Bispecific antibody fusion molecules targeting CD20 and CD3 and methods of use thereof

Abstract

The present disclosure provides bispecific and multispecific antibodies or antigen binding fragments thereof that specifically bind to CD3 and CD20. The antibodies are optionally fused with a CD58 peptide or portions thereof. Described herein are methods of making and using the bispecific antibodies for treatment of cancers that express CD20.

Description

Attorney Docket No. E0701.70002WO00 BISPECIFIC ANTIBODY FUSION MOLECULES TARGETING CD20 AND CD3 AND METHODS OF USE THEREOF RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 702,686, filed October 3, 2024, entitled “Bispecific Antibody Fusion Molecules Targeting CD20 and CD3 and Methods of Use Thereof”, and U.S. Provisional Application No.63 / 717,211, filed November 6, 2024, entitled “Bispecific Antibody Fusion Molecules Targeting CD20 and CD3 and Methods of Use Thereof,” the entire contents of each of which are incorporated herein by reference. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (E070170002WO00-SEQ-LJG.xml; Size: 536,259 bytes; and Date of Creation: October 2, 2025) are herein incorporated by reference in its entirety. FIELD

[0003] This disclosure relates to the pharmaceutical compositions of bispecific antibodies that bind to CD20 and CD3, as well as multispecific antibodies that bind to CD20, CD3, and CD2, and their use for treating CD20-expressing cancers. BACKGROUND

[0004] CD20 (also known as human B-lymphocyte-restricted differentiation antigen or Bp35) is a hydrophobic transmembrane protein on pre-B and mature B lymphocytes and has a molecular weight of approximately 35 kD (Valentine et al., (1989) J. biol. chem. 264(19):11282-11287 and Einfield et al., (1988) EMBO J.7(3) (711) 717). CD20 was found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs, and is expressed during early pre-B cell development, and remains present until plasma cell differentiation. CD20 is present on both normal B cells as well as malignant B cells. In particular, CD20 was expressed on greater than 90% of B-cell non-Hodgkin's lymphomas (NHLs) (Anderson et al., (1984) Blood 63(6):1424-1433), but not found on hematopoietic 1 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 stem cells, pre-B cells, normal plasma cells or other normal tissues (Tedder et al., (1985) J. Immunol. 135(2):973-979). Although the exact function of CD20 is unclear, CD20 is implicated in calcium mobilization and may function as a calcium channel (Tedder et al., J. Cell Biochem., 14D:195 (1990)). CD20 might be involved in the activation and differentiation of B-cells (Tedder et al., Eur. J. Immunol., 16(8):881-887 (1986)) and in the cell surface regulation of CD19 and the B cell receptor Kläsenser et al. Proc Natl Acad Sci U S A (2021)).

[0005] Given the expression of CD20 by unwanted cells, such as in B-cell lymphomas, this antigen is useful for targeting harmful CD20 positive cells (e.g., lymphoma cells). In essence, such targeting is generalized as follows: antibodies specific to CD20 surface antigen of B cells are administered to a patient. These anti-CD20 antibodies specifically bind to the CD20 antigen of both normal and unwanted (e.g., malignant and immunoreactive) B cells; the antibody bound to the CD20 surface antigen may lead to the destruction and depletion of the B cells.

[0006] The use of CD20 antibodies for the treatment of cancer is mentioned in WO2011090762, WO2011028952, and WO2014047231. The rituximab (RITUXAN®) antibody is a genetically engineered chimeric murine / human monoclonal antibody directed against CD20. Rituximab is the antibody called “C2B8” in U.S. Pat. No.5,736,137 (Anderson et al.). Rituximab is currently approved for the treatment of relapsed or refractory follicular lymphoma (Leget et al., Curr. Opin. Oncol., 10:548-551 (1998)). Reports indicate that with weekly infusions, rituximab resulted in overall response rates of 48%. However, many patients do not respond to rituximab treatment and responding patients taking rituximab eventually relapse and often develop resistance to rituximab treatment. Relapse and resistance, for example, to currently available therapies necessitate the discovery of new CD20-directed agents and therapies with greater cell killing potency and the potential to fully deplete CD20-positive B cells.

[0007] There is a continued need to develop agents that target CD20 that are efficacious and safe for human use. There is a need in the art for agents that target CD20 for the diagnosis and treatment of CD20 associated conditions, such as cancers. Further, there exists a need for methods and kits to identify whether a subject with CD20-expressing cancer will be 2 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 responsive to such antibody-based therapies. Provided herein are methods and compositions addressing this need. SUMMARY

[0008] The disclosure provides an antibody comprising a first antigen binding region that binds to CD20, a second antigen binding region that binds to CD20, and a third antigen binding region that binds to CD3, wherein the first antigen binding region that binds to CD20 comprises three heavy chain complementarity determining regions (VH1_CDR1, VH1_CDR2, VH1_CDR3) and three light chain complementarity determining regions (VL1_CDR1, VL1_CDR2, VL1_CDR3), wherein: a) VH1_CDR1 comprises the amino acid sequence of SEQ ID NO: 3; VH1_CDR2 comprises the amino acid sequence of SEQ ID NO: 10; VH1_CDR3 comprises the amino acid sequence of SEQ ID NO: 16; VL1_CDR1 comprises the amino acid sequence of SEQ ID NO: 21; VL1_CDR2 comprises the amino acid sequence of SEQ ID NO: 23; and VL1_CDR3 comprises the amino acid sequence of SEQ ID NO: 26; or b) VH1_CDR1 comprises the amino acid sequence of SEQ ID NO: 1; VH1_CDR2 comprises the amino acid sequence of SEQ ID NO: 8; VH1_CDR3 comprises the amino acid sequence of SEQ ID NO: 14; VL1_CDR1 comprises the amino acid sequence of SEQ ID NO: 20; VL1_CDR2 comprises the amino acid sequence of SEQ ID NO: 22; and VL1_CDR3 comprises the amino acid sequence of SEQ ID NO: 25; and wherein the second antigen binding region that binds to CD20 comprises three heavy chain complementarity determining regions (VH2_CDR1, VH2_CDR2, VH2_CDR3) and three light chain complementarity determining regions (VL2_CDR1, VL2_CDR2, VL2_CDR3), wherein: I) VH1_CDR1 comprises the amino acid sequence of SEQ ID NO: 3; VH1_CDR2 comprises the amino acid sequence of SEQ ID NO: 10; VH1_CDR3 comprises the amino acid sequence of SEQ ID NO: 16; VL1_CDR1 comprises the amino acid sequence of SEQ ID NO: 21; VL1_CDR2 comprises the amino acid sequence of SEQ ID NO: 23; and VL1_CDR3 comprises the amino acid sequence of SEQ ID NO: 26; or II) VH1_CDR1 comprises the amino acid sequence of SEQ ID NO: 1; VH1_CDR2 comprises the amino acid sequence of SEQ ID NO: 8; VH1_CDR3 comprises the amino acid sequence of SEQ ID NO: 14; VL1_CDR1 comprises the amino acid sequence of SEQ ID NO: 20; VL1_CDR2 comprises the amino acid sequence of SEQ ID NO: 22; and VL1_CDR3 comprises the amino acid sequence of SEQ ID NO: 25; and wherein the third antigen 3 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 binding region that binds to CD3 comprises three heavy chain complementarity determining regions (VH3_CDR1, VH3_CDR2, VH3_CDR3) and three light chain complementarity determining regions (VL3_CDR1, VL3_CDR2, VL3_CDR3), wherein i) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 4; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 24; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; ii) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 2; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; iii) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 12; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 18; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 27; v) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 6; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 13; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; v) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 6; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; vi) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; vii) 4 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 13; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 27; viii) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 13; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; ix) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 19; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 27; x) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; or xi) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 13; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15.

[0009] In some embodiments, the first antigen binding region that binds to CD20 comprises a first variable heavy chain region (VH1) and a first variable light chain region (VL1), wherein: a) VH1 comprises the amino acid sequence of SEQ ID NO: 32; and VL1 comprises the amino acid sequence of SEQ ID NO: 33; or b) VH1 comprises the amino acid sequence of SEQ ID NO: 28; and VL1 comprises the amino acid sequence of SEQ ID NO: 29; wherein the second antigen binding region that binds to CD20 comprises a second variable heavy chain region (VH2) and a second variable light chain region (VL2), wherein: 5 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 I) VH1 comprises the amino acid sequence of SEQ ID NO: 32; and VL1 comprises the amino acid sequence of SEQ ID NO: 33; or II) VH1 comprises the amino acid sequence of SEQ ID NO: 28; and VL1 comprises the amino acid sequence of SEQ ID NO: 29; wherein the third antigen binding region that binds to CD3 comprises a third variable heavy chain region (VH3) and a third variable light chain region (VL3), wherein: i) VH3 comprises the amino acid sequence of SEQ ID NO: 34; and VL3 comprises the amino acid sequence of SEQ ID NO: 35; ii) VH3 comprises the amino acid sequence of SEQ ID NO: 30; and VL3 comprises the amino acid sequence of SEQ ID NO: 31; iii) VH3 comprises the amino acid sequence of SEQ ID NO: 36; and VL3 comprises the amino acid sequence of SEQ ID NO: 37; iv) VH3 comprises the amino acid sequence of SEQ ID NO: 38; and VL3 comprises the amino acid sequence of SEQ ID NO: 39; v) VH3 comprises the amino acid sequence of SEQ ID NO: 38; and VL3 comprises the amino acid sequence of SEQ ID NO: 31; vi) VH3 comprises the amino acid sequence of SEQ ID NO: 38; and VL3 comprises the amino acid sequence of SEQ ID NO: 40; vii)VH3 comprises the amino acid sequence of SEQ ID NO: 41; and VL3 comprises the amino acid sequence of SEQ ID NO: 37; viii) VH3 comprises the amino acid sequence of SEQ ID NO: 42; and VL3 comprises the amino acid sequence of SEQ ID NO: 38; or ix) VH3 comprises the amino acid sequence of SEQ ID NO: 43; and VL3 comprises the amino acid sequence of SEQ ID NO: 38.

[0010] In some embodiments, the antibody comprises a first heavy chain polypeptide (H1) and a second heavy chain polypeptide (H2), a first light chain polypeptide (L1), a second light chain polypeptide (L2), and a third light chain polypeptide (L3), wherein a) the first heavy chain polypeptide (H1) comprises the second variable heavy chain region (VH2); b) the second heavy chain polypeptide (H2) comprises the third variable heavy chain region (VH3); c) the first variable heavy chain region (VH1) is located on either the first heavy chain polypeptide (H1) or the second heavy chain polypeptide (H2), d) the first light chain polypeptide (L1) comprises the first variable light chain region (VL1), e) the second light chain polypeptide (L2) comprises the second variable light chain region (VL2), and f) the third light chain polypeptide (L3) comprises the third variable light chain region (VL3).

[0011] In some embodiments, a polypeptide is fused to the N-terminus or the C-terminus of the first heavy chain polypeptide (H1), the second heavy chain polypeptide (H2), or the third light chain polypeptide (L3). 6 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0012] In some embodiments, the polypeptide is fused via a linker peptide. In some embodiments, the linker peptide comprises the amino acid sequence of any one of SEQ ID NOs: 96-101, 283-338, or 553-606.

[0013] In some embodiments, the polypeptide comprises CD58 or a fragment thereof. In some embodiments, CD58 comprises the amino acid sequence of any one of SEQ ID NOs: 95, 102, or 624-628. In some embodiments, CD58 comprises the amino acid sequence of SEQ ID NO: 95.

[0014] In some embodiments, i) the VH1 comprises the amino acid sequence of SEQ ID NO: 32; the VH2 comprises the amino acid sequence of SEQ ID NO: 32; the VH3 comprises the amino acid sequence of SEQ ID NO: 34; the VL1 comprises the amino acid sequence of SEQ ID NO: 33; the VL2 comprises the amino acid sequence of SEQ ID NO: 33; and the VL3 comprises the amino acid sequence of SEQ ID NO: 35; ii) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 30; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 31; iii) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 36; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 37; iv) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 38; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 39; v) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 28; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 31; vi) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 7 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 38; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 40; vii) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 41; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 37; viii) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 42; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 38; or ix) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 43; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 38.

[0015] In some embodiments, the antibody comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 107, an LC1 comprising the amino acid sequence of SEQ ID NO: 108, an HC2 comprising the amino acid sequence of SEQ ID NO: 109, and an LC2 comprising the amino acid sequence of SEQ ID NO: 110; an HC1 comprising the amino acid sequence of SEQ ID NO: 111, an LC1 comprising the amino acid sequence of SEQ ID NO: 112, an HC2 comprising the amino acid sequence of SEQ ID NO: 113, and an LC2 comprising the amino acid sequence of SEQ ID NO: 114; an HC1 comprising the amino acid sequence of SEQ ID NO: 115, an LC1 comprising the amino acid sequence of SEQ ID NO: 116, an HC2 comprising the amino acid sequence of SEQ ID NO: 117, and an LC2 comprising the amino acid sequence of SEQ ID NO: 118; an HC1 comprising the amino acid sequence of SEQ ID NO: 119, an LC1 comprising the amino acid sequence of SEQ ID NO: 120, an HC2 comprising the amino acid sequence of SEQ ID NO: 121, and an LC2 comprising the amino acid sequence of SEQ ID NO: 122; an HC1 comprising the amino acid sequence of SEQ ID NO: 123, an LC1 comprising the amino acid sequence of SEQ ID 8 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 NO: 124, an HC2 comprising the amino acid sequence of SEQ ID NO: 125, and an LC2 comprising the amino acid sequence of SEQ ID NO: 126; an HC1 comprising the amino acid sequence of SEQ ID NO: 127, an LC1 comprising the amino acid sequence of SEQ ID NO: 128, an HC2 comprising the amino acid sequence of SEQ ID NO: 129, and an LC2 comprising the amino acid sequence of SEQ ID NO: 130; an HC1 comprising the amino acid sequence of SEQ ID NO: 131, an LC1 comprising the amino acid sequence of SEQ ID NO: 132, an HC2 comprising the amino acid sequence of SEQ ID NO: 133, and an LC2 comprising the amino acid sequence of SEQ ID NO: 134; an HC1 comprising the amino acid sequence of SEQ ID NO: 135, an LC1 comprising the amino acid sequence of SEQ ID NO: 136, an HC2 comprising the amino acid sequence of SEQ ID NO: 137, and an LC2 comprising the amino acid sequence of SEQ ID NO: 138; an HC1 comprising the amino acid sequence of SEQ ID NO: 139, an LC1 comprising the amino acid sequence of SEQ ID NO: 140, an HC2 comprising the amino acid sequence of SEQ ID NO: 141, and an LC2 comprising the amino acid sequence of SEQ ID NO: 142; an HC1 comprising the amino acid sequence of SEQ ID NO: 143, an LC1 comprising the amino acid sequence of SEQ ID NO: 144, an HC2 comprising the amino acid sequence of SEQ ID NO: 145, and an LC2 comprising the amino acid sequence of SEQ ID NO: 146; an HC1 comprising the amino acid sequence of SEQ ID NO: 147, an LC1 comprising the amino acid sequence of SEQ ID NO: 148, an HC2 comprising the amino acid sequence of SEQ ID NO: 149, and an LC2 comprising the amino acid sequence of SEQ ID NO: 150; an HC1 comprising the amino acid sequence of SEQ ID NO: 151, an LC1 comprising the amino acid sequence of SEQ ID NO: 152, an HC2 comprising the amino acid sequence of SEQ ID NO: 153, and an LC2 comprising the amino acid sequence of SEQ ID NO: 154; an HC1 comprising the amino acid sequence of SEQ ID NO: 155, an LC1 comprising the amino acid sequence of SEQ ID NO: 156, an HC2 comprising the amino acid sequence of SEQ ID NO: 157, and an LC2 comprising the amino acid sequence of SEQ ID NO: 158; an HC1 comprising the amino acid sequence of SEQ ID NO: 159, an LC1 comprising the amino acid sequence of SEQ ID NO: 160, an HC2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC2 comprising the amino acid sequence of SEQ ID NO: 162; an HC1 comprising the amino acid sequence of SEQ ID NO: 163, an LC1 comprising the amino acid sequence of SEQ ID NO: 164, an HC2 comprising the amino acid sequence of SEQ ID NO: 165, and an LC2 9 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 comprising the amino acid sequence of SEQ ID NO: 166; an HC1 comprising the amino acid sequence of SEQ ID NO: 167, an LC1 comprising the amino acid sequence of SEQ ID NO: 168, an HC2 comprising the amino acid sequence of SEQ ID NO: 169, and an LC2 comprising the amino acid sequence of SEQ ID NO: 170; an HC1 comprising the amino acid sequence of SEQ ID NO: 171, an LC1 comprising the amino acid sequence of SEQ ID NO: 172, an HC2 comprising the amino acid sequence of SEQ ID NO: 173, and an LC2 comprising the amino acid sequence of SEQ ID NO: 174; an HC1 comprising the amino acid sequence of SEQ ID NO: 175, an LC1 comprising the amino acid sequence of SEQ ID NO: 176, an HC2 comprising the amino acid sequence of SEQ ID NO: 177, and an LC2 comprising the amino acid sequence of SEQ ID NO: 178; an HC1 comprising the amino acid sequence of SEQ ID NO: 179, an LC1 comprising the amino acid sequence of SEQ ID NO: 180, an HC2 comprising the amino acid sequence of SEQ ID NO: 181, and an LC2 comprising the amino acid sequence of SEQ ID NO: 182; an HC1 comprising the amino acid sequence of SEQ ID NO: 183, an LC1 comprising the amino acid sequence of SEQ ID NO: 184, an HC2 comprising the amino acid sequence of SEQ ID NO: 185, and an LC2 comprising the amino acid sequence of SEQ ID NO: 186; an HC1 comprising the amino acid sequence of SEQ ID NO: 187, an LC1 comprising the amino acid sequence of SEQ ID NO: 188, an HC2 comprising the amino acid sequence of SEQ ID NO: 189, and an LC2 comprising the amino acid sequence of SEQ ID NO: 190; an HC1 comprising the amino acid sequence of SEQ ID NO: 191, an LC1 comprising the amino acid sequence of SEQ ID NO: 192, an HC2 comprising the amino acid sequence of SEQ ID NO: 193, and an LC2 comprising the amino acid sequence of SEQ ID NO: 194; an HC1 comprising the amino acid sequence of SEQ ID NO: 195, an LC1 comprising the amino acid sequence of SEQ ID NO: 196, an HC2 comprising the amino acid sequence of SEQ ID NO: 197, and an LC2 comprising the amino acid sequence of SEQ ID NO: 198; an HC1 comprising the amino acid sequence of SEQ ID NO: 199, an LC1 comprising the amino acid sequence of SEQ ID NO: 200, an HC2 comprising the amino acid sequence of SEQ ID NO: 201, and an LC2 comprising the amino acid sequence of SEQ ID NO: 282; an HC1 comprising the amino acid sequence of SEQ ID NO: 202, an LC1 comprising the amino acid sequence of SEQ ID NO: 203, an HC2 comprising the amino acid sequence of SEQ ID NO: 204, and an LC2 comprising the amino acid sequence of SEQ ID NO: 205; an HC1 comprising the amino 10 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 acid sequence of SEQ ID NO: 206, an LC1 comprising the amino acid sequence of SEQ ID NO: 207, an HC2 comprising the amino acid sequence of SEQ ID NO: 208, and an LC2 comprising the amino acid sequence of SEQ ID NO: 209; an HC1 comprising the amino acid sequence of SEQ ID NO: 210, an LC1 comprising the amino acid sequence of SEQ ID NO: 211, an HC2 comprising the amino acid sequence of SEQ ID NO: 212, and an LC2 comprising the amino acid sequence of SEQ ID NO: 213; an HC1 comprising the amino acid sequence of SEQ ID NO: 214, an LC1 comprising the amino acid sequence of SEQ ID NO: 215, an HC2 comprising the amino acid sequence of SEQ ID NO: 216, and an LC2 comprising the amino acid sequence of SEQ ID NO: 217; an HC1 comprising the amino acid sequence of SEQ ID NO: 218, an LC1 comprising the amino acid sequence of SEQ ID NO: 219, an HC2 comprising the amino acid sequence of SEQ ID NO: 220, and an LC2 comprising the amino acid sequence of SEQ ID NO: 221; an HC1 comprising the amino acid sequence of SEQ ID NO: 222, an LC1 comprising the amino acid sequence of SEQ ID NO: 223, an HC2 comprising the amino acid sequence of SEQ ID NO: 224, and an LC2 comprising the amino acid sequence of SEQ ID NO: 225; an HC1 comprising the amino acid sequence of SEQ ID NO: 226, an LC1 comprising the amino acid sequence of SEQ ID NO: 227, an HC2 comprising the amino acid sequence of SEQ ID NO: 228, and an LC2 comprising the amino acid sequence of SEQ ID NO: 229; an HC1 comprising the amino acid sequence of SEQ ID NO: 230, an LC1 comprising the amino acid sequence of SEQ ID NO: 231, an HC2 comprising the amino acid sequence of SEQ ID NO: 232, and an LC2 comprising the amino acid sequence of SEQ ID NO: 233; an HC1 comprising the amino acid sequence of SEQ ID NO: 234, an LC1 comprising the amino acid sequence of SEQ ID NO: 235, an HC2 comprising the amino acid sequence of SEQ ID NO: 236, and an LC2 comprising the amino acid sequence of SEQ ID NO: 237; an HC1 comprising the amino acid sequence of SEQ ID NO: 238, an LC1 comprising the amino acid sequence of SEQ ID NO: 239, an HC2 comprising the amino acid sequence of SEQ ID NO: 240, and an LC2 comprising the amino acid sequence of SEQ ID NO: 241; an HC1 comprising the amino acid sequence of SEQ ID NO: 242, an LC1 comprising the amino acid sequence of SEQ ID NO: 243, an HC2 comprising the amino acid sequence of SEQ ID NO: 244, and an LC2 comprising the amino acid sequence of SEQ ID NO: 245; an HC1 comprising the amino acid sequence of SEQ ID NO: 246, an LC1 comprising the amino acid sequence of SEQ ID 11 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 NO: 247, an HC2 comprising the amino acid sequence of SEQ ID NO: 248, and an LC2 comprising the amino acid sequence of SEQ ID NO: 249; an HC1 comprising the amino acid sequence of SEQ ID NO: 250, an LC1 comprising the amino acid sequence of SEQ ID NO: 251, an HC2 comprising the amino acid sequence of SEQ ID NO: 252, and an LC2 comprising the amino acid sequence of SEQ ID NO: 253; an HC1 comprising the amino acid sequence of SEQ ID NO: 254, an LC1 comprising the amino acid sequence of SEQ ID NO: 255, an HC2 comprising the amino acid sequence of SEQ ID NO: 256, and an LC2 comprising the amino acid sequence of SEQ ID NO: 257; an HC1 comprising the amino acid sequence of SEQ ID NO: 258, an LC1 comprising the amino acid sequence of SEQ ID NO: 259, an HC2 comprising the amino acid sequence of SEQ ID NO: 260, and an LC2 comprising the amino acid sequence of SEQ ID NO: 261; an HC1 comprising the amino acid sequence of SEQ ID NO: 262, an LC1 comprising the amino acid sequence of SEQ ID NO: 263, an HC2 comprising the amino acid sequence of SEQ ID NO: 264, and an LC2 comprising the amino acid sequence of SEQ ID NO: 265; an HC1 comprising the amino acid sequence of SEQ ID NO: 266, an LC1 comprising the amino acid sequence of SEQ ID NO: 267, an HC2 comprising the amino acid sequence of SEQ ID NO: 268, and an LC2 comprising the amino acid sequence of SEQ ID NO: 269; an HC1 comprising the amino acid sequence of SEQ ID NO: 270, an LC1 comprising the amino acid sequence of SEQ ID NO: 271, an HC2 comprising the amino acid sequence of SEQ ID NO: 272, and an LC2 comprising the amino acid sequence of SEQ ID NO: 273; an HC1 comprising the amino acid sequence of SEQ ID NO: 374, an LC1 comprising the amino acid sequence of SEQ ID NO: 375, an HC2 comprising the amino acid sequence of SEQ ID NO: 376, and an LC2 comprising the amino acid sequence of SEQ ID NO: 377; or an HC1 comprising the amino acid sequence of SEQ ID NO: 378, an LC1 comprising the amino acid sequence of SEQ ID NO: 379, an HC2 comprising the amino acid sequence of SEQ ID NO: 380, and an LC2 comprising the amino acid sequence of SEQ ID NO: 381.

[0016] In some embodiments, the disclosure provides a polynucleotide comprising a nucleic acid sequence encoding an antibody provided herein. In some embodiments, the disclosure provides a vector comprising a polynucleotide provided herein. In some embodiments, the disclosure provides a pharmaceutical composition comprising an antibody, a polynucleotide, or a vector provided herein, and a pharmaceutically acceptable carrier. 12 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 In some embodiments, the present disclosure provides method for treating cancer that expresses CD20 in a subject, the method comprising administering an effective amount of the antibody, the polynucleotide, the vector, or the pharmaceutical composition described herein. In some embodiments, the cancer is multiple myeloma. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 shows schematic diagrams of bispecific 2:1 antibody designs with charged pair mutations, disulfide bond repositioning, and knob-into-hole mutations. VH1: first heavy chain variable region; VL1: first light chain variable region; VH2: second heavy chain variable region; VL2: second light chain variable region; CH1H1: first constant heavy chain region (CH1) having a constant region 1 domain (CH1H1); CL1: first constant light chain region; CH2H1: second constant heavy chain region (CH2) having a constant region 1 domain (CH2H1); CL2: second constant light chain region; CH1H2: first constant heavy chain region (CH1) having a constant region 2 domain (CH1H2); CH1H3: first constant heavy chain region (CH1) having a constant region 3 domain (CH1H3); CH2H2: second constant heavy chain region (CH2) having a constant region 2 domain (CH2H2); CH2H3: second constant heavy chain region (CH2) having a constant region 3 domain (CH2H3); H1H: first hinge region; H2H: second hinge region; H1: first heavy chain; H2: second heavy chain; L1: first light chain; L2: second light chain.

[0018] FIG.2 shows a schematic diagram of exemplary multispecific 2:1 antibody designs with charged pair mutations, disulfide bond repositioning, and knob-into-hole mutations comprising either an anti-CD20 arm or two tandem anti-CD20 arms, an anti-CD3 arm with an N-terminus anti-CD20 arm, and a CD58 peptide fused to a constant heavy chain region, a constant light chain region, or a variable light chain region.

[0019] FIG. 3 shows tumor cell cytotoxicity assays of representative 2:1 antibody designs compared to bispecific antibodies with matched anti-CD3 affinity in a co-culture assay of HT cells and peripheral blood mononuclear cells (PBMCs) at a 15:1 E:T ratio and treated with a serial dilution of biologics for 72 hours.

[0020] FIGs. 4A-4B shows in vivo tumor growth inhibition in a human peripheral blood mononuclear cell (PBMC)-engrafted cell line derived from a xenograft mouse model using 13 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 the WSU-DLCL2 DLBCL cell line. Line graphs in FIG. 4A indicate inhibition of WSU- DLCL2 tumor growth over time after treatment with representative 2:1 antibody designs compared to a clinical benchmark biosimilar comparator (2:1 bisp-comparator). FIG. 4B shows plasma concentration of biologics over time.

[0021] FIG. 5 shows CD20 expression level on cell lines and normal B cells isolated from PBMC.

[0022] FIG. 6 shows tumor cell cytotoxicity assays of representative 2:1 antibody designs compared to the bispecific antibody with matching anti-CD3 affinity in a co-culture assay of HT cells and peripheral blood mononuclear cells (PBMCs) at a 15:1 E:T ratio and treated with a serial dilution of biologics for 72 hours.

[0023] FIGs.7A-7F are a series of graphs depicting functional evaluations of representative 2:1 antibody designs compared to bispecific antibodies in a co-culture assay of HT cells and peripheral blood mononuclear cells (PBMCs) at a 15:1 E:T ratio and treated with a serial dilution of biologics for 72 hours. FIG.7A shows antibody-mediated cytolysis of tumor cells. FIG. 7B shows the B cell depletion. FIG. 7C shows the CD4 T cell expansion. FIG. 7D shows the CD8 T cell expansion. FIG. 7E shows the improvement of the potency of representative 2:1 antibodies compared to bispecific antibodies. FIG. 7F shows the improvement of the cytokine release of representative 2:1 antibodies compared to bispecific antibodies glofitamab and epcoritamab.

[0024] FIG. 8 shows the functional evaluation of representative 2:1 antibody designs compared to CD20-targeting bispecific antibodies or monoclonal antibodies in a co-culture assay of HT cells and B cell- depleted peripheral blood monoclonal cells (PBMCs) at a 10:1 E:T ratio and treated with a serial dilution of biologics for 72 hours.

[0025] FIG. 9A shows antibody binding to pan-T cells isolated from peripheral blood mononuclear cells (PBMCs). FIG. 9B shows structure activity relationship (SAR) of two- CD20 binding domains of 2:1 CD20-targeted fusion multispecifics in avidity-driven target binding in a standard ELISA using CD20-VLP. FIG.9C shows human CD3 binding kinetics (association, dissociation, and equilibrium constants) of CD20-targeted 2:1 bispecific fusions with CD2 costimulation using surface plasmon resonance (SPR) on a Biacore 8K+ system (Cytiva). 14 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0026] FIG. 10 shows structure activity relationship (SAR) of two-CD20 binding domains of 2:1 CD20-targeted fusion multispecifics over T cell activation potency in a T cell activation assay using Jurkat-NFAT-Luc reporter cell and Raji(CD20medium) or HT(CD20low) tumor cells at a 4:1 E:T ratio and treated with a serial dilution of biologics for 4-6 hours.

[0027] FIGs.11A-11D show the functional evaluation of representative 2:1 antibody designs compared to 1:1 antibody designs in a co-culture assay of HT cells and peripheral blood monoclonal cells (PBMCs) at a 15:1 E:T ratio and treated with a serial dilution of biologics for 72 hours. FIG.11A shows antibody-mediated cytolysis of tumor cells. FIG.11B shows the B cell depletion. FIG.11C shows the CD4 T cell expansion. FIG.11D shows the CD8 T cell expansion.

[0028] FIGs.12A-12D show the functional evaluation of representative 2:1 antibody design compared to 2:1 and 1:1 bispecific antibody designs in a co-culture assay of enriched tissue B cells and B cell-depleted peripheral blood monoclonal cells (PBMCs) at an 8:1 E:T ratio and treated with a serial dilution of biologics for 72 hours. FIG. 12A shows antibody- mediated cytolysis of plasmablast. FIG. 12B shows relative potency in the depletion of plasmablast. FIG.12C shows antibody-mediated cytolysis of activated memory B cells. FIG. 12D shows relative potency in the depletion of activated memory B cells.

[0029] FIG. 13 shows in vivo tumor growth inhibition in a human peripheral blood mononuclear cell (PBMC)-engrafted cell line derived from a xenograft mouse model using the WSU-DLCL2 DLBCL cell line. Dotplot indicates inhibition of WSU-DLCL2 tumor growth over time after treatment with representative 2:1 antibody designs compared to a clinical benchmark biosimilar comparator Glofitamab.

[0030] FIGs. 14A-14B relate to purity, size, and yield of representative fusion proteins, as determined by SDS-PAGE gel (FIG. 14A) and analytical size exclusion chromatography (FIG.14B).

[0031] FIGs. 15A-15L relate to the functional activity of 2:1 CD20-targeting Evolve molecules in vitro. FIGs. 15A-15C show killing of CD20-low expressing cancer cell (HT cells) induced by Evolve-37 (FIG.15A), Evolve-36 (FIG.15B), and Evolve-35 (FIG.15C). FIGs.15D-15F show killing of primary B cells induced by Evolve-37 (FIG.15D), Evolve- 36 (FIG. 15E), and Evolve-35 (FIG. 15F). FIGs. 15G-15I show CD4+ T-cell expansion induced by Evolve-37 (FIG.15G), Evolve-36 (FIG.15H) and Evolve-35 (FIG.15I). FIGs. 15 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 15J-15L show CD8+ T-cell expansion induced by Evolve-37 (FIG.15J), Evolve-36 (FIG. 15K), and Evolve-35 (FIG.15L).

[0032] FIGs. 16A-16B relate to killing of HT cells (FIG. 16A) and primary B cells (FIG. 16B) by Evolve-36, Evolve-43, Evolve-44, and Evolve-40. DETAILED DESCRIPTION

[0033] T cell retargeting (or T cell redirecting) bispecific and multispecific antibodies are a novel class of therapeutics capable of recruiting T cells to tumor cells and inducing tumor- specific (but MHC-independent) activation of T cell effector activities. The present disclosure is directed toward T cell retargeting bispecific and multispecific antibodies containing an antigen binding domain that targets the CD3 portion of the T cell receptor protein complex for T cell recruitment and an antigen binding domain that targets T cells to a CD20 antigen. This targeting design promotes the recruitment of T cell and positions it in close contact with a CD20-expressing cell, resulting in the formation of an immunological synapse, local T cell activation, and the subsequent destruction of the target cell, such as but not limited to a cancer cell, by perforin and granzyme released from T cell cytotoxic granules into the target cell.

[0034] As the CD3 binding affinity of the T cell retargeting bispecific and multispecific antibodies is crucial for the recruitment of T cells, the present disclosure also relates to the generation of a panel of antibodies that display different binding affinities. The affinity of the CD3 arm of a bispecific and multispecific antibody can significantly modify the functional activity of the bispecific and multispecific antibody. Thus, it is desirable and advantageous to have bispecific and multispecific antibodies with varied affinities.

[0035] Additionally, multispecific antibodies disclosed herein may have a cytokine or costimulatory molecule fusion peptide that acts as an antagonist to inhibit or block deleterious interactions or as an agonist to mimic or enhance physiological responses. Physiological responses include but are not limited to T cell activation, T cell proliferation, T cell persistence, and prevention of T cell exhaustion. These properties are advantageous over conventional CD3-bispecific antibodies or tumor-targeted co-stimulatory receptor agonists which do not optimally activate T cells and induce (or promote) T cell dysfunction. In accordance, cytokine and / or costimulatory fusion peptides are advantageous for enhancing the therapeutic potential of multispecific antibodies. In some embodiments, the costimulatory 16 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 molecule of the anti-CD20 / anti-CD3 multispecific antibodies of the present disclosure is CD58 or a fragment thereof.

[0036] Bispecific and multispecific T cell retargeting agents of the disclosure share the drug- like properties of human monoclonal antibodies. Further, T cell retargeting bispecific and multispecific antibodies are advantageous over other existing therapies (e.g., CAR-T therapies) because they provide an off-the-shelf product with a highly favorable safety profile (e.g., mitigation of cytokine release syndrome and reduced levels of tonic signaling leading to T cell dysfunction) and the possibility of dose titration and escalation.

[0037] Also provided herein are methods of identifying a human subject having a CD20 expressing cancer, such as B-cell leukemia and lymphoma, that is suitable for therapy with an anti-CD20 antibody or antigen binding fragment thereof.

[0038] ANTIBODY COMPOSITIONS AND STRUCTURES

[0039] In some embodiments, an antibody of the disclosure is bispecific (i.e. binds two distinct antigens) and has three antigen binding regions. In some embodiments, these antibodies are referred to as bispecific 2:1 antibodies. In some embodiments, an antibody of the disclosure is multispecific (i.e. binds three distinct antigens). In some embodiments, these antibodies are referred to as multispecific 2:1 antibodies.

[0040] In some embodiments, a bispecific antibody of the disclosure that binds two distinct antigens comprises a first antigen binding region that binds a first antigen, a second antigen binding region that binds the first antigen, and a third antigen binding region that binds a second antigen. For example, a bispecific antibody of the disclosure can bind CD3 and CD20 wherein the antibody has a first antigen binding region that binds CD3, a second antigen binding region that binds CD20, and a third antigen binding region that binds CD20. For example, a bispecific antibody of the disclosure can bind CD3 and CD20 wherein the antibody has a first antigen binding region that binds CD3, a second antigen binding region that binds CD3, and a third antigen binding region that binds CD20. For example, a bispecific antibody of the disclosure can bind CD3 and CD20 wherein the antibody has a first antigen binding region that binds CD20, a second antigen binding region that binds CD20, and a third antigen binding region that binds CD3. A schematic diagram of the antibody structure of the disclosure is shown in FIGs.1-2.

[0041] The present disclosure provides a multispecific antibody that binds two distinct 17 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 antigens comprises a first antigen binding region that binds a first antigen, a second antigen binding region that binds the first antigen, and a third antigen binding region that binds a second antigen and that further comprises a CD2 ligand, such as CD58. Multispecific antibodies of the disclosure further comprising a CD58 ligand (e.g. a CD58 polypeptide) can lead to CD2-directed costimulatory T cell activation.

[0042] In some embodiments, the antigen binding regions of the bispecific and multispecific antibodies of the disclosure comprise a variable light chain region and a variable heavy chain region. Variable light chain regions of the disclosure comprise three complementarity determining regions (CDR) and variable heavy chain regions of the disclosure also comprise three CDRs.

[0043] In some embodiments, an antibody having three antigen binding regions comprises the following domain structure: a) a first heavy chain polypeptide (H1) comprising a first variable region (VH1), a second variable region (VH2), and a constant region (CH1) having a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2) and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1), and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2), and b) a second heavy chain polypeptide (H2) comprising a third variable region (VH3), and a constant region (CH2) having a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2H3); and third light chain polypeptide (L3) comprising a variable region (VL3) and a constant region (CL3).

[0044] In some embodiments, an antibody having three antigen binding regions comprises the following domain structure: a) a first heavy chain polypeptide (H1) comprising a first variable region (VH1) and a constant region (CH1) having a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2) and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1) and a constant region (CL2), and b) a second heavy chain polypeptide (H2) comprising a second variable region (VH2), a third variable region (VH3), and a constant region (CH2) having a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2H3); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region 18 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 (CL2) and third light chain polypeptide (L3) comprising a variable region (VL3) and a constant region (CL3).

[0045] As used herein, the term “antibody” refers to an immunoglobulin (Ig) molecule and immunologically active portions of an immunoglobulin molecule, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By “specifically bind” or “immunoreacts with” “or directed against” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd> 10-6). Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies. The antibody may be from recombinant sources and / or produced in transgenic animals.

[0046] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0047] In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG1, IgG2, IgG4 and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Accordingly, in one embodiment, the antibody disclosed herein is an IgG antibody.

[0048] Antibodies may be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol.14, No.8 (April 17, 2000), pp.25-28).

[0049] The term "antibody fragment" as used herein is intended to include without limitation, Fv, Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, multispecific antibody fragments and Domain Antibodies. Antibodies can be 19 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

[0050] Techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the disclosure (see e.g., U.S. Patent No.4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246:1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.

[0051] As used herein, the term “epitope” refers to the site on an antigen that is recognized by the antibodies and fragments disclosed herein. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is < 1 micromolar; e.g., < 100 nM, preferably < 10 nM and more preferably < 1 nM.

[0052] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. The present disclosure also provides a bispecific antibody having a first antigen binding region that binds to a first antigen (e.g. CD3) and a second antigen binding region that binds to a second antigen (e.g. disease associated antigen such as CD20), and a third antigen binding region that binds to the second antigen (e.g. disease associated antigen such as CD20).

[0053] Multispecific antibodies are antibodies that have binding specificities for at least three different antigens. The present disclosure provides a multispecific antibody having a first antigen binding region that binds to a first antigen (e.g. CD3) and a second antigen binding region that binds to a second antigen (e.g. disease associated antigen such as CD20), and a third antigen binding region that binds to the second antigen (e.g. disease associated antigen 20 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 such as CD20) and further comprises a CD2 ligand, such as CD58.

[0054] Trispecific antibodies can be prepared. Tutt et al., J. Immunol.147:60 (1991). Antibody Variants

[0055] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the heavy chain heterodimerization, light chain heterodimerization, binding affinity, and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., light chain heterodimerization, heavy chain heterodimerization, antigen binding).

[0056] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic (negatively charged): Asp, Glu; (4) basic (positively charged): His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0057] Functional variants of the antibody or antigen-binding fragments described herein are also encompassed by the present disclosure. The term "functional variant" as used herein includes modifications or chemical equivalents of the amino acid and nucleic acid sequences disclosed herein that perform substantially the same function as the polypeptides or nucleic acid molecules disclosed herein in substantially the same way. For example, functional variants of polypeptides disclosed herein include, without limitation, conservative amino acid substitutions.

[0058] A "conservative amino acid substitution" as used herein, is one in which one amino acid residue is replaced with another amino acid residue that change an amino acid to a different amino acid with similar biochemical properties (e.g. charge, hydrophobicity and 21 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 size). Variants of polypeptides also include additions and deletions to the polypeptide sequences disclosed herein. In addition, variant nucleotide sequences include analogs and derivatives thereof. A variant of the binding proteins disclosed herein include proteins that bind to the same antigen or epitope as the binding proteins.

[0059] In some embodiments, the charged amino acid residue is a naturally occurring amino acid or a non-naturally occurring amino acid. In some embodiments, the naturally occurring charged amino acid residue is an arginine, a lysine, a histidine, a glutamic acid or an aspartic acid. Light Chain and Heavy Chain Substitution Variants

[0060] To generate a substantially homogeneous population of bispecific or multispecific antibodies with the correct pairing of heavy chain and light chains (i.e. cognate pairing or heterodimerization of a light chain with the heavy chain necessary to form the variable domain or antigen binding domain of the original antibody), the first heavy chain polypeptide (H1) has a strong preference for binding with the first light chain polypeptide (L1) relative to the second light chain polypeptide (L2); and the second heavy chain polypeptide (H2) has a strong preference for binding with the second light chain polypeptide (L2) relative to first light chain polypeptide (L1). In addition, the first heavy chain polypeptide (H1) and the second heavy chain polypeptide (H2) have a stronger preference for heterodimerization than homodimerization (i.e. heavy chain heterodimerization).

[0061] Antibody variants having one or more amino acid substitutions are provided herein. Exemplary substitutional mutagenesis sites include the charged substitution pairs shown in Tables 1-6. For bispecific and multispecific antibodies of the disclosure having a heavy or light chain with more than one variable region (i.e. two antigen binding regions), the numbering of heavy and light chains substitution variants and mutations will be according to the EU and / or Kabat numbering scheme. Table 1. Kappa Light Chain and Heavy Chain – Constant Domain Mutations Pairs22 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00All position information is reported using the EU numbering scheme. Wild type (WT) indicates the natural amino acid at the indicated position. Charge pairs with negative and positive charge residues could be reversed between heavy and light chains, where D or E (negative charge) are replaced by K or R (positive charge) and cognate chain K or R (positive charge) are replaced by D or E (negative charge). Table 2. Kappa Light Chain and Heavy Chain – Variable Domain Mutations PairsAll position information is reported using the Kabat numbering scheme. Wild type (WT) indicates the natural amino acid at the indicated position. Charged pairs with negative and positive charged residues could be reversed between heavy and light chains, where D or E (negative charged) are replaced by K or R (positive charged) and cognate chain K or R (positive charged) are replaced by D or E (negative charged). Table 3. Lambda Light Chain and Heavy Chain – Constant Domain Mutations PairsAll position information is reported using the Kabat numbering scheme. Charge pairs with negative and positive charge residues could be reversed between heavy and light chains, where D or E (negative charge) are replaced by K or R (positive charge) and cognate chain K or R (positive charge) are replaced by D or E (negative charge). Table 4. Lambda Light Chain and Heavy Chain – Variable Domain Mutations PairsAll position information is reported using the Kabat numbering scheme. 23 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 Charge pairs with negative and positive charge residues could be reversed between heavy and light chains, where D or E (negative charge) are replaced by K or R (positive charge) and cognate chain K or R (positive charge) are replaced by D or E (negative charge). Table 5. Kappa Constant Chain Cysteine Mutation PairsAll position information is reported using the EU numbering scheme. Table 6. Lambda Constant Chain Cysteine Mutation PairsAll position information is reported using the Kabat numbering scheme.

[0062] In certain embodiments, anti-CD3 / anti-CD20 bispecific antibodies, and anti- CD3 / anti-CD20 / CD58 multispecific antibodies of the present disclosure comprise antibody variants comprise substitutions in the variable heavy, variable light, constant heavy or constant light chain domains of the anti-CD3, anti-CD20 arms, or both, as discussed in PCT Application No. PCT / US2023 / 064728 and PCT Publication No. WO2019 / 104075 A1, which are incorporated by reference herein in their entirety.

[0063] In certain embodiments, the antibody variant comprises the “light chain pairing mutation set D” comprising the following substitutions: a) the heavy chain and light chain of a first antigen binding arm comprise the following: i) the amino acid at position 39 (Kabat numbering) of the VH1 is a K and the amino acid at position 38 (Kabat numbering) of the VL1 is a D; ii) the amino acid at position 147 (EU numbering) of the CH1H1 is a K and the amino acid at position 131 (EU numbering) of the CL1 is a D; iii) the amino acid at position 173 (EU numbering) of the CH1H1 is a C and the amino acid at position 162 (EU numbering) of the CL1 is a C; iv) the amino acid at position 220 (EU numbering) in the H1H is a S and the amino acid at position 214 (EU numbering) of the CL1 is a S; and b) the heavy chain and light chain of a second antigen binding arm comprise the following: i) the amino acid at position 39 (Kabat numbering) of the VH2 is a D and the amino acid at position 38 (Kabat 24 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 numbering) of the VL2 is a K; and ii) the amino acid at position 147 (EU numbering) of the CH2H1is a D and the amino acid at position 180 (EU numbering) of the CL2 is a R.

[0064] In certain embodiments, the antibody variant comprises the “light chain pairing mutation set D” comprising the following substitutions: a) the heavy chain and light chain of a first antigen binding arm comprise the following: i) the amino acid at position 39 (Kabat numbering) of the VH1 is a K and the amino acid at position 38 (Kabat numbering) of the VL1 is a D; ii) the amino acid at position 147 (EU numbering) of the CH1H1 is a K and the amino acid at position 131 (EU numbering) of the CL1 is a D; iii) the amino acid at position 173 (EU numbering) of the CH1H1 is a C and the amino acid at position 162 (EU numbering) of the CL1 is a C; iv) the amino acid at position 220 (EU numbering) in the H1H is a S and the amino acid at position 214 (EU numbering) of the CL1 is a S; and b) the heavy chain and light chain of a second antigen binding arm comprise the following: i)the amino acid at position 39 (Kabat numbering) of the VH2 is a D and the amino acid at position 38 (Kabat numbering) of the VL2 is a K; and ii) the amino acid at position 147 (EU numbering) of the CH2H1is a D and the amino acid at position 180 (EU numbering) of the CL2 is a R.

[0065] It can be desirable to modify an antibody disclosed herein with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating diseases and disorders. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and / or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922. (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0066] Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6.737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0067] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). 25 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0068] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No.6,194,551 , WO 99 / 51642, and Idusogie et al. J. Immunol.164: 4178-4184 (2000).

[0069] Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of 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, e.g., substitution of Fc region residue 434 (US Patent No.7,371 ,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No.5,648,260; U.S. Patent No.5,624,821 ; and WO 94 / 29351 concerning other examples of Fc region variants.

[0070] In some embodiments, antibodies may comprise a substitution mutation in the Fc region that reduces effector function. In some embodiments, the substitution mutation is an aglycosylation site mutation. In some embodiments, the aglycosylation site mutation is at amino acid residue 297 and amino acid substitutions at residues 234, 235, 237, 265 and 331 (EU numbering) to disrupt the Fc receptor binding interface. In some embodiments, the aglycosylation site mutation reduces effector function of the antibody.

[0071] In some embodiments, i) the H1H and / or the H2H has an A at positions 234 and 235 (EU numbering); or ii) the H1H and / or the H2H has an A at positions 234, 235 and 237 (EU numbering) iii) the H1H and / or the H2H has an A at positions 234 and 235 and G at position 329 (EU numbering). In some embodiments, i) the CH1H2 and / or the CH2H2 has an A at position 297 (EU numbering); ii) the CH1H2and / or the CH2H2has a G at position 297 (EU numbering); iii) the CH1H2 and / or the CH2H2 has a Q at position 297 (EU numbering); or iv) the CH1H2 and / or the CH2H2 has a S at position 297 (EU numbering). In some embodiments, the CH1H2and / or the CH2H2has an S at position 331 (EU numbering).

[0072] In some embodiments, the CH1H2 and / or the CH2H2 has an A at position 299 (EU numbering) so that N-linked glycosylation is eliminated thus abrogating effector function. 26 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0073] The use of knobs into holes as a method of producing multispecific antibodies is well known in the art. See U.S. Pat. No.5,731,168 granted 24 Mar.1998 assigned to Genentech, PCT Pub. No. WO2009089004 published 16 Jul.2009 and assigned to Amgen, and US Pat. Pub. No.20090182127 published 16 Jul.2009 and assigned to Novo Nordisk A / S. See also Marvin and Zhu, Acta Pharmacologica Sincia (2005) 26(6):649-658 and Kontermann (2005) Acta Pharacol. Sin., 26:1-9.

[0074] A “protuberance” refers to at least one amino acid side chain which projects from the interface of a first polypeptide and is therefore positionable in a compensatory cavity in the adjacent interface (i.e. the interface of a second polypeptide) so as to stabilize the heteromultimeric antibody, and thereby favor heteromultimeric antibody formation over homomultimeric antibody formation, for example. The protuberance may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the first polypeptide is altered to encode the protuberance. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the first polypeptide is replaced with nucleic acid encoding at least one “import” amino acid residue which has a larger side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding import residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the first polypeptide.

[0075] The preferred import residues for the formation of a protuberance are generally naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Most preferred are tryptophan and tyrosine. In one embodiment, the original residue for the formation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. Exemplary amino acid substitutions in the CH1H3or CH2H3domain for forming the protuberance include without limitation the T366W substitution.

[0076] A “cavity” refers to at least one amino acid side chain which is recessed from the interface of a second polypeptide and therefore accommodates a corresponding protuberance on the adjacent interface of a first polypeptide. The cavity may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the second polypeptide is altered to encode 27 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 the cavity. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one “import” amino acid residue which has a smaller side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding import residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the second polypeptide. The preferred import residues for the formation of a cavity are usually naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T) and valine (V). Most preferred are serine, alanine or threonine. In one embodiment, the original residue for the formation of the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. Exemplary amino acid substitutions in the CH1H3or CH2H3domain for generating the cavity include without limitation the T366S, L368A, Y407A, Y407T and Y407V substitutions. In certain embodiments, the knob half-antibody comprises T366W substitution, and the hole half-antibody comprises the T366S / L368A / Y407V substitutions.

[0077] In certain embodiments, the antibody variant comprises the following substitutions: the CH1H3 has a C at position 349, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH2H3has a C at position 354 and a W at position 366 (EU numbering).

[0078] In certain embodiments, the antibody variant comprises the following substitutions: the CH2H3has a C at position 349, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH1H3has a C at position 354 and a W at position 366 (EU numbering).

[0079] In certain embodiments, the antibody variant comprises the following substitutions: the CH1H3has a C at position 354, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH2H3 has a C at position 349 and a W at position 366 (EU numbering);

[0080] In certain embodiments, the antibody variant comprises the following substitutions: the CH2H3 has a C at position 354, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH1H3 has a C at position 349 and a W at position 366 (EU numbering). 28 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 Cluster of Differentiation 3 (CD3)

[0081] The present disclosure provides bispecific antibodies comprising a first antigen binding domain that binds to a CD3 expressed on a T cell, and a second and third antigen binding domain that binds to a CD20 antigen on the surface of a cancer cell. The present disclosure also provides multispecific antibodies comprising a first antigen binding domain that binds to a CD3 antigen expressed on a T cell, a second and third antigen binding domain that binds to a CD20 antigen on the surface of a cancer cell, and a CD2 ligand, such as CD58.

[0082] Antibodies of the disclosure are useful, for example, for treating or delaying the progression of a cell proliferative disorder (e.g., cancer expressing CD20).

[0083] The term “cluster of differentiation 3” or “CD3,” as used herein, refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans, cynomolgus monkey) and rodents (e.g., mice and rats), unless otherwise indicated, including, for example, CD3ε, CD3γ, CD3ζ, and CD3δ. CD3 is a cell surface complex expressed on T cells in association with the T cell receptor. The CD3 complex is required for the activation of CD8+ and CD4+ T lymphocytes. It is formed of three different but highly related chains: one CD3 gamma chain, one CD3 delta chain, and two CD3 epsilon chains, which associate with each other to form a CD3 epsilon / gamma heterodimer, and a CD3 epsilon / delta heterodimer. The two CD3 heterodimers, together with the T cell receptor (TCR) and the signal-transducing zeta chain homodimer form the T cell receptor complex.

[0084] The term encompasses “full-length” unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, human CD3ε protein (NCBI RefSeq No. NP_000724), which is 207 amino acids in length.

[0085] In some embodiments, the disclosure provides isolated antibodies that bind to CD3. In some embodiments, the disclosure provides antibodies that bind to CD3ε. In some instances the anti-CD3ε antibody binds to a human CD3ε polypeptide or a cynomolgus monkey (cyno) CD3ε polypeptide. In some instances, the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3ε polypeptide (SEQ ID NO: 419) or a cyno CD3ε polypeptide (SEQ ID NO: 420), respectively. In some instances, the anti-CD3 antibody binds to an epitope within a fragment of CD3ε (e.g., human CD3ε) consisting of amino acid 29 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 residues 1-26 or amino acid residues 1-27 of human CD3ε (SEQ ID NO: 419).

[0086] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0087] In some embodiments, alanine scanning mutagenesis was performed on the “SP34” anti-CD3ε antibody to produce affinity modulated anti-CD3ε antibodies of the disclosure.

[0088] In some embodiments, the first antigen binding region that binds to CD3 comprises any one of the VH and VL sequences listed in Table 7 or Table 7.1.

[0089] In some embodiments, an anti-CD3 antibody of the disclosure comprises: a) a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (VHCDR1), a VH complementarity determining region 2 (VHCDR2) and a VH complementarity determining region 3 (VHCDR3); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VLCDR1), a VL complementarity determining region 2 (VLCDR2) and a VL complementarity determining region 3 (VLCDR3). Tables 8 and 9 provide exemplary CDR sequences of the anti-CD3 antibodies provided herein (CDR sequences are determined according to Kabat). Table 7. Anti-CD3 Variable Heavy Chain and Variable Light Chain Domains30 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00Table 7.1. Additional Anti-CD3 Variable Heavy Chain and Light Chain Domains31 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00Table 8. Anti-CD3 Heavy Chain CDRsTable 9. Anti-CD3 Light Chain CDRs32 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0090] Provided herein are 2:1 bispecific antibodies comprising the following structure: a first heavy chain polypeptide (H1) comprising a first variable heavy region (VH1), a second variable heavy region (VH2), a first light chain polypeptide (L1) comprising a first variable light region (VL1), second heavy chain polypeptide (H2) comprising a third variable heavy region (VH3), a second light chain polypeptide (L2) comprising a second variable light region (VL2), and a third light chain polypeptide (L3) comprising a third variable light region (VL3) wherein the first antigen binding domain comprises VL1 and VH1, the second antigen binding domain comprises VL2 and VH2, the third antigen binding region comprises VL3 and VH3 and wherein the first antigen binding domain binds one of CD3 or CD20, the second antigen binding domain binds one of CD3 or CD20, and the third antigen binding domain binds one of CD3 or CD20.

[0091] In some embodiments, a 2:1 bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a first variable heavy region (VH1), a first light chain polypeptide (L1) comprising a first variable light region (VL1), second heavy chain polypeptide (H2) comprising a second variable heavy region (VH2), a third variable heavy region (VH3), a second light chain polypeptide (L2) comprising a second variable light region (VL2), and a third light chain polypeptide (L3) comprising a third variable light region (VL3) wherein the first antigen binding domain comprises VL1 and VH1, the second antigen binding domain comprises VL2 and VH2, the third antigen binding region comprises VL3 and VH3 and wherein the first antigen binding domain binds one of CD3 or CD20, the second antigen binding domain binds one of CD3 or CD20, and the third antigen binding domain binds one of CD3 or CD20.

[0092] Provided herein are 2:1 multispecific antibodies comprising the following structure: a first heavy chain polypeptide (H1) comprising a first variable heavy region (VH1), a second variable heavy region (VH2), a first light chain polypeptide (L1) comprising a first variable light region (VL1), second heavy chain polypeptide (H2) comprising a third variable heavy region (VH3), a second light chain polypeptide (L2) comprising a second variable light region (VL2), a third light chain polypeptide (L3) comprising a third variable light region (VL3), 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 and a CD2 ligand, such as CD58, wherein the first antigen binding domain comprises VL1 and VH1, the second antigen binding domain comprises VL2 and VH2, the third antigen binding region comprises VL3 and VH3 and wherein the first antigen binding domain binds one of CD3 or CD20, the second antigen binding domain binds one of CD3 or CD20, and the third antigen binding domain binds one of CD3 or CD20.

[0093] In some embodiments, a 2:1 multispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a first variable heavy region (VH1), a first light chain polypeptide (L1) comprising a first variable light region (VL1), second heavy chain polypeptide (H2) comprising a second variable heavy region (VH2), a third variable heavy region (VH3), a second light chain polypeptide (L2) comprising a second variable light region (VL2), a third light chain polypeptide (L3) comprising a third variable light region (VL3), and a CD2 ligand, such as CD58, wherein the first antigen binding domain comprises VL1 and VH1, the second antigen binding domain comprises VL2 and VH2, the third antigen binding region comprises VL3 and VH3 and wherein the first antigen binding domain binds one of CD3 or CD20, the second antigen binding domain binds one of CD3 or CD20, and the third antigen binding domain binds one of CD3 or CD20.

[0094] In some embodiments, the bispecific and multispecific antibodies of the disclosure comprise an antigen binding domain (e.g. binding to CD3) comprising any one of the VH1 and VL1 sequences listed in Table 7 and / or Table 7.1.

[0095] Tables 8 and 9 provide exemplary CDR sequences of the anti-CD3 antibodies provided herein.

[0096] In some embodiments, the bispecific or multispecific antibody comprises any one of the anti-CD3 antibodies of the disclosure.

[0097] In some embodiments, the binding affinity (KD) of the first antigen binding region of the bispecific antibody that binds to CD3 (e.g., human CD3ε (e.g., (SEQ ID NO: 419) or cynomolgus CD3ε (e.g., (SEQ ID NO: 422)) is about 0.001 nM to about 5000 nM. In some embodiments, the binding affinity to CD3 is about 0.001 nM to about 0.01 nM, about 0.01 to about 0.1 nM or about 0.1 to about 1nM. In some embodiments, the binding affinity is about 1 nM to about 1000 nM, about 10 nM to about 1000 nM or about 100nM to about 1000 nM.

[0098] In some embodiments, the binding affinity is about 50 nM to about 5000 nM, about 50 nM to about 4000 nM, about 50 nM to about 3000 nM, about 50 nM to about 2000 nM, 34 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 about 50 nM to about 1000 nM, about 50 nM to about 900 nM, about 50 nM to about 800 nM, about 50 nM to about 700 nM, about 50 nM to about 600 nM, about 50 nM to about 500 nM, about 50 nM to about 400 nM, about 50 nM to about 300 nM, about 50 nM to about 200 nM, about 50 nM to about 100 nM or about 50 nM to about 500 nM. In some embodiments, the binding affinity is about 50 nM to about 200 nM.

[0099] In some embodiments, the binding affinity is about 10 nM to about 20 nM, about 20 nM to about 30 nM, about 30 nM to about 40 nM, about 50 nM to about 60 nM, about 60 nM to about 70 nM, about 70 nM to about 80 nM, about 80 nM to about 90 nM, about 90 nM to about 100 nM, about 100 nM to about 110 nM, about 110 nM to about 120 nM, about 120 nM to about 130 nM, about 130 nM to about 140 nM, about 150 nM to about 160 nM, about 160 nM to about 170 nM, about 170 nM to about 180 nM, about 180 nM to about 190 nM, or about 190 nM to about 200 nM.

[0100] In some embodiments, the binding affinity is less than about 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 275 nM, 250 nM, 225 nM, 200 nM, 175 nM, 150 nM, 125 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9.5 nM, 9nM, 8.5 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM. CD20 Antigen

[0101] The present disclosure provides bispecific and multispecific antibodies comprising an antigen binding domain that binds to a CD3 expressed on a T cell, and an antigen binding domain that binds to a CD20 antigen on the surface of a cancer cell. Antibodies of the disclosure are useful, for example, for treating or delaying the progression of a cell proliferative disorder (e.g., cancer expressing CD20).

[0102] “CD20” as used herein refers to the human B-lymphocyte antigen CD20 (also known as CD20, human B-lymphocyte-restricted differentiation antigen, B-lymphocyte surface antigen B1, Leu-16, Bp35, BMS, and LFS; the sequence is characterized by the SwissProt database entry P11836), which is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes. (Valentine, M. A., et al., J. Biol. Chem.264(19) (198911282-11287; Tedder, T. F., et al, Proc. Natl. Acad. Sci. U.S.A.85 (1988) 208-12; Stamenkovic, I., et al., J. Exp. Med.167 35 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 (1988) 1975-80; Einfeld, D. A., et al., EMBO J.7 (1988) 711-7; Tedder, T. F., et al., J. Immunol.142 (1989) 2560-8). CD20 is found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs and is expressed during early pre-B cell development and remains until plasma cell differentiation. CD20 is present on both normal B cells as well as malignant B cells. In particular, CD20 is expressed on greater than 90% of B cell non-Hodgkin's lymphomas (NHL) (Anderson, K. C., et al., Blood 63(6) (1984) 1424- 1433) but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissues (Tedder, T. F., et al., J. Immunol.135(2)(1985) 973-979).

[0103] The 85 amino acid carboxyl-terminal region of the CD20 protein is located within the cytoplasm. The length of this region contrasts with that of other B cell-specific surface structures such as IgM, IgD, and IgG heavy chains or histocompatibility antigens class I1 a or ß chains, which have relatively short intracytoplasmic regions of 3, 3, 28, 15, and 16 amino acids, respectively (Komaromy, M., et al., NAR 11 (1983) 6775-6785). Of the last 61 carboxyl-terminal amino acids, 21 are acidic residues, whereas only 2 are basic, indicating that this region has a strong net negative charge. The GenBank Accession No. is NP- 690605. It is thought that CD20 might be involved in regulating an early step(s) in the activation and differentiation process of B cells (Tedder, T. F., et al., Eur. J. Immunol.16 (1986) 881-887) and could function as a calcium ion channel (Tedder. T. F., et al., J. Cell. Biochem.14D (1990) 195).

[0104] The terms “CD20” and “CD20 antigen” are used interchangeably herein, and include any variants, isoforms and species homologs of human CD20 which are naturally expressed by cells or are expressed on cells transfected with the CD20 gene. Binding of an antibody of the disclosure to the CD20 antigen mediate the killing of cells expressing CD20 (e.g., a tumor cell) by inactivating CD20. The killing of the cells expressing CD20 may occur by one or more of the following mechanisms: Cell death / apoptosis induction, ADCC and CDC.

[0105] Synonyms of CD20, as recognized in the art, include B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BMS, and LF5.

[0106] The term “anti-CD20 antibody” according to the disclosure is an antibody that binds specifically to CD20 antigen. Depending on binding properties and biological activities of anti-CD20 antibodies to the CD20 antigen, two types of anti-CD20 antibodies 36 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 (type I and type II anti-CD20 antibodies) can be distinguished according to Cragg, M. S., et al., Blood 103 (2004) 2738-2743; and Cragg, M. S., et al., Blood 101 (2003) 1045-1052.

[0107] The two different types of anti-CD20 antibodies differ significantly in their mode of CD20 binding and biological activities (Cragg, M. S., et al., Blood 103 (2004) 2738- 2743; and Cragg, M. S., et al., Blood 101 (2003) 1045-1052). Type I antibodies, as e.g. rituximab, are potent in complement mediated cytotoxicity, whereas type II antibodies, as e.g. Tositumomab (B1), 11B8, AT80 or humanized B-Ly1 antibodies, effectively initiate target cell death via caspase-independent apoptosis with concomitant phosphatidylserine exposure.

[0108] In some instances, CD20 may be expressed in low copy number on the target cell (e.g. tumor cell). For example, in some instances, CD20 is expressed or present at less than 35,000 copies per target cell. In some embodiments, the low copy number cell surface CD20 is present between 100 and 35,000 copies per target cell; between 100 and 30,000 copies per target cell; between 100 and 25,000 copies per target cell; between 100 and 20,000 copies per target cell; between 100 and 15,000 copies per target cell; between 100 and 10,000 copies per target cell; between 100 and 5,000 copies per target cell; between 100 and 2,000 copies per target cell; between 100 and 1,000 copies per target cell; or between 100 and 500 copies per target cell. Copy number of the cell surface CD20 can be determined, for example, using a standard Scratchcard plot.

[0109] This disclosure provides bispecific and multispecific antibodies comprising a binding region that binds CD3 and a binding region that binds CD20.

[0110] In some embodiments, a 2:1 bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a first variable heavy region (VH1), a second variable heavy region (VH2), a first light chain polypeptide (L1) comprising a first variable light region (VL1), second heavy chain polypeptide (H2) comprising a third variable heavy region (VH3), a second light chain polypeptide (L2) comprising a second variable light region (VL2), and a third light chain polypeptide (L3) comprising a third variable light region (VL3) wherein the first antigen binding domain comprises VL1 and VH1, the second antigen binding domain comprises VL2 and VH2, the third antigen binding region comprises VL3 and VH3 and wherein the first antigen binding domain binds one of CD3 or CD20, the second antigen binding domain binds one of CD3 or CD20, and the third antigen binding domain binds one 37 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 of CD3 or CD20.

[0111] In some embodiments, a 2:1 bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a first variable heavy region (VH1), a first light chain polypeptide (L1) comprising a first variable light region (VL1), second heavy chain polypeptide (H2) comprising a second variable heavy region (VH2), a third variable heavy region (VH3), a second light chain polypeptide (L2) comprising a second variable light region (VL2), and a third light chain polypeptide (L3) comprising a third variable light region (VL3) wherein the first antigen binding domain comprises VL1 and VH1, the second antigen binding domain comprises VL2 and VH2, the third antigen binding region comprises VL3 and VH3 and wherein the first antigen binding domain binds one of CD3 or CD20, the second antigen binding domain binds one of CD3 or CD20, and the third antigen binding domain binds one of CD3 or CD20.

[0112] Provided herein are 2:1 multispecific antibodies comprising the following structure: a first heavy chain polypeptide (H1) comprising a first variable heavy region (VH1), a second variable heavy region (VH2), a first light chain polypeptide (L1) comprising a first variable light region (VL1), second heavy chain polypeptide (H2) comprising a third variable heavy region (VH3), a second light chain polypeptide (L2) comprising a second variable light region (VL2), a third light chain polypeptide (L3) comprising a third variable light region (VL3), and a CD2 ligand, such as CD58, wherein the first antigen binding domain comprises VL1 and VH1, the second antigen binding domain comprises VL2 and VH2, the third antigen binding region comprises VL3 and VH3 and wherein the first antigen binding domain binds one of CD3 or CD20, the second antigen binding domain binds one of CD3 or CD20, and the third antigen binding domain binds one of CD3 or CD20.

[0113] In some embodiments, a 2:1 multispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a first variable heavy region (VH1), a first light chain polypeptide (L1) comprising a first variable light region (VL1), second heavy chain polypeptide (H2) comprising a second variable heavy region (VH2), a third variable heavy region (VH3), a second light chain polypeptide (L2) comprising a second variable light region (VL2), a third light chain polypeptide (L3) comprising a third variable light region (VL3), and a CD2 ligand, such as CD58, wherein the first antigen binding domain comprises VL1 and VH1, the second antigen binding domain comprises VL2 and VH2, the third antigen 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 binding region comprises VL3 and VH3 and wherein the first antigen binding domain binds one of CD3 or CD20, the second antigen binding domain binds one of CD3 or CD20, and the third antigen binding domain binds one of CD3 or CD20.

[0114] In some embodiments, a 2:1 multispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a first variable heavy region (VH1), a second variable heavy region (VH2), a first light chain polypeptide (L1) comprising a first variable light region (VL1), second heavy chain polypeptide (H2) comprising a third variable heavy region (VH3), a second light chain polypeptide (L2) comprising a second variable light region (VL2), and a third light chain polypeptide (L3) comprising a third variable light region (VL3) wherein the first antigen binding domain comprises VL1 and VH1, the second antigen binding domain comprises VL2 and VH2, the third antigen binding region comprises VL3 and VH3 and wherein the first antigen binding domain binds one of CD3 or CD20, the second antigen binding domain binds one of CD3or CD20, and the third antigen binding domain binds one of CD3 or CD20.

[0115] In some embodiments, a 2:1 multispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a first variable heavy region (VH1), a first light chain polypeptide (L1) comprising a first variable light region (VL1), second heavy chain polypeptide (H2) comprising a second variable heavy region (VH2), a third variable heavy region (VH3), a second light chain polypeptide (L2) comprising a second variable light region (VL2), and a third light chain polypeptide (L3) comprising a third variable light region (VL3) wherein the first antigen binding domain comprises VL1 and VH1, the second antigen binding domain comprises VL2 and VH2, the third antigen binding region comprises VL3 and VH3 and wherein the first antigen binding domain binds one of CD3 or CD20, the second antigen binding domain binds one of CD3 or CD20, and the third antigen binding domain binds one of CD3 or CD20.

[0116] For example, the second binding region that binds to CD20 can be derived from the binding regions of an anti-CD20 antibody. Exemplary anti-CD20 antibodies include but are not limited to obinutuzumab (GA101; GAZYVA® or GAZYVARO®), tositumomab, rituximab, ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab, PRO131921, ublituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in WO 2005 / 103081 ), 2F2 IgG1 (as disclosed in WO 2004 / 035607 and WO 2005 / 103081) and 39 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 2H7 IgG1 (as disclosed in WO 2004 / 056312).

[0117] In some embodiments, an antigen binding region that binds CD20 comprises any one of the VH and VL sequences listed in Table 10.

[0118] In some embodiments, a binding region that binds to CD20 comprises: a) a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (VHCDR1), a VH complementarity determining region 2 (VHCDR2) and a VH complementarity determining region 3 (VHCDR3); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VLCDR1), a VL complementarity determining region 2 (VLCDR2) and a VL complementarity determining region 3 (VLCDR3). Tables 11 and 12 provide exemplary CDR sequences of the anti-CD20 antibodies (CDR sequences are determined according to Kabat). Table 10. Anti-CD20 Variable Heavy Chain and Variable Light Chain DomainsTable 11. Anti-CD20 Heavy Chain CDRsTable 12. Anti-CD20 Light Chain CDRsFusion peptides

[0119] Provided herein is an antibody (e.g. multispecific antibody) that has a fusion peptide fused to the N-terminus or the C-terminus of the first heavy chain polypeptide or the second heavy chain polypeptide. 40 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0120] Critical to the initial T cell response is the capacity for T cells to detect foreign and mutated proteins through their T cell receptor. This response, often referred to as signal 1 of T cell activation, occurs when the T cell receptor engages a cell that displays a foreign or mutated protein fragment or antigen in a specific protein complex called the Major Histocompatibility Complex I (MHCI). The activation of the T cell receptor is by itself both activating and auto-regulatory to T cells. Strong binding of the TCR to an MHCI complex creates chronic activation of the TCR. This form of signal is associated with T cells that are reactive to self-antigens. T cells are programmed to inactivate when they experience this form of activation. T cells with TCR that bind weaker, but sufficient for activation, experience acute signaling with the potential to remain active and differentiate into memory T cells. This is emerging as an important consideration in the design the T cell therapeutics.

[0121] T cell cytokine activation, often referred to as signal 3, is important in T cell transitions, either from non-dividing to a state of rapid cell division or from one phenotypic state to another. T cell cytokine receptors bind to cytokines that are produced by immune and non-immune cells and depending on the cytokine and the state of the T cell at the time of receiving the cytokine signal can induce cell proliferation, can sustain vitality, or can induce differentiation of T cells into a specialized cell state appropriate for sustained activation or inactivation following infection.

[0122] One example is the transition that naïve cells experience through cytokines which can induce naïve T cells to proliferate and promote T cell differentiation into memory T cells. Exemplary cytokines include but are not limited to IL-2, IL-7, IL-10, IL-12, IL-15, IL-18 and IL-21.

[0123] Costimulatory receptor activation, referred to as signal 2 provides a context specific cell-to-cell reinforcement of T activation. The most recognized form of costimulation occurs when T cells interact with activated antigen presenting cells through the T cell costimulatory receptor CD28 with CD80 and CD86 ligands found on APCs. These interactions can “prime” specific T cells armed with T cell receptors responsive to pathogen or cancer proteins.

[0124] Less appreciated is costimulation induced at the site of infection and malignancies. This includes costimulation that acts through CD2 and NKG2D receptors responsive to ligands like CD58 and UL16 binding proteins (e.g. ULBP2 / 5 / 6) that are induced in immune cells and epithelial cells upon viral infection. These signals provide not only reinforcement 41 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 of T activation, but confirmation that the T cell’s lethal effector activities are targeted with single cell accuracy. While many costimulatory receptors have been discovered, the importance of each receptor’s specific context and the impact of concurrent signaling of multiple costimulatory receptors remains largely unknown and an area to greatly advance our understanding of T cell biology and creating possibilities for novel tumor-targeted T cell therapeutic development.

[0125] Costimulatory ligands include but are not limited to CD58, CD48, CD86, OX40L, 4-1BBL, GITRL, CD70, CD80, MR1, or ICOSLG.

[0126] CD58 is advantageous over other costimulatory ligands in that it is the primary costimulatory pathway available at the tumor site as tumor infiltrating T lymphocytes often lose expression of other costimulatory receptors like CD28, or due to the low immunogenicity of tumor cells, tumor cells do not sufficiently activate T cell, thus limiting the potential of inducible costimulatory receptors like 41BB.

[0127] As described previously, the anti-CD3ε antibodies of the disclosure induce varying levels of T cell receptor activation that confer alteration in T cell vitality and cytokine production. Accordingly, a fusion of the costimulatory ligand CD58 to the anti-CD3 / anti- CD20 antibody provides integrated costimulatory T cell activation via CD2 binding for optimal T cell activation.

[0128] In some embodiments, the bispecific or multispecific antibody has a peptide fused to the N-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific or multispecific antibody has a peptide fused to the C-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific or multispecific antibody has a polypeptide fused to the N-terminus of the second heavy chain polypeptide (H2). In some embodiments, the bispecific or multispecific antibody has a peptide fused to the C-terminus of the second heavy chain polypeptide (H2). In some embodiments, the bispecific or multispecific antibody has a peptide fused to the N-terminus of the first light chain polypeptide (L1). In some embodiments, the bispecific or multispecific antibody has a peptide fused to the C-terminus of the first light chain polypeptide (L1). In some embodiments, the bispecific or multispecific antibody has a polypeptide fused to the N-terminus of the second light chain polypeptide (L2). In some embodiments, the bispecific or multispecific antibody has a peptide fused to the C-terminus of the second light chain polypeptide (L2). In some 42 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 embodiments, the bispecific or multispecific antibody has a polypeptide fused to the N- terminus of the third light chain polypeptide (L3). In some embodiments, the bispecific or multispecific antibody has a peptide fused to the C-terminus of the third light chain polypeptide (L3). Exemplary peptides include but are not limited to IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 or portions thereof. Exemplary peptides include but are not limited to

[0129] CD58, CD48, CD86, OX40L, 4-1BBL, GITRL, CD70, CD80, MR1, or ICOSLG or portions thereof. Exemplary peptide sequences that are fused to the bispecific antibodies include but are not limited to those listed in Table 13.1 and Table 13.2. Table 13.1. Exemplary Fusion Peptide SequencesTable 13.2. Exemplary Fusion Peptide Sequences43 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0130] In some embodiments the polypeptide is fused directly to the bispecific antibody. In some embodiments, the polypeptide is fused indirectly through a linker. In some embodiments, the bispecific antibody fused with a peptide comprises a linker sequence. Exemplary linker sequences include but are not limited to those listed in Table 14.1 and Table 14.2. In some embodiments, a linker is a flexible linker. In some embodiments, a linker is a cleavable linker. In some embodiments, a linker is a non-cleavable linker. In some embodiments, a linker is a glycine-serine linker. In some embodiments, a linker comprises 2 amino acids to 150 amino acids. In some embodiments, a linker comprises 2 amino acids to 100 amino acids. In some embodiments, a linker comprises 2 amino acids to 75 amino acids. In some embodiments, a linker comprises 2 amino acids to 50 amino acids. In some embodiments, a linker comprises 2 amino acids to 25 amino acids. In some embodiments, a linker comprises 2 amino acids to 15 amino acids. In some embodiments, a linker comprises 2 amino acids to 10 amino acids. In some embodiments, a linker comprises 5 amino acids to 50 amino acids. In some embodiments, a linker comprises 10 amino acids to 50 amino acids. In some embodiments, a linker comprises 20 amino acids to 50 amino acids. In some embodiments, a linker comprises 30 amino acids to 50 amino acids. In some embodiments, a linker comprises about 10 amino acids. In some embodiments, a linker comprises about 20 amino acids. In some embodiments, a linker comprises about 30 amino acids. In some embodiments, a linker comprises about 40 amino acids. In some embodiments, a linker comprises about 50 amino acids. In some embodiments, a linker comprises the amino acid sequence of any one of SEQ ID NOs: 96-101, 283-338, or 553- 606. In some embodiments, a linker does not comprise a solvent-exposed serine. In some embodiments, a linker is not O-glycosylated. In some embodiments, a linker comprises the amino acid sequence of SEQ ID NO: 344. In some embodiments, a linker comprises the amino acid sequence of SEQ ID NO: 335. Table 14.1. Exemplary Linker Sequences 44 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00Table.14.2 Exemplary Linker Sequences45 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0046 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00Exemplary Bispecific and Multispecific Antibodies That Bind to CD3 and CD20

[0131] Provided herein are bispecific and multispecific antibodies comprising an antigen binding domain that binds a first antigen (e.g. CD3); a second antigen binding domain that binds to a CD20; and a third antigen binding domain that binds CD20.

[0132] Tables 8 and 9 provide exemplary CDR sequences of the anti-CD3ε antibodies provided herein. Tables 11 and 12 provide exemplary CDR sequences of the anti-CD20 antibodies provided herein.

[0133] In some embodiments, the bispecific and multispecific antibody of the disclosure comprises a first antigen binding domain (e.g. binding to CD3) comprising any one of the VH and VL sequences listed in Table 7 and / or Table 7.1 and a second antigen binding domain (e.g. binding to CD20) comprising any one of the VH and VL sequences listed in Table 10.

[0134] In some embodiments, the bispecific and multispecific antibodies of the disclosure comprises a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1); and a second heavy chain polypeptide (H2); a second light chain polypeptide (L2) and / or a third light chain (L3) comprising any one of the sequence listed in Table 15, Table 16, or Table 47 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 23.

[0135] In some embodiments, the bispecific and multispecific antibodies of the disclosure provided herein comprises a H1 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 15, Table 16, or Table 23. In some embodiments, the H1 comprises a C-terminal lysine (K) residue.

[0136] In some embodiments, the bispecific and multispecific antibodies of the disclosure provided herein comprises an L1 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 15, Table 16, or Table 23.

[0137] In some embodiments, the bispecific and multispecific antibodies of the disclosure provided herein comprises a H2 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 15, Table 16, or Table 23. In some embodiments, the H1 comprises a C-terminal lysine (K) residue.

[0138] In some embodiments, the bispecific and multispecific antibodies of the disclosure provided herein comprises a L2 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 15, Table 16, or Table 23. Table 15. Exemplary 2:1 Bispecific Antibodies that bind CD3 and CD2048 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0049 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0050 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00Table 16: Exemplary 2:1 Multispecific Antibodies that Bind CD3 and CD20 with a CD58 Fusion Peptide 51 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0052 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0053 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0054 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0055 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0056 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0057 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0058 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0059 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0060 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0061 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0062 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0063 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0064 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0065 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0066 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0067 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0068 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0069 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0070 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0071 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0072 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0073 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0074 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00Table 23: Exemplary 1:1 and 2:1 Multispecific Antibodies that Bind CD3 and CD20 optionally with a CD58 Fusion Peptide (CDRs according to Kabat in bold face; linker sequence underlined)75 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0076 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0077 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0078 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0079 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0080 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0081 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0082 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0083 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0084 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0085 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0086 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0087 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0088 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0089 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0090 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0091 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0092 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0093 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0094 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0095 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0096 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0097 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0098 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO0099 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00100 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00101 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00102 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00Underlined sequences are linker sequences

[0139] Each of the 2:1 multispecific antibodies provided in Table 23 further comprises a third light chain polypeptide (L3) comprising a third variable light chain region (VL3), wherein the L3 comprises a sequence that is identical to the L1 of the corresponding 2:1 multispecific antibody.

[0140] In some embodiments, for each of the 2:1 multispecific antibodies provided in Table 23, a linker sequence (underlined in Table 23) alternatively comprises the amino acid sequence of SEQ ID NO: 335.

[0141] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 107, an LC1 comprising the amino acid sequence of SEQ ID NO: 108, an HC2 comprising the amino acid sequence of SEQ ID NO: 109, and an LC2 comprising the amino acid sequence of SEQ ID NO: 110, and an LC3 comprising the amino acid sequence of SEQ ID NO: 108.

[0142] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 111, an LC1 comprising the amino acid sequence of SEQ ID NO: 112, an HC2 comprising the amino acid sequence of SEQ ID NO: 113, and an LC2 comprising the amino acid sequence of SEQ ID NO: 114, and an LC3 comprising the amino acid sequence of SEQ ID NO: 112.

[0143] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 115, an LC1 comprising the amino acid sequence of SEQ ID NO: 116, an HC2 comprising the amino acid sequence of SEQ ID NO: 117, and 103 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 an LC2 comprising the amino acid sequence of SEQ ID NO: 118, and an LC3 comprising the amino acid sequence of SEQ ID NO: 116.

[0144] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 119, an LC1 comprising the amino acid sequence of SEQ ID NO: 120, an HC2 comprising the amino acid sequence of SEQ ID NO: 121, and an LC2 comprising the amino acid sequence of SEQ ID NO: 122, and an LC3 comprising the amino acid sequence of SEQ ID NO: 120.

[0145] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 123, an LC1 comprising the amino acid sequence of SEQ ID NO: 124, an HC2 comprising the amino acid sequence of SEQ ID NO: 125, and an LC2 comprising the amino acid sequence of SEQ ID NO: 126, and an LC3 comprising the amino acid sequence of SEQ ID NO: 124.

[0146] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 127, an LC1 comprising the amino acid sequence of SEQ ID NO: 128, an HC2 comprising the amino acid sequence of SEQ ID NO: 129, and an LC2 comprising the amino acid sequence of SEQ ID NO: 130, and an LC3 comprising the amino acid sequence of SEQ ID NO: 128.

[0147] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 131, an LC1 comprising the amino acid sequence of SEQ ID NO: 132, an HC2 comprising the amino acid sequence of SEQ ID NO: 133, and an LC2 comprising the amino acid sequence of SEQ ID NO: 134, and an LC3 comprising the amino acid sequence of SEQ ID NO: 132.

[0148] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 135, an LC1 comprising the amino acid sequence of SEQ ID NO: 136, an HC2 comprising the amino acid sequence of SEQ ID NO: 137, and an LC2 comprising the amino acid sequence of SEQ ID NO: 138, and an LC3 comprising the amino acid sequence of SEQ ID NO: 136.

[0149] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 139, an LC1 comprising the amino acid sequence of SEQ ID NO: 140, an HC2 comprising the amino acid sequence of SEQ ID NO: 141, and 104 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 an LC2 comprising the amino acid sequence of SEQ ID NO: 142, and an LC3 comprising the amino acid sequence of SEQ ID NO: 140.

[0150] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 143, an LC1 comprising the amino acid sequence of SEQ ID NO: 144, an HC2 comprising the amino acid sequence of SEQ ID NO: 145, and an LC2 comprising the amino acid sequence of SEQ ID NO: 146, and an LC3 comprising the amino acid sequence of SEQ ID NO: 144.

[0151] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 147, an LC1 comprising the amino acid sequence of SEQ ID NO: 148, an HC2 comprising the amino acid sequence of SEQ ID NO: 149, and an LC2 comprising the amino acid sequence of SEQ ID NO: 150, and an LC3 comprising the amino acid sequence of SEQ ID NO: 148.

[0152] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 151, an LC1 comprising the amino acid sequence of SEQ ID NO: 152, an HC2 comprising the amino acid sequence of SEQ ID NO: 153, and an LC2 comprising the amino acid sequence of SEQ ID NO: 154, and an LC3 comprising the amino acid sequence of SEQ ID NO: 152.

[0153] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 155, an LC1 comprising the amino acid sequence of SEQ ID NO: 156, an HC2 comprising the amino acid sequence of SEQ ID NO: 157, and an LC2 comprising the amino acid sequence of SEQ ID NO: 158, and an LC3 comprising the amino acid sequence of SEQ ID NO: 156.

[0154] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 159, LC1 comprising the amino acid sequence of SEQ ID NO: 160, an HC2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC2 comprising the amino acid sequence of SEQ ID NO: 162, and an LC3 comprising the amino acid sequence of SEQ ID NO: 160.

[0155] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 163, an LC1 comprising the amino acid sequence of SEQ ID NO: 164, an HC2 comprising the amino acid sequence of SEQ ID NO: 165, and 105 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 an LC2 comprising the amino acid sequence of SEQ ID NO: 166, and an LC3 comprising the amino acid sequence of SEQ ID NO: 164.

[0156] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 167, an LC1 comprising the amino acid sequence of SEQ ID NO: 168, an HC2 comprising the amino acid sequence of SEQ ID NO: 169, and an LC2 comprising the amino acid sequence of SEQ ID NO: 170, and an LC3 comprising the amino acid sequence of SEQ ID NO: 168.

[0157] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 171, an LC1 comprising the amino acid sequence of SEQ ID NO: 172, an HC2 comprising the amino acid sequence of SEQ ID NO: 173, and an LC2 comprising the amino acid sequence of SEQ ID NO: 174, and an LC3 comprising the amino acid sequence of SEQ ID NO: 172.

[0158] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 175, an LC1 comprising the amino acid sequence of SEQ ID NO: 176, an HC2 comprising the amino acid sequence of SEQ ID NO: 177, and an LC2 comprising the amino acid sequence of SEQ ID NO: 178, and an LC3 comprising the amino acid sequence of SEQ ID NO: 176.

[0159] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 179, an LC1 comprising the amino acid sequence of SEQ ID NO: 180, an HC2 comprising the amino acid sequence of SEQ ID NO: 181, and an LC2 comprising the amino acid sequence of SEQ ID NO: 182, and an LC3 comprising the amino acid sequence of SEQ ID NO: 180.

[0160] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 183, an LC1 comprising the amino acid sequence of SEQ ID NO: 184, an HC2 comprising the amino acid sequence of SEQ ID NO: 185, and an LC2 comprising the amino acid sequence of SEQ ID NO: 186, and an LC3 comprising the amino acid sequence of SEQ ID NO: 184.

[0161] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 187, an LC1 comprising the amino acid sequence of SEQ ID NO: 188, an HC2 comprising the amino acid sequence of SEQ ID NO: 189, and 106 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 an LC2 comprising the amino acid sequence of SEQ ID NO: 190, and an LC3 comprising the amino acid sequence of SEQ ID NO: 188.

[0162] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 191, an LC1 comprising the amino acid sequence of SEQ ID NO: 192, an HC2 comprising the amino acid sequence of SEQ ID NO: 193, and an LC2 comprising the amino acid sequence of SEQ ID NO: 194, and an LC3 comprising the amino acid sequence of SEQ ID NO: 192.

[0163] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 195, an LC1 comprising the amino acid sequence of SEQ ID NO: 196, an HC2 comprising the amino acid sequence of SEQ ID NO: 197, and an LC2 comprising the amino acid sequence of SEQ ID NO: 198, and an LC3 comprising the amino acid sequence of SEQ ID NO: 196.

[0164] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 199, an LC1 comprising the amino acid sequence of SEQ ID NO: 200, an HC2 comprising the amino acid sequence of SEQ ID NO: 201, and an LC2 comprising the amino acid sequence of SEQ ID NO: 282, and an LC3 comprising the amino acid sequence of SEQ ID NO: 200.

[0165] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 202, an LC1 comprising the amino acid sequence of SEQ ID NO: 203, an HC2 comprising the amino acid sequence of SEQ ID NO: 204, and an LC2 comprising the amino acid sequence of SEQ ID NO: 205, and an LC3 comprising the amino acid sequence of SEQ ID NO: 203.

[0166] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 206, an LC1 comprising the amino acid sequence of SEQ ID NO: 207, an HC2 comprising the amino acid sequence of SEQ ID NO: 208, and an LC2 comprising the amino acid sequence of SEQ ID NO: 209, and an LC3 comprising the amino acid sequence of SEQ ID NO: 207.

[0167] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 210, an LC1 comprising the amino acid sequence of SEQ ID NO: 211, an HC2 comprising the amino acid sequence of SEQ ID NO: 212, and 107 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 an LC2 comprising the amino acid sequence of SEQ ID NO: 213, and an LC3 comprising the amino acid sequence of SEQ ID NO: 211.

[0168] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 214, an LC1 comprising the amino acid sequence of SEQ ID NO: 215, an HC2 comprising the amino acid sequence of SEQ ID NO: 216, and an LC2 comprising the amino acid sequence of SEQ ID NO: 217, and an LC3 comprising the amino acid sequence of SEQ ID NO: 215.

[0169] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 218, an LC1 comprising the amino acid sequence of SEQ ID NO: 219, an HC2 comprising the amino acid sequence of SEQ ID NO: 220, and an LC2 comprising the amino acid sequence of SEQ ID NO: 221, and an LC3 comprising the amino acid sequence of SEQ ID NO: 219.

[0170] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 222, an LC1 comprising the amino acid sequence of SEQ ID NO: 223, an HC2 comprising the amino acid sequence of SEQ ID NO: 224, and an LC2 comprising the amino acid sequence of SEQ ID NO: 225, and an LC3 comprising the amino acid sequence of SEQ ID NO: 223.

[0171] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 226, an LC1 comprising the amino acid sequence of SEQ ID NO: 227, an HC2 comprising the amino acid sequence of SEQ ID NO: 228, and an LC2 comprising the amino acid sequence of SEQ ID NO: 229, and an LC3 comprising the amino acid sequence of SEQ ID NO: 227.

[0172] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 230, an LC1 comprising the amino acid sequence of SEQ ID NO: 231, an HC2 comprising the amino acid sequence of SEQ ID NO: 232, and an LC2 comprising the amino acid sequence of SEQ ID NO: 233, and an LC3 comprising the amino acid sequence of SEQ ID NO: 231.

[0173] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 234, an LC1 comprising the amino acid sequence of SEQ ID NO: 235, an HC2 comprising the amino acid sequence of SEQ ID NO: 236, and 108 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 an LC2 comprising the amino acid sequence of SEQ ID NO: 237, and an LC3 comprising the amino acid sequence of SEQ ID NO: 235.

[0174] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 238, an LC1 comprising the amino acid sequence of SEQ ID NO: 239, an HC2 comprising the amino acid sequence of SEQ ID NO: 240, and an LC2 comprising the amino acid sequence of SEQ ID NO: 241, and an LC3 comprising the amino acid sequence of SEQ ID NO: 239.

[0175] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 242, an LC1 comprising the amino acid sequence of SEQ ID NO: 243, an HC2 comprising the amino acid sequence of SEQ ID NO: 244, and an LC2 comprising the amino acid sequence of SEQ ID NO: 245, and an LC3 comprising the amino acid sequence of SEQ ID NO: 243.

[0176] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 246, an LC1 comprising the amino acid sequence of SEQ ID NO: 247, an HC2 comprising the amino acid sequence of SEQ ID NO: 248, and an LC2 comprising the amino acid sequence of SEQ ID NO: 249, and an LC3 comprising the amino acid sequence of SEQ ID NO: 247.

[0177] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 250, an LC1 comprising the amino acid sequence of SEQ ID NO: 251, an HC2 comprising the amino acid sequence of SEQ ID NO: 252, and an LC2 comprising the amino acid sequence of SEQ ID NO: 253, and an LC3 comprising the amino acid sequence of SEQ ID NO: 251.

[0178] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 254, an LC1 comprising the amino acid sequence of SEQ ID NO: 255, an HC2 comprising the amino acid sequence of SEQ ID NO: 256, and an LC2 comprising the amino acid sequence of SEQ ID NO: 257, and an LC3 comprising the amino acid sequence of SEQ ID NO: 255.

[0179] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 258, an LC1 comprising the amino acid sequence of SEQ ID NO: 259, an HC2 comprising the amino acid sequence of SEQ ID NO: 260, and 109 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 an LC2 comprising the amino acid sequence of SEQ ID NO: 261, and an LC3 comprising the amino acid sequence of SEQ ID NO: 259.

[0180] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 262, an LC1 comprising the amino acid sequence of SEQ ID NO: 263, an HC2 comprising the amino acid sequence of SEQ ID NO: 264, and an LC2 comprising the amino acid sequence of SEQ ID NO: 265, and an LC3 comprising the amino acid sequence of SEQ ID NO: 263.

[0181] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 266, an LC1 comprising the amino acid sequence of SEQ ID NO: 267, an HC2 comprising the amino acid sequence of SEQ ID NO: 268, and an LC2 comprising the amino acid sequence of SEQ ID NO: 269, and an LC3 comprising the amino acid sequence of SEQ ID NO: 267.

[0182] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 270, an LC1 comprising the amino acid sequence of SEQ ID NO: 271, an HC2 comprising the amino acid sequence of SEQ ID NO: 272, and an LC2 comprising the amino acid sequence of SEQ ID NO: 273, and an LC3 comprising the amino acid sequence of SEQ ID NO: 271.

[0183] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 374, an LC1 comprising the amino acid sequence of SEQ ID NO: 375, an HC2 comprising the amino acid sequence of SEQ ID NO: 376, and an LC2 comprising the amino acid sequence of SEQ ID NO: 377, and an LC3 comprising the amino acid sequence of SEQ ID NO: 375.

[0184] In some embodiments, an antibody described herein comprises: an HC1 comprising the amino acid sequence of SEQ ID NO: 378, an LC1 comprising the amino acid sequence of SEQ ID NO: 379, an HC2 comprising the amino acid sequence of SEQ ID NO: 380, and an LC2 comprising the amino acid sequence of SEQ ID NO: 381, and an LC3 comprising the amino acid sequence of SEQ ID NO: 379.

[0185] Any one of the anti-CD3 / anti-CD20 bispecific and multispecific antibodies shown above in Table 15, Table 16, or Table 23 can be further modified by substituting any one of the anti-CD3ε antigen binding regions with any one of the anti-CD3ε binding regions shown in Tables 7-9. 110 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0186] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-1. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 107, a LC1 comprising the amino acid sequence of SEQ ID NO: 108, a HC2 comprising the amino acid sequence of SEQ ID NO: 109, a LC2 comprising the amino acid sequence of SEQ ID NO: 110, and a LC3 comprising the amino acid sequence of SEQ ID NO: 108. In some embodiments, the linker sequence in SEQ ID NO: 109 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti- CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 107, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 108, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 109, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 110, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 108.

[0187] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-21. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 187, a LC1 comprising the amino acid sequence of SEQ ID NO: 188, a HC2 comprising the amino acid sequence of SEQ ID NO: 189, a LC2 comprising the amino acid sequence of SEQ ID NO: 190, and a LC3 comprising the amino acid sequence of SEQ ID NO: 188. In some embodiments, one or both of the linker sequences in SEQ ID NO: 111 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 189 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 187, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 188, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 189, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 190, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 188.

[0188] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-26. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 206, a LC1 comprising the amino acid sequence of SEQ ID NO: 207, a HC2 comprising the amino acid sequence of SEQ ID NO: 208, a LC2 comprising the amino acid sequence of SEQ ID NO: 209, and a LC3 comprising the amino acid sequence of SEQ ID NO: 207. In some embodiments, the linker sequence in SEQ ID NO: 206 alternatively comprises the amino acid sequence of SEQ ID NO: 335 and / or the linker sequence in SEQ ID NO: 208 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 206, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 112 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 207, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 208, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 209, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 207.

[0189] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-29. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 218, a LC1 comprising the amino acid sequence of SEQ ID NO: 219, a HC2 comprising the amino acid sequence of SEQ ID NO: 220, a LC2 comprising the amino acid sequence of SEQ ID NO: 221, and a LC3 comprising the amino acid sequence of SEQ ID NO: 219. In some embodiments, the linker sequence in SEQ ID NO: 220 alternatively comprises the amino acid sequence of SEQ ID NO: 335 and / or the linker sequence in SEQ ID NO: 221 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 218, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 219, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 220, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 113 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 221, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 219.

[0190] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-35. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 242, a LC1 comprising the amino acid sequence of SEQ ID NO: 243, a HC2 comprising the amino acid sequence of SEQ ID NO: 244, a LC2 comprising the amino acid sequence of SEQ ID NO: 245, and a LC3 comprising the amino acid sequence of SEQ ID NO: 243. In some embodiments, one or both of the linker sequences in SEQ ID NO: 244 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 242, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 243, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 244, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 245, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 243.

[0191] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-36. In some embodiments, a 2:1 anti-CD20 / anti-CD3 114 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 246, a LC1 comprising the amino acid sequence of SEQ ID NO: 247, a HC2 comprising the amino acid sequence of SEQ ID NO: 248, a LC2 comprising the amino acid sequence of SEQ ID NO: 249, and a LC3 comprising the amino acid sequence of SEQ ID NO: 247. In some embodiments, one or both of the linker sequences in SEQ ID NO: 248 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 246, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 247, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 248, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 249, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 247.

[0192] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-37. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 250, a LC1 comprising the amino acid sequence of SEQ ID NO: 251, a HC2 comprising the amino acid sequence of SEQ ID NO: 252, a LC2 comprising the amino acid sequence of SEQ ID NO: 253, and LC3 comprising the amino acid sequence of SEQ ID NO: 251. In some embodiments, the linker sequence in SEQ ID NO: 250 alternatively comprises the amino acid sequence of SEQ ID NO: 335 and / or the linker sequence in SEQ ID NO: 252 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some 115 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 250, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 251, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 252, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 253, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 251.

[0193] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-39. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 258, a LC1 comprising the amino acid sequence of SEQ ID NO: 259, a HC2 comprising the amino acid sequence of SEQ ID NO: 260, a LC2 comprising the amino acid sequence of SEQ ID NO: 261, and a LC3 comprising the amino acid sequence of SEQ ID NO: 259. In some embodiments, the linker sequence in SEQ ID NO: 260 alternatively comprises the amino acid sequence of SEQ ID NO: 335 and / or the linker sequence in SEQ ID NO: 261 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 258, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 116 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 259, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 260, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 261, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 259.

[0194] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-40. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 262, a LC1 comprising the amino acid sequence of SEQ ID NO: 263, a HC2 comprising the amino acid sequence of SEQ ID NO: 264, a LC2 comprising the amino acid sequence of SEQ ID NO: 265, and a LC3 comprising the amino acid sequence of SEQ ID NO: 263. In some embodiments, the linker sequence in SEQ ID NO: 264 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti- CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 262, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 263, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 264, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 265, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 117 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 263.

[0195] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-41. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 266, a LC1 comprising the amino acid sequence of SEQ ID NO: 267, a HC2 comprising the amino acid sequence of SEQ ID NO: 268, a LC2 comprising the amino acid sequence of SEQ ID NO: 269, and LC3 comprising the amino acid sequence of SEQ ID NO: 267. In some embodiments, one or both of the linker sequences in SEQ ID NO: 268 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 266, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 267, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 268, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 269, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 267.

[0196] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-42. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 270, a LC1 comprising the amino acid sequence of SEQ ID NO: 118 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 271, a HC2 comprising the amino acid sequence of SEQ ID NO: 272, a LC2 comprising the amino acid sequence of SEQ ID NO: 273, and a LC3 comprising the amino acid sequence of SEQ ID NO: 271. In some embodiments, the linker sequence in SEQ ID NO: 272 alternatively comprises the amino acid sequence of SEQ ID NO: 335 and / or the linker sequence in SEQ ID NO: 273 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 270, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 271, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 272, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 273, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 271.

[0197] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-43. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 374, a LC1 comprising the amino acid sequence of SEQ ID NO: 375, a HC2 comprising the amino acid sequence of SEQ ID NO: 376, a LC2 comprising the amino acid sequence of SEQ ID NO: 377, and a LC3 comprising the amino acid sequence of SEQ ID NO: 375. In some embodiments, one or both of the linker sequences in SEQ ID NO: 376 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at 119 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 374, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 375, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 376, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 377, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 375.

[0198] In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule described herein is EVOLVE-44. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising the amino acid sequence of SEQ ID NO: 378, a LC1 comprising the amino acid sequence of SEQ ID NO: 379, a HC2 comprising the amino acid sequence of SEQ ID NO: 380, a LC2 comprising the amino acid sequence of SEQ ID NO: 381, and a LC3 comprising the amino acid sequence of SEQ ID NO: 379. In some embodiments, one or both of the linker sequences in SEQ ID NO: 380 alternatively comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a 2:1 anti-CD20 / anti-CD3 multispecific antibody fusion molecule comprises: a HC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 378, a LC1 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 379, a HC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 120 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 98%, at least 99%, or 100%) identical to SEQ ID NO: 380, a LC2 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 381, and a LC3 comprising an amino acid sequence at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 379. Methods of Production

[0199] Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a given target, such as, for example CD20, a disease associated antigen or other target, or against derivatives, fragments, analogs homologs or orthologs thereof. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0200] Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol.14, No.8 (April 17, 2000), pp.25-28).

[0201] In some embodiments, the antibodies of the disclosure are monoclonal antibodies. Monoclonal antibodies are generated, for example, by using the procedures set forth in the Examples provided herein. Antibodies are also generated, e.g., by immunizing BALB / c mice with combinations of cell transfectants expressing high levels of a given target on their surface. Hybridomas resulting from myeloma / B cell fusions are then screened for reactivity to the selected target.

[0202] Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an 121 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0203] The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59- 103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

[0204] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63)).

[0205] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in 122 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.

[0206] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0207] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0208] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the disclosure can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the disclosure serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the disclosure, or can be substituted for the variable domains of one antigen-combining site of an antibody of the disclosure to create a chimeric bivalent antibody. 123 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0209] Monoclonal antibodies of the disclosure include humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization is performed, e.g., by following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534- 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No.5,225,539). In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies also comprise, e.g., residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody includes substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also includes at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0210] Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed “human antibodies”, or “fully human antibodies” herein. Monoclonal antibodies can be prepared by using trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONALANTIBODIES ANDCANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see 124 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0211] In addition, human antibodies can also be produced using additional techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol.1365-93 (1995).

[0212] Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal’s endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94 / 02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host’s genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. An example of such a nonhuman animal is a mouse termed the XenomouseTMas disclosed in PCT publications WO 96 / 33735 and WO 96 / 34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and 125 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv (scFv) molecules.

[0213] An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No.5,939,598. It can be obtained by a method, which includes deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

[0214] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

[0215] In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen and a correlative method for selecting an antibody that binds specifically to the relevant epitope with high affinity are disclosed in PCT publication WO 99 / 53049.

[0216] The antibody can be expressed by a vector containing a DNA segment encoding the single chain antibody described above.

[0217] These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA. gene gun, catheters, etc. Vectors include chemical conjugates such as described in WO 93 / 64701, which has targeting moiety (e.g., a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g., polylysine), viral vector (e.g., a DNA or RNA viral vector), fusion proteins such as described in PCT / US 95 / 02140 (WO 95 / 22618) which is a fusion protein containing a target moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., a protamine), plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal or synthetic. 126 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0218] Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include moloney murine leukemia viruses. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) and Adeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

[0219] Pox viral vectors introduce the gene into the cell’s cytoplasm. Avipox virus vectors result in only a short term expression of the nucleic acid. Adenovirus vectors, adeno- associated virus vectors and herpes simplex virus (HSV) vectors are preferred for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The particular vector chosen will depend upon the target cell and the condition being treated. The introduction can be by standard techniques, e.g., infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, (Ca)2(PO4)3 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0220] The vector can be employed to target essentially any desired target cell. For example, stereotaxic injection can be used to direct the vectors (e.g., adenovirus, HSV) to a desired location. Additionally, the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System. A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the vector to the target cell. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol.266:292-305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration. 127 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0221] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for a first target such as CD3 or any fragment thereof. The second binding target is a disease associated antigen such as CD20 or any fragment thereof.

[0222] Multispecific antibodies are antibodies that have binding specificities for more than two antigens. In some embodiments, multispecific antibodies of the disclosure bind to at least three or at least four antigens. In the present case, one of the binding specificities is for a first target such as CD3 or any fragment thereof. Another binding specificity is for a second binding target such as a disease associated antigen such as CD20 or any fragment thereof. A third binding specificity CD2.

[0223] Methods for making bispecific and multispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific and multispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain / light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific or multispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93 / 08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0224] Bispecific, multispecific, and / or monovalent antibodies of the disclosure can be made using any of a variety of art-recognized techniques, including those disclosed in co- pending application WO 2012 / 023053, filed August 16, 2011, the contents of which are hereby incorporated by reference in their entirety. The methods described in WO 2012 / 023053 generate bispecific antibodies that are identical in structure to a human immunoglobulin. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant Kappa domain and second light chain variable region fused to a constant Lambda domain. Each combining site displays a different antigen specificity to which both the heavy and light chain contribute. The light chain variable regions can be of the Lambda or Kappa family and are preferably fused to a Lambda and Kappa constant domains, respectively. This is preferred in order to avoid the 128 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 generation of non-natural polypeptide junctions. However, it is also possible to obtain bispecific and multispecific antibodies of the disclosure by fusing a Kappa light chain variable domain to a constant Lambda domain for a first specificity and fusing a Lambda light chain variable domain to a constant Kappa domain for the second specificity. The bispecific antibodies described in WO 2012 / 023053 are referred to as IgGκλ antibodies or “κλ bodies,” a new fully human bispecific IgG format. This κλ-body format allows the affinity purification of a bispecific antibody that is undistinguishable from a standard IgG molecule with characteristics that are undistinguishable from a standard monoclonal antibody and, therefore, favorable as compared to previous formats.

[0225] An essential step of the method is the identification of two antibody Fv regions (each composed by a variable light chain and variable heavy chain domain) having different antigen specificities that share the same heavy chain variable domain. Numerous methods have been described for the generation of monoclonal antibodies and fragments thereof. (See, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Fully human antibodies are antibody molecules in which the sequence of both the light chain and the heavy chain, including the CDRs 1 and 2, arise from human genes. The CDR3 region can be of human origin or designed by synthetic means. Such antibodies are termed “human antibodies”, or “fully human antibodies” herein. Human monoclonal antibodies can be prepared by using the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0226] Monoclonal antibodies are generated, e.g., by immunizing an animal with a target antigen or an immunogenic fragment, derivative or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen, such that the target antigen is expressed and associated with the surface of 129 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 the transfected cells. A variety of techniques are well-known in the art for producing xenogenic non-human animals. For example, see U.S. Pat. No.6,075,181 and No.6,150,584, which is hereby incorporated by reference in its entirety.

[0227] Alternatively, the antibodies are obtained by screening a library that contains antibody or antigen binding domain sequences for binding to the target antigen. This library is prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (i.e., “phage displayed library”). Alternatively, a library can be prepared in yeast as protein or peptide fusions to a cell wall protein on the surface of yeast cells and encoding DNA sequences contained within the yeast cells (i.e. “yeast display library”).

[0228] Hybridomas resulting from myeloma / B cell fusions are then screened for reactivity to the target antigen. Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0229] Although not strictly impossible, the serendipitous identification of different antibodies having the same heavy chain variable domain but directed against different antigens is highly unlikely. Indeed, in most cases the heavy chain contributes largely to the antigen binding surface and is also the most variable in sequence. In particular the CDR3 on the heavy chain is the most diverse CDR in sequence, length and structure. Thus, two antibodies specific for different antigens will almost invariably carry different heavy chain variable domains.

[0230] The methods disclosed in application WO 2012 / 023053 overcomes this limitation and greatly facilitates the isolation of antibodies having the same heavy chain variable domain by the use of antibody libraries in which the heavy chain variable domain is the same for all the library members and thus the diversity is confined to the light chain variable domain. Such libraries are described, for example, in applications WO 2010 / 135558 and WO 2011 / 084255, each of which is hereby incorporated by reference in its entirety. However, as the light chain 130 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 variable domain is expressed in conjunction with the heavy variable domain, both domains can contribute to antigen binding. To further facilitate the process, antibody libraries containing the same heavy chain variable domain and either a diversity of Lambda variable light chains or Kappa variable light chains can be used in parallel for in vitro selection of antibodies against different antigens. This approach enables the identification of two antibodies having a common heavy chain but one carrying a Lambda light chain variable domain and the other a Kappa light chain variable domain that can be used as building blocks for the generation of a bispecific or multispecific antibody in the full immunoglobulin format of the disclosure. The bispecific and multispecific antibodies of the disclosure can be of different Isotypes and their Fc portion can be modified in order to alter the bind properties to different Fc receptors and in this way modify the effectors functions of the antibody as well as it pharmacokinetic properties. Numerous methods for the modification of the Fc portion have been described and are applicable to antibodies of the disclosure. (see for example Strohl, WR Curr Opin Biotechnol 2009 (6):685-91; U.S. Pat. No. 6,528,624; PCT / US2009 / 0191199 filed Jan 9, 2009). The methods of the disclosure can also be used to generate bispecific and multispecific antibodies and antibody mixtures in a F(ab’)2 format that lacks the Fc portion.

[0231] The common heavy chain and two different light chains are co-expressed into a single cell to allow for the assembly of a bispecific or multispecific antibody of the disclosure. If all the polypeptides get expressed at the same level and get assembled equally well to form an immunoglobulin molecule then the ratio of monospecific (same light chains) and bispecific (two different light chains) should be 50%. However, it is likely that different light chains are expressed at different levels and / or do not assemble with the same efficiency. Therefore, a means to modulate the relative expression of the different polypeptides is used to compensate for their intrinsic expression characteristics or different propensities to assemble with the common heavy chain. This modulation can be achieved via promoter strength, the use of internal ribosome entry sites (IRES) featuring different efficiencies or other types of regulatory elements that can act at transcriptional or translational levels as well as acting on mRNA stability. Different promoters of different strength could include CMV (Immediate- early Cytomegalovirus virus promoter); EF1-1α (Human elongation factor 1α-subunit promoter); Ubc (Human ubiquitin C promoter); SV40 (Simian virus 40 promoter). Different 131 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 IRES have also been described from mammalian and viral origin. (See e.g., Hellen CU and Sarnow P. Genes Dev 200115: 1593–612). These IRES can greatly differ in their length and ribosome recruiting efficiency. Furthermore, it is possible to further tune the activity by introducing multiple copies of an IRES (Stephen et al. 2000 Proc Natl Acad Sci USA 97: 1536-1541). The modulation of the expression can also be achieved by multiple sequential transfections of cells to increase the copy number of individual genes expressing one or the other light chain and thus modify their relative expressions. The Examples provided herein demonstrate that controlling the relative expression of the different chains is critical for maximizing the assembly and overall yield of the bispecific antibody.

[0232] The co-expression of the heavy chain and two light chains generates a mixture of three different antibodies into the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. The latter has to be purified from the mixture to obtain the molecule of interest. The method described herein greatly facilitates this purification procedure by the use of affinity chromatography media that specifically interact with the Kappa or Lambda light chain constant domains such as the CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland). This multi-step affinity chromatography purification approach is efficient and generally applicable to antibodies of the disclosure. This is in sharp contrast to specific purification methods that have to be developed and optimized for each bispecific antibodies derived from quadromas or other cell lines expressing antibody mixtures. Indeed, if the biochemical characteristics of the different antibodies in the mixtures are similar, their separation using standard chromatography technique such as ion exchange chromatography can be challenging or not possible at all.

[0233] Other suitable purification methods include those disclosed in application PCT / IB2012 / 003028, filed on October 19, 2012, published as WO2013 / 088259, the contents of which are hereby incorporated by reference in their entirety.

[0234] In other embodiments of producing bispecific or multispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. 132 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0235] According to another approach described in WO 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface includes at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0236] Techniques for generating bispecific or multispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0237] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab’ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VLand VHdomains of another fragment, thereby forming two antigen- 133 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 binding sites. Another strategy for making bispecific antibody fragments by the use of single- chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol.152:5368 (1994).

[0238] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol.147:60 (1991).

[0239] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the disclosure. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

[0240] Heteroconjugate antibodies are also within the scope of the present disclosure. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (see U.S. Patent No.4,676,980), and for treatment of HIV infection (see WO 91 / 00360; WO 92 / 200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

[0241] It can be desirable to modify the antibody of the disclosure with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer and / or other diseases and disorders associated with aberrant CD20 expression and / or activity. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and / or increased complement-mediated cell killing and 134 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

[0242] The disclosure also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0243] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include212Bi,131I,131In,90Y, and186Re.

[0244] Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See WO94 / 11026).

[0245] Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies of the disclosure. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. 135 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0246] Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. The preferred binding is, however, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present disclosure, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).

[0247] Preferred linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N- hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2- pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC- SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

[0248] The linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT 136 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.

[0249] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No.5,013,556.

[0250] Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present disclosure can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Methods of Use

[0251] Any of the bispecific or multispecific antibodies of the disclosure (e.g. anti-CD3 and anti-CD20 antibody; anti-CD3 and anti-CD20 antibody with CD58 fusion peptide) an may be used in therapeutic methods. In one aspect, a bispecific or multispecific antibody may be for use as a medicament is provided. In further aspects, a bispecific or multispecific antibody for use in treating or delaying progression of a cell proliferative disorder (e.g., cancer). In certain embodiments, a bispecific or multispecific antibody for use in a method of treatment is provided. In certain embodiments, the disclosure provides a bispecific or multispecific antibody for use in a method of treating an individual having a cell proliferative disorder comprising administering to the individual an effective amount of the bispecific or multispecific antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. In further embodiments, the disclosure provides a bispecific or multispecific antibody for use in enhancing immune function in an individual having a cell 137 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 proliferative disorder. In certain embodiments, the disclosure provides a bispecific or multispecific antibody for use in a method of enhancing immune function in an individual having a cell proliferative disorder comprising administering to the individual an effective of the bispecific or multispecific antibody to activate effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells), expand (increase) an effector cell population, reduce a target cell population, and / or kill a target cell (e.g., target tumor cell). An "individual" according to any of the above embodiments may be a human.

[0252] In some embodiments, Bispecific and multispecific antibodies of the disclosure can optionally further comprise a CD2 ligand, such as CD58. Bispecific and multispecific antibodies of the disclosure further comprising a CD58 ligand (e.g. a CD58 polypeptide) can lead to CD2-directed costimulatory T cell activation.

[0253] In a further aspect, the disclosure provides for the use of bispecific or multispecific antibodies of the disclosure (e.g. anti-CD3 and anti-CD20 antibody; anti-CD3 and anti-CD20 antibody with CD58 fusion peptide) in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of a cell proliferative disorder (e.g., cancer,). In a further embodiment, the medicament is for use in a method of treating a cell proliferative disorder or an autoimmune disorder comprising administering to an individual having a cell proliferative disorder or an autoimmune disorder an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. In a further embodiment, the medicament is for activating effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells), expanding (increasing) an effector cell population, reducing a target cell (e.g., a cell expressing CD20) population, and / or killing target cells (e.g., target tumor cells) in the individual. In a further embodiment, the medicament is for use in a method of enhancing immune function in an individual having a cell proliferative disorder or an autoimmune disorder comprising administering to the individual an amount effective of the medicament to activate effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells), expand (increase) an effector cell population, reduce a target cell (e.g., a cell expressing CD20) population, and / or kill a target cell (e.g., target tumor cell). An "individual" according to any of the above embodiments may be a human. 138 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0254] In a further aspect, the disclosure provides a method for treating a cell proliferative disorder (e.g., cancer). In one embodiment, the method comprises administering to an individual having such a cell proliferative disorder an effective amount of a bispecific or multispecific antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. An "individual" according to any of the above embodiments may be a human. In a further aspect, the disclosure provides a method for enhancing immune function in an individual having a cell proliferative disorder or an autoimmune disorder in an individual having a cell proliferative disorder or an autoimmune disorder. In one embodiment, the method comprises administering to the individual an effective amount of a bispecific or multispecific antibody to activate effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells), expand (increase) an effector cell population, reduce a target cell (e.g., a cell expressing CD20) population, and / or kill a target cell (e.g., target tumor cell).

[0255] In some embodiments, the disclosure provides a method for treating a cancer that expresses CD20. In some embodiments, the cancer that expressed CD20 is a hematological cancer, such as a B cell cancer (for example, mature B-cell lymphoma). In some embodiments, the method of treating a cancer comprises administering an effective amount of the bispecific antibody of the disclosure (e.g. anti-CD3ε and anti-CD20 antibody; anti- CD3ε and anti-CD20 antibody with CD58 fusion peptide). In a further aspect of the embodiment, the mature B-cell lymphoma is a Non-Hodgkin's Lymphoma (NHL). In a further aspect of the embodiment, the NHL is selected from the group comprising: germinal-center B-cell-like (GCB) DLBCL, activated B-cell like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (W), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, Splenic marginal zone lymphoma, Hairy cell leukemia, Splenic lymphoma / leukemia, unclassifiable, Splenic diffuse red pulp small B-cell lymphoma, Hairy cell leukemia variant, Waldenstrom macroglobulinemia, Heavy chain diseases, a Heavy chain disease, γ Heavy chain disease, μ Heavy chain disease, Plasma cell myeloma, Solitary plasmacytoma of bone, [Extraosseous plasmacytoma, Extranodal marginal zone lymphoma 139 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 of mucosa-associated lymphoid tissue (MALT lymphoma), Nodal marginal zone lymphoma, Pediatric nodal marginal zone lymphoma, Pediatric follicular lymphoma, Primary cutaneous follicle centre lymphoma, T-cell / histiocyte rich large B-cell lymphoma, Primary DLBCL of the CNS, Primary cutaneous DLBCL, leg type, EBV-positive DLBCL of the elderly, DLBCL associated with chronic inflammation, Lymphomatoid granulomatosis, Primary mediastinal (thymic) large B-cell lymphoma, Intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, Plasmablastic lymphoma, Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, Primary effusion lymphoma: B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma. In some embodiments of the disclosure, the method comprises treating a cancer comprising germinal-center B-cell like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (WM), central nervous system lymphoma (CNSL), or Burkitt's lymphoma (BL).

[0256] In some embodiments, the disclosure provides a method for treating an inflammatory disease or an immunological disease. Exemplary inflammatory or immunological diseases include but are not limited to neurological diseases, pulmonological diseases, rheumatological diseases, hematological diseases, dermatological diseases, nephrological diseases and endocrinological diseases. Exemplary neurological disease include but are not limited to myasthenia gravis, relapsing-remitting multiple sclerosis (RMS), primary progressive multiple sclerosis (PPMS) and neuromyelitis optica. Exemplary pulmonological diseases include but are not limited to progressive pulmonary fibrosis including either usual interstitial pneumonia (UIP) and non-specific interstitial pneumonia (NSIP), and idiopathic pulmonary fibrosis. Exemplary rheumatological diseases include but are not limited to ANCA-associated vasculitis, Behcet’s syndrome, Castelman’s disease, cryoglobulinemia, granulomatosis with polyangiitis (GPA), IgG4-mediated diseases, juvenile idiopathic arthritis, microscopic polyangiitis (MPA), myositis, rheumatoid 140 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 arthritis, SLE, Sjogren’s syndrome, and Systemic sclerosis / scleroderma. Exemplary hematological diseases include but are not limited to antiphospholipid syndrome, autoimmune hemolytic anemia, immune thrombocytopenic purpura. Exemplary dermatological diseases include but are not limited to acquired angioedema with C1 deficiency, bullous pemphigoid and pemphigus vulgaris. Exemplary nephrological diseases include but are not limited to IgA nephropathy, membranous nephropathy and nephrotic syndrome. Exemplary endocrinological diseases include but are not limited to autoimmune hepatitis, Graves’ disease and Type 1 Diabetes.

[0257] In some embodiments, the method of treating a cancer comprises administering an effective amount of the bispecific or multispecific antibody of the disclosure (e.g. anti-CD3 and anti-CD20 antibody; anti-CD3 and anti-CD20 antibody with CD58 fusion peptide).

[0258] In some embodiments, the cancer expresses CD20 at a high level. In some embodiments CD20-expressing cancer include white blood cell cancers, such as B-cell lymphoma or leukemia.

[0259] In some embodiments, a method of identifying a subject to receive a CD20 therapy is provided. The method comprises testing a cancer sample for a high level of expression of CD20. In some embodiments, the cancer is B-cell leukemia or lymphoma.

[0260] The method further comprises comparing a level of CD20 expression in the cancer sample to a level of CD20 expression in healthy / control / reference tissue to determine if the level of CD20 expression in the cancer sample is higher. If the level of CD20 expression is higher in the cancer sample, the subject receives a CD20 antagonist. In some embodiments, the CD20 antagonist is one or more of the antibodies provided herein. In some embodiments, any method for measuring the level of CD20 can be employed. In some embodiments, this is any amount greater than a negative amount in a staining assay. In some embodiments, this is any amount above a level present in surrounding healthy / control / reference tissue or corresponding tissue from a healthy / control / reference subject. In some embodiments, high levels of expression of CD20 is defined in comparison to an expression level of CD20 in a non-cancer sample.

[0261] In some embodiments, the expression level of CD20 in the cancer sample is at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% higher than the expression level of CD20 in the non-cancer sample. In some embodiments, the expression level of CD20 141 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 in the cancer sample is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% higher than the expression level of CD20 in the non-cancer sample. In some embodiments, the expression level of CD20 in the cancer sample is at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475% or 500% higher than the expression level of CD20 in the non-cancer sample. In some embodiments, the expression level of CD20 in the cancer sample is at least 2 fold, 3, fold, 4 fold, 5, fold, 6 fold, 7, fold 8, fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold or 20 fold higher than the expression level of CD20 in the non-cancer sample.

[0262] In some embodiments, the healthy / control / reference sample is a sample from a normal tissue. In some embodiments, the normal tissue is a tissue that is an adjacent tissue to the cancer in the subject. For example, when assaying for expression via staining, the levels of the protein are above those understood to be “negative” to one of skill in the art for the particular assay. Exemplary assays include but are not limited to RNA based assays, FACS, and IHC, each with their appropriate levels of positive and negative results. Any method of detecting the level of a protein in a sample is contemplated. One skilled in the art can select a suitable method depending on the type of sample being analyzed and the identity and number of proteins being detected. Nonlimiting exemplary such methods include immunohistochemistry, ELISA, Western blotting, multiplex analyte detection (using, for example, Luminex technology), mass spectrometry, etc. Similarly, any method of detecting the level of an mRNA in a sample is contemplated. One skilled in the art can select a suitable method depending on the type of sample being analyzed and the identity and number of mRNAs being detected. Nonlimiting exemplary such methods include RT-PCR, quantitative RT-PCR and microarray-based methods, etc.

[0263] A CD20-positive cancer according to any of the above embodiments may be, e.g., CD20-positive multiple myeloma. In some embodiments, a CD20-positive cancer is a cancer that receives an anti-CD20 immunohistochemistry (IHC) or in situ hybridization (ISH) score greater than “0,” which corresponds to very weak or no staining in >90% of tumor cells. In another embodiment, a CD20-positive cancer expresses CD20 at a 1+, 2+ or 3+ level. In some embodiments, a CD20-positive cancer is a cancer that expresses CD20 according to a reverse- 142 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 transcriptase PCR (RT-PCR) assay that detects CD20 mRNA. In some embodiments, the RT- PCR is quantitative RT-PCR.

[0264] In a further aspect, the disclosure provides pharmaceutical formulations comprising any of the bispecific or multispecific antibodies provided herein, e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the bispecific or multispecific antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the bispecific or multispecific antibodies provided herein and at least one additional therapeutic agent, for example, as described herein.

[0265] Antibodies of the disclosure can be used either alone or in combination with other agents in a therapy. For instance, an antibody of the disclosure may be co-administered with at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is a chemotherapeutic agent, growth inhibitory agent, cytotoxic agent, agent used in radiation therapy, anti-angiogenesis agent, apoptotic agent, anti-tubulin agent, or other agent, such as a epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1 / EGFR inhibitor (e.g., erlotinib (Tarceva™), platelet derived growth factor inhibitor (e.g., Gleevec™ (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferon, cytokine, antibody other than the anti-CD3 antibody of the disclosure, such as an antibody that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR- beta, BlyS, APRIL, BCMA VEGF, or VEGF receptor(s), TRAIL / Apo2, PD-1 (e.g. Nivolumab, Pembrolizumab, Cemiplimab), PD-L1 (Atezolizumab, Avelumab, Durvalumab), PD-L2, or another bioactive or organic chemical agent. In some embodiments, the disclosure provides a method wherein the additional therapeutic agent is a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone.

[0266] In some aspects, the additional therapeutic agent is a checkpoint inhibitor. The term “inhibition” or “inhibitor” includes a reduction in a certain parameter, e.g., an activity, of a given molecule, e.g., an immune checkpoint inhibitor. For example, inhibition of an activity, e.g., an activity of, e.g., PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta, of at least 5%, 10%, 20%, 30%, 40% or more is included by this term. The level of inhibition need not be 100%. 143 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0267] In some aspects, the checkpoint inhibitor is a PD-1 inhibitor. In some aspects, the PD-1 inhibitor is an anti-PD1 antibody. In some aspects, the PD-1 inhibitor is an anti PD-1 monoclonal antibody. Exemplary anti-PD-1 monoclonal antibodies include, but are not limited to cemiplimab (Libtayo), nivolumab (Opdivo), pembrolizumab (Keytruda). In some aspects, the checkpoint inhibitor is a PD-L1 inhibitor. Exemplary PD-L1 inhibitors include but are not limited to avelumab (Bavencio), durvalumab (Imfinzi) and atezolizumab (Tecentriq).

[0268] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the disclosure can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent or agents. In one embodiment, administration of the bispecific or multispecific antibody and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other. Bispecific or multispecific antibodies of the disclosure can also be used in combination with radiation therapy.

[0269] In some aspects, the additional therapeutic agent is a chimeric antigen receptor (CAR) T cell therapy. In some embodiments, the CAR-T cell therapy specifically binds CD19. Exemplary CAR-T cell therapies that specifically bind CD19 include but are not limited to BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), YESCARTA™ (axicabtagene ciloleucel), ABECMA® (idecabtagene vicleucel), or CARVYKTI™ (ciltacabtagene autoleucel).

[0270] An antibody of the disclosure (and / or any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibody is administered by subcutaneous administration. In some embodiments, an anti-CD3 antibody administered by subcutaneous injection exhibits a less toxic response in a patient than the same anti-CD3 antibody administered by intravenous injection. Dosing can be by any suitable route, for example, by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or 144 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0271] Antibodies of the disclosure would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0272] For the prevention or treatment of disease, the appropriate dosage of an antibody of the disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.In some embodiments, the methods may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery, chemotherapy, gene therapy, DMA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and / or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some 145 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy may be a separate administration of one or more of the therapeutic agents described above.

[0273] It will be appreciated that administration of therapeutic entities in accordance with the disclosure will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol.32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

[0274] Therapeutic formulations of the disclosure, which include an antibody of the disclosure, are used to treat or alleviate a symptom associated with a cancer, such as, by way of non-limiting example, white blood cell cancers including multiple myelomas. The present disclosure also provides methods of treating or alleviating a symptom associated with a cancer. A therapeutic regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a cancer, using standard methods. 146 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0275] Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the antibody confers a clinical benefit.

[0276] Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.

[0277] Antibodies directed against a target such as CD3 or CD20, or a combination thereof (or a fragment thereof), may be used in methods known within the art relating to the localization and / or quantitation of these targets, e.g., for use in measuring levels of these targets within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies specific to any of these targets, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharmacologically active compounds (referred to hereinafter as “Therapeutics”).

[0278] An antibody of the disclosure can be used to isolate a particular target using standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. Antibodies of the disclosure (or a fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include125I,131I,35S or3H. 147 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0279] Antibodies of the disclosure, including polyclonal, monoclonal, humanized and fully human antibodies, may be used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology associated with aberrant expression or activation of a given target in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Administration of the antibody may abrogate or inhibit or interfere with the signaling function of the target. Administration of the antibody may abrogate or inhibit or interfere with the binding of the target with an endogenous ligand to which it naturally binds.

[0280] A therapeutically effective amount of an antibody of the disclosure relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the disclosure may be, by way of nonlimiting example, from about 0.001 mg / kg body weight to about 50 mg / kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.

[0281] Antibodies or a fragment thereof of the disclosure can be administered for the treatment of a variety of diseases and disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol.4), 1991, M. Dekker, New York.

[0282] Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized 148 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 chemically and / or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0283] The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

[0284] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0285] Sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0286] An antibody according to the disclosure can be used as an agent for detecting the presence of a given target (or a protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. Antibodies are polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) is used. The 149 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the disclosure can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Pharmaceutical Compositions

[0287] The antibodies of the disclosure (also referred to herein as “active compounds”), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, 150 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0288] A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0289] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene 151 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0290] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0291] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. 152 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0292] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0293] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0294] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0295] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No.4,522,811.

[0296] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to 153 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0297] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0298] The disclosure will be further described in the following examples, which do not limit the scope of the disclosure described in the claims. Definitions

[0299] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well- known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0300] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0301] The term “CD20-positive cancer” refers to a cancer comprising cells that express CD20. In some embodiments, expression of CD20 may be on the cell surface. In some 154 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 embodiments, expression of CD20 on the cell surface is determined, for example, using antibodies to CD20 in a method such as immunohistochemistry, FACS, etc. Alternatively, CD20 mRNA expression is considered to correlate to CD20 expression on the cell surface and can be determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR).

[0302] The term “CD20-positive cell” refers to a cell that expresses CD20.

[0303] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non- Hodgkin's lymphoma), myeloma ( including multiple myeloma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including triple negative (ER− / PR− / Her2−) breast cancer), colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0304] The phrase “reference sample”, “reference cell”, or “reference tissue”, denote a sample with at least one known characteristic that can be used as a comparison to a sample with at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. A reference sample can be used to establish a level of protein and / or mRNA that is present in, for example, healthy tissue, in contrast to a level of protein and / or mRNA present in the sample with unknown characteristics. In some embodiments, the reference sample comes from the same subject, but is from a different part of the subject than that being tested. In some embodiments, the reference sample is from a tissue area surrounding or adjacent to the cancer. In some embodiments, the reference sample is not from the subject being tested, but is a sample from a subject known to have, or not to have, a disorder in question (for example, a particular cancer or CD20 related disorder). In some embodiments, the reference sample is from the same subject, but from a point in time before the subject developed cancer. In some embodiments, the reference sample is from a 155 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 benign cancer sample (for example, benign breast cancer sample), from the same or a different subject. When a negative reference sample is used for comparison, the level of expression or amount of the molecule in question in the negative reference sample will indicate a level at which one of skill in the art will appreciate, given the present disclosure, that there is no and / or a low level of the molecule. When a positive reference sample is used for comparison, the level of expression or amount of the molecule in question in the positive reference sample will indicate a level at which one of skill in the art will appreciate, given the present disclosure, that there is a level of the molecule.

[0305] A “disease” or “disorder” as used herein refers to a condition where treatment is needed and / or desired.

[0306] The term “tumor cell”, “cancer cell”, “cancer”, “tumor”, and / or “neoplasm”, unless otherwise designated, are used herein interchangeably and refer to a cell (or cells) exhibiting an uncontrolled growth and / or abnormal increased cell survival and / or inhibition of apoptosis which interferes with the normal functioning of bodily organs and systems. Included in this definition are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. The terms “cancer” and “tumor” encompass solid and hematological / lymphatic cancers and also encompass malignant, pre-malignant, and benign growth, such as dysplasia. Also, included in this definition are cells having abnormal proliferation that is not impeded (e g immune evasion and immune escape mechanisms) by the immune system (e.g. virus infected cells). Exemplary tumor cells include, but are not limited to: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; 156 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B- cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non- cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0307] In some embodiments, the term cancer can encompass: a white blood cell cancer such as for example, B-cell lymphoma or leukemia. When desired, the difference between a “cancer” and a “cancer cell” can be denoted by the use of the explicit use of the phrase “cancer cell”; however, the term “cancer” will encompass concepts such as the subject having cancer, and multicellular tumors, as well as single cancer cells.

[0308] An “increase or decrease” refers to a statistically significant increase or decrease respectively. As will be clear to the skilled person, “modulating” can also involve effecting a change (which can either be an increase or a decrease) in affinity, avidity, specificity and / or selectivity of a target or antigen, for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; effecting a change (which can either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of an antibody, bispecific or multispecific polypeptide agent. This can be determined in any suitable manner and / or using any suitable assay known per se or described herein, depending on the target involved.

[0309] As used herein, “an immune response” is meant to encompass cellular and / or humoral immune responses that are sufficient to inhibit or prevent onset or ameliorate the symptoms of disease (for example, cancer or cancer metastasis). “An immune response” can encompass aspects of both the innate and adaptive immune systems. 157 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0310] As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. “Treatment” as used herein, covers any administration or application of a therapeutic for disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (for example, metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total). Also encompassed by “treatment” is a reduction of pathological consequence of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. In-line with the above, the term treatment does not require one-hundred percent removal of all aspects of the disorder.

[0311] “Ameliorating” means a lessening or improvement of one or more symptoms as compared to not administering a CD20 antibody. “Ameliorating” also includes shortening or reduction in duration of a symptom.

[0312] The term “biological sample” means a quantity of a substance from a living thing or formerly living thing. Such substances include, but are not limited to, blood, (for example, whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen.

[0313] The term “control” refers to a composition known to not contain an analyte (“negative control”) or to contain analyte (“positive control”). A positive control can comprise a known concentration of analyte. “Control,” “positive control,” and “calibrator” may be used interchangeably herein to refer to a composition comprising a known concentration of analyte. A “positive control” can be used to establish assay performance characteristics and is a useful indicator of the integrity of reagents (for example, analytes).

[0314] “Predetermined cutoff” and “predetermined level” refer generally to an assay cutoff value that is used to assess diagnostic / prognostic / therapeutic efficacy results by comparing the assay results against the predetermined cutoff / level, where the predetermined cutoff / level already has been linked or associated with various clinical parameters (for example, severity of disease, progression / nonprogression / improvement, etc.). While the present disclosure may 158 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 provide exemplary predetermined levels, it is well-known that cutoff values may vary depending on the nature of the immunoassay (for example, antibodies employed, etc.). It further is well within the skill of one of ordinary skill in the art to adapt the disclosure herein for other immunoassays to obtain immunoassay-specific cutoff values for those other immunoassays based on this disclosure. Whereas the precise value of the predetermined cutoff / level may vary between assays, correlations as described herein (if any) may be generally applicable.

[0315] The terms “inhibition” or “inhibit” refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and / or amount as compared to a reference. In some embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater. In some embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In some embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater. In some embodiments, the amount noted above is inhibited or decreased over a period of time, relative to a control dose (such as a placebo) over the same period of time.

[0316] As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and / or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

[0317] “Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. Unless otherwise specified, the terms “reduce”, “inhibit”, or “prevent” do not denote or require complete prevention over all time.

[0318] As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody which 159 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.

[0319] The term "antibody binding region" refers to a region of the antigen, which comprises the epitope to which the antibody binds. An antibody binding region may be determined by epitope binning using biolayer interferometry, by alanine scan, or by domain shuffle assays (using antigen constructs in which regions of the antigen are exchanged with that of another species and determining whether the antibody still binds to the antigen or not). The amino acids within the antibody binding region that are involved in the interaction with the antibody may be determined by hydrogen / deuterium exchange mass spectrometry and / or by crystallography of the antibody bound to its antigen.

[0320] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By “specifically bind” or “immunoreacts with” or “immunospecifically bind” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd > 10-6). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab’and F(ab')2fragments, scFvs, and an Fabexpression library.

[0321] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG1, IgG2, IgG4 and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[0322] The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique 160 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

[0323] The term “antigen binding region” or “antigen-binding site” or “binding portion” refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions,” are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”. Thus, the term “FR” refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” Various methods are known in the art for numbering the amino acids sequences of antibodies and identification of the complementary determining regions. For example, the Kabat numbering system (See Kabat, E.A., et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)) or the IMGT numbering system (See IMGT®, the international ImMunoGeneTics information system®. Available online: http: / / www.imgt.org / ). The IMGT numbering system is routinely used and accepted as a reliable and accurate system in the art to determine amino acid positions in coding sequences, alignment of alleles, and to easily compare sequences in immunoglobulin (IG) and T cell receptor (TR) from all vertebrate species. The accuracy and the consistency of the IMGT data are based on IMGT-ONTOLOGY, the first, and so far unique, ontology for immunogenetics and immunoinformatics (See Lefranc. M.P. et al., Biomolecules, 2014 Dec; 4(4), 1102-1139). IMGT tools and databases run against IMGT reference directories built from a large repository of sequences. In the IMGT system the IG V-DOMAIN and IG C-DOMAIN are delimited taking into account the exon delimitation, 161 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 whenever appropriate. Therefore, the availability of more sequences to the IMGT database, the IMGT exon numbering system can be and “is used” by those skilled in the art reliably to determine amino acid positions in coding sequences and for alignment of alleles. Additionally, correspondences between the IMGT unique numbering with other numberings (i.e., Kabat) are available in the IMGT Scientific chart (See Lefranc. M.P. et al., Biomolecules, 2014 Dec; 4(4), 1102-1139).

[0324] The term "hypervariable region" or “variable region” refers to the amino acid residues of an antibody that are typically responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., around about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the VHwhen numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and / or those residues from a "hypervariable loop" (e.g., residues 24- 34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (HI), 52-56 (H2) and 95-101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol.196:901-917 (1987)); and / or those residues from a "hypervariable loop" VCDR (e.g., residues 27-38 (LI), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (HI), 56-65 (H2) and 105-120 (H3) in the VH when numbered in accordance with the IMGT numbering system; Lefranc, M.P. et al. Nucl. Acids Res.27:209-212 (1999), Ruiz, M. e al. Nucl. Acids Res.28:219-221 (2000)). Optionally, the antibody has symmetrical insertions at one or more of the following points 28, 36 (LI), 63, 74-75 (L2) and 123 (L3) in the VL, and 28, 36 (HI), 63, 74-75 (H2) and 123 (H3) in the VH when numbered in accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol.309:657-670 (2001)).

[0325] As used herein, the term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T cell receptor. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies may be raised against N-terminal or C-terminal peptides of a polypeptide. An 162 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 antibody is the to specifically bind an antigen when the dissociation constant is ≤ 1 µM; e.g., ≤ 100 nM, preferably ≤ 10 nM and more preferably ≤ 1 nM.

[0326] As used herein, the terms “immunological binding,” and “immunological binding properties” refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of Koff / Konenables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present disclosure is the to specifically bind to its target, when the equilibrium binding constant (Kd) is ≤1 µM, e.g., ≤ 100 nM, preferably ≤ 10 nM, and more preferably ≤ 1 nM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0327] The term “isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides in accordance with the disclosure include the nucleic acid molecules encoding the heavy chain immunoglobulin molecules, and nucleic acid molecules encoding the light chain immunoglobulin molecules described herein.

[0328] The term “isolated protein” referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the “isolated protein” (1) is not associated with proteins found 163 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 in nature, (2) is free of other proteins from the same source, e.g., free of marine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.

[0329] The term “polypeptide” is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus. Polypeptides in accordance with the disclosure comprise the heavy chain immunoglobulin molecules, and the light chain immunoglobulin molecules described herein, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.

[0330] The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.

[0331] The term “operably linked” as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0332] The term “control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. The term “polynucleotide” as referred to herein means a polymeric boron of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA. 164 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0333] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers (e.g., D- amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present disclosure. Examples of unconventional amino acids include: 4 hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε -N- acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0334] As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.

[0335] Preferably, residue positions which are not identical differ by conservative amino acid substitutions.

[0336] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic- hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic- aspartic, and asparagine-glutamine.

[0337] As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present disclosure, 165 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that 166 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the disclosure.

[0338] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally- occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991).

[0339] As used herein, the terms “label” or “labeled” refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,3H,14C,15N,35S,90Y,99Tc,111In,125I,131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p- galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term “pharmaceutical agent or drug” as used herein refers to a 167 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0340] Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw- Hill, San Francisco (1985)).

[0341] As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.

[0342] Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0343] The term patient includes human and veterinary subjects. EXAMPLES Example 1: Development of CD3 and CD20 bispecific and multispecific antibodies Methods Antibody Expression and Purification

[0344] DNA sequences corresponding to antibody heavy and light chains were synthesized in pDT5 vector (ATUM, Newark CA). Plasmid DNA (1 µg / ml) were transfected into Expi293 cells (ThermoFisher) according to manufacturer’s protocols. Cells were grown in flasks with rotation (125 rpm) at 37 °C with 8 % CO2. Four to five days post-transfection, the conditioned media was harvested from the cells by centrifugation (3000 xg) for 30 minutes and filtered using 0.45 um filter. Antibodies were then purified using an AKTA Avant chromatography system (Cytiva) and a tandem purification method using HiTrap Mabselect Protein A and 168 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 HiLoad Superdex 200 pg columns (Cytiva). Antibodies were stored in PBS, pH 7.4 at 4 °C following purification and prior to analysis. Mass Spectrometry

[0345] The intact mass of the purified fusion proteins and antibody chains was confirmed by Xevo G2-XS QTof Quadrupole Time-of-Flight Mass Spectrometry coupled to a Acquity UHPLC system (Waters) equipped with a Protein BEH C4 (300 Å 1.7 µm) column. Antibody samples were deglycosylated first using rapid PNGase F enzyme (New England Biolabs) in reducing and non-reducing conditions following supplier’s protocols. Reaction mixture was diluted to 1:10 in 50% Acetonitrile containing 0.1% Formic acid and 2 μL of which was injected into LC-MS. The total ion chromatogram and m / z data of the proteins were acquired with gradient run of 10 to 70 mL HPLC grade acetonitrile over 12 min. Mass of the protein samples was deconvoluted from total ion chromatogram using BYOS software from Protein Metrics. Differential scanning calorimetry (DSC)

[0346] The thermal stability of the antibodies with respect to Tonset and Tms were determined by differential scanning calorimetry (DSC) using nanoDSC from TA Instrument. Samples were prepared in 1xPBS at a concentration of 1 mg / mL and each sample was run in duplicate and scanned at 1 ⁰C / min from 25 to 95 ⁰C. A buffer background was subtracted from each individual scan leaving the partial molar heat capacity, which was normalized using the moles of the protein in the active cell volume. Data was analyzed from each individual scan using NanoAnalyzer program provided by the supplier. Thermal forced aggregation assay

[0347] The fusion protein and antibody samples in 2 mg / mL concentration were subjected to thermal denaturation for 2 h at varying degrees of temperatures, from 35 °C to 90 °C at 5 °C interval. Following cooling down the samples to 4 °C, the denatured aggregated protein fractions were removed from the samples by centrifugation at 10,000 xg for 5 min. The cleared samples were analyzed by analytical size exclusion chromatography (aSEC) to 169 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 determine the remaining intact protein fractions from integrated peak area of the chromatograms. Biacore 8K kinetic binding analysis

[0348] Analysis was performed on Biacore 8K+ system (Cytiva). Biotinylated human CD3-epsilon and CD3-delta heterodimer (Acro Biosystems) was captured on the streptavidin sensor chip (Cytiva) with the RU values ranging from 50-70. Protein samples at different concentrations diluted in the running buffer (10 mM HEPES, 150 mM NaCl, 0.05% v / v Surfactant P20, pH 7.4) were then injected to each channel at a flow rate of 30 μL / min for the multi-cycle kinetics / affinity analysis. The binding kinetics were carried out at 25 °C and the association contact and dissociation times used were 60-120 seconds and 120-420 seconds. The chip surface was regenerated by injecting 10 mM glycine, pH 1.5, at a flow rate of 30 μL / min for 60 sec. The kinetic and affinity constants are determined by fitting the data to a 1:1 binding model using the Biacore Insight Evaluation Software (Cytiva). CD20 target binding by Enzyme Linked Immunosorbent Assay (ELISA)

[0349] Binding of target antigen is performed by standard ELISA using CD20-VLP. Expi293 cells were transfected with human CD20-ORF clone (NM_021950) from Origene, and Gag, Pol and REV vectors and the VLP was purified using MembranePro Expression kit (Thermofisher). CD20-VLP was immobilized on Nunc Maxisorp 96-well plate in 200 ng of total protein concentration per well in bicarbonate buffer overnight at 4 °C. Plates were treated with a serial dilution of biologics with appropriate controls in the binding buffer, and following washing treated with a HRP conjugated secondary anti-human IgG (G18-145). Luminescence substrate from LumiGlo Peroxidase Chemiluminescent Substrate Kit (SeraCare) following the manufacturers’ protocols was added to the plate and the luminescence signal was measured using Ensight plate reader (Perkin Elmer). The data was analyzed using four-parameter logistic (4-PL) non-linear regression analysis for data fitting and to obtain EC50 values using Prism software (GraphPad). Hematological cancer cell lines 170 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0350] Hematological cancer cell lines were ordered from ATCC: Toledo (Diffuse Large B Cell Lymphoma, DLBLC), Raji (Burkitt’s lymphoma), JeKo-1 (mantle cell lymphoma), WSU-DLCL2 (DLBCL), HT (large B cell lymphoma), and NALM-6 (Acute lymphoblastic leukemia, ALL). JeKo-1, Raji, WSU-DLCL2, and NALM-6 cells were engineered to overexpress GFP in-house. Jurkat-NFAT-Luciferase Reporter Assay for T cell activation

[0351] EVOLVE bispecific and control bispecifics were evaluated for NFAT activation using the NFAT luciferase reporter Jurkat cell line and CD20 expressing Raji cell line in coculture assay. Engagement of T cell antigen receptor (TCR) / CD3 complex in T cells leads to intracellular signaling events and the transcriptional activation of the Nuclear Factor of Activated T cells (NFAT) pathway. Human tumor cells were seeded at 40,000 cells per well in 96- well tissue culture plates (Perkin Elmer) and mixed with 100,000 NFAT Luciferase Reporter Jurkat cells (Signosis) in 2.5:1 E:T ratio and the cells were treated with serial dilution of biologics and incubated for 4-6 hours at 37 °C with 5 % CO2. Upon addition of Bio-Glo reagent (Promega), luminescence was measured using luminescent plate reader (Ensight, Perkin Elmer). PBMC Mediated Cytotoxicity Assays

[0352] The efficacy of the bispecific antibodies to mediate tumor cell lysis was evaluated in PBMC-tumor cell co-culture assays. PBMC from healthy human donors were added at 120,000 per well in 96-well cell-repellent plates (Greiner Bio-One), and CellTrace Violet (Invitrogen) - labeled or GFP-expressing human tumor cells were seeded at 8,000 cells per well, in the presence or absence of serial dilution of antibodies and incubated at 37oC. Research grade Mosunetuzumab, Glofitamab, and Epcoritamab were purchased from Ichorbio or Medchem Express or generated in-house. Research grade Odronextamab was purchased from Ichorbio. After 3 days of incubation, the plate was centrifugated and the supernatant was collected and frozen for further cytokine analysis. The cell pellets were stained with Zombie NIR™ Fixable Viability Kit (BioLegend), followed by Fc receptor blocking and surface marker staining. Flow antibodies against surface cell markers (CD3, CD4, CD8, CD19) were purchased from BioLegend and BD Bioscience, and the stained cells 171 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 were analyzed by flow cytometry. Numbers of tumor cells, CD4 T cells, CD8 T cells, and B cells were analyzed in FlowJo software. Cytolysis of tumor cells, depletion of B cells, CD4 T cells and CD8 T cells proliferation curves were generated by either plotting the cell counts or normalized cell counts versus bispecific antibodies concentration using Prism software (GraphPad). Four-parameter non-linear regression analysis was used to obtain EC50s using Prism software (GraphPad). Quantification of cytokine release by Enzyme Linked Immunosorbent Assay (ELISA)

[0353] Cytokine (IFNγ, IL-2, IL-6, and TNFa) were measured using LEGEND MAX ELISA kit (BioLegend) and LumiGlo Peroxidase Chemiluminescent Substrate Kit (SeraCare) following the manufacturers’ protocols. Luminescence was measured using Ensight plate reader (Perkin Elmer). The cytokine values were interpolated using Excel. Four-parameter non-linear regression analysis was used to obtain EC50s and maximal cytokine release using Prism software (GraphPad). Tumor regression study with human PBMC adoptive transfer model in NSG mice

[0354] The efficacy of 1:1 and 2:1 CD20 targeted fusion multispecifics was evaluated in a xenograft study using human PBMC adoptive transfer and diffuse large B cell lymphoma cell line, WSU-DLCL2. Tumor growth inhibition of representative CD20 targeting EVOLVE multispecifics was examined using WSU-DLBCL2 cells, with adoptive transfer of human PBMC in a xenograft NSG mouse model. At day 0, 10 x 106WSU-DLBCL2 cells were implanted subcutaneously into left flank of NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ, Jackson Laboratory). Tumor growth was monitored using electronic calipers and volume were calculated according to the formula: π / 6 × (length × width2). On day 15, when tumors reached an average size of 300 mm3, mice were randomized into different groups with similar mean tumor volumes and subjected to intravenous injection of 15 x 106human PBMCs. Two days after PBMCs engraftment, mice were treated with 1:1, 2:1 CD20 targeted biologics or vehicle at 0.25 mg / kg molar equivalent intravenous doses and continue with once in a week dosing till day 45. Tumor volume of the mice was monitored twice a week by electronic caliper measurement.172 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00

[0355] The schematics of 2:1 CD20 targeted bispecifics and bispecific fusions described here are represented in FIGs.1-2 and are composed of the following: (1) the CD20 targeting antibody Ofatumumab, (2) CD3 binding antibody consisting of previously described SP34 antibody variants (Table 1), (3) knob-in-hole mutations in the heavy chain as previously described (International Publication No. WO 2023 / 178,357), (4) mutations corresponding to set D the enforce light chain heterodimerization (Table 2) as described previously (International Publication No. WO 2023 / 178,357), protein linker, and full length CD58. Table 20. Nomenclature and mutations associated with CD3 variantsTable 21. Light chain mutation set D

[0356] CD20 targeted bispecifics and bispecific fusions were produced in transient Expi293 system or transient CHO system. The purity, size and yield of the representative fusion proteins were determined by SDS-PAGE gel and analytical size exclusion chromatography, as shown in FIGs.14A and 14B, respectively.

[0357] The differential binding to CD3 epsilon / delta heterodimer among different 2:1 bispecifics and multispecific costimulatory fusions were confirmed using surface plasmon resonance (SPR) for which the kinetic constants are summarized in Table 22. The binding of 173 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 target antigen was confirmed by ELISA using CD20-Virus-like Particles and the EC50values are summarized in Table 22. Table 22: Association, dissociation, and equilibrium constants for CD3 binding of CD20-targeted 2:1 bispecific fusions

[0358] The cytolytic activity of CD20-targeted EVOLVE molecules were evaluated in a co-culture assay using CD20-low expressing HT cell line and human peripheral blood mononuclear cells (PBMCs). All EVOLVE molecules engineered with either one (Evolve-1) or two CD20-binding arms (Evolve-26, -21, and -29), regardless of the CD3-binding arm affinity, dose-dependently killed HT cells (FIG. 3). While the potency of Evolve-1, a 1:1 EVOLVE molecule, was weaker than that of Glofitamab, all three 2:1 EVOLVE molecules demonstrated stronger potency than Glofitamab. The efficacy of 1:1 and 2:1 CD20 targeted fusion multispecifics was evaluated in xenograft study using human PBMC adoptive transfer and diffuse large B cell lymphoma cell line, WSU-DLCL2. FIG.4A shows the mean tumor volume of different treated groups and vehicle control.2:1 CD20-targeted fusion multispecifics and 1:1 CD20-targeted fusion multispecifics show superior tumor growth inhibition over the clinical 2:1 bispecific comparator Glofitamab. The dot plot in FIG.13 shows the mean tumor growth of different 2:1 CD20-targeted fusion multispecifics treated groups and 2:1 bispecific comparator BsAb-1. One of the 2:1 CD20-targeted fusion multispecifics, Evolve-36, showed superior tumor growth inhibition over clinical 2:1 bispecific comparator. Blood samples were collected from mice between 2 and 168 hours and plasma samples were analyzed to 174 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 quantify biologic exposure . The concentration of biologics was determined by an MSD assay. FIG.4B shows the plasma concentration of biologics over time. The rapid clearance of Evolve-29 from circulation is in line with the poorer tumor growth inhibition observed with this molecule. The molecules Evolve-21, Evolve-26 and the comparator Glofitamab showed similar PK profiles in this model. Example 2: A highly potent 2:1 CD20-targeted CD2 co-stimulatory T cell engager engineered for the treatment of B cell malignancies and B cell autoimmune diseases

[0359] T cell engager CD3-bispecifics have improved the treatment of patients with B cell lymphomas leading to recent approvals of the CD20-targeted CD3 bispecifics Glofitamab, Mosunetuzumab, and Epcoritamab for patients with relapsed, refractory diseases. Glofitamab with its 2:1 molecular format has the potential for increased CD20 binding and superior antitumor response compared to conventional 1:1 bispecifics. Recently, drug combinations of CD3-bispecifics and tumor-targeted costimulatory agonists, CD19-4- 1BBL and CD19-CD28, have shown to be an attractive strategy in preclinical models to enhance Glofitamab’s efficacy, and are being investigated in the clinic but have challenges to establish optimal dose scheduling and dose levels which ensure simultaneous CD3 and costimulatory pathway activation. T cell costimulation using CD2 is an alternative strategy which may have significant benefits compared to 4-1BB and CD28. Methods

[0360] Here we report on the development of a next-generation 2:1 format CD20-targeted fusion multispecific with integrated CD2-costimulation to improve CD3-mediated T cell receptor activation and sustain T cell-mediated cytotoxicity by increasing avidity-driven binding and the potential for the formation of alternative tumor-T cell synapses, to achieve superior performance to clinical benchmarks. Results

[0361] A CD20-targeted fusion multispecific with 2:1 molecular format and integrated CD2-costimulatory agonist shows superior cytotoxicity in both high and low CD20 expressing cell lines, representative of human B lineage sub-population found in lymphoid 175 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 tissues. In an in vitro co-culture assay with tumor cells and Human PBMCs, EVOLVE205 lead candidates (e.g., Evlolve-36, Evolve-37, Evolve-39) display improved human T cell activation, increased T cell expansion, and superior tumor cell killing without a substantial increase in cytokine release compared to 1:1 and 2:1 clinical benchmark bispecifics. Importantly, the tumor-killing potency of the CD20-targeted fusion multispecific was improved by up to 60-fold compared to Glofitamab and Epcoritamab, and by over 1,000-fold compared to B cell depleting therapeutics (BCDTs) such as Rituximab. These data suggest the potential of CD20-targeted fusion multispecifics to establish an improved therapeutic index relative to these clinical benchmarks. In a PBMC-engrafted xenograft NSG mouse model with WSU-DLCL2 large B cell lymphoma cell line, CD20-targeted fusion multispecific demonstrated superior tumor control to Glofitamab.

[0362] CD2 costimulation integrated CD20-targeted fusion multispecifics may provide sustained and potent T cell activation potentially to induce durable anti-tumor response in patients, while demonstrating good developability and favorable IgG-like pharmacokinetics in mice. CD20-targeted fusion multispecific is a highly differentiated CD20-targeted immunotherapy capable of providing deep and durable tissue and peripheral B cell depletion with favorable cytokine release profile compared to clinically available CD20-targeted TCE therapies and BCDTs for the treatment of B cell lymphomas and B cell- mediated autoimmune diseases. Example 3. EVOLVE 205, a highly potent 2:1 CD20-targeted CD2 co-stimulatory T cell engager engineered for the treatment of B cell malignancies and B cell autoimmune diseases

[0363] Experiments in this Example were performed in part as described in Example 1. Additional methods are provided below. B- and T-cell isolation from healthy donor PBMC and tissue B cell enrichment from tonsil mononuclear cells ...

Claims

Attorney Docket No. E0701.70002WO00 CLAIMS What is claimed is:

1. An antibody comprising a first antigen binding region that binds to CD20, a second antigen binding region that binds to CD20, and a third antigen binding region that binds to CD3, wherein the first antigen binding region that binds to CD20 comprises three heavy chain complementarity determining regions (VH1_CDR1, VH1_CDR2, VH1_CDR3) and three light chain complementarity determining regions (VL1_CDR1, VL1_CDR2, VL1_CDR3), wherein a) VH1_CDR1 comprises the amino acid sequence of SEQ ID NO: 3; VH1_CDR2 comprises the amino acid sequence of SEQ ID NO: 10; VH1_CDR3 comprises the amino acid sequence of SEQ ID NO: 16; VL1_CDR1 comprises the amino acid sequence of SEQ ID NO: 21; VL1_CDR2 comprises the amino acid sequence of SEQ ID NO: 23; and VL1_CDR3 comprises the amino acid sequence of SEQ ID NO: 26; or b) VH1_CDR1 comprises the amino acid sequence of SEQ ID NO: 1; VH1_CDR2 comprises the amino acid sequence of SEQ ID NO: 8; VH1_CDR3 comprises the amino acid sequence of SEQ ID NO: 14; VL1_CDR1 comprises the amino acid sequence of SEQ ID NO: 20; VL1_CDR2 comprises the amino acid sequence of SEQ ID NO: 22; and VL1_CDR3 comprises the amino acid sequence of SEQ ID NO: 25; and wherein the second antigen binding region that binds to CD20 comprises three heavy chain complementarity determining regions (VH2_CDR1, VH2_CDR2, VH2_CDR3) and three light chain complementarity determining regions (VL2_CDR1, VL2_CDR2, VL2_CDR3), wherein I) VH2_CDR1 comprises the amino acid sequence of SEQ ID NO: 3; VH2_CDR2 comprises the amino acid sequence of SEQ ID NO: 10; VH2_CDR3 comprises the amino acid sequence of SEQ ID NO: 16; VL2_CDR1 comprises the amino acid sequence of SEQ ID NO: 21; VL2_CDR2 comprises the amino acid sequence of SEQ ID NO: 23; and VL2_CDR3 comprises the amino acid sequence of SEQ ID NO: 26; or 206 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 II) VH2_CDR1 comprises the amino acid sequence of SEQ ID NO: 1; VH2_CDR2 comprises the amino acid sequence of SEQ ID NO: 8; VH2_CDR3 comprises the amino acid sequence of SEQ ID NO: 14; VL2_CDR1 comprises the amino acid sequence of SEQ ID NO: 20; VL2_CDR2 comprises the amino acid sequence of SEQ ID NO: 22; and VL2_CDR3 comprises the amino acid sequence of SEQ ID NO: 25; and wherein the third antigen binding region that binds to CD3 comprises three heavy chain complementarity determining regions (VH3_CDR1, VH3_CDR2, VH3_CDR3) and three light chain complementarity determining regions (VL3_CDR1, VL3_CDR2, VL3_CDR3), wherein i) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 4; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 24; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; ii) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 2; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; iii) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 12; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 18; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 27; iv) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 6; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; 207 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 13; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; v) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 6; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; vi) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; vii) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 13; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 27; viii) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 13; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; ix) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 19; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; 208 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 27; x) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 11; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO: 15; or xi) VH3_CDR1 comprises the amino acid sequence of SEQ ID NO: 7; VH3_CDR2 comprises the amino acid sequence of SEQ ID NO: 13; VH3_CDR3 comprises the amino acid sequence of SEQ ID NO: 17; VL3_CDR1 comprises the amino acid sequence of SEQ ID NO: 5; VL3_CDR2 comprises the amino acid sequence of SEQ ID NO: 9; and VL3_CDR3 comprises the amino acid sequence of SEQ ID NO:

15.

2. The antibody of claim 1, wherein the first antigen binding region that binds to CD20 comprises a first variable heavy chain region (VH1) and a first variable light chain region (VL1), wherein: a) VH1 comprises the amino acid sequence of SEQ ID NO: 32; and VL1 comprises the amino acid sequence of SEQ ID NO: 33; or b) VH1 comprises the amino acid sequence of SEQ ID NO: 28; and VL1 comprises the amino acid sequence of SEQ ID NO: 29; wherein the second antigen binding region that binds to CD20 comprises a second variable heavy chain region (VH2) and a second variable light chain region (VL2), wherein: I) VH2 comprises the amino acid sequence of SEQ ID NO: 32; and VL2 comprises the amino acid sequence of SEQ ID NO: 33; or II) VH2 comprises the amino acid sequence of SEQ ID NO: 28; and VL2 comprises the amino acid sequence of SEQ ID NO: 29; wherein the third antigen binding region that binds to CD3 comprises a third variable heavy chain region (VH3) and a third variable light chain region (VL3), wherein: i) VH3 comprises the amino acid sequence of SEQ ID NO: 34; and 209 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 VL3 comprises the amino acid sequence of SEQ ID NO: 35; ii) VH3 comprises the amino acid sequence of SEQ ID NO: 30; and VL3 comprises the amino acid sequence of SEQ ID NO: 31; iii) VH3 comprises the amino acid sequence of SEQ ID NO: 36; and VL3 comprises the amino acid sequence of SEQ ID NO: 37; iv) VH3 comprises the amino acid sequence of SEQ ID NO: 38; and VL3 comprises the amino acid sequence of SEQ ID NO: 39; v) VH3 comprises the amino acid sequence of SEQ ID NO: 38; and VL3 comprises the amino acid sequence of SEQ ID NO: 31; vi) VH3 comprises the amino acid sequence of SEQ ID NO: 38; and VL3 comprises the amino acid sequence of SEQ ID NO: 40; vii) VH3 comprises the amino acid sequence of SEQ ID NO: 41; and VL3 comprises the amino acid sequence of SEQ ID NO: 37; viii) VH3 comprises the amino acid sequence of SEQ ID NO: 42; and VL3 comprises the amino acid sequence of SEQ ID NO: 38; or ix) VH3 comprises the amino acid sequence of SEQ ID NO: 43; and VL3 comprises the amino acid sequence of SEQ ID NO:

38.

3. The antibody of claim 2, wherein the antibody comprises a first heavy chain polypeptide (H1) and a second heavy chain polypeptide (H2), a first light chain polypeptide (L1), a second light chain polypeptide (L2), and a third light chain polypeptide (L3), wherein a) the first heavy chain polypeptide (H1) comprises the second variable heavy chain region (VH2); b) the second heavy chain polypeptide (H2) comprises the third variable heavy chain region (VH3); c) the first variable heavy chain region (VH1) is located on either the first heavy chain polypeptide (H1) or the second heavy chain polypeptide (H2), d) the first light chain polypeptide (L1) comprises the first variable light chain region (VL1), 210 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 e) the second light chain polypeptide (L2) comprises the second variable light chain region (VL2), and f) the third light chain polypeptide (L3) comprises the third variable light chain region (VL3).

4. The antibody of claim 3, wherein a polypeptide is fused to the N-terminus or the C- terminus of the first heavy chain polypeptide (H1), the second heavy chain polypeptide (H2), or the third light chain polypeptide (L3).

5. The antibody of claim 4, wherein the polypeptide is fused via a linker peptide.

6. The antibody of claim 5, wherein the linker peptide comprises the amino acid sequence of any one of SEQ ID NOs: 96-101, 283-338, 344, or 553-606.

7. The antibody of any one of claims 4-6, wherein the polypeptide comprises CD58 or a fragment thereof.

8. The antibody of claim 7, wherein CD58 comprises the amino acid sequence of any one of SEQ ID NOs: 95, 102, or 624-628.

9. The antibody of claim 7 or 8, wherein CD58 comprises the amino acid sequence of SEQ ID NO:

95.

10. The antibody of any one of claims 2-9, wherein i) the VH1 comprises the amino acid sequence of SEQ ID NO: 32; the VH2 comprises the amino acid sequence of SEQ ID NO: 32; the VH3 comprises the amino acid sequence of SEQ ID NO: 34; the VL1 comprises the amino acid sequence of SEQ ID NO: 33; the VL2 comprises the amino acid sequence of SEQ ID NO: 33; and the VL3 comprises the amino acid sequence of SEQ ID NO: 35; ii) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; 211 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 30; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 31; iii) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 36; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 37; iv) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 38; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 39; v) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 28; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 31; vi) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 38; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 40; vii) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; 212 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 the VH3 comprises the amino acid sequence of SEQ ID NO: 41; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 37; viii) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 42; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO: 38; or ix) the VH1 comprises the amino acid sequence of SEQ ID NO: 28; the VH2 comprises the amino acid sequence of SEQ ID NO: 28; the VH3 comprises the amino acid sequence of SEQ ID NO: 43; the VL1 comprises the amino acid sequence of SEQ ID NO: 29; the VL2 comprises the amino acid sequence of SEQ ID NO: 29; and the VL3 comprises the amino acid sequence of SEQ ID NO:

38.

11. The antibody of any one of claims 1-10, wherein the antibody comprises: (1) an HC1 comprising the amino acid sequence of SEQ ID NO: 107, an LC1 comprising the amino acid sequence of SEQ ID NO: 108, an HC2 comprising the amino acid sequence of SEQ ID NO: 109, an LC2 comprising the amino acid sequence of SEQ ID NO: 110, and an LC3 comprising the amino acid sequence of SEQ ID NO: XX; (2) an HC1 comprising the amino acid sequence of SEQ ID NO: 111, an LC1 comprising the amino acid sequence of SEQ ID NO: 112, an HC2 comprising the amino acid sequence of SEQ ID NO: 113, and an LC2 comprising the amino acid sequence of SEQ ID NO: 114; (3) an HC1 comprising the amino acid sequence of SEQ ID NO: 115, an LC1 comprising the amino acid sequence of SEQ ID NO: 116, an HC2 comprising the amino acid sequence of SEQ ID NO: 117, and an LC2 comprising the amino acid sequence of SEQ ID NO: 118; 213 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 (4) an HC1 comprising the amino acid sequence of SEQ ID NO: 119, an LC1 comprising the amino acid sequence of SEQ ID NO: 120, an HC2 comprising the amino acid sequence of SEQ ID NO: 121, and an LC2 comprising the amino acid sequence of SEQ ID NO: 122; (5) an HC1 comprising the amino acid sequence of SEQ ID NO: 123, an LC1 comprising the amino acid sequence of SEQ ID NO: 124, an HC2 comprising the amino acid sequence of SEQ ID NO: 125, and an LC2 comprising the amino acid sequence of SEQ ID NO: 126; (6) an HC1 comprising the amino acid sequence of SEQ ID NO: 127, an LC1 comprising the amino acid sequence of SEQ ID NO: 128, an HC2 comprising the amino acid sequence of SEQ ID NO: 129, and an LC2 comprising the amino acid sequence of SEQ ID NO: 130; (7) an HC1 comprising the amino acid sequence of SEQ ID NO: 131, an LC1 comprising the amino acid sequence of SEQ ID NO: 132, an HC2 comprising the amino acid sequence of SEQ ID NO: 133, and an LC2 comprising the amino acid sequence of SEQ ID NO: 134; (8) an HC1 comprising the amino acid sequence of SEQ ID NO: 135, an LC1 comprising the amino acid sequence of SEQ ID NO: 136, an HC2 comprising the amino acid sequence of SEQ ID NO: 137, and an LC2 comprising the amino acid sequence of SEQ ID NO: 138; (9) an HC1 comprising the amino acid sequence of SEQ ID NO: 139, an LC1 comprising the amino acid sequence of SEQ ID NO: 140, an HC2 comprising the amino acid sequence of SEQ ID NO: 141, and an LC2 comprising the amino acid sequence of SEQ ID NO: 142; (10) an HC1 comprising the amino acid sequence of SEQ ID NO: 143, an LC1 comprising the amino acid sequence of SEQ ID NO: 144, an HC2 comprising the amino acid sequence of SEQ ID NO: 145, and an LC2 comprising the amino acid sequence of SEQ ID NO: 146; (11) an HC1 comprising the amino acid sequence of SEQ ID NO: 147, 214 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 an LC1 comprising the amino acid sequence of SEQ ID NO: 148, an HC2 comprising the amino acid sequence of SEQ ID NO: 149, and an LC2 comprising the amino acid sequence of SEQ ID NO: 150; (12) an HC1 comprising the amino acid sequence of SEQ ID NO: 151, an LC1 comprising the amino acid sequence of SEQ ID NO: 152, an HC2 comprising the amino acid sequence of SEQ ID NO: 153, and an LC2 comprising the amino acid sequence of SEQ ID NO: 154; (13) an HC1 comprising the amino acid sequence of SEQ ID NO: 155, an LC1 comprising the amino acid sequence of SEQ ID NO: 156, an HC2 comprising the amino acid sequence of SEQ ID NO: 157, and an LC2 comprising the amino acid sequence of SEQ ID NO: 158; (14) an HC1 comprising the amino acid sequence of SEQ ID NO: 159, an LC1 comprising the amino acid sequence of SEQ ID NO: 160, an HC2 comprising the amino acid sequence of SEQ ID NO: 161, and an LC2 comprising the amino acid sequence of SEQ ID NO: 162; (15) an HC1 comprising the amino acid sequence of SEQ ID NO: 163, an LC1 comprising the amino acid sequence of SEQ ID NO: 164, an HC2 comprising the amino acid sequence of SEQ ID NO: 165, and an LC2 comprising the amino acid sequence of SEQ ID NO: 166; (16) an HC1 comprising the amino acid sequence of SEQ ID NO: 167, an LC1 comprising the amino acid sequence of SEQ ID NO: 168, an HC2 comprising the amino acid sequence of SEQ ID NO: 169, and an LC2 comprising the amino acid sequence of SEQ ID NO: 170; (17) an HC1 comprising the amino acid sequence of SEQ ID NO: 171, an LC1 comprising the amino acid sequence of SEQ ID NO: 172, 215 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 an HC2 comprising the amino acid sequence of SEQ ID NO: 173, and an LC2 comprising the amino acid sequence of SEQ ID NO: 174; (18) an HC1 comprising the amino acid sequence of SEQ ID NO: 175, an LC1 comprising the amino acid sequence of SEQ ID NO: 176, an HC2 comprising the amino acid sequence of SEQ ID NO: 177, and an LC2 comprising the amino acid sequence of SEQ ID NO: 178; (19) an HC1 comprising the amino acid sequence of SEQ ID NO: 179, an LC1 comprising the amino acid sequence of SEQ ID NO: 180, an HC2 comprising the amino acid sequence of SEQ ID NO: 181, and an LC2 comprising the amino acid sequence of SEQ ID NO: 182; (20) an HC1 comprising the amino acid sequence of SEQ ID NO: 183, an LC1 comprising the amino acid sequence of SEQ ID NO: 184, an HC2 comprising the amino acid sequence of SEQ ID NO: 185, and an LC2 comprising the amino acid sequence of SEQ ID NO: 186; (21) an HC1 comprising the amino acid sequence of SEQ ID NO: 187, an LC1 comprising the amino acid sequence of SEQ ID NO: 188, an HC2 comprising the amino acid sequence of SEQ ID NO: 189, and an LC2 comprising the amino acid sequence of SEQ ID NO: 190; (22) an HC1 comprising the amino acid sequence of SEQ ID NO: 191, an LC1 comprising the amino acid sequence of SEQ ID NO: 192, an HC2 comprising the amino acid sequence of SEQ ID NO: 193, and an LC2 comprising the amino acid sequence of SEQ ID NO: 194; (23) an HC1 comprising the amino acid sequence of SEQ ID NO: 195, an LC1 comprising the amino acid sequence of SEQ ID NO: 196, an HC2 comprising the amino acid sequence of SEQ ID NO: 197, and 216 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 an LC2 comprising the amino acid sequence of SEQ ID NO: 198; (24) an HC1 comprising the amino acid sequence of SEQ ID NO: 199, an LC1 comprising the amino acid sequence of SEQ ID NO: 200, an HC2 comprising the amino acid sequence of SEQ ID NO: 201, and an LC2 comprising the amino acid sequence of SEQ ID NO: 282; (25) an HC1 comprising the amino acid sequence of SEQ ID NO: 202, an LC1 comprising the amino acid sequence of SEQ ID NO: 203, an HC2 comprising the amino acid sequence of SEQ ID NO: 204, and an LC2 comprising the amino acid sequence of SEQ ID NO: 205; (26) an HC1 comprising the amino acid sequence of SEQ ID NO: 206, an LC1 comprising the amino acid sequence of SEQ ID NO: 207, an HC2 comprising the amino acid sequence of SEQ ID NO: 208, and an LC2 comprising the amino acid sequence of SEQ ID NO: 209; (27) an HC1 comprising the amino acid sequence of SEQ ID NO: 210, an LC1 comprising the amino acid sequence of SEQ ID NO: 211, an HC2 comprising the amino acid sequence of SEQ ID NO: 212, and an LC2 comprising the amino acid sequence of SEQ ID NO: 213; (28) an HC1 comprising the amino acid sequence of SEQ ID NO: 214, an LC1 comprising the amino acid sequence of SEQ ID NO: 215, an HC2 comprising the amino acid sequence of SEQ ID NO: 216, and an LC2 comprising the amino acid sequence of SEQ ID NO: 217; (29) an HC1 comprising the amino acid sequence of SEQ ID NO: 218, an LC1 comprising the amino acid sequence of SEQ ID NO: 219, an HC2 comprising the amino acid sequence of SEQ ID NO: 220, and an LC2 comprising the amino acid sequence of SEQ ID NO: 221; 217 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 (30) an HC1 comprising the amino acid sequence of SEQ ID NO: 222, an LC1 comprising the amino acid sequence of SEQ ID NO: 223, an HC2 comprising the amino acid sequence of SEQ ID NO: 224, and an LC2 comprising the amino acid sequence of SEQ ID NO: 225; (31) an HC1 comprising the amino acid sequence of SEQ ID NO: 226, an LC1 comprising the amino acid sequence of SEQ ID NO: 227, an HC2 comprising the amino acid sequence of SEQ ID NO: 228, and an LC2 comprising the amino acid sequence of SEQ ID NO: 229; (32) an HC1 comprising the amino acid sequence of SEQ ID NO: 230, an LC1 comprising the amino acid sequence of SEQ ID NO: 231, an HC2 comprising the amino acid sequence of SEQ ID NO: 232, and an LC2 comprising the amino acid sequence of SEQ ID NO: 233; (33) an HC1 comprising the amino acid sequence of SEQ ID NO: 234, an LC1 comprising the amino acid sequence of SEQ ID NO: 235, an HC2 comprising the amino acid sequence of SEQ ID NO: 236, and an LC2 comprising the amino acid sequence of SEQ ID NO: 237; (34) an HC1 comprising the amino acid sequence of SEQ ID NO: 238, an LC1 comprising the amino acid sequence of SEQ ID NO: 239, an HC2 comprising the amino acid sequence of SEQ ID NO: 240, and an LC2 comprising the amino acid sequence of SEQ ID NO: 241; (35) an HC1 comprising the amino acid sequence of SEQ ID NO: 242, an LC1 comprising the amino acid sequence of SEQ ID NO: 243, an HC2 comprising the amino acid sequence of SEQ ID NO: 244, and an LC2 comprising the amino acid sequence of SEQ ID NO: 245; 218 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 (36) an HC1 comprising the amino acid sequence of SEQ ID NO: 246, an LC1 comprising the amino acid sequence of SEQ ID NO: 247, an HC2 comprising the amino acid sequence of SEQ ID NO: 248, and an LC2 comprising the amino acid sequence of SEQ ID NO: 249; (37) an HC1 comprising the amino acid sequence of SEQ ID NO: 250, an LC1 comprising the amino acid sequence of SEQ ID NO: 251, an HC2 comprising the amino acid sequence of SEQ ID NO: 252, and an LC2 comprising the amino acid sequence of SEQ ID NO: 253; (38) an HC1 comprising the amino acid sequence of SEQ ID NO: 254, an LC1 comprising the amino acid sequence of SEQ ID NO: 255, an HC2 comprising the amino acid sequence of SEQ ID NO: 256, and an LC2 comprising the amino acid sequence of SEQ ID NO: 257; (39) an HC1 comprising the amino acid sequence of SEQ ID NO: 258, an LC1 comprising the amino acid sequence of SEQ ID NO: 259, an HC2 comprising the amino acid sequence of SEQ ID NO: 260, and an LC2 comprising the amino acid sequence of SEQ ID NO: 261; (40) an HC1 comprising the amino acid sequence of SEQ ID NO: 262, an LC1 comprising the amino acid sequence of SEQ ID NO: 263, an HC2 comprising the amino acid sequence of SEQ ID NO: 264, and an LC2 comprising the amino acid sequence of SEQ ID NO: 265; (41) an HC1 comprising the amino acid sequence of SEQ ID NO: 266, an LC1 comprising the amino acid sequence of SEQ ID NO: 267, an HC2 comprising the amino acid sequence of SEQ ID NO: 268, and an LC2 comprising the amino acid sequence of SEQ ID NO: 269; 219 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 (42) an HC1 comprising the amino acid sequence of SEQ ID NO: 270, an LC1 comprising the amino acid sequence of SEQ ID NO: 271, an HC2 comprising the amino acid sequence of SEQ ID NO: 272, and an LC2 comprising the amino acid sequence of SEQ ID NO: 273; (43) an HC1 comprising the amino acid sequence of SEQ ID NO: 374, an LC1 comprising the amino acid sequence of SEQ ID NO: 375, an HC2 comprising the amino acid sequence of SEQ ID NO: 376, and an LC2 comprising the amino acid sequence of SEQ ID NO: 377; or (44) an HC1 comprising the amino acid sequence of SEQ ID NO: 378, an LC1 comprising the amino acid sequence of SEQ ID NO: 379, an HC2 comprising the amino acid sequence of SEQ ID NO: 380, and an LC2 comprising the amino acid sequence of SEQ ID NO:

381.

12. A polynucleotide comprising a nucleic acid sequence encoding the antibody of any one of claims 1-11.

13. A vector comprising the polynucleotide of claim 12.

14. A pharmaceutical composition comprising the antibody of any one of claims 1-11, the polynucleotide of claim 12, or the vector of claim 13, and a pharmaceutically acceptable carrier.

15. A method for treating cancer that expresses CD20 in a subject, the method comprising administering an effective amount of the antibody of any one of claims 1-11, the polynucleotide of claim 12, the vector of claim 13, or the pharmaceutical composition of claim 14. 220 305009757 v1 #14387171v1Attorney Docket No. E0701.70002WO00 16. The method of claim 15, wherein the cancer is multiple myeloma. 221 305009757 v1 #14387171v1

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