Anti-CD20 x Anti-CD28 combination therapy

Abstract

Provided herein are a novel anti-CD20 × anti-CD28 antibody and a method for using such an antibody for the treatment of B-cell malignancies. The target anti-CD20 × anti-CD28 antibody can agonist-conjugate to the CD28 costimulatory molecule on T cells and to CD20 on B cells. Therefore, such an antibody enhances antitumor activity at tumor sites. The target antibody used herein is particularly useful for the treatment of B-cell malignancies in combination with other anticancer agents (e.g., anti-CD3 × anti-CD20 × anti-CD79b antibody).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 579,267, filed on 28 August 2023, titled "Anti-CD20 × Anti-CD28 Combination Therapy," which is incorporated herein by reference in its entirety.

[0002] Array List This application includes an array list submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy was created on 22 August 2024, named 067461-5318-WO_SL.xml, and has a size of 640,102 bytes. [Background technology]

[0003] The CD20 molecule (also known as the human B lymphocyte restricted differentiation antigen or Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD present in progenitor (pre-) B lymphocytes and mature B lymphocytes. CD20 is found on the surface of over 90% of B cells derived from peripheral blood or lymphoid organs, is expressed in the early stages of B cell development, and persists until differentiation into plasma cells. CD20 is present not only in normal B cells but also in malignant B cells, including those in certain B-cell lymphomas and leukemias. Specifically, CD20 is expressed in over 90% of B-cell non-Hodgkin lymphomas (NHL), but is not found in hematopoietic stem cells, progenitor (pro-) B cells, normal plasma cells, or other normal tissues.

[0004] CD28 is a type I transmembrane protein expressed on the surface of T cells, assembled as a homodimer, and possessing a single extracellular Ig-V-like domain. CD28 is a receptor for the CD80(B7.1) and CD86(B7.2) proteins and is activated by CD80 or CD86 expressed on the APC. Binding of CD28 to CD80 or CD86 provides a crucial co-stimulatory signal for T cell activation and survival. T cell stimulation via CD28, in addition to the T cell receptor (TCR), provides a potent signal for the production of various interleukins. CD28 enhances cellular signaling, including pathways regulated by the NFκB transcription factor after TCR activation. CD28 co-signaling is important for effective T cell activation, including T cell differentiation, proliferation, cytokine release, and cell death.

[0005] Antibody-based therapies have been successfully used to treat a variety of diseases, including cancer. An increasingly widespread area of ​​research is the design of a single immunoglobulin molecule that simultaneously binds to two different antigens. Such alternative antibody forms that bind two different antigens are often called bispecific antibodies. One special technique for bispecific antibodies involves manipulating the first binding domain to bind to CD28 and the second binding domain to bind to an antigen associated with or increased on cancer cells (e.g., CD20), so that the bispecific antibody redirects T cells to destroy cancer cells. Bispecific antigen-binding molecules that bind both CD28 and target antigens (such as CD20) would be useful in therapeutic scenarios where specific targeting and T-cell-mediated killing of cells expressing the target antigen are desired. There is also a need for anti-CD20 × anti-CD28 antibodies that are safe for use in pharmaceutical compositions. [Overview of the project]

[0006] Provided herein are a novel anti-CD20 × anti-CD28 antibody and a method for using such an antibody for the treatment of B-cell malignancies. The target anti-CD20 × anti-CD28 antibody can bind to the CD28 costimulatory molecule on T cells and to CD20 on B cells. The target antibody used herein is particularly useful for the treatment of B-cell malignancies in combination with other T-cell-related antibodies (e.g., anti-CD3 × anti-CD20 × anti-CD79b antibody).

[0007] In one embodiment, provided herein is an antibody or antibody fragment comprising the following: (a) A first antigen-binding domain that binds to CD20. Hereinafter, the first antigen-binding domain includes the following: (i) Heavy chain complementarity determination regions 1 (vhCDR1), vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 439, and light chain complementarity determination regions 1 (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 443; (ii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 447, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 451; (iii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 455, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 459; (iv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 463, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 467; (v) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 471, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 472; (vi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 473, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 474; (vii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 475, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 472; (viii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 477, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 478; (ix) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 479, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 480; (x) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 481, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 482; (xi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 483, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 484; (xii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 487, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 467; (xiii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 489, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 490; (xiv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 491, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 492; (xv) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 495, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 496; or (xvi) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 497, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 498; and (b) A second antigen-binding domain that binds to CD28. Hereinafter, the second antigen-binding domain includes the following: (i) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 1, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 5; (ii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 11, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 5; (iii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 1, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 19; (iv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 11, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 19; (v) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 15, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 5; (vi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 15, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 19; (vii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 63, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 75; (viii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 67, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 75; (ix) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 63, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 79; (x) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 67, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 111; (xi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 71, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 75; (xii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 71, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 79; (xiii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 320, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 324; (xiv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 327, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 331; (xv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 335, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 339; (xvi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 343, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 347; (xvii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 351, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 355; (xviii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 359, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 355; (xix) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 367, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 371; (xx) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 375, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 380; (xxi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 391, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 395; (xxii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 399, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 403; (xxiii) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 407, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 411; (xxiv) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 415, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 419; (xxv) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 423, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 5; or (xxvi) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 431, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 435.

[0008] In another embodiment, provided herein is an antibody or an antibody fragment thereof comprising a first antigen binding domain that binds to CD20 and a second antigen binding domain that binds to CD28, wherein: (a) the first antigen binding domain comprises those set forth below: (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 441; and vhCDR3 having the amino acid sequence of SEQ ID NO: 442; and vlCDR1 having the amino acid sequence of SEQ ID NO: 444, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 446; (ii) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 449; and vhCDR3 having the amino acid sequence of SEQ ID NO: 450; and vlCDR1 having the amino acid sequence of SEQ ID NO: 452, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 454; (iii) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 457; and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; and vlCDR1 having the amino acid sequence of SEQ ID NO: 460, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 462; or (iv) vhCDR1 having the amino acid sequence of SEQ ID NO: 464; vhCDR2 having the amino acid sequence of SEQ ID NO: 465; and vhCDR3 having the amino acid sequence of SEQ ID NO: 466; and vlCDR1 having the amino acid sequence of SEQ ID NO: 356, vlCDR2 having the amino acid sequence of SEQ ID NO: 357, and vlCDR3 having the amino acid sequence of SEQ ID NO: 470; and (b) the second antigen-binding domain comprises any of the following: (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; (ii) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; (iii) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; (iv) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; (v) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or (vi) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22.

[0009] In another embodiment, provided herein is an antibody or antibody fragment comprising a first antigen-binding domain that binds to CD20 and a second antigen-binding domain that binds to CD28, wherein: (a) The first antibody-binding domain includes the following: (i) VH1 having the amino acid sequence of SEQ ID NO: 439; and VL1 having the amino acid sequence of SEQ ID NO: 443; (ii) VH1 having the amino acid sequence of SEQ ID NO: 447; and VL1 having the amino acid sequence of SEQ ID NO: 451; (iii) VH1 having the amino acid sequence of SEQ ID NO: 455; and VL1 having the amino acid sequence of SEQ ID NO: 459; (iv) VH1 having the amino acid sequence of SEQ ID NO: 463; and VL1 having the amino acid sequence of SEQ ID NO: 467; (v) VH1 having the amino acid sequence of SEQ ID NO: 471; and VL1 having the amino acid sequence of SEQ ID NO: 472; (vi) VH1 having the amino acid sequence of SEQ ID NO: 473; and VL1 having the amino acid sequence of SEQ ID NO: 474; (vii) VH1 having the amino acid sequence of SEQ ID NO: 475; and VL1 having the amino acid sequence of SEQ ID NO: 472; (viii) VH1 having the amino acid sequence of SEQ ID NO: 477; and VL1 having the amino acid sequence of SEQ ID NO: 478; (ix) VH1 having the amino acid sequence of SEQ ID NO: 479; and VL1 having the amino acid sequence of SEQ ID NO: 480; (x) VH1 having the amino acid sequence of SEQ ID NO: 481; and VL1 having the amino acid sequence of SEQ ID NO: 482; (xi) VH1 having the amino acid sequence of SEQ ID NO: 483; and VL1 having the amino acid sequence of SEQ ID NO: 484; (xii) VH1 having the amino acid sequence of SEQ ID NO: 487; and VL1 having the amino acid sequence of SEQ ID NO: 467; (xiii) VH1 having the amino acid sequence of SEQ ID NO: 489; and VL1 having the amino acid sequence of SEQ ID NO: 490; (xiv) VH1 having the amino acid sequence of SEQ ID NO: 491; and VL1 having the amino acid sequence of SEQ ID NO: 492; (xv) VH1 having the amino acid sequence of SEQ ID NO: 495; and VL1 having the amino acid sequence of SEQ ID NO: 496; or, (xvi) VH1 having the amino acid sequence of SEQ ID NO: 497; and VL1 having the amino acid sequence of SEQ ID NO: 498. And, (b) The second antigen-binding domain includes the following: (i) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 5; (ii) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 5; (iii) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 19; (iv) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 19; (v) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 5; (vi) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 19; (vii) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 75; (viii) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 75; (ix) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 79; (x) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 111; (xi) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 75; (xii) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 79; (xiii) VH2 having the amino acid sequence of SEQ ID NO: 320; and VL2 having the amino acid sequence of SEQ ID NO: 324; (xiv) VH2 having the amino acid sequence of SEQ ID NO: 327; and VL2 having the amino acid sequence of SEQ ID NO: 331; (xv) VH2 having the amino acid sequence of SEQ ID NO: 335; and VL2 having the amino acid sequence of SEQ ID NO: 339; (xvi) VH2 having the amino acid sequence of SEQ ID NO: 343; and VL2 having the amino acid sequence of SEQ ID NO: 347; (xvii) VH2 having the amino acid sequence of SEQ ID NO: 351; and VL2 having the amino acid sequence of SEQ ID NO: 355; (xviii) VH2 having the amino acid sequence of SEQ ID NO: 359; and VL2 having the amino acid sequence of SEQ ID NO: 355; (xix) VH2 having the amino acid sequence of SEQ ID NO: 367; and VL2 having the amino acid sequence of SEQ ID NO: 371; (xx) VH2 having the amino acid sequence of SEQ ID NO: 375; and VL2 having the amino acid sequence of SEQ ID NO: 380; or, (xxi) VH2 having the amino acid sequence of SEQ ID NO: 391; and VL2 having the amino acid sequence of SEQ ID NO: 395; (xxii) VH2 having the amino acid sequence of SEQ ID NO: 399; and VL2 having the amino acid sequence of SEQ ID NO: 403; (xxiii) VH2 having the amino acid sequence of SEQ ID NO: 407; and VL2 having the amino acid sequence of SEQ ID NO: 411; (xxiv) VH2 having the amino acid sequence of SEQ ID NO: 415; and VL2 having the amino acid sequence of SEQ ID NO: 419; (xxv) VH2 having the amino acid sequence of SEQ ID NO: 423; and VL2 having the amino acid sequence of SEQ ID NO: 5; or, (xxvi) VH2 having the amino acid sequence of SEQ ID NO: 431; and VL2 having the amino acid sequence of SEQ ID NO: 435.

[0010] In another embodiment, provided herein is an antibody or antibody fragment comprising a first antigen-binding domain that binds to CD20 and a second antigen-binding domain that binds to CD28, wherein: (a) The first antigen-binding domain includes the following: (i) VH1 having the amino acid sequence of SEQ ID NO: 439; and VL1 having the amino acid sequence of SEQ ID NO: 443; (ii) VH1 having the amino acid sequence of SEQ ID NO: 447; and VL1 having the amino acid sequence of SEQ ID NO: 451; (iii) VH1 having the amino acid sequence of SEQ ID NO: 455; and VL1 having the amino acid sequence of SEQ ID NO: 459; or (iv) VH1 having the amino acid sequence of SEQ ID NO: 463; and VL1 having the amino acid sequence of SEQ ID NO: 467. And, (b) The second antigen-binding domain includes the following: (i) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 5; (ii) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 5; (iii) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 19; (iv) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 19; (v) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 5; (vi) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 19; (vii) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 75; (viii) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 75; (ix) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 79; (x) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 79; (xi) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 75; or, (xii) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 79.

[0011] In one embodiment, provided herein is an antibody or antibody fragment comprising a first antigen-binding domain that binds to CD20 and a second antigen-binding domain that binds to CD28, wherein: (a) The first antigen-binding domain includes vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 457; and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; and also includes vlCDR1 having the amino acid sequence of SEQ ID NO: 460, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 462; and, (b) The second antigen-binding domain includes vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22.

[0012] In one embodiment, provided herein is an antibody or antibody fragment comprising a first antigen-binding domain that binds to CD20 and a second antigen-binding domain that binds to CD28, wherein: (a) The first antigen-binding domain comprises VH1 having the amino acid sequence of SEQ ID NO: 455; and VL1 having the amino acid sequence of SEQ ID NO: 459; (b) The second antigen-binding domain includes VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 79.

[0013] In another embodiment, provided herein are antibodies or antibody fragments, including those described below: a) A first monomer comprising VH1-CH1-hinge-CH2-CH3 (from N-terminus to C-terminus), wherein VH1 is a first variable heavy chain domain and CH2-CH3 is a first Fc domain; and, b)i) A single-chain variable fragment (scFv) comprising a second variable heavy chain domain (VH2), a linker, and a second variable light chain domain (VL2), and ii) A second monomer comprising a second Fc domain, wherein scFv is covalently bonded to the N-terminus of the second Fc domain using a domain linker; c) A light chain comprising VL1-CL (from the N-terminus to the C-terminus), where VL1 is the first variable light chain domain and CL is the constant light chain domain. (Here, the above VH1 and VL1 combine to form a first antigen-binding domain, and the above VH2 and VL2 combine to form a second binding domain, and, Here, the first antigen-binding domain binds to CD20, and the second antigen-binding domain binds to CD28.

[0014] Furthermore, this specification provides a method for treating B-cell malignancies. In one embodiment, a method for treating a B-cell malignancy in a patient requiring treatment for the B-cell malignancy, comprising administering to the patient a combination of an anti-CD20 × anti-CD28 antibody or an antibody fragment and a T-cell engager. Here: (A) The anti-CD20 × anti-CD28 antibody or fragment thereof comprises the following: (a)(i) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 441; and vhCDR3 having the amino acid sequence of SEQ ID NO: 442; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 444, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 446; (ii) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 449; and vhCDR3 having the amino acid sequence of SEQ ID NO: 450; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 452, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 454; (iii) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 457; and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; and vlCDR1 having the amino acid sequence of SEQ ID NO: 460, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 462; or (iv) including vhCDR1 having the amino acid sequence of SEQ ID NO: 464; vhCDR2 having the amino acid sequence of SEQ ID NO: 465; and vhCDR3 having the amino acid sequence of SEQ ID NO: 466; and vlCDR1 having the amino acid sequence of SEQ ID NO: 356, vlCDR2 having the amino acid sequence of SEQ ID NO: 357, and vlCDR3 having the amino acid sequence of SEQ ID NO: 470; The first antigen-binding domain that binds to CD20; and, (b)(i) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; (ii) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; (iii) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; (iv) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; (v) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or (vi) including vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; The second antigen-binding domain that binds to CD28. (B) The T cell engager is an anti-CD79b × anti-CD20 × anti-CD3 antibody or a fragment of that antibody.

[0015] In one embodiment, provided herein is a method for treating a B-cell malignancy in a patient requiring treatment for the B-cell malignancy, the method comprising administering to the patient the following: A)i)(a) VH1 comprising vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 457; and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; and (b) VL1 comprising vlCDR1 having the amino acid sequence of SEQ ID NO: 460; vlCDR2 having the amino acid sequence of SEQ ID NO: 445; and vlCDR3 having the amino acid sequence of SEQ ID NO: 462. A first antigen-binding domain, and a second antigen-binding domain comprising: (a) VH2 comprising vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (b) VL2 comprising vlCDR1 having the amino acid sequence of SEQ ID NO: 6; vlCDR2 having the amino acid sequence of SEQ ID NO: 7; and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; and B) a T cell engager (where the T cell engager is an anti-CD79b x anti-CD20 x anti-CD3 antibody).In some embodiments, the anti-CD79b × anti-CD20 × anti-CD3 antibody or antibody fragment includes: (a) an antigen-binding domain that binds to CD79b, comprising vhCDR1 having the amino acid sequence of SEQ ID NO: 524, vhCDR2 having the amino acid sequence of SEQ ID NO: 525, and vhCDR3 having the amino acid sequence of SEQ ID NO: 526; and vlCDR1 having the amino acid sequence of SEQ ID NO: 528, vlCDR2 having the amino acid sequence of SEQ ID NO: 529, and vlCDR3 having the amino acid sequence of SEQ ID NO: 530; and (b) an antigen-binding domain that binds to CD79b, comprising vhCDR1 having the amino acid sequence of SEQ ID NO: 540, vhCDR2 having the amino acid sequence of SEQ ID NO: 541, and the amino acid sequence of SEQ ID NO: 542. (c) an antigen-binding domain that binds to CD20, comprising vhCDR3 having an amino acid sequence; and vlCDR1 having the amino acid sequence of SEQ ID NO 544, vlCDR2 having the amino acid sequence of SEQ ID NO 545, and vlCDR3 having the amino acid sequence of SEQ ID NO 546; and an antigen-binding domain that binds to CD3, comprising vhCDR1 having the amino acid sequence of SEQ ID NO 532; vhCDR2 having the amino acid sequence of SEQ ID NO 533, and vhCDR3 having the amino acid sequence of SEQ ID NO 534; and vlCDR1 having the amino acid sequence of SEQ ID NO 536, vlCDR2 having the amino acid sequence of SEQ ID NO 537, and vlCDR3 having the amino acid sequence of SEQ ID NO 538.

[0016] In another embodiment, provided herein is a method for promoting T cell proliferation, comprising contacting cells with the following: A) i) a first antigen-binding domain having VH1 comprising vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 457; and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; and (b) VL1 comprising vlCDR1 having the amino acid sequence of SEQ ID NO: 460; vlCDR2 having the amino acid sequence of SEQ ID NO: 445; and vlCDR3 having the amino acid sequence of SEQ ID NO: 462; Furthermore, ii) comprising a second antigen-binding domain: (a) VH2 comprising vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (b) VL2 comprising vlCDR1 having the amino acid sequence of SEQ ID NO: 6; vlCDR2 having the amino acid sequence of SEQ ID NO: 7; and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; and B) a T cell engager comprising a T cell engager wherein the T cell engager is an anti-CD79b × anti-CD20 × anti-CD3 antibody.In some embodiments, the anti-CD79b × anti-CD20 × anti-CD3 antibody or antibody fragment includes: (a) an antigen-binding domain that binds to CD79b, comprising vhCDR1 having the amino acid sequence of SEQ ID NO: 524, vhCDR2 having the amino acid sequence of SEQ ID NO: 525, and vhCDR3 having the amino acid sequence of SEQ ID NO: 526; and vlCDR1 having the amino acid sequence of SEQ ID NO: 528, vlCDR2 having the amino acid sequence of SEQ ID NO: 529, and vlCDR3 having the amino acid sequence of SEQ ID NO: 530; and (b) an antigen-binding domain that binds to CD79b, comprising vhCDR1 having the amino acid sequence of SEQ ID NO: 540, vhCDR2 having the amino acid sequence of SEQ ID NO: 541, and the amino acid sequence of SEQ ID NO: 542. (c) an antigen-binding domain that binds to CD20, comprising vhCDR3 having an amino acid sequence; and vlCDR1 having the amino acid sequence of SEQ ID NO 544, vlCDR2 having the amino acid sequence of SEQ ID NO 545, and vlCDR3 having the amino acid sequence of SEQ ID NO 546; and an antigen-binding domain that binds to CD3, comprising vhCDR1 having the amino acid sequence of SEQ ID NO 532; vhCDR2 having the amino acid sequence of SEQ ID NO 533, and vhCDR3 having the amino acid sequence of SEQ ID NO 534; and vlCDR1 having the amino acid sequence of SEQ ID NO 536, vlCDR2 having the amino acid sequence of SEQ ID NO 537, and vlCDR3 having the amino acid sequence of SEQ ID NO 538.

[0017] In some embodiments, the anti-CD79b × anti-CD20 × anti-CD3 antibody comprises i) a first monomer having the amino acid sequence of SEQ ID NO: 520; ii) a second monomer having the amino acid sequence of SEQ ID NO: 521; and iii) a light chain having the amino acid sequence of SEQ ID NO: 522.

[0018] In some embodiments, the anti-CD20 × anti-CD28 antibody is a heterodimer antibody comprising i) a first monomer having the amino acid sequence of SEQ ID NO: 461; ii) a second monomer having the amino acid sequence of SEQ ID NO: 469; and iii) a light chain having the amino acid sequence of SEQ ID NO: 429. [Brief explanation of the drawing]

[0019] [Figure 1A] The sequences of human, mouse, and cynomolgus monkey CD28 are shown. Such CD28 is useful for developing cross-reactive CD28 antigen-binding domains to facilitate clinical development. [Figure 1B] The sequences of human, mouse, and cynomolgus monkey CD28 are shown. Such CD28 is useful for developing cross-reactive CD28 antigen-binding domains to facilitate clinical development. [Figure 2] The sequences of human, mouse, and cynomolgus monkey CD20 are shown. Such CD20 is useful for developing cross-reactive CD20 antigen-binding domains to facilitate clinical development. [Figure 3A] This shows useful pairs of heterodimerized mutants (including asymmetric mutants and pI mutants). [Figure 3B] This shows useful pairs of heterodimerized mutants (including asymmetric mutants and pI mutants). [Figure 3C] This shows useful pairs of heterodimerized mutants (including asymmetric mutants and pI mutants). [Figure 3D] This shows useful pairs of heterodimerized mutants (including asymmetric mutants and pI mutants). [Figure 3E] This shows useful pairs of heterodimerized mutants (including asymmetric mutants and pI mutants). [Figure 3F] A useful pair of heterodimerized mutants (including asymmetric and pI mutants) is shown. Some mutants lack a corresponding "monomer 2" mutant. Such mutants can be used individually as monomers in bispecific antibodies (e.g., CD20×CD28bsAb), or as pI mutants included in the non-scFv side of a configuration utilizing scFv as a component, where a suitable charged scFv linker can be used as the second monomer utilizing scFv as the CD28-binding domain. Suitable charged linkers are shown in Figure 6. [Figure 4]A table is shown listing the constant regions of isoelectronically distributed mutant antibodies and their respective substitutions. pI_(-) indicates a mutant with a lower pI, and pI_(+) indicates a mutant with a higher pI. These mutants can be arbitrarily and independently combined with other mutants, including the heterodimerizing mutants outlined herein. [Figure 5] This section describes useful excision variants (also called "knockout" or "KO" variants) that excise FcγR binding. In some embodiments, such excision variants are contained in the Fc domains of both monomers of the target antibody described herein. In other embodiments, the excision variant is contained in only one of the variant Fc domains. [Figure 6] As described herein, we present a number of charged scFv linkers that find use in increasing or decreasing the pI of target heterodimer bispecific antibodies (e.g., CD20 × CD28 bispecific antibody (bsAb)) that utilize one or more scFv as components. (+H) positive linkers find particular use herein, especially with anti-CD28 VL and VH sequences as shown herein. A single prior art scFv linker having a monovalent charge is referred to as "Whitlow" from Whitlow et al., Protein Engineering 6(8):989-995 (1993). It should be noted that this linker was used to reduce aggregation in scFv and enhance proteolytic stability. Such charged scFv linkers may be used in any of the target antibody forms disclosed herein that include scFv (e.g., 1+1Fab-scFv-Fc form and 2+1Fab2-scFv-Fc form). [Figure 7A] Numerous exemplary domain linkers are shown. In some embodiments, these linkers find applications in linking single-stranded Fv to Fc chains. In some embodiments, these linkers may be combined in any orientation. For example, the GGGGS linker (SEQ ID NO: 57) may be combined with a “bottom half-hinge” linker at the N-terminus or C-terminus. [Figure 7B]Numerous exemplary domain linkers are shown. In some embodiments, these linkers find applications in linking single-stranded Fv to Fc chains. In some embodiments, these linkers may be combined in any orientation. For example, the GGGGS linker (SEQ ID NO: 57) may be combined with a “bottom half-hinge” linker at the N-terminus or C-terminus. [Figure 8] This invention presents a bispecific antibody platform particularly useful for the CD20 × CD28 bispecific antibody (bsAb). While the platform is described in the context of a 1+1Fab-scFv-Fc configuration, it is also adaptable to use with other bispecific antibody configurations. [Figure 9] This specification shows various heterodimer asymmetric mutant amino acid substitutions that can be used with the heterodimer antibodies described herein. [Figure 10A]Several useful heterodimer CD20×CD28bsAb backbone sequences based on human IgG1 with the variable domain removed are shown. Heterodimer Fc backbone 1 is based on human IgG1 (356E / 358M allotype) and includes the L368D / K370S asymmetric mutant and the Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, the S364K / E357Q asymmetric mutant on the second heterodimer Fc chain, and the E233P / L234V / L235A / G236del / S267K excision mutant on both chains. The heterodimer Fc backbone 2 is based on human IgG1 (356E / 358M allotype) and includes L368D / K370S asymmetric mutants and Q295E / N384D / Q418E / N421D pI mutants on the first heterodimer Fc chain, an S364K asymmetric mutant on the second heterodimer Fc chain, and E233P / L234V / L235A / G236del / S267K excision mutants on both chains. Heterodimer Fc backbone 3 is based on human IgG1 (356E / 358M allotype) and includes L368E / K370S asymmetric mutants and Q295E / N384D / Q418E / N421D pI mutants on the first heterodimer Fc chain, S364K asymmetric mutants on the second heterodimer Fc chain, and E233P / L234V / L235A / G236del / S267K excision mutants on both chains. The heterodimer Fc backbone 4 is based on human IgG1 (356E / 358M allotype) and includes the K360E / Q362E / T411E asymmetric mutant and the Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, the D401K asymmetric mutant on the second heterodimer Fc chain, and the E233P / L234V / L235A / G236del / S267K excision mutant on both chains. Each of these main chains contains a sequence that is 90, 95, 98, and 99% identical (as defined herein) to the referenced sequence, and / or a sequence containing one, two, three, four, five, six, seven, eight, nine, or ten additional amino acid substitutions (which, as those skilled in the art will understand, already contain numerous amino acid modifications compared to parent human IgG1 (or IgG2 or IgG4, depending on the main chain) compared to the “parent” in the figure).In other words, the cited backbone may contain, or substitute for, further amino acid modifications (generally amino acid substitutions) in addition to the asymmetric, pI, and excised mutants included in the backbone of this figure. Furthermore, the backbone shown herein may contain deletions of C-terminal glycine (G446_) and / or lysine (K447_). Deletions of C-terminal glycine and / or lysine may be intentionally manipulated to reduce heterogeneity or in the context of certain bispecificity forms such as mAb-scFv forms. Furthermore, deletions of C-terminal glycine and / or lysine may occur spontaneously, for example, during manufacturing or storage. [Figure 10B]Several useful heterodimer CD20×CD28bsAb backbone sequences based on human IgG1 with the variable domain removed are shown. Heterodimer Fc backbone 5 is based on human IgG1 (356D / 358L allotype) and includes the L368D / K370S asymmetric mutant and the Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, the S364K / E357Q asymmetric mutant on the second heterodimer Fc chain, and the E233P / L234V / L235A / G236del / S267K excision mutant on both chains. The heterodimer Fc backbone 6 is based on human IgG1 (356E / 358M allotype) and includes the L368D / K370S asymmetric mutant and the Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, the S364K / E357Q asymmetric mutant on the second heterodimer Fc chain, the E233P / L234V / L235A / G236del / S267K excision mutant, and the N297A mutant which removes glycosylation on both chains. The heterodimer Fc backbone 7 is based on human IgG1 (356E / 358M allotype) and includes the L368D / K370S asymmetric mutant and the Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, the S364K / E357Q asymmetric mutant on the second heterodimer Fc chain, the E233P / L234V / L235A / G236del / S267K excision mutant, and the N297S mutant which removes glycosylation on both chains. The heterodimer Fc backbone 8 is based on human IgG4 and includes the L368D / K370S asymmetric mutant and Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, the S364K / E357Q asymmetric mutant on the second heterodimer Fc chain, and the S228P (according to EU numbering, S241P in Kabat) mutant, which removes the exchange of Fab arms on both chains (as known in the art).Each of these backchains contains sequences that are 90, 95, 98, and 99% identical (as defined herein) to the cited sequences, and / or sequences containing one, two, three, four, five, six, seven, eight, nine, or ten additional amino acid substitutions (already containing numerous amino acid modifications compared to parent human IgG1 (or IgG2 or IgG4, depending on the backchain), as will be understood by those skilled in the art, compared to the “parent” in the figure). That is, the cited backchains may contain further amino acid modifications (generally amino acid substitutions) in addition to, or as a substitute for, the asymmetric, pI, and excised variants contained within the backchains in this figure. Furthermore, the backchains shown herein may contain deletions of C-terminal glycine (G446_) and / or lysine (K447_). Deletions of C-terminal glycine and / or lysine may be intentionally manipulated to reduce heterogeneity or in the context of certain bispecificity forms such as mAb-scFv. Furthermore, C-terminal glycine and / or lysine deletions may occur spontaneously, for example, during manufacturing or storage. [Figure 10C]Several useful heterodimer CD20×CD28bsAb backbone sequences based on human IgG1 with the variable domain removed are shown. Heterodimer Fc backbone 9 is based on human IgG2 and includes the L368D / K370S asymmetric mutant and the Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, and the S364K / E357Q asymmetric mutant on the second heterodimer Fc chain. Heterodimer Fc backbone 10 is based on human IgG2 and includes the L368D / K370S asymmetric mutant and the Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, the S364K / E357Q asymmetric mutant on the second heterodimer Fc chain, and the S267K excision mutant on both chains. The heterodimer Fc main chain 11 is based on human IgG1 (356E / 358M allotype) and includes the L368D / K370S asymmetric mutant and the Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, the S364K / E357Q asymmetric mutant on the second heterodimer Fc chain, and the E233P / L234V / L235A / G236del / S267K excision mutant and the M428L / N434S Xtend mutant on both chains. The heterodimer Fc backbone 12 is based on human IgG1 (356E / 358M allotype) and includes the L368D / K370S asymmetric mutant on the first heterodimer Fc chain, the S364K / E357Q asymmetric mutant and the P217R / P229R / N276K pI mutant on the second heterodimer Fc chain, as well as the E233P / L234V / L235A / G236del / S267K excision mutant on both chains. Each of these main chains contains a sequence that is 90, 95, 98, and 99% identical (as defined herein) to the referenced sequence, and / or a sequence containing one, two, three, four, five, six, seven, eight, nine, or ten additional amino acid substitutions (which, as those skilled in the art will understand, already contain numerous amino acid modifications compared to parent human IgG1 (or IgG2 or IgG4, depending on the main chain) compared to the “parent” in the figure).In other words, the cited backbone may contain, or substitute for, further amino acid modifications (generally amino acid substitutions) in addition to the asymmetric, pI, and excised mutants included in the backbone of this figure. Furthermore, the backbone shown herein may contain deletions of C-terminal glycine (G446_) and / or lysine (K447_). Deletions of C-terminal glycine and / or lysine may be intentionally manipulated to reduce heterogeneity or in the context of certain bispecificity forms such as mAb-scFv forms. Furthermore, deletions of C-terminal glycine and / or lysine may occur spontaneously, for example, during manufacturing or storage. [Figure 10D]Several useful heterodimer CD20×CD28bsAb backbone sequences based on human IgG1 with the variable domain removed are shown. Heterodimer Fc backbone 13 is based on human IgG1 (356E / 358M allotype) and includes the T366W asymmetric mutant on the first heterodimer Fc chain, the T366S / L368A / Y407V asymmetric mutant and the H435R / Y436F purified mutant on the second heterodimer Fc chain, as well as the L234A / L235A / D265S excised mutant on both chains. The heterodimer Fc backbone 14 is based on human IgG1 (356E / 358M allotype) and includes the T366W asymmetric mutant on the first heterodimer Fc chain, the T366S / L368A / Y407V asymmetric mutant and the H435R / Y436F purified mutant on the second heterodimer Fc chain, as well as the L234A / L235A / D265S excision mutant and the M252Y / S254T / T256E half-life extension mutant on both chains. The heterodimer Fc backbone 15 is based on human IgG1 (356D / 358L allotype) and includes the L368D / K370S asymmetric mutant and the Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, the S364K / E357Q asymmetric mutant on the second heterodimer Fc chain, and the E233P / L234V / L235A / G236del / S267K excision mutant and the M428L / N434S Xtend mutant on both chains. The heterodimer Fc backbone 16 is based on human IgG1 (356E / 358M allotype) and includes the L368D / K370S asymmetric mutant and the Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, the S364K / E357Q asymmetric mutant on the second heterodimer Fc chain, and the E233P / L234V / L235A / G236del / S267K excision mutant and the M428L / N434A Xtend mutant on both chains.Each of these backchains contains sequences that are 90, 95, 98, and 99% identical (as defined herein) to the cited sequences, and / or sequences containing one, two, three, four, five, six, seven, eight, nine, or ten additional amino acid substitutions (already containing numerous amino acid modifications compared to parent human IgG1 (or IgG2 or IgG4, depending on the backchain), as will be understood by those skilled in the art, compared to the “parent” in the figure). That is, the cited backchains may contain further amino acid modifications (generally amino acid substitutions) in addition to, or as a substitute for, the asymmetric, pI, and excised variants contained within the backchains in this figure. Furthermore, the backchains shown herein may contain deletions of C-terminal glycine (G446_) and / or lysine (K447_). Deletions of C-terminal glycine and / or lysine may be intentionally manipulated to reduce heterogeneity or in the context of certain bispecificity forms such as mAb-scFv. Furthermore, C-terminal glycine and / or lysine deletions may occur spontaneously, for example, during manufacturing or storage. [Figure 10E]Several useful heterodimer CD20×CD28bsAb backbone sequences based on human IgG1 with the variable domain removed are shown. Heterodimer Fc backbone 17 is based on human IgG1 (356D / 358L allotype) and includes the L368D / K370S asymmetric mutant and the Q295E / N384D / Q418E / N421D pI mutant on the first heterodimer Fc chain, the S364K / E357Q skew mutant on the second heterodimer Fc chain, and the E233P / L234V / L235A / G236del / S267K excision mutant and the M428L / N434A Xtend mutant on both chains. Each of these backchains contains sequences that are 90, 95, 98, and 99% identical (as defined herein) to the cited sequences, and / or sequences containing one, two, three, four, five, six, seven, eight, nine, or ten additional amino acid substitutions (already containing numerous amino acid modifications compared to parent human IgG1 (or IgG2 or IgG4, depending on the backchain), as will be understood by those skilled in the art, compared to the “parent” in the figure). That is, the cited backchains may contain further amino acid modifications (generally amino acid substitutions) in addition to, or as a substitute for, the asymmetric, pI, and excised variants contained within the backchains in this figure. Furthermore, the backchains shown herein may contain deletions of C-terminal glycine (G446_) and / or lysine (K447_). Deletions of C-terminal glycine and / or lysine may be intentionally manipulated to reduce heterogeneity or in the context of certain bispecificity forms such as mAb-scFv. Furthermore, C-terminal glycine and / or lysine deletions may occur spontaneously, for example, during manufacturing or storage. [Figure 11]Exemplary sequences of the heterodimer CD20×CD28bsAb backbone used in the 2+1mAb-scFv format are shown. The format shown here is based on heterodimer Fc backbone 1 as shown in Figure X, except that it further includes G446_ on monomer 1(-) and G446_ / K447_ on monomer 2(+). It should be noted that any additional backbone shown in Figure X may be adapted for use in the 2+1mAb-scFv format, with or without K447_ on one or both strands. It should be noted that these sequences may further include the M428L / N434S variant or the M252Y / S254T / T256E half-life extension variant. [Figure 12] The sequence of "CH1" found to be used in the embodiment of CD20×CD28bsAb is shown. [Figure 13] This shows the arrangement of "hinge" components in CD20×CD28bsAb as found to be used in the embodiment. [Figure 14] This shows the constant domain of the congeneral light chain that is found to be used in the control CD20×CD28bsAb, which utilizes the Fab-binding domain. [Figure 15] The variable heavy and light chain sequences of 1A7, an exemplary phage-derived CD28-binding domain, and the sequence of XENP28428, an anti-CD28 mAb based on the IgG1 backbone with 1A7 and the E233P / L234V / L235A / G236del / S267K excision mutant, are shown. CDRs are underlined, and slashes indicate the boundary(s) between the variable region and the constant domain. As is true for all sequences described herein, including CDRs, the precise identification of CDR locations may vary slightly depending on the numbering system used, as shown in Table 2. Therefore, this specification includes not only underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, for all sequences in the figures, these VH and VL sequences may be used in either scFv or Fab format. [Figure 16]The sequence of the affinity-optimized variable heavy chain domain from anti-CD28 clone 1A7 is shown. It should be noted that the variable heavy chain domain can be paired with any of the other variable light chain domains shown herein, including, for example, SEQ ID NOs: 19, 75, 79, and 200-305 (e.g., 1A7_H1.1_L1.71 used in XENP39583). [Figure 17] The sequence of the affinity-optimized variable light chain domain from anti-CD28 clone 1A7 is shown. It should be noted that the variable light chain domain can be paired with any of the other variable heavy chain domains shown herein, including, for example, SEQ ID NOs: 1, 11, 15, 63, 67, 71, and 131-199 (e.g., 1A7_H1.1_L1.71 used in XENP39583). [Figure 18A] Exemplary affinity-optimized 1A7VH / VH pair sequences are shown. Note that these pairs may be formalized as Fabs or scFv. Furthermore, in scFv form, these pairs may be formalized as VHVL orientation or VLVH orientation. [Figure 18B] Exemplary affinity-optimized 1A7VH / VH pair sequences are shown. Note that these pairs may be formalized as Fabs or scFv. Furthermore, in scFv form, these pairs may be formalized as VHVL orientation or VLVH orientation. [Figure 18C] Exemplary affinity-optimized 1A7VH / VH pair sequences are shown. Note that these pairs may be formalized as Fabs or scFv. Furthermore, in scFv form, these pairs may be formalized as VHVL orientation or VLVH orientation. [Figure 19]To enable the “stapling” of scFv, the sequence of an exemplary variable heavy chain domain from cysteine-introduced anti-CD28 clone 1A7 is shown. It should be noted that the variable heavy chain domain can be paired with any of the other variable light chain domains shown in Figures 17, 20, and 23, including SEQ ID NOs: 19, 75, 79, and 200-305 (e.g., used in XENP42157, XENP42160, and XENP42163). [Figure 20] To enable the “stapling” of scFv, the sequence of an exemplary variable light chain domain from cysteine-introduced anti-CD28 clone 1A7 is shown. It should be noted that the variable light chain domain can be paired with any of the other variable heavy chain domains shown in Figures 15, 16, 19, and 22, including sequence numbers 1, 11, 15, 63, 67, 71, and 131-199 (for example, used in XENP42157, XENP42160, and XENP42163). [Figure 21A] An exemplary arrangement of staple 1A7VH / VH pairs is shown. These pairs may be formalized with VHVL or VLVH orientation. [Figure 21B] An exemplary arrangement of staple 1A7VH / VH pairs is shown. These pairs may be formalized with VHVL or VLVH orientation. [Figure 22A] Additional sequences for a variable heavy chain domain optimized for affinity from anti-CD28 clone 1A7 are shown. It should be noted that the variable heavy chain domain can be paired with any of the other variable light chain domains shown herein, including sequence numbers 19, 75, 79, and 200-305. [Figure 22B] Additional sequences for a variable heavy chain domain optimized for affinity from anti-CD28 clone 1A7 are shown. It should be noted that the variable heavy chain domain can be paired with any of the other variable light chain domains shown herein, including sequence numbers 19, 75, 79, and 200-305. [Figure 22C]Additional sequences for a variable heavy chain domain optimized for affinity from anti-CD28 clone 1A7 are shown. It should be noted that the variable heavy chain domain can be paired with any of the other variable light chain domains shown herein, including sequence numbers 19, 75, 79, and 200-305. [Figure 22D] Additional sequences for a variable heavy chain domain optimized for affinity from anti-CD28 clone 1A7 are shown. It should be noted that the variable heavy chain domain can be paired with any of the other variable light chain domains shown herein, including sequence numbers 19, 75, 79, and 200-305. [Figure 22E] Additional sequences for a variable heavy chain domain optimized for affinity from anti-CD28 clone 1A7 are shown. It should be noted that the variable heavy chain domain can be paired with any of the other variable light chain domains shown herein, including sequence numbers 19, 75, 79, and 200-305. [Figure 22F] Additional sequences for a variable heavy chain domain optimized for affinity from anti-CD28 clone 1A7 are shown. It should be noted that the variable heavy chain domain can be paired with any of the other variable light chain domains shown herein, including sequence numbers 19, 75, 79, and 200-305. [Figure 23A] The following shows additional sequences of affinity-optimized variable light chain domains from anti-CD28 clone 1A7. It should be noted that the variable light chain domains can be paired with any of the other variable heavy chain domains shown herein, including sequence numbers 1, 11, 15, 63, 67, 71, and 131-199. [Figure 23B] The following shows additional sequences of affinity-optimized variable light chain domains from anti-CD28 clone 1A7. It should be noted that the variable light chain domains can be paired with any of the other variable heavy chain domains shown herein, including sequence numbers 1, 11, 15, 63, 67, 71, and 131-199. [Figure 23C]The following shows additional sequences of affinity-optimized variable light chain domains from anti-CD28 clone 1A7. It should be noted that the variable light chain domains can be paired with any of the other variable heavy chain domains shown herein, including sequence numbers 1, 11, 15, 63, 67, 71, and 131-199. [Figure 23D] The following shows additional sequences of affinity-optimized variable light chain domains from anti-CD28 clone 1A7. It should be noted that the variable light chain domains can be paired with any of the other variable heavy chain domains shown herein, including sequence numbers 1, 11, 15, 63, 67, 71, and 131-199. [Figure 23E] The following shows additional sequences of affinity-optimized variable light chain domains from anti-CD28 clone 1A7. It should be noted that the variable light chain domains can be paired with any of the other variable heavy chain domains shown herein, including sequence numbers 1, 11, 15, 63, 67, 71, and 131-199. [Figure 23F] The following shows additional sequences of affinity-optimized variable light chain domains from anti-CD28 clone 1A7. It should be noted that the variable light chain domains can be paired with any of the other variable heavy chain domains shown herein, including sequence numbers 1, 11, 15, 63, 67, 71, and 131-199. [Figure 23G] The following shows additional sequences of affinity-optimized variable light chain domains from anti-CD28 clone 1A7. It should be noted that the variable light chain domains can be paired with any of the other variable heavy chain domains shown herein, including sequence numbers 1, 11, 15, 63, 67, 71, and 131-199. [Figure 23H] The following shows additional sequences of affinity-optimized variable light chain domains from anti-CD28 clone 1A7. It should be noted that the variable light chain domains can be paired with any of the other variable heavy chain domains shown herein, including sequence numbers 1, 11, 15, 63, 67, 71, and 131-199. [Figure 23I]The following shows additional sequences of affinity-optimized variable light chain domains from anti-CD28 clone 1A7. It should be noted that the variable light chain domains can be paired with any of the other variable heavy chain domains shown herein, including sequence numbers 1, 11, 15, 63, 67, 71, and 131-199. [Figure 24A] The consensus framework region (FR) and complementarity-determining region (CDR) (according to Kabat) of the variable heavy chain domain variants and variable light chain domain variants of anti-CD28 clone 1A7 are shown. These common sequences may further include "staple" modifications to enable use in "staple" scFv. [Figure 24B] The consensus framework region (FR) and complementarity-determining region (CDR) (according to Kabat) of the variable heavy chain domain variants and variable light chain domain variants of anti-CD28 clone 1A7 are shown. These common sequences may further include "staple" modifications to enable use in "staple" scFv. [Figure 25] The binding affinities of exemplary affinity operations, specifically 1A7VH / VL pairs and scFv (1+1Fab-scFv-Fc bsAb form), are shown. [Figure 26A] The variable heavy chain and variable light chain sequences of additional CD28-binding domains found to be used in the CD20×CD28bsAb of the present invention are shown. As is true for all sequences described herein and including CDRs, the precise identification of the CDR location may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 26B]The variable heavy chain and variable light chain sequences of additional CD28-binding domains found to be used in the CD20×CD28bsAb of the present invention are shown. As is true for all sequences described herein and including CDRs, the precise identification of the CDR location may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 26C] The variable heavy chain and variable light chain sequences of additional CD28-binding domains found to be used in the CD20×CD28bsAb of the present invention are shown. As is true for all sequences described herein and including CDRs, the precise identification of the CDR location may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 26D] The variable heavy chain and variable light chain sequences of additional CD28-binding domains found to be used in the CD20×CD28bsAb of the present invention are shown. As is true for all sequences described herein and including CDRs, the precise identification of the CDR location may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 26E]The variable heavy chain and variable light chain sequences of additional CD28-binding domains found to be used in the CD20×CD28bsAb of the present invention are shown. As is true for all sequences described herein and including CDRs, the precise identification of the CDR location may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 26F] The variable heavy chain and variable light chain sequences of additional CD28-binding domains found to be used in the CD20×CD28bsAb of the present invention are shown. As is true for all sequences described herein and including CDRs, the precise identification of the CDR location may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 26G] The variable heavy chain and variable light chain sequences of additional CD28-binding domains found to be used in the CD20×CD28bsAb of the present invention are shown. As is true for all sequences described herein and including CDRs, the precise identification of the CDR location may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 26H]The variable heavy chain and variable light chain sequences of additional CD28-binding domains found to be used in the CD20×CD28bsAb of the present invention are shown. As is true for all sequences described herein and including CDRs, the precise identification of the CDR location may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 27] The sequences of XENP27181, a bivalent anti-CD28 mAb based on the IgG1 backbone containing the HuTN228 binding domain and the E233P / L234V / L235A / G236del / S267K excision mutant, and XENP29154, a proprietary version of TGN1412, are shown. [Figure 28] This shows exemplary binding of a bivalent anti-CD28 mAb based on a phage-derived clone on human PBMCs. The data demonstrate that the phage campaign generated a CD28-binding domain with weaker maximum binding than prior art HuTN228 (which relates to the humanized CD28-binding domain described in Example 1A). [Figure 29A] The present invention exhibits a bispecific form, the "1+1Fab-scFv-Fc" form, in which the first Fab arm binds to the first antigen and the second scFv arm binds to the second antigen. The 1+1Fab-scFv-Fc form comprises a first monomer containing a first heavy chain variable region (VH1) covalently (optionally via a linker) to the N-terminus of the first heterodimer Fc backbone, a second monomer containing a single-stranded Fv covalently (optionally via a linker) to the N-terminus of the second corresponding heterodimer Fc backbone, and a third monomer containing a light chain variable region covalently bound to the light chain constant domain (where the light chain variable region is complementary to VH1). [Figure 29B]The present invention presents a bispecific form. It presents a "2+1Fab2-scFv-Fc" form having a first Fab arm and a second Fab-scFv arm, where Fab binds to the first antigen and scFv binds to the second antigen. The 2+1Fab2-scFv-Fc form includes a first monomer containing a first heavy chain variable region (VH1) covalently (optionally via a linker) to the N-terminus of a first heterodimer Fc backbone, a second monomer containing VH1 covalently (optionally via a linker) to a single chain Fv covalently (optionally via a linker) to the N-terminus of a second corresponding heterodimer Fc backbone, and a third monomer containing a light chain variable region covalently (where the light chain variable region is complementary to VH1) to a light chain constant domain. [Figure 29C] The present invention presents a bispecific form. It presents a "1+1 common light chain" or "1+1 CLC" form having a first Fc containing a first Fab arm for binding a first antigen, and a second Fc containing a second Fab arm for binding a second antigen. The 1+1 CLC form comprises a first monomer containing VH1-CH1-hinge-CH2-CH3, a second monomer containing VH2-CH1-hinge-CH2-CH3, and a third monomer containing VL-CL. VL pairs with VH1 to form a binding domain having a first antigen-binding specificity, and VL pairs with VH2 to form a binding domain having a second antigen-binding specificity. [Figure 29D] The present invention presents a bispecific form. It presents a "2+1 common light chain" or "2+1 CLC" form, each having a first Fc containing two Fab arms that bind to a first antigen, and a second Fc containing one Fab arm that binds to a second antigen. The 2+1 CLC form includes a first monomer containing VH1-CH1-hinge-VH1-CH1-hinge-CH2-CH3, a second monomer containing VH2-CH1-hinge-CH2-CH3, and a third monomer containing VL-CL. VL pairs with VH1 to form a binding domain having a first antigen-binding specificity, and VL pairs with VH2 to form a binding domain having a second antigen-binding specificity. The present invention presents a bispecific form. [Figure 29E]The present invention presents a bispecific form. It presents a "2+1mAb-scFv" form having a first Fc containing an N-terminal Fab arm for binding to a first antigen, and a second Fc containing an N-terminal Fab arm for binding to a first antigen and a C-terminal scFv for binding to a second antigen. The 2+1mAb-scFv form comprises a first monomer containing VH1-CH1-hinge-CH2-CH3, a second monomer containing VH1-CH1-hinge-CH2-CH3-scFv, and a third monomer containing VL-CL. VL pairs with VH1 to form a binding domain having binding specificity to the first antigen. [Figure 29F] The present invention presents a dual-specificity form, the "2+1mAb-scFv" form, comprising a first monomer containing VH1-CH1-linker-VH2-CH1-hinge-CH2-CH3 (N-terminus to C-terminus) (where CH2-CH3 is the first heterodimer Fc domain), a second monomer containing scFv-linker-CH2-CH3 (N-terminus to C-terminus) (where CH2-CH3 is the second heterodimer Fc domain complementary to the first heterodimer Fc domain, and scFv has the first antigen specificity), and a third monomer which is a common light chain containing VL-CL (N-terminus to C-terminus) (where VL pairs with VH1 and VH2 of the first monomer to form two antigen-binding domains, each having specificity for the second antigen-binding domain). [Figure 29G] An additional bispecific form, bispecific scFv, is shown. [Figure 29H] An additional bispecific form, unilateral scFv-mAb, is shown. [Figure 29I] An additional bispecific form, scFv-mAb, is shown. [Figure 29J] Additional bispecific forms and bispecific mAbs are shown. [Figure 29K] An additional bispecific form, uniarm-centered scFv, is shown. [Figure 29L] An additional dual-specificity form, mAb-Fv, is shown. [Figure 29M] An additional bispecific form, center-Fv, is shown. [Figure 29N]An additional double singularity form, a trifoliate, is shown. [Figure 30A] The variable heavy and variable light chain sequences of the CD20 binding domain used in the CD20×CD28bsAb of the present invention are shown. As is the case with all sequences described herein and including CDRs, the precise identification of the CDR position may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, as with all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 30B] The variable heavy and variable light chain sequences of the CD20 binding domain used in the CD20×CD28bsAb of the present invention are shown. As is the case with all sequences described herein and including CDRs, the precise identification of the CDR position may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, as with all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 30C] The variable heavy and variable light chain sequences of the CD20 binding domain used in the CD20×CD28bsAb of the present invention are shown. As is the case with all sequences described herein and including CDRs, the precise identification of the CDR position may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, as with all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 30D]The variable heavy and variable light chain sequences of the CD20 binding domain used in the CD20×CD28bsAb of the present invention are shown. As is the case with all sequences described herein and including CDRs, the precise identification of the CDR position may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, as with all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 30E] The variable heavy and variable light chain sequences of the CD20 binding domain used in the CD20×CD28bsAb of the present invention are shown. As is the case with all sequences described herein and including CDRs, the precise identification of the CDR position may vary slightly depending on the numbering used, as shown in Table 2, and therefore, this specification includes not only the underlined CDRs but also CDRs contained within VH and VL domains using other numbering systems. Furthermore, as with all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab format. [Figure 31]A shows the classical T cell / APC interaction. B shows the replication of the classical T cell / APC interaction by combining a CD3 bispecific antibody and a CD28 bispecific antibody. The classical T cell / APC interaction has a first signal (signal 1) due to the reactivity of the TCR with peptide-MHC, and a second signal (signal 2) due to the crosslinking of CD28 by CD80 / CD86 expressed on the APC, which together fully activate the T cell. In contrast, treatment with CD3 bispecificity provides only the first signal. The CD28 signal may be provided by CD28 bispecificity aimed at promoting activation and proliferation by CD28 costimulation. In some embodiments, TAA1 and TAA2 may be different antigens. In some embodiments, TAA1 and TAA2 may be the same antigen but have different epitopes. In some embodiments, TAA1 and TAA2 may be the same antigen and have the same epitope. [Figure 32A] The sequence of an exemplary CD79b×CD20×CD3 trispecific antibody, which may be combined with the CD20×CD28bsAb of the present invention, is shown. [Figure 32B] The sequence of an exemplary CD79b×CD20×CD3 trispecific antibody, which may be combined with the CD20×CD28bsAb of the present invention, is shown. [Figure 33A]Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33B] Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33C]Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33D] Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33E]Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33F] Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33G]Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33H] Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33I]Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33J] Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33K]Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33L] Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33M]Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33N] Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 33O]Exemplary CD20×CD28bsAb sequences of the 1+1Fab-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 34] Exemplary CD20×CD28bsAb sequences of the 2+1Fab2-scFv-Fc form are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may utilize variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. In addition, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 35A]Exemplary CD20×CD28bsAb sequences in the 2+1 stack Fab2-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other suitable backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 35B] Exemplary CD20×CD28bsAb sequences in the 2+1 stack Fab2-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other suitable backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 35C]Exemplary CD20×CD28bsAb sequences in the 2+1 stack Fab2-scFv-Fc format are shown. Some of these sequences utilize platform X, others utilize platform J, but other suitable backlines, including those shown in Figure 10, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 36A] Exemplary CD20×CD28bsAb sequences in 2+1mAb-scFv format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 11, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 36B]Exemplary CD20×CD28bsAb sequences in 2+1mAb-scFv format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 11, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 36C] Exemplary CD20×CD28bsAb sequences in 2+1mAb-scFv format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 11, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 36D]Exemplary CD20×CD28bsAb sequences in 2+1mAb-scFv format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 11, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 36E] Exemplary CD20×CD28bsAb sequences in 2+1mAb-scFv format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 11, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 36F]Exemplary CD20×CD28bsAb sequences in 2+1mAb-scFv format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 11, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 36G] Exemplary CD20×CD28bsAb sequences in 2+1mAb-scFv format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 11, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 36H]Exemplary CD20×CD28bsAb sequences in 2+1mAb-scFv format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 11, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 36I] Exemplary CD20×CD28bsAb sequences in 2+1mAb-scFv format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 11, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 36J]Exemplary CD20×CD28bsAb sequences in 2+1mAb-scFv format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 11, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 36K] Exemplary CD20×CD28bsAb sequences in 2+1mAb-scFv format are shown. Some of these sequences utilize platform X, others utilize platform J, but other preferred backlines, including those shown in Figure 11, may be used. CDR is underlined, and slashes indicate the boundary(s) between the variable region and other domains. It should be noted that CD20×CD28bsAb can be 90, 95, 98, and 99% identical (as defined herein) and / or may be available as variable region sequences, Fc region sequences, and constant domain sequences containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. Furthermore, each sequence outlined herein may or may not contain the M428L / N434S, M428L / N434A, or M252Y / S254T / T256E variants in one or preferably both Fc domains, the presence of which results in a longer half-life in serum. [Figure 37] This shows the release of IFNγ from air-dried human PBMCs treated with XENP28428 (anti-CD28 clone 1A7), TGN1412 (XENP29154), or negative control PBS. [Figure 38] A) This shows the induction of IL2 secretion by T cells cultured with Carnaval and treated with CD79b×CD20×CD3 (CD28 titration) in combination with XENP42157 or XENP42160 CD20×CD28bsAb based on the Platform J+ staple linker. B) This shows the induction of IL2 secretion by T cells cultured with OCI-Ly-10 and treated with CD79b×CD20×CD3 (CD28 titration) in combination with XENP42157 or XENP42160 CD20×CD28bsAb based on the Platform J+ staple linker. C) This shows the induction of IL2 secretion by T cells cultured with Ramos cancer cells and treated with CD79b×CD20×CD3 (CD28 titration) in combination with XENP42157 or XENP42160 CD20×CD28bsAb based on the Platform J+ staple linker. [Figure 39] A) This shows the induction of IL2 secretion by T cells cultured with Carnaval and treated with CD79b×CD20×CD3 (CD3 titration) in combination with XENP42157 or XENP42160 CD20×CD28bsAb based on the Platform J+ staple linker. B) This shows the induction of IL2 secretion by T cells cultured with OCI-Ly-10 and treated with CD79b×CD20×CD3 (CD3 titration) in combination with XENP42157 or XENP42160 CD20×CD28bsAb based on the Platform J+ staple linker. C) This shows the induction of IL2 secretion by T cells cultured with Ramos cancer cells and treated with CD79b×CD20×CD3 (CD3 titration) in combination with XENP42157 or XENP42160 CD20×CD28bsAb based on the Platform J+ staple linker. [Figure 40A]This study demonstrates the effects of CD79b×CD20×CD20×CD3 and CD20×CD28 on T cell activation using Pan-T cells from healthy donors and tumor cells expressing CD79b and CD20 (CARNAVAL and OCI-Ly10). T cell activation measured by cytotoxicity analysis is also presented. [Figure 40B] This study demonstrates the effects of CD79b×CD20×CD20×CD3 and CD20×CD28 on T cell activation using Pan-T cells from healthy donors and tumor cells expressing CD79b and CD20 (CARNAVAL and OCI-Ly10). T cell activation measured with CD25 expression is also presented. [Figure 40C] This study demonstrates the effects of CD79b×CD20×CD20×CD3 combined with CD20×CD28 on T cell activation using Pan-T cells from healthy donors and tumor cells expressing CD79b and CD20 (CARNAVAL and OCI-Ly10). It also shows a synergistic effect plot of CD20×CD28bsAb combined with CD79b×CD20×CD3 on T cell-mediated cytotoxicity. [Figure 41] This study demonstrates the efficacy of combined therapy with CD20×CD28 and CD79b×CD20×CD3 in inhibiting the proliferation of SC OCI-Ly10 DLBCL xenografts established in T-cell humanized mice. [Figure 42A] This study demonstrates the effects of CD79b×CD20×CD3 combined with CD20×CD28 on T cell activation in Pan-T cells from healthy donors and tumor cells expressing low levels of CD79b and CD20 (WILL-2 and NALM6). The WILL2 cell line was developed using six donors, and the NALM6 cell line used three donors. T cell activation, as measured by cytotoxicity analysis, is presented. [Figure 42B]This study demonstrates the effects of CD79b×CD20×CD3 combined with CD20×CD28 on T cell activation using Pan-T cells from healthy donors and tumor cells expressing low levels of CD79b and CD20 (WILL-2 and NALM6). The WILL2 cell line was developed using six donors, and the NALM6 cell line used three donors. T cell activation measured by CD25 expression is also presented. [Figure 42C] This study demonstrates the effects of CD20×CD28 combined with CD79b×CD20×CD3 on T cell activation using Pan-T cells from healthy donors and tumor cells expressing low levels of CD79b and CD20 (WILL-2 and NALM6). The WILL2 cell line used six donors, and the NALM6 cell line used three donors. The synergistic effect plot of CD20×CD28bsAb combined with CD79b×CD20×CD3 on T-cell-mediated cytotoxicity is shown. White dots represent negative effects, and black dots represent positive effects. [Figure 42D] This study demonstrates the effects of CD20×CD28 combined with CD79b×CD20×CD3 on T cell activation using Pan-T cells from healthy donors and tumor cells expressing low levels of CD79b and CD20 (WILL-2 and NALM6). The WILL2 cell line used six donors, and the NALM6 cell line used three donors. The synergistic effect plot of CD20×CD28bsAb combined with CD79b×CD20×CD3 on T-cell-mediated cytotoxicity is shown. White dots represent negative effects, and black dots represent positive effects. [Figure 43A] The effects of CD79b×CD20×CD3 combined with CD20×CD28 on T cell activation, measured at 72 hours, are presented using Pan-T cells from healthy donors and tumor cells expressing CD79b and CD20 (CARNAVAL and OCI-Ly10). The activation (%CD25+) and proliferation of CD4 and CD8 T cells are also shown. [Figure 43B]The effects of CD79b×CD20×CD3 combined with CD20×CD28 on T cell activation, measured at 72 hours, are presented using Pan-T cells from healthy donors and tumor cells expressing CD79b and CD20 (CARNAVAL and OCI-Ly10). Cytokine release is also described. [Figure 44A] This study demonstrates the effects of CD20×CD28, combined with CD79b×CD20×CD3, on the proliferation of Pan-T cells, CD8 T cells, and CD4 T cells. [Figure 44B] This study demonstrates the effects of CD20×CD28, combined with CD79b×CD20×CD3, on the proliferation of Pan-T cells, CD8 T cells, and CD4 T cells. [Figure 44C] This study shows the effects of CD20×CD28, combined with CD79b×CD20×CD3, on the activation of CD8 T cells and CD4 T cells (%CD25+ cells). [Figure 44D] This shows the effect of CD20×CD28, combined with CD79b×CD20×CD3, on the differentiation of CD4 and CD8 T cells. [Figure 44E] This shows the effect of CD20×CD28, combined with CD79b×CD20×CD3, on the differentiation of CD4 and CD8 T cells. [Figure 44F] This shows the effect of CD20×CD28, combined with CD79b×CD20×CD3, on the differentiation of CD4 and CD8 T cells. [Figure 44G] This shows the effect of CD20×CD28 combined with CD79b×CD20×CD3 on cytokine release. [Modes for carrying out the invention]

[0020] I. Overview The CD20 antigen is selectively expressed in mature B cells and is a therapeutic target for B-cell malignancies.

[0021] T cell activation in cancer therapy is a widely studied topic. T cells require multiple signals for complete activation and differentiation. As shown in Figure 31A, signal 1, enhanced by the recognition of peptide-MHC (pMHC) complexes by the T cell receptor (TCR), is absolutely essential for T cell activation. Signal 2, which synergizes with and amplifies signal 1, is typically mediated by the interaction of CD28 ligands CD80 and CD86 with CD28 itself. CD28 engagement alone is inactive, but when combined with the activation of signal 1, it enhances T cell activation, survival, and proliferation signals such as IL-2 secretion. Since CD80 and CD86 are spontaneously expressed by specialized antigen-presenting cells (APCs), the degree of CD28 costimulation in tumor settings can be highly variable. Therefore, the present invention is directed toward a novel classification of tumor-targeted anti-CD20 × anti-CD28 antibodies that mimic the CD80 / CD86 engagement of CD28, thereby providing a source of signal 2. Notably, signal 1 can be provided by the innate tumor cell recognition of TCR:pMHC or by a combination with a CD28 bispecific CD3 T cell engager (e.g., anti-CD3 × anti-CD20 × anti-CD79b) that can mimic signal 1.

[0022] Accordingly, provided herein are novel anti-CD20 × anti-CD28 (also known as "αCD20 × αCD28" or sometimes "CD20 × CD28") antibodies and methods for using such antibodies for the treatment of B-cell malignancies. In many cases, these antibodies are heterodimers. The αCD20 × αCD28 antibodies in question can bind to the CD28 costimulatory molecule on T cells and target CD20 on malignant B cells that express CD20. Thus, such antibodies selectively enhance antitumor activity at CD20-expressing tumor sites while minimizing peripheral toxicity. The antibodies in question provided herein are particularly useful for enhancing antitumor activity when used as monotherapy or in combination with other anticancer therapies, as described in more detail herein.

[0023] Accordingly, in one embodiment, what is provided herein is a heterodimer antibody or fragment thereof that binds to two different antigens, for example, the antibody is "bispecific" in that it binds to two different target antigens, generally CD20 and CD28, as described below. These heterodimer antibodies can bind to their respective target antigens either monovalent (e.g., having a single antigen-binding domain) or bivalent (having two antigen-binding domains, each independently binding to an antigen). In some embodiments, the heterodimer antibodies provided herein contain one CD28-binding domain and one CD20-binding domain (e.g., a heterodimer antibody of the "1+1Fab-scFv-Fc" form described herein, which is therefore bispecific and bivalent). In other embodiments, the heterodimer antibodies provided herein contain one CD28-binding domain and two CD20-binding domains (e.g., the "2+1Fab2-scFv-Fc" type heterodimer antibodies described herein, which therefore contain three antigen-binding domains (ABDs) and are thus bispecific but trivalent). The heterodimer antibodies used herein are based on the use of different monomers containing amino acid substitutions (i.e., "asymmetric mutants") that "asymmetrize" the formation of heterodimers compared to homodimers, as outlined in more detail below. In some embodiments, the heterodimer antibodies are also conjugated with a purified mutant (e.g., a "pI mutant") that allows for the simple purification of the heterodimer apart from the homodimer, as also outlined below. The heterodimer antibodies provided generally rely on the use of engineered or mutant Fc domains that can self-assemble in the producing cell to produce heterodimer proteins, and on methods for generating and purifying such heterodimer proteins.

[0024] II. Nomenclature The nomenclature for specific antigen-binding domains (e.g., TROP2 and CD28 binding domains) uses the format "Hx.xx_Ly.yy", where the numbers are unique identifiers for specific variable chain sequences. For example, the CD28 binding domain "1A7[CD28_H1_L1]" (Figure 15) contains the variable heavy chain domain H1 and the variable light chain domain L1. When these sequences are used as scFv, "H1_L1" is used. The designation indicates that the binding domain contains a variable heavy chain domain "H1" and a variable light chain domain "L1," and is VH-linker-VL oriented (N-terminus to C-terminus). A molecule in which the sequences of the heavy chain variable domain and the light chain variable domain are identical but in the reverse order (VL-linker-VH orientation (N-terminus to C-terminus)) would be named "L1_H1." Similarly, as is evident from the sequence list and figures, different constructs can "mix and harmonize" the heavy and light chains.

[0025] III. Definition Some definitions are provided below to help you understand this application more fully. Such definitions are intended to encompass grammatical equivalents.

[0026] "B lymphocyte antigen CD20", "CD20", "MS4A1", "B1", "Bp35", "CD20", "CVID5", "LEU-16", "MS4A2", "S7, transmembrane 4-domain A1", and "FMC7" (e.g., Genebank acceptance numbers NP_068769 (human), NP_690605 (human), NP_690606 (human)) refer to B lymphocyte surface molecules that play a role in B cell development and differentiation into plasma cells. In humans, CD20 is encoded by the MS4A1 gene. CD20 is present in B cell development from late pro-B cells to memory cells. CD20 is found in B cell lymphoma, pilocytic cell leukemia, B cell chronic lymphocytic leukemia, and melanoma cancer stem cells. An exemplary CD20 sequence is shown in Figure 2. Unless otherwise noted, references to CD20 refer to human CD20 sequences.

[0027] In this specification, “CD28,” “Surface Antigen Classification 28,” and “Tp44” (e.g., Genebank acceptance numbers NP_001230006 (human), NP_001230007 (human), NP_006130 (human), and NP_031668 (mouse)) refer to B7 receptors expressed on T cells that provide costimulatory signals necessary for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation via CD28 provides a potent signal for the production of various interleukins. CD28 is a receptor for the CD80 (B7.1) and CD86 (B7.2) proteins. CD28 contains an intercellular domain with a YMNM motif (SEQ ID NO: 517) that is important for the recruitment of SH2 domain-containing proteins, particularly PI3K. CD28 also contains two proline-rich motifs that can bind SH3-containing proteins. A typical sequence of CD28 is shown in Figure 1. Unless otherwise specified, references to CD28 refer to the human CD28 sequence.

[0028] As used herein, “excision” means a reduction or removal of activity. Therefore, for example, “excision of FcγR binding” means that the Fc region amino acid variant has less than 50% of the starting binding compared to an Fc region without the particular variant. FcγR binding is evaluated using binding analysis such as surface plasmon resonance (SPR) analysis, such as Biacore, or biolayer interferometry (BLI), and excision is defined as a loss of more than 50, 70, 80, 90, 95, or 98% of binding activity. Those specifically used for excision of FcγR binding are shown in Figure 5, and they are generally included in the bispecific antibodies disclosed herein.

[0029] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated response in which nonspecific cytotoxic cells expressing FcγR recognize a bound antibody on target cells, subsequently lysing the target cells. ADCC activity is correlated with binding to FcγRIIIa, with increased binding to FcγRIIIa leading to increased ADCC activity.

[0030] As used herein, “ADCP” or “antibody-dependent cell-mediated phagocytosis” refers to a cell-mediated response in which nonspecific phagocytic cells expressing FcγR recognize a bound antibody on a target cell, subsequently causing phagocytosis of the target cell.

[0031] As used herein, the term “antibody” is used in general terms. The antibodies provided herein can take many forms, including conventional antibodies as described herein, as well as antibody derivatives, fragments, and mimetic forms described herein.

[0032] Conventional immunoglobulin (Ig) antibodies are "Y"-shaped tetramers. Each tetramer typically contains two pairs of identical polypeptide chains, each pair having one "light chain" monomer (typically with a molecular weight of about 25 kDa) and one "heavy chain" monomer (typically with a molecular weight of about 50–70 kDa).

[0033] Non-limiting examples of suitable antibody fragments include, but are not limited to, bispecific antibodies and single-chain molecules, and Fab, F(ab')2, Fc, Fabc, and Fv molecules, single-chain (Sc) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions of antibody chains or CDRs with other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy chain and one light chain, monovalent fragments consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain, bivalent fragments containing two Fab fragments disulfide-bonded at a hinge region, Fd fragments essentially consisting of a VH domain and a CH1 domain, Fv fragments essentially consisting of a VL domain and a VH domain on one arm of an antibody, dAb fragments essentially consisting of a VH domain, camelid antibodies or nanobodies, and isolated complementarity-determining regions (CDRs).

[0034] Other useful antibody formats, as used herein, include, but are not limited to, the “1+1Fab-scFv-Fc”, “2+1Fab2-scFv-Fc”, “2+1Stack Fab2-scFv-Fc”, and “2+1mAb-scFv” formats (Figure 29). Additional useful antibody formats include, but are not limited to, antibodies in the “1+1 Common Light Chain”, “2+1 Common Light Chain”, “mAb-Fv”, “mAb-scFv”, “Center-Fv”, “Single-Armed scFv-mAb”, “scFv-mAb”, “Dual cFv”, and “Triproxy” formats (Figure 29). See also US20180127501A1, incorporated herein by reference, particularly the relevant portions concerning antibody formats (see, for example, Figure 2 of US20180127501A1).

[0035] Antibody heavy chains typically contain a variable heavy chain (VH) domain including vhCDR1-3 and an Fc domain containing a CH2-CH3 monomer. In some embodiments, the antibody heavy chain includes a hinge and a CH1 domain. Conventional antibody heavy chains are monomers structured from the N-terminus to the C-terminus, such as VH-CH1-hinge-CH2-CH3. The CH1-hinge-CH2-CH3 is collectively called the "constant domain" or "constant region" of the antibody heavy chain, and there are five different types or "isotypes": IgA, IgD, IgG, IgE, and IgM.

[0036] In some embodiments, the antibodies provided herein include several subtypes, including but not limited to IgG1, IgG2, IgG3, and IgG4, which are constant domains of the IgG isotype. IgG subtypes of immunoglobulins have several immunoglobulin domains in their heavy chain. In this specification, “immunoglobulin (Ig) domain” means a region of immunoglobulin having a distinct tertiary structure. Of particular interest in the present invention are the heavy chain domains, which include constant heavy chain (CH) domains and hinge domains. In the context of IgG antibodies, each IgG isotype has three CH regions. Therefore, in the context of IgG, the “CH” domains are as follows: “CH1” refers to positions 118–215 according to the Kabat EU index; “Hinge” refers to positions 216–230 according to the Kabat EU index; “CH2” refers to positions 231–340 according to the Kabat EU index; and “CH3” refers to positions 341–447 according to the Kabat EU index. As shown in Table 1, the precise numbering and arrangement of heavy chain domains may differ between different numbering systems. As described herein and below, pI variants may reside in one or more CH regions, as well as in the hinge region, which will be discussed later.

[0037] It should be noted that IgG1 has different allotypes, exhibiting polymorphism in 356(D or E) and 358(L or M). While the sequences presented herein use the 356E / 358M allotype, other allotypes are also included herein. That is, any sequence containing an IgG1 Fc domain included herein may have 356D / 358L instead of the 356E / 358M allotype. It should be understood that therapeutic antibodies may also include isotype and / or subtype hybrids. For example, as shown in U.S. Publication 2009 / 0163699 incorporated by reference, the antibodies of the present invention include human IgG1 / G2 hybrids in some embodiments.

[0038] As used herein, “Fc” or “Fc region” or “Fc domain” means the polypeptide constituting the constant region of an antibody, and optionally includes all or part of the hinge, except for all or part of the first constant region immunoglobulin domain (e.g., CH1). In the case of IgG, the Fc domain includes the immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3), and optionally all or part of the hinge region between CH1 (Cγ1) and CH2 (Cγ2). Thus, optionally, the Fc domain includes CH2-CH3 and hinge-CH2-CH3 (N-terminus to C-terminus). In some embodiments, the Fc domain is from IgG1, IgG2, IgG3, or IgG4, and IgG1 hinge-CH2-CH3 and IgG4 hinge-CH2-CH3 are found to have special uses in many embodiments. Furthermore, in the case of the human IgG1 Fc domain, the hinge may include the C220S amino acid substitution. Furthermore, in the case of the human IgG4 Fc domain, the hinge may include the S228P amino acid substitution. While the boundaries of the Fc region may differ, the human IgG heavy chain Fc region is typically defined as encompassing residues E216, C226, or A231 to its carboxyl terminus, with numbering following EU indices such as Kabat. In some embodiments, as described in more detail below, amino acid modifications are made to the Fc region, for example, to alter the binding to one or more FcγR or FcRn.

[0039] In this specification, “heavy chain constant region” means the CH1-hinge-CH2-CH3 portion of an antibody (or fragment thereof) excluding the variable heavy chain domain. In the EU numbering of human IgG1, this corresponds to amino acids 118-447. In this specification, “heavy chain constant region fragment” means a heavy chain constant region that, from either the N-terminus or C-terminus, or both, contains fewer amino acids but still retains the ability to form dimers with another heavy chain constant region.

[0040] Another type of heavy chain domain is the hinge region. In this specification, “hinge,” “hinge region,” “antibody hinge region,” or “hinge domain” refers to a flexible polypeptide containing amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain terminates at EU215, and the IgG CH2 domain begins at residue EU231. Therefore, in the case of IgG, the antibody hinge is defined herein as encompassing positions 216 (E216 in IgG1) through 230 (P230 in IgG1). In some cases, a “hinge fragment” is used, which contains fewer amino acids at either the N-terminus or C-terminus, or both, of the hinge domain. As described herein, pI variants can also be created in the hinge region. Many of the antibodies herein have at least one cysteine ​​at position 220 (hinge region) according to EU numbering substituted with serine. Generally, this modification is on the "scFv monomer" side (where the 1+1 or 2+1 form is used) for most of the sequences described herein, but it can also be on the "Fab monomer" side, or both, to reduce disulfide formation. Specifically, the sequences described herein include those in which one or both of these cysteines are substituted (C220S).

[0041] As those skilled in the art will understand, the precise numbering and arrangement of heavy chain constant domains (i.e., CH1 domain, hinge domain, CH2 domain, and CH3 domain) can vary between different numbering systems. A useful comparison of EU and Kabat heavy chain constant domain numbering is provided below; please refer to Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, and Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda (these are incorporated in their entirety by reference).

[0042] [Table 1]

[0043] Antibody light chains generally consist of two domains: a variable light chain domain (VL) containing light chain CDR vlCDR1-3, and a constant light chain region (often called CL or Cκ). Antibody light chains are typically structured from the N-terminus to the C-terminus, such as VL-CL.

[0044] In this specification, “antigen-binding domain” or “ABD” means a set of six complementary determinant regions (CDRs) that, when present as part of a polypeptide sequence, specifically bind to a target antigen (e.g., CD20 or CD28) as discussed herein. As is known in the art, these CDRs generally exist as a first set of variable heavy chain CDRs (vhCDR or VHCDR) and a second set of variable light chain CDRs (vlCDR or VLCDR), each comprising three CDRs: vhCDR1, vhCDR2, vhCDR3 variable heavy chain CDRs and vlCDR1, vlCDR2, and vlCDR3 variable light chain CDRs. The CDRs are located within the variable heavy chain domains (vhCDR1-3) and the variable light chain domains (vlCDR1-3). The variable heavy chain domains and variable light chain domains form the Fv region.

[0045] The present invention provides a set of numerous different CDRs. In this case, a “complete set of CDRs” includes three variable light chain CDRs and three variable heavy chain CDRs, e.g., vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2, and vhCDR3. Each of these may be a larger variable light chain domain or part of a variable heavy chain domain. Furthermore, as outlined in more detail herein, the variable heavy and variable light chain domains may reside on separate polypeptide chains when heavy and light chains are used (e.g., when Fab is used), or on a single polypeptide chain in the case of scFv sequences.

[0046] As those skilled in the art will understand, the exact numbering and arrangement of CDRs may differ between different numbering systems. However, it should be understood that the disclosure of variable heavy chain sequences and / or variable light chain sequences includes the disclosure of the relevant (unique) CDRs. Thus, the disclosure of each variable heavy chain region is a disclosure of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light chain region is a disclosure of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3). A useful comparison of CDR numbering is as follows:

[0047] [Table 2]

[0048] Throughout this specification, the Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 in the light chain variable region and residues 1-113 in the heavy chain variable region), and the EU numbering system is used for the Fc region.

[0049] CDRs contribute to the formation of antigen-binding sites, or more specifically, antigen-binding domains and antibody epitope-binding sites. An "epitope" refers to a determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule, also known as a paratope. Epitopes are clusters of molecules, such as amino acid or sugar side chains, and typically possess specific structural and charge properties. A single antigen may have multiple epitopes.

[0050] Antibodies that recognize the same epitope can be identified by simple immunoanalysis, which shows that one antibody has the ability to inhibit another antibody from binding to the target antigen, for example, by "binning." As outlined below, the present invention includes not only the antigen-binding domains and antibodies listed herein, but also those that compete for binding with the epitopes bound by the listed antigen-binding domains.

[0051] In some embodiments, the six CDRs of the antigen-binding domain are contributed by a variable heavy chain domain and a variable light chain domain. In the “Fab” form, the set of six CDRs consists of two different polypeptide sequences, a variable heavy chain domain (vh or VH; including vhCDR1, vhCDR2, and vhCDR3), and a variable light chain domain (vl or VL; including vlCDR1, vlCDR2, and vlCDR3), where the C-terminus of the vh domain binds to the N-terminus of the CH1 domain of the heavy chain, and the C-terminus of the vl domain binds to the N-terminus of the constant light chain domain (thus forming a light chain). In the scFv form, the vh and vl domains are generally covalently bound to a single polypeptide sequence through the use of a linker ("scFv linker"), as outlined herein, which may be either vh-linker-vl or vl-linker-vh (starting from the N-terminus). Generally, the C-terminus of the scFv domain is bound to the N-terminus of all or part of the hinge in the second monomer.

[0052] As used herein, “variable region” or “variable domain” means a region of immunoglobulin containing one or more Ig domains substantially encoded by any of the Vκ, Vλ, and / or VH genes, which constitute the kappa, lambda, and heavy chain immunoglobulin loci, respectively, and containing antigen-constituting CDRs. Thus, a “variable heavy chain domain” pairs with a “variable light chain domain” to form an antigen-binding domain (“ABD”). Furthermore, each variable domain contains three hypervariable regions (“complementary determining regions,” “CDRs”) (vhCDR1, vhCDR2, and vhCDR3 in the case of a variable heavy chain domain; vlCDR1, vlCDR2, and vlCDR3 in the case of a variable light chain domain) and four framework (FR) regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0053] As used herein, “Fab” or “Fab region” generally refers to an antibody region comprising VH, CH1, VL, and CL immunoglobulin domains present on two distinct polypeptide chains (e.g., VH-CH1 on one chain and VL-CL on the other). Fab may refer to this region alone or, in the context of the bispecific antibody of the present invention, to this region. In the context of Fab, Fab includes the Fv region in addition to the CH1 and CL domains.

[0054] As used herein, “Fv,” “Fv fragment,” or “Fv region” means an antibody region containing the VL and VH domains. The Fv region can take the form of both Fab (two distinct polypeptides, generally including the constant region outlined above, as described above) and single-stranded Fv (scFv), in which the vl and vh domains are contained within a single peptide and are generally linked by a linker, as discussed herein.

[0055] In this specification, “single-stranded Fv” or “scFv” means a variable heavy-chain domain that covalently binds to a variable light-chain domain using generally scFv linkers, as discussed herein, to form an scFv domain or scFv domain. The scFv domain can be in either direction from the N-terminus to the C-terminus (vh-linker-vl, or vl-linker-vh). In the sequences shown in the sequence listings and figures, the order of the vh and vl domains is indicated in the name; for example, H.X_L.Y means vh-linker-vl from the N-terminus to the C-terminus, and L.Y_H.X means vl-linker-vh.

[0056] Some embodiments of the target antibodies provided herein include at least one scFv domain, which includes a variable heavy chain domain and a variable light chain domain, which are not naturally occurring but are generally linked by an scFv linker. As outlined herein, the scFv domain is generally oriented as VH-scFv linker-VL from the N-terminus to the C-terminus, but this can be reversed to VL-scFv linker-VH for any scFv domain (or one constructed using vh and vl sequences from Fabs), and depending on the form, may have any linker at one or both ends.

[0057] In this specification, “modification” or “mutation” means an amino acid substitution, insertion, and / or deletion in a polypeptide sequence, or an alteration of a portion chemically bound to a protein. For example, a modification may be a change in a carbohydrate bound to a protein or a PEG structure. In this specification, “amino acid modification” means an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. For clarity, unless otherwise specified, amino acid modifications always refer to amino acids encoded by DNA, such as the 20 amino acids that have codons in DNA and RNA.

[0058] In this specification, “amino acid substitution” or “substitution” means the substitution of an amino acid at a specific position in a parent polypeptide sequence with a different amino acid. Specifically, in some embodiments, a substitution is a substitution of an amino acid that is not naturally present in an organism or is not naturally present at a specific position in any organism. For example, substitution E272Y refers to a mutant polypeptide in which glutamic acid at position 272 is substituted with tyrosine, in this case the Fc mutant. To clarify, a protein that has been manipulated to change the nucleic acid coding sequence but not the starting amino acid (for example, replacing CGG (which codes for arginine) with CGA (which also codes for arginine) to increase the expression level in a host organism) is not an “amino acid substitution.” In other words, if a new gene has been created that codes for the same protein, but the protein has the same amino acid at the specific position where it started, it is not an amino acid substitution.

[0059] As used herein, “amino acid insertion” or “insertion” means the addition of an amino acid sequence to a specific position in the parent polypeptide sequence. For example, -233E or 233E specifies the insertion of glutamic acid after position 233 and before position 234. Furthermore, -233ADE or A233ADE specifies the insertion of AlaAspGlu after position 233 and before position 234.

[0060] As used herein, "amino acid deletion" or "deletion" means the removal of an amino acid sequence at a specific position in the parent polypeptide sequence. For example, E233-, E233#, E233_, E233(), and E233del all indicate the deletion of glutamic acid at position 233. Furthermore, EDA233- or EDA233# specifies the deletion of a sequence beginning at position 233, GluAspAla.

[0061] As used herein, “mutant protein,” “protein variant,” or “variant” means a protein that differs from the parent protein by at least one amino acid modification. A mutant protein has at least one amino acid modification compared to the parent protein, but not so many that it would not align with the parent protein using the adjustment programs described below. Generally, mutant proteins (such as the mutant Fc domain outlined herein) are generally at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the parent protein using the adjustment programs described below, such as BLAST. See Altschul, SF et al., (1990) “Basic Local Alignment Search Tool,” J.Mol.Biol.215:403-10, “BLAST” algorithm, https: / / blast.ncbi.nlm.nih.gov / Blast.cgi.

[0062] As used herein, “Fc variant” or “variant Fc” means a protein that contains amino acid modifications in its Fc domain. These modifications may be additions, deletions, or substitutions. Fc variants are defined according to the amino acid modifications that constitute them. Thus, N434S or 434S is an Fc variant in which the 434th position is replaced with serine, relative to the parent Fc polypeptide, where the numbering follows the EU index. Similarly, M428L / N434S defines an Fc variant with the M428L and N434S substitutions relative to the parent Fc polypeptide. In cases where the identity of the WT amino acids cannot be determined, the aforementioned variant is called 428L / 434S. It should be noted that the order in which substitutions are provided is arbitrary; for example, 428L / 434S may be the same Fc variant as 434S / 428L. For all positions discussed herein relating to antibodies or their derivatives and fragments (e.g., Fc domains), unless otherwise specified, amino acid position numbering follows the EU index. The “EU index,” or “EU index in Kabat,” or “EU numbering” scheme refers to the EU antibody numbering (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, which is incorporated herein by reference in its entirety). Modifications may be additions, deletions, or substitutions.

[0063] Generally, mutant Fc domains have at least approximately 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with the corresponding parental human IgG Fc domain (using the identity algorithm described later, and in one embodiment, using the BLAST algorithm known in the art with initial value parameters). Alternatively, mutant Fc domains may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the parental Fc domain. Alternatively, the mutant Fc domain may have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the parent Fc domain. Furthermore, as discussed herein, the mutant Fc domains described herein still retain the ability to form dimers with other Fc domains, as measured using known techniques described herein, such as non-denaturing gel electrophoresis.

[0064] As used herein, “protein” means at least two covalently bonded amino acids and includes proteins, polypeptides, oligopeptides, and peptides. Furthermore, polypeptides constituting the antibodies of the present invention may include synthetic derivatization, glycosylation, PEGylation, cyclic permutation, cyclization, linking to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels to one or more side chains or terminals.

[0065] As used herein, "IgG variant modification" or "isotype modification" means an amino acid modification in which one amino acid of one IgG isotype is replaced with a corresponding amino acid of a different, aligned IgG isotype. For example, since IgG1 contains tyrosine and IgG2 contains phenylalanine at the EU296 position, the F296Y substitution in IgG2 is considered an IgG variant modification.

[0066] As used herein, “non-spontaneous modification” means a non-isotype amino acid modification. For example, since no human IgG contains serine at position 434, a 434S substitution in IgG1, IgG2, IgG3, or IgG4 (or a hybrid thereof) is considered a non-spontaneous modification.

[0067] As used herein, “amino acid” and “amino acid identity” mean one of the 20 naturally occurring amino acids encoded by DNA and RNA.

[0068] As used herein, “effector function” means a biochemical event resulting from the interaction between an antibody Fc region and an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0069] As used herein, “IgG Fc ligand” means any biologically derived molecule, preferably polypeptide, that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex. Fc ligands include, but are not limited to, FcγRI, FcγRII, FcγRIII, FcRn, C1q, C3, mannan-binding lectins, mannose receptors, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors homologous to FcγR. Fc ligands may include undiscovered molecules that bind Fc. Specific IgG Fc ligands are FcRn and the Fcγ receptor. As used herein, “Fc ligand” means any biologically derived molecule, preferably polypeptide, that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.

[0070] As used herein, “Fc gamma receptor,” “FcγR,” or “Fc gamma R” means any member of the family of proteins encoded by the FcγR gene that ligates to the Fc region of an IgG antibody. In humans, this family includes, but is not limited to, FcγRI(CD64) including isotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32) including isotypes FcγRIIa (including allotypes H131 and R131), FcγRIIb (including allotypes FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16) including isotypes FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2), as well as any undiscovered human FcγR, or isotypes or allotypes of FcγR. FcγR may originate from any organism, including, but is not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγR includes, but is not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγR, or FcγR isotype, or allotype.

[0071] As used herein, “FcRn” or “neonatal Fc receptor” means a protein that encapsulates the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn may originate from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, a functional FcRn protein often comprises two polypeptides, called the heavy chain and the light chain. The light chain is β2-microglobulin, and the heavy chain is encoded by the FcRn gene. In this specification, unless otherwise stated, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and β-2-microglobulin. Various FcRn variants are used to increase binding to the FcRn receptor and, in some cases, to increase its serum half-life. An “FcRn variant” is an amino acid modification that contributes to increased binding to the FcRn receptor, and preferred FcRn variants are listed below.

[0072] As used herein, “parent polypeptide” means the starting polypeptide that has been modified to produce the mutant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or modified form of a naturally occurring polypeptide. Thus, as used herein, “parent immunoglobulin” means the unmodified immunoglobulin polypeptide that has been modified to produce the mutant, and as used herein, “parent antibody” means the unmodified antibody that has been modified to produce the mutant antibody. It should be noted that “parent antibody” includes known commercially available recombinant antibodies, such as those outlined below. In this context, “parent Fc domain” would be relative to the described mutant. Thus, “mutant human IgG1 Fc domain” is compared to the parent Fc domain of human IgG1, “mutant human IgG4 Fc domain” is compared to the parent Fc domain of human IgG4, and so on.

[0073] In the context of the monomers of the heterodimer antibodies of the present invention as used herein, “chain-like” means that the heterodimerizing variants are incorporated into each monomer so as to retain the ability to “match” in order to form a heterodimer, similar to two strands of “matching” DNA. For example, if several pI variants are manipulated into monomer A (e.g., to increase pI), the stereoisomers, which are similarly available “charge pairs,” do not interfere with the pI variants. For example, the charged variants that increase pI are placed on the same “chain” or “monomer” to retain both functionalities. Similarly, for “asymmetric” variants that form a pair, as outlined in more detail below, those skilled in the art will consider pI when determining which chain or monomer one of the pairs will be placed in so asymmetric pI also maximizes pI segregation.

[0074] This specification provides numerous antibody domains (e.g., Fc domains) that have sequence identity with human antibody domains. Sequence identity between two similar sequences (e.g., antibody variable domains) can be measured by algorithms such as those described below: Smith, TF & Waterman, MS (1981) “Comparison Of Biosequences,” Adv.Appl.Math.2:482 (Local Homology Algorithm); Needleman, SB & Wunsch, CD (1970) “A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins,” J.Mol.Biol.48:443 (Homologous Algorithm Adjustment Algorithm); Pearson, WR & Lipman, DJ (1988) “Improved Tools For Biological Sequence Comparison,” Proc.Natl.Acad.Sci.(USA)85:2444 (Similarity Search Method); or Altschul, SF et al, (1990) “Basic Local Alignment Search Tool, J.Mol.Biol.215:403-10, “BLAST” algorithm (see https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). When using any of the aforementioned algorithms, default parameters (window length, gap penalty, etc.) are used. In one embodiment, sequence identity is performed using the BLAST algorithm with default parameters.

[0075] The antibody of the present invention is generally isolated or recombinant. When used to describe the various polypeptides disclosed herein, "isolated" means a polypeptide that has been identified, separated and / or recovered from a cell or cell culture in which it is expressed. Ordinarily, an isolated polypeptide will be prepared through at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities. "Recombinant" means an antibody produced using recombinant nucleic acid technology in a heterologous host cell, and can also be isolated.

[0076] "Specific binding", or "specifically binds to" or "is specific for" a particular antigen or epitope, means a binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining the binding of a molecule as compared to the binding of a control molecule, which is generally a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target.

[0077] Specific binding to a particular antigen or epitope can be exhibited, for example, by an antibody having a KD for the antigen or epitope of at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M, or higher, wherein KD refers to the dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5000-fold, 10000-fold or more higher than the KD for a control molecule relative to the antigen or epitope.

[0078] Furthermore, specific binding to a particular antigen or epitope can be demonstrated, for example, by an antibody whose KA or Ka for the antigen or epitope is at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5000-fold, 10000-fold, or more compared to the control. Binding affinity is generally measured using Biacore, SPR, or BLI analysis.

[0079] IV. Anti-CD28 x anti-CD20 antibody In one embodiment, a novel anti-CD20 × anti-CD28 antibody is provided herein. In some embodiments, the anti-CD20 × anti-CD28 antibody described herein can bind to the CD28 costimulatory molecule on T cells and CD20 on B cells in B-cell malignancies. Such an antibody selectively enhances antitumor activity against B cells expressing CD20 in such B-cell malignancies. In embodiments, the target antibody used herein is particularly useful for the treatment of such B-cell malignancies, for example, in combination with other anticancer therapies including multivalent antibodies.

[0080] The anti-CD20 × anti-CD28 antibody is polyvalent and contains at least two antigen-binding domains (ABDs), where at least one antigen-binding domain is a CD20-binding domain and at least one antigen-binding domain is a CD28-binding domain. Any suitable CD20-binding domain and CD28-binding domain may be included in the target anti-CD20 × anti-CD28 antibody, for example, provided herein, which contains the CD20-binding domain and CD28-binding domain.

[0081] The antigen-binding domains provided herein generally include variable heavy chain domains (VH) having vhCDR1, vhCDR2, and vhCDR3, and variable light chain domains (VL) having vlCDR1, vlCDR2, and vlCDR3.

[0082] Furthermore, as mentioned above, the numbering used in the sequence list and figures for CDR identification is Kabat, but a different numbering system can be used, which will result in a change in the amino acid sequence of the CDR, as shown in Table 2.

[0083] All of the variable heavy and light chain domains listed herein can be further modified. As outlined herein, in some embodiments, a set of six CDRs may have 0, 1, 2, 3, 4, or 5 amino acid modifications (amino acid substitutions may find specific uses), as well as modifications to the framework regions of the variable heavy and light chain domains, provided that the framework (excluding the CDRs) retains at least about 80, 85, or 90% identity with a human germline sequence selected from those listed in Figure 1 of U.S. Patent No. 7,657,380 (this figure and legend are incorporated herein by reference in their entirety). Thus, for example, the same CDR described herein can be combined with different framework sequences from human germline sequences, provided that the framework regions retain at least 80, 85, 90, 95, or 99% identity with a human germline sequence selected from those listed in Figure 1 of U.S. Patent No. 7,657,380. Alternatively, a CDR may have amino acid modifications (e.g., one, two, three, four, or five amino acid modifications in a set of CDRs, i.e., any combination of CDRs may be modified as long as the framework region maintains at least 80, 85, 90, 95, or 99% identity with a human germline sequence selected from those described in Figure 1 of U.S. Patent No. 7,657,380).

[0084] As will be understood by those skilled in the art, any set of six CDRs, or VH and VL domains, can be in scFv or Fab form, which are then attached to the heavy and light chain constant domains (where the heavy chain constant domains constitute variants, including those within the CH1 and Fc domains).

[0085] Furthermore, in embodiments in which the target antibody contains scFv, the scFv may be oriented from the N-terminus to the C-terminus as VH-scFv linker-VL or VL-scFv linker-VH. In some embodiments, one or more ABDs are generally Fabs containing a VH domain on one protein chain (generally as a component of the heavy chain) and a VL domain on another protein chain (generally as a component of the light chain). An exemplary scFv linker for use with the target antibody is shown in Figure 6.

[0086] Useful CD20-binding domains and CD28-binding domains that can be included in the target anti-CD20 × anti-CD28 antibody are described in further detail herein.

[0087] In some embodiments, the anti-CD20 × anti-CD28 antibody is a bispecific antibody. In some embodiments, the anti-CD20 × anti-CD28 antibody is a divalent antibody. In some embodiments, the anti-CD20 × anti-CD28 antibody is a trivalent antibody. In some embodiments, the anti-CD20 × anti-CD28 antibody is a bispecific divalent antibody. In some embodiments, the anti-CD20 × anti-CD28 antibody contains one CD28-binding domain and one CD20-binding domain. In exemplary embodiments, the anti-CD20 × anti-CD28 antibody is a bispecific trivalent antibody. In some embodiments, the anti-CD20 × anti-CD28 antibody contains one CD28-binding domain and two CD20-binding domains.

[0088] The anti-CD20 × anti-CD28 antibodies used herein may be any useful form, including, for example, standard immunoglobulins, as well as the “1+1Fab-scFv-Fc”, “2+1Fab2-scFv-Fc”, “2+1Stack Fab2-scFv-Fc”, and “2+1mAb-scFv” forms described herein (Figure 29). Additional useful forms, but not limited to these, include: “1+1 Common Light Chain” and “2+1 Common Light Chain”, “mAb-Fv”, “mAb-scFv”, “Center-Fv”, “Single-Armed scFv-mAb”, “scFv-mAb”, “Dual scFv”, and the “Three-Pronged” form provided herein (see, for example, Figure 29). See also US20180127501A1, incorporated herein by reference, particularly the relevant parts concerning antibody forms (see, for example, Figure 2). In some embodiments, the anti-CD20 × anti-CD28 antibody is a heterodimer bispecific antibody comprising a mutant Fc domain having one of the heterodimerizing asymmetric mutants, pI mutants, and / or excision mutants described herein. See, for example, Figure 8.

[0089] It should be noted that, unless otherwise specified herein, the order of antigens in the name does not confer structure. That is, in anti-CD20 × anti-CD281 + 1Fab-scFv-Fc antibody, scFv can bind to CD20 or CD28, but in some cases, the order, as shown, identifies the structure.

[0090] The anti-CD20 × anti-CD28 antibodies provided herein further comprise different antibody domains. As described herein and known in the art, the antibodies described herein comprise different domains within the heavy and light chains, which may overlap. These domains include, but are not limited to, Fc domains, CH1 domains, CH2 domains, CH3 domains, hinge domains, heavy chain constant domains (CH1-hinge-Fc domain, or CH1-hinge-CH2-CH3), variable heavy chain domains, variable light chain domains, light chain constant domains, Fab domains, and scFv domains.

[0091] As shown herein, there are numerous suitable linkers (for use as either domain linkers or scFv linkers) that can be used to covalently bond cited domains (e.g., scFv, Fabs, Fc domains, VH domains, VL domains, etc.) containing conventional peptide bonds, generated by recombinant technology. An exemplary linker for linking domains of a target antibody to each other is shown in Figure 7. In some embodiments, the linker peptide may primarily contain the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be long enough to bond so that the two molecules adopt the correct conformations relative to each other so that they retain the desired activity. In one embodiment, the linker is about 1 to 50 amino acids long, preferably about 1 to 30 amino acids long. In one embodiment, linkers of 1 to 20 amino acids long may be used, and in some embodiments, the use of about 5 to about 10 amino acids is found. Useful linkers include, for example, glycine-serine polymers (where n is an integer of at least 1 (and generally 3-4)) including (GS)n, (GSGGS)n (SEQ ID NO: 518), (GGGGS)n (SEQ ID NO: 57), and (GGGS)n (SEQ ID NO: 519), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, various non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers.

[0092] Other linker sequences may include any sequence of any length from the CL / CH1 domain, rather than all residues from the CL / CH1 domain. For example, the first 5-12 amino acid residues of the CL / CH1 domain. Linkers may originate from immunoglobulin light chains, such as Cκ or Cλ. Linkers may originate from immunoglobulin heavy chains of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may also be sequences derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), hinge region sequences, and other native sequences from other proteins.

[0093] In some embodiments, the linker is a “domain linker” used to join any two domains together, as outlined herein. For example, in the 2+1Fab2-scFv-Fc form, there may be a domain linker that joins the C-terminus of the CH1 domain of Fab to the N-terminus of scFv, accompanied by another arbitrary domain linker (in many embodiments, Hinge is used as this domain linker) that joins the C-terminus of scFv to the CH2 domain. Any suitable linker may be used, but in many embodiments, glycine-serine polymers are utilized as domain linkers, including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 518), (GGGGS)n (SEQ ID NO: 57), and (GGGS)n (SEQ ID NO: 519) (where n is an integer of at least 1 (and generally 3-4-5)), as well as any peptide sequence that allows recombination of two domains having sufficient length and flexibility to allow each domain to retain its biological function. In some cases, and with due consideration to the "chain-like" nature as outlined below, charged domain linkers can be used, such as those used in some embodiments of the scFv linker. An exemplary useful domain linker is shown in Figure 7.

[0094] In some embodiments, the linker is an scFv linker used to covalently bond the VH domain and the VL domain, as discussed herein. Often, the scFv linker is a charged scFv linker, many of which are shown in Figure 6. Provided herein, therefore, is a charged scFv linker for facilitating separation in pI between a first monomer and a second monomer. That is, by incorporating either a positive or negative (or both in the case of a scaffold using scFvs on different monomers) charged scFv linker, this allows monomers containing the charged linker to change pI without causing further changes to the Fc domain. These charged linkers can be replaced with any scFv, including a standard linker. Again, as will be understood by those skilled in the art, the charged scFv linker is used on the correct “chain” or monomers according to the desired change in pI. For example, as discussed herein, to produce a heterodimer antibody of the form 1+1Fab-scFv-Fc, the original pI of the Fv region of each of the desired antigen-binding domains is calculated, one is selected to produce an scFv, and depending on the pI, either a positive or negative linker is selected. Charged domain linkers can also be used to increase the pI separation of the monomers of the present invention, and thus those included in Figure 6 may be used in any embodiment of this specification in which a linker is utilized. In some embodiments, the scFv is a “staple-treated” scFv that includes a “staple linker”. “Staple-treated” scFv exhibiting improved stability and / or reduced aggregation are described in further detail herein. Exemplary staple linkers useful for inclusion in such “staple-treated” scFv are provided in Figure 6.

[0095] In some embodiments, when scFv is contained in an anti-CD20 × anti-CD28 antibody (e.g., a 1+1 Fab-scFv-Fc type antibody, or a 2+1 Fab2-scFv-Fc type antibody), the scFv includes a “staple” modification that improves the stability of the scFv and / or reduces aggregation. In exemplary embodiments, such a “stapled” scFv includes: a) a first disulfide bond between a structurally preserved surface-exposed VH cysteine ​​and a first scFv linker cysteine; b) a second disulfide bond between a structurally preserved surface-exposed VL cysteine ​​and a second scFv linker cysteine; or c) a first disulfide bond between a structurally preserved surface-exposed VH cysteine ​​and a first scFv linker cysteine, and a second disulfide bond between a structurally preserved surface-exposed VL cysteine ​​and a second scFv linker cysteine. An exemplary scFv “staple linker” for inclusion in the “stapled” scFv is shown in Figure 6. A method for preparing “stapled” scFv is described in WO2021 / 030657 (which is incorporated in whole by reference, including the appropriate parts relating to the method for preparing “stapled” scFv and “stapled” scFv compositions).

[0096] Exemplary target anti-CD20×anti-CD28 antibodies are shown, for example, in Figures 33-36. During cell culture production of the anti-CD20×anti-CD28 antibodies provided herein, a C-terminal lysine residue, or a C-terminal lysine and glycine residue, may be cleaved from the heavy chain monomer, thereby resulting in mutants having a C-terminal "truncate." See, for example, Jiang et al., Journal of Pharmaceutical Sciences 105:2066-2072 (2016). Thus, in some embodiments provided herein, the anti-CD20×anti-CD28 antibody is a mutant of the anti-CD20×anti-CD28 antibody depicted in Figures 33-36, comprising a deletion of a C-terminal lysine (-K) residue, or a lysine and glycine (-GK) residue, in "chain 1" and / or "chain 2." In some embodiments, the deletions are G446del and / or K447del. In some embodiments, the anti-CD20×anti-CD28 antibody is manipulated to include the G446del and / or K447del modification in one or both of the Fc domains of the anti-CD20×anti-CD28 antibody described herein. In some embodiments, the anti-CD20×anti-CD28 antibody contains the naturally occurring G446del and / or K447del modification in one or both of the Fc domains compared to the anti-CD20×anti-CD28 antibody provided herein.

[0097] The embodiment of the anti-CD20 × anti-CD28 antibody is described in more detail below.

[0098] A. CD28 binding domain The anti-CD20 × anti-CD28 antibodies provided herein include at least one CD28-binding domain. Any suitable CD28-binding domain may be included in the anti-CD20 × anti-CD28 antibodies provided herein. In exemplary embodiments, the CD28-binding domain is a repulsive CD28ABD that advantageously provides T-cell costimulatory activity.

[0099] As those skilled in the art will understand, a preferred CD28 binding domain may include a set of six CDRs, as indicated by the underlined portion in the figures, or, if a different numbering scheme is used, as described herein, and as shown in Table 2, as identified using other modifications within the variable heavy chain (VH) domain and variable light chain (VL) domain sequences shown in Figures 15-24 and 26. A preferred CD28ABD may also include these sequences and the entire VH and VL sequences, as shown in the figures, used as scFv or Fabs.

[0100] In some embodiments, CD28ABD comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, where VH is selected from the amino acid sequences represented by SEQ ID NOs: 1, 11, 15, 63, 67, 71, 132-196, 320, 327, 335, 343, 351, 359, 367, 375, 383, 391, 399, 407, 415, 423, and 431, or variants thereof; and where VL is selected from the amino acid sequences represented by SEQ ID NOs: 5, 19, 75, 79, 200-305, 324, 331, 339, 347, 355, 371, 379, 387, 395, 403, 411, 419, and 435, or variants thereof.

[0101] In addition to the set of parent CDRs disclosed in the Figures and Sequence Lists that form the ABD against CD28, provided herein are mutant CD28ABDs having a CDR that includes at least one modification of the CD28ABD CDRs disclosed herein (e.g., Figures 15-18, 21, and 26, and the Sequence List). In one embodiment, the CD28ABD of the target anti-CD20 × anti-CD28 antibody includes a set of six CDRs having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 amino acid modifications compared to the six CD28ABD CDRs described herein, including in the Figures and Sequence Lists. In an exemplary embodiment, the target anti-CD20 × anti-CD28 antibody CD28ABD comprises a set of six CDRs having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 amino acid modifications, compared to six CDRs of CD28ABD having either VH or VL as follows: (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 79; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 111; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 79; or (i) VH having the amino acid sequence of SEQ ID NO: 320; and (ii) VL having the amino acid sequence of SEQ ID NO: 324; or (i) VH having the amino acid sequence of SEQ ID NO: 327; and (ii) VL having the amino acid sequence of SEQ ID NO: 331; or (i) VH having the amino acid sequence of SEQ ID NO: 335; and (ii) VL having the amino acid sequence of SEQ ID NO: 339; or (i) VH having the amino acid sequence of SEQ ID NO: 343; and (ii) VL having the amino acid sequence of SEQ ID NO: 347; or (i) VH having the amino acid sequence of SEQ ID NO: 351; and (ii) VL having the amino acid sequence of SEQ ID NO: 355; or (i) VH having the amino acid sequence of SEQ ID NO: 359; and (ii) VL having the amino acid sequence of SEQ ID NO: 355; or (i) VH having the amino acid sequence of SEQ ID NO: 367; and (ii) VL having the amino acid sequence of SEQ ID NO: 371; or (i) VH having the amino acid sequence of SEQ ID NO: 375; and (ii) VL having the amino acid sequence of SEQ ID NO: 380; or (i) VH having the amino acid sequence of SEQ ID NO: 391; and (ii) VL having the amino acid sequence of SEQ ID NO: 395; or (i) VH having the amino acid sequence of SEQ ID NO: 399; and (ii) VL having the amino acid sequence of SEQ ID NO: 403; or (i) VH having the amino acid sequence of SEQ ID NO: 407; and (ii) VL having the amino acid sequence of SEQ ID NO: 411; or (i) VH having the amino acid sequence of SEQ ID NO: 415; and (ii) VL having the amino acid sequence of SEQ ID NO: 419; or (i) VH having the amino acid sequence of SEQ ID NO: 423; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 431; and (ii) VL having the amino acid sequence of SEQ ID NO: 435. In certain embodiments, the target anti-CD20 × anti-CD28 antibody CD28ABD can be conjugated to the CD28 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet analysis), the latter of which is particularly used in many embodiments. In certain embodiments, CD28ABD can be conjugated to human CD28 antigen (see Figure 1).

[0102] In some embodiments, the CD28ABD of the target anti-CD20 × anti-CD28 antibody comprises six CDRs that are at least 90%, 95%, 97%, 98%, or 99% identical to six CDRs of the CD28ABD described herein, including figures and sequence lists. In exemplary embodiments, the CD28ABD of the target anti-CD20 × anti-CD28 antibody comprises six CDRs that are at least 90%, 95%, 97%, 98%, or 99% identical to six CDRs of the CD28ABD having either VH or VL as follows: (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 79; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 111; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 79; or (i) VH having the amino acid sequence of SEQ ID NO: 320; and (ii) VL having the amino acid sequence of SEQ ID NO: 324; or (i) VH having the amino acid sequence of SEQ ID NO: 327; and (ii) VL having the amino acid sequence of SEQ ID NO: 331; or (i) VH having the amino acid sequence of SEQ ID NO: 335; and (ii) VL having the amino acid sequence of SEQ ID NO: 339; or (i) VH having the amino acid sequence of SEQ ID NO: 343; and (ii) VL having the amino acid sequence of SEQ ID NO: 347; or (i) VH having the amino acid sequence of SEQ ID NO: 351; and (ii) VL having the amino acid sequence of SEQ ID NO: 355; or (i) VH having the amino acid sequence of SEQ ID NO: 359; and (ii) VL having the amino acid sequence of SEQ ID NO: 355; or (i) VH having the amino acid sequence of SEQ ID NO: 367; and (ii) VL having the amino acid sequence of SEQ ID NO: 371; or (i) VH having the amino acid sequence of SEQ ID NO: 375; and (ii) VL having the amino acid sequence of SEQ ID NO: 380; or (i) VH having the amino acid sequence of SEQ ID NO: 391; and (ii) VL having the amino acid sequence of SEQ ID NO: 395; or (i) VH having the amino acid sequence of SEQ ID NO: 399; and (ii) VL having the amino acid sequence of SEQ ID NO: 403; or (i) VH having the amino acid sequence of SEQ ID NO: 407; and (ii) VL having the amino acid sequence of SEQ ID NO: 411; or (i) VH having the amino acid sequence of SEQ ID NO: 415; and (ii) VL having the amino acid sequence of SEQ ID NO: 419; or (i) VH having the amino acid sequence of SEQ ID NO: 423; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 431; and (ii) VL having the amino acid sequence of SEQ ID NO: 435 (Figures 15, 18, 21, and 26). In certain embodiments, CD28ABD can bind to CD28 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biological interference, e.g., Octet analysis), the latter of which is particularly used in many embodiments. In certain embodiments, CD28ABD can bind to human CD28 antigen (see Figure 1).

[0103] In some embodiments, the anti-CD20 × anti-CD28 antibody comprises CD28ABD, which includes a variable heavy chain domain and / or a variable light chain domain, which are variants of the VH and VL domains of CD28ABD disclosed herein. In one embodiment, the variable VH domain and / or variable VL domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from the VH domain and / or VL domain of CD28ABD described herein, including figures and sequence listings. In exemplary embodiments, the variant VH domain and / or variant VL domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes from the VH and / or VL domain of CD28ABD having either VH or VL as follows: (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 79; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 111; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 79; or (i) VH having the amino acid sequence of SEQ ID NO: 320; and (ii) VL having the amino acid sequence of SEQ ID NO: 324; or (i) VH having the amino acid sequence of SEQ ID NO: 327; and (ii) VL having the amino acid sequence of SEQ ID NO: 331; or (i) VH having the amino acid sequence of SEQ ID NO: 335; and (ii) VL having the amino acid sequence of SEQ ID NO: 339; or (i) VH having the amino acid sequence of SEQ ID NO: 343; and (ii) VL having the amino acid sequence of SEQ ID NO: 347; or (i) VH having the amino acid sequence of SEQ ID NO: 351; and (ii) VL having the amino acid sequence of SEQ ID NO: 355; or (i) VH having the amino acid sequence of SEQ ID NO: 359; and (ii) VL having the amino acid sequence of SEQ ID NO: 355; or (i) VH having the amino acid sequence of SEQ ID NO: 367; and (ii) VL having the amino acid sequence of SEQ ID NO: 371; or (i) VH having the amino acid sequence of SEQ ID NO: 375; and (ii) VL having the amino acid sequence of SEQ ID NO: 380; or (i) VH having the amino acid sequence of SEQ ID NO: 391; and (ii) VL having the amino acid sequence of SEQ ID NO: 395; or (i) VH having the amino acid sequence of SEQ ID NO: 399; and (ii) VL having the amino acid sequence of SEQ ID NO: 403; or (i) VH having the amino acid sequence of SEQ ID NO: 407; and (ii) VL having the amino acid sequence of SEQ ID NO: 411; or (i) VH having the amino acid sequence of SEQ ID NO: 415; and (ii) VL having the amino acid sequence of SEQ ID NO: 419; or (i) VH having the amino acid sequence of SEQ ID NO: 423; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 431; and (ii) VL having the amino acid sequence of SEQ ID NO: 435 (Figures 15, 18, 21, and 26). In some embodiments, the alteration is located within the VH domain. In some embodiments, the alteration is located within the VL domain. In some embodiments, the alteration is located within both the VH and VL domains. In exemplary embodiments, CD28ABD is an scFv, and the amino acid alteration introduces a cysteine ​​residue into the VH framework region and / or VL framework region (FR1-FR4) to enable “stapling” of the scFv, as described herein. See also WO2021 / 030657 (the whole of which, including relevant parts relating to methods for producing “stapled” scFv and “stapled” scFv compositions, is incorporated by reference). Exemplary “stapled” CD28-binding domains and VH / VL domains that may be contained in the subject anti-CD20 × anti-CD28 antibodies shown herein are shown in Figures 19-21. In certain embodiments, the anti-CD20 × anti-CD28 antibody CD28ABD can bind to CD28 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biological interference, e.g., Octet analysis), the latter of which is particularly used in many embodiments. In certain embodiments, CD28ABD can bind to human CD28 antigen (see Figure 1).

[0104] In one embodiment, the CD28 antigen-binding domain of an anti-CD20 × anti-CD28 antibody includes a variable heavy chain domain (VH) having vhCDR1-3 (i.e., vhCDR1-3) of 1A7_H1sp (SEQ ID NO: 63, Figure 19). In some embodiments, the CD28 antigen-binding domain of an anti-CD20 × anti-CD28 antibody further includes any of the CD28-binding domain variable light chain domains provided herein. In exemplary embodiments, the variable light chain domain is 1A7_L1.71sp (SEQ ID NO: 79, Figure 20) or a variant thereof. In certain embodiments, the CD28ABD of an anti-CD20 × anti-CD28 antibody can bind to the CD28 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biological layer interferometry, e.g., Octet analysis), the latter of which is found to be particularly useful in many embodiments. In certain embodiments, the CD28ABD of the anti-CD20 × anti-CD28 antibody can bind to the human CD28 antigen (see Figure 1). In exemplary embodiments, the anti-CD20 × anti-CD28 antibody is an antibody in the form of "1+1Fab-scFv-Fc", "2+1Fab2-scFv-Fc", "2+1 stack Fab2-scFv-Fc", or "2+1mAb-scFv".

[0105] In one embodiment, the CD28ABD of the anti-CD20 × anti-CD28 antibody includes variable heavy chain domains (VH) having vhCDR1-3, which have 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 amino acid modifications compared to vhCDR1-3 of 1A7_H1sp (SEQ ID NO: 63, Figure 19). In some embodiments, the CD28 antigen-binding domain of the anti-CD20 × anti-CD28 antibody further includes any of the CD28-binding domain variable light chain domains provided herein. In exemplary embodiments, the variable light chain domain is 1A7_L1.71sp (SEQ ID NO: 79, Figure 20) or a variant thereof. In certain embodiments, the CD28ABD of the anti-CD20 × anti-CD28 antibody can conjugate to the CD28 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biological interferometry, e.g., Octet analysis), the latter of which is found to be particularly useful in many embodiments. In certain embodiments, the CD28ABD of the anti-CD20 × anti-CD28 antibody can conjugate to the human CD28 antigen (see Figure 1). In certain embodiments, the CD28ABD of the anti-CD20 × anti-CD28 antibody can conjugate to the human CD28 antigen (see Figure 1). In exemplary embodiments, the anti-CD20 × anti-CD28 antibody is an antibody in the form of "1+1Fab-scFv-Fc", "2+1Fab2-scFv-Fc", "2+1 stack Fab2-scFv-Fc", or "2+1mAb-scFv".

[0106] In some embodiments, the CD28ABD of the anti-CD20 × anti-CD28 antibody includes a variable heavy chain domain (VH) having vhCDR1-3 of 1A7_H1sp (SEQ ID NO: 63, Figure 19) and vhCDR1-3 that are at least 90%, 95%, 97%, 98%, or 99% identical. In some embodiments, the CD28 antigen-binding domain of the anti-CD20 × anti-CD28 antibody further includes one of the CD28-binding domain variable light chain domains provided herein. In exemplary embodiments, the variable light chain domain is 1A7_L1.71sp (SEQ ID NO: 79, Figure 20) or a variant thereof. In certain embodiments, the CD28ABD of the anti-CD20 × anti-CD28 antibody is capable of binding to CD28 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet analysis), the latter of which is found to be particularly useful in many embodiments. In certain embodiments, the CD28ABD of the anti-CD20 × anti-CD28 antibody can bind to the human CD28 antigen (see Figure 1). In exemplary embodiments, the anti-CD20 × anti-CD28 antibody is an antibody in the form of "1+1Fab-scFv-Fc", "2+1Fab2-scFv-Fc", "2+1 stack Fab2-scFv-Fc", or "2+1mAb-scFv".

[0107] In exemplary embodiments, the CD28-binding domain of the anti-CD20 × anti-CD28 antibody is scFv. In some embodiments, the anti-CD28scFv comprises one of the CD28ABD VH and / or VL, or variants thereof, as described herein. In some embodiments, the anti-CD28scFv of the anti-CD20 × anti-CD28 antibody comprises one or more “staple” modifications that improve the stability of the scFv or reduce aggregation. In some embodiments, the anti-CD20 × anti-CD28 antibody comprises a “stapled” scFv, where the stapled scFv comprises: a) a first disulfide bond between a structurally preserved surface-exposed VH cysteine ​​and a first scFv linker cysteine; b) a second disulfide bond between a structurally preserved surface-exposed VL cysteine ​​and a second scFv linker cysteine; or c) a first disulfide bond between a structurally preserved surface-exposed VH cysteine ​​and a first scFv linker cysteine, and a second disulfide bond between a structurally preserved surface-exposed VL cysteine ​​and a second scFv linker cysteine. In exemplary embodiments, the “stapled” scFv is a variant of any of the CD28ABD described herein, wherein the variant CD28ABD includes: a) amino acid substitution for introducing surface-exposed VH cysteine; b) amino acid substitution for introducing surface-exposed VL cysteine; or c) amino acid substitution for introducing both surface-exposed VH cysteine ​​and surface-exposed VH cysteine. In some embodiments, the amino acid change(s) are located in the VH framework region and / or VL framework region (FR1, FR2, FR3, or FR4). Exemplary scFv “staple linkers” for inclusion in the “stapled” scFv are provided in Figure 6. Methods for preparing the “stapled” scFv are described, for example, in WO2021 / 030657 (the whole of which, including appropriate parts relating to methods for preparing the “stapled” scFv and “stapled” scFv compositions, is incorporated by reference).In exemplary embodiments, the anti-CD20 × anti-CD28 antibody includes a “stapled” scFv of CD28ABD having one of the following VH and VL: (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 79; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 111; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 79; or (i) VH having the amino acid sequence of SEQ ID NO: 320; and (ii) VL having the amino acid sequence of SEQ ID NO: 324; or (i) VH having the amino acid sequence of SEQ ID NO: 327; and (ii) VL having the amino acid sequence of SEQ ID NO: 331; or (i) VH having the amino acid sequence of SEQ ID NO: 335; and (ii) VL having the amino acid sequence of SEQ ID NO: 339; or (i) VH having the amino acid sequence of SEQ ID NO: 343; and (ii) VL having the amino acid sequence of SEQ ID NO: 347; or (i) VH having the amino acid sequence of SEQ ID NO: 351; and (ii) VL having the amino acid sequence of SEQ ID NO: 355; or (i) VH having the amino acid sequence of SEQ ID NO: 359; and (ii) VL having the amino acid sequence of SEQ ID NO: 355; or (i) VH having the amino acid sequence of SEQ ID NO: 367; and (ii) VL having the amino acid sequence of SEQ ID NO: 371; or (i) VH having the amino acid sequence of SEQ ID NO: 375; and (ii) VL having the amino acid sequence of SEQ ID NO: 380; or (i) VH having the amino acid sequence of SEQ ID NO: 391; and (ii) VL having the amino acid sequence of SEQ ID NO: 395; or (i) VH having the amino acid sequence of SEQ ID NO: 399; and (ii) VL having the amino acid sequence of SEQ ID NO: 403; or (i) VH having the amino acid sequence of SEQ ID NO: 407; and (ii) VL having the amino acid sequence of SEQ ID NO: 411; or (i) VH having the amino acid sequence of SEQ ID NO: 415; and (ii) VL having the amino acid sequence of SEQ ID NO: 419; or (i) VH having the amino acid sequence of SEQ ID NO: 423; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 431; and (ii) VL having the amino acid sequence of SEQ ID NO: 435 (Figures 15, 18, and 26). Examples of "stapled" CD28-binding domains include, but are not limited to, those shown in Figure 21.

[0108] In some embodiments, the anti-CD20 × anti-CD28 antibody includes a CD28-binding domain comprising a CDR selected from the following: (i) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 1, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 5; (ii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 11, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 5; (iii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 1, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 19; (iv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 11, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 19; (v) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 15, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 5; (vi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 15, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 19; (vii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 63, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 75; (viii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 67, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 75; (ix) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 63, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 79; (x) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 67, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 111; (xi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 71, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 75; (xii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 71, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 79; (xiii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 320, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 324; (xiv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 327, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 331; (xv) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 335, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 339; (xvi) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 343, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 347; (xvii) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 351, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 355; (xviii) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 359, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 355; (xix) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 367, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 371; (xx) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 375, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 380; (xxi) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 391, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 395; (xxii) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 399, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 403; (xxiii) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 407, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 411; (xxiv) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 415, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 419; (xxv) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 423, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 5; or (xxvi) vhCDR1, vhCDR2 and vhCDR3 of a variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 431, and light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of a variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 435.

[0109] In some embodiments, the anti-CD20×anti-CD28 antibody comprises a CD28 binding domain comprising CDRs selected from the following: (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22.

[0110] In some embodiments, the anti-CD20 × anti-CD28 antibody includes a CD28-binding domain comprising VH and VL selected from the following: (i) VH having an amino acid sequence that is at least about 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 1; and (ii) VL having an amino acid sequence that is at least about 95% identical to SEQ ID NO: 5; or (i) VH having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 11; and (ii) VL having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 5; or (i) VH having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 1; and (ii) VL having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 19; or (i) VH having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 11; and (ii) VL having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 19; or (i) VH having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 15; and (ii) VL having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 5; or (i) VH having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 15; and (ii) VL having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 19; or (i) VH having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 63; and (ii) VL having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 75; or (i) VH having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 67; and (ii) VL having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 75; or (i) VH having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 63; and (ii) VL having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 79; or (i) VH having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 67; and (ii) VL having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 111; or (i) VH having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 71; and (ii) VL having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 75; or (i) VH having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 71; and (ii) VL having an amino acid sequence that is at least approximately 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 79.

[0111] In some embodiments, CD28ABD includes VH which is at least 90%, 95%, 97%, 98%, or 99% identical to one of the following: SEQ ID NOs: 1, 11, 15, 63, 67, 71, 132-196, 320, 327, 335, 343, 351, 359, 367, 375, 383, 391, 399, 407, 415, 423, and 431. In some embodiments, CD28ABD includes VL which is at least 90%, 95%, 97%, 98%, or 99% identical to one of the following: 5, 19, 75, 79, 200-305, 324, 331, 339, 347, 355, 371, 379, 387, 395, 403, 411, 419, and 435. In certain embodiments, CD28ABD can bind to CD28 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biological interference, e.g., Octet analysis), the latter of which is particularly used in many embodiments. In certain embodiments, CD28ABD can bind to human CD28 antigen (see Figure 1).

[0112] In some embodiments, the anti-CD20 × anti-CD28 antibody includes a CD28-binding domain comprising VH and VL selected from the following: (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 79; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 111; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 79.

[0113] In some embodiments, the CD28-binding domain in the anti-CD20 × anti-CD28 antibody includes vhCDR1 having the amino acid sequence of SEQ ID NO: 307, vhCDR2 having the amino acid sequence of SEQ ID NO: 309, and / or vhCDR3 having the amino acid sequence of SEQ ID NO: 311. In some embodiments, the CD28-binding domain in the anti-CD20 × anti-CD28 antibody includes vlCDR1 having the amino acid sequence of SEQ ID NO: 314, vlCDR2 having the amino acid sequence of SEQ ID NO: 316, and / or vlCDR3 having the amino acid sequence of SEQ ID NO: 318.

[0114] B CD20 binding domain CD20 binding domain. In some embodiments, a target antibody containing such a CD20 antigen-binding domain (e.g., an anti-CD20 × anti-CD28 bispecific antibody) advantageously targets cells expressing CD20.

[0115] As will be appreciated by those skilled in the art, a suitable CD20 binding domain may comprise a set of six CDRs, as shown as underlined CDRs in the sequence listing and Figure 30, or, if a different numbering scheme is used, as CDRs identified using other adjustments within the variable heavy chain (VH) domain and variable light chain (VL) domain sequences of those shown in Figure 30 and the sequence listing, as described herein and as shown in Table 2 (see Table 2). Suitable CD20ABDs may also comprise the entire VH and VL sequences, as shown in these sequences and figures, used as an scFv or as a Fab domain.

[0116] In some embodiments, the CD20 antigen binding domain comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH is selected from the amino acid sequences represented by SEQ ID NOs: 439, 447, 455, 463, 471, 473, 475, 477, 479, 481, 483, 487, 489, 491, 495, and 497; and wherein the VL is selected from the amino acid sequences represented by SEQ ID NOs: 443, 451, 459, 467, 472, 474, 476, 478, 480, 482, 484, 490, 492, 496, and 498.

[0117] In one embodiment, the CD20 antigen binding domain of the anti-CD20×anti-CD28 antibody comprises the six CDRs (i.e., vhCDR1-3 and vlCDR1-3) of the CD20ABD described herein, including in the figures and the sequence listing. In an exemplary embodiment, the CD20ABD is one of the following CD20ABDs: CD20-A_H1L1, CD20-A_H1.202_L1.113, CD20-B_H1L1, CD20-C, rituximab, obinutuzumab, ibritumomab, ocrelizumab, mosunetuzumab, ublituximab, belzutumab, glofitamab, epcoritamab, TRU-015_SMIP, odronextamab, Ripertamab, and ocaratuzumab (Figure 30).

[0118] In addition to the set of parent CDRs disclosed in the Figures and Sequence List that form ABDs against CD20, provided herein are variant CD20ABDs having CDRs that include at least one modification of the CD20ABD CDRs disclosed herein. In one embodiment, CD20ABD includes a set of six CDRs having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 amino acid modifications compared to the six CD20ABD CDRs described herein, including the Figures and Sequence List. In an exemplary embodiment, CD20ABD of an anti-CD20 × anti-CD28 antibody includes a set of six CDRs having 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 amino acid modifications compared to the six CD20ABD CDRs having either VH or VL as follows: (i) VH having the amino acid sequence of SEQ ID NO: 439; and (ii) VL having the amino acid sequence of SEQ ID NO: 443; or (i) VH having the amino acid sequence of SEQ ID NO: 447; and (ii) VL having the amino acid sequence of SEQ ID NO: 451; or (i) VH having the amino acid sequence of SEQ ID NO: 455; and (ii) VL having the amino acid sequence of SEQ ID NO: 459; or (i) VH having the amino acid sequence of SEQ ID NO: 463; and (ii) VL having the amino acid sequence of SEQ ID NO: 467; or (i) VH having the amino acid sequence of SEQ ID NO: 471; and (ii) VL having the amino acid sequence of SEQ ID NO: 472; or (i) VH having the amino acid sequence of SEQ ID NO: 473; and (ii) VL having the amino acid sequence of SEQ ID NO: 474; or (i) VH having the amino acid sequence of SEQ ID NO: 475; and (ii) VL having the amino acid sequence of SEQ ID NO: 472; or (i) VH having the amino acid sequence of SEQ ID NO: 477; and (ii) VL having the amino acid sequence of SEQ ID NO: 478; or (i) VH having the amino acid sequence of SEQ ID NO: 479; and (ii) VL having the amino acid sequence of SEQ ID NO: 480; or (i) VH having the amino acid sequence of SEQ ID NO: 481; and (ii) VL having the amino acid sequence of SEQ ID NO: 482; or (i) VH having the amino acid sequence of SEQ ID NO: 483; and (ii) VL having the amino acid sequence of SEQ ID NO: 484; or (i) VH having the amino acid sequence of SEQ ID NO: 487; and (ii) VL having the amino acid sequence of SEQ ID NO: 467; or (i) VH having the amino acid sequence of SEQ ID NO: 489; and (ii) VL having the amino acid sequence of SEQ ID NO: 490; or (i) VH having the amino acid sequence of SEQ ID NO: 491; and (ii) VL having the amino acid sequence of SEQ ID NO: 492; or (i) VH having the amino acid sequence of SEQ ID NO: 495; and (ii) VL having the amino acid sequence of SEQ ID NO: 496; or (i) VH having the amino acid sequence of SEQ ID NO: 497; and (ii) VL having the amino acid sequence of SEQ ID NO: 498 (Figure 30).

[0119] In certain embodiments, the mutant CD20ABD can be conjugated to the CD20 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet analysis), the latter of which is particularly used in many embodiments. In certain embodiments, CD20ABD can be conjugated to the human CD20 antigen (see Figure 2).

[0120] In one embodiment, CD20ABD of an anti-CD20 × anti-CD28 antibody comprises six CDRs that are at least 90%, 95%, 97%, 98%, or 99% identical to six CDRs of CD20ABD described herein, including figures and sequence lists. In an exemplary embodiment, CD20ABD comprises six CDRs that are at least 90%, 95%, 97%, 98%, or 99% identical to six CDRs of CD20ABD having either VH or VL as follows: (i) VH having the amino acid sequence of SEQ ID NO: 439; and (ii) VL having the amino acid sequence of SEQ ID NO: 443; or (i) VH having the amino acid sequence of SEQ ID NO: 447; and (ii) VL having the amino acid sequence of SEQ ID NO: 451; or (i) VH having the amino acid sequence of SEQ ID NO: 455; and (ii) VL having the amino acid sequence of SEQ ID NO: 459; or (i) VH having the amino acid sequence of SEQ ID NO: 463; and (ii) VL having the amino acid sequence of SEQ ID NO: 467; or (i) VH having the amino acid sequence of SEQ ID NO: 471; and (ii) VL having the amino acid sequence of SEQ ID NO: 472; or (i) VH having the amino acid sequence of SEQ ID NO: 473; and (ii) VL having the amino acid sequence of SEQ ID NO: 474; or (i) VH having the amino acid sequence of SEQ ID NO: 475; and (ii) VL having the amino acid sequence of SEQ ID NO: 472; or (i) VH having the amino acid sequence of SEQ ID NO: 477; and (ii) VL having the amino acid sequence of SEQ ID NO: 478; or (i) VH having the amino acid sequence of SEQ ID NO: 479; and (ii) VL having the amino acid sequence of SEQ ID NO: 480; or (i) VH having the amino acid sequence of SEQ ID NO: 481; and (ii) VL having the amino acid sequence of SEQ ID NO: 482; or (i) VH having the amino acid sequence of SEQ ID NO: 483; and (ii) VL having the amino acid sequence of SEQ ID NO: 484; or (i) VH having the amino acid sequence of SEQ ID NO: 487; and (ii) VL having the amino acid sequence of SEQ ID NO: 467; or (i) VH having the amino acid sequence of SEQ ID NO: 489; and (ii) VL having the amino acid sequence of SEQ ID NO: 490; or (i) VH having the amino acid sequence of SEQ ID NO: 491; and (ii) VL having the amino acid sequence of SEQ ID NO: 492; or (i) VH having the amino acid sequence of SEQ ID NO: 495; and (ii) VL having the amino acid sequence of SEQ ID NO: 496; or (i) VH having the amino acid sequence of SEQ ID NO: 497; and (ii) VL having the amino acid sequence of SEQ ID NO: 498 (Figure 30).

[0121] In certain embodiments, CD20ABD can bind to the CD20 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biological interference, e.g., Octet analysis), the latter of which is particularly used in many embodiments. In certain embodiments, CD20ABD can bind to human CD20 antigen (see Figure 2).

[0122] In another exemplary embodiment, the CD20ABD of the anti-CD20 × anti-CD28 antibody comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain of any one of the CD20ABDs described herein, including the figures and sequence listings. In the exemplary embodiment, the CD20ABD comprises one of the following VH and VL: (i) VH having the amino acid sequence of SEQ ID NO: 439; and (ii) VL having the amino acid sequence of SEQ ID NO: 443; or (i) VH having the amino acid sequence of SEQ ID NO: 447; and (ii) VL having the amino acid sequence of SEQ ID NO: 451; or (i) VH having the amino acid sequence of SEQ ID NO: 455; and (ii) VL having the amino acid sequence of SEQ ID NO: 459; or (i) VH having the amino acid sequence of SEQ ID NO: 463; and (ii) VL having the amino acid sequence of SEQ ID NO: 467; or (i) VH having the amino acid sequence of SEQ ID NO: 471; and (ii) VL having the amino acid sequence of SEQ ID NO: 472; or (i) VH having the amino acid sequence of SEQ ID NO: 473; and (ii) VL having the amino acid sequence of SEQ ID NO: 474; or (i) VH having the amino acid sequence of SEQ ID NO: 475; and (ii) VL having the amino acid sequence of SEQ ID NO: 472; or (i) VH having the amino acid sequence of SEQ ID NO: 477; and (ii) VL having the amino acid sequence of SEQ ID NO: 478; or (i) VH having the amino acid sequence of SEQ ID NO: 479; and (ii) VL having the amino acid sequence of SEQ ID NO: 480; or (i) VH having the amino acid sequence of SEQ ID NO: 481; and (ii) VL having the amino acid sequence of SEQ ID NO: 482; or (i) VH having the amino acid sequence of SEQ ID NO: 483; and (ii) VL having the amino acid sequence of SEQ ID NO: 484; or (i) VH having the amino acid sequence of SEQ ID NO: 487; and (ii) VL having the amino acid sequence of SEQ ID NO: 467; or (i) VH having the amino acid sequence of SEQ ID NO: 489; and (ii) VL having the amino acid sequence of SEQ ID NO: 490; or (i) VH having the amino acid sequence of SEQ ID NO: 491; and (ii) VL having the amino acid sequence of SEQ ID NO: 492; or (i) VH having the amino acid sequence of SEQ ID NO: 495; and (ii) VL having the amino acid sequence of SEQ ID NO: 496; or (i) VH having the amino acid sequence of SEQ ID NO: 497; and (ii) VL having the amino acid sequence of SEQ ID NO: 498 (Figure 30).

[0123] In addition to the variable heavy and / or variable light domains of the parent CD20ABD disclosed herein, provided herein are CD20ABDs comprising variable heavy and / or variable light domains, which are variants of the VH and VL domains of the CD20ABD disclosed herein. In one embodiment, the variant VH and / or VL domains have one, two, three, four, five, six, seven, eight, nine, or ten amino acid changes from the VH and / or VL domains of the CD20ABD described herein, including figures and sequence lists. In an exemplary embodiment, the variant VH and / or VL domains have one, two, three, four, five, six, seven, eight, nine, or ten amino acid changes from the VH and / or VL domains of the CD20ABD having one of the following VH and VL: (i) VH having the amino acid sequence of SEQ ID NO: 439; and (ii) VL having the amino acid sequence of SEQ ID NO: 443; or (i) VH having the amino acid sequence of SEQ ID NO: 447; and (ii) VL having the amino acid sequence of SEQ ID NO: 451; or (i) VH having the amino acid sequence of SEQ ID NO: 455; and (ii) VL having the amino acid sequence of SEQ ID NO: 459; or (i) VH having the amino acid sequence of SEQ ID NO: 463; and (ii) VL having the amino acid sequence of SEQ ID NO: 467; or (i) VH having the amino acid sequence of SEQ ID NO: 471; and (ii) VL having the amino acid sequence of SEQ ID NO: 472; or (i) VH having the amino acid sequence of SEQ ID NO: 473; and (ii) VL having the amino acid sequence of SEQ ID NO: 474; or (i) VH having the amino acid sequence of SEQ ID NO: 475; and (ii) VL having the amino acid sequence of SEQ ID NO: 472; or (i) VH having the amino acid sequence of SEQ ID NO: 477; and (ii) VL having the amino acid sequence of SEQ ID NO: 478; or (i) VH having the amino acid sequence of SEQ ID NO: 479; and (ii) VL having the amino acid sequence of SEQ ID NO: 480; or (i) VH having the amino acid sequence of SEQ ID NO: 481; and (ii) VL having the amino acid sequence of SEQ ID NO: 482; or (i) VH having the amino acid sequence of SEQ ID NO: 483; and (ii) VL having the amino acid sequence of SEQ ID NO: 484; or (i) VH having the amino acid sequence of SEQ ID NO: 487; and (ii) VL having the amino acid sequence of SEQ ID NO: 467; or (i) VH having the amino acid sequence of SEQ ID NO: 489; and (ii) VL having the amino acid sequence of SEQ ID NO: 490; or (i) VH having the amino acid sequence of SEQ ID NO: 491; and (ii) VL having the amino acid sequence of SEQ ID NO: 492; or (i) VH having the amino acid sequence of SEQ ID NO: 495; and (ii) VL having the amino acid sequence of SEQ ID NO: 496; or (i) VH having the amino acid sequence of SEQ ID NO: 497; and (ii) VL having the amino acid sequence of SEQ ID NO: 498 (Figure 30).

[0124] In some embodiments, the alteration is located in the VH domain shown in Figure 30. In some embodiments, the alteration is located in the VL domain shown in Figure 30. In some embodiments, the alteration is located in both the VH and VL domains shown in Figure 30. In some embodiments, the amino acid alteration(s) is located in the VH framework region and / or the VL framework region (FR1, FR2, FR3, or FR4). In certain embodiments, the CD20ABD anti-CD20 × anti-CD28 antibody can bind to CD20 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biological layer interferometry, e.g., Octet analysis) analysis, the latter of which is found to be particularly useful in many embodiments. In certain embodiments, the CD20ABD can bind to the human CD20 antigen.

[0125] In one embodiment, the mutant VH domain and / or VL domain is at least 90%, 95%, 97%, 98%, or 99% identical to the VH and / or VL of CD20ABD described herein, including in the figures and sequence listings. In an exemplary embodiment, the mutant VH domain and / or VL domain is at least 90%, 95%, 97%, 98%, or 99% identical to the VH and / or VL of CD20ABD having one of the following VH and VL: (i) VH having the amino acid sequence of SEQ ID NO: 439; and (ii) VL having the amino acid sequence of SEQ ID NO: 443; or (i) VH having the amino acid sequence of SEQ ID NO: 447; and (ii) VL having the amino acid sequence of SEQ ID NO: 451; or (i) VH having the amino acid sequence of SEQ ID NO: 455; and (ii) VL having the amino acid sequence of SEQ ID NO: 459; or (i) VH having the amino acid sequence of SEQ ID NO: 463; and (ii) VL having the amino acid sequence of SEQ ID NO: 467; or (i) VH having the amino acid sequence of SEQ ID NO: 471; and (ii) VL having the amino acid sequence of SEQ ID NO: 472; or (i) VH having the amino acid sequence of SEQ ID NO: 473; and (ii) VL having the amino acid sequence of SEQ ID NO: 474; or (i) VH having the amino acid sequence of SEQ ID NO: 475; and (ii) VL having the amino acid sequence of SEQ ID NO: 472; or (i) VH having the amino acid sequence of SEQ ID NO: 477; and (ii) VL having the amino acid sequence of SEQ ID NO: 478; or (i) VH having the amino acid sequence of SEQ ID NO: 479; and (ii) VL having the amino acid sequence of SEQ ID NO: 480; or (i) VH having the amino acid sequence of SEQ ID NO: 481; and (ii) VL having the amino acid sequence of SEQ ID NO: 482; or (i) VH having the amino acid sequence of SEQ ID NO: 483; and (ii) VL having the amino acid sequence of SEQ ID NO: 484; or (i) VH having the amino acid sequence of SEQ ID NO: 487; and (ii) VL having the amino acid sequence of SEQ ID NO: 467; or (i) VH having the amino acid sequence of SEQ ID NO: 489; and (ii) VL having the amino acid sequence of SEQ ID NO: 490; or (i) VH having the amino acid sequence of SEQ ID NO: 491; and (ii) VL having the amino acid sequence of SEQ ID NO: 492; or (i) VH having the amino acid sequence of SEQ ID NO: 495; and (ii) VL having the amino acid sequence of SEQ ID NO: 496; or (i) VH having the amino acid sequence of SEQ ID NO: 497; and (ii) VL having the amino acid sequence of SEQ ID NO: 498 (Figure 30).

[0126] In some embodiments, CD20ABD includes VH, which is at least 90%, 95%, 97%, 98%, or 99% identical to the VH domain shown in Figure 30. In some embodiments, CD20ABD includes VL, which is at least 90%, 95%, 97%, 98%, or 99% identical to the VL domain shown in Figure 30. In some embodiments, CD20ABD includes VH and VL, which are at least 90%, 95%, 97%, 98%, or 99% identical to the VH and VL domains shown in Figure 30. In certain embodiments, CD20ABD of an anti-CD20 × anti-CD28 antibody can bind to CD20 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biological interferometry, e.g., Octet analysis) analysis, the latter of which is found to be particularly useful in many embodiments. In certain embodiments, CD20ABD can be used to bind human CD20 antigen.

[0127] In some embodiments, the anti-CD20 × anti-CD28 antibody is a bivalent antibody containing one CD20-binding domain (e.g., a 1+1Fab-scFv-Fc type antibody). In other embodiments, the anti-CD20 × anti-CD28 antibody is a trivalent antibody containing two CD20-binding domains (e.g., a 2+1Fab2-scFv-Fc type antibody).

[0128] In some embodiments, the CD20-binding domain CDR(s) are selected from the following: (i) Heavy chain complementarity determination regions 1 (vhCDR1), vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 439, and light chain complementarity determination regions 1 (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 443; (ii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 447, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 451; (iii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 455, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 459; (iv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 463, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 467; (v) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 471, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 472; (vi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 473, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 474; (vii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 475, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 472; (viii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 477, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 478; (ix) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 479, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 480; (x) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 481, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 482; (xi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 483, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 484; (xii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 487, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 467; (xiii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 489, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 490; (xiv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 491, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 492; (xv) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 495, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 496; or (xvi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 497, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 498.

[0129] In some embodiments, the CD20-binding domain CDR(s) are selected from the following: (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 441; and vhCDR3 having the amino acid sequence of SEQ ID NO: 442; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 444, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 446; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 449; and vhCDR3 having the amino acid sequence of SEQ ID NO: 450; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 452, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 454; (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 457; and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 460, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 462; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 464; vhCDR2 having the amino acid sequence of SEQ ID NO: 465; and vhCDR3 having the amino acid sequence of SEQ ID NO: 466; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 356, vlCDR2 having the amino acid sequence of SEQ ID NO: 357, and vlCDR3 having the amino acid sequence of SEQ ID NO: 470.

[0130] In some embodiments, the VH and VL of the CD20 binding domain(s) are selected from the following: (i) VH having an amino acid sequence that is at least approximately 95% identical to SEQ ID NO: 439; and (ii) VL having an amino acid sequence that is at least approximately 95% identical to SEQ ID NO: 443; or (i) VH having an amino acid sequence that is at least approximately 95% identical to SEQ ID NO: 447; and (ii) VL having an amino acid sequence that is at least approximately 95% identical to SEQ ID NO: 451; or (i) VH having an amino acid sequence that is at least approximately 95% identical to SEQ ID NO: 455; and (ii) VL having an amino acid sequence that is at least approximately 95% identical to SEQ ID NO: 459; or (i) VH having an amino acid sequence that is at least approximately 95% identical to SEQ ID NO: 463; and (ii) VL having an amino acid sequence that is at least approximately 95% identical to SEQ ID NO: 467.

[0131] In some embodiments, the VH and VL of the CD20 binding domain(s) are selected from the following: (i) VH having the amino acid sequence of SEQ ID NO: 439; and (ii) VL having the amino acid sequence of SEQ ID NO: 443; or (i) VH having the amino acid sequence of SEQ ID NO: 447; and (ii) VL having the amino acid sequence of SEQ ID NO: 451; or (i) VH having the amino acid sequence of SEQ ID NO: 455; and (ii) VL having the amino acid sequence of SEQ ID NO: 459; or (i) VH having the amino acid sequence of SEQ ID NO: 463; and (ii) VL having the amino acid sequence of SEQ ID NO: 467.

[0132] C. Chimeric antibodies and humanized antibodies In certain embodiments, the antibodies provided herein include heavy chain variable regions derived from specific germline heavy chain immunoglobulin genes and / or light chain variable regions derived from specific germline light chain immunoglobulin genes. For example, such antibodies may include human antibodies that are “products” or “derived” of a specific germline sequence and contain a heavy chain variable region or a light chain variable region. Human antibodies that are “products” or “derived” of a human germline immunoglobulin sequence may be identified in this way by comparing the amino acid sequence of the human antibody with the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that has the sequence closest to the sequence of the human antibody (i.e., the highest identity %) (using the methods outlined herein). Human antibodies that are “products” or “derived” of a specific human germline immunoglobulin sequence may include amino acid differences compared to the germline sequence, for example, due to naturally occurring somatic mutations or the intentional introduction of site-directed mutations. However, humanized antibodies are typically at least 90% identical to the amino acid sequence encoded by human germline immunoglobulin genes and contain amino acid residues that identify them as antibodies derived from human sequences when compared to germline immunoglobulin amino acid sequences of other species (e.g., mouse germline sequences). In certain cases, humanized antibodies may be at least 95%, 96%, 97%, 98%, or 99%, or even at least 96%, 97%, 98%, or 99%, identical to the amino acid sequence encoded by germline immunoglobulin genes. Typically, humanized antibodies derived from a particular human germline sequence will not differ from the amino acid sequence encoded by human germline immunoglobulin genes by more than 10 to 20 amino acids (before the introduction of any asymmetric, pI, and excision variants as defined herein; i.e., before the introduction of the variants of the present invention, the number of variants is generally small).In certain cases, humanized antibodies may exhibit differences of five or fewer amino acids from the amino acid sequence encoded by germline immunoglobulin genes (again, before the introduction of any asymmetric, pI, and excision mutants as defined herein; i.e., before the introduction of the mutants of the present invention, the number of mutants is generally small).

[0133] In one embodiment, the parent antibody is affinity-matured as is known in the art. Structure-based methods may be employed for humanization and affinity maturation, for example, as described in USSN 11 / 004,590. Selection-based methods may be employed to humanize and / or affinity mature the variable region of an antibody, including, but not limited to, the following methods: Wu et al., 1999, J.Mol.Biol.294:151-162; Baca et al., 1997, J.Biol.Chem.272(16):10678-10684; Rosok et al., 1996, J.Biol.Chem.271(37):22611-22618; Rader et al., 1998, Proc.Natl.Acad.Sci.USA 95:8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759 (all of which are incorporated in their entirety by reference). Other humanization methods may involve joining only a portion of the CDR, and include, but are not limited to, the following methods: USSN09 / 810,510; Tan et al.,2002,J.Immunol.169:1119-1125; De Pascalis et al.,2002,J.Immunol.169:3076-3084 (all of which are incorporated by reference in their entirety).

[0134] D. Heterodimer antibodies In exemplary embodiments, the anti-CD20 × anti-CD28 antibody provided herein is a heterodimer bispecific antibody comprising two mutant Fc domain sequences. Such mutant Fc domains include amino acid modifications to facilitate the self-assembly and / or purification of the heterodimer antibody.

[0135] An ongoing challenge in antibody technology is the desire for "bispecific" antibodies that can simultaneously bind to two different antigens, and thus generally bring different antigens into close proximity, resulting in novel functionalities and therapeutic applications. Generally, these antibodies are produced by incorporating the heavy and light chain genes into host cells. This generally results in the formation of the desired heterodimer (AB) and two homodimers (AA and BB (without the problem of light chain heterodimers)). However, a major obstacle in the formation of bispecific antibodies is the difficulty in opting for the formation of the desired heterodimer antibody rather than homodimers, and / or purifying the heterodimer antibody while avoiding homodimers.

[0136] Numerous mechanisms can be used to generate the target heterodimer antibodies. Furthermore, as will be understood by those skilled in the art, these different mechanisms can be combined to ensure high heterodimerization. Amino acid modifications that facilitate the production and purification of heterodimers are collectively called "heterodimerizing mutants." As will be discussed later, heterodimerizing mutants include not only "pI mutants" that enable the purification of heterodimers from homodimers, but also "asymmetric" mutants (e.g., the "knob and hole" mutants and "charged pair" mutants described later). Useful mechanisms of heterodimerization, as commonly described in U.S. Patent No. 9,605,084 (which is incorporated herein by reference in its entirety), and for which a specific discussion of heterodimerizing mutants will be discussed later, include the “knobs and holes” ("KIHs") described in U.S. Patent No. 9,605,084, the “electrostatic steering” or “charge pairs” described in U.S. Patent No. 9,605,084, the pI mutants described in U.S. Patent No. 9,605,084, and additional common Fc mutants outlined in U.S. Patent No. 9,605,084 and below.

[0137] The following provides further details on heterodimerization variants useful for the formation and purification of target heterodimer antibodies (e.g., bispecific antibodies).

[0138] 1. Asymmetric mutants In some embodiments, the heterodimer antibody includes asymmetric variants, which are one or more amino acid modifications in the first Fc domain (A) and / or the second Fc domain (B), that prefer the formation of an Fc heterodimer (an Fc dimer containing a first Fc domain and a second Fc domain; AB) to an Fc homodimer (an Fc dimer containing two first Fc domains or two second Fc domains; AA or BB). Suitable scuba riants are included in Figure 29, and in Figures 3 and 9, of U.S. Publication No. 2016 / 0355608 (which is incorporated herein by reference in whole and in particular for its disclosure of scuba riants).

[0139] A particular type of asymmetric mutant is generally referred to in the art as “knob and hole” and refers to an amino acid manipulation that produces a steric effect that favors heterodimerization and rejects homodimerization, as described in USSN 61 / 596,846, Ridgway et al., Protein Engineering 9(7):617(1996), Atwell et al., J.Mol.Biol.1997 270:26; U.S. Patent No. 8,216,805 (all of which are incorporated herein by reference in their entirety, particularly with regard to the disclosure of “knob and hole” mutations). This is sometimes referred to herein as “steric variant.” The figure illustrates numerous “monomer A-monomer B” pairs that depend on “knob and hole.” Furthermore, as described in Merchant et al., Nature Biotech. 16:677 (1998), these “knob and hole” mutations can be combined with disulfide bonds to further favor the formation of Fc heterodimers.

[0140] Another method used for heterodimer formation is sometimes called “electrostatic steering,” as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010) (the entire text of which is incorporated herein by reference). This may also be referred to herein as “charge pairing.” In this embodiment, electrostatics are used to make the formation asymmetric for heterodimerization. As will be understood by those skilled in the art, these may also affect pI and therefore purification, and thus in some cases, they may be considered pI variants. However, since they are generated to force heterodimerization and are not used as a purification method, they are classified as “asymmetric variants.” These include, but are not limited to, D221E / P228E / L368E paired with D221R / P228R / K409R (for example, these are a "monomer-compatible set"), and C220E / P228E / 368E paired with C220R / E224R / P228R / K409R.

[0141] In some embodiments, asymmetric mutants simultaneously favor heterodimerization based on both the "knob and hole" mechanism and the "electrostatic steering" mechanism. In some embodiments, the heterodimer antibody contains one or more sets of such heterodimerizing asymmetric mutants. These mutants form a "pair" of "sets," where one set of the pair is incorporated into the first monomer and the other set of the pair is incorporated into the second monomer. It should be noted that these sets do not necessarily behave as "knob in hole" mutants, where residues on one monomer correspond one-to-one with residues on the other monomer. Rather, these sets may instead form an interface between the two monomers that promotes heterodimerization and inhibits homodimerization, resulting in a proportion of heterodimers spontaneously formed under biological conditions exceeding 90% (homodimer A / A 25%:heterodimer A / B 50%:homodimer B / B 25%) rather than the expected 50%. Exemplary heterodimerized "asymmetric" mutants are shown in Figures 3 and 9. Such "asymmetric" mutants include, but are not limited to, the following: S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411T / E360E / Q362E:D401K;L368D / K370S:S364K / E357L;K370S:S364K / E357Q (EU numbering).

[0142] In exemplary embodiments, the heterodimer antibody includes:S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411T / E360E / Q362E:D401K;L368D / K370S:S364K / E357L;K370S:S364K / E357Q; or T366S / L368A / Y407V:T366W (optionally including cross-linked disulfides, T366S / L368A / Y407V / Y349C:T366W / S354C) a set of "asymmetric" mutant amino acid substitutions (EU numbered). In exemplary embodiments, the heterodimer antibody contains the amino acid substitution set “S364K / E357Q:L368D / K370S”. Nomenclature, the “S364K / E357Q:L368D / K370S” pair means that one monomer contains an Fc domain with the amino acid substitutions S364K and E357Q, and the other monomer contains an Fc domain with the amino acid substitutions L368D and K370S. As above, the “chain-like” nature of these pairs depends on the start pI.

[0143] In some embodiments, the Fc heterodimerization domain resulting in heterodimerization of two different Fc domains includes, but is not limited to, the following amino acid variants within the Fc domain: S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411T / E360E / Q362E:D401K;L368D / K370 S:S364K / E357L;K370S:S364K / E357Q; or T366S / L368A / Y407V:T366W (optionally including cross-linked disulfides, T366S / L368A / Y407V / Y349C:T366W / S354C, or T366S / L368A / Y407V / Y354C:T366W / S349C) sets of "asymmetric" mutant amino acid substitutions (EU numbered), and others shown in Figure 1. Generally, these heterodimerized Fc variants are created in the human IgG1, IgG2, or IgG4 backbone.

[0144] In some embodiments, the asymmetric variants provided herein may, independently and arbitrarily, be incorporated into one or both of the Fc domains of a heterodimer antibody, along with any other modifications, including but not limited to other asymmetric variants (see, for example, Figure 37 of U.S. Patent Publication No. 2012 / 0149876 (incorporated herein by reference), particularly for the disclosure of asymmetric variants), pI variants, isotype variants, FcRn variants, excision variants, etc. Furthermore, individual modifications may also, independently and arbitrarily, be included in or excluded from the heterodimer antibody.

[0145] In some embodiments, the asymmetric variants outlined herein may be incorporated independently and optionally into one or both heavy chain monomers, along with any pI variant (or other variants such as Fc variants, FcRn variants, etc.), and may be included in or excluded from the heterodimer antibody of interest, independently and optionally.

[0146] 2. Purified mutants In some embodiments, the heterodimer antibody includes purified variants that favorably enable the separation of the heterodimer protein (e.g., an anti-CD20 × anti-CD28 bispecific antibody) from the homodimer protein.

[0147] Several basic mechanisms can facilitate the purification of heterodimer antibodies. For example, by modifying one or both of the antibody heavy chain monomers A and B so that each monomer has a different pI, heterodimer AB antibodies can be isoelectrically focused and purified from monomer AA and BB proteins. Alternatively, several scaffold formats, such as the "1+1Fab-scFv-Fc" and "2+1Fab2-scFv-Fc" formats, allow for size-based separation. As mentioned above, it is also possible to make heterodimer formation "asymmetric" than homodimer formation using asymmetric mutants. Therefore, combinations of heterodimer-asymmetric mutants and purification mutants are particularly useful in the heterodimer antibodies used herein.

[0148] Furthermore, as will be described in more detail below, depending on the structure of the heterodimeric antibody, purified variants and / or domain linkers contained within the constant region and / or Fc region of the monomer may be used. In some embodiments, the heterodimeric antibody may also include additional modifications for alternative functionality, which may result in pI changes such as Fc variants, FcRn variants, and KO variants.

[0149] In some embodiments, the heterodimeric antibodies of interest provided herein include at least one monomer having one or more modifications that alter the pI of the monomer (i.e., a “pI variant”). Generally, as will be understood by those skilled in the art, there are two common types of pI variants: those that increase the pI of the protein (basicity changes) and those that decrease the pI of the protein (acidicity changes). As described herein, all combinations of these variants are possible. That is, one monomer may be wild-type or a variant that does not exhibit a significantly different pI from the wild-type, while the other may be more basic or more acidic. Alternatively, each monomer may be altered so that one becomes more basic and the other more acidic.

[0150] Depending on the heterodimer antibody configuration, pI variants may be contained within the monomer's constant domain and / or Fc domain, or a charged linker, either a domain linker or an scFv linker, may be used. That is, antibody configurations utilizing scFv(s), such as "1+1Fab-scFv-Fc," may include a charged scFv linker (either positive or negative) to provide a further increase in pI for purification purposes. As those skilled in the art will understand, some 1+1Fab-scFv-Fc and 2+1Fab2-scFv-Fc configurations are useful with only a charged scFv linker and without additional pI adjustment; however, the present invention also provides pI variants and / or charged domain linkers present in one or both monomers. Furthermore, additional amino acid manipulations for alternative functionality, such as Fc variants, FcRn variants, and KO variants, may also provide pI changes.

[0151] In heterodimer antibodies that utilize pI as a separation mechanism to enable the purification of heterodimer proteins, amino acid variants are introduced into one or both monomeric polypeptides. That is, the pI of one monomer (referred to herein as "monomer A" for simplicity) may be manipulated while avoiding monomer B, or both monomers may be altered by increasing the pI of monomer A and decreasing the pI of monomer B. As will be outlined in more detail below, changes in the pI of one or both monomers can be achieved by removing or adding charged residues (e.g., replacing a neutral amino acid with a positively or negatively charged amino acid residue, e.g., glycine to glutamic acid), changing the charge of a charged residue from positive or negative to the opposite charge (e.g., aspartic acid to lysine), or changing a charged residue to a neutral residue (e.g., loss of charge; lysine to serine). Numerous examples of these variants are shown in Figures 3 and 4.

[0152] Therefore, in some embodiments, the target heterodimer antibody includes an amino acid modification in the constant region that alters the isoelectric point (pI) of at least one, if not both, of the monomers of the dimer protein in order to form a “pI antibody” by incorporating an amino acid substitution (“pI variant” or “pI substitution”) into one or both monomers. As shown herein, separation of heterodimers from two homodimers is possible if the pI of the two monomers is down to 0.1 pH units, and any pI of 0.2, 0.3, 0.4, or 0.5 or higher can all be used in the present invention.

[0153] As those skilled in the art will understand, the number of pI variants to be present in each monomer or both monomers(s) to obtain good separation will depend in part on the starting pIs of the constituents, e.g., in the 1+1Fab-scFv-Fc, 2+1Fab2-scFv-Fc, 1+1CLC, and 2+1CLC forms, scFv(1+1Fab-scFv-Fc, 2+1Fab2-scFv-Fc) and the starting pI of the target Fab(s). That is, the Fv sequences of the two target antigens are calculated and a decision is made therefrom to determine which monomer to operate on or in which "direction" (e.g., more positive or more negative) to operate on. As is known in the art, different Fvs will have different starting pIs utilized in this invention. Generally, as outlined herein, the pI is manipulated such that the sum of the pI differences of each monomer is at least about 0.1log, and preferably 0.2 to 0.5 as outlined herein.

[0154] When pI variants are used to achieve heterodimerization, a more modular approach is provided for designing and purifying bispecific proteins (including antibodies) by utilizing the constant region(s) of the heavy chain(s). Therefore, in some embodiments, heterodimerizing variants (including asymmetric and pI heterodimerizing variants) are not located in variable regions that would require the manipulation of individual antibodies. Furthermore, in some embodiments, the potential for immunogenicity due to pI variants is significantly reduced by incorporating pI variants from different IgG isotypes so that pI is modified without introducing significant immunogenicity. Therefore, a further issue to be addressed is the elucidation of low-pI constant domains with high human sequence content, e.g., minimizing or avoiding non-human residues at any specific location. By utilizing isoparticulate substitutions (e.g., Asn to Asp; and Gln to Glu) instead of, or in addition to, isotype substitutions, the potential for immunogenicity due to pI variants is significantly reduced.

[0155] As will be discussed later, the secondary benefits that can result from this pI manipulation are also the extension of the serum half-life and the increase in FcRn binding. Specifically, as described in U.S. Patent Publication US2012 / 0028304 (which is incorporated in its entirety by reference), lowering the pI of the antibody constant domain (including those found in antibodies and Fc fusions) increases the serum retention time in vivo. These pI variants for extending the serum half-life also promote pI changes for purification.

[0156] Furthermore, it should be noted that pI variants offer additional advantages in the analysis and quality control processes of bispecific antibodies, because the ability to remove, minimize, or identify homodimers when they are present is crucial. Similarly, the ability to reliably test the reproducibility of heterodimeric antibody production is also important.

[0157] Generally, specific embodiments of use rely on a set of mutants, including asymmetric mutants that promote heterodimerization rather than homodimerization, in addition to pI mutants that increase the pI difference between the two monomers to facilitate the purification of heterodimers from homodimers.

[0158] Exemplary combinations of pI variants are shown in Figures 4 and 5, and in Figure 30 of U.S. Patent Publication No. 2016 / 0355608, all of which are incorporated herein by reference in their entirety, particularly with respect to the disclosure of pI variants. Preferred combinations of pI variants are shown in Figures 3 and 4. As outlined herein and shown in the figures, these variations relate to IgG1, but all isotypes can be varied in this way, as can isotype hybrids. R133E and R133Q may also be used when the heavy chain constant domain is derived from IgG2-4.

[0159] In one embodiment, a preferred combination of pI variants has one monomer (negative Fab side) containing the 208D / 295E / 384D / 418E / 421D variant (N208D / Q295E / N384D / Q418E / N421D when relative to human IgG1), and a second monomer (positive scFv side) containing a positively charged scFv linker containing (GKPGS)4 (SEQ ID NO: 42). However, as will be understood by those skilled in the art, the first monomer contains a CH1 domain including position 208. Therefore, in constructs that do not contain a CH1 domain (e.g., in the case of an antibody that does not utilize a CH1 domain in one of its domains), a preferred negative pI variant Fc set contains the 295E / 384D / 418E / 421D variant (Q295E / N384D / Q418E / N421D when relative to human IgG1).

[0160] Therefore, in some embodiments, one monomer has a set of substitutions from Figure 8, and the other monomer has a charged linker (which may be in the form of a charged scFv linker, since the monomer constitutes an scFv, or in the form of a charged domain linker, as defined by its form, and can be selected from those depicted in Figure 6).

[0161] In some embodiments, modifications are made at the hinge of the Fc domain, including positions 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, and 230 based on EU numbering. Thus, pI mutations, and in particular substitutions, can be made at one or more of positions 216–230, with one, two, three, four, or five mutations finding use. Again, any possible combination, either alone or with other pI variants in other domains, is conceivable.

[0162] Specific substitutions used to lower the pI of the hinge domain include, but are not limited to, deletions at position 221, unnatural valine or threonine at position 222, deletion at position 223, unnatural glutamate at position 224, deletion at position 225, deletion at position 235, and deletion or unnatural alanine at position 236. In some cases, only pI substitutions occur in the hinge domain, while in other domains, these substitutions are added in any combination to other pI variants.

[0163] In some embodiments, mutations may be made in the CH2 region including positions 233, 234, 235, 236, 274, 296, 300, 309, 320, 322, 326, 327, 334, and 339, based on EU numbering. It should be noted that changes at positions 233–236 may be made to increase the effector function in the IgG2 backbone (along with 327A). Here again, all possible combinations of these 14 positions are possible. For example, antibodies provided herein may contain mutant Fc domains having one, two, three, four, five, six, seven, eight, nine, or ten CH2 pI substitutions.

[0164] Specific substitutions used to reduce the pI of the CH2 domain include, but are not limited to, unnatural glutamine or glutamic acid at position 274, unnatural phenylalanine at position 296, unnatural phenylalanine at position 300, unnatural valine at position 309, unnatural glutamic acid at position 320, unnatural glutamic acid at position 322, unnatural glutamic acid at position 326, unnatural glycine at position 327, unnatural glutamic acid at position 334, unnatural threonine at position 339, and all possible combinations within CH2 and with other domains.

[0165] In this embodiment, modifications can be independently and arbitrarily selected from positions 355, 359, 362, 384, 389, 392, 397, 418, 419, 444, and 447 (EU numbering) in the CH3 region. Certain substitutions found to be used in reducing the pI of the CH3 domain include, but are not limited to, unnatural glutamine or glutamic acid at position 355, unnatural serine at position 384, unnatural asparagine or glutamic acid at position 392, unnatural methionine at position 397, unnatural glutamic acid at position 419, unnatural glutamic acid at position 359, unnatural glutamic acid at position 362, unnatural glutamic acid at position 389, unnatural glutamic acid at position 418, unnatural glutamic acid at position 444, and deletion or unnatural aspartic acid at position 447.

[0166] In some embodiments, anti-CD20 × anti-CD28 antibodies contain an amino acid substitution in one of their Fc domains that reduces binding to protein A. Such purified variants produce heterodimers with asymmetric binding to protein A, which can then be used for pH gradient separation of the heterodimer population from the homodimer population. Exemplary purified amino acid substitutions that reduce binding to protein A include, but are not limited to, H435R and Y436F (IgG1 CH3 domain, EU numbered). See, for example, US2010331527 (which is incorporated by reference in whole for relevant disclosures, particularly regarding Fc domain modifications for reducing protein A binding).

[0167] 3. Isotype variants Furthermore, many embodiments of the target heterodimer antibodies rely on the "transfer" of pI amino acids at specific positions from one IgG isotype to another, thus reducing or eliminating the possibility of introducing undesirable immunogenicity into the variant. Many of these are shown in Figure 21 of U.S. Patent Publication No. 2014 / 0370013 (incorporated herein by reference). Specifically, IgG1 is a common isotype for therapeutic antibodies for various reasons, including high effector function. However, the heavy chain constant region of IgG1 has a higher pI than that of IgG2 (8.10 vs. 7.31). By introducing IgG2 residues into the IgG1 backbone at specific positions, the pI of the resulting monomer is reduced (or increased), and it also exhibits a longer serum half-life. For example, IgG1 has glycine at position 137 (pI 5.97), while IgG2 has glutamic acid (pI 3.22). Introducing glutamate will likely affect the pI of the resulting protein. As will be discussed later, a significant impact on the pI of mutant antibodies generally requires numerous amino acid substitutions. However, it should be noted that, as will be discussed later, even changes in the IgG2 molecule can lead to an increase in serum half-life.

[0168] In other embodiments, as will be further described later, non-isotype amino acid changes are made to reduce the overall charge state of the resulting protein (for example, by changing amino acids with higher pI to amino acids with lower pI) or to enable structural adaptation for stability, etc.

[0169] Furthermore, significant changes can be observed in each monomer of the heterodimer by manipulating the pI of both the heavy chain and light chain constant domains. As discussed herein, by differentiating the pI of the two monomers by at least 0.5, separation by ion exchange chromatography, isoelectric focusing, or other isoelectric-sensitive methods can be enabled.

[0170] 4. Calculation of pI The pI of each monomer of the antibodies provided herein may depend on the pI of the mutant heavy chain constant domain, as well as the pI of the entire monomer, including the mutant heavy chain constant domain and its fusion partner. Therefore, in some embodiments, the pI change is calculated based on the mutant heavy chain constant domain using the table in Figure 19 of U.S. Patent Publication No. 2014 / 0370013. As discussed herein, the choice of monomer to manipulate is generally determined by the intrinsic pI of the Fv and scaffold regions. Alternatively, the pI of each monomer can be compared.

[0171] 5. pI variants that also confer better FcRn in vivo binding. If a pI variant reduces monomeric pI, it may have the additional benefit of improving serum retention in vivo.

[0172] Although still under investigation, the Fc region is thought to have a long in vivo half-life because its binding to FcRn at pH 6 within the endosome sequesters Fc (Ghetie and Ward, 1997 Immunol Today. 18(12):592-598, all incorporated by reference). The endosomal compartment then reuses Fc on the cell surface. Once this compartment opens to the extracellular space, a higher pH (approximately 7.4) induces the release of Fc into the bloodstream. In mice, Dall' Acqua et al. showed that Fc mutants with increased FcRn binding at pH 6 and pH 7.4 actually decreased serum concentrations and had the same half-life as wild-type Fc (Dall' Acqua et al. 2002, J.Immunol. 169:5171-5180, all incorporated by reference). The increased affinity of Fc for FcRn at pH 7.4 is thought to have prevented the re-release of Fc into the bloodstream. Therefore, mutations in Fc that would lengthen the half-life of Fc in vivo would ideally increase FcRn binding at lower pH levels while allowing Fc release at higher pH levels. The amino acid histidine changes its charge in the pH range of 6.0 to 7.4. Therefore, it is not surprising that His residues are located in key positions in the Fc / FcRn complex.

[0173] Recently, it has been suggested that antibodies with a variable region having a low isoelectric point may also have a longer serum half-life (Igawa et al. 23(5):385-392, all incorporated by reference). However, the mechanism is still not well understood. Furthermore, the variable region differs among antibodies. Variants of the constant region with reduced pI and extended half-life would provide a more modular approach to improving the pharmacokinetic properties of antibodies, as described herein.

[0174] E. Additional Fc variants for additional function In addition to the heterodimerized mutants mentioned above, there are many useful Fc amino acid modifications that can be performed for various reasons, including, but not limited to, altering the binding to one or more FcγR receptors or altering the binding to FcRn receptors, as will be discussed later.

[0175] Therefore, the antibodies provided herein (heterodimers as well as homodimers) may include such amino acid modifications, with or without the presence of heterodimerized variants (e.g., pI variants and steric variants) outlined herein. Each set of variants may, independently and arbitrarily, be included in or excluded from any particular heterodimer protein.

[0176] 1. FcγR and FcRn variants Therefore, there are numerous useful Fc substitutions that can be performed to alter binding to one or more FcγR receptors. In certain embodiments, the target antibody contains modifications (i.e., "FcγR variants") that alter binding to one or more FcγR receptors. Substitutions that result in increased binding, as well as decreased binding, can also be useful. For example, it is known that increased binding to FcγRIIIa generally increases ADCC (antibody-dependent cell-mediated cytotoxicity; a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize the bound antibody on target cells, resulting in the lysis of target cells). Similarly, decreasing binding to FcγRIIb (an inhibitory receptor) may also be beneficial in certain situations. Amino acid substitutions found to be used in the target antibodies include those described in U.S. Patent No. 8,188,321 (particularly Figure 41) and No. 8,084,582, and U.S. Published Patent Publications 20060235208 and 20070148170 (all of which are expressly incorporated herein by reference in whole with respect to the variants disclosed in their specifications, particularly those affecting Fcγ receptor binding). Specific variants that have found use include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 243A, 243L, 264A, 264V, and 299T. Such modifications may be present in one or both of the Fc domains of the target antibody.

[0177] In some embodiments, the target antibody includes one or more Fc modifications that increase its serum half-life. Fc substitutions for which utilization has been found to increase binding to the FcRn receptor and increase serum half-life include, but are not limited to, 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I or V / 434S, 436V / 428L, 259I / 308F / 428L, and M252Y / S254T / T256E, which are specifically disclosed in USSN 12 / 341,769 (which is incorporated herein by reference in its entirety). Such modifications may be contained in one or both of the Fc domains of the target antibody.

[0178] 2. Excision mutants In some embodiments, heterodimer antibodies include one or more modifications that reduce or eliminate the normal binding of the Fc domain to one or more or all of the Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) in order to avoid additional mechanisms of action. Such modifications are referred to as “FcγR excision variants” or “Fc knockout (FcKO or KO)” variants. In these embodiments, for some therapeutic applications, it is desirable to reduce or eliminate the normal binding of the Fc domain to one or more or all of the Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) in order to avoid additional mechanisms of action. That is, for example, in many embodiments, particularly in the use of bispecific antibodies that monovalently bind CD28, it is generally desirable to excise FcγRIIIa binding in order to eliminate or significantly reduce ADCC activity. In some embodiments, at least one of the Fc domains of the target antibodies described herein contains one or more Fcγ receptor excision mutants. In some embodiments, both Fc domains of the target antibodies described herein contain one or more Fcγ receptor excision mutants. These excision mutants are shown in Figure 5, and each may be independently and optionally included or excluded. In preferred embodiments, excision mutants selected from the group including: L234A / L235A / D265S, G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del. It should be noted that the excision mutants referred to herein excise FcγR binding but generally do not excise FcRn binding.

[0179] As is known in the art, the Fc domain of human IgG1 has the highest binding to the Fcγ receptor, and therefore excised mutants can be used when the constant domain (or Fc domain) in the backbone of a heterodimeric antibody is IgG1. Alternatively, in addition to excised mutants in the IgG1 background, mutations at glycosylation position 297 (generally A or S) can significantly reduce binding to FcγRIIIa, for example. Human IgG2 and IgG4 have naturally reduced binding to the Fcγ receptor, and therefore their backchains can be used with or without excised mutants.

[0180] F. Combination of heterodimer and Fc mutant As those skilled in the art will understand, all of the cited heterodimerizing mutants (including asymmetric and / or purified mutants) can be combined independently and in any way, as long as they retain their "chain" or "monomer splitting" properties. Furthermore, all of these mutants can be combined into any form of heterodimerization.

[0181] While embodiments that find specific uses for pI variants are shown in the figure, other combinations can be generated by following the basic rule of changing the pI difference between the two monomers to facilitate purification.

[0182] Furthermore, any heterodimerized mutant (asymmetric mutant and purified mutant) can be independently and arbitrarily combined with Fc excision mutants, Fc mutants, or FcRn mutants, as generally outlined herein.

[0183] Exemplary combinations of variants included in some embodiments of antibodies in heterodimer 1+1Fab-scFv-Fc, 2+1Fab2-scFv-Fc, 2+1 stack Fab2-scFv-Fc, and 2+1mAb-scFv forms are shown in Figure 8. In some embodiments, the heterodimer antibody includes a combination of variants as shown in Figure 8. In certain embodiments, the antibody is a heterodimer 1+1Fab-scFv-Fc, 2+1Fab2-scFv-Fc, 2+1 stack Fab2-scFv-Fc, and 2+1mAb-scFv form antibody, including a "platform X" combination of variants shown in Figure 8. In certain embodiments, the antibody is a "platform Y" combination of variants shown in Figure 8. 」 These are heterodimer antibodies in the form of 1+1Fab-scFv-Fc, 2+1Fab2-scFv-Fc, 2+1 stacked Fab2-scFv-Fc, and 2+1mAb-scFv, including combinations of these features.

[0184] G. Useful antibody formats As will be understood by those skilled in the art and will be discussed in more detail below, the heterodimer bispecific antibodies provided herein can generally take on several different configurations, as shown in Figure 29.

[0185] As those skilled in the art will understand, the heterodimer form of the present invention may not only be bispecific but also have different valencies. That is, the heterodimer antibody of the present invention may be bivalent and bispecific, or trivalent and bispecific, where the first antigen is bound by two binding domains and the second antigen is bound by a second binding domain. As outlined herein, when CD28 is one of the target antigens, it is preferable that CD28 binds only monovalently.

[0186] The present invention utilizes a CD28-binding domain in combination with a CD20-binding domain. As will be understood by those skilled in the art, any group of anti-CD28 CDRs, anti-CD28 variable light chain domains and variable heavy chain domains, Fabs, and scFvs, as shown in any of the figures (see in particular Figures 15-24 and 26), can be used. Similarly, any of the CD20 antigen-binding domains can be used, whether or not they are used independently or in any combination, as shown in any of the figures (e.g., Figure 30), including CDRs, variable light chain domains and variable heavy chain domains, Fabs, and scFvs.

[0187] 1.1+1Fab-scFv-Fc format One heterodimer antibody form that finds particular use among the target anti-CD20 × anti-CD28 antibodies provided herein is the “1+1Fab-scFv-Fc” form, or “bottle opener” form, as shown in Figure 29A. The 1+1Fab-scFv-Fc form antibody comprises a first monomer which is a “normal” heavy chain (VH1-CH1-hinge-CH2-CH3), where VH1 is the first variable heavy chain domain and CH2-CH3 is the first Fc domain. The 1+1Fab-scFv-Fc also comprises a light chain which includes a first variable light chain domain VL1 and a constant light chain domain CL. The light chain interacts with VH1-CH1 of the first monomer to form a first antigen-binding domain which is Fab. The second monomer of the antibody comprises a second binding domain which is a single-chain Fv (hereinafter defined as “scFv”) and a second Fc domain. scFv comprises a second variable heavy chain domain (VH2) and a second variable light chain domain (VL2), where VH2 is conjugated to VL2 using a chargeable scFv linker (see, for example, Figure 6). scFv is conjugated to the heavy chain using a domain linker (see, for example, Figure 7). The two monomers are joined by the use of amino acid variants (e.g., the heterodimerizing variants described above) in constant regions (e.g., the Fc domain, CH1 domain, and / or hinge region) that promote the formation of a heterodimer antibody, as described in more detail below. This structure is sometimes referred to herein as the “bottle opener” form because it roughly resembles a bottle opener visually. In some embodiments, the 1+1Fab-scFv-Fc form antibody is a bivalent antibody.

[0188] The "1+1Fab-scFv-Fc" configuration of the present invention has several distinct advantages. As is known in the art, antibody analogs that rely on two scFv constructs often have stability and aggregation problems, but in the present invention, this can be mitigated by adding a "conventional" heavy-chain and light-chain pair. Furthermore, in contrast to configurations that rely on two heavy chains and two light chains, there is no problem with incorrect heavy-chain and light-chain pairings (e.g., a pair of heavy chain 1 and light chain 2).

[0189] In some embodiments of the 1+1Fab-scFv-Fc antibody, one of the first or second antigen-binding domains is a CD28-binding domain, and the other binding domain is a CD20-binding domain. In some embodiments of 1+1Fab-scFv-Fc, scFv binds to CD28, and Fab binds to CD20. An exemplary anti-CD20 × anti-CD28 bispecific antibody of the 1+1Fab-scFv-Fc form is shown in Figure 33.

[0190] In some embodiments, the first and second Fc domains of the 1+1Fab-scFv-Fc antibody are mutant Fc domains containing heterodimerization asymmetric variants (e.g., a set of amino acid substitutions as shown in Figures 3 and 9). Particularly useful heterodimerized asymmetric mutants include: S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411T / E360E / Q362E:D401K;L368D / K370S:S364K / E357L;K370S:S364K / E357Q;T366S / L368A / Y407V:T366W, and T366S / L368A / Y407V / Y349C:T366W / S354C (EU numbering). In an exemplary embodiment, one of the first or second mutant Fc domains comprises the heterodimerization asymmetric mutant L368D / K370S, and the other of the first or second mutant Fc domains comprises the heterodimerization asymmetric mutant S364K / E357Q, where the numbering follows EU numbering.

[0191] In some embodiments, the mutant Fc domain includes excised mutants (including those shown in Figure 5). In some embodiments, each of the first and second mutant Fc domains includes the excised mutants E233P / L234V / L235A / G236_ / S267K, where the numbering follows EU numbering.

[0192] In some embodiments, the steady domain (CH1-hinge-CH2-CH3) of the first monomer includes pI mutants (including those shown in Figure 4). In exemplary embodiments, the steady domain (CH1-hinge-CH2-CH3) of the first monomer includes pI mutants N208D / Q295E / N384D / Q418E / N421D, where the numbering follows EU numbering.

[0193] In exemplary embodiments, the 1+1Fab-scFv-Fc form antibody comprises a “Platform X” amino acid variant as shown in Figure 8. In such embodiments, the CH1-hinge-CH2-CH3 of the first monomer comprises the amino acid variant L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K, and the second Fc domain comprises the amino acid variant S364K / E357Q / E233P / L234V / L235A / G236del / S267K, where the numbering follows EU numbering.

[0194] In some embodiments, the scFv of the 1+1Fab-scFv-Fc form antibody provided herein comprises a charged scFv linker (including that shown in Figure 6). In some embodiments, the 1+1Fab-scFv-Fc form antibody provided herein comprises the FcRn variant M428L / N434S, where the numbering follows EU numbering.

[0195] In exemplary embodiments of the 1+1 Fab-scFv-Fc form antibody having a "Platform X" variant, the first Fc domain comprises the heterodimerizing asymmetric mutant L368D / K370S, the second Fc domain comprises the heterodimerizing asymmetric mutant S364K / E357Q, each of the first and second Fc domains comprises the excision mutant E233P / L234V / L235A / G236_ / S267K, and the constant domain (CH1-hinge-CH2-CH3) of the first monomer comprises the pI mutant N208D / Q295E / N384D / Q418E / N421D, where numbering follows EU numbering. In some embodiments, the scFv of the 1+1 Fab-scFv-Fc form antibody provided herein comprises the (GKPGS)4 (SEQ ID NO: 42) charged scFv linker. In some embodiments, the 1+1Fab-scFv-Fc form antibody provided herein comprises the FcRn variant M428L / N434S, where the numbering follows EU numbering. In some embodiments, the scFv of the 1+1Fab-scFv-Fc form antibody provided herein comprises a charged scFv linker (including one shown in Figure 6).

[0196] In exemplary embodiments, the 1+1Fab-scFv-Fc form antibody includes a “Platform Y” amino acid modification as shown in Figure 8. In such embodiments, the first Fc domain includes the amino acid substitution L234A / L235A / D265S / T366S / L368A / Y407V / H435R / Y436F, and the first Fc domain includes the amino acid substitution L234A / L235A / D265S / LT366W, where the numbering follows EU numbering.

[0197] In exemplary embodiments of the 1+1 Fab-scFv-Fc form antibody having a platform Y variant, the first Fc domain comprises the heterodimerizing asymmetric variant T366S / L368A / Y407V, the second Fc domain comprises the heterodimerizing asymmetric variant T366W, each of the first and second variant Fc domains comprises the excision variant L234A / L235A / D265S, and the second Fc domain comprises the purified variant H435R / Y436F, where numbering follows EU numbering. In some embodiments, the scFv of the 1+1 Fab-scFv-Fc form antibody provided herein comprises the (GKPGS)4 (SEQ ID NO: 42) charged scFv linker. In some embodiments, the 1+1 Fab-scFv-Fc form antibody provided herein comprises the FcRn variant M252Y / S254T / T256E, where numbering follows EU numbering. In some embodiments, the scFv of the 1+1Fab-scFv-Fc antibody provided herein includes a charged scFv linker (including one shown in Figure 6).

[0198] In exemplary embodiments, the 1+1 Fab-scFv-Fc antibody having the platform J variant further comprises a “staple-treated” scFv (e.g., a “staple-treated” anti-CD28scFv). “Staple-treated” scFv exhibiting improved stability and / or reduced aggregation are described in further detail herein. An exemplary staple linker useful for inclusion in such a “staple-treated” scFv is provided in Figure 6. In exemplary embodiments, the staple linker is GGGSGGSGGCPPCGGSGG (SEQ ID NO: 56).

[0199] In some embodiments, one of the first or second binding domains binds CD20, and the other binding domain binds CD28. Any suitable CD28-binding domain may be included in a target 1+1Fab-scFv-Fc antibody containing any of the CD28-binding domains provided herein.

[0200] In some embodiments of the 1+1Fab-scFv-Fc format, the anti-CD28ABD has a CDR selected from the following: (i) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 1, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 5; (ii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 11, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 5; (iii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 1, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 19; (iv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 11, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 19; (v) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 15, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 5; (vi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 15, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 19; (vii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 63, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 75; (viii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 67, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 75; (ix) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 63, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 79; (x) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 67, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 111; (xi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 71, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 75; (xii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 71, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 79; (xiii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 320, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 324; (xiv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 327, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 331; (xv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 335, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 339; (xvi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 343, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 347; (xvii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 351, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 355; (xviii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 359, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 355; (xix) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 367, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 371; (xx) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 375, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 380; (xxi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 391, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 395; (xxii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 399, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 403; (xxiii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 407, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 411; (xxiv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 415, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 419; (xxv) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 423, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 5; or (xxvi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 431, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 435.

[0201] In some embodiments of the 1+1Fab-scFv-Fc format, the anti-CD28ABD has a CDR selected from the following: (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22.

[0202] In some embodiments of the 1+1Fab-scFv-Fc form, the anti-CD28ABD has a VH domain and a VL domain, selected from those described below or their variants: (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 1; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 11; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 15; and (ii) VL having the amino acid sequence of SEQ ID NO: 19; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 63; and (ii) VL having the amino acid sequence of SEQ ID NO: 79; or (i) VH having the amino acid sequence of SEQ ID NO: 67; and (ii) VL having the amino acid sequence of SEQ ID NO: 111; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 75; or (i) VH having the amino acid sequence of SEQ ID NO: 71; and (ii) VL having the amino acid sequence of SEQ ID NO: 79; or (i) VH having the amino acid sequence of SEQ ID NO: 320; and (ii) VL having the amino acid sequence of SEQ ID NO: 324; or (i) VH having the amino acid sequence of SEQ ID NO: 327; and (ii) VL having the amino acid sequence of SEQ ID NO: 331; or (i) VH having the amino acid sequence of SEQ ID NO: 335; and (ii) VL having the amino acid sequence of SEQ ID NO: 339; or (i) VH having the amino acid sequence of SEQ ID NO: 343; and (ii) VL having the amino acid sequence of SEQ ID NO: 347; or (i) VH having the amino acid sequence of SEQ ID NO: 351; and (ii) VL having the amino acid sequence of SEQ ID NO: 355; or (i) VH having the amino acid sequence of SEQ ID NO: 359; and (ii) VL having the amino acid sequence of SEQ ID NO: 355; or (i) VH having the amino acid sequence of SEQ ID NO: 367; and (ii) VL having the amino acid sequence of SEQ ID NO: 371; or (i) VH having the amino acid sequence of SEQ ID NO: 375; and (ii) VL having the amino acid sequence of SEQ ID NO: 380; or (i) VH having the amino acid sequence of SEQ ID NO: 391; and (ii) VL having the amino acid sequence of SEQ ID NO: 395; or (i) VH having the amino acid sequence of SEQ ID NO: 399; and (ii) VL having the amino acid sequence of SEQ ID NO: 403; or (i) VH having the amino acid sequence of SEQ ID NO: 407; and (ii) VL having the amino acid sequence of SEQ ID NO: 411; or (i) VH having the amino acid sequence of SEQ ID NO: 415; and (ii) VL having the amino acid sequence of SEQ ID NO: 419; or (i) VH having the amino acid sequence of SEQ ID NO: 423; and (ii) VL having the amino acid sequence of SEQ ID NO: 5; or (i) VH having the amino acid sequence of SEQ ID NO: 431; and (ii) VL having the amino acid sequence of SEQ ID NO: 435.

[0203] In some embodiments, one of the first or second binding domains of the 1+1Fab-scFv-Fc antibody conjugates CD20. In some embodiments of the 1+1Fab-scFv-Fc form, the anti-CD20ABD has a CDR selected from the following: (i) Heavy chain complementarity determination regions 1 (vhCDR1), vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 439, and light chain complementarity determination regions 1 (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 443; (ii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 447, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 451; (iii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 455, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 459; (iv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 463, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 467; (v) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 471, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 472; (vi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 473, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 474; (vii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 475, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 472; (viii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 477, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 478; (ix) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 479, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 480; (x) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 481, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 482; (xi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 483, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 484; (xii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 487, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 467; (xiii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 489, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 490; (xiv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 491, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 492; (xv) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 495, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 496; or (xvi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH) having the amino acid sequence of SEQ ID NO: 497, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL) having the amino acid sequence of SEQ ID NO: 498.

[0204] In some embodiments of the 1+1Fab-scFv-Fc format, the anti-CD20ABD has a CDR selected from the following: (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 441; and vhCDR3 having the amino acid sequence of SEQ ID NO: 442; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 444, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 446; or (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 449; and vhCDR3 having the amino acid sequence of SEQ ID NO: 450; and (ii) vlCDR1 having the amino acid sequence of SEQ ID NO: 452, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid seque...

Claims

1. Antibodies or antibody fragments, including those listed below: (a) A first antigen-binding domain that binds to CD20, including the following: (i) Heavy chain complementarity determination regions 1 (vhCDR1), vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 439, and light chain complementarity determination regions 1 (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 443; (ii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 447, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 451; (iii) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 455, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 459; (iv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 463, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 467; (v) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 471, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 472; (vi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 473, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 474; (vii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 475, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 472; (viiii) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 477, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 478; (ix) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 479, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 480; (x) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 481, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 482; (xi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 483, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 484; (xi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 487, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 467; (xiiii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 489, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 490; (xiv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 491, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 492; (xv) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 495, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 496; or (xvi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH1) having the amino acid sequence of SEQ ID NO: 497, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL1) having the amino acid sequence of SEQ ID NO: 498; Furthermore (b) A second antigen-binding domain that binds to CD28, including the following: (i) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 1, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 5; (ii) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 11, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 5; (iii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 1, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 19; (iv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 11, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 19; (v) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 15, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 5; (vi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 15, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 19; (vii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 63, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 75; (viiii) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 67, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 75; (ix) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 63, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 79; (x) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 67, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 111; (xi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 71, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 75; (xi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 71, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 79; (xiiii) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 320, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 324; (xiv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 327, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 331; (xv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 335, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 339; (xvi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 343, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 347; (xvii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 351, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 355; (xviiii) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 359, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 355; (xix) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 367, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 371; (xx) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 375, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 380; (xxi) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 391, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 395; (xxii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 399, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 403; (xxiii) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 407, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 411; (xxiv) vhCDR1, vhCDR2, and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 415, and the light chain complementarity determining regions (vlCDR1), vlCDR2, and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 419; (xxv) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 423, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO: 5; or (xxvi) vhCDR1, vhCDR2 and vhCDR3 of the variable heavy chain domain (VH2) having the amino acid sequence of SEQ ID NO: 431, and the light chain complementarity determining regions (vlCDR1), vlCDR2 and vlCDR3 of the variable light chain domain (VL2) having the amino acid sequence of SEQ ID NO:

435.

2. (a) The first antigen-binding domain is (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 441; and vhCDR3 having the amino acid sequence of SEQ ID NO: 442; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 444, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 446; (ii) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 449; and vhCDR3 having the amino acid sequence of SEQ ID NO: 450; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 452, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 454; (iii) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 457; and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; and vlCDR1 having the amino acid sequence of SEQ ID NO: 460, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 462; or, (iv) vhCDR1 having the amino acid sequence of SEQ ID NO: 464; vhCDR2 having the amino acid sequence of SEQ ID NO: 465; and vhCDR3 having the amino acid sequence of SEQ ID NO: 466; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 356, vlCDR2 having the amino acid sequence of SEQ ID NO: 357, and vlCDR3 having the amino acid sequence of SEQ ID NO: 470; including, and, (b) The second antigen-binding domain is (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; (ii) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; (iii) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; (iv) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; (v) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or, (vi) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; The antibody or antibody fragment according to claim 1, comprising:

3. (a) The first antigen-binding domain is (i) VH1 having the amino acid sequence of SEQ ID NO: 439; and VL1 having the amino acid sequence of SEQ ID NO: 443; (ii) VH1 having the amino acid sequence of SEQ ID NO: 447; and VL1 having the amino acid sequence of SEQ ID NO: 451; (iii) VH1 having the amino acid sequence of SEQ ID NO: 455; and VL1 having the amino acid sequence of SEQ ID NO: 459; (iv) VH1 having the amino acid sequence of SEQ ID NO: 463; and VL1 having the amino acid sequence of SEQ ID NO: 467; (v) VH1 having the amino acid sequence of SEQ ID NO: 471; and VL1 having the amino acid sequence of SEQ ID NO: 472; (vi) VH1 having the amino acid sequence of SEQ ID NO: 473; and VL1 having the amino acid sequence of SEQ ID NO: 474; (vii) VH1 having the amino acid sequence of SEQ ID NO: 475; and VL1 having the amino acid sequence of SEQ ID NO: 472; (viiii) VH1 having the amino acid sequence of SEQ ID NO: 477; and VL1 having the amino acid sequence of SEQ ID NO: 478; (ix) VH1 having the amino acid sequence of SEQ ID NO: 479; and VL1 having the amino acid sequence of SEQ ID NO: 480; (x) VH1 having the amino acid sequence of SEQ ID NO: 481; and VL1 having the amino acid sequence of SEQ ID NO: 482; (xi) VH1 having the amino acid sequence of SEQ ID NO: 483; and VL1 having the amino acid sequence of SEQ ID NO: 484; (xi) VH1 having the amino acid sequence of SEQ ID NO: 487; and VL1 having the amino acid sequence of SEQ ID NO: 467; (xiiii) VH1 having the amino acid sequence of SEQ ID NO: 489; and VL1 having the amino acid sequence of SEQ ID NO: 490; (xiv) VH1 having the amino acid sequence of SEQ ID NO: 491; and VL1 having the amino acid sequence of SEQ ID NO: 492; (xv) VH1 having the amino acid sequence of SEQ ID NO: 495; and VL1 having the amino acid sequence of SEQ ID NO: 496; or, (xvi) VH1 having the amino acid sequence of SEQ ID NO: 497; and VL1 having the amino acid sequence of SEQ ID NO: 498; including, and, (b) The second antigen-binding domain is (i) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 5; (ii) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 5; (iii) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 19; (iv) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 19; (v) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 5; (vi) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 19; (vii) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 75; (viiii) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 75; (ix) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 79; (x) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 111; (xi) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 75; (xi) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 79; (xiii) VH2 having the amino acid sequence of SEQ ID NO: 320; and VL2 having the amino acid sequence of SEQ ID NO: 324; (xiv) VH2 having the amino acid sequence of SEQ ID NO: 327; and VL2 having the amino acid sequence of SEQ ID NO: 331; (xv) VH2 having the amino acid sequence of SEQ ID NO: 335; and VL2 having the amino acid sequence of SEQ ID NO: 339; (xvi) VH2 having the amino acid sequence of SEQ ID NO: 343; and VL2 having the amino acid sequence of SEQ ID NO: 347; (xvii) VH2 having the amino acid sequence of SEQ ID NO: 351; and VL2 having the amino acid sequence of SEQ ID NO: 355; (xviiii) VH2 having the amino acid sequence of SEQ ID NO: 359; and VL2 having the amino acid sequence of SEQ ID NO: 355; (xix) VH2 having the amino acid sequence of SEQ ID NO: 367; and VL2 having the amino acid sequence of SEQ ID NO: 371; (xx) VH2 having the amino acid sequence of SEQ ID NO: 375; and VL2 having the amino acid sequence of SEQ ID NO: 380; or, (xxi) VH2 having the amino acid sequence of SEQ ID NO: 391; and VL2 having the amino acid sequence of SEQ ID NO: 395; (xxii) VH2 having the amino acid sequence of SEQ ID NO: 399; and VL2 having the amino acid sequence of SEQ ID NO: 403; (xxiii) VH2 having the amino acid sequence of SEQ ID NO: 407; and VL2 having the amino acid sequence of SEQ ID NO: 411; (xxiv) VH2 having the amino acid sequence of SEQ ID NO: 415; and VL2 having the amino acid sequence of SEQ ID NO: 419; (xxv) VH2 having the amino acid sequence of SEQ ID NO: 423; and VL2 having the amino acid sequence of SEQ ID NO: 5; or, (xxvi) VH2 having the amino acid sequence of SEQ ID NO: 431; and VL2 having the amino acid sequence of SEQ ID NO: 435; The antibody or antibody fragment according to claim 1, comprising:

4. (a) The first antigen-binding domain is (i) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 439; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 443; (ii) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 447; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 451; (iii) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 455; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 459; (iv) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 463; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 467; (v) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 471; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 472; (vi) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 473; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 474; (vii) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 475; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 472; (viiii) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 477; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 478; (ix) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 479; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 480; (x) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 481; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 482; (xi) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 483; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 484; (xi) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 487; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 467; (xiiii) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 489; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 490; (xiv) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 491; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 492; (xv) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 495; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 496; or (xvi) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 497; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 498; including, and, (b) The second antigen-binding domain is (i) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 5; (ii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 11; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 5; (iii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 19; (iv) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 11; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 19; (v) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 15; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 5; (vi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 15; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 19; (vii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 63; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 75; (viiii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 67; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 75; (ix) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 63; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 79; (x) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 67; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 111; (xi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 71; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 75; (xi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 71; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 79; (xiiii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 320; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 324; (xiv) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 327; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 331; (xv) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 335; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 339; (xvi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 343; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 347; (xvii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 351; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 355; (xviiii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 359; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 355; (xix) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 367; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 371; (xx) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 375; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 380; or (xxi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 391; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 395; (xxii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 399; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 403; (xxiii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 407; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 411; (xxiv) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 415; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 419; (xxv) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 423; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 5; or (xxvi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 431; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 435; The antibody or antibody fragment according to claim 1, comprising:

5. (a) The first antigen-binding domain is (i) VH1 having the amino acid sequence of SEQ ID NO: 439; and VL1 having the amino acid sequence of SEQ ID NO: 443; (ii) VH1 having the amino acid sequence of SEQ ID NO: 447; and VL1 having the amino acid sequence of SEQ ID NO: 451; (iii) VH1 having the amino acid sequence of SEQ ID NO: 455; and VL1 having the amino acid sequence of SEQ ID NO: 459; or, (iv) VH1 having the amino acid sequence of SEQ ID NO: 463; and VL1 having the amino acid sequence of SEQ ID NO: 467; including, and, (b) The second antigen-binding domain is (i) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 5; (ii) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 5; (iii) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 19; (iv) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 19; (v) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 5; (vi) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 19; (vii) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 75; (viiii) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 75; (ix) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 79; (x) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 79; (xi) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 75; or, (xi) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 79; The antibody or antibody fragment according to claim 1, comprising:

6. The antibody or antibody fragment according to claim 1, wherein the first antigen-binding domain is a bispecific antibody, Fab, F(ab')2, Fc, Fabc, Fv, scFv, or a protein scaffold.

7. The antibody or antibody fragment according to claim 1, wherein the second antigen-binding domain is a bispecific antibody, Fab, F(ab')2, Fc, Fabc, Fv, scFv, or a protein scaffold.

8. The antibody or antibody fragment according to claim 1, wherein the antibody is a humanized antibody or an antibody fragment.

9. The antibody or antibody fragment according to claim 1, wherein the antibody is an IgG antibody or an antibody fragment.

10. The antibody or antibody fragment according to claim 1, wherein the antibody comprises a first Fc domain and a second Fc domain.

11. The antibody or antibody fragment according to claim 10, wherein one of the first and second Fc domains comprises the amino acid substitution T366W, and the other of the first and second Fc domains comprises one or more amino acid substitutions T366S / L368A / Y407V (wherein the numbering follows EU numbering).

12. The antibody or antibody fragment according to claim 10, wherein the first and second Fc domains comprise one or more amino acid substitutions L234A / L235A / D265S (wherein numbering follows EU numbering).

13. The antibody or antibody fragment according to claim 10, wherein the first or second Fc domain comprises one or more amino acid substitutions H435R / Y436F (where numbering follows EU numbering).

14. The antibody or antibody fragment according to claim 10, wherein the first and second Fc domains comprise amino acid substitutions selected from: S364K / E357Q;L368D / K370S;S364K;L368D / K370S;S364K;L368E / K370S;D401K;T411E / K360E / Q362E;T366W; and T366S / L368A / Y407V (where the numbering follows EU numbering).

15. The antibody or antibody fragment according to claim 1, wherein the first antigen-binding domain is Fab and the second antigen-binding domain is a stapled scFv.

16. (a) The first antigen-binding domains include vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 457; and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 460, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 462; including, and, (b) The second antigen-binding domains include vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; The antibody or antibody fragment according to claim 1, comprising:

17. (a) The first antigen-binding domain is VH1 having the amino acid sequence of SEQ ID NO: 455; and VL1 having the amino acid sequence of SEQ ID NO: 459; including, and, (b) The second antigen-binding domains are VH2 having the amino acid sequence of SEQ ID NO: 63 and VL2 having the amino acid sequence of SEQ ID NO: 79; The antibody or antibody fragment according to claim 1, comprising:

18. A nucleic acid sequence encoding an antibody or antibody fragment according to claim 2.

19. An expression vector comprising the nucleic acid sequence described in claim 18.

20. A host cell comprising the expression vector according to claim 19.

21. Antibodies or antibody fragments, including those listed below: a) The first monomer comprising VH1-CH1-hinge-CH2-CH3 (from N-terminus to C-terminus), wherein VH1 is a first variable heavy chain domain and CH2-CH3 is a first Fc domain; And, b) i) A single-chain variable fragment (scFv) comprising a second variable heavy chain domain (VH2), a linker, and a second variable light chain domain (VL2), ii) A second Fc domain wherein scFv is covalently bonded to the N-terminus of the second Fc domain using a domain linker; A second monomer containing; c) A light chain comprising VL1-CL (from N-terminus to C-terminus), wherein VL1 is a first variable light chain domain and CL is a constant light chain domain; (Here, VH1 and VL1 combine to form a first antigen-binding domain, VH2 and VL2 combine to form a second binding domain, and, Here, the first antigen-binding domain binds to CD20, and the second antigen-binding domain binds to CD28.

22. (a) The first antigen-binding domain is (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 441; and vhCDR3 having the amino acid sequence of SEQ ID NO: 442; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 444, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 446; (ii) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 449; and vhCDR3 having the amino acid sequence of SEQ ID NO: 450; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 452, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 454; (iii) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 457; and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; and vlCDR1 having the amino acid sequence of SEQ ID NO: 460, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 462; or, (iv) vhCDR1 having the amino acid sequence of SEQ ID NO: 464; vhCDR2 having the amino acid sequence of SEQ ID NO: 465; and vhCDR3 having the amino acid sequence of SEQ ID NO: 466; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 356, vlCDR2 having the amino acid sequence of SEQ ID NO: 357, and vlCDR3 having the amino acid sequence of SEQ ID NO: 470; including, and, (b) The second antigen-binding domain is (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; (ii) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; (iii) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; (iv) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; (v) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or, (vi) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; The antibody or antibody fragment according to claim 21, comprising:

23. (a) The first antigen-binding domain is (i) VH1 having the amino acid sequence of SEQ ID NO: 439; and VL1 having the amino acid sequence of SEQ ID NO: 443; (ii) VH1 having the amino acid sequence of SEQ ID NO: 447; and VL1 having the amino acid sequence of SEQ ID NO: 451; (iii) VH1 having the amino acid sequence of SEQ ID NO: 455; and VL1 having the amino acid sequence of SEQ ID NO: 459; (iv) VH1 having the amino acid sequence of SEQ ID NO: 463; and VL1 having the amino acid sequence of SEQ ID NO: 467; (v) VH1 having the amino acid sequence of SEQ ID NO: 471; and VL1 having the amino acid sequence of SEQ ID NO: 472; (vi) VH1 having the amino acid sequence of SEQ ID NO: 473; and VL1 having the amino acid sequence of SEQ ID NO: 474; (vii) VH1 having the amino acid sequence of SEQ ID NO: 475; and VL1 having the amino acid sequence of SEQ ID NO: 472; (viiii) VH1 having the amino acid sequence of SEQ ID NO: 477; and VL1 having the amino acid sequence of SEQ ID NO: 478; (ix) VH1 having the amino acid sequence of SEQ ID NO: 479; and VL1 having the amino acid sequence of SEQ ID NO: 480; (x) VH1 having the amino acid sequence of SEQ ID NO: 481; and VL1 having the amino acid sequence of SEQ ID NO: 482; (xi) VH1 having the amino acid sequence of SEQ ID NO: 483; and VL1 having the amino acid sequence of SEQ ID NO: 484; (xi) VH1 having the amino acid sequence of SEQ ID NO: 487; and VL1 having the amino acid sequence of SEQ ID NO: 467; (xiiii) VH1 having the amino acid sequence of SEQ ID NO: 489; and VL1 having the amino acid sequence of SEQ ID NO: 490; (xiv) VH1 having the amino acid sequence of SEQ ID NO: 491; and VL1 having the amino acid sequence of SEQ ID NO: 492; (xv) VH1 having the amino acid sequence of SEQ ID NO: 495; and VL1 having the amino acid sequence of SEQ ID NO: 496; or, (xvi) VH1 having the amino acid sequence of SEQ ID NO: 497; and VL1 having the amino acid sequence of SEQ ID NO: 498; including, and, (b) The second antigen-binding domain is (i) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 5; (ii) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 5; (iii) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 19; (iv) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 19; (v) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 5; (vi) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 19; (vii) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 75; (viiii) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 75; (ix) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 79; (x) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 111; (xi) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 75; (xi) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 79; (xiii) VH2 having the amino acid sequence of SEQ ID NO: 320; and VL2 having the amino acid sequence of SEQ ID NO: 324; (xiv) VH2 having the amino acid sequence of SEQ ID NO: 327; and VL2 having the amino acid sequence of SEQ ID NO: 331; (xv) VH2 having the amino acid sequence of SEQ ID NO: 335; and VL2 having the amino acid sequence of SEQ ID NO: 339; (xvi) VH2 having the amino acid sequence of SEQ ID NO: 343; and VL2 having the amino acid sequence of SEQ ID NO: 347; (xvii) VH2 having the amino acid sequence of SEQ ID NO: 351; and VL2 having the amino acid sequence of SEQ ID NO: 355; (xviiii) VH2 having the amino acid sequence of SEQ ID NO: 359; and VL2 having the amino acid sequence of SEQ ID NO: 355; (xix) VH2 having the amino acid sequence of SEQ ID NO: 367; and VL2 having the amino acid sequence of SEQ ID NO: 371; (xx) VH2 having the amino acid sequence of SEQ ID NO: 375; and VL2 having the amino acid sequence of SEQ ID NO: 380; or, (xxi) VH2 having the amino acid sequence of SEQ ID NO: 391; and VL2 having the amino acid sequence of SEQ ID NO: 395; (xxii) VH2 having the amino acid sequence of SEQ ID NO: 399; and VL2 having the amino acid sequence of SEQ ID NO: 403; (xxiii) VH2 having the amino acid sequence of SEQ ID NO: 407; and VL2 having the amino acid sequence of SEQ ID NO: 411; (xxiv) VH2 having the amino acid sequence of SEQ ID NO: 415; and VL2 having the amino acid sequence of SEQ ID NO: 419; (xxv) VH2 having the amino acid sequence of SEQ ID NO: 423; and VL2 having the amino acid sequence of SEQ ID NO: 5; or, (xxvi) VH2 having the amino acid sequence of SEQ ID NO: 431; and VL2 having the amino acid sequence of SEQ ID NO: 435; The antibody or antibody fragment according to claim 21, comprising:

24. (a) The first antigen-binding domain is (i) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 439; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 443; (ii) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 447; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 451; (iii) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 455; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 459; (iv) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 463; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 467; (v) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 471; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 472; (vi) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 473; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 474; (vii) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 475; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 472; (viiii) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 477; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 478; (ix) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 479; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 480; (x) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 481; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 482; (xi) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 483; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 484; (xi) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 487; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 467; (xiiii) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 489; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 490; (xiv) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 491; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 492; (xv) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 495; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 496; or (xvi) VH1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 497; and VL1 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 498; including, and, (b) The second antigen-binding domain is (i) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 5; (ii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 11; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 5; (iii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 19; (iv) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 11; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 19; (v) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 15; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 5; (vi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 15; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 19; (vii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 63; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 75; (viiii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 67; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 75; (ix) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 63; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 79; (x) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 67; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 111; (xi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 71; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 75; (xi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 71; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 79; (xiiii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 320; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 324; (xiv) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 327; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 331; (xv) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 335; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 339; (xvi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 343; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 347; (xvii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 351; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 355; (xviiii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 359; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 355; (xix) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 367; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 371; (xx) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 375; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 380; or (xxi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 391; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 395; (xxii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 399; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 403; (xxiii) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 407; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 411; (xxiv) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 415; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 419; (xxv) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 423; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 5; or (xxvi) VH2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 431; and VL2 having an amino acid sequence that is at least 95% identical to SEQ ID NO: 435; The antibody or antibody fragment according to claim 21, comprising:

25. (a) The first antigen-binding domain is (i) VH1 having the amino acid sequence of SEQ ID NO: 439; and VL1 having the amino acid sequence of SEQ ID NO: 443; (ii) VH1 having the amino acid sequence of SEQ ID NO: 447; and VL1 having the amino acid sequence of SEQ ID NO: 451; (iii) VH1 having the amino acid sequence of SEQ ID NO: 455; and VL1 having the amino acid sequence of SEQ ID NO: 459; or, (iv) VH1 having the amino acid sequence of SEQ ID NO: 463; and VL1 having the amino acid sequence of SEQ ID NO: 467; including, and, (b) The second antigen-binding domain is (i) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 5; (ii) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 5; (iii) VH2 having the amino acid sequence of SEQ ID NO: 1; and VL2 having the amino acid sequence of SEQ ID NO: 19; (iv) VH2 having the amino acid sequence of SEQ ID NO: 11; and VL2 having the amino acid sequence of SEQ ID NO: 19; (v) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 5; (vi) VH2 having the amino acid sequence of SEQ ID NO: 15; and VL2 having the amino acid sequence of SEQ ID NO: 19; (vii) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 75; (viiii) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 75; (ix) VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 79; (x) VH2 having the amino acid sequence of SEQ ID NO: 67; and VL2 having the amino acid sequence of SEQ ID NO: 79; (xi) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 75; or, (xi) VH2 having the amino acid sequence of SEQ ID NO: 71; and VL2 having the amino acid sequence of SEQ ID NO: 79; The antibody or antibody fragment according to claim 21, comprising:

26. (a) The first monomer is selected from the amino acid sequences represented by SEQ ID NOs: 405, 422, 427, 436, 453, 456, 461, and 485; b) The second monomer is selected from the amino acid sequences represented by SEQ ID NOs: 406, 417, 418, 421, 428, 430, 432, 434, 438, 448, 468, 469, and 486; and, c) The light chain is selected from the amino acid sequences represented by SEQ ID NOs: 334, 366, 426, and 429. The antibody or antibody fragment according to claim 21.

27. The antibody or antibody fragment according to claim 21, wherein the antibody is a humanized antibody or an antibody fragment.

28. The antibody or antibody fragment according to claim 21, wherein the antibody is an IgG antibody or an antibody fragment.

29. The antibody or antibody fragment according to claim 21, wherein one of the first and second Fc domains comprises an amino acid substitution T366W, and the other of the first and second Fc domains comprises one or more amino acid substitutions T366S / L368A / Y407V (wherein the numbering follows EU numbering).

30. The antibody or antibody fragment according to claim 21, wherein the first and second Fc domains comprise one or more amino acid substitutions L234A / L235A / D265S (where the numbering follows EU numbering).

31. The antibody or antibody fragment according to claim 21, wherein the first or second Fc domain comprises one or more amino acid substitutions H435R / Y436F (where the numbering follows EU numbering).

32. The antibody or antibody fragment according to claim 21, wherein the first and second Fc domains comprise amino acid substitutions selected from: S364K / E357Q;L368D / K370S;S364K;L368D / K370S;S364K;L368E / K370S;D401K;T411E / K360E / Q362E;T366W; and T366S / L368A / Y407V (where the numbering follows EU numbering).

33. (a) The first antigen-binding domain comprises vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 457; and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; and vlCDR1 having the amino acid sequence of SEQ ID NO: 460, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 462; and, (b) The second antigen-binding domain includes vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; The antibody or antibody fragment according to claim 21.

34. (a) The first antigen-binding domain comprises VH1 having the amino acid sequence of SEQ ID NO: 455; and VL1 having the amino acid sequence of SEQ ID NO: 459; (b) The second antigen-binding domain comprises VH2 having the amino acid sequence of SEQ ID NO: 63; and VL2 having the amino acid sequence of SEQ ID NO: 79; The antibody or antibody fragment according to claim 21.

35. The antibody or antibody fragment according to claim 21, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 461, the second monomer comprises the amino acid sequence of SEQ ID NO: 469, and the light chain comprises the amino acid sequence of SEQ ID NO:

429.

36. A method for treating a B-cell malignancy in a patient requiring treatment of a B-cell malignancy, comprising administering to the patient a combination of an anti-CD20 × anti-CD28 antibody or an antibody fragment and a T-cell engager, wherein: (A) The anti-CD20 × anti-CD28 antibody or a fragment thereof (a)(i) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 441; and vhCDR3 having the amino acid sequence of SEQ ID NO: 442; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 444, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 446; (ii) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 449; and vhCDR3 having the amino acid sequence of SEQ ID NO: 450; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 452, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 454; (iii) vhCDR1 having the amino acid sequence of SEQ ID NO: 440; vhCDR2 having the amino acid sequence of SEQ ID NO: 457; and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; and vlCDR1 having the amino acid sequence of SEQ ID NO: 460, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 462; or, (iv) vhCDR1 having the amino acid sequence of SEQ ID NO: 464; vhCDR2 having the amino acid sequence of SEQ ID NO: 465; and vhCDR3 having the amino acid sequence of SEQ ID NO: 466; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 356, vlCDR2 having the amino acid sequence of SEQ ID NO: 357, and vlCDR3 having the amino acid sequence of SEQ ID NO: 470; A first antigen-binding domain that binds to CD20, including, and (b) (i) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; (ii) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; (iii) vhCDR1 having the amino acid sequence of SEQ ID NO: 2; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; (iv) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 3; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; (v) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 8; or, (vi) vhCDR1 having the amino acid sequence of SEQ ID NO: 12; vhCDR2 having the amino acid sequence of SEQ ID NO: 17; and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; as well as vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO: 22; A second antigen-binding domain that binds to CD28, including the above; including, and, (B) The T cell engager is an anti-CD79b × anti-CD20 × anti-CD3 antibody or a fragment of that antibody; The aforementioned method.

37. The anti-CD20 × anti-CD28 antibody or the antibody fragment is (i) a first antigen-binding domain that binds to CD20, comprising vhCDR1 having the amino acid sequence of SEQ ID NO: 440, vhCDR2 having the amino acid sequence of SEQ ID NO: 457, and vhCDR3 having the amino acid sequence of SEQ ID NO: 458; and vlCDR1 having the amino acid sequence of SEQ ID NO: 460, vlCDR2 having the amino acid sequence of SEQ ID NO: 445, and vlCDR3 having the amino acid sequence of SEQ ID NO: 462; and The method according to claim 36, comprising (ii) a second antigen-binding domain that binds to CD28, including vhCDR1 having the amino acid sequence of SEQ ID NO: 2, vhCDR2 having the amino acid sequence of SEQ ID NO: 3, and vhCDR3 having the amino acid sequence of SEQ ID NO: 4; and vlCDR1 having the amino acid sequence of SEQ ID NO: 6, vlCDR2 having the amino acid sequence of SEQ ID NO: 7, and vlCDR3 having the amino acid sequence of SEQ ID NO:

22.

38. The anti-CD79b × anti-CD20 × anti-CD3 antibody or the antibody fragment is (a) an antigen-binding domain that binds to CD79b, comprising: vhCDR1 having the amino acid sequence of SEQ ID NO: 524, vhCDR2 having the amino acid sequence of SEQ ID NO: 525, and vhCDR3 having the amino acid sequence of SEQ ID NO: 526; and vlCDR1 having the amino acid sequence of SEQ ID NO: 528, vlCDR2 having the amino acid sequence of SEQ ID NO: 529, and vlCDR3 having the amino acid sequence of SEQ ID NO: 530; (b) an antigen-binding domain that binds to CD20, comprising vhCDR1 having the amino acid sequence of SEQ ID NO: 540, vhCDR2 having the amino acid sequence of SEQ ID NO: 541, and vhCDR3 having the amino acid sequence of SEQ ID NO: 542; and vlCDR1 having the amino acid sequence of SEQ ID NO: 544, vlCDR2 having the amino acid sequence of SEQ ID NO: 545, and vlCDR3 having the amino acid sequence of SEQ ID NO: 546; and The method according to claim 36, comprising (c) an antigen-binding domain that binds to CD3, including vhCDR1 having the amino acid sequence of SEQ ID NO: 532, vhCDR2 having the amino acid sequence of SEQ ID NO: 533, and vhCDR3 having the amino acid sequence of SEQ ID NO: 534; and vlCDR1 having the amino acid sequence of SEQ ID NO: 536, vlCDR2 having the amino acid sequence of SEQ ID NO: 537, and vlCDR3 having the amino acid sequence of SEQ ID NO:

538.

39. The anti-CD20 × anti-CD28 antibody or the antibody fragment is (a) A CD20 antigen-binding domain comprising VH1 having the amino acid sequence of SEQ ID NO: 455; and VL1 having the amino acid sequence of SEQ ID NO: 459; and The method according to claim 36, comprising a CD28 antigen-binding domain having (b) VH2 having the amino acid sequence of SEQ ID NO: 63 and VL2 having the amino acid sequence of SEQ ID NO:

79.

40. The anti-CD79b × anti-CD20 × anti-CD3 antibody or the antibody fragment is (a) A CD79b antigen-binding domain comprising VH1 having the amino acid sequence of SEQ ID NO: 523 and VL1 having the amino acid sequence of SEQ ID NO: 527; (b) A CD20 antigen-binding domain comprising VH2 having the amino acid sequence of SEQ ID NO: 539; and VL2 having the amino acid sequence of SEQ ID NO: 543; The method according to claim 36, comprising a CD3 antigen-binding domain having (c) VH2 having the amino acid sequence of SEQ ID NO: 531 and VL2 having the amino acid sequence of SEQ ID NO:

535.

41. (a) The anti-CD20 × anti-CD28 antibody or the antibody fragment is (i) A CD20 antigen-binding domain comprising VH1 having the amino acid sequence of SEQ ID NO: 455; and VL1 having the amino acid sequence of SEQ ID NO: 459; and (ii) A CD28 antigen-binding domain comprising VH2 having the amino acid sequence of SEQ ID NO: 63 and VL2 having the amino acid sequence of SEQ ID NO: 79; including, and, (b) The anti-CD79b × anti-CD20 × anti-CD3 antibody or the antibody fragment is (i) CD79b antigen-binding domains comprising VH1 having the amino acid sequence of SEQ ID NO: 523 and VL1 having the amino acid sequence of SEQ ID NO: 527; (ii) CD20 antigen-binding domains comprising VH2 having the amino acid sequence of SEQ ID NO: 539 and VL2 having the amino acid sequence of SEQ ID NO: 543, and (iii) CD3 antigen-binding domains comprising VH2 having the amino acid sequence of SEQ ID NO: 531 and VL2 having the amino acid sequence of SEQ ID NO: 535; The method according to claim 36, including the method described in claim 36.

42. The anti-CD20 × anti-CD28 antibody or the antibody fragment is (a) A first monomeric polypeptide having the amino acid sequence of SEQ ID NO: 461; (b) A second monomeric polypeptide having the amino acid sequence of SEQ ID NO: 469; and (c) The method according to claim 36, comprising a light chain polypeptide having the amino acid sequence of SEQ ID NO:

429.

43. The anti-CD79b × anti-CD20 × anti-CD3 antibody or the antibody fragment is (a) A first monomeric polypeptide having the amino acid sequence of SEQ ID NO: 520; (b) A second monomeric polypeptide having the amino acid sequence of SEQ ID NO: 521; and (c) The method according to claim 36, comprising a light chain polypeptide having the amino acid sequence of SEQ ID NO:

522.

44. (a) The anti-CD20 × anti-CD28 antibody or the antibody fragment is (i) A first monomeric polypeptide having the amino acid sequence of SEQ ID NO: 461; (ii) A second monomeric polypeptide having the amino acid sequence of SEQ ID NO: 469; (iii) A first light chain polypeptide having the amino acid sequence of SEQ ID NO: 429; including, and, (b) The anti-CD79b × anti-CD20 × anti-CD3 antibody or the antibody fragment is (i) A fourth monomeric polypeptide having the amino acid sequence of SEQ ID NO: 520; (ii) A fifth monomeric polypeptide having the amino acid sequence of SEQ ID NO: 521; and (iii) A second light chain polypeptide having the amino acid sequence of SEQ ID NO: 522; The method according to claim 36, including the method described in claim 36.

45. The method according to claim 36, wherein the anti-CD20 × anti-CD28 antibody or an antibody fragment thereof, and the anti-CD79b × anti-CD20 × anti-CD3 antibody or an antibody fragment thereof are administered together in a single composition or separately in two or more different compositions.

46. The method according to claim 36, wherein the anti-CD20 × anti-CD28 antibody or an antibody fragment thereof, and the anti-CD79b × anti-CD20 × anti-CD3 antibody or an antibody fragment thereof are administered subcutaneously or intravenously.

47. The method according to claim 36, wherein the anti-CD20 × anti-CD28 antibody or an antibody fragment thereof, and the anti-CD79b × anti-CD20 × anti-CD3 antibody or an antibody fragment thereof are administered simultaneously or sequentially.

48. The method according to claim 36, wherein the B-cell malignant tumor is B-cell lymphoma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Waldenström macroglobulinemia (WM), multiple myeloma (MM), mucosa-associated lymphoid tissue (MALT) lymphoma, Hodgkin lymphoma, Burkitt lymphoma, pilocytic cell leukemia, or plasmacytoma.

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