Antibodies targeting immunosuppressive B cells

Abstract

Described herein are complex binding molecules comprising (i) a CD38 antigen binding moiety that binds to CD38, (ii) a CD19 antigen binding moiety that binds to CD19, and (iii) a variant Fc region that comprises one or more mutations compared to the wild-type Fc region, wherein the variant Fc region exhibits reduced effector function compared to the wild-type Fc region. Also described are methods of using the complex binding molecules in methods of treating cancer.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 237,065, filed August 25, 2021, which is incorporated by reference in its entirety for all purposes. [Background technology]

[0002] Although antibody therapeutics have been successfully used to treat a variety of diseases, their application may be limited in terms of clinical efficacy for complex diseases such as cancer. Engineering antibody-based therapeutics to alter target binding affinity and valency provides a potential route towards achieving increased efficacy and improving treatment outcomes. Thus, bispecific or multivalent antibodies provide a potential approach to solving the challenges linked to the multifactorial nature of complex diseases. By binding two different antigen molecules, or different epitopes of the same antigen, bispecific antibodies provide greater functionality and offer diverse uses as targeting agents for the treatment of numerous diseases. Summary of the Invention

[0003] The dynamic relationship between cancer biology and the immune system is a factor associated with clinical outcomes. The immune response plays a significant role in regulating the tumor microenvironment during cancer development. Thus, immune cells, such as T cells and B cells, act as modulators and effectors of cancer progression or metastasis. Notably, immunosuppressive cells play a key role in antitumor immune responses, where immunosuppression is commonly associated with tumor growth and invasion and correlates with negative outcomes. While B cells are known to positively regulate immune responses, a population of immunosuppressive B cells functions to suppress antitumor immune responses and promote tumor growth.

[0004] Provided herein are specific binding molecules that target immunosuppressive B cell populations with bispecific or multivalent targeting molecules. Targeting immunosuppressive B cell populations offers a route for therapeutic intervention against cancer that effectively modulates antitumor immune responses to improve treatment outcomes (as opposed to, for example, selective depletion of epithelial cancer cell populations). The binding molecules provided herein can include bispecific antibodies that bind to B cell lineage surface markers (e.g., CD19, CD138, IgA, and / or CD20) and immunosuppressive B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, and / or latent TGF-β (e.g., TGF-β LAP)). In certain specific embodiments, the bispecific antibodies bind to CD19 and CD38, and thus have selectivity for specific immunosuppressive B cell populations.

[0005] In certain instances, bispecific or multivalent targeting molecules target immunosuppressive B cell populations (e.g., thereby reducing immunosuppression) to promote tumor clearance or inhibit tumor growth compared to direct targeting of tumor cells. In such cases, antibody-induced cell death or cytotoxicity in target cells is undesirable without direct targeting of tumor cells, and furthermore, antibody-induced cell death or cytotoxicity in target cells (e.g., not tumor cells) may lead to undesired side effects (e.g., lymphopenia).

[0006] Provided herein is a complex binding molecule comprising: (i) a CD38 antigen binding component that binds CD38; (ii) a CD19 antigen binding component that binds CD19; and (iii) a variant Fc region that comprises one or more mutations compared to the wild-type Fc region, wherein the variant Fc region exhibits an altered effector function compared to the wild-type Fc region. In some embodiments, the altered effector function is selected from the list consisting of decreased antibody-dependent cell-mediated cytotoxicity (ADCC), decreased complement-dependent cytotoxicity (CDC), decreased affinity for C1q, and any combination thereof. In some embodiments, the altered effector function is selected from the list consisting of increased antibody-dependent cell-mediated cytotoxicity (ADCC), decreased complement-dependent cytotoxicity (CDC), decreased affinity for C1q, and any combination thereof.

[0007] In some embodiments, the variant Fc region comprises an IgG1 Fc region, and the one or more mutations are: (a) 297A, 297Q, 297G, or 297D; (b) 279F, 279K, or 279L; (c) 228P; (d) 235A, 235E, 235G, 235Q, 235R, or 235S; (e) 237A, 237E, 237K, 237N, or 237R; (f) 234A, 234V, or 234F; (g) 233P; (h) 328A; (i) 327Q or 327T; (j) 329A, 329G, 329Y, or 329R; (k) 328P; 331S, (l) 236F or 236R, (m) 238A, 238E, 238G, 238H, 238I, 238V, 238W, or 238Y, (n) 248A, (o) 254D, 254E, 254G, 254H, 254I, 254N, 254P, 254Q, 254T, or 254V, (p) 255N, (q) 256H, 256K, 256R, or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y, or 265A, (t) 267G, 267H, 267I, or 267K, (u) 268K, ( v) 269N or 269Q, (w) 270A, 270G, 270M, or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (l l) 380D, (mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235E, (vv) L234A and L235A, (ww) L234A, L235A, and G237A, (xx) L234A, L235A, and P329G, (yy) L234F, L235E,and P331S, (zz)L234A, L235E, and G237A, (aaa)L234A, L235E, G237A, and P331S, (bbb)L234A, L235A, G237A, P238S, H268A, A330S, and P331S, (ccc)L234A, L235A, and P329A, (ddd)G236R and L328R, (eee)G237A, (fff)F241A, (ggg) V264A, (hhh) D265A, (iii) D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (lll) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S, or (ppp) any combination of (a) to (uu).

[0008] In some embodiments, the one or more mutations relative to the wild-type Fc region include or consist of L234A, L235A, and P329G according to EU numbering. In some embodiments, the one or more mutations relative to the wild-type Fc region include or consist of L234A, L235A, G237A, A330S, and P331S according to EU numbering. In some embodiments, the one or more mutations relative to the wild-type Fc region are selected from the group consisting of N297A / Q / G, L235A / G237A / E318A, L234A / L235A, G236R / L328R, S298G / T299A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, L234A / L235A / P329G, V234A / G237A / P238S / H268A / V309L / A330S / P331S, and L234F / L235E / D265A.

[0009] In some embodiments, the variant Fc region is selected from Table 1. In some embodiments, the one or more mutations relative to the wild-type Fc region include L234A, L235E, G237A, A330S, and / or P331S, according to EU numbering. In some embodiments, the one or more mutations relative to the wild-type Fc region include L234A, L235E, G237A, A330S, and P331S, according to EU numbering. In some embodiments, the one or more mutations relative to the wild-type Fc region include K322A, according to EU numbering. In some embodiments, the one or more mutations relative to the wild-type Fc region consist of K322A, according to EU numbering. In some embodiments, the one or more mutations relative to the wild-type Fc region consist of S329D and I332E, according to EU numbering.

[0010] In some embodiments, the CD38 antigen binding component comprises: a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 71 to 75; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 81 to 85 or 151 to 155; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95; d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125; The CD19 antigen-binding component g) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15; h) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 21 to 25; i) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35; j) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; k) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and l) comprising a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125.

[0011] In some embodiments, the CD38 antigen binding component comprises an HCDR2 amino acid sequence comprising an amino acid sequence set forth in SEQ ID NO: 151-155. In some embodiments, the CD38 antigen binding component comprises an HCDR2 amino acid sequence comprising an amino acid sequence set forth in SEQ ID NO: 154. In some embodiments, the CD38 antigen binding component comprises an HCDR2 amino acid sequence comprising any one of the amino acid sequences set forth in SEQ ID NO: 81-85. In some embodiments, the anti-CD38 immunoglobulin heavy chain variable region comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 3 or 5, and the anti-CD38 immunoglobulin light chain variable region comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 4. In some embodiments, the anti-CD38 immunoglobulin heavy chain variable region comprises an amino acid sequence identical to SEQ ID NO: 3 or 5, and the anti-CD38 immunoglobulin light chain variable region comprises an amino acid sequence identical to SEQ ID NO: 4. In some embodiments, the anti-CD19 immunoglobulin heavy chain variable region comprises an amino acid sequence having at least about 90% identity to SEQ ID NO:1, 6, or 7, and the anti-CD19 immunoglobulin light chain variable region comprises an amino acid sequence having at least about 90% identity to SEQ ID NO:2. In some embodiments, the anti-CD19 immunoglobulin heavy chain variable region comprises an amino acid sequence identical to SEQ ID NO:1, 6, or 7, and the anti-CD19 immunoglobulin light chain variable region comprises an amino acid sequence identical to SEQ ID NO:2.

[0012] In some embodiments, the anti-CD38 immunoglobulin heavy chain variable region further comprises an immunoglobulin heavy chain constant region, the anti-CD38 immunoglobulin heavy chain constant region comprising one or more amino acid substitutions that do not support homodimerization of the anti-CD38 immunoglobulin heavy chain constant region but promote heterodimerization of the anti-CD38 immunoglobulin heavy chain constant region with a non-anti-CD38 immunoglobulin heavy chain constant region. In some embodiments, the anti-CD38 immunoglobulin heavy chain constant region comprises a T366W substitution (EU numbering) or a T366S / L368A / Y407V substitution (EU numbering) such that heterodimerization of the anti-CD38 immunoglobulin heavy chain constant region and the non-anti-CD38 immunoglobulin heavy chain constant region is supported relative to homodimerization of the anti-CD38 immunoglobulin heavy chain. In some embodiments, the anti-CD19 immunoglobulin heavy chain variable region further comprises an immunoglobulin heavy chain constant region, the anti-CD19 immunoglobulin heavy chain constant region comprising one or more amino acid substitutions that do not support homodimerization of the anti-CD19 immunoglobulin heavy chain constant region but promote heterodimerization of a second heavy chain constant region with a non-anti-CD19 immunoglobulin heavy chain constant region. In some embodiments, the anti-CD19 immunoglobulin heavy chain constant region comprises a T366W substitution (EU numbering) or a T366S / L368A / Y407V substitution (EU numbering) such that heterodimerization of the anti-CD19 immunoglobulin heavy chain constant region and the non-anti-CD19 immunoglobulin heavy chain constant region is supported relative to homodimerization of the anti-CD19 immunoglobulin heavy chain.

[0013] In some embodiments, the anti-CD38 immunoglobulin light chain variable region further comprises an immunoglobulin light chain constant region. In some embodiments, the CD19 antigen binding component comprises a heavy chain immunoglobulin sequence set forth in SEQ ID NO: 301 or 304 and a light chain immunoglobulin sequence set forth in SEQ ID NO: 213, and the CD38 binding component comprises a heavy chain immunoglobulin sequence set forth in SEQ ID NO: 302, 303, 305-310 and a light chain immunoglobulin sequence set forth in SEQ ID NO: 213. In some embodiments, the anti-CD19 immunoglobulin heavy chain variable region comprises an A84S or A108L substitution according to the Kabat numbering. In some embodiments, the anti-CD38 immunoglobulin light chain variable region comprises a W32H substitution according to the Kabat numbering.

[0014] In some embodiments, a single bispecific binding molecule is formed from a CD38 antigen-binding component and a CD19 antigen-binding component. In some embodiments, the complex binding molecule is a common light chain bispecific antibody. In some embodiments, the bispecific binding molecule is part of a composition that includes a pharma- ceutically acceptable diluent, carrier, or excipient.

[0015] Also provided herein is a complex binding molecule comprising: (i) a CD38 antigen binding moiety that binds CD38; (ii) a CD19 antigen binding moiety that binds CD19; and (iii) a variant Fc region that comprises one or more mutations compared to a wild-type Fc region, wherein the variant Fc region exhibits an altered effector function compared to the wild-type Fc region; and wherein the CD38 antigen binding moiety: a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 71 to 75; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 81 to 85 or 151 to 155; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95; d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125; The CD19 antigen-binding component g) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15; h) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 21 to 25; i) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35; j) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; k) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 111 to 115; and l) A complex-binding molecule is provided, comprising a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 121-125.

[0016] Further provided herein is a complex binding molecule comprising: (i) a CD38 antigen binding moiety that binds CD38; (ii) a CD19 antigen binding moiety that binds CD19; and (iii) a variant Fc region that comprises one or more mutations selected from Table 1, wherein the CD38 antigen binding moiety is a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 71 to 75; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 81 to 85 or 151 to 155; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95; d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125; The CD19 antigen-binding component g) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15; h) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 21 to 25; i) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35; j) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; k) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 111 to 115; and l) A complex-binding molecule is provided, comprising a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 121-125.

[0017] 1. A complex binding molecule comprising: (i) a CD38 antigen binding moiety that binds CD38; (ii) a CD19 antigen binding moiety that binds CD19; and (iii) a variant Fc region that comprises an L234A, L235E, G237A, A330S, and / or P331S mutation (EU numbering), wherein the CD38 antigen binding moiety is a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 71 to 75; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 81 to 85 or 151 to 155; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95; d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125; The CD19 antigen-binding component g) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15; h) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 21 to 25; i) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35; j) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; k) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and l) A complex-binding molecule is provided, comprising a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 121-125.

[0018] Also provided is a complex binding molecule comprising: (i) a CD38 antigen binding moiety that binds to CD38; (ii) a CD19 antigen binding moiety that binds to CD19; and (iii) a variant Fc region that comprises a 322A mutation (EU numbering), wherein the CD38 antigen binding moiety is a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 71 to 75; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 81 to 85 or 151 to 155; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 91 to 95; d) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125; The CD19 antigen-binding component g) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15; h) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 21 to 25; i) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35; j) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; k) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 111 to 115; and l) A complex-binding molecule is provided, comprising a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 121-125. In some embodiments, the variant Fc region reduces ADCC by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or more compared to an antibody comprising a non-variant Fc region.

[0019] In some embodiments, the variant Fc region reduces CDC by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or more compared to an antibody comprising a non-variant Fc region.

[0020] Methods of treating an individual suffering from a cancer or tumor are provided, comprising administering to an individual suffering from a cancer or tumor a complex binding molecule described herein, thereby treating the cancer or tumor.

[0021] In some embodiments, the cancer or tumor is a hematological cancer. In some embodiments, the hematological cancer is a B-cell malignancy. In some embodiments, the B-cell malignancy is a B-cell acute lymphocytic leukemia. In some embodiments, the B-cell malignancy is a chronic lymphocytic leukemia, a small lymphocytic lymphoma, a mantle cell lymphoma, or a non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, follicular lymphoma). In some embodiments, the hematological cancer is a plasma malignancy. In some embodiments, the plasma malignancy is multiple myeloma. In some embodiments, the hematological cancer expresses CD19 and CD38.

[0022] In some embodiments, the cancer or tumor is a solid tissue cancer. In some embodiments, the solid tissue cancer comprises breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, stomach cancer, esophageal cancer, skin cancer, colon cancer, or head and neck cancer. In some embodiments, the breast cancer is triple negative breast cancer, the lung cancer is non-small cell lung cancer, the head and neck cancer is head and neck squamous cell carcinoma, the kidney cancer is renal cell carcinoma, the brain tumor is glioblastoma multiforme, or the skin cancer is melanoma.

[0023] Also provided is a method of reducing tumor infiltrating B cells in, adjacent to, or surrounding a tumor in an individual afflicted with a tumor or cancer, comprising the step of reducing tumor infiltrating B cells in the tumor by administering to the individual afflicted with a tumor or cancer a complex binding molecule as described herein.

[0024] Provided is a method of reducing immunosuppressive B cells in, adjacent to, or surrounding a tumor in an individual afflicted with a tumor or cancer, comprising reducing immunosuppressive B cells in the tumor by administering to the individual afflicted with a tumor or cancer a complex binding molecule as described herein.

[0025] Further provided is a method of inhibiting the function of immunosuppressive B cells in, adjacent to, or around a tumor in an individual suffering from a tumor or cancer, comprising administering to the individual suffering from a tumor or cancer a complex binding molecule as described herein, thereby reducing immunosuppression by immunosuppressive B cells in the tumor. Also provided is a method of inhibiting the function of immunosuppressive B cells in, adjacent to, or around a tumor, comprising contacting the immunosuppressive B cells with a complex binding molecule as described herein, thereby reducing immunosuppression by immunosuppressive B cells in the tumor.

[0026] In some embodiments, the function of the immunosuppressive B cells comprises release of IL-10, IL-35, TGF-β, or a combination thereof. In some embodiments, the tumor infiltrating B cells or immunosuppressive B cells comprise CD19 positive B cells, CD38+ positive B cells, CD19·CD38 double positive B cells, or a combination thereof.

[0027] Also provided are methods of making the complex binding molecules described herein, comprising incubating cells in cell culture medium under conditions sufficient to allow expression, assembly, and secretion of the complex binding molecules into the cell culture medium, hi some embodiments, the method comprises isolating and purifying the molecules from the cell culture medium.

[0028] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0029] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0030] [Figure 1] 1 illustrates the structure of a common light chain bispecific IgG. [Diagram 2] Illustrates the structure of Fab-Fc:scFv-Fc bispecific IgG. [Diagram 3] 1 illustrates the structure of Fab-Fc-Fab:Fc bispecific IgG. [Figure 4] Fab-Fc-scFv: Illustrates the structure of a Fab-Fc-scFv bispecific IgG. [Diagram 5] Illustrates the structure of Fab-Fc-scFv:Fc bispecific IgG. [Figure 6] 1 illustrates the structure of Fab-Fc-Fab:Fab-Fc bispecific IgG. [Figure 7] Illustrates the structure of scFv-Fab-Fc: scFv-Fab-Fc bispecific IgG. [Figure 8] Fab-Fab-Fc: Fab-Fab-Fc bispecific IgG structure. [Figure 9] Fab-Fc-Fab: Fab-Fc-Fab bispecific IgG structure is illustrated. [Figure 10] 1 illustrates the structure of Fab-Fc-scFv: Fab-Fc bispecific IgG. [Figure 11] 1 illustrates the structure of scFv-Fab-Fc:Fc bispecific IgG. [Figure 12A] FIG. 1 shows binding data of antibodies to Daudi cells. [Figure 12B] FIG. 1 shows binding data of antibodies to Daudi cells. [Figure 13A] FIG. 13 shows binding data of antibodies to REH cells. [Figure 13B] FIG. 13 shows binding data of antibodies to REH cells. [Figure 14A] FIG. 1 shows antibody binding data to CD19-transfected HEK293 cells. [Figure 14B] FIG. 1 shows antibody binding data to CD19-transfected HEK293 cells. [Figure 15A] FIG. 1 shows antibody binding data to CD38-transfected HEK293 cells. [Figure 15B] FIG. 1 shows antibody binding data for CD38-transfected HEK293 cells. [Figure 16A] FIG. 1 shows antibody binding data to non-transfected CHO cells. [Figure 16B] FIG. 1 shows antibody binding data to non-transfected CHO cells. [Figure 17A] FIG. 1 shows data on direct apoptosis of Daudi cells for antibody test articles. [Figure 17B] FIG. 1 shows data on direct apoptosis of Daudi cells for antibody test articles. [Figure 18A] FIG. 13 shows data on crosslinking-induced apoptosis in Daudi cells for antibody test substances. [Figure 18B] FIG. 13 shows data on crosslinking-induced apoptosis in Daudi cells for antibody test substances. [Figure 19A] FIG. 1 shows ADCC data for three donors across antibody test articles. [Figure 19B] FIG. 1 shows ADCC data for three donors across antibody test articles. [Figure 19C] FIG. 1 shows ADCC data for three donors across antibody test articles. [Figure 20A] FIG. 1 shows ADCC data for three donors across antibody test articles. [Figure 20B]FIG. 1 shows ADCC data for three donors across antibody test articles. [Figure 20C] FIG. 1 shows ADCC data for three donors across antibody test articles. [Figure 21A] FIG. 1 shows CDC profiles across test articles. [Figure 21B] FIG. 1 shows CDC profiles across test articles. [Figure 22] FIG. 1 shows ADCP data across antibody test articles. [Figure 23] FIG. 13 shows RBC binding data across antibody test articles. [Figure 24A] FIG. 1 shows the hemagglutination profile for antibody test articles. [Figure 24B] FIG. 1 shows the hemagglutination profile for antibody test articles. [Diagram 25] FIG. 13 shows hemolysis data across antibody test articles. [Figure 26A] FIG. 1 shows ADCC data for three donors across antibody test articles, including those with variants. [Figure 26B] FIG. 1 shows ADCC data for three donors across antibody test articles, including those with variants. [Figure 26C] FIG. 1 shows ADCC data for three donors across antibody test articles, including those with variants. [Figure 26D] FIG. 1 shows ADCC data for three donors across antibody test articles, including those with variants. [Figure 26E] FIG. 1 shows ADCC data for three donors across antibody test articles, including those with variants. [Figure 26F] FIG. 1 shows ADCC data for three donors across antibody test articles, including those with variants. [Figure 26G] FIG. 1 shows ADCC data for three donors across antibody test articles, including those with variants. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Immunosuppressive B cell populations that suppress anti-tumor immune responses can generally be defined by the presence of more than one cell surface biomarker. Thus, therapeutics that effectively and specifically target immunosuppressive B cells can be used to prevent and / or remove immunosuppression in, adjacent to, or around a tumor or within the tumor environment. Provided herein are complex binding molecules that target immunosuppressive B cells. Also provided are complex binding molecules that include a first binding moiety configured to bind to a first target and a second binding moiety configured to bind to a second target, where the first target includes a B cell lineage surface marker and the second target includes an inhibitory B cell surface marker. Disclosed herein are multivalent antibodies that specifically bind to B cell populations associated with negative regulation or immunosuppression of anti-tumor responses. Immunosuppressive B cells can include or be defined by the cell surface biomarkers CD19 and CD38. The bispecific antibodies provided herein can target both CD19 and CD38 to inhibit the function of immunosuppressive B cells. In certain cases, the function of immunosuppressive B cells includes the release of IL10, IL35, TGF-β, or a combination thereof. Multivalent or bispecific antibodies targeting CD19 and CD38 can also be used to treat tumorigenic conditions and / or cancers associated with immunosuppressive B cells and / or immune dysfunction.

[0032] The terms "immunosuppression", "immunodepression", or "negative immune regulation" as used herein refer to a reduction or suppression of immune system function, i.e., immune suppression generally refers to a state in which immune system function is reduced or absent. In certain cases, immune suppression generally refers to a state in which immune system function is reduced or absent against a tumor or in, around, or adjacent to the tumor microenvironment. The overall immune response may be suppressed, the immune response in a local or specific area may be reduced, or a specific population of immunologically active lymphocytes may be selectively affected. Antigen-specific immune suppression may be the result of deletion or suppression of a specific population of antigen-specific cells, or the result of improved regulation of the immune response by antigen-specific suppressor cells. Reference to immunosuppressive B cells refers to B cells or B cell populations that exert a negative regulation on the immune response, and may be identified by specific surface markers associated with such populations, such as CD38. In certain cases, immunosuppression can be identified by the presence or release of IL-10, IL-35, TGF-β, or a combination thereof. In certain cases, immunosuppression can be identified by the presence or release of IL-10, IL-35, TGF-β, or a combination thereof by B cells.

[0033] As used herein, the term "cancer" refers to or can describe a physiological condition in mammals that is typically characterized by unregulated cell growth.Cancer can also include, but is not limited to, blood tumors and / or solid tumors.Cancer can refer to diseases of blood, bone, organs, skin tissue, and vascular system, including, but not limited to, bladder, blood, bone, brain, breast, cervix, breast, colon, endometrium, esophagus, eye, head, kidney, liver, lung, lymph nodes, mouth, cervix, ovary, pancreas, prostate, rectum, skin, stomach, testes, pharynx, and uterus. Specific cancers include leukemia (acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, mature B-cell tumors (small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmocytic lymphoma (Waldenstrom's giant cell tumor) ball), proteinemia, or low-grade lymphoma), splenic marginal zone lymphoma, plasma cell myeloma, plasma cell leukemia, plasmacytoma, peri-implant immunoglobulin deposition, heavy chain disease, extranodal marginal zone B-cell lymphoma, MALT lymphoma), nodal marginal zone B-cell lymphoma (NMZL), gastrointestinal tumors (e.g., gastrointestinal stromal tumor (GIST)), follicular lymphoma, mantle cell lymphoma / leukemia, diffuse B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, and Burkitt's lymphoma (Burkitt's lymphoma), mature T-cell and natural killer cell (NK) tumors (prolymphocytic leukemia, T-cell large lymphocy ... leukemia, invasive NK cell leukemia, adult T cell leukemia / lymphoma, extranodal NK / T cell lymphoma, enteropathic T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides (Sézary syndrome), primary Skin degenerative large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma not otherwise specified and degenerative large cell lymphomalymphoma), Hodgkin's lymphoma (nodular sclerosis, mixed cell type, lymphocyte-rich, lymphocyte-depleted or non-depleted, nodular lymphocytic), myeloma (multiple myeloma, inert myeloma myeloma, smoldering myeloma), chronic myeloproliferative disorders, myelodysplastic syndromes / myeloproliferative disorders, myelodysplastic syndromes, lymphoproliferative disorders associated with immunodeficiency, histiocytic and dendritic cell neoplasms, leukocytosis, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant giant cell tumor, myeloma bone disease, osteosarcoma, breast cancer (hormone-dependent and non-hormone-dependent), gynecological cancers (pediatric cervical, endometrial, fallopian tube, gestational trophoblastic disease, ovarian, peritoneal, uterine, vaginal, and vulvar), basal cell carcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma, protuberous dermatofibrosarcoma, Merkel cell carcinoma, Kaposi's sarcoma, astrocytoma, hairy cell astrocytoma, embryonic hair growth neuroepithelial neoplasia neoplasia), oligodendroglioma, ependymoma, glioblastoma multiforme, mixed glioma, oligodendroglial astrocytoma, medulloblastoma, retinoblastoma, neuroblastoma, embryonal tissue tumor, teratoma, malignant mesothelioma (peritoneal mesothelioma, pericardial mesothelioma, pleural mesothelioma), gastric-entero-pancreatic or gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor, pancreatic endocrine tumor (PET), colorectal adenocarcinoma, knot rectangular cancer, invasive neuroendocrine tumor, leiomyosarcoma, mucinous adenocarcinoma, signet ring cell adenocarcinoma, hepatocellular carcinoma, hepatobiliary liver cancer These include, but are not limited to, hepatoblastoma, hemangioma, hepatic adenoma, focal nodular hyperplasia (nodular regenerative hyperplasia, hamartoma), non-small cell lung cancer (NSCLC) (squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma), small cell lung carcinoma, thyroid carcinoma, prostate cancer (hormone refractory, non-androgen dependent sex, androgen dependent, hormone insensitive), renal cell carcinoma and soft tissue sarcomas (fibrosarcoma, malignant fibrous histiocytoma, dermatofibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor / neurofibrosarcoma, extraskeletal osteosarcoma).

[0034] The term "CD19" or "cluster of differentiation 19" (also known as B4, T cell surface antigen Leu-12, and CVID3) refers to a B cell lineage surface biomarker or transmembrane protein encoded in humans by the gene CD19. CD19 functions as a coreceptor for the B cell antigen receptor complex (BCR) on B lymphocytes, and can reduce the threshold for activation of downstream signaling pathways and for inducing a B cell response to antigen. Structurally, the CD19 amino acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity over a sequence length of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 amino acids, or over the entire length of the polypeptide, to the amino acid sequence of, for example, GenBank Accession No. NM_001178098.2→NP_001171569.1 or NM_001770.6→NP_001761.3. Structurally, a CD19 nucleic acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity over a sequence length of at least 300, 500, 750, 1000, 1250, 1500 nucleic acids, or over the entire length of the polynucleotide, for example, to the amino acid sequence of GenBank Accession No. NG_007275.1 or NCBI Gene ID 930. Sequence alignment can be performed using any alignment algorithm known in the art, for example, BLAST or ALIGN set to default settings.

[0035] The term "CD38" or "cluster of differentiation 38" (also known as ADPRC1) refers to a B cell surface biomarker or transmembrane protein encoded in humans by the gene CD38. CD38 can function in B cell signaling leading to cell activation and proliferation. Structurally, the CD38 amino acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity over a sequence length of at least 50, 100, 150, 200, 250 amino acids, or over the entire length of the polypeptide, to the amino acid sequence of, for example, GenBank Accession No. NM_001775.4→NP_001766.2. Structurally, a CD19 nucleic acid sequence has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity over a sequence length of at least 300, 500, 750 nucleic acids, or over the entire length of the polynucleotide, for example, to a nucleic acid sequence of GenBank Accession No. NC_000004.12 or NCBI Gene ID 952. Sequence alignment can be performed using any alignment algorithm known in the art, for example, BLAST or ALIGN set to default settings.

[0036] The term "antibody" herein is used in the broadest sense and includes multivalent or bispecific antibodies and monoclonal antibodies, such as intact antibodies and functional (antigen-binding) antibody fragments thereof, such as fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, such as single-chain variable fragments (sFv or scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies, such as bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full length antibodies, including antibodies of any class or subclass, including IgG, and its subclasses IgM, IgE, IgA, and IgD. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region.

[0037] Among the antibodies provided are multispecific or multivalent antibodies (e.g., bispecific and polyreactive antibodies) and antibody fragments thereof. Antibodies include antibody conjugates, and molecules that contain antibodies, such as chimeric molecules. Thus, antibodies include full length and native antibodies, as well as fragments and portions thereof that retain their binding specificity, including, but not limited to, any specific binding portion thereof, such as those having any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM), and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including, but not limited to, Fab, F(ab')2, Fv, and scFv (single chain or related entities). Monoclonal antibodies are generally in a composition of substantially homogeneous antibodies, such that every individual antibody contained within a monoclonal antibody composition is identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies can include a human IgG1 constant region or a human IgG4 constant region.

[0038] The terms "complementarity determining region" and "CDR", which are synonymous with "hypervariable region" or "HVR", are known in the art and refer to non-contiguous sequences of amino acids in antibody variable regions that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of heavy and light chains. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.The precise amino acid sequence boundaries of a given CDR or FR can be determined by 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); MacCallum et al., "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 (1996) ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan;27(1):55-77 (the "IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, 2001 Jun 8;309(3):657-70 (the "Aho" numbering scheme); and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modelling antibodies on the WEB", Protein Eng. 2000 Dec;13(12):819-24 (the "AbM" numbering scheme).In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0039] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering of both the Kabat scheme and the Chothia scheme is based on the length of the most common antibody region sequence, with insertions applied by the insertion letter, e.g., "30a", and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in differential numbering. The Contact scheme is based on the analysis of complex crystal structures, and is similar in many ways to the Chothia numbering scheme.

[0040] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) have an overall similar structure, with each domain containing four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). H or V L The domain may be sufficient to confer antigen-binding specificity. Furthermore, an antibody that binds to a particular antigen may be able to derive a V domain from the antibody that binds to the antigen. H or V L The domains can be used to screen libraries of complementary VL or VH domains, respectively (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0041] Among the antibodies provided are antibody fragments. An "antibody fragment" can refer to a molecule other than an intact antibody that includes a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv or sFv), and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment that includes a variable heavy chain region and / or a variable light chain region, such as an scFv. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment that includes a non-naturally occurring configuration, such as having two or more antibody regions or chains joined by a synthetic linker, e.g., a polypeptide linker, and / or is not produced by enzymatic digestion of a naturally occurring intact antibody.

[0042] As used herein, a molecule, peptide, polypeptide, antibody, or antibody fragment may be referred to as "bispecific" or "bispecific," including grammatical equivalents. A bispecific molecule has the ability to specifically bind to at least two structurally distinct targets. Specific binding can be achieved by two distinct binding moieties that are structurally distinct at the molecular level, including, but not limited to, distinct, non-identical amino acid sequences, or by binding with high affinity (e.g., about 1×10 -6This can be the result of a single binding moiety that can specifically bind to two structurally distinct targets with a KD of less than 100 kD. A molecule, peptide, polypeptide, antibody, or antibody fragment referred to as "multispecific" refers to a molecule that has the ability to specifically bind to at least three structurally distinct targets. A "bispecific antibody", including grammatical equivalents, refers to a bispecific molecule that preserves at least one fragment of an antibody, such as a variable region, heavy or light chain, or one or more complementarity determining regions from an antibody molecule, that can specifically bind to a target. A "multispecific antibody", including grammatical equivalents, refers to a multispecific molecule that preserves at least one fragment of an antibody, such as a variable region, heavy or light chain, or one or more complementarity determining regions from an antibody molecule, that can specifically bind to a target.

[0043] "Linker" is also referred to herein as "linker sequence", "spacer", "tethering sequence" or grammatical equivalents thereof. "Linker" as referred to herein connects two separate molecules that themselves have target binding, catalytic activity, or are naturally expressed and assembled as separate polypeptides. For example, two separate binding moieties or heavy / light chains are paired. Numerous strategies can be used to covalently link the molecules. These include, but are not limited to, polypeptide linkages between the N-terminus and C-terminus of a protein or protein domain, linkages via disulfide bonds, and linkages via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond generated by recombinant techniques or peptide synthesis. The linker peptide may primarily comprise the amino acid residues Gly, Ser, Ala, or Thr. The linker peptide must be of sufficient length to link the two molecules so that they assume the correct conformation relative to each other and thus retain the desired activity. In one embodiment, the linker is about 1-50 amino acids long or about 1-30 amino acids long. In one embodiment, a linker of 1-20 amino acids long may be used. Useful linkers include glycine-serine polymers, such as (GS)n, (GSGGS)n (SEQ ID NO: 224), (GGGGS)n (SEQ ID NO: 225), and (GGGS)n (SEQ ID NO: 226), where n is an integer of at least 1, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Exemplary linkers for linking antibody fragments or single chain variable fragments can include AAEPKSS (SEQ ID NO: 227), AAEPKSSDKTHTCPPCP (SEQ ID NO: 228), GGGG (SEQ ID NO: 229), or GGGGDKTHTCPPCP (SEQ ID NO: 230). Alternatively, various non-proteinaceous polymers can be used as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.

[0044] A "fragment-based" bispecific antibody, or a bispecific antibody comprising a "single-chain variable fragment" or "scFv" of the present disclosure, may refer to a single-chain antibody or fragment thereof, comprising two binding moieties and a linker connecting the two binding moieties. The linker may be a polypeptide linker or other suitable and flexible linker that does not inhibit binding of either targeting moiety. Fragment-based bispecific antibody formats include tandem VHH antibodies, tandem scFv, scFv-Fab, F(ab)2, dual affinity retargeting antibodies (DART). Such fragment-based antibodies can be further engineered to include additional binding moieties with specificity for a given target, e.g., A2:B1, A1:B2, or A2:B2, or with fragments of the Fc region to improve pharmacokinetics or promote ADCC, ADCP, or CDC.

[0045] "Binding moiety" refers to the portion of a molecule, peptide, polypeptide, antibody, or antibody fragment that mediates specific binding to a recited target, antigen, or epitope. By way of example, the binding portion of an antibody may comprise a heavy / light chain variable region pair, or one or more complementarity determining regions (CDRs).

[0046] A "target" as referred to herein refers to a portion of a molecule that is associated with the binding portion of a molecule, peptide, polypeptide, antibody, or antibody fragment. A target can include an amino acid sequence and / or a carbohydrate, lipid, or other chemical entity. An "antigen" is a target that includes a portion that can be bound by an adaptive immune molecule, such as an antibody or antibody fragment, a B cell receptor, or a T cell receptor.

[0047] The "valency" of a bispecific or multispecific molecule refers to the number of targets to which the recited molecule, peptide, polypeptide, antibody, or antibody fragment can bind. For example, a monovalent molecule can bind one molecule of a specific target, a bivalent molecule can bind two molecules, and a tetravalent molecule can bind four targets. For example, a bispecific and bivalent molecule is a molecule that can bind two targets, and two structurally different targets. For example, a bispecific and bivalent molecule, when contacted with a solution containing target A and target B, can bind to A2, B2, or A:B.

[0048] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from a non-human CDR and all or substantially all FR amino acid residues are derived from a human FR. A humanized antibody can optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody typically refers to a variant of a non-human antibody that has undergone humanization to retain the specificity and affinity of the parent non-human antibody while reducing immunogenicity to humans. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0049] Among the antibodies provided are human antibodies. A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or a non-human source utilizing a human antibody repertoire, including a human antibody library, or other human antibody coding sequence. The term excludes humanized forms of non-human antibodies that contain a non-human antigen-binding region, such as those in which all or substantially all CDRs are non-human. Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus or that is extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin locus is totally inactivated. Human antibodies may also be derived from human antibody libraries, including phage display and cell-free libraries, that contain antibody coding sequences derived from a human repertoire.

[0050] "ADCC" or "antibody-dependent cell-mediated cytotoxicity" as used herein refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibodies on target cells, resulting in subsequent lysis of the target cells. ADCC can be correlated with binding to FcγRIIIa, and increased binding to FcγRIIIa leads to an increase in ADCC activity. "ADCP" or antibody-dependent cell-mediated phagocytosis as used herein can refer to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibodies on target cells, resulting in subsequent phagocytosis of the target cells.

[0051] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including the antibodies and antibody chains provided and other peptides, such as linkers and connecting peptides, can contain amino acid residues, including natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, a polypeptide can contain modifications to the native or natural sequence, so long as the protein maintains the desired activity. These modifications can be deliberate, such as through site-directed mutagenesis, or can be accidental, such as through mutations of the host that produces the protein or errors due to PCR amplification.

[0052] Percent sequence identity (%) to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, without considering any conservative substitutions as part of sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence identity percentage. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways known in the art, for example, using computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software, which are publicly available. Suitable parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes herein, values ​​of percent amino acid sequence identity are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office, Washington DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.In the situation where ALIGN-2 is employed for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to a given amino acid sequence B) is calculated as 100 x fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be recognized that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0053] Amino acid sequence variants of the antibodies provided herein can be envisioned and envisioned. Variants typically differ from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the present invention and evaluating one or more biological activities of the polypeptide as described herein and / or using any of a number of known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody, and amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct has the desired characteristics, e.g., antigen binding. Antibody variants with one or more amino acid substitutions can be provided. Sites of interest for substitutional mutagenesis include CDRs and FRs. Amino acid substitutions can be introduced into an antibody of interest and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0054] The present disclosure also provides "immunoconjugates," "antibody conjugates," or "antibody-drug conjugates," which refer to an antibody conjugated to one or more heterologous molecules. For example, an immunoconjugate can include one or more cytotoxic agents, such as an antibody conjugated to a chemotherapeutic agent or drug, a growth inhibitory agent, a protein domain, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope. In some embodiments, an immunoconjugate can include a complex binding molecule disclosed herein, or a fragment thereof (e.g., an scFv).

[0055] The antibodies described herein can be encoded by nucleic acids. A nucleic acid is a type of polynucleotide that contains two or more nucleotide bases. In certain embodiments, a nucleic acid is a component of a vector that can be used to transfer a polynucleotide encoding a polypeptide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. One type of vector is a genome-integrating vector, or "integrating vector," which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. A vector capable of directing the expression of a gene operably linked thereto is referred to herein as an "expression vector." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In an expression vector, regulatory elements such as promoters, enhancers, polyadenylation signals, and the like, used to control transcription can be derived from mammalian, microorganism, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from viruses such as lentivirus, retrovirus, adenovirus, and adeno-associated virus may be employed. Plasmid vectors may be linearized for integration into chromosomal locations. Vectors may contain sequences that direct site-specific integration (e.g., AttP-AttB recombination) into a defined location or a limited set of sites in the genome. Additionally, vectors may contain sequences derived from transposable elements.

[0056] As used herein, the terms "homologous", "homology", or "percent homology" as used herein to describe an amino acid sequence or a nucleic acid sequence compared to a reference sequence can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990; revised in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula is incorporated into the basic local alignment search tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent sequence homology can be determined using the most recent version of BLAST as of the filing date of this application.

[0057] Nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform, or otherwise render suitable cells transgenic for the nucleic acid, thereby enabling production of the antibody for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large scale cell culture are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production." Mabs. 2010 Sep-Oct;2(5):466-477. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a cell line useful for producing antibodies and is a Chinese Hamster Ovary (CHO) cell, an NS0 mouse myeloma cell, or a PER.C6® cell. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of a cell useful for producing the antibody. In certain embodiments, described herein is a method of making an antibody, comprising culturing a cell comprising a nucleic acid encoding the antibody under in vitro conditions sufficient to allow production and secretion of the antibody.

[0058] As used herein, the terms "individual," "patient," or "subject" refer to an individual who has been diagnosed with, is suspected of having, or is at risk of developing at least one disease for which the described compositions and methods are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In certain embodiments, the individual is a human.

[0059] As used herein, the term "about" used to modify a particular number refers to that number plus or minus 10%. The term "about" used to modify a range refers to a range extending from minus 10% of its minimum value to plus 10% of its maximum value.

[0060] As used herein, the term "treatment" or "treating" is used in reference to a pharmaceutical or other intervention regimen used to obtain a beneficial or desired outcome in a recipient. Beneficial or desired outcome includes, but is not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit may refer to the eradication or amelioration of symptoms or of the underlying disorder being treated. Therapeutic benefit may also be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, even though the subject may still be afflicted by the underlying disorder. Prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or illness, delaying or eliminating the onset of symptoms of a disease or illness, slowing, halting, or reversing the progression of a disease or illness, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease or reporting one or more of the physiological symptoms of a disease may receive treatment even if a diagnosis of the disease has not been made. One of skill in the art will recognize that not all of a given population of individuals who are candidates for treatment will respond, or will respond equally, to the treatment, and such individuals will be considered to have been treated.

[0061] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0062] bispecific molecule Provided herein is a bispecific, multivalent, or complex binding molecule comprising a first binding moiety configured to bind to a first target and a second binding moiety configured to bind to a second target, where the first target comprises a B cell lineage surface marker and the second target comprises an inhibitory B cell surface marker. The immunosuppressive B cell or B cell population can comprise a B cell lineage surface biomarker and an inhibitory B cell surface biomarker. The B cell lineage surface marker can comprise CD19, CD138, IgA, or CD45. The immunosuppressive B cell surface marker can comprise IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-βLAP). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38. In certain embodiments, the complex binding molecule binds to CD38 and CD19.

[0063] A bispecific, multivalent, or complex binding molecule has the ability to specifically bind to at least two structurally distinct targets. Specific binding can be the result of two separate binding moieties that are structurally distinct at the molecular level, including but not limited to distinct non-identical amino acid sequences, or a single binding moiety that can specifically bind to two structurally distinct targets. A molecule, peptide, polypeptide, antibody, or antibody fragment referred to as "multispecific," "multivalent," or "bispecific" can refer to a molecule that has the ability to specifically bind to at least two structurally distinct targets. In some embodiments, the first or second binding component of the complex binding molecule comprises a polypeptide. In certain embodiments, the first or second binding component consists of a polypeptide. In some embodiments, the first and second binding components of the complex binding molecule comprise a polypeptide. In certain embodiments, the first and second binding components consist of a polypeptide. In certain embodiments, the polypeptide of the first or second binding component comprises an amino acid sequence of at least 100 amino acid residues in length. In certain embodiments, the polypeptides of the first and second binding moieties comprise amino acid sequences at least 100 amino acid residues in length.

[0064] A bispecific molecule may be a bispecific antibody that preserves at least one fragment of an antibody capable of specifically binding to a target, such as a variable region, a heavy or light chain, or one or more complementarity determining regions from an antibody molecule. In some embodiments, the complex binding molecule described herein is a bispecific antibody and / or a dual antigen-binding fragment thereof. A bispecific antibody has the ability to bind to two structurally distinct targets or antigens. In some embodiments, a bispecific antibody comprises a first binding component configured to bind to a first target and a second binding component configured to bind to a second target, the first target comprising a B cell lineage surface marker (e.g., CD19, CD138, IgA, or CD45) and the second target comprising an inhibitory B cell surface marker (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-βLAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD 19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0065] The immunosuppressive B cell or immunosuppressive B cell population can include cell surface biomarkers CD19 and CD38. Further disclosed herein are bispecific antibodies targeting CD19 and CD38. In some embodiments, the CD19 binding component comprises a variable heavy chain (VH) comprising SEQ ID NO:1. In certain embodiments, the CD19 binding component comprises a VH CDR1 region comprising any one of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15. In certain embodiments, the CD19 binding component comprises a VH CDR2 region comprising any one of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25. In certain embodiments, the CD19 binding component comprises a VH CDR3 region comprising any one of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35.

[0066] In some embodiments, the CD19 binding component comprises a variable light chain (VL) comprising SEQ ID NO:2. In certain embodiments, the CD19 binding component comprises a VL CDR1 region comprising any one of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, or SEQ ID NO:45. In certain embodiments, the CD19 binding component comprises a VL CDR2 region comprising any one of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, or SEQ ID NO:55. In certain embodiments, the CD19 binding component comprises a VL CDR3 region comprising any one of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, or SEQ ID NO:65.

[0067] In some embodiments, the bispecific antibody comprises a first binding moiety, wherein the first binding moiety comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 21-25, an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 31-35, an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 41-45, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 51-55, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61-65.

[0068] In some embodiments, the bispecific antibody comprises a CD19 binding component, wherein the CD19 binding component comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 11, an HCDR2 amino acid sequence set forth in SEQ ID NO: 21, an HCDR3 amino acid sequence set forth in SEQ ID NO: 31, an LCDR1 amino acid sequence set forth in SEQ ID NO: 41, an LCDR2 amino acid sequence set forth in SEQ ID NO: 51, and / or an LCDR3 amino acid sequence set forth in SEQ ID NO: 61.

[0069] In some embodiments, the bispecific antibody comprises a CD19 binding component, and the first CD19 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 12, the HCDR2 amino acid sequence set forth in SEQ ID NO: 22, the HCDR3 amino acid sequence set forth in SEQ ID NO: 32, the LCDR1 amino acid sequence set forth in SEQ ID NO: 42, the LCDR2 amino acid sequence set forth in SEQ ID NO: 52, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO: 62.

[0070] In some embodiments, the bispecific antibody comprises a CD19 binding component, wherein the CD19 binding component comprises an HCDR1 amino acid sequence set forth in SEQ ID NO: 15, an HCDR2 amino acid sequence set forth in SEQ ID NO: 25, an HCDR3 amino acid sequence set forth in SEQ ID NO: 35, an LCDR1 amino acid sequence set forth in SEQ ID NO: 45, an LCDR2 amino acid sequence set forth in SEQ ID NO: 55, and / or an LCDR3 amino acid sequence set forth in SEQ ID NO: 65.

[0071] In some embodiments, the CD19 binding comprises variable heavy and light chains or CDRs corresponding to or derived from inebilizumab, tafasitamab, taplitumomab, obexelimab, blinatumomab, cortuximab, denintuzumab, or loncastuximab, MOR208, MEDI-551, XmAb 5871, MDX-1342, or AFM11.

[0072] In some embodiments, the CD38 binding component comprises a variable heavy chain (VH) comprising SEQ ID NO: 3. In certain embodiments, the CD19 binding component comprises a VH CDR1 region comprising any one of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75. In certain embodiments, the CD19 binding component comprises a VH CDR2 region comprising any one of SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, or SEQ ID NO: 85. In certain embodiments, the CD19 binding component comprises a VH CDR3 region comprising any one of SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, or SEQ ID NO: 95.

[0073] In some embodiments, the CD38 binding component comprises a variable light chain (VL) comprising SEQ ID NO: 4. In certain embodiments, the CD19 binding component comprises a VL CDR1 region comprising any one of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, or SEQ ID NO: 105. In certain embodiments, the CD19 binding component comprises a VL CDR2 region comprising any one of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 115. In certain embodiments, the CD19 binding component comprises a VL CDR3 region comprising any one of SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, or SEQ ID NO: 125.

[0074] In some embodiments, the bispecific antibody comprises a CD38 binding component, wherein the CD38 binding component comprises an HCDR1 amino acid sequence set forth in SEQ ID NO:71, an HCDR2 amino acid sequence set forth in SEQ ID NO:81, an HCDR3 amino acid sequence set forth in SEQ ID NO:91, an LCDR1 amino acid sequence set forth in SEQ ID NO:101, an LCDR2 amino acid sequence set forth in SEQ ID NO:111, and / or an LCDR3 amino acid sequence set forth in SEQ ID NO:121.

[0075] In some embodiments, the bispecific antibody comprises a CD38 binding component, wherein the CD38 binding component comprises an HCDR1 amino acid sequence set forth in SEQ ID NO:72, an HCDR2 amino acid sequence set forth in SEQ ID NO:82, an HCDR3 amino acid sequence set forth in SEQ ID NO:92, an LCDR1 amino acid sequence set forth in SEQ ID NO:102, an LCDR2 amino acid sequence set forth in SEQ ID NO:112, and / or an LCDR3 amino acid sequence set forth in SEQ ID NO:122.

[0076] In some embodiments, the bispecific antibody comprises a CD38 binding component, wherein the CD38 binding component comprises an HCDR1 amino acid sequence set forth in SEQ ID NO:75, an HCDR2 amino acid sequence set forth in SEQ ID NO:85, an HCDR3 amino acid sequence set forth in SEQ ID NO:95, an LCDR1 amino acid sequence set forth in SEQ ID NO:105, an LCDR2 amino acid sequence set forth in SEQ ID NO:115, and / or an LCDR3 amino acid sequence set forth in SEQ ID NO:125.

[0077] In some embodiments (e.g., any of the preceding embodiments), the CDR-H2 of the CD38 binding component comprises amino acid residues P(X1)LG(X2)A (SEQ ID NO: 150), where X1 and X2 tolerate amino acid substitutions while maintaining binding to CD38. In certain embodiments, X1 and X2 are selected from amino acids that reduce the hydrophobicity of the CDRH2 amino acid sequence. In certain embodiments, amino acids that reduce hydrophobicity include H, Q, T, N, S, G, A, R, K, D, or E. In certain embodiments, X1 is H and X2 is T.

[0078] In some embodiments, the bispecific antibody comprises a CD38 binding component and a CD19 binding component, the CD38 binding component comprises a VH amino acid sequence and a VL amino acid sequence, the VH amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:3, and the VL comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:4, and the CD19 binding component comprises a VH amino acid sequence and a VL amino acid sequence, the VH amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:1, and the VL comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:2.

[0079] In some embodiments, the bispecific antibody comprises a CD38 binding component and a CD19 binding component, the CD38 binding component comprises a VH amino acid sequence and a VL amino acid sequence, the VH amino acid sequence comprises an amino acid sequence identical to SEQ ID NO:3 and the VL comprises an amino acid sequence identical to SEQ ID NO:4, and the CD19 binding component comprises a VH amino acid sequence and a VL amino acid sequence, the VH amino acid sequence comprises an amino acid sequence identical to SEQ ID NO:1 and the VL comprises an amino acid sequence identical to SEQ ID NO:2.

[0080] In some embodiments, the bispecific antibody comprises a CD38 binding component and a CD19 binding component, wherein the CD38 binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:3, 215, or 218-223, and the VL comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:4 or 223, and the CD19 binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:1, 201, or 216-217, and the VL comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:2. In some embodiments, the CD19 binding component comprises a VH amino acid sequence that comprises substitutions at A84 and A108. In some embodiments, the substitutions comprise A84S and A108L.

[0081] In some embodiments, the bispecific antibody comprises a CD38 binding component and a CD19 binding component, wherein the CD38 binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence identical to SEQ ID NO: 3, 215, or 218-223, and the VL comprises an amino acid sequence identical to SEQ ID NO: 4 or 223, and the CD19 binding component comprises a VH amino acid sequence and a VL amino acid sequence, wherein the VH amino acid sequence comprises an amino acid sequence identical to SEQ ID NO: 1, 201, 216-217, and the VL comprises an amino acid sequence identical to SEQ ID NO: 2. In some embodiments, the CD19 binding component comprises a VH amino acid sequence comprising substitutions at A84 and A108. In some embodiments, the substitutions comprise A84S and A108L.

[0082] In some embodiments, the bispecific antibody comprises a CD38 binding component and a CD19 binding component, wherein the CD38 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:71, the HCDR2 amino acid sequence set forth in SEQ ID NO:81, the HCDR3 amino acid sequence set forth in SEQ ID NO:91, the LCDR1 amino acid sequence set forth in SEQ ID NO:101, the LCDR2 amino acid sequence set forth in SEQ ID NO:111, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:121, and wherein the CD19 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:11, the HCDR2 amino acid sequence set forth in SEQ ID NO:21, the HCDR3 amino acid sequence set forth in SEQ ID NO:31, the LCDR1 amino acid sequence set forth in SEQ ID NO:41, the LCDR2 amino acid sequence set forth in SEQ ID NO:51, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:61.

[0083] In some embodiments, the bispecific antibody comprises a CD38 binding component and a CD19 binding component, wherein the CD38 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:72, the HCDR2 amino acid sequence set forth in SEQ ID NO:82, the HCDR3 amino acid sequence set forth in SEQ ID NO:92, the LCDR1 amino acid sequence set forth in SEQ ID NO:102, the LCDR2 amino acid sequence set forth in SEQ ID NO:112, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:122, and wherein the CD19 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:12, the HCDR2 amino acid sequence set forth in SEQ ID NO:22, the HCDR3 amino acid sequence set forth in SEQ ID NO:32, the LCDR1 amino acid sequence set forth in SEQ ID NO:42, the LCDR2 amino acid sequence set forth in SEQ ID NO:52, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:62.

[0084] In some embodiments, the bispecific antibody comprises a CD38 binding component and a CD19 binding component, wherein the CD38 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:75, the HCDR2 amino acid sequence set forth in SEQ ID NO:85, the HCDR3 amino acid sequence set forth in SEQ ID NO:95, the LCDR1 amino acid sequence set forth in SEQ ID NO:105, the LCDR2 amino acid sequence set forth in SEQ ID NO:115, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:125, and wherein the CD19 binding component comprises the HCDR1 amino acid sequence set forth in SEQ ID NO:15, the HCDR2 amino acid sequence set forth in SEQ ID NO:25, the HCDR3 amino acid sequence set forth in SEQ ID NO:35, the LCDR1 amino acid sequence set forth in SEQ ID NO:45, the LCDR2 amino acid sequence set forth in SEQ ID NO:55, and / or the LCDR3 amino acid sequence set forth in SEQ ID NO:65.

[0085] In some embodiments, the CD38 binding component comprises variable heavy and light chains or CDRs corresponding to or derived from daratumumab or isatuximab.

[0086] Substitutions, insertions, or deletions may occur within one or more CDRs, where the substitutions, insertions, or deletions do not substantially reduce the binding of the antibody to the antigen. For example, conservative substitutions that do not substantially reduce binding affinity may be made in the CDRs. Such modifications may be outside the CDR "hot spots". In some embodiments of variant VH and VL sequences, each CDR is unaltered. Amino acid sequence insertions and deletions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions and deletions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions at the N- or C-terminus of the antibody to enzymes (e.g., for ADEPT) or polypeptides that increase the serum half-life of the antibody. An example of an intrasequence insertion variant of an antibody molecule includes an insertion of three amino acids in the light chain. An example of a terminal deletion includes an antibody with a deletion of seven or fewer amino acids at the end of the light chain.

[0087] Modifications (e.g., substitutions) may be made in the CDRs, for example, to improve the affinity of the antibody. Such modifications may be made in CDRs that encode codons with high mutation rates during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and the resulting variants may be tested for binding affinity. Affinity maturation (e.g., using error-prone PCR, chain shuffling, randomization of CDRs, or oligonucleotide-directed mutagenesis) can be used to improve the affinity of the antibody (see, e.g., Hoogenboom et al., Methods in Molecular Biology 178:1-37 (2001)). CDR residues involved in antigen binding may be specifically identified, for example, using alanine scanning mutagenesis or modeling (see, e.g., Cunningham and Wells Science, 244:1081-1085 (1989)). CDR-H3 and CDR-L3 are often targeted in particular.Alternatively or additionally, there is a crystal structure of antigen-antibody complex to identify the contact points between antibody and antigen.Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution.Variants can be screened to determine whether they contain desired properties.

[0088] Antibodies can be modified to increase or decrease their glycosylation (e.g., by modifying the amino acid sequence to create or remove one or more glycosylation sites). The carbohydrate attached to the Fc region of the antibody can be modified. Native antibodies from mammalian cells typically contain a branched, biantennary oligosaccharide attached by an N-linkage to Asn297 of the CH2 domain of the Fc region (see, e.g., Wright et al., TIBTECH 15:26-32 (1997)). The oligosaccharide can be a variety of carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, fucose attached to GlcNAc at the base of the biantennary oligosaccharide structure. Modification of the oligosaccharide in the antibody can be performed, for example, to generate antibody variants with specific improved properties. The antibody glycosylation variants can have improved ADCC and / or CDC functions. In some embodiments, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures attached to Asn297 (see, e.g., WO 08 / 077546). Asn297 refers to an asparagine residue located at about position 297 (EU numbering of Fc region residues; see, e.g., Edelman et al., Proc Natl Acad Sci USA. 1969 May; 63(1):78-85) in the Fc region. However, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294-300, due to minor sequence variations in the antibody. Such fucosylation variants can have improved ADCC function (see, e.g., Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004) and Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004)).Cell lines, e.g., knockout cell lines, and methods for their use can be used to produce defucosylated antibodies, e.g., Lec13 CHO cells deficient in protein fucosylation and α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (see, e.g., Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. 94(4):680-688 (2006)). Other antibody glycosylation variants are also included (see, e.g., U.S. Pat. No. 6,602,684).

[0089] In some embodiments, the conjugate binding molecules provided herein have an affinity for the antibody target of about 10 μM, 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K D The target of the antibody can be a CD19 target, a CD38 target, or a target that includes both CD19 and CD38. D can be measured by any suitable assay. In certain embodiments, KD can be measured using a surface plasmon resonance assay (e.g., using a BIACORE®-2000 or BIACORE®-3000 or Octet).

[0090] The antibodies can have an extended half-life and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., U.S. Patent Application Publication No. 2005 / 0014934). Such antibodies can include an Fc region with one or more substitutions that improve binding of the Fc region to FcRn, including those with substitutions at one or more of the Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 according to the EU numbering system (see, e.g., U.S. Patent No. 7,371,826). Other examples of Fc region variants are also contemplated (see, for example, Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, and WO 94 / 29351).

[0091] In some embodiments, it may be desirable to create cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with a cysteine ​​residue. In some embodiments, the substituted residues are present at accessible sites of the antibody. The reactive thiol group can be placed at the site for conjugation to other moieties, such as a drug moiety or a linker drug moiety, to create an immunoconjugate. In some embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region.

[0092] In some embodiments, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties that are known and available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules.

[0093] Complex binding molecules or bispecific antibodies may vary based on the binding moieties associated with these molecules, and there are also several different formats that can be deployed and are contemplated herein. Complex binding molecules or bispecific antibodies can include antibody fragments, substantially intact antibodies, or combinations thereof. In some embodiments, the first or second binding moiety comprises an immunoglobulin heavy and light chain pair, an scFv, F(ab), F(ab')2, a single domain antibody, a variable region fragment from an immunoglobulin neoantigen receptor (VNAR), or a variable region derived from a heavy chain antibody (VHH). In certain embodiments, the first and second binding moieties comprise an immunoglobulin heavy and light chain pair, an scFv, F(ab), F(ab')2, a single domain antibody, a variable region fragment from an immunoglobulin neoantigen receptor (VNAR), or a variable region derived from a heavy chain antibody (VHH). In some embodiments, the first or second binding moiety comprises an immunoglobulin heavy and light chain pair. In certain embodiments, the first and second binding moieties comprise an immunoglobulin heavy and light chain pair. In some embodiments, the first or second binding moiety comprises an scFv. In certain embodiments, the first and second binding moieties comprise an scFv.

[0094] Bispecific antibodies according to the present disclosure comprise intact or nearly intact antibody molecules, and may be asymmetric or symmetric.

[0095] Asymmetric bispecific antibodies generally include a heavy chain / light chain (HC / LC) pair from an antibody specific for target A and a HC / LC pair from an antibody specific for target B, creating a heterobifunctional antibody. Heterobifunctional antibodies such as these face the problem of non-productive formation of molecules during production. HC / LC-A:HC / LC-B is desirable, but is usually thermodynamically or statistically unfavorable from all possible combinations. Several schemes have been introduced to circumvent this problem. In some cases, the HC / LC pair from an antibody with specificity for A and the HC / LC pair from an antibody with specificity for B further include mutations to the FC region to increase the probability of forming an antibody with HC / LC-A:HC / LC-B. This can be achieved by engineering structural features that promote the formation of heterodimers between HC-A and HC-B, such as a "knob" for the FC region of HC-A and a "hole" for HC-B, or vice versa. Another scheme to promote HC-A:HC-B heterodimers is to engineer amino acid residues in the FC portions of HC-A and HC-B to contain charge pairs that promote electrostatic interactions between HC-B and HC-A. Another scheme to address the chain association problem is to ligate the variable region of one of the HC / LC pairs into a single chain binding molecule (e.g., V HHor scFv), resulting in one half of the molecule containing a classical HC / LC pair and the other containing the HC constant region fused or otherwise connected to the single chain binding molecule. Further modifications can be made to promote proper HC / LC pairing, including engineering mutations to the HC and LC of either A or B to favor the formation of the proper HC / LC pair, and CrossMab technology that exploits the exchange of the corresponding constant regions of the HC / LC pair. Symmetric bispecific antibodies avoid the problem of chain association by not relying on the formation of heterobifunctional molecules. Such examples include, among others, dual variable domain molecules containing stacked variable regions of different specificities, IgG-scFv molecules containing scFvs of different specificities fused to the c-terminus of the heavy chain of a classical antibody molecule, (scFV)4-FC containing two scFvs connected by the Fc region of an Ig (Fc dimerizes to create a bispecific tetravalent molecule), DART-Fc, and two-in-one.

[0096] The structure of the complex binding molecule or bispecific antibody can be envisioned and designed to modify the functionality or binding properties of the complex binding molecule or bispecific antibody (see, e.g., "Bispecific antibodies: a mechanistic review of the pipeline.", Nat Rev Drug Discovery. 2019 Aug;18(8):585-608) (see, e.g., "The making of bispecific antibodies", MAbs. 2017 Feb-Mar;9(2):182-212). For example, the bispecific antibody can be selected from one of the following formats: common light chain bispecific IgG, Fab-Fc:scFv-Fc bispecific IgG, Fab-Fc-Fab:Fc bispecific IgG, Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG, Fab-Fc-scFv:Fc bispecific IgG, Fab-Fc-Fab:Fab-Fc bispecific IgG, scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG, Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG, Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG, and Fab-Fc-scFv:Fab-Fc bispecific IgG.

[0097] Common light chain bispecific IgG Bispecific antibodies with a common light chain bispecific IgG structure can be used in the present invention. Figure 1 illustrates a bispecific antibody with a common light chain bispecific IgG structure. This structure includes a first and a second IgG heavy chain. Each heavy chain includes a VH, CH1, CH2, and CH3 domain. The first heavy chain includes VH 102, CH1 104, CH2 106, and CH3 108. The second heavy chain includes VH 112, CH1 114, CH2 116, and CH3 118. The common light chain bispecific IgG structure also includes a light chain that includes a VL domain 120 and a CL domain 122. Generally, the first heavy chain includes a sequence derived from a heavy chain of an antibody with a first specificity, and the second heavy chain includes a heavy chain from an antibody with a second specificity. The light chains paired with the first and second heavy chains are identical and can be derived from the light chains of antibodies with either specificity or different specificities. A heavy chain may be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 130). A heavy chain may be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 134 and / or 136). A common light chain bispecific IgG structure may include first and second heavy chain molecules that further include a mutation in the CH3 domain that facilitates linkage of the first and second heavy chains and / or prevents linkage of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutation may prevent linkage of two first heavy chain molecules or two second heavy chain molecules physically (e.g., steric hindrance, "knobs"-into-"holes") or biochemically (e.g., electrostatic interactions). Exemplary knob-into-hole mutations can include T366W (EU numbering) in one heavy chain and T366S / L368A / Y407V (EU numbering) in another heavy chain. Exemplary mutations that facilitate linkage of first and second heavy chain molecules are disclosed, for example, in WO2009089004, U.S. Patent No. 8,642,745, U.S. Patent Application Publication No. 20140322756, and "The making of bispecific antibodies," MAbs. 2017 Feb-Mar;9(2):182-212.The common light chain bispecific IgG structure may also include a carbohydrate molecule 140 linked thereto, or additional modifications thereof.

[0098] Bispecific antibodies having a common light chain bispecific IgG structure can target a B cell lineage surface marker (e.g., CD19, CD138, IgA, or CD45) and an inhibitory B cell surface marker (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-β LAP)). In some embodiments, the first heavy chain is configured to bind to a B cell lineage surface marker and the second heavy chain is configured to bind to an inhibitory B cell surface marker. In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0099] In some embodiments, the first heavy chain comprises a VH sequence that comprises a CD19 binding component and the second heavy chain comprises a VH sequence that comprises a CD38 binding component. In certain embodiments, the heavy chain CD19 binding component comprises SEQ ID NO:201, SEQ ID NO:1, a variant that comprises a mutation at one or both of A84 and A108 of SEQ ID NO:201, and the heavy chain CD38 binding component comprises SEQ ID NO:202, 215, 218-221. In certain embodiments, the variant comprises the mutations A84S and A108L. In some embodiments, the bispecific antibody comprises a common light chain. In certain embodiments, the common light chain sequence comprises a CD19 binding component (e.g., SEQ ID NO:2). In certain embodiments, the common light chain sequence comprises a CD38 binding component (e.g., SEQ ID NO:4 or SEQ ID NO:222).

[0100] BS1 as described herein comprises a common light chain format having a CD19 binding component configured to bind CD19 and a CD38 binding component configured to bind CD38, wherein the CD19 binding component comprises an antibody or antigen-binding fragment thereof, wherein the CD38 binding component comprises an antibody or antigen-binding fragment thereof, wherein the CD38 antibody or antigen-binding fragment comprises an anti-CD38 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, and wherein the CD19 antibody or antigen-binding fragment comprises an anti-CD38 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region. and a light chain variable region paired with an anti-CD19 immunoglobulin heavy chain variable region, the CD38 antibody or antigen-binding component comprising: a) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 71-75; b) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 81-85 or 150-155; c) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 91-95; d) a heavy chain complementarity determining region 4 (HCDR5) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 101-105. a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 111-115; e) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 111-115; and / or f) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 121-125; and a CD19 antigen-binding component comprising g) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 11-15; h) an amino acid sequence set forth in any one of SEQ ID NOs: 21-25. i) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 31 to 35; j) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 105; k) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111 to 115; and / or l) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121 to 125.In some embodiments, the CD38 antigen binding component comprises an HCDR2 amino acid sequence comprising the sequence P-X1-LG-X2-A (SEQ ID NO: 156), where X1 and X2 are each selected from the group consisting of H, Q, T, N, S, G, A, R, K, D, or E. In certain embodiments, X1 is H and X2 is T. In some embodiments, the CD19 heavy chain sequence comprises an A84S and / or A108L substitution. In some embodiments, the CD38 light chain comprises a W32H substitution.

[0101] Fab-Fc: scFv-Fc bispecific IgG Bispecific antibodies having a Fab-Fc:scFv-Fc bispecific IgG structure can be used in the present invention. Figure 2 illustrates a bispecific antibody having a Fab-Fc:scFv-Fc bispecific IgG structure. This structure comprises a first heavy chain molecule and a modified second IgG heavy chain molecule comprising a single chain variable fragment. The first heavy chain comprises, from N-terminus to C-terminus, VH 202, CH1 204, CH2 206, and CH3 208, respectively. The modified second heavy chain comprises, from N-terminus to C-terminus, single chain variable fragment (scFv) 210, CH2 216, and CH3 218, respectively. The single chain variable fragment (scFv) can comprise a first domain 212, or a fragment thereof, corresponding to the variable light chain domain, a second domain 214, or a fragment thereof, corresponding to the variable heavy chain, and a linker polypeptide 215. The Fab-Fc:scFv-Fc bispecific IgG structure also includes a light chain that includes a VL domain 220 and a CL domain 222. The first heavy chain can be covalently linked to the light chain molecule via a covalent bond (e.g., disulfide bond 230). The first heavy chain can be linked to the modified second heavy chain via one or more covalent bonds (e.g., disulfide bonds 234 and / or 236). The Fab-Fc:scFv-Fc bispecific IgG structure can include a first and a modified second heavy chain molecule that further includes a mutation in the CH3 domain that facilitates linking of the first and second heavy chains and / or prevents linking of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutation can prevent linking of the two first heavy chain molecules or the two second heavy chain molecules physically (e.g., steric hindrance) or biochemically (e.g., electrostatic interactions). Exemplary mutations that facilitate linkage of the first and second heavy chain molecules are disclosed, for example, in US Patent Publication No. 20140322756 and in "The making of bispecific antibodies," MAbs. 2017 Feb-Mar;9(2):182-212. The Fab-Fc:scFv-Fc bispecific IgG structure can also include a carbohydrate molecule 240 linked thereto, or additional modifications thereof.

[0102] Bispecific antibodies having a Fab-Fc:scFv-Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45, e.g., CD19, CD38, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-βLAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0103] A Fab-Fc:scFv-Fc bispecific IgG structure can be engineered such that the first antigen-binding site targets CD19 and the second antigen-binding site targets CD38. In some embodiments, the first heavy chain comprises a VH sequence that includes a CD19 binding component, and the second heavy chain comprises a single chain variable fragment (scFv) sequence that includes a CD38 binding component. In certain embodiments, the heavy chain that includes a CD38 single chain variable fragment comprises SEQ ID NO: 205 or SEQ ID NO: 206. In certain embodiments, the VL sequence includes a CD19 binding component. In certain embodiments, the single chain variable fragment (scFv) sequence that includes a CD38 binding component includes a CD38 binding component that corresponds to an antibody heavy and light chain variable sequence, or a CD38 binding fragment thereof. In some embodiments, the first heavy chain comprises a VH sequence that includes a CD38 binding component, and the second heavy chain comprises a single chain variable fragment (scFv) sequence that includes a CD19 binding component. In certain embodiments, the heavy chain comprising the CD19 single chain variable fragment comprises SEQ ID NO: 203, SEQ ID NO: 204, or SEQ ID NO: 217. In certain embodiments, the single chain variable fragment (scFv) sequence comprising a CD19 binding component comprises a CD19 binding component corresponding to an antibody heavy and light chain variable sequence, or a CD19 binding fragment thereof.

[0104] A Fab-Fc:scFv-Fc bispecific IgG structure can be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD19. In some embodiments, the first heavy chain comprises a VH sequence that includes a CD38-binding component, and the second heavy chain comprises a single-chain variable fragment (scFv) sequence that includes a CD19-binding component. In certain embodiments, the VL sequence includes a CD38-binding component. In certain embodiments, the single-chain variable fragment (scFv) sequence that includes a CD19-binding component includes a CD19-binding component that corresponds to an antibody heavy and light chain variable sequence, or a CD19-binding fragment thereof.

[0105] The BS2 described herein comprises a CD19 binding component configured to bind CD19 and a CD38 binding component configured to bind CD38, wherein the CD19 binding component comprises an antibody or antigen-binding fragment thereof, wherein the CD38 binding component comprises an antibody or antigen-binding fragment thereof, wherein the CD38 antigen-binding component comprises a Fab that binds to CD38 comprising an anti-CD38 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, wherein the CD19 antigen-binding component comprises an scFv that binds to CD19 comprising an anti-CD19 immunoglobulin heavy chain variable region paired with an anti-CD38 immunoglobulin light chain variable region, wherein the CD38 binding component comprises an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 71-75, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 81-85, or 150-155, or an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 91-95. the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 101-105, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 111-115, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 121-125; the CD19-binding component comprises an immunoglobulin heavy chain comprising an HCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 11-15, an HCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 21-25, and an HCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 31-35; and the immunoglobulin light chain comprises an LCDR1 amino acid sequence set forth in any one of SEQ ID NOs: 41-45, an LCDR2 amino acid sequence set forth in any one of SEQ ID NOs: 51-55, and / or an LCDR3 amino acid sequence set forth in any one of SEQ ID NOs: 61-65. In some embodiments, the CD38 antigen binding component comprises an HCDR2 amino acid sequence comprising the sequence P-X1-LG-X2-A (SEQ ID NO: 156), where X1 and X2 are selected from the group consisting of H, Q, T, N, S, G, A, R, K, D, or E. In certain embodiments, X1 is H and X2 is T. In some embodiments, the CD19 heavy chain sequence comprises an A84S and / or A108L substitution.In some embodiments, the CD38 light chain comprises a W32H substitution.

[0106] Fab-Fc-Fab:Fc bispecific IgG Engineered bispecific antibodies with a Fab-Fc-Fab:Fc bispecific IgG structure can be used in the present invention. Figure 3 illustrates a bispecific antibody with a Fab-Fc-Fab:Fc bispecific IgG structure. This structure comprises a first heavy chain molecule and a modified IgG heavy chain molecule. The first heavy chain comprises, from N-terminus to C-terminus, a VH domain 302, a CH1 domain 304, a CH2 domain 306, a CH3 domain 308, a linker 310, a second VH domain 312, and a second CH1 domain 314, respectively. The modified heavy chain comprises, from N-terminus to C-terminus, a CH2 domain 316, and a CH3 domain 318, respectively. The Fab-Fc-Fab:Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 320 and a CL domain 322. The Fab-Fc-Fab:Fc bispecific IgG structure also comprises a second light chain comprising a VL domain 324 and a CL domain 326. The heavy chain may be covalently linked to the light chain molecule via a covalent bond (e.g., disulfide bond 330). The first heavy chain may also be covalently linked to the first and second chain molecules via a covalent bond (e.g., disulfide bond 332). The heavy and light chains may be linked such that the VH and CH1 domains of the first heavy chain pair with the VL and CL domains of the first light chain. The first heavy and second light chains may be linked such that the second VH and second CH1 domains of the first heavy chain pair with the VL and CL domains of the second light chain. The first heavy chain may be linked to the modified second heavy chain via one or more covalent bonds (e.g., disulfide bonds 334 and / or 336). The Fab-Fc-Fab:Fc bispecific IgG structure can comprise a first and a modified second heavy chain molecule that further comprises a mutation in the CH3 domain that facilitates linkage of the first and second heavy chains and / or prevents linkage of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutation can prevent linkage of the two first heavy chain molecules or the two second heavy chain molecules physically (e.g. steric hindrance) or biochemically (e.g. electrostatic interactions).Exemplary mutations that facilitate linkage of the first and second heavy chain molecules are disclosed, for example, in US Patent Publication No. 20140322756 and in "The making of bispecific antibodies," MAbs. 2017 Feb-Mar;9(2):182-212. The Fab-Fc-Fab:Fc bispecific IgG structure can also include a carbohydrate molecule 340 linked thereto, or additional modifications thereof.

[0107] Bispecific antibodies having a Fab-Fc-Fab:Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45, e.g., CD19, CD38, IgA, or CD45), and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-βLAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0108] A Fab-Fc-Fab:Fc bispecific IgG structure can be engineered such that a first antigen binding site targets CD19 and a second antigen binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 302) and VL domain (e.g., 320) comprise a CD19 binding component, and the second VH domain (e.g., 312) and VL domain (e.g., 324) comprise a CD38 binding component. In some embodiments, the Fab-Fc-Fab heavy chain comprises SEQ ID NO:207 and the Fc heavy chain comprises SEQ ID NO:208.

[0109] Fab-Fc-Fab:Fc bispecific IgG structures can also be engineered such that a first antigen binding site targets CD38 and a second antigen binding site targets CD 19. In some embodiments, the first heavy chain VH domain (e.g., 302) and VL domain (e.g., 320) comprise a CD38 binding component, and the second VH domain (e.g., 312) and VL domain (e.g., 324) comprise a CD19 binding component.

[0110] Fab-Fc-scFv: Fab-Fc-scFv bispecific IgG Engineered bispecific antibodies having a Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG structure can be used in the present invention. Figure 4 illustrates a bispecific antibody having a Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG structure. This structure comprises two first heavy chain molecules. The first heavy chain comprises, from N-terminus to C-terminus, a VH domain 402, a CH1 domain 404, a CH2 domain 406, a CH3 domain 408, a linker 410, and a single chain variable fragment (scFv) 412. The single chain variable fragment (scFv) can comprise a first domain 414 or a fragment thereof corresponding to a variable light chain domain, a second domain 416 or a fragment thereof corresponding to a variable heavy chain, and a second linker polypeptide 415. The Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG structure also comprises a first light chain comprising a VL domain 420 and a CL domain 422. A heavy chain may be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 430). A heavy chain may be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 434 and / or 436). The Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG structure may also include a carbohydrate molecule 440 or additional modifications thereof linked thereto.

[0111] Bispecific antibodies having a Fab-Fc-scFv:Fab-Fc-scFv bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45, e.g., CD19, CD38, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-βLAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0112] Fab-Fc-scFv: Fab-Fc-scFv bispecific IgG structures can be engineered such that a first antigen binding site targets CD19 and a second antigen binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 402) and VL domain (e.g., 420) comprise a CD19 binding component, and the single chain variable fragment (scFv) (e.g., 412) sequence comprises a CD38 binding component. In certain embodiments, the single chain variable fragment (scFv) sequence comprising a CD38 binding component comprises a CD38 binding component corresponding to the antibody heavy and light chain variable sequences, or a CD38 binding fragment thereof.

[0113] Fab-Fc-scFv: Fab-Fc-scFv bispecific IgG structures can also be engineered such that a first antigen binding site targets CD38 and a second antigen binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 402) and VL domain (e.g., 420) comprise a CD38 binding component, and the single chain variable fragment (scFv) (e.g., 412) sequence comprises a CD19 binding component. In certain embodiments, the single chain variable fragment (scFv) sequence comprising a CD19 binding component comprises a CD19 binding component corresponding to the antibody heavy and light chain variable sequences, or a CD19 binding fragment thereof. In some embodiments, the Fab-Fc-scFv heavy chain comprises SEQ ID NO: 209.

[0114] Fab-Fc-scFv:Fc bispecific IgG Engineered bispecific antibodies with a Fab-Fc-scFv:Fc bispecific IgG structure can be used in the present invention. Figure 5 illustrates a bispecific antibody with a Fab-Fc-scFv:Fc bispecific IgG structure. This structure comprises a first heavy chain molecule and a second IgG heavy chain molecule. The first heavy chain comprises, from N-terminus to C-terminus, a VH domain 502, a CH1 domain 504, a CH2 domain 506, a CH3 domain 508, a linker 510, and a single chain variable fragment (scFv) 512. The single chain variable fragment (scFv) can comprise a first domain 514 or a fragment thereof corresponding to a variable light chain domain, a second domain 516 or a fragment thereof corresponding to a variable heavy chain, and a second linker polypeptide 515. The Fab-Fc-scFv:Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 520 and a CL domain 522. The Fab-Fc-scFv:Fc bispecific IgG structure also includes a second light chain that includes a VL domain 524 and a CL domain 526. The heavy chain can be covalently linked to the light chain molecule via a covalent bond (e.g., disulfide bond 530). The heavy chain can be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 534 and / or 536). The Fab-Fc-scFv:Fc bispecific IgG structure can include a first and a modified second heavy chain molecule that further includes a mutation in the CH3 domain that facilitates linking of the first and second heavy chains and / or prevents linking of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutation can prevent linking of the two heavy chain molecules or the two second heavy chain molecules physically (e.g., steric hindrance) or biochemically (e.g., electrostatic interactions). Exemplary mutations that facilitate linkage of the first and second heavy chain molecules are disclosed, for example, in US Patent Publication No. 20140322756 and in "The making of bispecific antibodies," MAbs. 2017 Feb-Mar;9(2):182-212. The Fab-Fc-scFv:Fc bispecific IgG structure can also include a carbohydrate molecule 540 linked thereto, or additional modifications thereof.

[0115] Bispecific antibodies having a Fab-Fc-scFv:Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45, e.g., CD19, CD38, IgA, or CD45), and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-βLAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0116] The Fab-Fc-scFv:Fc bispecific IgG structure can be engineered such that a first antigen binding site targets CD19 and a second antigen binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 502) and VL domain (e.g., 520) comprise a CD19 binding component, and the single chain variable fragment (scFv) (e.g., 512) sequence comprises a CD38 binding component. In certain embodiments, the single chain variable fragment (scFv) sequence that comprises a CD38 binding component comprises a CD38 binding component corresponding to the antibody heavy and light chain variable sequences, or a CD38 binding fragment thereof.

[0117] Fab-Fc-scFv:Fc bispecific IgG structures can also be engineered such that a first antigen binding site targets CD38 and a second antigen binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 502) and VL domain (e.g., 520) comprise a CD38 binding component, and the single chain variable fragment (scFv) (e.g., 512) sequence comprises a CD19 binding component. In certain embodiments, the single chain variable fragment (scFv) sequence that comprises a CD19 binding component comprises a CD19 binding component corresponding to the antibody heavy and light chain variable sequences, or a CD19 binding fragment thereof.

[0118] Fab-Fc-Fab: A Fab-Fc bispecific IgG Engineered bispecific antibodies with a Fab-Fc-Fab:Fab-Fc bispecific IgG structure can be used in the present invention. Figure 6 illustrates a bispecific antibody with a Fab-Fc-Fab:Fab-Fc bispecific IgG structure. This structure comprises a first heavy chain molecule and a second IgG heavy chain molecule. The first heavy chain comprises, from N-terminus to C-terminus, a VH domain 602, a CH1 domain 604, a CH2 domain 606, a CH3 domain 608, a linker 610, a second VH domain 612, and a second CH1 domain 614, respectively. The second heavy chain comprises, from N-terminus to C-terminus, a VH domain 652, a CH1 domain 654, a CH2 domain 656, and a CH3 domain 658, respectively, similar to those of the first heavy chain. The Fab-Fc-Fab:Fab-Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 620 and a CL domain 622. The Fab-Fc-Fab:Fab-Fc bispecific IgG structure also includes a second light chain comprising a VL domain 624 and a CL domain 626. The heavy chain may be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 630). The first heavy chain and the first light chain may be linked such that the VH domain and the CH1 domain of the first heavy chain pair with the VL domain and the CL domain of the first light chain. The first heavy chain and the second light chain may be linked such that the second VH domain and the second CH1 domain of the first heavy chain pair with the VL domain and the CL domain of the second light chain. The heavy chain may be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 634 and / or 636). The Fab-Fc-Fab:Fab-Fc bispecific IgG structure can comprise first and second heavy chain molecules that further comprise a mutation in the CH3 domain that facilitates linkage of the first and second heavy chains and / or prevents linkage of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutation can prevent linkage of the two first heavy chain molecules or the two second heavy chain molecules physically (e.g. steric hindrance) or biochemically (e.g. electrostatic interactions).Exemplary mutations that facilitate linkage of the first and second heavy chain molecules are disclosed, for example, in US Patent Publication No. 20140322756 and in "The making of bispecific antibodies", MAbs. 2017 Feb-Mar;9(2):182-212. The Fab-Fc-Fab:Fab-Fc bispecific IgG structure can also include a carbohydrate molecule or additional modifications thereof linked thereto.

[0119] Bispecific antibodies having a Fab-Fc-Fab:Fab-Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-β LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0120] A Fab-Fc-Fab:Fab-Fc bispecific IgG structure can be engineered such that a first antigen binding site targets CD19 and a second antigen binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 602) and VL domain (e.g., 620) comprise a CD19 binding component, and the second VH domain (e.g., 612) and VL domain (e.g., 624) comprise a CD38 binding component.

[0121] Fab-Fc-Fab:Fab-Fc bispecific IgG structures can also be engineered such that a first antigen binding site targets CD38 and a second antigen binding site targets CD 19. In some embodiments, the first heavy chain VH domain (e.g., 602) and VL domain (e.g., 620) comprise a CD38 binding component, and the second VH domain (e.g., 612) and VL domain (e.g., 624) comprise a CD19 binding component.

[0122] scFv-Fab-Fc: scFv-Fab-Fc bispecific IgG Engineered bispecific antibodies having a scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can be used in the present invention. Figure 7 illustrates a bispecific antibody having a scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure. This structure comprises two first heavy chain molecules. The first heavy chain comprises, from N-terminus to C-terminus, a single chain variable fragment (scFv) 712, a linker 710, a VH domain 702, a CH1 domain 704, a CH2 domain 706, and a CH3 domain 708, respectively. The single chain variable fragment (scFv) can comprise a first domain 714 or a fragment thereof corresponding to a variable light chain domain, a second domain 716 or a fragment thereof corresponding to a variable heavy chain, and a second linker polypeptide 715. The scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 720 and a CL domain 722. A heavy chain may be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 730). A heavy chain may be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 734 and / or 736). The ScFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure may also include a carbohydrate molecule 740 linked thereto, or additional modifications thereof.

[0123] Bispecific antibodies having a scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-β LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0124] The scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can be engineered such that a first antigen binding site targets CD19 and a second antigen binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 702) and VL domain (e.g., 720) comprise a CD19 binding component, and the single chain variable fragment (scFv) (e.g., 712) sequence comprises a CD38 binding component. In certain embodiments, the single chain variable fragment (scFv) sequence that comprises a CD38 binding component comprises a CD38 binding component corresponding to the antibody heavy and light chain variable sequences, or a CD38 binding fragment thereof.

[0125] The scFv-Fab-Fc:scFv-Fab-Fc bispecific IgG structure can also be engineered such that a first antigen binding site targets CD38 and a second antigen binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 702) and VL domain (e.g., 720) comprise a CD38 binding component, and the single chain variable fragment (scFv) (e.g., 712) sequence comprises a CD19 binding component. In certain embodiments, the single chain variable fragment (scFv) sequence that comprises a CD19 binding component comprises a CD19 binding component corresponding to the antibody heavy and light chain variable sequences, or a CD19 binding fragment thereof.

[0126] Fab-Fab-Fc: Fab-Fab-Fc bispecific IgG Engineered bispecific antibodies with a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure can be used in the present invention. Figure 8 illustrates a bispecific antibody with a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure. This structure comprises two heavy chain molecules. The heavy chains each comprise, from N-terminus to C-terminus, an additional VH domain 812, and an additional CH1 domain 814, a linker 810, a VH domain 802, a CH1 domain 804, a CH2 domain 806, and a CH3 domain 808. The Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 820 and a CL domain 822. The Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure also comprises a second light chain comprising a VL domain 824 and a CL domain 826. The heavy chain molecules can be covalently linked to the light chain molecules via a covalent bond (e.g., a disulfide bond 830). The heavy chain and the first light chain may be linked such that the VH and CH1 domains of the heavy chain pair with the VL and CL domains of the first light chain. The heavy chain and the second light chain may be linked such that the additional VH and additional CH1 domains of the heavy chain pair with the VL and CL domains of the second light chain. The heavy chain may be linked to the modified second heavy chain via one or more covalent bonds (e.g., disulfide bonds 834 and / or 836). The Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure may also include a carbohydrate molecule 840 linked thereto, or additional modifications thereof.

[0127] Bispecific antibodies having a Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-β LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0128] A Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structure can be engineered such that a first antigen binding site targets CD19 and a second antigen binding site targets CD38. In some embodiments, the first VH domain (e.g., 802) and VL domain (e.g., 820) comprise a CD19 binding component, and the second VH domain (e.g., 812) and VL domain (e.g., 824) comprise a CD38 binding component.

[0129] Fab-Fab-Fc:Fab-Fab-Fc bispecific IgG structures can also be engineered such that a first antigen binding site targets CD38 and a second antigen binding site targets CD 19. In some embodiments, the VH domain (e.g., 802) and the VL domain (e.g., 820) comprise a CD38 binding component, and the second VH domain (e.g., 812) and the VL domain (e.g., 824) comprise a CD19 binding component.

[0130] Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG Engineered bispecific antibodies with a Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure can be used in the present invention. Figure 9 illustrates a bispecific antibody with a Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure. This structure comprises two heavy chain molecules and two light chain molecules. The heavy chains each comprise, from N-terminus to C-terminus, a VH domain 902, a CH1 domain 904, a CH2 domain 906, a CH3 domain 908, a linker 910, a second VH domain 912, and a second CH1 domain 914. The Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure also comprises a first light chain comprising a VL domain 920 and a CL domain 922. The Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure also comprises a second light chain comprising a VL domain 924 and a CL domain 926. The heavy chain may be covalently linked to a light chain molecule via a covalent bond (e.g., disulfide bond 930). The heavy chain and a first light chain may be linked such that the VH and CH1 domains of the heavy chain pair with the VL and CL domains of the first light chain. The heavy chain and a second light chain may be linked such that the second VH and second CH1 domains of the heavy chain pair with the VL and CL domains of the second light chain. The heavy chain may also be covalently linked to another heavy chain molecule via a covalent bond (e.g., disulfide bonds 930 and 936). The Fab-Fc-Fab bispecific IgG structure may also include a carbohydrate molecule 940 linked thereto, or additional modifications thereof.

[0131] Bispecific antibodies having a Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-β LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0132] A Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structure can be engineered such that a first antigen binding site targets CD19 and a second antigen binding site targets CD38. In some embodiments, the first VH domain (e.g., 902) and the VL domain (e.g., 920) comprise a CD19 binding component, and the second VH domain (e.g., 912) and the VL domain (e.g., 924) comprise a CD38 binding component.

[0133] Fab-Fc-Fab:Fab-Fc-Fab bispecific IgG structures can also be engineered such that a first antigen binding site targets CD38 and a second antigen binding site targets CD 19. In some embodiments, the VH domain (e.g., 902) and the VL domain (e.g., 920) comprise a CD38 binding component, and the second VH domain (e.g., 912) and the VL domain (e.g., 924) comprise a CD19 binding component.

[0134] Fab-Fc-scFv: Fab-Fc bispecific IgG Engineered bispecific antibodies with a Fab-Fc-scFv:Fab-Fc bispecific IgG structure can be used in the present invention. Figure 10 demonstrates a bispecific antibody with a Fab-Fc-scFv:Fab-Fc bispecific IgG structure. This structure comprises a first heavy chain molecule and a second IgG heavy chain molecule. The first heavy chain comprises, from N-terminus to C-terminus, a VH domain 1002, a CH1 domain 1004, a CH2 domain 1006, a CH3 domain 1008, a linker 1010, and a single chain variable fragment (scFv) 1012. The single chain variable fragment (scFv) can comprise a first domain 1014 or a fragment thereof corresponding to a variable light chain domain, a second domain 1016 or a fragment thereof corresponding to a variable heavy chain, and a second linker polypeptide 1015. The second heavy chain, like that of the first heavy chain, comprises, from N-terminus to C-terminus, a VH domain 1002, a CH1 domain 1004, a CH2 domain 1004, and a CH3 domain 1008, respectively. The Fab-Fc-scFv:Fab-Fc bispecific IgG structure also comprises a first light chain comprising a VL domain 1020 and a CL domain 1022. The heavy chain may be covalently linked to the light chain molecule via a covalent bond (e.g., disulfide bond 1030). The heavy chain may be linked to another heavy chain via one or more covalent bonds (e.g., disulfide bonds 1034 and / or 1036). The Fab-Fc-scFv:Fab-Fc bispecific IgG structure can include first and second heavy chain molecules that further include a mutation in the CH3 domain that facilitates linking of the first and second heavy chains and / or prevents linking of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutation can physically (e.g., steric hindrance) or biochemically (e.g., electrostatic interactions) prevent linking of the two first heavy chain molecules or the two second heavy chain molecules. Exemplary mutations that facilitate linking of the first and second heavy chain molecules are disclosed, for example, in US Patent Publication No. 20140322756 and in "The making of bispecific antibodies", MAbs. 2017 Feb-Mar;9(2):182-212.The Fab-Fc-scFv:Fab-Fc bispecific IgG structure may also include a carbohydrate molecule 1040 linked thereto or additional modifications thereof.

[0135] Bispecific antibodies having a Fab-Fc-scFv:Fab-Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-β LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0136] Fab-Fc-scFv: Fab-Fc bispecific IgG structures can be engineered such that a first antigen binding site targets CD19 and a second antigen binding site targets CD38. In some embodiments, the first heavy chain VH domain (e.g., 1002) and VL domain (e.g., 1020) comprise a CD19 binding component, and the single chain variable fragment (scFv) (e.g., 1012) sequence comprises a CD38 binding component. In certain embodiments, the single chain variable fragment (scFv) sequence that comprises a CD38 binding component comprises a CD38 binding component corresponding to the antibody heavy and light chain variable sequences, or a CD38 binding fragment thereof.

[0137] Fab-Fc-scFv: Fab-Fc bispecific IgG structures can also be engineered such that a first antigen binding site targets CD38 and a second antigen binding site targets CD19. In some embodiments, the first heavy chain VH domain (e.g., 1002) and VL domain (e.g., 1020) comprise a CD38 binding component, and the single chain variable fragment (scFv) (e.g., 1012) sequence comprises a CD19 binding component. In certain embodiments, the single chain variable fragment (scFv) sequence that comprises a CD19 binding component comprises a CD19 binding component corresponding to the antibody heavy and light chain variable sequences, or a CD19 binding fragment thereof.

[0138] scFv-Fab-Fc:Fc bispecific IgG Engineered bispecific antibodies with scFv-Fab-Fc:Fc bispecific IgG structure can be used in the present invention. Figure 11 demonstrates a bispecific antibody with scFv-Fab-Fc:Fc bispecific IgG structure. This structure includes a first heavy chain molecule including an scFv, VH, and Fc region, and a second heavy chain molecule including Fc. The scFv-Fab-Fc:Fc bispecific IgG structure can include first and second heavy chain molecules that further include a mutation in the CH3 domain that facilitates linkage of the first and second heavy chains and / or prevents linkage of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutation can facilitate the association of the first heavy chain molecule to the second heavy chain molecule physically (e.g., knob-in-hole structure) or biochemically (e.g., electrostatic interaction). The ScFv-Fab-Fc:Fc bispecific IgG structure comprises a light chain molecule associated with a first heavy chain molecule creating a first antigen binding site. The second antigen binding site is provided by an scFv fragment linked to the N-terminus of the first heavy chain. Exemplary mutations that facilitate linking of the first and second heavy chain molecules are disclosed, for example, in US Patent Publication No. 20140322756 and in "The making of bispecific antibodies", MAbs. 2017 Feb-Mar;9(2):182-212. The scFv-Fab-Fc:Fc bispecific IgG structure can also include a carbohydrate molecule 1140 linked thereto, or additional modifications thereof.

[0139] Bispecific antibodies having a scFv-Fab-Fc:Fc bispecific IgG structure can target B cell lineage surface markers (e.g., CD19, CD138, IgA, or CD45) and inhibitory B cell surface markers (e.g., IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-β LAP)). In some embodiments, the B cell lineage surface marker comprises CD19. In certain embodiments, the B cell lineage surface marker consists of CD19. In some embodiments, the inhibitory B cell surface marker comprises CD38. In certain embodiments, the inhibitory B cell surface marker consists of CD38.

[0140] The scFv-Fab-Fc:Fc bispecific IgG structure can be engineered such that a first antigen-binding site targets CD19 and a second antigen-binding site targets CD38. In some embodiments, the first heavy chain VH and VL domains comprise a CD19-binding component and the single chain variable fragment (scFv) sequence comprises a CD38-binding component. In certain embodiments, the single chain variable fragment (scFv) sequence comprises a CD38-binding component corresponding to the antibody heavy and light chain variable sequences, or a CD38-binding fragment thereof.

[0141] The scFv-Fab-Fc:Fc bispecific IgG structure can also be engineered such that a first antigen-binding site targets CD38 and a second antigen-binding site targets CD19. In some embodiments, the heavy chain VH and VL domains comprise a CD38-binding component and the single chain variable fragment (scFv) sequence comprises a CD19-binding component. In certain embodiments, the single chain variable fragment (scFv) sequence that comprises a CD19-binding component comprises a CD19-binding component that corresponds to the antibody heavy and light chain variable sequences, or a CD19-binding fragment thereof.

[0142] In certain embodiments, the first heavy chain molecule comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 212. In certain embodiments, the first heavy chain molecule comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 212.

[0143] In certain embodiments, the first light chain molecule comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 213. In certain embodiments, the first light chain molecule comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 213.

[0144] In certain embodiments, the second heavy chain molecule comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 214. In certain embodiments, the first heavy chain molecule comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 214.

[0145] Fc variants In some embodiments, one or more amino acid modifications are introduced into the fragment crystallizable (Fc) region of a human or humanized antibody, thereby generating an Fc region variant. The Fc region may include the C-terminal region of an immunoglobulin heavy chain, including the hinge region, the CH2 domain, the CH3 domain, or any combination thereof. As used herein, Fc region includes native sequence Fc regions and variant Fc regions. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4) that includes an amino acid modification (e.g., substitution, addition, or deletion) at one or more amino acid positions.

[0146] In some embodiments, the variant Fc region comprises at least one amino acid modification in the Fc region. Combinations of amino acid modifications are also useful. For example, the variant Fc region may comprise, for example, 2, 3, 4, 5 substitutions therein, for example, at specific Fc region positions identified herein.

[0147] In some embodiments, the antibodies described herein have modified effector functions compared to human IgG. In some embodiments, the modified effector functions are reduced effector functions. In some embodiments, the modified effector functions are increased effector functions. Effector functions generally refer to biological events resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Non-limiting effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. In some cases, antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (e.g., natural killer cells, neutrophils, macrophages) recognize bound antibodies on target cells, resulting in the subsequent lysis of the target cells. Complement dependent cytotoxicity (CDC) sometimes refers to the lysis of a target cell in the presence of complement, the complement pathway being initiated by the binding of C1q to target-bound antibody.

[0148] In certain cases, it is beneficial to reduce the effector functions of the antibodies described herein. In some cases, modifications in the Fc region generate Fc variants with (a) reduced antibody-dependent cell-mediated cytotoxicity (ADCC), (b) reduced complement-mediated cytotoxicity (CDC), and / or (c) reduced affinity for C1q. In some embodiments, the Fc region is 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 270, 272, 276, 278, 2 The variant Fc region is modified to decrease antibody-dependent cellular cytotoxicity (ADCC), decrease antibody-dependent cell-mediated phagocytosis (ADCP), decrease complement-mediated cytotoxicity (CDC), and / or decrease affinity for C1q by modifying one or more amino acids at positions 31, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439 (EU numbering). In some embodiments, the variant Fc region is selected from Table 1. In some embodiments, the variant Fc region comprises one or more of the mutations in Table 1.

[0149] [Table 1-1]

[0150] [Table 1-2]

[0151] Non-limiting examples of in vitro assays for examining the ADCC activity of molecules of interest are described in US Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assay methods can be employed (e.g., ACTI™ and CytoTox96® non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0152] In some embodiments, the variant Fc region exhibits reduced ADCC by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or more compared to an antibody comprising a non-variant Fc region, i.e., an antibody having the same sequence identity except for substitutions that reduce ADCC (such as human IgG1). In some embodiments, the variant Fc region exhibits reduced CDC by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or more compared to an antibody comprising a non-variant Fc region, i.e., an antibody having the same sequence identity except for substitutions that reduce CDC (such as human IgG1).

[0153] In certain embodiments, the variant Fc region exhibits ADCC that is reduced by about 10% to about 100%. In certain embodiments, the variant Fc region is from about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% or about 90% to about 100%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100% decreased ADCC. In certain embodiments, the variant Fc region exhibits ADCC that is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In certain embodiments, the variant Fc region exhibits ADCC that is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0154] In certain embodiments, the variant Fc region exhibits between about 10% and about 100% reduced CDC. In certain embodiments, the variant Fc region comprises about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 7 ...50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 100%, about 30% to about 100 In one embodiment, the present invention shows a CDC reduction of about 100%, about 30% to about 90%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100%. In certain embodiments, the variant Fc region exhibits reduced CDC by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In certain embodiments, the variant Fc region exhibits reduced CDC by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0155] In some embodiments, the variant Fc region exhibits reduced effector function compared to wild-type human IgG1. Non-limiting examples of Fc mutations in IgG1 that in certain cases reduce ADCC and / or CDC include substitutions at one or more of positions 231, 232, 234, 235, 236, 237, 238, 239, 264, 265, 267, 269, 270, 297, 299, 318, 320, 322, 325, 327, 328, 329, 330, and 331 in IgG1, where the numbering system of the constant region is that of the EU index as set forth by Kabat.

[0156] In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises an N297A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises an N297Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises an N297D substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a D265A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises an S228P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises an L235A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises an L237A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises an L234A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises an E233P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises an L234V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a C236 deletion according to the Kabat numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a P238A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises an A327Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a P329A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a P329G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a L235E substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a P331S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a L234F substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 235G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 235Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 235R substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 235S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 236F substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 236R substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 237E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 237K substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 237N substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 237R substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 238A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 238E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 238G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 238H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that includes a 238I substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 238V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 238W substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 238Y substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 248A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 254D substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 254E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 254G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 254H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 254I substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 254N substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 254P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 254Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 254T substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 254V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 255N substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 256H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 256K substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 256R substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 256V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 264S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 265H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 265K substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 265S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 265Y substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 267G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 267H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 267I substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 267K substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 268K substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 269N substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 269Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 270A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 270G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising a 270M substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 270N substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 271T substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 272N substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 279F substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 279K substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 279L substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 292E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 292F substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 292G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 292I substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 293S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 301W substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 304E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 311E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 311G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 311S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that includes a 316F substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 327T substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 328V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 329Y substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 330R substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 339E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 339L substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 343I substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 343V substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 373A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 373G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 373S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 376E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 376W substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 376Y substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 380D substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 382D substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 382P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 385P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 424H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 424M substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that includes a 424V substitution according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 434I substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 438G substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 439E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 439H substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 439Q substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 440A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 440D substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 440E substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 440F substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 440M substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 440T Fc region substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises a 440V substitution according to the EU numbering system.

[0157] In some embodiments, the variant Fc region comprises an IgG1 Fc region L234A, L235E, G237A, A330S, and / or P331S according to EU numbering. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising E233P according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG4 Fc region comprising S228P and L235E. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L235E according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A and L235A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235A, and G237A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235A, and P329G according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234F, L235E, and P331S according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235E, and G237A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235E, G237A, and P331S according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235A, G237A, P238S, H268A, A330S, and P331S (IgG1) according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising L234A, L235A, and P329A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising G236R and L328R according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region comprising G237A according to the EU numbering system.In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises F241A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises V264A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises D265A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises D265A and N297A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises D265A and N297G according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises D270A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises N297A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises N297G according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises N297D according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises N297Q according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises P329A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises P329G according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises P329R according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises A330L according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises P331A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG1 Fc region that comprises P331S according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2 Fc region, hi some embodiments, the variant Fc region comprises an IgG4 Fc region.In some embodiments, the variant Fc region comprises an IgG4 Fc region comprising S228P according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG4 Fc region comprising S228P, F234A, and L235A according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2-IgG4 cross-subclass (IgG2 / G4) Fc region. In some embodiments, the variant Fc region comprises an IgG2-IgG3 cross-subclass Fc region. In some embodiments, the variant Fc region comprises an IgG2 Fc region comprising H268Q, V309L, A330S, and P331S according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2 Fc region comprising V234A, G237A, P238S, H268A, V309L, A330S, and P331S according to the EU numbering system. In some embodiments, the antibody comprises an Fc region that comprises high mannose glycosylation.

[0158] In some embodiments, the one or more mutations relative to the wild-type Fc region include or consist of L234A, L235A, and P329G according to EU numbering. In some embodiments, the one or more mutations relative to the wild-type Fc region include or consist of L234A, L235A, G237A, A330S, and P331S according to EU numbering. In some embodiments, the one or more mutations relative to the wild-type Fc region are selected from the group consisting of N297A / Q / G, L235A / G237A / E318A, L234A / L235A, G236R / L328R, S298G / T299A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, L234A / L235A / P329G, V234A / G237A / P238S / H268A / V309L / A330S / P331S, and L234F / L235E / D265A.

[0159] In some embodiments, the variant Fc region comprises an IgG4 Fc region that comprises an S228P substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG4 Fc region that comprises an A330S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG4 Fc region that comprises a P331S substitution according to the EU numbering system.

[0160] In some embodiments, the variant Fc region comprises an IgG2 Fc region that comprises an A330S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2 Fc region that comprises a P331S substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2 Fc region that comprises a 234A substitution according to the EU numbering system. In some embodiments, the variant Fc region comprises an IgG2 Fc region that comprises a 237A substitution according to the EU numbering system.

[0161] In some embodiments, the variant Fc region comprises an IgG1 Fc region, and the one or more mutations are: (a) 297A, 297Q, 297G, or 297D; (b) 279F, 279K, or 279L; (c) 228P; (d) 235A, 235E, 235G, 235Q, 235R, or 235S; (e) 237A, 237E, 237K, 237N, or 237R; (f) 234A, 234V, or 234F; (g) 233P; (h) 328A; (i) 327Q or 327T; (j) 329A, 329G, 329Y, or 329R; (k) 328P; 331S, (l) 236F or 236R, (m) 238A, 238E, 238G, 238H, 238I, 238V, 238W, or 238Y, (n) 248A, (o) 254D, 254E, 254G, 254H, 254I, 254N, 254P, 254Q, 254T, or 254V, (p) 255N, (q) 256H, 256K, 256R, or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y, or 265A, (t) 267G, 267H, 267I, or 267K, (u) 268K, ( v) 269N or 269Q, (w) 270A, 270G, 270M, or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (l l) 380D, (mm) 382D or 382P, (nn) 385P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235E, (vv) L234A and L235A, (ww) L234A, L235A, and G237A, (xx) L234A, L235A, and P329G, (yy) L234F, L235E,and P331S, (zz)L234A, L235E, and G237A, (aaa)L234A, L235E, G237A, and P331S, (bbb)L234A, L235A, G237A, P238S, H268A, A330S, and P331S, (ccc)L234A, L235A, and P329A, (ddd)G236R and L328R, (eee)G237A, (fff)F241A, (ggg) V264A, (hhh) D265A, (iii) D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (lll) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S, and P331S, or (ppp) any combination of (a) to (uu).

[0162] In some embodiments, the variant Fc region comprises the amino acid sequence set forth in SEQ ID NO: 311. In some embodiments, in the complex binding molecule, the CD19 antigen binding component comprises a heavy chain immunoglobulin sequence set forth in SEQ ID NO: 301 or 304, and the CD38 binding component comprises a heavy chain immunoglobulin sequence set forth in SEQ ID NO: 302, 303, 305-310.

[0163] Framework Area Mutations or back mutations to the germline sequences made within the framework regions of the heavy and light chains may be advantageous in improving the pharmacokinetic and pharmacodynamic properties of the CD19 and CD38 binding molecules described herein. In certain cases, mutations or back mutations to the germline sequences made within the heavy and / or light chains improve the stability of the CD19 and CD38 binding molecules (e.g., the bispecific antibodies described herein). In certain cases, mutations or back mutations to the germline sequences made within the heavy and / or light chains reduce the immunogenicity of the CD19 and CD38 binding molecules (e.g., the bispecific antibodies described herein). Thus, in some embodiments, the framework regions of the heavy and / or light chains contain 1, 2, 3, 4, 5, 8, or 10 mutations or back mutations back to the germline sequences. In some embodiments, the framework regions of the heavy and / or light chains contain from 1 mutation or back mutation back to the germline sequences to 10 mutations or back mutations back to the germline sequences. In some embodiments, the framework regions of the heavy and / or light chains contain at least one mutation or backmutation back to the germline sequence. In some embodiments, the framework regions of the heavy and / or light chains contain up to 10 mutations or backmutations back to the germline sequence. In some embodiments, the framework regions of the heavy and / or light chains contain from 1 mutation or backmutation back to the germline sequence to 2 mutations or backmutations back to the germline sequence, from 1 mutation or backmutation back to the germline sequence to 3 mutations or backmutations back to the germline sequence, from 1 mutation or backmutation back to the germline sequence to 4 mutations or backmutations back to the germline sequence, from 1 mutation or backmutation back to the germline sequence to 5 mutations or backmutations back to the germline sequence, from 1 mutation or backmutation back to the germline sequence to 8 mutations or backmutations back to the germline sequence, from 1 mutation or backmutation back to the germline sequence to 10 mutations or backmutations back to the germline sequence.2 mutations or reversions back to the germline sequence up to 3 mutations or reversions back to the germline sequence, 2 mutations or reversions back to the germline sequence up to 4 mutations or reversions back to the germline sequence, 2 mutations or reversions back to the germline sequence up to 5 mutations or reversions back to the germline sequence, 2 mutations or reversions back to the germline sequence up to 8 mutations or reversions back to the germline sequence, 2 mutations or reversions back to the germline sequence up to 10 mutations or reversions back to the germline sequence, 3 mutations or reversions back to the germline sequence up to 4 mutations or reversions back to the germline sequence, 3 mutations or reversions back to the germline sequence up to 5 mutations or reversions back to the germline sequence, 3 mutations or reversions back to the germline sequence up to 4 mutations or reversions back to the germline sequence, In some embodiments, the framework regions of the heavy and / or light chains include up to 8 mutations or reversions back to the germline sequence, 3 mutations or reversions back to the germline sequence up to 10 mutations or reversions back to the germline sequence, 4 mutations or reversions back to the germline sequence up to 5 mutations or reversions back to the germline sequence, 4 mutations or reversions back to the germline sequence up to 8 mutations or reversions back to the germline sequence, 4 mutations or reversions back to the germline sequence up to 10 mutations or reversions back to the germline sequence, 5 mutations or reversions back to the germline sequence up to 8 mutations or reversions back to the germline sequence, 5 mutations or reversions back to the germline sequence up to 10 mutations or reversions back to the germline sequence, or 8 mutations or reversions back to the germline sequence up to 10 mutations or reversions back to the germline sequence. In some embodiments, the framework regions of the heavy and / or light chains include up to 1 mutation or reversion back to the germline sequence, 2 mutations or reversions back to the germline sequence, 3 mutations or reversions back to the germline sequence,The heavy chain framework region may include 4 mutations or backmutations back to the germline sequence, 5 mutations or backmutations back to the germline sequence, 8 mutations or backmutations back to the germline sequence, or 10 mutations or backmutations back to the germline sequence. In some embodiments, the CD38 binding portion comprises a heavy chain framework region set forth in SEQ ID NO:5. In some embodiments, the CD38 binding portion comprises a heavy chain framework region set forth in SEQ ID NO:6 or 7.

[0164] Pharmaceutically acceptable excipients, carriers, and diluents The composition comprising the complex binding molecule of the present disclosure is contained in a pharmaceutical composition comprising one or more pharma- ceutically acceptable excipients, carriers, and diluents. In certain embodiments, the antibody of the present disclosure is administered suspended in a sterile and / or isotonic solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of a buffer, e.g., acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, e.g., glycine or arginine; an antioxidant, e.g., ascorbic acid, methionine, or a chelating agent, e.g., EDTA or EGTA.

[0165] Subcutaneous formulations for administration of antibodies can include one or more of the following: a buffer, such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris), a surfactant, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188, a polyol / disaccharide / polysaccharide, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40, an amino acid, such as glycine or arginine, an antioxidant, such as ascorbic acid, methionine, or a chelating agent, such as EDTA or EGTA. Additionally, a compound or molecule that relieves pain at the injection site can be included, such as hyaluronidase at a concentration of about 2,000 U / ml to about 12,000 U / ml.

[0166] In certain embodiments, the complex binding molecules of the present disclosure are lyophilized for transport / storage and reconstituted prior to administration. In certain embodiments, the lyophilized antibody formulation includes a bulking agent, such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation can be contained in a vial composed of glass or other suitable non-reactive material. The antibody, when formulated, whether or not reconstituted, can be buffered to a particular pH, generally a pH below 7.0. In certain embodiments, the pH can be between 4.5-6.5, 4.5-6.0, 4.5-5.5, 4.5-5.0, or 5.0-6.0.

[0167] Also described herein are kits comprising one or more of the complex binding molecules described herein in a suitable container and one or more additional components selected from instructions for use, diluents, excipients, carriers, and devices for administration.

[0168] In certain embodiments, described herein is a method of preparing a cancer treatment, comprising mixing one or more pharma- ceutically acceptable excipients, carriers, or diluents with a complex binding molecule of the present disclosure. In certain embodiments, described herein is a method of preparing a cancer treatment for storage or transportation, comprising lyophilizing one or more antibodies of the present disclosure.

[0169] Production and manufacturing Nucleic acids encoding the complex binding molecules (e.g., bispecific antibodies) described herein can be used to infect, transfect, transform, or otherwise make suitable cells transgenic for the nucleic acid, thereby allowing the production of the complex binding molecules for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell culture are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production." Mabs. 2010 Sep-Oct;2(5):466-477.

[0170] In certain embodiments, the nucleic acid sequence encodes a complex-binding molecule or a bispecific antibody disclosed herein. In certain embodiments, the polynucleotide sequence encoding the complex-binding molecule is operably linked to a eukaryotic regulatory sequence. In some embodiments, the cell comprises the nucleic acid sequence.

[0171] In some embodiments, the cell comprises a nucleic acid encoding a complex-binding molecule disclosed herein. In certain embodiments, the cell comprises a prokaryotic cell. In certain embodiments, the prokaryotic cell is an E. coli cell. In certain embodiments, the cell comprises a eukaryotic cell. In certain embodiments, the eukaryotic cell is a Chinese Hamster Ovary (CHO) cell, an NS0 mouse myeloma cell, or a human PER.C6 cell.

[0172] In certain embodiments, described herein are methods of making a complex binding molecule, comprising culturing a cell comprising a nucleic acid encoding the complex binding molecule under in vitro conditions sufficient to allow production and secretion of the complex binding molecule.

[0173] In certain embodiments, described herein is a master cell bank comprising (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location, and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol. In certain embodiments, the master cell bank comprises (a) a CHO cell line comprising a nucleic acid encoding a complex-binding molecule integrated at a genomic location, and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol. In certain embodiments, the master cell bank is contained in a suitable vial or container that can withstand freezing with liquid nitrogen.

[0174] Also described herein are methods of making the complex binding molecules described herein. Such methods include incubating cells or cell lines containing nucleic acids encoding the complex binding molecules in cell culture medium under conditions sufficient to allow expression and secretion of the complex binding molecules, and further harvesting the complex binding molecules from the cell culture medium. Harvesting can further include one or more purification steps to remove viable cells, cell debris, uncomplexed binding molecule proteins or polypeptides, undesired salts, buffers, and medium components. In certain embodiments, the additional purification steps include centrifugation, ultracentrifugation, Protein A, Protein G, Protein A / G, or Protein L purification, and / or ion exchange chromatography.

[0175] How to use Suppression of immune responses by immunoregulatory cells can promote tumor growth, migration, and metastasis. Immunosuppression or negative immune regulation can include processes or pathways that result in a total or partial reduction of immune responses. Immunosuppression can be systemic or localized to a specific site (e.g., tumor microenvironment), tissue, or region of a subject's or patient's body. Although B cells are primarily known as positive immune modulators through the production of antibodies that promote neutralization of pathogens, certain B cell populations can function to suppress or negatively regulate immune responses. Such B cell populations can be defined by the expression of more than one cell surface biomarker. Immunosuppressive B cells or B cell populations can include B cell lineage surface biomarkers and inhibitory B cell surface biomarkers. B cell lineage surface markers can include CD19, CD138, IgA, or CD45. B cell surface markers can include IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or latent TGF-β (e.g., TGF-βLAP). Immunosuppressive B cells or immunosuppressive B cell populations can function to suppress immune responses by suppressing a diverse set of cell subtypes, including T cells, through the secretion of anti-inflammatory mediators such as cytokines. Immunosuppressive B cells can also function in attenuating immune responses by negatively regulating lymphoid structures and / or promoting the conversion of T cells into regulatory T cells. Thus, methods are disclosed herein for targeting immunosuppressive B cell populations to effectively modulate responses.

[0176] Targeting immunosuppressive B cells or B cell populations can result in positive regulation of immune activation or immune response against tumors or tumorigenic cells. Provided herein is a method of treating an individual suffering from a cancer or tumor, comprising administering to the individual suffering from a cancer or tumor a complex binding molecule disclosed herein. Also provided herein is a method of reducing immunosuppressive B cells in, adjacent to, or around a tumor in an individual suffering from a cancer, comprising administering to the individual suffering from a tumor or cancer a complex binding molecule disclosed herein, thereby reducing immunosuppressive B cells in, adjacent to, or around a tumor. Also disclosed is a method of contacting immunosuppressive B cells in a subject with a complex binding molecule, comprising administering to the subject a complex binding molecule. In certain embodiments, the subject has a tumor or cancer.

[0177] The type, subtype, or form of tumor or cancer may be an important factor in treatment strategies and methods. In some embodiments, the cancer or tumor is a blood cancer. In some embodiments, the cancer or tumor is a solid tissue cancer. In some embodiments, the cancer comprises breast cancer, prostate cancer, pancreatic cancer, lung cancer, kidney cancer, stomach cancer, esophageal cancer, skin cancer, colon cancer, or head and neck cancer.

[0178] Immunosuppressive B cells can suppress anti-tumor immune responses. In some embodiments, the tumor or cancer comprises B cells comprising B cell lineage surface biomarkers and inhibitory B cell surface biomarkers. The B cell lineage surface markers can comprise CD19, CD138, IgA, or CD45. The B cell surface markers can comprise IgD, CD1, CD5, CD21, CD24, CD38, HM13, SLAMF7, AQP3, or TGFB. In some embodiments, the B cell surface markers comprise CD19 (e.g., CD19+) and CD38 (e.g., CD38+). In some embodiments, the tumor-infiltrating B cells or immunosuppressive B cells comprise CD19+, CD38+ B cells.

[0179] In certain embodiments, bispecific antibodies useful for treating cancer or tumors are disclosed herein. Treatment refers to a method that attempts to improve or ameliorate the condition being treated. With respect to cancer, treatment includes, but is not limited to, reducing tumor volume, reducing tumor volume growth, increasing progression-free survival, or increasing overall life expectancy. In certain embodiments, treatment affects the remission of the cancer being treated. In certain embodiments, treatment includes use as a prophylactic or maintenance dose intended to prevent the recurrence or progression of a previously treated cancer or tumor. It will be understood by those skilled in the art that not all individuals will respond equally or reliably to a given treatment, but these individuals are nevertheless considered to be treated.

[0180] In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor is a hematological cancer or tumor. In certain embodiments, the cancer or tumor comprises tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, and liver. In certain embodiments, tumors that can be treated with the antibodies of the invention comprise adenoma, adenocarcinoma, hemangiosarcoma, astrocytoma, epithelial carcinoma, embryonal tumor, glioblastoma, glioma, hemangioendothelioma, hemangioma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and / or teratoma. In certain embodiments, the tumor / cancer is selected from the group consisting of acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, Bartholin's adenocarcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Ewing's sarcoma, and the like. sarcoma), focal nodular hyperplasia, gastrinoma, germ line tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinite, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung carcinoma, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelioma, nerve sheath tumor, medulloblastoma, medullary epithelium The tumor is selected from the group consisting of ovarian carcinoma, mesothelioma, mucosal epithelial carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian carcinoma, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, prostate carcinoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal / vulvar carcinoma, VIPpoma, and Wilms' tumor.In certain embodiments, the tumors / cancers treated with one or more antibodies of the present disclosure include brain tumors, head and neck cancer, colon cancer, acute myeloid leukemia, pre-B cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably grade II, III, or IV astrocytoma, glioblastoma, glioblastoma multiforme, small cell carcinoma, and non-small cell carcinoma, preferably non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic adenocarcinoma, androgen-dependent metastatic adenocarcinoma, prostate cancer, and breast cancer, preferably breast ductal carcinoma, and / or breast cancer. In certain embodiments, the cancers treated with the antibodies of the present disclosure include glioblastoma. In certain embodiments, the cancers treated with the antibodies of the present disclosure include pancreatic cancer. In certain embodiments, the cancers treated with the antibodies of the present disclosure include ovarian cancer. In certain embodiments, the cancers treated with the antibodies of the present disclosure include lung cancer. In certain embodiments, the cancers treated with the antibodies of the present disclosure include prostate cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure comprises colon cancer. In certain embodiments, the cancer treated comprises glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In certain embodiments, the cancer is refractory to other treatments. In certain embodiments, the cancer treated is recurrent. In certain embodiments, the cancer comprises recurrent / refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer.

[0181] In certain embodiments, the cancer and / or tumor treated with the conjugate binding molecules herein is a mature B-cell neoplasm: chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, follicular lymphoma), mucosa-associated lymphoid tissue (MALT) lymphoma, mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma and Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, or primary central nervous system lymphoma.

[0182] In certain embodiments, the cancer and / or tumor treated with the conjugate binding molecules herein is a T-cell neoplasm, such as T-cell non-Hodgkin's lymphoma, T-cell ALL, mycosis fungoides, anaplastic large cell lymphoma, peripheral T-cell lymphoma, T-lymphocytic leukemia (T-ALL), acute myeloblastic leukemia, acute monocytic leukemia, and the like.

[0183] In certain embodiments, the antibody can be administered to a subject in need of the antibody by any route suitable for administration of a pharmaceutical composition containing the antibody, such as, for example, subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered subcutaneously. In certain embodiments, the antibody is administered intratumorally. In certain embodiments, the antibody is administered on a suitable dosing schedule, such as weekly, twice weekly, monthly, twice monthly, once every two weeks, once every three weeks, or once monthly. In certain embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1 mg / kg and about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 30 mg / kg. A therapeutically effective amount includes an amount sufficient to ameliorate one or more symptoms associated with the disease or affliction being treated. EXAMPLES

[0184] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0185] Example 1: Octet binding data Biolayer interferometry was used to determine the binding affinity of parental and bispecific antibodies. Binding experiments were performed on an Octet Red96 at 25°C using an assay buffer consisting of 0.1% BSA, 1XPBS, 0.02% Tween-20, 0.05% NaN3. Antibodies were loaded onto anti-hIgG Fc Capture biosensors for 300 seconds. Ligand-loaded sensors were immersed in serial dilutions of antigen (starting at 300 nM: 2-fold serial dilutions for CD19 and 3-fold serial dilutions for CD38) for association (200 seconds for CD19, 150 seconds for CD38) followed by dissociation (600 seconds for CD19, 400 seconds for CD38). Rate constants were calculated using a monovalent (1:1) binding model.

[0186] The parent test substances included:

[0187] 851A=anti-CD19 3C10

[0188] 851B=Anti-CD19 3C10 heavy chain & Anti-CD38 003 light chain

[0189] 851C=anti-CD38 003 heavy chain & anti-CD19 3C10 light chain

[0190] 851D=Anti-CD38 003

[0191] 851E=anti-CD19 3C10(scFv-Fc)2

[0192] 851F=anti-CD38 003(scFv-Fc)2

[0193] The two parental antibodies (851A / 851E) with anti-CD19 3C10 VH and VL bound to CD19 with similar KD. Substitution of anti-CD19 3C10 VL with anti-CD38 VL (851B) resulted in an approximately 5-fold reduction in binding to CD19. As expected, the parental antibody (851D / 851F) with anti-CD38 003 VH and VL did not bind to CD19.

[0194] Table 2 shows the binding data. Two pasrental antibodies with anti-CD38 003 VH and VL (851D / 851F) bound to CD38 with similar KD. Substitution of anti-CD38 003 VL with anti-CD19 VL (851C) resulted in a significant reduction in binding to CD38. As expected, the parental antibodies with anti-CD19 VH and VL (851A / 851E) did not bind to CD38, nor did 851B. This data indicates that only anti-CD38 003 VL can function as a common light chain for anti-CD19 3C10 VH.

[0195] [Table 2]

[0196] Bispecific antibody (format) test articles included:

[0197] BS1 = 003HC:3C10HC:003LC (common light chain) in a ratio of 1:1:2

[0198] BS1b = 003HC:3C10HC:003LC (common light chain) in a ratio of 2:1:2

[0199] BS2 = 003Knob:3C10scFvHole:003LC (Fab-Fc:scFv-Fc bispecific IgG1) at a ratio of 1:1:1

[0200] BS2b = 003Knob:3C10scFvHole:003LC (Fab-Fc:scFv-Fc bispecific IgG1) in a ratio of 4:1:4

[0201] BS3 = 3C10scFv-003Fab-FcKnob:FcHole:003LC) at a ratio of 1:1:1 (scFv-Fab-Fc:Fc bispecific IgG1)

[0202] BS4 = 003Fab-FcKnob-3C10scFv:FcHole (Fab-Fc-scFv:Fc bispecific IgG1) at a ratio of 1:1:1

[0203] BS4b = 003Fab-FcKnob-3C10scFv:FcHole (Fab-Fc-scFv:Fc bispecific IgG1) at a ratio of 4:1:4

[0204] CM1 = 3C10Hole:VZVKnob:003LC anti-CD19 control antibody at a ratio of 1:1:2

[0205] CM1b=1:3:3 ratio 3C10Hole:VZVKnob:003LC

[0206] CM2 = 1:1:2 ratio of 003Knob:VZVHole:003LC anti-CD38 control antibody

[0207] CM2b=3:1:3 ratio 003Knob:VZVHole:003LC

[0208] Table 3 shows the binding data for the bispecific test articles in single antigen format. Bispecific antibodies BS1 / BS2 / BS4 bound both target antigens with KDs within 4-fold of the parent antibodies (shown in grey shading). BS3 bound only to CD19 and not to CD38, suggesting either that the anti-CD38 Fab binding site was blocked by the anti-CD19 scFv N-terminal fusion or that anti-CD38 requires a free VH N-terminus for binding. One-arm control antibodies (CM1, CM2) bound only to their intended target antigens.

[0209] [Table 3]

[0210] In the two-antigen format, antibodies were loaded onto anti-hIgG Fc Capture biosensors for 300 seconds. The ligand-loaded sensors were saturated with 500 nM of the first antigen for 500 seconds, followed by 300 nM of the second antigen for 240 seconds. Rate constants were calculated using a monovalent (1:1) binding model. Table 5 shows that bispecific antibodies BS1 / BS2 / BS4 were able to simultaneously bind both target antigens with ka (1 / Ms) within 2-fold of the parent antibodies (851B, 851D, and 851E). As with the one-antigen format, BS3 bound only to CD19 and not to CD38.

[0211] [Table 4-1]

[0212] [Table 4-2]

[0213] The variants were further tested for their binding ability to CD19 and / or CD38. Binding experiments were performed on Octet Red at 25°C. Antibodies were loaded onto anti-hIgG Fc Capture (AHC) biosensors for 300 seconds. The ligand-loaded sensors were immersed in two-fold serial dilutions of antigens (CD19 and CD38) (starting at 300 nM) for 240 seconds for CD19 and 150 seconds for CD38 for association, followed by 600 seconds for CD19 and 130 seconds for CD38 for dissociation. Rate constants were calculated using a monovalent (1:1) binding model. Table 5 shows the binding of anti-CD38 CDRH2 variants. Table 6 shows the binding of the CD38 light chain W32H variant. Table 8 shows the binding of the CD19 heavy chain framework mutant A84S A108L.

[0214] [Table 5]

[0215] [Table 6]

[0216] [Table 7]

[0217] Example 2: Cell binding assay Cell binding assay protocol: Five cell lines (HEK293-CD19, HEK293-CD38, HEK293-CD19 / CD38, Daudi, and REH) were incubated in triplicate with test articles at 133 nM followed by 3-fold serial dilutions (total of 7 points) in addition to an untreated control. HEK293 cell lines were transiently transfected.

[0218] Studies were performed to evaluate cell surface expression of CD19 and CD38 on Daudi, Raji, and REH cell lines. Cells were stained in triplicate with commercially available antibodies conjugated to PE, washed, and acquired by flow cytometry. To quantify molecule expression on the cell surface, a standard curve was generated to interpolate MFI to molecule number / cell values ​​using the Quantum Simply Cellular anti-mouse IgG kit from Bangs Laboratories (catalog #815-A) (Table 8).

[0219] [Table 8]

[0220] Figure 12A shows the binding of parental antibodies (851A, 851B, 851D) as well as two control bispecific antibodies (each with one arm against CD19 or CD38 and the other arm against Varicella-Zoster Virus) to Daudi cells. Considering that Daudi cells have about 1 million copies of CD38 on their surface but only about 200,000 copies of CD19, Figure 12A shows that anti-CD38 851D and 38K-VZVH bind efficiently, whereas anti-CD19 851A, 851B, 19H-VZVK bind only moderately. Note that 851D, which has two CD38-binding Fabs, binds about 5 times better than 38K-VZVH, which has only one binding Fab against CD38.

[0221] Figure 12B shows the binding of bispecific antibodies BS1, BS2, and BS4 to Daudi cells. The avidity of the bispecific antibodies that bind to both CD38 and CD19 is revealed by comparing their binding to 38K-VZVH, which binds only to CD38.

[0222] Figure 13A shows the binding of parental antibodies (851A, 851B, 851D) as well as two control bispecific antibodies (each with one arm against CD19 or CD38, and the other arm against Varicella-Zoster Virus) to REH cells. Considering that REH cells have about 300,000 copies of CD38 on their surface but only about 50,000 copies of CD19, Figure 13A shows that anti-CD38 851D and 38K-VZVH bind efficiently, whereas anti-CD19 851A, 851B, 19H-VZVK bind only moderately. The magnitude of MFI is significantly less compared to Daudi cells due to the low expression levels of both CD38 and CD19 on REH cells (Figures 2A, 2B). Note that 851D, which has two CD38 binding Fabs, binds approximately 5-fold better than 38K-VZVH, which has only one binding Fab to CD38.

[0223] Figure 13B shows the binding of bispecific antibodies BS1, BS2, and BS4 to REH cells. The avidity of the bispecific antibodies that bind to both CD38 and CD19 is revealed by comparing their binding to 38K-VZVH, which binds only to CD38.

[0224] Figure 14A shows the binding of parental antibodies (851A, 851B, 851D) and two control bispecific antibodies (38K-VZVH, 19H-VZVK) to CD19-transfected HEK293 cells. As expected, the two anti-CD38 antibodies do not bind to these cells. Note that 851A and 851B, which have two CD19-binding Fabs, bind significantly better than 19H-VZVK, which has only one binding Fab to CD19.

[0225] Figure 14B shows the binding of bispecific antibodies BS1, BS2, and BS4 to CD19 transfected HEK293 cells. BS2 and BS$ bind slightly better than BS1. Because BS1 has an anti-CD38 light chain, BS2 and BS4 bind CD19 approximately 10 times better than BS1 (see Octet data table).

[0226] Figure 15A shows the binding of parental antibodies (851A, 851B, 851D) and two control bispecific antibodies (38K-VZVH, 19H-VZVK) to CD38-transfected HEK293 cells. As expected, the three anti-CD19 antibodies do not bind to these cells. Note that 851D, which has two CD38-binding Fabs, binds better than 38K-VZVH, which has only one binding Fab for CD38.

[0227] FIG. 15B shows binding of bispecific antibodies BS1, BS2, and BS4 to CD38-transfected HEK293 cells.

[0228] Cell Binding Study Protocol - Nonspecific Background Binding: A study was performed to evaluate the binding of three parental monoclonal antibodies (anti-CD19 clones 851A and 851B, and anti-CD38 clone 851D), a human IgG1 isotype control, and daratumumab to CHO-S and Expi293T cell lines. The two cell lines were stained with a viability dye and then incubated in triplicate with the test article at the highest concentration of 1,250 nM followed by 5-fold serial dilutions (4 points total) in addition to an untreated control as well as a no treatment, no secondary control.

[0229] Figure 16A shows the binding of parental antibodies (851A, 851B, 851D) to non-transfected CHO-S cells. Non-specific binding was observed for all three parental antibodies starting at 250 nM, and was more pronounced with anti-CD38 851D.

[0230] Figure 16B shows binding of parental antibodies (851A, 851B, 851D) to non-transfected Expi293T cells. Non-specific binding was observed for all three parental antibodies starting at 250 nM, and was more pronounced with anti-CD38 851D.

[0231] Example 3: Direct and cross-linking apoptosis For the assessment of direct apoptosis, cells were treated with test substances and incubated for 48 hours at 37°C / 5% CO2. For the assessment of crosslinking-induced apoptosis, cells were incubated with test substances for 30 minutes on ice, followed by the addition of 5 μg / mL rabbit anti-human Fc gamma specific F(ab')2. Cells were then incubated for 48 hours at 37°C / 5% CO2. After incubation, cells were washed, stained with Annexin V, and then resuspended in Annexin V buffer containing a dead cell labeling reagent (propidium iodide; PI) before flow cytometry acquisition. Early apoptotic cells were defined as Annexin V+ / PI- single cells, and late apoptotic / necrotic cells were defined as Annexin V+ / PI+ single cells. The sum of Annexin V+ / PI- and Annexin V+ / PI- was defined as the total apoptotic / necrotic cells. The percentage of Annexin V+ / PI- cells or Annexin V+ / PI+ was plotted to compare the various apoptotic conditions.

[0232] For direct apoptosis assessment, test articles were tested in triplicate at a final top concentration of 33 nM, followed by seven 5-fold serial dilutions, in addition to an untreated control. For crosslinking-induced apoptosis, individual test articles (BS1, BS2, BS4, 851A, 851B, and 851D) and combinations of test articles (851A and 851D, 851B and 851D, 38K-VZVH and 19H-VZVK), in addition to daratumumab and IgG1 isotype controls, were tested in triplicate at a final top concentration of 33 nM, followed by seven 5-fold serial dilutions, in addition to an untreated control. As a positive control for Annexin V staining, cells were treated with 5 mM staurosporine.

[0233] Figure 17A shows direct apoptosis in Daudi cells for parental antibodies (851A, 851B, 851D), two control bispecific antibodies (38K-VZVH, 19H-VZVK), daratumumab and an IgG1 isotype control. Daratumumab exhibited the highest level of apoptosis. Both anti-CD19 parents (851A, 851B) exhibited low levels of apoptosis compared to daratumumab. The two bispecific controls and the anti-CD38 parent antibody 851D did not show any discernible direct apoptosis.

[0234] Figure 17B shows direct apoptosis in Daudi cells for bispecific antibodies BS1, BS2, BS4, daratumumab, and IgG1 isotype control. The BS1 and BS2 formats showed significantly higher levels of direct apoptosis compared to daratumumab. The bispecific format BS4 showed a similar level of direct apoptosis to the parent anti-CD19 851A / 851B antibody (compare Figure 17A), which may be due to the inability of the BS4 format to bring CD19 and CD38 into close proximity to initiate apoptosis.

[0235] FIG. 18A shows cross-linking-induced apoptosis in Daudi cells for parental antibodies (851A, 851B, 851D), two combinations of parental antibodies (851A+851D, 851B+851D), daratumumab, and an IgG1 isotype control. Cross-linking increased the level of daratumumab-driven apoptosis. Cross-linking significantly increased the level of apoptosis in anti-CD38 851D, which did not show direct apoptosis. Cross-linking of anti-CD19 parental antibodies 851A and 851B increased the level of apoptosis less than CD38 antibodies, likely due to the low CD19 levels in Daudi cells compared to CD38 (see Table 9). Cross-linking of anti-CD19 851A or 851B in combination with anti-CD38 851D did not increase the level of apoptosis compared to 851D alone.

[0236] FIG. 18B shows cross-linking-induced apoptosis in Daudi cells for bispecific antibodies BS1, BS2, BS4, (38K-VZVH+19H-VZVK), daratumumab, and an IgG1 isotype control. When cross-linked, the BS1 and BS2 formats showed a level of apoptosis comparable to daratumumab. Notably, the bispecific format BS4 showed a level of cross-linking-induced apoptosis comparable to BS1, BS2, and daratumumab, and in the absence of cross-linking, BS4 showed no apoptosis (see FIG. 6B). The combination of the two control antibodies, 38K-VZVH and 19H-VZVK, exhibited significant apoptosis, but less than either of the bispecific formats, demonstrating the advantage of including anti-CD19 and anti-CD38 binding sites in a single antibody over separate antibodies.

[0237] Example 4: Cytotoxicity Daudi target cells were treated with a dose response of test article and incubated for 15 min at 37° C. / 5% CO2. In addition to a 0 nM control, test articles were tested at a final top concentration of 133 nM followed by seven point 5-fold serial dilutions. Daratumumab and an IgG1 isotype control were used as positive and negative controls.

[0238] Pretreated target cells were co-cultured with human PBMCs from n=3 donors (E:T 25:1). PBMCs were "primed" overnight with 100 U / mL IL-2. PBMCs were labeled with ViaFluor 405 (VF405). Samples were incubated for 4 hours at 37°C / 5% CO2 before flow cytometric analysis for cytotoxicity. For cytotoxicity analysis, cells were stained with propidium iodide (PI) and analyzed by high-throughput flow cytometry. The percentage of PI+ cells within the VF405- population was analyzed as an index of target cell cytotoxicity.

[0239] Figures 19A, 19B, and 19C show antibody-dependent cellular cytotoxicity (ADCC) for three donors. Results were similar for all three donors. The three bispecific formats BS1, BS2, BS4, and daratumumab exhibited similar levels of ADCC. The anti-CD19 bispecific control 19H-VZVK did not induce ADCC and was comparable to the IgG1 control antibody (see Table 9), likely due to the low levels of CD19 on the target Daudi cells. In contrast, the anti-CD38 bispecific control 38K-VZVH exhibited ADCC comparable to the bispecifics and daratumumab, likely due to the much higher levels of CD38 on Daudi cells compared to CD19.

[0240] Figures 20A, 20B, and 20C show ADCC for three donors. Results were similar for all three donors. The three bispecific formats BS1, BS2, and BS4 exhibited similar levels of ADCC. The defucosylated versions of BS1, BS2, and BS4 showed approximately 10-fold increase in ADCC compared to the fucosylated versions.

[0241] Complement-dependent cytotoxicity (CDC) assays were also performed. Target cells were treated with the following test articles: BS1, BS2, 38K-VZVH, 19H-VZVH, the combination 38K-VZVH / 19H-VZVH, as well as dose response of the controls Darazalex, anti-CD20, WT IgG1 Tafasitamab, and human IgG1 isotype control. All were tested at a top concentration of 133 nM followed by 5-fold serial dilutions for a total of seven points, in addition to an untreated control. After 15 minutes of incubation at 37°C, 5% CO2, complement was added to the treated cells at a final concentration of 25%. Cells were then incubated with complement for an additional 2 hours at 37°C, 5% CO2. After incubation with complement, cells were washed and resuspended with 5 μg / mL of the dead cell labeling reagent, propidium iodide (PI), and acquired by high-throughput flow cytometry.

[0242] Figures 21A and 21B show the results of a complement-dependent cytotoxicity (CDC) assay. The positive technical control, anti-CD20, induced robust dose-dependent CDC activity. 38K-VZVH and 19H-VZVH (either alone or in combination), anti-CD19 tafasitamab (wt IgG1), and human IgG1 isotype control did not induce any CDC activity. Darzalex, BS1, and BS2 all showed CDC activity (although not at the same magnitude as anti-CD20, as expected from the literature). The maximum cytotoxicity of Darzalex was higher than that of both BS1 and BS2.

[0243] Antibody-dependent cellular phagocytosis (ADCP) was further assayed by pHrodo Green AM (pHG)-labeled Raji cells treated with a dose response of test substances and incubated at 37°C, 5% CO2 for 15 min. pHG is a pH-sensitive dye that is only weakly fluorescent at neutral pH, but highly fluorescent at low pH in mature phagosomes of macrophages. pHG-labeled Raji target cells with anti-CD20 antibody and IgG1 isotype control were used as positive and negative controls, with a top concentration of 133 nM, seven 5-fold serial dilutions, and a 0 nM control. Pretreated target cells were co-cultured (E:T 1:2) with human macrophages (differentiated in vitro from monocytes) from n=3 donors. Macrophages were labeled with Cell Trace Violet (CTV). Samples were incubated at 37°C, 5% CO2 for 4 h before flow cytometric analysis of phagocytosis. The percentage of pHGhi / CTV+ cells was analyzed as an index of target cell phagocytosis. The percentage was plotted against the logarithm of the test article concentration on an XY chart, and the data was fitted to a four-parameter nonlinear regression curve from which the EC50 was calculated.

[0244] FIG. 22 shows the results of an antibody-dependent cellular phagocytosis (ADCP) assay using Raji cells and donor macrophages as targets. The positive control, anti-CD20, demonstrated dose-dependent phagocytosis in all three donors after 4 hours (maximum phagocytosis between 5-10%). The negative control, IgG1 isotype control, demonstrated no dose-dependent phagocytosis in all three donors after 4 hours. Darazalex demonstrated dose-dependent phagocytosis in all three donors after 4 hours (maximum phagocytosis between 4-10%). BS-1, BS-2, defucosylated BS-1, and defucosylated BS-2 showed slight dose-dependent phagocytosis, with the defucosylated format resulting in increased ADCP.

[0245] Example 5: Interaction with RBCs A flow cytometry-based red blood cell (RBC) binding study was performed to evaluate the binding of test articles to red blood cells from n=3 cynomolgus monkeys and n=3 human donors. Whole blood was washed with 1X PBS and then diluted 20-fold with PBS before treatment with test articles. In addition to a 0 nM control, bispecifics (BS1, BS2), parental monoclonals (851A, 851D), and controls (anti-CD38 Darazalex, recombinant anti-CD19 Tafasitamab, IgG1 isotype control, anti-CD47 conjugated to Alexa Fluor 647) were tested in triplicate at a top final concentration of 133 nM followed by 5-fold serial dilutions for a total of seven points. Single-arm controls (38K-VZVH, 19H-VZVK) were tested in combination, both with a top concentration of 133 nM and the same dose response.

[0246] After 30 minutes incubation on ice with primary antibody, cells were washed and stained with 5ug / mL of secondary antibody (Alexa Fluor 647-labeled goat anti-human Fcγ F(ab')2) to detect binding of test article on red blood cells. No secondary antibody was used for anti-CD47-A647 stained cells. After another 30 minutes incubation on ice with secondary antibody, stained cells were washed, diluted, and acquired by high-throughput flow cytometry. AlexaFluor 647 geometric mean fluorescence intensity (MFI) of single cell populations was calculated. The MFI of AF647 was plotted on an XY chart, MFI was graphed against the logarithm of concentration, and the data was fitted to a nonlinear regression curve from which the EC50 was calculated.

[0247] Figure 23 shows that AF647-conjugated anti-CD47 showed dose-response binding curves with red blood cells from all three human donors. Darzalex also showed a dose-dependent increase in binding in all three donors, but the maximum MFI was an order of magnitude lower than anti-CD47. Anti-CD38 851D showed the second highest maximum MFI after Darzalex, followed by BS1, BS2, the combination of 38K-VZVH&19H-VZVK, and anti-CD19 tafasitamab. Finally, anti-CD19 851A and the IgG1 isotype showed only a slight increase in MFI only at the highest concentrations.

[0248] In vitro hemagglutination assays were performed on red blood cells from a total of three healthy (n=3) cynomolgus monkey (Cyno) donors and three healthy (n=3) human donors. Whole blood was obtained on the day of the study and examined for clotting. Blood was then washed with PBS and diluted 1:50 to obtain "whole blood matrix." Whole blood matrix was plated in 96-well round-bottom plates and treated in triplicate with test articles (BS1, BS2, 38K-VZVH+19H-VZVK, 851A, and 851D), controls (tafasitamab with wild-type IgG1), darazalex, and human IgG1 isotype control), or positive technical control (IGM-55.5) in PBS at a top final concentration of 133 nM, followed by six point 5-fold serial dilutions, in addition to a 0 nM control. After 1 hour of incubation at 37°C and 5% CO2, the plates were photographed to confirm the level of hemagglutination. Using the photograph as a reference, each well was scored on a unique hemagglutination scale from 0 to 5. The specific sign of each score is somewhat relative to the individual donor.

[0249] FIG. 24A shows the results of the hemagglutination assay for human donor 3. The positive control, anti-CD47, induced hemagglutination in all three human donors starting between 0.04 and 1.1 nM. BS1, BS2, 38K-VZVH+19H-VZVK, Darazalex, Tafasitamab, and the human IgG1 isotype control all showed no induction of hemagglutination at any concentration in all three donors. Both monoclonal antibodies 851A (anti-CD19) and 851D (anti-CD38) induced hemagglutination in all three donors starting at 0.2 or 1.1 nM, respectively, with responses similar in magnitude to the technical control (anti-CD47). In contrast to the parent monoclonal antibodies, BS1 and BS2 showed no induction of hemagglutination at any concentration.

[0250] FIG. 24B shows the results of the hemagglutination assay for cynomolgus donor 3. The positive control IGM-55.5 (anti-little i antigen IgM antibody) induced hemagglutination in all three cynomolgus donors starting at 0.04 or 0.2 nM. BS1, BS2, 38K-VZVH+19H-VZVK, Darazalex, Tafasitamab, and the human IgG1 isotype control all showed no induction of hemagglutination at any concentration in all three donors. Both monoclonal antibodies 851A (anti-CD19) and 851D (anti-CD38) induced hemagglutination in all three donors starting at 1.1 nM each. In contrast to the parent monoclonal antibodies, BS1 and BS2 showed no induction of hemagglutination at any concentration.

[0251] An in vitro hemolysis assay was also performed on red blood cells from three (n=3) healthy cynomolgus monkeys (cyno) and three (n=3) healthy human donors. Whole blood was obtained on the day of testing and examined for clotting. Blood was washed with PBS and diluted 1:10 to obtain a "whole blood matrix." Whole blood matrix was treated with test articles and controls in PBS. In addition to a 0 nM control, bispecifics (BS1, BS2), parental monoclonals (851A, 851D), and controls (anti-CD38 Darazalex, recombinant anti-CD19 Tafasitamab, IgG1 isotype control) were tested in triplicate at a top final concentration of 133 nM followed by 5-fold serial dilutions for a total of seven points. Single arm controls (38K-VZVH, 19H-VZVK) were tested in combination, both with a top concentration of 133 nM and the same dose response. Saponin was tested at a top concentration of 0.1% with a total of seven 3-fold serial dilutions. After 1 hour incubation at 37°C, 5% CO2, plates were centrifuged and supernatants were collected. Supernatants were analyzed for optical density (OD) at 540 nm by plate reader. The positive control, saponin, induced dose-dependent hemolysis starting at 0.001% up to 0.10% in all species and donors. None of the test substances induced hemolysis at any of the concentrations tested.

[0252] Figure 25 shows that all test articles did not induce hemolysis at any of the concentrations tested. The positive control, saponin, induced dose-dependent hemolysis starting at 0.001% up to 0.10% in all species and donors.

[0253] Example 6: FCR variants reduce ADCC in CD38- and CD19-binding bispecific antibodies B cells isolated from healthy human peripheral blood mononuclear cells (PBMCs) were treated with a dose response of test article and incubated for 15 minutes. Raji and Daudi target cells were similarly treated with a dose response of Rituxan, Darzalex, or IgG1 isotype control and incubated for 15 minutes at 37° C., 5% CO2. In addition to the 0 nM control, N=5 test articles and n=3 controls (Rituxan, Darzalex, and human IgG1 isotype) were tested at a maximum final concentration of 133 nM followed by seven 5-fold serial dilutions.

[0254] Pretreated target cells were co-cultured with human PBMCs from n=3 donors (E:T 25:1). PBMCs were primed overnight with 100U / mL IL-2. PBMCs were labeled with ViaFluor 405. Samples were incubated for 4 hours at 37°C and 5% CO2. Test articles included BS1, defucosylated BS1, BS1 with Fc variants ("dead Fc", e.g., SEQ ID NOs: 301 and 302), as well as controls Rituxan, Darazalex, and human IgG1 isotype. BS1 already showed a good low ADCC profile, so it was interesting in that in each case the variant Fc (dead Fc) was further reduced and / or decreased.

[0255] For cytotoxicity analysis, cells were stained with propidium iodide (PI) and analyzed by high-throughput flow cytometry. The percentage of PI+ cells within the VF405- population was analyzed as an index of target cell cytotoxicity. Figure 26A shows the level of ADCC on Raji and Daudi control target cells using PBMCs from donor 3. Figure 26B shows the level of ADCC on target B cells from donor 1 using PBMCs from donor 1. Figure 26C shows the level of ADCC on target B cells from donor 3 using PBMCs from donor 1. Figure 26D shows the level of ADCC on target B cells from donor 1 using PBMCs from donor 2. Figure 26E shows the level of ADCC on target B cells from donor 3 using PBMCs from donor 2. Figure 26F shows the level of ADCC on target B cells from donor 1 using PBMCs from donor 3. Figure 26G shows the level of ADCC on target B cells from donor 3 using PBMC from donor 3. Because BS1 demonstrated a good ADCC profile (e.g., low ADCC), it was unexpected that the ADCC of BS1 could be further reduced using a variant Fc (e.g., a "death" Fc containing mutations S239D and I332E). Such further reduction could be advantageous in therapeutic treatment by further reducing the possibility of immunological adverse events. This is especially true in a possible mechanism of action where unwanted cells non-tumor cells (i.e., CD19xCD38 inhibitory B cells) could be specifically targeted.

[0256] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

[0257]

Table 9-1

[0258]

Table 9-2

[0259]

Table 9-3

[0260]

Table 9-4

[0261]

Table 9-5

[0262]

Table 9-6

[0263]

Table 9-7

[0264]

Table 9-8

[0265]

Table 9-9

[0266]

Table 9-10

Claims

1. 1. A complex binding molecule comprising: (i) a CD38 antigen binding component that binds to CD38; (ii) a CD19 antigen binding component that binds to CD19; and (iii) a variant Fc region comprising one or more mutations compared to a wild-type Fc region, wherein the variant Fc region exhibits an altered effector function compared to the wild-type Fc region, wherein the altered effector function is selected from the list consisting of decreased antibody-dependent cell-mediated cytotoxicity (ADCC), decreased complement-dependent cytotoxicity (CDC), decreased affinity for C1q, and any combination thereof.

2. The complex-binding molecule described in claim 1, wherein the variant Fc region is selected from Table 1.

3. wherein the one or more mutations compared to a wild-type Fc region are:

3. The complex binding molecule of claim 2, selected from the group consisting of N297A / Q / G, L235A / G237A / E318A, L234A / L235A, G236R / L328R, S298G / T299A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, L234A / L235A / P329G, V234A / G237A / P238S / H268A / V309L / A330S / P331S, and L234F / L235E / D265A.

4. the CD38 antigen binding component a) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 71-75; b) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 81-85 or 151-155; c) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 91-95; d) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101-105; e) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111-115; and f) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121-125; the CD19 antigen-binding component g) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 11-15; h) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 21-25; i) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 31-35; j) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 101-105; k) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 111-115; and 1) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 121-125; 5. The complex-binding molecule of claim 4, wherein the CD38 antigen-binding component comprises an HCDR2 amino acid sequence comprising an amino acid sequence set forth in SEQ ID NOs: 151-155.

6. The complex-binding molecule of claim 5, wherein the CD38 antigen-binding component comprises an HCDR2 amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO:

154.

7. The complex-binding molecule of claim 4, wherein the CD38 antigen-binding component comprises an HCDR2 amino acid sequence comprising any one of the amino acid sequences set forth in SEQ ID NOs: 81-85.

8. The complex-binding molecule of claim 1, wherein the CD38 antigen-binding component comprises an anti-CD38 immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 90% identity to SEQ ID NO: 3 or 5, the anti-CD38 immunoglobulin light chain variable region comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 4, the CD19 antigen-binding component comprises an anti-CD19 immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 90% identity to SEQ ID NO: 1, 6, or 7, and the anti-CD19 immunoglobulin light chain variable region comprises an amino acid sequence having at least about 90% identity to SEQ ID NO:

2.

9. The complex binding molecule described in claim 1, wherein the anti-CD38 immunoglobulin heavy chain variable region further comprises an immunoglobulin heavy chain constant region, and the anti-CD38 immunoglobulin heavy chain constant region comprises one or more amino acid substitutions that do not support homodimerization of the anti-CD38 immunoglobulin heavy chain constant region but promote heterodimerization of the anti-CD38 immunoglobulin heavy chain constant region with a non-anti-CD38 immunoglobulin heavy chain constant region.

10. The complex-binding molecule of claim 1, wherein the anti-CD19 immunoglobulin heavy chain variable region further comprises an immunoglobulin heavy chain constant region, and the anti-CD19 immunoglobulin heavy chain constant region comprises one or more amino acid substitutions that do not support homodimerization of the anti-CD19 immunoglobulin heavy chain constant region but promote heterodimerization of a second heavy chain constant region with a non-anti-CD19 immunoglobulin heavy chain constant region.

11. A complex-binding molecule described in any one of claims 1 to 10, which is a common light chain bispecific antibody.

12. A composition comprising a complex binding molecule according to any one of claims 1 to 10, further comprising a pharmaceutically acceptable diluent, carrier, or excipient.

13. A nucleic acid or multiple nucleic acids comprising a polynucleotide sequence encoding a complex-binding molecule according to any one of claims 1 to 10.

14. A complex-binding molecule according to any one of claims 1 to 10, wherein the variant Fc region reduces ADCC by at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% or more compared to an antibody comprising a non-variant Fc region.

15. A complex-binding molecule according to any one of claims 1 to 10, wherein the variant Fc region reduces CDC by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or more compared to an antibody comprising a non-variant Fc region.

16. A complex binding molecule according to any one of claims 1 to 10 for use in treating an individual suffering from cancer or a tumor, wherein the cancer or tumor is a blood cancer or a solid tissue cancer.

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