4.

[0384] [Table 43]

[0385] In one embodiment, e.g., in the formats of Figures 1B-1W, 1Y-1AH, 2B-2L, and 2N-2V, the combination of pI mutations has one Fc region (negative Fab side) containing the 208D / 295E / 384D / 418E / 421D mutations (N208D / Q295E / N384D / Q418E / N421D when compared to human IgG1) and a second Fc region (positive scFv side) containing a positively charged scFv linker, e.g., L36 (as described in Section 7.4.3). However, as will be understood by those skilled in the art, the first Fc region contains a CH1 domain comprising position 208. Thus, in a construct that does not include a CH1 domain (e.g., for an MBM that does not utilize a CH1 domain as one of its domains, e.g., in the format shown in Figure 2K), an exemplary negative pI mutated Fc set can include 295E / 384D / 418E / 421D mutations (Q295E / N384D / Q418E / N421D when compared to human IgG1).

[0386] In one embodiment, the first Fc region has a set of substitutions from Table 6, and the second Fc region is linked to a charged linker (eg, selected from those described in Section 7.4.3).

[0387] In some embodiments, a CD19 binding molecule of the present disclosure comprises a first Fc region and a second Fc region. In some embodiments, the first Fc region comprises the following mutations: N208D, Q295E, N384D, Q418E, and N421D. In some embodiments, the second Fc region comprises the following mutations: N208D, Q295E, N384D, Q418E, and N421D.

[0388] Isotype Variation Furthermore, many embodiments of the present disclosure rely on the "incorporation" of pI amino acids at specific positions from one IgG isotype to another, thus reducing or eliminating the possibility of introducing undesirable immunogenicity into the variant. Some of these are shown in Figure 21 of U.S. Patent Application Publication No. 2014 / 0370013. Specifically, IgG1 is a common isotype for therapeutic antibodies for a variety of reasons, including high effector function. However, the heavy chain constant region of IgG1 has a higher pI than that of IgG2 (8.10 vs. 7.31). By introducing IgG2 residues into the IgG1 backbone at specific positions, the pI of the resulting Fc region is lowered (or increased), further exhibiting a longer serum half-life. For example, IgG1 has a glycine at position 137 (pI 5.97), while IgG2 has a glutamic acid (pI 3.22); incorporating glutamic acid affects the pI of the resulting protein. As described below, several amino acid substitutions are generally required to significantly affect the pI of the mutant antibody. However, it should be noted that even changes in the IgG2 molecule can allow for increased serum half-life, as described below.

[0389] In other embodiments, non-isotypic amino acid changes are made to reduce the overall charge state of the resulting protein (e.g., by changing from higher pI amino acids to lower pI amino acids) or to allow for structural tuning for stability, as further described below.

[0390] Furthermore, by pI engineering both the heavy and light chain constant domains of a CD19 binding molecule comprising two half antibodies, significant changes in each half antibody can be observed, with the pI of the two half antibodies differing by at least 0.5, which may allow for separation by ion exchange chromatography or isoelectric focusing or other methods sensitive to isoelectric point.

[0391] 7.4.1.5.5. Calculation of pI The pI of a half antibody comprising an Fc region and an ABM or ABM chain can depend on the pI of the variant heavy chain constant domain and the pI of the entire half antibody comprising the variant heavy chain constant domain and the ABM or ABM chain. Thus, in one embodiment, the change in pI is calculated based on the variant heavy chain constant domain using the chart in Figure 19 of U.S. Patent Application Publication No. 2014 / 0370013. As described herein, which half antibody to engineer is generally determined by the intrinsic pI of the half antibody. Alternatively, the pI of each half antibody can be compared.

[0392] 7.4.1.5.6. pI Mutations That Also Confer Good FcRn Binding in Vivo If the pI mutation decreases the pI of the Fc region, it may have the added benefit of improving serum retention in vivo.

[0393] Because Fc is sequestered by binding to FcRn at pH 6 in endosomes, pI-mutated Fc regions are thought to confer a longer half-life to antigen-binding molecules in vivo (Ghetie and Ward, 1997, Immunol Today. 18(12):592-598). The endosomal compartment then recycles Fc to the cell surface. Once the compartment opens to the extracellular space, a higher pH of approximately 7.4 induces Fc release into the blood. In mice, Dall' Acqua et al. showed that Fc mutants with increased FcRn binding at pH 6 and pH 7.4 actually had reduced serum concentrations and the same half-life as wild-type Fc (Dall' Acqua et al. 2002, J. Immunol. 169:5171-5180). The increased affinity of Fc for FcRn at pH 7.4 is thought to prevent Fc release into the blood. Therefore, Fc mutations that increase the in vivo half-life of Fc would ideally increase FcRn binding at lower pH while still allowing Fc release at higher pH. The amino acid histidine changes its charge state in the pH range of 6.0 to 7.4. Therefore, it is not surprising to find His residues at key positions in the Fc / FcRn complex.

[0394] It has been suggested that antibodies with variable regions with lower isoelectric points may also have longer serum half-lives (Igawa et al., 2010, PEDS. 23(5):385-392). However, this mechanism remains poorly understood. Furthermore, variable regions vary from antibody to antibody. Constant region variants with reduced pI and extended half-lives, as described herein, would provide a more modular approach to improving the pharmacokinetic properties of CD19 binding molecules.

[0395] 7.4.1.5.7.Polar crosslinking Heterodimerization of polypeptide chains of CD19-binding molecules containing an Fc domain, such as MBM, can be increased by introducing modifications based on the principle of "polar bridges," which cause residues at the binding interface of two polypeptide chains to interact with residues of similar (or complementary) physical properties in the heterodimeric form, but with residues of different physical properties in the homodimeric form. In particular, these modifications are designed so that polar residues interact with polar residues, while hydrophobic residues interact with hydrophobic residues, in heterodimer formation. In contrast, in homodimer formation, residues are modified so that polar residues interact with hydrophobic residues. The favorable interactions in the heterodimeric form and the unfavorable interactions in the homodimeric form combine to make the Fc region more likely to form heterodimers than homodimers.

[0396] In exemplary embodiments, the modifications are made at one or more of residues 364, 368, 399, 405, 409, and 411 of the CH3 domain.

[0397] In some embodiments, one or more modifications selected from the group consisting of S364L, T366V, L368Q, N399K, F405S, K409F, and R411K are introduced into one of the two CH3 domains. One or more modifications selected from the group consisting of Y407F, K409Q, and T411N may be introduced into the second CH3 domain.

[0398] In another embodiment, one or more modifications selected from the group consisting of S364L, T366V, L368Q, D399K, F405S, K409F and T411K are introduced into one CH3 domain, while one or more modifications selected from the group consisting of Y407F, K409Q and T411D are introduced into a second CH3 domain.

[0399] In one exemplary embodiment, the original threonine residue at position 366 in one CH3 domain is substituted with a valine, while the original tyrosine residue at position 407 in the other CH3 domain is substituted with a phenylalanine.

[0400] In another exemplary embodiment, the original serine residue at position 364 of one CH3 domain is substituted with leucine, while the original leucine residue at position 368 of the same CH3 domain is substituted with glutamine.

[0401] In yet another exemplary embodiment, the original phenylalanine residue at position 405 of one CH3 domain is substituted with serine and the original lysine residue at position 409 of this CH3 domain is substituted with phenylalanine, while the original lysine residue at position 409 of the other CH3 domain is substituted with glutamine.

[0402] In yet another exemplary embodiment, the original aspartic acid residue at position 399 in one CH3 domain is substituted with lysine and the original threonine residue at position 411 in the same CH3 domain is substituted with lysine, while the original threonine residue at position 411 in the other CH3 domain is substituted with aspartic acid.

[0403] The amino acid substitutions described herein can be introduced into the CH3 domain using well-known techniques (see, for example, McPherson, ed., 1991, Directed Mutagenesis: a Practical Approach; Adelman et al., 1983, DNA, 2:183). Polar cross-linking techniques are described, for example, in WO 2006 / 106905, WO 2009 / 089004, and Gunasekaran et al., 2010, JBC 285:19637-19646.

[0404] Further polar bridge modifications are described, for example, in PCT Publication No. WO 2014 / 145806 (e.g., Figure 6 of WO 2014 / 145806), PCT Publication No. WO 2014 / 110601, and PCT Publication Nos. WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, and WO 2016 / 182751. Examples of polar bridge variants include constant chains containing N208D, Q295E, N384D, Q418E, and N421D modifications.

[0405] In any of the embodiments described herein, the CH3 domain may be further modified to introduce a pair of cysteine ​​residues, as described in Section 7.4.1.3.

[0406] Further techniques for promoting heterodimerization are described, for example, in WO 2016 / 105450, WO 2016 / 086186, WO 2016 / 086189, WO 2016 / 086196, WO 2016 / 141378, and WO 2014 / 145806, and WO 2014 / 110601. Any of these techniques may be used with the CD19 binding molecules described herein.

[0407] 7.4.1.6. Combinations of Heterodimerization Mutations and Other Fc Mutations As will be appreciated by those skilled in the art, all of the listed heterodimerization mutations (including distortions and / or pI mutations) can be optionally and independently combined in any way, so long as the Fc regions of the Fc domains retain their ability to dimerize. Furthermore, all of these mutations can be combined into any heterodimerization format.

[0408] In the case of pI mutations, particularly useful embodiments are shown in Table 6, but other combinations can be generated following the basic rule of altering the pI difference between the two Fc regions in an Fc heterodimer to facilitate purification.

[0409] Additionally, any of the heterodimerization mutations, distortions and pI may be independently and optionally combined with Fc ablation mutations, Fc mutations, FcRn mutations, as generally outlined herein.

[0410] In one embodiment, the particular combination of distortion and pI mutations utilized in the present disclosure is T366S / L368A / Y407V:T366W (optionally including the bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C), with one Fc region comprising Q295E / N384D / Q418E / N481D and the other comprising a positively charged scFv linker (if the format comprises an scFv domain). As will be appreciated by those skilled in the art, "knobs-in-holes" mutations do not alter the pI and therefore can be used on either Fc region in an Fc heterodimer.

[0411] In one embodiment, the first and second Fc regions utilized in the present disclosure comprise the amino acid substitutions S364K / E357Q:L368D / K370S, wherein the first and / or second Fc region comprises the deletion mutant substitutions 233P / L234V / L235A / G236del / S267K, and the first and / or second Fc region comprises the pI mutant substitutions N208D / Q295E / N384D / Q418E / N421D (pl_(-)_isosteric_A).

[0412] Hinge Area A CD19 binding molecule may also include, for example, a hinge region that links the antigen-binding domain to the Fc region. The hinge region may be a natural or modified hinge region. The hinge region is typically found at the N-terminus of the Fc region.

[0413] A native hinge region is the hinge region normally found between the Fab and Fc domains in a natural antibody. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges may include hinge regions derived from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat hinge regions. Other modified hinge regions may include a complete hinge region derived from an antibody of a different class or subclass than that of the heavy chain Fc region. Alternatively, a modified hinge region may include a portion of a native hinge or a repeating unit in which each unit in the repeat is derived from a native hinge region. In a further alternative, the native hinge region may be modified by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting appropriately placed residues to cysteine ​​residues. By such means, the number of cysteine ​​residues in the hinge region may be increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. Altering the number of cysteine ​​residues in the hinge region can, for example, facilitate assembly of the light and heavy chains or increase or decrease the stability of the CD19 binding molecule. Other modified hinge regions can be entirely synthetic and designed to have desired properties, such as length, cysteine ​​composition, and flexibility.

[0414] Several modified hinge regions are described, for example, in U.S. Pat. No. 5,677,425, WO 9915549, WO 2005003170, WO 2005003169, WO 2005003170, WO 9825971 and WO 2005003171.

[0415] Examples of suitable hinge sequences are shown in Table 7.

[0416] [Table 44]

[0417] In one embodiment, the heavy chain Fc region has an intact hinge region at its N-terminus.

[0418] In one embodiment, the heavy chain Fc region and hinge region are derived from IgG4, and the hinge region contains the modified sequence CPPC (SEQ ID NO: 55). The core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 65) compared to IgG1, which contains the sequence CPPC (SEQ ID NO: 55). The serine residues present in the IgG4 sequence provide increased flexibility in this region, and therefore some of the molecules form disulfide bonds within the same protein chain (intrachain disulfides) rather than cross-linking to other heavy chains in the IgG molecule to form interchain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108). Changing the serine residues to proline to obtain the same core sequence as IgG1 allows complete formation of interchain disulfides in the IgG4 hinge region, thus reducing heterogeneity of the purified product. This modified isotype is called IgG4P.

[0419] ABM Linker In certain embodiments, the present disclosure provides CD19 binding molecules in which two or more components of an ABM (e.g., the VH and VL of an scFv), two or more ABMs, or an ABM and a non-ABM domain (e.g., a dimerization domain such as an Fc region) are linked to each other by a peptide linker. Such linkers are referred to herein as "ABM linkers."

[0420] Peptide linkers can range from 2 amino acids to 60 or more amino acids, and in certain aspects, peptide linkers range from 3 amino acids to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, or 12 to 20 amino acids. In certain embodiments, peptide linkers are 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, 40 amino acids, 41 amino acids, 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 ​​amino acids, 49 amino acids or 50 amino acids in length.

[0421] Charged and / or flexible linkers may be used.

[0422] Examples of flexible ABM linkers that can be used in CD19 binding molecules include those disclosed by Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. A particularly useful flexible linker is (GGGGS)n (also referred to as (GS)n) (SEQ ID NO: 78). In certain embodiments, n is any number from 1 to 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or any range bounded by any two of the above numbers, such as 1-5, 2-5, 3-6, 2-4, 1-4, etc., and so forth.

[0423] Other examples of ABM linkers suitable for use in the CD19 binding molecules of the present disclosure are shown in Table 8 below.

[0424] [Table 45]

[0425] [Table 46]

[0426] In various aspects, the present disclosure provides CD19 binding molecules comprising one or more ABM linkers. Each ABM linker can range from 2 amino acids to 60 amino acids in length, e.g., 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, or 12 to 20 amino acids in length, optionally selected from Table 8 above. In specific embodiments, the CD19 binding molecule comprises two, three, four, five, or six ABM linkers. The ABM linkers can be on one, two, three, four, or even more polypeptide chains of the CD19 binding molecule.

[0427] 7.5. Bispecific binding molecular forms An exemplary BBM configuration is shown in FIG. 1. FIG. 1A shows the components of the BBM configuration shown in FIGS. 1B-1AH. The scFv, Fab, scFab, non-immunoglobulin-based ABM, and Fc domain can have the properties described for these components in Sections 7.3 and 7.4, respectively. The components of the BBM configuration shown in FIG. 1 can be associated with each other by any of the means described in Sections 7.3 and 7.4 (e.g., by direct bonding, ABM linkers, disulfide bonds, Fc domains modified with knob-in-hole interactions, etc.). The orientation and association of the various components shown in FIG. 1 are exemplary only; as will be understood by one of skill in the art, other orientations and associations may be suitable (e.g., as described in Sections 7.3 and 7.4).

[0428] BBM is not limited to the form shown in Figure 1. Other forms that can be used are known to those skilled in the art. See, for example, WO 2014 / 145806; WO 2017 / 124002; Liu et al., 2017, Front Immunol. 8:38; Brinkmann & Kontermann, 2017, mAbs 9:2,182-212; U.S. Patent Application Publication No. 2016 / 0355600; Klein et al., 2016, MAbs 8(6):1010-20; and U.S. Patent Application Publication No. 2017 / 0145116.

[0429] 7.5.1. Exemplary Bivalent BBM BBMs can be bivalent, i.e., they have two antigen-binding domains, one that binds to CD19 (ABM1) and one that binds to a second target antigen (ABM2), such as a component of the TCR complex.

[0430] Exemplary bivalent BBM forms are shown in Figures 1B-1F.

[0431] As shown in Figures 1B-1D, a BBM can comprise two half antibodies, one containing one ABM and the other containing one ABM, and the two half antibodies are paired via their Fc domains.

[0432] In the embodiment of Figure 1B, the first (or left) half antibody comprises a Fab and an Fc region, and the second (or right) half antibody comprises a Fab and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0433] In the embodiment of Figure 1C, the first (or left) half antibody comprises a Fab and an Fc region, and the second (or right) half antibody comprises an scFv and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0434] In the embodiment of Figure ID, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises an scFab and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0435] As shown in Figures 1E-1F, a bivalent BBM can include two ABMs attached to one Fc region of an Fc domain.

[0436] In the embodiment of FIG. 1E, the BBM comprises a Fab, scFv and Fc domain, where the scFv is located between the Fab and Fc domains.

[0437] In the embodiment of FIG. 1F, the BBM (in the "one-arm scFv-mAb" configuration) comprises a Fab, scFv, and Fc domain, where the Fab is located between the scFv and Fc domain.

[0438] In the forms shown in Figures 1B-1F, each of X and Y represents either ABM1 or ABM2, with the proviso that a BBM includes one ABM1 and one ABM2. Thus, the present disclosure provides a bivalent BBM as shown in any one of Figures 1B-1F, where X is ABM1 and Y is ABM2 (this form of the ABM is conveniently referred to as "B1"). The present disclosure also provides a bivalent BBM as shown in any one of Figures 1B-1F, where X is ABM2 and Y is ABM1 (this form of the ABM is conveniently referred to as "B2").

[0439] 7.5.2. Exemplary Trivalent BBM The BBM can be trivalent, i.e., it has three antigen-binding domains, one or two of which bind to CD19 (ABM1) and one or two of which bind to a second target antigen (ABM2), e.g., a component of the TCR complex.

[0440] Exemplary trivalent BBM morphologies are shown in Figures 1G-1Z.

[0441] As shown in Figures 1G to 1N, 1Q to 1W, and 1Y to 1Z, a BBM contains two half antibodies, one containing two ABMs and the other containing one ABM, and these two half antibodies are paired via the Fc domain.

[0442] In the embodiment of Figure 1G, the first (or left) half antibody comprises a Fab and an Fc region, and the second (or right) half antibody comprises an scFab, a Fab, and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0443] In the embodiment of Figure 1H, the first (or left) half antibody comprises a Fab and an Fc region, and the second (or right) half antibody comprises a Fab, an scFv, and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0444] In the embodiment of Figure 1I, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises two Fab and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0445] In the embodiment of Figure 1J, the first (or left) half antibody comprises two Fab and Fc regions, and the second (or right) half antibody comprises a Fab and Fc region, and the first and second half antibodies associate via the Fc regions to form an Fc domain.

[0446] In the embodiment of Figure 1K, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises two scFvs and an Fc region, and the first and second half antibodies associate via their Fc regions to form an Fc domain.

[0447] In the embodiment of Figure 1L, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises an scFv, an Fab, and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0448] In the embodiment of Figure 1M, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises a Fab, an scFv, and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0449] In the embodiment of Figure 1N, the first (or left) half antibody comprises a diabody-type binding domain and an Fc region, and the second (or right) half antibody comprises a Fab and an Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0450] In the embodiment of Figure 1Q, the first (or left) half antibody comprises a Fab and an Fc region, and the second (or right) half antibody comprises a Fab, an Fc region, and an scFv, and the first and second half antibodies are associated by their Fc regions forming an Fc domain.

[0451] In the embodiment of Figure 1R, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises an Fab, an Fc region, and an scFv, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0452] In the embodiment of Figure 1S, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises an scFv, an Fc region, and a second scFv, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0453] In the embodiment of Figure 1T, the first (or left) half antibody comprises an scFv, an Fc region, and an Fab, and the second (or right) half antibody comprises an Fab and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0454] In the embodiment of Figure 1U, the first (or left) half antibody comprises two Fab and Fc regions, and the second (or right) half antibody comprises a non-immunoglobulin-based ABM and Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0455] In the embodiment of Figure 1V, the first (or left) half antibody comprises a Fab, scFv, and Fc region, and the second (or right) half antibody comprises a non-immunoglobulin-based ABM and Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0456] In the embodiment of Figure 1W, the first (or left) half antibody comprises a Fab and an Fc region, and the second (or right) half antibody comprises an scFv, a non-immunoglobulin-based ABM, and an Fc region, and the first and second half antibodies are associated via the Fc region to form an Fc domain.

[0457] In the embodiment of Figure 1Y, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises a Fab, an scFv, and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0458] In the embodiment of Figure 1Z, the first (or left) half antibody comprises a Fab, an Fc region, and an scFab, and the second (or right) half antibody comprises a Fab and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0459] Alternatively, as shown in Figures 1O and 1P, a trivalent BBM can comprise two half antibodies, each comprising one complete ABM (Fab in Figures 1O and 1P) and a portion of another ABM (one VH, the other VL), which are paired via the Fc domain, where the VH and VL associate to form a complete antigen-binding Fv domain.

[0460] The BBM can be a single chain, as shown in Figure 1X, which comprises three scFv domains linked via linkers.

[0461] In the embodiments shown in Figures 1G-1Z, each of X, Y, and A represents either ABM1 or ABM2, with the proviso that the BBM comprises at least an ABM1 and at least one ABM2. Thus, a trivalent BBM comprises one or two ABM1s and one or two ABM2s. In some embodiments, a trivalent BBM comprises two ABM1s and one ABM2. In other embodiments, a trivalent BBM of the present disclosure comprises one ABM1 and two ABM2s.

[0462] Thus, the present disclosure provides a trivalent BBM as shown in any one of Figures 1G-1Z, where X is ABM1, Y is ABM1, and A is ABM2 (this form of ABM is conveniently referred to as "T1").

[0463] The present disclosure further provides a trivalent BBM as shown in any one of Figures 1G-1Z, where X is ABM1, Y is ABM2, and A is ABM1 (this form of ABM is conveniently referred to as "T2").

[0464] The present disclosure further provides a trivalent BBM as shown in any one of Figures 1G-1Z, where X is ABM2, Y is ABM1, and A is ABM1 (this form of ABM is conveniently referred to as "T3").

[0465] The present disclosure further provides a trivalent BBM as shown in any one of Figures 1G-1Z, where X is ABM1, Y is ABM2, and A is ABM2 (this form of ABM is conveniently referred to as "T4").

[0466] The present disclosure further provides a trivalent BBM as shown in any one of Figures 1G-1Z, where X is ABM2, Y is ABM1, and A is ABM2 (this form of ABM is conveniently referred to as "T5").

[0467] The present disclosure further provides a trivalent BBM as shown in any one of Figures 1G-1Z, where X is ABM2, Y is ABM2, and A is ABM1 (this form of ABM is conveniently referred to as "T6").

[0468] 7.5.3. Exemplary 4-valent BBM The BBM can be tetravalent, i.e., the BBM has four antigen-binding domains, one, two, or three of which bind to CD19 (ABM1) and one, two, or three of which bind to a second target antigen (ABM2), e.g., a component of the TCR complex.

[0469] Exemplary tetravalent BBM configurations are shown in Figures 1AA-1AH.

[0470] As shown in Figures 1AA-1AH, a tetravalent BBM can comprise two half antibodies, each containing two complete ABMs, and these two half antibodies are paired via their Fc domains.

[0471] In the embodiment of Figure 1AA, the first (or left) half antibody comprises a Fab, an Fc region, and an scFab, and the second (or right) half antibody comprises a Fab, an Fc region, and an scFab, and the first and second half antibodies are associated via the Fc region to form an Fc domain.

[0472] In the embodiment of Figure 1AB, the first (or left) half antibody comprises a Fab, scFv, and Fc region, and the second (or right) half antibody comprises a Fab, scFv, and Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0473] In the embodiment of Figure 1AC, the first (or left) half antibody comprises an scFv, a Fab, and an Fc region, and the second (or right) half antibody comprises an scFv, a Fab, and an Fc region, and the first and second half antibodies associate via the Fc region to form an Fc domain.

[0474] In the embodiment of Figure 1AD, the first (or left) half antibody comprises a Fab, an Fc region, and a second Fab, and the second (or right) half antibody comprises a Fab, an Fc region, and a second Fab, the first and second half antibodies being associated by their Fc regions forming an Fc domain.

[0475] In the embodiment of Figure 1AE, the first (or left) half antibody comprises an scFv, a second scFv, and an Fc region, and the second (or right) half antibody comprises an scFv, a second scFv, and an Fc region, and the first and second half antibodies are associated by their Fc regions forming an Fc domain.

[0476] In the embodiment of Figure 1AF, the first (or left) half antibody comprises a Fab, scFv, and Fc region, and the second (or right) half antibody comprises a Fab, scFv, and Fc region, and the first and second half antibodies are associated by their Fc regions forming an Fc domain.

[0477] In the embodiment of Figure 1AG, the first (or left) half antibody comprises a Fab, an Fc region, and an scFv, and the second (or right) half antibody comprises an scFv, an Fc region, and a Fab, and the first and second half antibodies are associated by their Fc regions to form an Fc domain.

[0478] In the embodiment of Figure 1AH, the first (or left) half antibody comprises an scFv, an Fc region, and an Fab, and the second (or right) half antibody comprises an scFv, an Fc region, and an Fab, and the first and second half antibodies are associated by their Fc regions forming an Fc domain.

[0479] In the embodiments shown in Figures 1AA-1AH, each of X, Y, A, and B represents an ABM1 or ABM2, in any order, provided that the BBM comprises at least one ABM1 and at least one ABM2. Thus, a tetravalent ABM will comprise one, two, or three ABM1s and one, two, or ABM2s. In some embodiments, a tetravalent BBM will comprise three ABM1s and one ABM2. In other embodiments, a tetravalent BBM will comprise two ABM1s and two ABM2s. In yet other embodiments, a tetravalent BBM will comprise one ABM1 and three ABM2s.

[0480] Thus, the present disclosure provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where X is ABM1 and Y, A, and B each comprise ABM2 (this form of ABM is conveniently referred to as "Tv 1").

[0481] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where Y is ABM1 and each of X, A, and B is ABM2 (this form of ABM is conveniently referred to as "Tv 2").

[0482] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where A is ABM1 and each of X, Y, and B is ABM2 (this form of ABM is conveniently referred to as "Tv 3").

[0483] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where B is ABM1 and each of X, Y, and A is ABM2 (this form of ABM is conveniently referred to as "Tv 4").

[0484] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where X and Y are both ABM1 and A and B are both ABM2 (this form of ABM is conveniently referred to as "Tv 5").

[0485] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where X and A are both ABM1 and Y and B are both ABM2 (this form of ABM is conveniently referred to as "Tv 6").

[0486] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where X and B are both ABM1 and Y and A are both ABM2 (this form of ABM is conveniently designated as "Tv 7").

[0487] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AG, where Y and A are both ABM1 and X and B are both ABM2 (this form of ABM is conveniently designated as "Tv 8").

[0488] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where Y and B are both ABM1 and X and A are both ABM2 (this form of ABM is conveniently designated as "Tv 9").

[0489] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where A and B are both ABM1 and X and Y are both ABM2 (this form of ABM is conveniently designated as "Tv 10").

[0490] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where each of X, Y, and A is ABM1 and B is ABM2 (this form of ABM is conveniently designated as "Tv 11").

[0491] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where each of X, Y, and B is ABM1 and A is ABM2 (this form of ABM is conveniently designated as "Tv 12").

[0492] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where each of X, A, and B is ABM1 and Y is ABM2 (this form of ABM is conveniently designated as "Tv 13").

[0493] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH, where each of Y, A, and B is ABM1 and X is ABM2 (this form of ABM is conveniently designated as "Tv 14").

[0494] 7.6. Trispecific binding molecular morphology Exemplary TBM configurations are shown in Figure 2. Figure 2A illustrates the components of the TBM configurations shown in Figures 2B-1V. The scFv, Fab, non-immunoglobulin-based ABM, and Fc can each have the characteristics described for these components in Sections 7.3 and 7.4. The components of the TBM configurations shown in Figure 2 can be associated with each other by any of the means described in Sections 7.3 and 7.4 (e.g., by direct bonding, ABM linkers, disulfide bonds, Fc domains modified with knob-in-hole interactions, etc.). The orientations and associations of the various components shown in Figure 2 are merely exemplary; as one of skill in the art will appreciate, other orientations and associations may be suitable (e.g., as described in Sections 7.3 and 7.4).

[0495] TBMs are not limited to the configuration shown in Figure 2. Other configurations that can be used are known to those skilled in the art. See, e.g., WO 2014 / 145806; WO 2017 / 124002; Liu et al., 2017, Front Immunol. 8:38; Brinkmann & Kontermann, 2017, mAbs 9:2,182-212; U.S. Patent Application Publication No. 2016 / 0355600; Klein et al., 2016, MAbs 8(6):1010-20; and U.S. Patent Application Publication No. 2017 / 0145116.

[0496] 7.6.1. Exemplary Trivalent TBM The TBM of the present disclosure can be trivalent, i.e., the TBM has three antigen-binding domains, one that binds to CD19, one that binds to a component of the TCR complex, and one that binds to either CD2 or a TAA.

[0497] Exemplary trivalent TBM morphologies are shown in Figures 2B-2P.

[0498] As shown in Figures 2B-2K and 2N-2P, a TBM can comprise two half antibodies, one containing two ABMs and the other containing one ABM, and these two half antibodies are paired via the Fc domain.

[0499] In the embodiment of Figure 2B, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises a Fab, an scFv, and an Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0500] In the embodiment of Figure 2C, the first (or left) half antibody comprises two Fab and Fc regions, and the second (or right) half antibody comprises an Fab and an Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0501] In the embodiment of Figure 2D, the first (or left) half antibody comprises a Fab, scFv, and Fc region, and the second (or right) half antibody comprises a Fab and Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0502] In the embodiment of Figure 2E, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises two Fab and an Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0503] In the embodiment of Figure 2F, the first (or left) half antibody comprises an scFv, an Fc region, and an Fab, and the second (or right) half antibody comprises an Fab and an Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0504] In the embodiment of Figure 2G, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises a Fab Fc region and an scFv, and the first and second half antibodies are associated by their Fc regions forming an Fc domain.

[0505] In the embodiment of Figure 2H, the first (or left) half antibody comprises two Fab and Fc regions, and the second (or right) half antibody comprises a non-immunoglobulin-based Fab and Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0506] In the embodiment of Figure 2I, the first (or left) half antibody comprises a Fab, scFv, and Fc region, and the second (or right) half antibody comprises a non-immunoglobulin-based ABM and Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0507] In the embodiment of Figure 2J, the first (or left) half antibody comprises a Fab and an Fc region, and the second (or right) half antibody comprises an scFv, a non-immunoglobulin-based ABM, and an Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0508] In the embodiment of Figure 2K, the first (or left) half antibody comprises an scFv and an Fc region, and the second (or right) half antibody comprises an scFv, an Fc region, and a second scFv, and the first and second half antibodies are associated by their Fc regions forming an Fc domain.

[0509] In the embodiment of Figure 2N, the first (or left) half antibody comprises a Fab, an Fc region, and an scFv, and the second (or right) half antibody comprises a Fab and an Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0510] In the embodiment of Figure 2O, the first (or left) half antibody comprises a Fab, an Fc region, and an scFab, and the second (or right) half antibody comprises a Fab and an Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0511] In the embodiment of Figure 2P, the first (or left) half antibody comprises a Fab, a non-immunoglobulin-based ABM, and an Fc region, and the second (or right) half antibody comprises an scFv and an Fc region, and the first and second half antibodies are associated by the Fc regions forming an Fc domain.

[0512] Alternatively, as shown in Figure 2L, a trivalent TBM can comprise two half antibodies, each comprising one complete ABM and a portion of another ABM (one VH, the other VL), which are paired via the Fc domain, where the VH and VL associate to form a complete antigen-binding Fv domain.

[0513] The TBM can be a single chain, as shown in Figure 2M, which contains three scFv domains linked via linkers.

[0514] In each of the configurations shown in Figures 2B-2P, each of the domains designated X, Y, and Z represents, although not necessarily in that order, ABM1, ABM2, or ABM3. In other words, X can be ABM1, ABM2, or ABM3, Y can be ABM1, ABM2, or ABM3, and Z can be ABM1, ABM2, or ABM3, provided that a TBM includes one ABM1, one ABM2, and one ABM3.

[0515] Thus, the present disclosure provides a trivalent TBM as shown in any one of Figures 2B-2P, where X is ABM1, Y is ABM3, and Z is ABM2 (this form of ABM is conveniently referred to as "T1").

[0516] The present disclosure also provides a trivalent TBM as shown in any one of Figures 2B-2P, where X is ABM1, Y is ABM2, and Z is ABM3 (this form of ABM is conveniently referred to as "T2").

[0517] The present disclosure further provides a trivalent TBM as shown in any one of Figures 2B-2P, where X is ABM3, Y is ABM1, and Z is ABM2 (this form of ABM is conveniently referred to as "T3").

[0518] The present disclosure still further provides a trivalent TBM as shown in any one of Figures 2B-2P, where X is ABM3, Y is ABM2, and Z is ABM1 (this form of ABM is conveniently referred to as "T4").

[0519] The present disclosure still further provides a trivalent TBM as shown in any one of Figures 2B-2P, where X is ABM2, Y is ABM1, and Z is ABM3 (this form of ABM is conveniently referred to as "T5").

[0520] The present disclosure still further provides a trivalent TBM as shown in any one of Figures 2B-2P, where X is ABM2, Y is ABM3, and Z is ABM1 (this form of ABM is conveniently referred to as "T6").

[0521] 7.6.2. Exemplary Tetravalent TBM The TBM of the present disclosure can be tetravalent, i.e., the TBM has four antigen-binding domains, one or two of which bind to CD19, one or two of which bind to a component of the TCR complex, and one or two of which bind to CD2 or a TAA.

[0522] Exemplary tetravalent TBM forms are shown in Figures 2Q-2S.

[0523] As shown in Figures 2Q to 2S, a tetravalent TBM can contain two half antibodies, each containing two complete ABMs, and these two half antibodies are paired via their Fc domains.

[0524] In the embodiment of Figure 2Q, the first (or left) half antibody comprises a Fab, an Fc region, and a second Fab, and the second (or right) half antibody comprises a Fab, an Fc region, and a second Fab, the first and second half antibodies being associated by their Fc regions forming an Fc domain.

[0525] In the embodiment of Figure 2R, the first (or left) half antibody comprises a Fab, an Fc region, and an scFv, and the second (or right) half antibody comprises a Fab, an Fc region, and an scFv, and the first and second half antibodies are associated by their Fc regions forming an Fc domain.

[0526] In the embodiment of Figure 2S, the first (or left) half antibody comprises a Fab, an Fc region, and an scFv, and the second (or right) half antibody comprises an scFv, an Fc region, and a Fab, and the first and second half antibodies are associated by their Fc regions forming an Fc domain.

[0527] In the configurations shown in Figures 2Q-2S, each of X, Y, Z, and A represents an ABM1, ABM2, or ABM3, not necessarily in that order, provided that a TBM includes at least one ABM1, at least one ABM2, and at least one ABM3. Thus, a tetravalent ABM will include two ABMs: one for CD19, a component of the TCR complex, and one for CD2 or a TAA. In some cases, a tetravalent TBM has two CD19 ABMs.

[0528] 7.6.3. Exemplary Pentavalent TBM The TBM of the present disclosure can be pentavalent, i.e., the TBM has five antigen-binding domains, one, two, or three of which bind to CD19, one, two, or three of which bind to a component of the TCR complex, and one, two, or three of which bind to CD2 or a TAA.

[0529] An exemplary pentavalent TBM configuration is shown in Figure 2T.

[0530] As shown in Figure 2T, a pentavalent TBM can contain two half antibodies, one containing two complete ABMs and the other containing one complete ABM, and these two half antibodies are paired via the Fc domain.

[0531] In the embodiment of Figure 2T, the first (or left) half antibody comprises a Fab, an scFv, and an Fc region, and the second (or right) half antibody comprises a Fab, an Fc region, and an scFv, and the first and second half antibodies are associated by their Fc regions forming an Fc domain.

[0532] In the configuration shown in Figure 2T, each of X, Y, Z, A, and B represents an ABM1, ABM2, or ABM3, not necessarily in that order, provided that the TBM includes at least one ABM1, one ABM2, and one ABM3. Thus, a pentavalent TBM can include two ABMs for CD19, a component of the TCR complex, and two of CD2 or a TAA, or three ABMs for CD19, a component of the TCR complex, and one of CD2 or a TAA. In some cases, a pentavalent TBM has two or three CD19 ABMs. In some embodiments, a pentavalent TBM has three ABMs, one ABM2, and one ABM3.

[0533] 7.6.4. Exemplary Hexavalent TBM The TBM of the present disclosure can be hexavalent, i.e., the TBM has six antigen-binding domains, one, two, three, or four of which bind to CD19, one, two, three, or four of which bind to a component of the TCR complex, and one, two, three, or four of which bind to CD2 or a TAA.

[0534] Exemplary hexavalent TBM morphologies are shown in Figures 2U-2V.

[0535] As shown in Figures 2U-2V, a pentavalent TBM can contain two half antibodies, one of which contains two complete ABMs and the other of which contains one complete ABM, and these two half antibodies are paired via the Fc domain.

[0536] In the embodiment of Figure 2U, the first (or left) half antibody comprises a Fab, a second Fab, an Fc region, and an scFv, and the second (or right) half antibody comprises a Fab, a second Fab, an Fc region, and an scFv, and the first and second half antibodies are associated by their Fc regions forming an Fc domain.

[0537] In the embodiment of Figure 2V, the first (or left) half antibody comprises a first Fv, a second Fv, a third Fv, and an Fc region, and the second (or right) half antibody comprises a first Fv, a second Fv, a third Fv, and an Fc region, where the first and second half antibodies are associated by their Fc regions forming an Fc domain.

[0538] In the configurations shown in Figures 2U-2V, each of X, Y, Z, A, B, and C represents ABM1, ABM2, or ABM3, not necessarily in that order, provided that a TBM includes at least one ABM1, one ABM2, and one ABM3. Thus, a hexavalent TBM may include (i) two ABMs for each of CD19, a component of the TCR complex, and CD2 or a TAA; (ii) three ABMs for CD19, a component of the TCR complex, and one of CD2 or a TAA; or (iii) four ABMs for CD19, a component of the TCR complex, and one of CD2 or a TAA. For example, a hexavalent ABM may include three ABMs for CD19, two ABMs for CD2 or a TAA, and one ABM for a component of the TCR complex. As another example, a hexavalent TBM can include three ABMs for CD19, two ABMs for components of the TCR complex, and one ABM for CD2 or a TAA. In some cases, a hexavalent TBM has two, three, or four CD19 ABMs. In some embodiments, a hexavalent TBM has three CD19 ABMs. In other embodiments, a hexavalent TBM has four CD19 ABMs.

[0539] 7.7.TCR ABM The MBM of the present disclosure comprises an ABM that specifically binds CD19 and an ABM2 specific for a different antigen. In the BBM, type 1 TBM, and type 2 TBM of the present disclosure, ABM2 can bind to components of the TCR complex. The TCR is a disulfide-linked, membrane-anchored heterodimeric protein typically composed of hypervariable alpha (α) and beta (β) chains expressed as part of a complex with an invariant CD3 chain molecule. T cells that express this receptor are called α:β (or αβ) T cells; however, a minority of T cells express an alternative receptor, formed by variable gamma (γ) and delta (δ) chains, and are called γδ T cells.

[0540] In one embodiment, the MBM comprises an ABM that specifically binds to CD3.

[0541] 7.7.1.CD3 ABM The MBM may include an ABM that specifically binds to CD3. The term "CD3" refers to the T cell receptor differentiation cluster 3 coreceptor (or coreceptor complex or polypeptide chain of the coreceptor complex). The amino acid sequence of the polypeptide chain of human CD3 is provided in NCBI accession numbers P04234, P07766, and P09693. CD3 proteins may also include mutant forms. CD3 proteins may also include fragments. CD3 proteins may also include post-translational modifications of the CD3 amino acid sequence. Post-translational modifications include, but are not limited to, N-linked and O-linked glycosylation.

[0542] In some embodiments, the MBM can comprise an ABM that is an anti-CD3 antibody (e.g., as described in U.S. Patent Application Publication Nos. 2016 / 0355600, WO 2014 / 110601, and WO 2014 / 145806) or an antigen-binding domain thereof. Exemplary anti-CD3 VH, VL, and scFV sequences that can be used in the MBM are provided in Table 12A.

[0543] [Table 47]

[0544] [Table 48]

[0545] [Table 49]

[0546] [Table 50]

[0547] [Table 51]

[0548] [Table 52]

[0549] Kabat numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5 th The CDR sequences for several CD3 binding agents as defined by the National Institutes of Health (Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), the Chothia numbering scheme (Al-Lazikani et al., 1997, J. Mol. Biol 273:927-948), and a combination of Kabat and Chothia numbering are provided in Tables 12B-12D, respectively.

[0550] [Table 53]

[0551] [Table 54]

[0552] Table 55

[0553] Table 56

[0554] Table 57

[0555] Table 58

[0556] Table 59

[0557] Table 60

[0558] Table 61

[0559] Table 62

[0560] Table 63

[0561] Table 64

[0562] [Table 65]

[0563] [Table 66]

[0564] [Table 67]

[0565] In some embodiments, MBM may comprise a CD3 ABM comprising any of the CDRs of CD3-1 through CD3-130 as defined by Kabat numbering (e.g., as listed in Table 12B). In other embodiments, MBM may comprise a CD3 ABM comprising any of the CDRs of CD3-1 through CD3-130 as defined by Chothia numbering (e.g., as listed in Table 12C). In yet other embodiments, MBM may comprise a CD3 ABM comprising any of the CDRs of CD3-1 through CD3-130 as defined by a combination of Kabat and Chothia numbering (e.g., as listed in Table 12D).

[0566] In some embodiments, the CD3 ABM comprises CDR sequences of CD3-1. In some embodiments, the CD3 ABM comprises CDR sequences of CD3-2. In some embodiments, the CD3 ABM comprises CDR sequences of CD3-3. In some embodiments, the CD3 ABM comprises CDR sequences of CD3-4. In some embodiments, the CD3 ABM comprises CDR sequences of CD3-5. In some embodiments, the CD3 ABM comprises CDR sequences of CD3-6. In some embodiments, the CD3 ABM comprises CDR sequences of CD3-7. In some embodiments, the CD3 ABM comprises CDR sequences of CD3-8. In some embodiments, the CD3 ABM comprises CDR sequences of CD3-9. In some embodiments, the CD3 ABM comprises CDR sequences of CD3-10. In some embodiments, the CD3 ABM comprises CDR sequences of CD3-11. In some embodiments, the CD3 ABM comprises CDR sequences of CD3-12. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-13. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-14. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-15. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-16. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-17. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-18. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-19. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-20. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-21. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-22. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-23. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-24. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-25. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-26. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-27.In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-28. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-29. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-30. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-31. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-32. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-33. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-34. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-35. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-36. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-37. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-38. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-39. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-40. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-41. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-42. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-43. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-44. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-45. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-46. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-47. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-48. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-49. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-50. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-51. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-52. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-53. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-54.In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-55. In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-56. In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-57. In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-58. In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-59. In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-60. In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-61. In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-62. In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-63. In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-64. In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-65. In some embodiments, the CD3 ABM comprises a CDR sequence of CD3-66. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-67. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-68. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-69. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-70. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-71. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-72. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-73. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-74. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-75. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-76. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-77. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-78. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-79. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-80. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-81.In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-82. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-83. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-84. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-85. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-86. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-87. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-88. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-89. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-90. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-91. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-92. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-93. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-94. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-95. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-96. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-97. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-98. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-99. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-100. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-101. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-102. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-103. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-104. In some embodiments, the CD3 ABM comprises the CDR sequence of CD3-105. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-106. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-107.In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-108. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-109. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-110. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-111. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-112. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-113. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-114. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-115. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-116. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-117. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-118. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-119. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-120. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-121. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-122. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-123. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-124. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-125. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-126. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-127. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-126. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-127. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-128. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-129. In some embodiments, the CD3 ABM comprises the CDR sequences of CD3-130.

[0567] MBM may comprise the complete heavy and light chain variable sequences of any of CD3-1 through CD3-130. In some embodiments, MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-1. In some embodiments, MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-1. In some embodiments, MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-2. In some embodiments, MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-3. In some embodiments, MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-4. In some embodiments, MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-5. In some embodiments, MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-6. In some embodiments, MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-7. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-8. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-9. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-10. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-11. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-12. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-13. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-14. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-15. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-16. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-17. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-18. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-19. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-20.In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-21. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-22. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-23. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-24. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-25. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-26. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-27. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-28. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-129. In some embodiments, the MBM comprises a CD3 ABM comprising the VH and VL sequences of CD3-130.

[0568] In addition to the CDR sets set forth in Tables 12B-12D (i.e., the six CDR sets for each of CD3-1 through CD3-130), the present disclosure provides variant CDR sets. In one embodiment, the six CDR sets may have one, two, three, four, or five amino acid changes compared to the CDR sets set forth in Tables 12B-12D, so long as the CD3 ABM is still capable of binding to the target antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay) assays.

[0569] In addition to the variable heavy and variable light domains disclosed in Table 12A that form ABMs against CD3, the present disclosure provides variant VH and VL domains. In one embodiment, the variant VH and VL domains can each have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to the VH and VL domains set forth in Table 12A, so long as the ABMs are still capable of binding to the target antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (bio-layer interferometry, e.g., Octet assay) assays. In another embodiment, the variant VH and VL are at least 90, 95, 97, 98, or 99% identical to the respective VH or VL disclosed in Table 12A, so long as the ABMs are still capable of binding to the target antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (bio-layer interferometry, e.g., Octet assay) assays.

[0570] In some embodiments, the MBM may comprise an ABM that is a CD3 binding molecule or an antigen-binding domain thereof as described in WO2020 / 052692.

[0571] VH and VL sequences (amino acid sequences and nucleotide sequences encoding amino acid sequences) can be "mix and matched" to create other CD3 ABMs. Such "mix and matched" CD3 ABMs can be tested using binding assays known in the art (e.g., FACS assays). When mixing and matching chains, a VH sequence from a particular VH / VL pairing must be replaced with a structurally similar VH sequence. A VL sequence from a particular VH / VL pairing must be replaced with a structurally similar VL sequence.

[0572] In some embodiments, the antigen-binding domain that specifically binds to human CD3 is non-immunoglobulin-based and instead is derived from a non-antibody scaffold protein, such as one of the non-antibody scaffold proteins described in Section 7.3.2. In one embodiment, the antigen-binding domain that specifically binds to human CD3 comprises Affilin-144160, described in WO 2017 / 013136. Affilin-144160 has the following amino acid sequence: [ka]

[0573] TCR-α / β Antibody Binding Matrix The MBM can include an ABM that specifically binds to the TCR-α chain, the TCR-β chain, or the TCR-αβ dimer. Exemplary anti-TCR-α / β antibodies are known (see, e.g., U.S. Patent Application Publication No. 2012 / 0034221; Borst et al., 1990, Hum Immunol. 29(3):175-88, which describes antibody BMA031). The VH, VL, and Kabat CDR sequences of antibody BMA031 are provided in Table 13.

[0574] [Table 68]

[0575] In some embodiments, the TCR ABM may comprise the CDR sequences of antibody BMA031. In other embodiments, the TCR ABM may comprise the VH and VL sequences of antibody BMA031.

[0576] TCR-γ / δ ABM The MBM can comprise an ABM that specifically binds to a TCR-γ chain, a TCR-δ chain, or a TCR-γδ dimer. Exemplary anti-TCR-γ / δ antibodies are known (see, e.g., U.S. Pat. No. 5,980,892, which describes δTCS1, produced by the hybridoma deposited with the ATCC under Accession No. HB 9578).

[0577] 7.8.CD2 ABM Immunoglobulin-based CD2 ABM A type 1 TBM can include an ABM that is an anti-CD2 antibody or its antigen-binding domain. Exemplary anti-CD2 antibodies are known (see, e.g., U.S. Patent No. 6,849,258, Chinese Patent Application Publication No. 102827281A, U.S. Patent Application Publication No. 2003 / 0139579 A1, and U.S. Patent No. 5,795,572). Table 14 provides exemplary CDR, VH, and VL sequences that can be included in an anti-CD2 antibody or antigen-binding fragment thereof for use in the MBM of the present disclosure.

[0578] [Table 69]

[0579] [Table 70]

[0580] In some embodiments, the CD2 ABM comprises the CDR sequences of CD2-1 (SEQ ID NOs: 312-317). In some embodiments, the CD2 ABM comprises the heavy and light chain variable sequences of CD2-1 (SEQ ID NOs: 318 and 319, respectively). In some embodiments, the CD2 ABM comprises the heavy and light chain variable sequences of hu1CD2-1 (SEQ ID NOs: 320 and 321, respectively). In some embodiments, the CD2 ABM comprises the heavy and light chain variable sequences of hu2CD2-1 (SEQ ID NOs: 318 and 321, respectively).

[0581] In other embodiments, the CD2 ABM may comprise the CDR sequences of antibody 9D1, described in Chinese Patent Application Publication No. 102827281A, produced by a hybridoma deposited at the Chinese Culture Collection Committee General Microbiology Center on May 16, 2012 under accession number CGMCC 6132. In other embodiments, the CD2 ABM may comprise the CDR sequences of antibody LO-CD2b, described in U.S. Patent Application Publication No. 2003 / 0139579 A1, produced by a hybridoma deposited at the American Type Culture Collection on June 22, 1999 under accession number PTA-802. In yet another embodiment, the CD2 ABM may comprise the CDR sequences of the CD2 SFv-Ig described in U.S. Pat. No. 5,795,572, produced by expression of a construct cloned into recombinant E. coli deposited with the ATCC on April 9, 1993 under accession number 69277.

[0582] In other embodiments, the CD2 ABM can comprise the VH and VL sequences of antibody 9D1. In other embodiments, the CD2 ABM can comprise the VH and VL sequences of antibody LO-CD2b. In yet other embodiments, the CD2 ABM can comprise the VH and VL sequences of CD2 SFv-Ig produced by expression of a construct cloned into recombinant E. coli having ATCC Accession No. 69277.

[0583] 7.8.2.CD58-based CD2 ABM In certain embodiments, the present disclosure provides a type 1 TBM that includes a ligand, CD2 ABM. The CD2 ABM specifically binds to human CD2, the natural ligand of which is CD58, also known as LFA-3. The CD58 / LFA-3 protein is a glycoprotein expressed on the surface of various cell types (Dustin et al., 1991, Annu. Rev. Immunol. 9:27) and plays a role in mediating T cell interactions with APCs in both antigen-dependent and antigen-independent manners (Wallner et al., 1987, J. Exp. Med. 166:923). Thus, in certain embodiments, the CD2 ABM is a CD58 moiety. As used herein, a CD58 portion includes an amino acid sequence that has at least 70% sequence identity to the CD2-binding portion of CD58, e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD2-binding portion of CD58. The sequence of human CD58 has Uniprot identification number P19256 (www.uniprot.org / uniprot / P19256). It has been established that a CD58 fragment comprising amino acid residues 30-123 of full-length CD58 (i.e., the sequence designated CD58-6 in Table 15 below) is sufficient for binding to CD2 (Wang et al., 1999, Cell 97:791-803). Thus, in certain embodiments, the CD58 portion comprises an amino acid sequence comprising at least 70% sequence identity with amino acids 30-123 of CD58, e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence designated as CD58-6.

[0584] The interaction between CD58 and CD2 has been mapped using X-ray crystallography and molecular modeling. Substitutions of residues E25, K29, K30, K32, D33, K34, E37, D84, and K87 (numbering relative to the mature polypeptide) result in reduced binding to CD2. Ikemizu et al., 1999, Proc. Natl. Acad. Sci. USA 96:4289-94. Thus, in some embodiments, the CD58 moiety retains wild-type residues at E25, K29, K30, K32, D33, K34, E37, D84, and K87.

[0585] In contrast, the following substitutions (numbering is relative to the full-length polypeptide) did not affect binding to CD2: F29S; V37K; V49Q; V86K; T113S; and L121G. Thus, a CD58 portion can contain one, two, three, four, five, or all six of the foregoing substitutions.

[0586] In some embodiments, the CD58 portion is engineered to contain a pair of cysteine ​​substitutions that create a disulfide bridge upon recombinant expression. Exemplary amino acid pairs (numbering relative to the full-length polypeptide) that can be substituted for cysteines to form a disulfide bridge upon expression are (a) a V45C substitution and an M105C substitution; (b) a V54C substitution and a G88C substitution; (c) a V45C substitution and an M114C substitution; and (d) a W56C substitution and an L90C substitution.

[0587] Exemplary CD58 moieties are provided in Table 15 below.

[0588] [Table 71]

[0589] [Table 72]

[0590] CD48-based CD2 ABMIn certain aspects, the present disclosure provides an MBM comprising a CD2 ABM that is a CD48 portion. As used herein, a CD48 portion comprises an amino acid sequence that comprises at least 70% sequence identity to the CD2-binding portion of CD48, e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD2-binding portion of CD48. The sequence of human CD48 has Uniprot identification number P09326 (www.uniprot.org / uniprot / P09326), which includes the signal peptide (amino acids 1-26) and the GPI anchor (amino acids 221-243). In certain embodiments, the CD48 portion includes an amino acid sequence that has at least 70% sequence identity (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to the amino acid sequence consisting of amino acids 27-220 of Uniprot identification number P09326. Human CD48 has an Ig-like C2-type I domain (amino acids 29-127 of Uniprot Identification Number P09326) and an Ig-like C2-type II domain (amino acids 132-212 of Uniprot Identification Number P09326). Thus, in some embodiments, the CD48 portion comprises an amino acid sequence that comprises at least 70% sequence identity (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to an amino acid sequence consisting of amino acids 29-212 of Uniprot Identification No. P09326, the C2-type I domain (amino acids 29-127 of Uniprot Identification No. P09326), and / or the Ig-like C2-type 2 domain (amino acids 132-212 of Uniprot Identification No. P09326).The CD48 portion, in some embodiments, can include one or more naturally occurring variants relative to the sequence of Uniprot Identification Number P09326. For example, the CD48 portion can include an E102Q substitution. As another example, the CD48 portion can include an amino acid sequence corresponding to a CD-48 isoform or CD2-binding portion thereof, e.g., an isoform having Uniprot Identification Number P09326-2 or a CD2-binding portion thereof.

[0591] 7.9. Tumor-associated antigen ABM Type 2 TBM can include an ABM that specifically binds to a tumor-associated antigen (TAA). In some embodiments, the TAA is a human TAA. This antigen may or may not be present in normal cells. In certain embodiments, the TAA is preferentially expressed or upregulated in tumor cells compared to normal cells. In other embodiments, the TAA is a lineage marker.

[0592] In certain embodiments, the TAA is expressed in or upregulated in cancerous B cells relative to normal B cells, hi other embodiments, the TAA is a B cell lineage marker.

[0593] It is expected that any type of B cell malignancy can be targeted by the MBMs of the present disclosure. Exemplary types of B cell malignancies that can be targeted include Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), and multiple myeloma. Examples of NHL include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma (Waldenstrom's macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, primary mediastinal large B-cell lymphoma, mediastinal gray zone lymphoma (MGZL), splenic marginal zone B-cell lymphoma, MALT-type extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, and primary effusion lymphoma.

[0594] Examples of TAAs other than CD19 that may be targeted by MBM (e.g., TBM) include BCMA, CD20, CD22, CD123, CD33, CLL1, CD138 (also known as syndecan-1, SDC1), CS1, CD38, CD133, FLT3, CD52, TNFRSF13C (TNF receptor superfamily member 13C, also referred to in the art as BAFFR: B-cell activating factor receptor), TNFRSF13B (TNF receptor superfamily member 13B), and TNFRSF13C (TNF receptor superfamily member 13C). , also referred to in the art as TACI: transmembrane activator and CAML interactor), CXCR4 (CXC motif chemokine receptor 4), PD-L1 (programmed death-ligand 1), LY9 (lymphocyte antigen 9, also referred to in the art as CD229), CD200, FCGR2B (Fc fragment of IgG receptor IIb, also referred to in the art as CD32b), CD21, CD23, CD24, CD40L, CD72, CD79a, and CD79b. In some embodiments, the TAA is BCMA. In some embodiments, the TAA is CD20. In some embodiments, the TAA is CD22. In some embodiments, the TAA is CD123. In some embodiments, the TAA is CD33. In some embodiments, the TAA is CLL1. In some embodiments, the TAA is CD138. In some embodiments, the TAA is CS1. In some embodiments, the TAA is CD38. In some embodiments, the TAA is CD133. In some embodiments, the TAA is FLT3. In some embodiments, the TAA is CD52. In some embodiments, the TAA is TNFRSF13C. In some embodiments, the TAA is TNFRSF13B. In some embodiments, the TAA is CXCR4. In some embodiments, the TAA is PD-L1. In some embodiments, the TAA is LY9. In some embodiments, the TAA is CD200. In some embodiments, the TAA is CD21. In some embodiments, the TAA is CD23. In some embodiments, the TAA is CD24. In some embodiments, the TAA is CD40L. In some embodiments, the TAA is CD72.In some embodiments, the TAA is CD79a. In some embodiments, the TAA is CD79b.

[0595] The TAA-binding ABM can comprise, for example, an anti-TAA antibody or antigen-binding fragment thereof. The anti-TAA antibody or antigen-binding fragment can comprise, for example, the CDR sequences of an antibody listed in Table 16. In some embodiments, the anti-TAA antibody or antigen-binding domain thereof has the heavy and light chain variable region sequences of an antibody listed in Table 16.

[0596] [Table 73]

[0597] [Table 74]

[0598] [Table 75]

[0599] [Table 76]

[0600] In certain embodiments, the TAA is selected from BCMA and CD20. In some embodiments, the TAA is BCMA. "BCMA" refers to B-cell maturation antigen. BCMA (also known as TNFRSF17, BCM, or CD269) is a member of the tumor necrosis receptor (TNFR) family and is primarily expressed on terminally differentiated B cells, e.g., memory B cells and plasma cells. Its ligands include B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). The protein BCMA is encoded by the gene TNFRSF17. An exemplary BCMA sequence is available in the Uniprot database under accession number Q02223.

[0601] In certain embodiments, the type 2 TBM comprises an ABM3 that specifically binds BCMA, e.g., an anti-BCMA antibody or antigen-binding domain thereof. The anti-BCMA antibody or antigen-binding domain thereof may comprise, for example, a CDR, VH, VL, or scFV sequence set forth in Tables 17A-17G.

[0602] [Table 77]

[0603] [Table 78]

[0604] [Table 79]

[0605] [Table 80]

[0606] [Table 81]

[0607] [Table 82]

[0608] [Table 83]

[0609] [Table 84]

[0610] [Table 85]

[0611] Table 86

[0612] Table 87

[0613] Table 88

[0614] Table 89

[0615] Table 90

[0616] Table 91

[0617] Table 92

[0618] Table 93

[0619] Table 94

[0620] Table 95

[0621] Table 96

[0622] Table 97

[0623] Table 98

[0624] Table 99

[0625] Table 100

[0626] Table 101

[0627] In some embodiments, the ABM comprises a CDR sequence of BCMA-1. In some embodiments, the ABM comprises a CDR sequence of BCMA-2. In some embodiments, the ABM comprises a CDR sequence of BCMA-3. In some embodiments, the ABM comprises a CDR sequence of BCMA-4. In some embodiments, the ABM comprises a CDR sequence of BCMA-5. In some embodiments, the ABM comprises a CDR sequence of BCMA-6. In some embodiments, the ABM comprises a CDR sequence of BCMA-7. In some embodiments, the ABM comprises a CDR sequence of BCMA-8. In some embodiments, the ABM comprises a CDR sequence of BCMA-9. In some embodiments, the ABM comprises a CDR sequence of BCMA-10. In some embodiments, the ABM comprises a CDR sequence of BCMA-11. In some embodiments, the ABM comprises a CDR sequence of BCMA-12. In some embodiments, the ABM comprises a CDR sequence of BCMA-13. In some embodiments, the ABM comprises a CDR sequence of BCMA-14. In some embodiments, the ABM comprises a CDR sequence of BCMA-15. In some embodiments, the ABM comprises a CDR sequence of BCMA-16. In some embodiments, the ABM comprises a CDR sequence of BCMA-17. In some embodiments, the ABM comprises a CDR sequence of BCMA-18. In some embodiments, the ABM comprises a CDR sequence of BCMA-19. In some embodiments, the ABM comprises a CDR sequence of BCMA-20. In some embodiments, the ABM comprises a CDR sequence of BCMA-21. In some embodiments, the ABM comprises a CDR sequence of BCMA-22. In some embodiments, the ABM comprises a CDR sequence of BCMA-23. In some embodiments, the ABM comprises a CDR sequence of BCMA-24. In some embodiments...

Claims

[Claim 1] The invention described in the specification.