Bispecific antibodies that bind to CD28 and nectin-4

Abstract

Multispecific antibodies that bind to an effector associated antigen (EAA), such as CD28, and a disease associated antigen (DAA), such as Nectin-4, are provided, along with methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use to treat disorders, such as cancer, where modulation of effector cell activity against target cells can be used as a treatment modality.

Description

Attorney Ref.62571.00009WO01 (RND-0002-WO) BISPECIFIC ANTIBODIES THAT BIND TO CD28 AND NECTIN-4 CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of the filing date of U.S. Provisional Application Serial No.63 / 643,345, filed on May 6, 2024, the disclosure of which is incorporated by reference herein in its entirety. This application also claims priority benefit of the filing date of U.S. Provisional Application Serial No.63 / 695,765, filed on September 17, 2024, the disclosure of which is incorporated by reference herein in its entirety. FIELD OF THE INVENTION

[0002] The present invention concerns multispecific antibodies that bind to an effector associated antigen (EAA), such as CD28, and a disease associated antigen (DAA), such as Nectin-4. The invention further concerns methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and their use to treat disorders, such as cancer, where modulation of effector cell activity against target cells can be used as a treatment modality. BACKGROUND OF THE INVENTION Cluster of differentiation 28 (CD28)

[0003] Cell surface proteins expressed on immune effector cells may be referred to as effector- associated antigens (EAAs). One example of an EAA is CD28, also known as TP44 (UniProt KB No. P10747). CD28 is a cell surface glycoprotein expressed as a disulfide linked homodimer with a molecular weight of approximately 44 kDa. It is expressed by naïve T cells and generally at least 80% of CD4+ T cells and 50% of CD8+ T cells in adult human peripheral blood. Weng et al, Trends Immunol. 2009 Jul;30(7):306-12; Esensten et al, Immunity 2016 May 17;44(5):973-88. Unlike many other costimulatory molecules, CD28 expression is constitutive. CD28 is a powerful costimulatory receptor and activation through interaction with its natural ligands CD80 and CD86 potentiates T-cell activation, survival, proliferation and effector functions initiated by ligation of the T-cell receptor (TCR). Esensten et al, Immunity 2016 May 17;44(5):973-88; Chen et al, Nat Rev Immunol. 2013 Apr; 13(4):227-42. CD28 can be negatively regulated by the coinhibitory receptor, CTLA4, which interacts with both CD80 and CD86 with a higher affinity than CD28, effectively out-competing it for binding. Esensten et al, Immunity 2016 May 17;44(5):973-88. In the tumor microenvironment elevated CTLA4 expression and / or limited presence of CD80 and CD86 can impair CD28 signaling and therefore T-cell function. Bispecific antibodies containing a CD28 agonist domain and a tumor targeting domain have the potential to functionally replace signaling through the endogenous ligands CD80 and CD86 within the tumor microenvironment, leading to enhanced T cell activation in the presence of TCR stimulus and supporting a robust T cell-mediated anti-tumor response.Attorney Ref.62571.00009WO01 (RND-0002-WO) Nectin-4

[0004] Target molecules expressed on diseased cells may be referred to as disease-associated antigens (DAAs). An example of a DAA includes Nectin-4, also known as nectin cell adhesion molecule 4 (UniProt KB No. Q96NY8). Nectin-4 is a surface expressed tumor-associated glycoprotein with a molecular weight of 55.5 kDa. It is a member of the nectin immunoglobulin-like molecules, a four member structurally related family of proteins which contribute to adherens junctions and tight junctions. Three other family members, (Nectin-1, Nectin-2, and Nectin-3), are highly expressed in a variety of adult tissues, while Nectin-4 is primarily expressed during embryogenesis and is only found in a limited number of adult tissues, including skin, esophagus and bladder. Nectin-4 may be overexpressed by a wide range of solid tumors, including bladder, breast, gastric, lung, ovarian and pancreatic cancers among others. Challita-Eid et al., Cancer Res.2016, May 15;76(10):3003-13; Deng et al., Cancer Cell Int. 2019 Apr 23;19:106; Derycke et al., Am J Clin Pathol. 2010 Nov;134(5):835- 45; Nishiwada et al., J Exp Clin Cancer Res. 2015 Mar 28;34(1):30; Takano et al., Cancer Res. 2009 Aug 15;69(16):6694-703; Zhang et al., Oncol Lett. 2018 Jun;15(6):8789-8795. It is a clinically validated therapeutic target in urothelial cancer and its expression has been associated with poor patient outcomes in other solid tumors such as gastric and pancreatic cancers. Deng et al., Cancer Cell Int.2019 Apr 23;19:106; Nishiwada et al., J Exp Clin Cancer Res. 2015 Mar 28;34(1):30; Zhang et al., Oncol Lett.2018 Jun;15(6):8789-8795. SUMMARY OF THE INVENTION

[0005] Aspects of the present disclosure include antibodies that bind to CD28, comprising a variable region comprising: (a) a heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 1, 2, 3, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67; and / or (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 9, 10, 11, 12, 13, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85; and / or (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 18, 19, 20, 21, 22, 86, 87, 88, 89, 90, 91, and 92; and a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44.

[0006] In some embodiments, the variable region of an antibody comprises: (a) a heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 1, 2, 3, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67; and (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 9, 10, 11, 12, 13, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85; and (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 18, 19, 20, 21, 22, 86, 87, 88, 89, 90, 91, and 92; and a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0007] In some embodiments, the variable region of an antibody comprises: (a) a heavy chain variable region sequence comprising: a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 11; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 13; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 12; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 86; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or a heavy chain CDR1 sequence comprising SEQ ID NO: 58; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 71; a heavy chain CDR3 sequence comprising SEQ ID NO: 90; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 72; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 86; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3Attorney Ref.62571.00009WO01 (RND-0002-WO) sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 74; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or a heavy chain CDR1 sequence comprising SEQ ID NO: 59; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 75; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 76; a heavy chain CDR3 sequence comprising SEQ ID NO: 90; or a heavy chain CDR1 sequence comprising SEQ ID NO: 60; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 77; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 78; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 77; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 71; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 61; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; aAttorney Ref.62571.00009WO01 (RND-0002-WO) heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 81; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 82; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 63; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprisingAttorney Ref.62571.00009WO01 (RND-0002-WO) SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 64; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 84; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 92; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 92; or a heavy chain CDR1 sequence comprising SEQ ID NO: 65; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 66; a heavy chain CDR2 sequence comprising SEQ ID NO: 85; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 67; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; and a light chain variable region sequence comprising: (1) a light chain CDR1 sequence comprising SEQ ID NO: 28, a light chain CDR2 sequence comprising SEQ ID NO: 36, and a light chain CDR3 sequence comprising SEQ ID NO: 44.

[0008] In some embodiments, the variable region of an antibody comprises: (a) a heavy chain variable region sequence comprising: a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 11; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 13; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 12; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; and (b) a light chain variable region sequence comprising: (1) a light chain CDR1 sequence comprising SEQ ID NO: 28, a light chain CDR2 sequence comprising SEQ ID NO: 36, and a light chain CDR3 sequence comprising SEQ ID NO: 44.

[0009] In some embodiments, the heavy chain variable region CDR1, CDR2 and CDR3 sequences are present in a human VL framework.

[0010] In some embodiments, an antibody comprises a heavy chain variable region sequence having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 93-100, 107, 121-237, and 333-334. In some embodiments, an antibody comprises a heavy chain variable region sequence selectedAttorney Ref.62571.00009WO01 (RND-0002-WO) from the group consisting of SEQ ID NOs: 93-100, 107, 121-237, and 333-334. In some embodiments, an antibody comprises a heavy chain variable region sequence of SEQ ID NO: 93. In some embodiments, an antibody comprises a heavy chain variable region sequence of SEQ ID NO: 94. In some embodiments, an antibody comprises a heavy chain variable region sequence of SEQ ID NO: 95. In some embodiments, an antibody comprises a heavy chain variable region sequence of SEQ ID NO: 96. In some embodiments, an antibody comprises a heavy chain variable region sequence of SEQ ID NO: 97. In some embodiments, an antibody comprises a heavy chain variable region sequence of SEQ ID NO: 98. In some embodiments, an antibody comprises a heavy chain variable region sequence of SEQ ID NO: 99. In some embodiments, an antibody comprises a heavy chain variable region sequence of SEQ ID NO: 100.

[0011] In some embodiments, the light chain variable region CDR1, CDR2 and CDR3 sequences are present in a human VL framework.

[0012] In some embodiments, an antibody comprises a light chain variable region having at least 95% sequence identity to SEQ ID NO: 110. In some embodiments, an antibody comprises a light chain variable region sequence comprising the sequence of SEQ ID NO: 110.

[0013] Aspects of the present disclosure include antibodies that bind to Nectin-4, comprising a variable region comprising: (a) a heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 4, and 286-291; and / or (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 10, 83, and 292-294; and / or (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 23, and 295-300; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44.

[0014] In some embodiments, the variable region of an antibody comprises: (a) a heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 4, and 286-291; and (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 10, 83, and 292-294; and (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 23, and 295-300; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44.

[0015] In some embodiments, the variable region of an antibody comprises: (a) a heavy chain variable region sequence comprising: (1) a heavy chain CDR1 sequence comprising SEQ ID NO: 4, a CDR2 sequence comprising SEQ ID NO: 10, and a CDR3 sequence comprising SEQ ID NO: 23; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; or a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 292; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; or a heavy chain CDR1 sequence comprising SEQ ID NO: 287; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chainAttorney Ref.62571.00009WO01 (RND-0002-WO) CDR3 sequence comprising SEQ ID NO: 297; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; or a heavy chain CDR1 sequence comprising SEQ ID NO: 288; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 298; or a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 299; or a heavy chain CDR1 sequence comprising SEQ ID NO: 288; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 298; or a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; or a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 293; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; or a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or a heavy chain CDR1 sequence comprising SEQ ID NO: 290; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or a heavy chain CDR1 sequence comprising SEQ ID NO: 291; a heavy chain CDR2 sequence comprising SEQ ID NO: 294; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44.

[0016] In some embodiments, the heavy chain variable region CDR1, CDR2 and CDR3 sequences are present in a human VH framework.

[0017] In some embodiments, an antibody comprises a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 110. In some embodiments, an antibody comprises a light chain variable region comprising the sequence of SEQ ID NO: 110.

[0018] Aspects of the present disclosure include multi-specific antibodies comprising: a first binding unit that binds to CD28, comprising: (a) a first heavy chain variable region sequence comprising: (1) aAttorney Ref.62571.00009WO01 (RND-0002-WO) CDR1 sequence selected from the group comprising SEQ ID NOs: 1, 2, 3, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67; and / or (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 9, 10, 11, 12, 13, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85; and / or (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 18, 19, 20, 21, 22, 86, 87, 88, 89, 90, 91, and 92; and (b) a first light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44; and a second binding unit that binds to a disease-associated antigen (DAA).

[0019] In some embodiments, a multi-specific antibody comprises a first binding unit comprising: (a) a heavy chain variable region sequence comprising:(1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 1, 2, 3, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67; and (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 9, 10, 11, 12, 13, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85; and (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 18, 19, 20, 21, 22, 86, 87, 88, 89, 90, 91, and 92; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44.

[0020] In some embodiments, a multi-specific antibody comprises a first binding unit comprising: (a) a heavy chain variable region sequence comprising: a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 11; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 13; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 12; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 86; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or a heavy chain CDR1 sequence comprising SEQ ID NO: 58; a heavyAttorney Ref.62571.00009WO01 (RND-0002-WO) chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 71; a heavy chain CDR3 sequence comprising SEQ ID NO: 90; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 72; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 86; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 74; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or a heavy chain CDR1 sequence comprising SEQ ID NO: 59; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 75; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 76; a heavy chain CDR3 sequence comprising SEQ ID NO: 90; or a heavy chain CDR1 sequence comprising SEQ ID NO: 60; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ IDAttorney Ref.62571.00009WO01 (RND-0002-WO) NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 77; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 78; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 77; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 71; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 61; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 81; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 82; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequenceAttorney Ref.62571.00009WO01 (RND-0002-WO) comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 63; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 64; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 84; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 92; or a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 92; or a heavy chain CDR1 sequence comprising SEQ ID NO: 65; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 66; a heavy chain CDR2 sequence comprising SEQ ID NO: 85; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 67; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; and a light chain variable region sequence comprising: (1) a light chain CDR1 sequence comprising SEQ ID NO: 28, a light chain CDR2 sequence comprising SEQ ID NO: 36, and a light chain CDR3 sequence comprising SEQ ID NO: 44.

[0021] In some embodiments, a multi-specific antibody comprises a first binding unit comprising: (a) a heavy chain variable region sequence comprising: a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ IDAttorney Ref.62571.00009WO01 (RND-0002-WO) NO: 19; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 11; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 13; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 12; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; and (b) a light chain variable region sequence comprising: (1) a light chain CDR1 sequence comprising SEQ ID NO: 28, a light chain CDR2 sequence comprising SEQ ID NO: 36, and a light chain CDR3 sequence comprising SEQ ID NO: 44.

[0022] In some embodiments, the CDR1, CDR2, and CDR3 sequences in the heavy chain variable region of the first binding unit are present in a human VH framework; and the CDR1, CDR2, and CDR3 sequences in the light chain variable region of the first binding unit are present in a human VL framework.

[0023] In some embodiments, a multi-specific antibody comprises a heavy chain variable region sequence of the first binding unit comprising at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 93-100, 107, 121-237, and 333-334; and a light chain variable region sequence of the first binding unit comprising at least 95% sequence identity to SEQ ID NO: 110. In some embodiments, a multi-specific antibody comprises a heavy chain variable region sequence of the first binding unit selected from the group consisting of SEQ ID NOs: 93-100, 107, 121-237, and 333-334; and a light chain variable region sequence of the first binding unit comprising the sequence of SEQ ID NO: 110. In some embodiments, a multi-specific antibody comprises a heavy chain variable region sequence of the first binding unit comprising the sequence of SEQ ID NO: 93; or a heavy chain variable region sequence of the first binding unit comprising the sequence of SEQ ID NO: 94; or a heavy chain variable region sequence of the first binding unit comprising the sequence of SEQ ID NO: 95; or a heavy chain variable region sequence of the first binding unit comprising the sequence of SEQ ID NO: 96; or a heavy chain variable region sequence of the first binding unit comprising the sequence of SEQ ID NO: 97; or a heavy chain variable region sequence of the first binding unit comprising the sequence of SEQ ID NO: 98; or a heavy chain variable region sequence of the first binding unit comprising the sequence of SEQ ID NO: 99; or a heavy chain variable region sequence of the first binding unit comprising the sequence of SEQ ID NO: 100; and a light chain variable region sequence of the first binding unit comprising the sequence of SEQ ID NO: 110.

[0024] In some embodiments, the DAA of a multi-specific antibody is a tumor associated antigen (TAA) or a tumor specific antigen (TSA). In some embodiments, the TAA is Nectin-4.

[0025] Aspects of the present disclosure include multi-specific antibodies that bind to Nectin-4, comprising a first binding unit that binds to Nectin-4, comprising: (a) a first heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 4, andAttorney Ref.62571.00009WO01 (RND-0002-WO) 286-291; and / or (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 10, 83, and 292-294; and / or (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 23, and 295- 300; and (b) a first light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44; and a second binding unit that binds to an effector-associated antigen (EAA).

[0026] In some embodiments, an antibody comprises a first binding unit comprising: (a) a heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 4, and 286-291; and (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 10, 83, and 292-294; and (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 23, and 295-300; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44.

[0027] In some embodiments, an antibody comprises a first binding unit comprising: (a) a heavy chain variable region sequence comprising: (1) a heavy chain CDR1 sequence comprising SEQ ID NO: 4, a CDR2 sequence comprising SEQ ID NO: 10, and a CDR3 sequence comprising SEQ ID NO: 23; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; or a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 292; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; or a heavy chain CDR1 sequence comprising SEQ ID NO: 287; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 297; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; or a heavy chain CDR1 sequence comprising SEQ ID NO: 288; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 298; or a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 299; or a heavy chain CDR1 sequence comprising SEQ ID NO: 288; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 298; or a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; or a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 293; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; or a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or a heavyAttorney Ref.62571.00009WO01 (RND-0002-WO) chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or a heavy chain CDR1 sequence comprising SEQ ID NO: 290; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or a heavy chain CDR1 sequence comprising SEQ ID NO: 291; a heavy chain CDR2 sequence comprising SEQ ID NO: 294; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44.

[0028] In some embodiments, the CDR1, CDR2, and CDR3 sequences in the heavy chain variable region of the first binding unit are present in a human VH framework; and the CDR1, CDR2, and CDR3 sequences in the light chain variable region of the first binding unit are present in a human VL framework.

[0029] In some embodiments, an antibody comprises a heavy chain variable region sequence of the first binding unit comprising at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 101-102, and 301-317; and a light chain variable region sequence of the first binding unit comprising at least 95% sequence identity to SEQ ID NO: 110. In some embodiments, an antibody comprises a heavy chain variable region sequence of the first binding unit selected from the group consisting of SEQ ID NOs: 101-102, and 301-317; and a light chain variable region sequence of the first binding unit comprising SEQ ID NO: 110.

[0030] In some embodiments, the EAA of a multi-specific antibody is a cell surface protein on an immune effector cell. In some embodiments, the EAA is a T-cell antigen. In some embodiments, the T- cell antigen of a multi-specific antibody is CD3. In some embodiments, the T-cell antigen is CD28.

[0031] In some embodiments, the light chain of a multi-specific antibody comprises a light chain constant region (CL). In some embodiments, the light chain is a kappa LC or a lambda LC.

[0032] In some embodiments, a multi-specific antibody comprises a heavy chain constant region sequence.

[0033] In some embodiments, the heavy chain constant region sequence of a multi-specific antibody is a wild type human IgG1 constant region sequence. In some embodiments, the heavy chain constant region sequence of a multi-specific antibody is a variant human IgG1 constant region sequence comprising an L234A mutation, an L235A mutation, a P329G mutation, or any combination thereof. In some embodiments, a multi-specific antibody comprises a variant human IgG1 constant region sequence comprising an S354C mutation or a Y349C mutation. In some embodiments, a multi-specificAttorney Ref.62571.00009WO01 (RND-0002-WO) antibody comprises a variant human IgG1 constant region sequence comprising a T366W mutation, an S354C mutation, or both a T366W mutation and an S354C mutation. In some embodiments, a multi- specific antibody comprises a variant human IgG1 constant region sequence comprising a T366S mutation, an L368A mutation, a Y407V mutation, a Y349C mutation, or any combination thereof.

[0034] In some embodiments, the heavy chain constant region sequence of a multi-specific antibody is a wild type human IgG4 constant region sequence. In some embodiments, a multi-specific antibody comprises a variant human IgG4 constant region sequence comprising an S228P mutation, an F234A mutation, an L235A mutation, or any combination thereof. In some embodiments, a multi-specific antibody comprises a variant human IgG4 constant region sequence comprising an S354C mutation or a Y349C mutation. In some embodiments, a multi-specific antibody comprises a variant human IgG4 constant region sequence comprising a T366W mutation, an S354C mutation, or both a T366W mutation and an S354C mutation. In some embodiments, a multi-specific antibody comprises a variant human IgG4 constant region sequence comprising a T366S mutation, an L368A mutation, a Y407V mutation, a Y349C mutation, or any combination thereof. In some embodiments, a multi-specific antibody comprises a variant human IgG4 constant region sequence comprising an L445P mutation.

[0035] Aspects of the present disclosure include multi-specific antibodies that bind to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 240; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 241.

[0036] Aspects of the present disclosure include multi-specific antibodies that bind to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 243; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 244.

[0037] Aspects of the present disclosure include multi-specific antibodies that bind to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 245; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 246.

[0038] Aspects of the present disclosure include multi-specific antibodies that bind to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 247; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 248.

[0039] Aspects of the present disclosure include multi-specific antibodies that bind to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) aAttorney Ref.62571.00009WO01 (RND-0002-WO) second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 249; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 250.

[0040] Aspects of the present disclosure include multi-specific antibodies that bind to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 251; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 252.

[0041] Aspects of the present disclosure include multi-specific antibodies that bind to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 253; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 254.

[0042] Aspects of the present disclosure include multi-specific antibodies that bind to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 263; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 264.

[0043] Aspects of the present disclosure include multi-specific antibodies that bind to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 265; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 266.

[0044] Aspects of the present disclosure include multi-specific antibodies that bind to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 267; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 268.

[0045] Aspects of the present disclosure include multi-specific antibodies that bind to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 256; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 256.

[0046] Aspects of the present disclosure include multi-specific antibodies that bind to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunitAttorney Ref.62571.00009WO01 (RND-0002-WO) comprising SEQ ID NO: 257; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 257.

[0047] Aspects of the present disclosure include multi-specific antibodies that bind to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 259; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 259.

[0048] Aspects of the present disclosure include multi-specific antibodies that bind to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 260; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 260.

[0049] Aspects of the present disclosure include multi-specific antibodies that bind to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 261; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 261.

[0050] Aspects of the present disclosure include multi-specific antibodies that bind to Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 258; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 258.

[0051] Aspects of the present disclosure include multi-specific antibodies that bind to Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 262; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 262.

[0052] Aspects of the present disclosure include multi-specific antibodies that bind to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 329; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 329.

[0053] Aspects of the present disclosure include multi-specific antibodies that bind to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 335; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 335.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0054] Aspects of the present disclosure include multi-specific antibodies that bind to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 336; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 336.

[0055] Aspects of the present disclosure include multi-specific antibodies that bind to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 337; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 337.

[0056] Aspects of the present disclosure include antibodies comprising means for binding to CD28. Aspects of the invention include antibodies comprising means for binding to Nectin-4.

[0057] Aspects of the present disclosure include multi-specific antibodies comprising: a first binding unit comprising means for binding to CD28; and a second binding unit that binds to a disease-associated antigen (DAA). In some embodiments, the DAA is a TAA or a TSA. In some embodiments, the TAA is Nectin-4.

[0058] Aspects of the present disclosure include multi-specific antibodies comprising: a first binding unit comprising means for binding to Nectin-4; and a second binding unit that binds to an effector- associated antigen (EAA). In some embodiments, the EAA of a multi-specific antibody is CD28.

[0059] Aspects of the present disclosure include multi-specific antibodies comprising: a first binding unit comprising means for binding to CD28; and a second binding unit comprising means for binding to Nectin-4.

[0060] Aspects of the present disclosure include bispecific antibodies comprising: a first binding unit comprising means for binding to CD28; and a second binding unit comprising means for binding to Nectin-4.

[0061] Aspects of the present disclosure include pharmaceutical compositions comprising an antibody as described herein.

[0062] Aspects of the present disclosure include polynucleotides encoding an antibody as described herein, vectors comprising such polynucleotides, and cells comprising such vectors.

[0063] Aspects of the present disclosure include methods of producing an antibody as described herein, the methods comprising growing a cell as described herein under conditions permissive for expression of the antibody, and isolating the antibody from the cell and / or a cell culture medium in which the cell is grown.

[0064] Aspects of the present disclosure include methods of making an antibody as described herein, the methods comprising immunizing a transgenic animal with CD28 and identifying CD28-binding heavy chain sequences. Aspects of the invention include methods of making an antibody as describedAttorney Ref.62571.00009WO01 (RND-0002-WO) herein, the methods comprising immunizing a transgenic animal with Nectin-4and identifying Nectin- 4-binding heavy chain sequences.

[0065] Aspects of the present disclosure include methods of treatment, comprising administering to an individual in need an effective dose of an antibody or a pharmaceutical composition as described herein.

[0066] Aspects of the present disclosure include use of an antibody as described herein in the preparation of a medicament for the treatment of a disease or disorder in an individual in need.

[0067] Aspects of the present disclosure include an antibody or pharmaceutical composition as described herein, for use in therapy in an individual in need.

[0068] Aspects of the present disclosure include methods of treatment, comprising treatment of a disorder comprising administering to a subject with said disorder an antibody or pharmaceutical composition as described herein. In some embodiments, the disorder is cancer. In some embodiments, the cancer is a solid tumor cancer.

[0069] In some embodiments, the solid tumor cancer is bladder cancer, urothelial bladder cancer, breast cancer, kidney cancer, pancreatic cancer, penile cancer, prostate cancer, lung cancer, a head and neck cancer, small-cell lung cancer, non-small cell lung cancer (NSCLC), gastric cancer, cervical cancer, ovarian cancer, esophageal cancer, genitourinary cancer, ovarian cancer, skin cancer, liver cancer, brain cancer, testicular cancer, colon cancer, rectal cancer, colorectal cancer, vulval cancer, thyroid cancer, melanoma, or endometrial cancer.

[0070] In some embodiments, the solid tumor cancer is a carcinoma. In some embodiments, the carcinoma is adenocarcinoma, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), squamous carcinoma of the lung, epithelial squamous cell carcinoma, transitional cell carcinoma, non melanoma skin cancer, renal cell carcinoma (RCC), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), hepatocellular carcinoma (HCC), hepatic carcinoma, skin carcinoma, anal carcinoma, penile carcinoma, esophageal carcinoma, salivary gland carcinoma, nasopharyngeal carcinoma, laryngeal carcinomas, choriocarcinoma, urinary tract carcinoma, endometrial carcinoma, high grade serous ovarian carcinoma, or uterine carcinoma. In some embodiments, the adenocarcinoma is adenocarcinoma of the lung, or pancreatic ductal adenocarcinoma.

[0071] In some embodiments, the solid tumor cancer is a blastoma. In some embodiments, the blastoma is hepatoblastoma, medulloblastoma, nephroblastoma, Wilms tumor, neuroblastoma, glioblastoma multiforme, pleuropulmonary blastoma, retinoblastoma, arrhenoblastoma, or pancreatoblastoma.

[0072] In some embodiments, the solid tumor cancer is a sarcoma. In some embodiments, the sarcoma is angiosarcoma, chondrosarcoma, clear cell sarcoma, epithelioid sarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumors, Kaposi sarcoma, leiomyosarcoma, liposarcoma, myeloid sarcoma, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, spindle cell sarcoma, synovial sarcoma, or soft tissue sarcoma. In some embodiments, the sarcoma results from Schwannoma or oligodendroglioma.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0073] In some embodiments, the cancer is a liquid tumor cancer. In some embodiments, the liquid tumor cancer is a hematologic malignancy. In some embodiments, the hematologic malignancy is a lymphoma. In some embodiments, the lymphoma is a Hodgkin’s lymphoma, a non-Hodgkin’s lymphoma, T-cell lymphoma, nonspecific peripheral T-cell lymphoma (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), adult T-cell leukemia / lymphoma (ATLL), cutaneous T-cell lymphoma (CTCL), natural killer / T-cell lymphoma, or cutaneous lymphoma. In some embodiments, the hematologic malignancy is a leukemia. In some embodiments, the leukemia is acute myeloid leukemia, T-cell acute lymphoblastic leukemia (T-ALL), or mixed-phenotype acute leukemia. In some embodiments, the hematologic malignancy is acute hematologic malignancy, myeloma, or multiple myeloma.

[0074] In some embodiments, the cancer is metastatic cancer.

[0075] Aspects of the present disclosure include methods of treatment of a disorder characterized by expression of Nectin-4, comprising administering to a subject with said disorder an antibody or pharmaceutical composition as described herein.

[0076] In some embodiments, the disorder is cancer. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is bladder cancer, breast cancer, kidney cancer, pancreatic cancer, penile cancer, prostate cancer, lung cancer, a head and neck cancer, gastric cancer, esophageal cancer, genitourinary cancer, ovarian cancer, or endometrial cancer. In some embodiments, the solid tumor cancer is bladder cancer. In some embodiments, the solid tumor cancer is breast cancer. In some embodiments, the solid tumor cancer is ovarian cancer. In some embodiments, the solid tumor cancer is esophageal cancer. In some embodiments, the solid tumor cancer is gastric cancer.

[0077] In some embodiments, methods of treatment as described herein further comprise administering a Signal 1 molecule. In some embodiments, the Signal 1 molecule comprises a multi-specific antibody that binds to an EAA on an effector cell and a DAA on a target cell. In some embodiments, the EAA that the multi-specific antibody binds to is a CD3 protein. In some embodiments, the EAA that the multi-specific antibody binds to is a peptide MHC complex. In some embodiments, the EAA that the multi-specific antibody binds to is a TAA or a TSA. In some embodiments, the TAA that the multi- specific antibody binds to is Nectin-4. In some embodiments, the multi-specific antibody is a Nectin-4 x CD3 antibody.

[0078] In some embodiments, methods of treatment as described herein further comprise administering a cosignaling molecule. In some embodiments, the cosignaling molecule is a costimulatory cosignaling molecule. In some embodiments, the cosignaling molecule is a coinhibitory cosignaling molecule.

[0079] In some embodiments, methods of treatment as described herein further comprise administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a checkpoint inhibitor antibody. In some embodiments, the additional therapeutic agent comprises an antibody-drug conjugate (ADC). In some embodiments, the additional therapeutic agent comprises aAttorney Ref.62571.00009WO01 (RND-0002-WO) chemotherapeutic drug. In some embodiments, the additional therapeutic agent comprises an oncolytic virus.

[0080] Aspects of the present disclosure include kits for treating a disease or disorder in an individual in need, comprising an antibody or pharmaceutical composition as described herein, and instructions for use. In some embodiments, a kit as described herein further comprises at least one additional reagent. In some embodiments, the at least one additional reagent comprises a multi-specific antibody that binds to CD28 and Nectin-4, a checkpoint inhibitor antibody, an antibody-drug conjugate (ADC), a chemotherapeutic drug, a Signal 1 molecule, a cosignaling molecule, an oncolytic virus, or any combination thereof.

[0081] These and further aspects will be further explained in the rest of the disclosure, including the Examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG. 1, Panels A and B are diagrams showing examples of a mechanism for activating cytotoxicity against a target cell using a multispecific antibody.

[0083] FIG. 2 is a table showing melting temperatures and onset-of-aggregation temperature measurements of the indicated antibody constructs.

[0084] FIGS.3A-3C are graphs showing CD28 binding kinetics of the indicated antibody constructs. FIG.3D is a table summarizing the binding data for the indicated antibody constructs.

[0085] FIGS. 4A-4B provide a series of graphs showing binding kinetics of the indicated antibody constructs to human and cyno Nectin-4.

[0086] FIG. 4C is a table summarizing the binding kinetics data of the indicated antibody constructs to human and cyno Nectin-4.

[0087] FIGS. 5A-5B provide a series of graphs showing binding kinetics of the indicated antibody constructs to CD28.

[0088] FIG. 5C is a table summarizing the binding kinetics data of the indicated antibody constructs to CD28.

[0089] FIG.6, Panels A-C provide a series of graphs showing cell binding data for the indicated cell types and the indicated antibody constructs. FIG.6, Panel D is a table summarizing cell binding EC50 values for the indicated antibody constructs to the indicated cell types.

[0090] FIG. 7A provides a series of graphs showing ELISA binding data of the indicated antibody constructs to the indicated recombinant protein targets. FIG.7B is a table summarizing the EC50 ELISA binding data of the indicated antibody constructs to the indicated recombinant protein targets.

[0091] FIG. 8 is a table summarizing Nectin-4 antigen density, assessed on human tumor cell lines, primary human cells, and disassociated tumor cells.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0092] FIG. 9, Panel A is a graph showing interferon gamma production as a function of antibody construct for the indicated molecules. FIG. 9, Panel B is a graph showing T-cell proliferation as a function of antibody construct for the indicated molecules.

[0093] FIG.10, Panels A, B, and C, are graphs showing cytokine concentration produced in response to effector cell activation for the indicated cell types, and the indicated antibody constructs.

[0094] FIG. 11, Panels A and B, are graphs showing cytotoxicity activity of the indicated antibody constructs at the indicated E:T ratios, in bladder cancer target cells.

[0095] FIG. 12 , Panels A and B, are graphs showing cytotoxicity activity of the indicated antibody construct combinations at the indicated E:T ratios in T47D cells.

[0096] FIG.13, Panels A, B, and C, are graphs showing cytokine concentration produced in response to treatment of the indicated cell types with the indicated antibody constructs, at the indicated E:T ratio.

[0097] FIG. 14, Panels A and B, are graphs showing percent target cell cytotoxicity of the indicated cell type as a function of concentration of the indicated antibody constructs.

[0098] FIG. 15, Panels A and B, are graphs showing percent target cell cytotoxicity of the indicated cell type as a function of concentration of the indicated antibody constructs.

[0099] FIG.16, Panels A and B, are graphs showing T-cell proliferation as a function of concentration of the indicated antibody constructs, at the indicated E:T ratio.

[0100] FIG.17 is a graph showing that CD28 x Nectin-4 bispecific antibodies enhance expression of the T-cell surface activation marker CD25.

[0101] FIG. 18 is a graph showing that CD28 x Nectin-4 bispecific antibodies show similar cytotoxicity activity whether in an IgG4 or IgG1 format.

[0102] FIG. 19 is a graph showing that the CD28 targeting arms of the indicated CD28 x Nectin-4 bispecific antibodies behave similarly when paired with different DAA targeting arms.

[0103] FIG. 20 is a graph showing that the CD28 targeting arms of the indicated CD28 x Nectin-4 bispecific antibodies behave similarly when paired with different DAA targeting arms (e.g., a FRα targeting arm).

[0104] FIG.21 is a graph showing the cytotoxicity activity of a CD3 x Nectin-4 bispecific antibody.

[0105] FIG. 22A provides a series of graphs showing binding kinetics of the indicated antibody construct to human and cyno Nectin-4. FIG.22A also includes a table summarizing the binding kinetics data. FIG.22B is a table summarizing epitope binning activity.

[0106] FIGS.23A-23C provide a series of graphs showing binding kinetics of the indicated antibody construct to human Nectin-4. FIG 23D is a table summarizing the binding data for the indicated antibody constructs.

[0107] FIG. 24A is a graph showing binding kinetics of the indicated antibodies to the 5T4-positive target cell line T47D. FIG.24B is a graph showing binding kinetics of the indicated antibody constructs to the FRα -positive target cell line T47D.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0108] FIGS.25A-25C are a series of graphs showing binding kinetics of the non-CD28 binding unit of the indicated antibody constructs. FIG.25D is a table summarizing the binding data for the indicated antibody constructs.

[0109] FIGS. 26A-26B provide a series of graphs showing tumor growth inhibition by the indicated CD28 x Nectin-4 antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0110] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0111] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0112] Unless indicated otherwise, antibody residues herein are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0113] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[0114] All references cited throughout the disclosure, including patent applications and publications, are incorporated by reference herein in their entirety.Attorney Ref.62571.00009WO01 (RND-0002-WO) I. Definitions

[0115] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0116] By “comprising” it is meant that the recited elements are required in the composition / method / kit, but other elements may be included to form the composition / method / kit etc. within the scope of the claim.

[0117] By “consisting essentially of”, it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention.

[0118] By “consisting of”, it is meant the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim.

[0119] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies mean residue numbering by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies mean residue numbering by the EU numbering system.

[0120] Antibodies, also referred to as immunoglobulins, conventionally comprise at least one heavy chain and one light chain, where the amino terminal domain of the heavy and light chains is variable in sequence, hence is commonly referred to as a variable region domain, or a variable heavy (VH) or variable light (VL) domain. The two domains conventionally associate to form a specific binding region, although as will be discussed here, specific binding can also be obtained with heavy chain-only variable sequences, and a variety of non-natural configurations of antibodies are known and used in the art.

[0121] A “functional” or “biologically active” antibody or antigen-binding molecule (including heavy chain-only antibodies and multi-specific (e.g., bispecific) three-chain antibody-like molecules is one capable of exerting one or more of its natural activities in structural, regulatory, biochemical or biophysical events. For example, a functional antibody or other binding molecule, e.g., a bispecific, may have the ability to specifically bind an antigen and the binding may in turn elicit or alter a cellular or molecular event such as signal transduction or enzymatic activity. A functional antibody or other binding molecule, e.g., a bispecific, may also block ligand activation of a receptor or act as an agonist or antagonist. The capability of an antibody or other binding molecule, e.g., a bispecific, to exert one orAttorney Ref.62571.00009WO01 (RND-0002-WO) more of its natural activities depends on several factors, including proper folding and assembly of the polypeptide chains.

[0122] The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multi-specific antibodies (e.g., bispecific antibodies), heavy chain-only antibodies, three chain antibodies, single chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0123] The term antibody may reference a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptides, i.e., a polypeptide that comprises an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule, including engineered subclasses with altered Fc portions that provide for reduced or enhanced effector cell activity. Light chains of the subject antibodies can be kappa light chains (Vkappa) or lambda light chains (Vlambda). The immunoglobulins can be derived from any species. In one aspect, the immunoglobulin is of largely human origin.

[0124] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies in accordance with the present disclosure can be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, and can also be made via recombinant protein production methods (see, e.g., U.S. Patent No. 4,816,567), for example.

[0125] The term “variable”, as used in connection with antibodies, refers to the fact that certain portions of the antibody variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held togetherAttorney Ref.62571.00009WO01 (RND-0002-WO) in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

[0126] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity-determining region” or “CDR” (e.g., residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues from a “hypervariable loop” residues 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol.196:901-917 (1987)). In some embodiments, “CDR” means a complementarity-determining region of an antibody as defined in Lefranc, MP et al., IMGT, the International ImMunoGeneTics database, Nucleic Acids Res., 27:209- 212 (1999). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region / CDR residues as herein defined.

[0127] Exemplary CDR designations are shown herein; however, one of skill in the art will understand that a number of definitions of the CDRs are commonly in use, including the Kabat definition (see “Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions.” Mol Immunol.2010;47:694–700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of the structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature.1989; 342:877–883). Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001;309:657–670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B-cell epitopes.” J Immunol. 2008;181:6230–6235; Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132–143; and Padlanet al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133–139., each of which is herein specifically incorporated by reference.

[0128] An “intact antibody chain” as used herein is one comprising a full-length variable region and a full-length constant region (Fc). An intact “conventional” antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, hinge, CH2 and CH3 for secreted IgG. Other isotypes, such as IgM or IgA may have different CH domains. The constant domains may be native sequence constant domains (e.g., human nativeAttorney Ref.62571.00009WO01 (RND-0002-WO) sequence constant domains) or amino acid sequence variants thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors. Constant region variants include those that alter the effector profile, binding to Fc receptors, and the like.

[0129] Depending on the amino acid sequence of the Fc (constant domain) of their heavy chains, antibodies and various antigen-binding proteins can be provided as different classes. There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains that correspond to the different classes of antibodies may be referenced as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modifications or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005 / 0048572; US 2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two types, called κ (kappa) and λ (lambda), based on the amino acid sequences of their constant domains. Antibodies in accordance with embodiments of the present disclosure can comprise kappa light chain sequences or lambda light chain sequences.

[0130] A “functional Fc region” possesses an “effector function” of a native-sequence Fc region. Non- limiting examples of effector functions include C1q binding; CDC; Fc-receptor binding; ADCC; ADCP; down-regulation of cell-surface receptors (e.g., B-cell receptor), etc. Such effector functions generally require the Fc region to interact with a receptor, e.g., the FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors, and the low affinity FcRn receptor; and can be assessed using various assays known in the art. A “dead” or “silenced” Fc is one that has been mutated to retain activity with respect to, for example, prolonging serum half-life, but which does not activate a high affinity Fc receptor, or which has a reduced affinity to an Fc receptor.

[0131] A “native-sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include, for example, a native-sequence human IgG1 Fc region (non-A and A allotypes); native-sequence human IgG2 Fc region; native-sequence human IgG3 Fc region; and native-sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0132] A “variant Fc region” comprises an amino acid sequence that differs from that of a native- sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to aboutAttorney Ref.62571.00009WO01 (RND-0002-WO) ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native-sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native-sequence Fc region and / or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0133] Variant Fc sequences may include three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med.178:661 (1993) and Canfield and Morrison, J. Exp. Med.173:1483 (1991)). Substitution into human IgG1 or IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, Armour KL. et al., 1999 Eur J Immunol.29(8):2613-24; and Shields RL. et al., 2001. J Biol Chem.276(9):6591- 604). The human IgG1 Fc amino acid sequence (UniProt KB No. P01857) is provided herein as SEQ ID NO: 282. The human IgG4 Fc amino acid sequence (UniProt KB No. P01861) is provided herein as SEQ ID NO: 283. Silenced IgG1 is described, for example, in Boesch, A.W., et al., “Highly parallel characterization of IgG Fc binding interactions.” MAbs, 2014.6(4): p.915-27, the disclosure of which is incorporated herein by reference in its entirety.

[0134] Other Fc variants are possible, including, without limitation, those in which a region capable of forming a disulfide bond is added or deleted, or in which certain amino acid residues are eliminated at the N-terminal end of a native Fc, or a methionine residue is added thereto. Thus, in some embodiments, one or more Fc portions of an antibody can comprise one or more mutations in the hinge region to eliminate disulfide bonding, or one or more mutations in the Fc region to add additional disulfide bonding between the heavy chain subunits, as described further herein. In yet another embodiment, the hinge region of an Fc can be removed entirely. In still another embodiment, an antibody can comprise an Fc variant.

[0135] Further, an Fc variant can be constructed to remove or substantially reduce effector functions by substituting (mutating), deleting or adding amino acid residues to effect complement binding or Fc receptor binding. For example, and not limitation, a deletion may occur in a complement-binding site, such as a C1q-binding site. Techniques for preparing such sequence derivatives of the immunoglobulin Fc fragment are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478. In addition, the Fc domain may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

[0136] In some embodiments, an antibody comprises a variant human IgG1 CH3 domain sequence comprising a T366W mutation, which can optionally be referred to herein as an IgG1 CH3 knob sequence. In some embodiments, an antibody comprises a variant human IgG1 CH3 domain sequence comprising a T366S mutation, an L368A mutation, and a Y407V mutation, which can optionally be referred to herein as an IgG1 CH3 hole sequence. The IgG1 CH3 mutations described herein can beAttorney Ref.62571.00009WO01 (RND-0002-WO) utilized in any suitable manner so as to place a “knob” on a first heavy chain constant region of a first monomer in an antibody dimer, and a “hole” on a second heavy chain constant region of a second monomer in an antibody dimer, thereby facilitating proper pairing (heterodimerization) of the desired pair of heavy chain polypeptide subunits in the antibody.

[0137] In some embodiments, an antibody comprises a variant human IgG1 domain sequence comprising an S354C mutation, which can optionally be referred to herein as an IgG1 CH3 disulfide sequence. In some embodiments, an antibody comprises a variant human IgG1 domain sequence comprising a Y349C mutation, which can optionally be referred to herein as an IgG1 CH3 disulfide sequence. The IgG1 disulfide sequences described herein can be utilized in any suitable manner to facilitate formation of a disulfide bond between two heavy chain subunits of an IgG1 antibody, thereby improving stability of the IgG1 antibody. In some embodiments, a first heavy chain polypeptide subunit of an antibody comprises a first IgG1 CH3 disulfide sequence (e.g., comprises an S354C mutation) and a second heavy chain polypeptide subunit of the antibody comprises a second IgG1 CH3 disulfide sequence (e.g., a Y349C mutation), such that a disulfide bond can be formed between these two mutations when the first and second heavy chain polypeptide subunits are paired.

[0138] In some embodiments, an antibody comprises a variant human IgG1 domain sequence comprising an L234A mutation, an L235A mutation, and a P329G mutation, which can optionally be referred to herein as a silenced IgG1 sequence, referring to the reduction or elimination of effector function activity of an antibody comprising these mutations.

[0139] In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG1 Fc region comprising a P329G mutation, an L234A mutation, an L235A mutation, and a T366W mutation (knob). In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG1 Fc region comprising a P329G mutation, an L234A mutation, an L235A mutation, a T366S mutation, an L368A mutation, and a Y407V mutation (hole).

[0140] In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG1 Fc region comprising a P329G mutation, an L234A mutation, an L235A mutation, a T366W mutation (knob), and an S354C mutation. In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG1 Fc region comprising a P329G mutation, an L234A mutation, an L235A mutation, a T366S mutation, an L368A mutation, a Y407V mutation (hole), and a Y349C mutation.

[0141] In some embodiments, an antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation, which can optionally be referred to herein as an IgG4 CH3 knob sequence. In some embodiments, an antibody comprises a variant human IgG4 CH3 domain sequence comprising a T366S mutation, an L368A mutation, and a Y407V mutation, which can optionally be referred to herein as an IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein can be utilized in any suitable manner so as to place a “knob” on a first heavy chain constant region of a first monomer in an antibody dimer, and a “hole” on a second heavy chain constant region of a secondAttorney Ref.62571.00009WO01 (RND-0002-WO) monomer in an antibody dimer, thereby facilitating proper pairing (heterodimerization) of the desired pair of heavy chain polypeptide subunits in the antibody.

[0142] In some embodiments, an antibody comprises a variant human IgG4 domain sequence comprising an S354C mutation, which can optionally be referred to herein as an IgG4 CH3 disulfide sequence. In some embodiments, an antibody comprises a variant human IgG4 domain sequence comprising a Y349C mutation, which can optionally be referred to herein as an IgG4 CH3 disulfide sequence. The IgG4 CH3 disulfide sequences described herein can be utilized in any suitable manner to facilitate formation of a disulfide bond between two heavy chain subunits of an IgG4 antibody, thereby improving stability of the IgG4 antibody. In some embodiments, a first heavy chain polypeptide subunit of an antibody comprises a first IgG4 CH3 disulfide sequence (e.g., comprises an S354C mutation) and a second heavy chain polypeptide subunit of the antibody comprises a second IgG4 CH3 disulfide sequence (e.g., a Y349C mutation), such that a disulfide bond can be formed between these two mutations when the first and second heavy chain polypeptide subunits are paired.

[0143] In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, and an L235A mutation, which can optionally be referred to herein as a silenced IgG4 sequence, referring to the reduction or elimination of effector function activity of an antibody comprising these mutations.

[0144] In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235A mutation, and a T366W mutation (knob). In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235A mutation, a T366S mutation, an L368A mutation, and a Y407V mutation (hole).

[0145] In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235A mutation, a T366W mutation (knob), and an S354C mutation. In some embodiments, an antibody comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising an S228P mutation, an F234A mutation, an L235A mutation, a T366S mutation, an L368A mutation, a Y407V mutation (hole), and a Y349C mutation.

[0146] In some embodiments, an antibody comprises a mutation that reduces or prevents clipping (also referred to as cleaving) by carboxypeptidases. As provided, for example, in Xu et al., mAbs 2019 Oct;11(7):1289-1299, instability in the C-terminal region of the Fc region in certain antibody sequences, namely IgG1 and IgG4 Fc region sequences, under acidic conditions can lead to cleavage and aggregation. To address this, mutating residue L445 can be used to reduce fragmentation and resulting aggregation. In some embodiments, an antibody comprises a human IgG4 Fc region sequence comprising an L445P mutation on one or both of the heavy chain polypeptide subunits that reduces or prevents clipping by carboxypeptidases.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0147] The term “Fc-region-comprising antibody” refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Accordingly, an antibody having an Fc region according to this disclosure can comprise an antibody with or without K447.

[0148] Aspects of the present disclosure include antibodies comprising a variable region in a monovalent or bivalent configuration. As used herein, the term “monovalent configuration” as used in reference to a variable region domain or binding unit means that only one variable region domain or binding unit is present, having a single binding site. In contrast, the term “bivalent configuration” as used in reference to a variable region domain or binding unit means that two variable region domains or binding units are present (each having a single binding site), and are connected by a linker sequence. Non-limiting examples of linker sequences are discussed further herein, and include, without limitation, GS linker sequences of various lengths. When a variable region or binding unit is in a bivalent configuration, each of the two variable region domains or binding units can bind to the same antigen, or to different antigens (e.g., to different epitopes on the same protein; to two different proteins, etc.). However, unless specifically noted otherwise, a variable region or binding unit denoted as being in a “bivalent configuration” is understood to contain two identical variable region domains or binding units, connected by a linker sequence, wherein each of the two identical variable region domains or binding units binds to the same target antigen.

[0149] Aspects of the present disclosure include antibodies having multi-specific configurations, which include, without limitation, bispecific, trispecific, etc. A large variety of methods and protein configurations are known and used in connection with bispecific monoclonal antibodies (BsMAB), tri- specific antibodies, etc.

[0150] Various methods for the production of multivalent artificial antibodies have been developed by recombinantly fusing variable domains of two or more antibodies. In some embodiments, a first and a second antigen-binding domain on a polypeptide are connected by a polypeptide linker. One non- limiting example of such a polypeptide linker is a GS linker, having an amino acid sequence of four glycine residues, followed by one serine residue, and wherein the sequence is repeated n times, where n is an integer ranging from 1 to about 10, such as 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 331) (n=1) and GGGGSGGGGS (SEQ ID NO: 332) (n=2). Other suitable linkers can also be used, and are described, for example, in Chen et al., Adv Drug Deliv Rev.2013 October 15; 65(10): 1357-69, the disclosure of which is incorporated herein by reference in its entirety.

[0151] The term “three-chain antibody-like molecule” is used herein to refer to antibody-like molecules comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy and one light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains, that form a heavy chain / lightAttorney Ref.62571.00009WO01 (RND-0002-WO) chain pair. The heavy chain of this heavy chain / light chain pair comprises an antigen-binding region (e.g., a VH domain), a CH1 domain, and optionally further comprises additional CH domains (e.g., optionally further comprises a hinge region, a CH2, a CH3, and / or a CH4 domain). The light chain of this heavy chain / light chain pair comprises an antigen-binding region (e.g., a VL domain) and optionally further comprises a CL domain. This heavy chain / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy-chain only antibody comprising a heavy chain constant region (e.g., an Fc portion) comprising a hinge region and / or CH2 and / or CH3 and / or CH4 domains, in the absence of a CH1 domain, and one or more antigen binding domains (e.g., two antigen binding domains) that binds an epitope of a second antigen or a different epitope of the first antigen, where such binding domain is derived from or has sequence identity with the variable region of an antibody heavy or light chain. Parts of such variable region may be encoded by VHand / or VLgene segments, D and JHgene segments, or JLgene segments. The variable region may be encoded by rearranged VHDJH, VLDJH, VHJL, or VLJLgene segments.

[0152] A three-chain antibody-like molecule binding compound makes use of a “heavy chain only antibody” or “heavy chain antibody” or “heavy chain polypeptide” for one of its two arms, which, as used herein, means a single chain antibody comprising a heavy chain constant region comprising a hinge region and / or CH2 and / or CH3 and / or CH4, but no CH1 domain. The other arm of a three-chain antibody-like molecule binding compound makes use of a heavy chain / light chain pair, as described above. In one embodiment, the heavy chain antibody arm is composed of an antigen-binding domain, at least part of a hinge region and CH2 and CH3 domains. In another embodiment, the heavy chain antibody arm is composed of an antigen-binding domain, at least part of a hinge region and a CH2 domain. In a further embodiment, the heavy chain antibody arm is composed of an antigen-binding domain, at least part of a hinge region and a CH3 domain. Heavy chain antibodies in which the CH2 and / or CH3 domain is truncated are also included herein. In a further embodiment, the heavy chain antibody arm is composed of an antigen binding domain, and at least one CH domain selected from a CH2, CH3, or CH4 domain, but no hinge region. The heavy chain only antibody can be in the form of a dimer, in which two heavy chains are disulfide bonded or otherwise covalently or non-covalently attached to each other, and can optionally include an asymmetric interface (e.g., a knobs-in-holes (KiH) interface) between one or more of the CH domains to facilitate proper pairing between polypeptide chains. The heavy-chain antibody may belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD and IgE subclass, are also included herein. In a particular embodiment, the heavy chain antibody is of the IgG1, IgG2, IgG3, or IgG4 subtype, in particular the IgG1 subtype or the IgG4 subtype. Non-limiting examples of a three-chain antibody-like molecule binding compound are described in, for example, WO2017 / 223111 and WO2018 / 052503, the disclosures of which are incorporated herein by reference in their entirety.

[0153] Heavy-chain antibodies constitute about one fourth of the IgG antibodies produced by the camelids, e.g., camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). TheseAttorney Ref.62571.00009WO01 (RND-0002-WO) antibodies are formed by two heavy chains but are devoid of light chains. As a consequence, the variable antigen binding part is referred to as the VHH domain and it represents the smallest naturally occurring, intact, antigen-binding site, being only around 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy chain antibodies with a high specificity and affinity can be generated against a variety of antigens through immunization (van der Linden, R. H., et al. Biochim. Biophys. Acta.1431, 37-46 (1999)) and the VHH portion can be readily cloned and expressed in yeast (Frenken, L. G. J., et al. J. Biotechnol. 78, 11-21 (2000)). Their levels of expression, solubility and stability are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, M. A. et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies, termed VNAR. (Nuttall et al. Eur. J. Biochem. 270, 3543-3554 (2003); Nuttall et al. Function and Bioinformatics 55, 187-197 (2004); Dooley et al., Molecular Immunology 40, 25-33 (2003)).

[0154] The term “CD28” includes a CD28 protein of any human or non-human animal species, and specifically includes human CD28 (UniProt KB No. P10747) as well as CD28 of non-human mammals. CD28 is a cell surface glycoprotein expressed as a disulfide linked homodimer with a molecular weight of approximately 44 kDa. It is expressed by naïve T cells and generally at least 80% of CD4+ T cells and 50% of CD8+ T cells in adult human peripheral blood. Weng et al, Trends Immunol. 2009 Jul;30(7):306-12; Esensten et al, Immunity 2016 May 17;44(5):973-88.

[0155] The term “human CD28” as used herein includes any variants, isoforms and species homologs of human CD28 (UniProt KB No. P10747), regardless of its source or mode of preparation. Thus, “human CD28” includes human CD28 naturally expressed by cells and human CD28 expressed on cells transfected with the human CD28 gene.

[0156] The term “Nectin-4” includes a Nectin-4 protein of any human or non-human animal species, and specifically includes human Nectin-4 (UniProt KB No. Q96NY8) as well as Nectin-4 of non-human mammals. Nectin-4, also known as nectin cell adhesion molecule 4, is a tumor-associated antigen with a molecular weight of 55.5 kDa. Nectin-4 may be expressed by various types of cancer cells, such as for example, bladder cancer cells, breast cancer cells, and pancreatic cancer cells. Associated with low survival rates in patients, it has been designated as a cancer-specific biomarker, as Nectin-4 expression by non-cancerous, normal human cells is limited.

[0157] The term “human Nectin-4” as used herein includes any variants, isoforms and species homologs of human Nectin-4 (UniProt KB No. Q96NY8), regardless of its source or mode of preparation. Thus, “human Nectin-4” includes human Nectin-4 naturally expressed by cells and human Nectin-4 expressed on cells transfected with the human Nectin-4 gene.

[0158] The terms “anti-CD28 antibody”, “anti-human CD28 antibody”, “CD28 antibody”, or any variations thereof, are used interchangeably herein to refer to an antibody as defined herein, immunospecifically binding to CD28, including human CD28, as defined herein. The definitionAttorney Ref.62571.00009WO01 (RND-0002-WO) includes, without limitation, human antibodies produced by transgenic animals, such as transgenic rats or transgenic mice, expressing human immunoglobulin genes.

[0159] The terms “anti-Nectin-4 antibody”, “anti-human Nectin-4 antibody”, “Nectin-4 antibody”, or any variations thereof, are used interchangeably herein to refer to an antibody as defined herein, immunospecifically binding to Nectin-4, including human Nectin-4, as defined herein. The definition includes, without limitation, human antibodies produced by transgenic animals, such as transgenic rats or transgenic mice, expressing human immunoglobulin genes.

[0160] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.

[0161] An “isolated” antibody is one which has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0162] Antibodies of the present disclosure include multi-specific antibodies having more than one binding specificity. The term “multi-specific” specifically includes “bispecific” and “trispecific,” as well as higher-order independent specific binding affinities, such as higher-order polyepitopic specificity, as well as tetravalent antibodies and antibody fragments. The terms “multi-specific antibody,” “multi-specific heavy chain-only antibody,” and “multi-specific heavy chain antibody,” are used herein in the broadest sense and cover all antibodies with more than one binding specificity.

[0163] Aspects of the present disclosure include multi-specific antibodies that form, assist in forming, or act upon an immunological synapse between an effector cell and a target cell. Such multi-specificAttorney Ref.62571.00009WO01 (RND-0002-WO) antibodies contain at least one arm with a first binding unit that binds to an effector-associated antigen (EAA), such as CD28, on an effector cell (a “CD28-binding arm” or a “CD28 binding unit”), and at least one additional arm with a second binding unit that binds to a disease-associated antigen (DAA) on a target cell (a “DAA-binding arm” or a “DAA binding unit”). Non-limiting examples of DAAs include antigens expressed by a target cell, such as, for example, tumor-associated antigens (TAAs), tumor- specific antigens (TSAs), targeting antigens, integrins, pathogen antigens, and the like. In some embodiments, the DAA is a TAA such as, for example, Nectin-4, 5T4 (Uniprot KB No. Q13641, also referred to as Trophoblast glycoprotein, 5T4 oncofetal antigen), or FRα (Uniprot KB No. P15328, also referred to as Folate receptor alpha, FRalpha and FOLR1). In some embodiments, a multi-specific antibody comprises a first binding unit that binds to CD28 and a second binding unit that binds to a disease-associated antigen (DAA). In some embodiments, a multi-specific antibody comprises a first binding unit that binds to Nectin-4 and a second binding unit that binds to an effector-associated antigen (EAA). Non-limiting examples of EAAs include cell surface proteins expressed by any immune effector cell, such as, for example, CD3 expressed by T-cells.

[0164] Aspects of the present disclosure include multi-specific antibodies containing a CD28-binding arm as described above. In some embodiments, the CD28-binding arm contains a first binding unit that binds to CD28. Such multi-specific anti-CD28 antibodies of the present disclosure specifically include antibodies immunospecifically binding to two or more non-overlapping epitopes on a CD28 protein, such as a human CD28 (i.e., bivalent and biparatopic). The multi-specific anti-CD28 antibodies of the present disclosure also specifically include antibodies immunospecifically binding to an epitope on a CD28 protein, such as human CD28, and to an epitope on a different protein, such as, for example, a DAA, such as, for example, human Nectin-4.

[0165] Aspects of the present disclosure include multi-specific antibodies containing at least one arm with a first binding unit that binds to a disease-associated antigen (DAA) on a target cell, such as Nectin- 4, (the “Nectin-4-binding arm”), and at least one additional arm with a second binding unit that binds to an effector-associated antigen (EAA) on an effector cell (the “EAA-binding arm”). Non-limiting examples of EAAs include cell surface proteins expressed by any immune effector cell, such as, for example, CD28 expressed by T-cells, or CD3 expressed by T-cells. Such multi-specific anti-Nectin-4 antibodies of the present disclosure specifically include antibodies immunospecifically binding to two or more non-overlapping epitopes on a Nectin-4 protein, such as a human Nectin-4 (i.e., bivalent and biparatopic). The multi-specific anti-Nectin-4 antibodies of the present disclosure also specifically include antibodies immunospecifically binding to an epitope on a Nectin-4 protein, such as human Nectin-4, and to an epitope on a different protein, such as, for example, an EAA, such as CD28.

[0166] Aspects of the present disclosure include monospecific antibodies, having one binding specificity. Monospecific antibodies specifically include antibodies comprising a single binding specificity, as well as antibodies comprising more than one binding unit having the same bindingAttorney Ref.62571.00009WO01 (RND-0002-WO) specificity. The term “monospecific antibody” is used herein in the broadest sense and cover all antibodies with one binding specificity.

[0167] Aspects of the present disclosure include monospecific antibodies that bind to CD28. The monospecific anti-CD28 antibodies of the present disclosure specifically include antibodies immunospecifically binding to one epitope on a CD28 protein, such as a human CD28 protein (monovalent and monospecific). The monospecific anti-CD28 antibodies of the present disclosure also specifically include antibodies having more than one binding unit (e.g., multivalent antibodies) immunospecifically binding to an epitope on a CD28 protein, such as human CD28. For example, a monospecific antibody in accordance with embodiments of the present disclosure can include two identical heavy chain variable regions, each paired with an identical light chain variable region, to form two identical binding domains, or binding units, that bind to the same epitope on a CD28 protein (i.e., bivalent and monospecific).

[0168] Aspects of the present disclosure include monospecific antibodies that bind to Nectin-4. The monospecific anti-Nectin-4 antibodies of the present disclosure specifically include antibodies immunospecifically binding to one epitope on a Nectin-4 protein, such as a human Nectin-4 protein (monovalent and monospecific). The monospecific anti-Nectin-4 antibodies of the present disclosure also specifically include antibodies having more than one binding unit (e.g., multivalent antibodies) immunospecifically binding to an epitope on a Nectin-4 protein, such as human Nectin-4. For example, a monospecific antibody in accordance with embodiments of the present disclosure can include two identical heavy chain variable regions, each paired with an identical light chain variable region, to form two identical binding domains, or binding units, that bind to the same epitope on a Nectin-4 protein (i.e., bivalent and monospecific).

[0169] An “epitope” is the site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes.

[0170] “Epitope mapping” is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens. Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three-dimensional structure.

[0171] “Polyepitopic specificity” refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). As noted above, the present disclosure specifically includes anti-CD28 antibodies with polyepitopic specificities, i.e., anti-CD28 antibodies binding to one or more non-overlapping epitopes on a CD28 protein, such as a human CD28; and anti-CD28 antibodies binding to one or more epitopes on a CD28 protein and to an epitope on a different protein, such as, for example, a Nectin-4 protein. The present disclosure also specifically includes anti-Nectin-4 antibodies with polyepitopic specificities, i.e., anti-Nectin-4 antibodies binding to one or more non-overlappingAttorney Ref.62571.00009WO01 (RND-0002-WO) epitopes on a Nectin-4 protein, such as a human Nectin-4; and anti-Nectin-4 antibodies binding to one or more epitopes on a Nectin-4 protein and to an epitope on a different protein, such as, for example, a CD28 protein. The term “non-overlapping epitope(s)” or “non-competitive epitope(s)” of an antigen is defined herein to mean epitope(s) that are recognized by one member of a pair of antigen-specific antibodies but not the other member. Pairs of antibodies, or antigen-binding regions targeting the same antigen on a multi-specific antibody, recognizing non-overlapping epitopes, do not compete for binding to that antigen and are able to bind that antigen simultaneously.

[0172] An antibody binds “essentially the same epitope” as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in all number of different formats, using either labeled antigen or labeled antibody. Usually, the antigen is immobilized on a 96-well plate, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive or enzyme labels.

[0173] The term “valent” as used herein refers to a specified number of binding sites in an antibody molecule.

[0174] A “monovalent” antibody has one binding site. Thus, a monovalent antibody is also monospecific.

[0175] A “multi-valent” antibody has two or more binding sites. Thus, the terms “bivalent”, “trivalent”, and “tetravalent” refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Thus, a bispecific antibody according to the present disclosure is at least bivalent and may be trivalent, tetravalent, or otherwise multi-valent. A bivalent antibody in accordance with embodiments of the present disclosure may have two binding sites to the same epitope (i.e., bivalent, monoparatopic), or to two different epitopes (i.e., bivalent, biparatopic).

[0176] A large variety of methods and protein configurations are known and used for the preparation of bispecific monoclonal antibodies (BsMAB), tri-specific antibodies, and the like.

[0177] The term “three-chain antibody like molecule” is used herein to refer to antibody-like molecules comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise, consist essentially of, or consist of one heavy chain and one light chain of a monoclonal antibody, or functional antigen-binding fragments of such antibody chains, comprising an antigen-binding region and at least one CH domain. This heavy chain / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises, consists essentially of, or consists of a heavy chain-only antibody comprising an Fc portion comprising CH2 and / or CH3 and / or CH4 domains, in the absence of a CH1 domain, and an antigen binding domain that binds an epitope of a second antigen or a different epitope of the first antigen, where such binding domain is derived from or has sequence identity with the variable region of an antibody heavy or light chain. Parts of such variable region may be encoded by VHand / or VLgene segments, D and JHgene segments, or JLgene segments. The variable region mayAttorney Ref.62571.00009WO01 (RND-0002-WO) be encoded by rearranged VHDJH, VLDJH, VHJL, or VLJLgene segments. A three-chain antibody like molecule protein makes use of a heavy chain-only antibody as hereinabove defined.

[0178] The term “chimeric antigen receptor” or “CAR” is used herein in the broadest sense to refer to an engineered receptor, which grafts a desired binding specificity (e.g., the antigen-binding region of a monoclonal antibody or other ligand) to membrane-spanning and intracellular-signaling domains. Typically, the receptor is used to graft the specificity of a monoclonal antibody onto a T-cell to create a chimeric antigen receptor (CAR). (Sadelain et al., Cancer Discov (2013) 3 (4): 388–398; and Jackson et al., Nature Reviews Clinical Oncology, 2016; 13:370–383). CAR-T cells are T-cells that have been genetically engineered to produce an artificial T-cell receptor for use in immunotherapy. In one embodiment, “CAR-T cell” means a therapeutic T-cell expressing a transgene encoding one or more chimeric antigen receptors comprised minimally of an extracellular domain, a transmembrane domain, and at least one cytosolic domain.

[0179] The binding units of the antibodies described herein can be utilized in the production of T-cell engager molecules (e.g., bispecific T-cell engagers, aka BiTE molecules) as well as CAR-T structures. T-cell engager molecules are described, for example, in Huehls et al., Bispecific T cell engagers for cancer immunotherapy, Immunol Cell Biol. 2015 Mar; 93(3):290-296. CAR-T structures comprising single-domain antibodies as a binding (targeting) domain are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386.

[0180] The term “human antibody” is used herein to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences, e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. The term “human antibody” specifically includes antibodies having human heavy chain variable region sequences, produced by transgenic animals, such as transgenic rats or mice.

[0181] By a “chimeric antibody” or a “chimeric immunoglobulin” is meant an immunoglobulin molecule comprising amino acid sequences from at least two different Ig loci, e.g., a transgenic antibody comprising a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies with non-human Fc-regions or artificial Fc-regions, and human idiotypes. Such immunoglobulins can be isolated from animals of the present disclosure that have been engineered to produce such chimeric antibodies.

[0182] As used herein, the term “effector cell” refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Some effector cells express specific Fc receptors and carry out specific immune functions. In some embodiments, an effector cell such as a natural killer cell is capable of inducing antibody- dependent cellular cytotoxicity (ADCC). For example, monocytes and macrophages, which express FcR, are involved in specific killing of target cells and presenting antigens to other components of theAttorney Ref.62571.00009WO01 (RND-0002-WO) immune system, or binding to cells that present antigens. In some embodiments, an effector cell may phagocytose a target antigen or target cell.

[0183] “Human effector cells” are leukocytes that express receptors such as T-cell receptors or FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include, without limitation, natural killer (NK) cells, monocytes, cytotoxic T-cells and neutrophils. The effector cells may be isolated from a native source thereof, e.g., from blood or PBMCs as described herein.

[0184] The term “immune cell” is used herein in the broadest sense, including, without limitation, cells of myeloid or lymphoid origin, for instance lymphocytes (such as B-cells and T-cells including cytolytic T-cells (CTLs)), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils.

[0185] Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptor; BCR), etc.

[0186] “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0187] “Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g. an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano- Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0188] “Binding affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). AffinityAttorney Ref.62571.00009WO01 (RND-0002-WO) can be measured by common methods known in the art. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound.

[0189] As used herein, the “Kd” or “Kd value” refers to a dissociation constant determined by BioLayer Interferometry, using an OCTET® QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetics mode. For example, anti-mouse Fc sensors are loaded with mouse-Fc fused antigen and then dipped into antibody-containing wells to measure concentration dependent association rates (kon). Antibody dissociation rates (koff) are measured in the final step, where the sensors are dipped into wells containing buffer only. The Kd is the ratio of koff / kon. (For further details see, Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0190] The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

[0191] The terms “combination treatment” or “combination therapy” are used herein to broadly refer to methods involving administration of one or more additional therapeutic agent(s) before, after or concurrently with administration of antibodies or pharmaceutical compositions described herein. In some embodiments, an additional therapeutic agents may comprise, for example, other effector cell engaging therapies that function by modulating (e.g., increasing) an activation signal of the effector cell (e.g., the T-cell) towards a target cell. In some embodiments, additional therapeutic agents may comprise or further comprise administration of a checkpoint inhibitor antibody, an antibody-drug conjugate, a chemotherapeutic drug, a costimulatory molecule, or an oncolytic virus.

[0192] An “antibody-drug conjugate” (ADC) or immunoconjugate means an antibody, or antigen- binding fragment thereof, conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In some embodiments, the ADC comprises an antibody as described herein (Ab), a linker (L), and a drug moiety (D). In some embodiments, the ADC has the formula Ab-(L-D)p, where p is an integer that ranges from 1 to 8.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0193] A “Signal 1 molecule” means a molecule provided to effector cells (e.g., T-cells) to activate a signal in response to interaction with a target cell (e.g., tumor cells). A “Signal 2 molecule” means a molecule provided to effector cells (e.g., T-cells) to provide Signal 2 activation in addition to Signal 1 activation, amplifying the overall signal in response to interaction with a target cell. In some embodiments, a Signal 2 molecule can be used in instances where Signal 1 activation alone is insufficient to eliminate target cells (e.g., tumor cells).

[0194] A “cosignaling molecule” means a molecule provided to effector cells to modulate Signal 2 activation provided by a Signal 2 molecule. A cosignaling molecule can be used in instances where Signal 2 activation is insufficient to eliminate target cells (e.g., tumor cells), or instances where Signal 2 activation is causing excess cytotoxicity of the T-cell towards the target cells. A cosignaling molecule may be a costimulatory cosignaling molecule (amplifies Signal 2 activation) or a coinhibitory cosignaling molecule (counteracts a Signal 2 activation). Non-limiting examples of costimulatory cosignaling molecules include CD28, CD137 (4-1BB), ICOS, OX40, CD40L, CD2, and CD27. Non- limiting examples of coinhibitory cosignaling molecules include PD-1, CTLA-4, TIM3, LAG3 and TIGIT. Cosignaling molecules are further described, for example, in Chen et al., Nat Rev Immunol. 2013 Apr; 13(4):227-242.

[0195] An “oncolytic virus” is a virus that promotes effector cell (e.g., T-cell) activation by inducing tumor lysis. A non-limiting example of an approved oncolytic virus is T-vec (Talimogene laherparepvec). Oncolytic viruses are further described, for example, in Su et al., Transl Oncol. 2022 Nov; 25: 101530.

[0196] A “therapeutically effective amount” is intended for an amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a “therapeutically effective amount” is an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with a disease or which improves resistance to a disorder.

[0197] The term “characterized by expression of a DAA” broadly refers to any disease or disorder in which expression of the DAA is associated with or involved with one or more pathological processes that are characteristic of the disease or disorder. For example, in some embodiments, where the DAA is, for example, Nectin-4, such disorders include, but are not limited to, cancer.

[0198] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In an embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having cancer, individuals with autoimmune diseases, with pathogen infections, and the like. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mouse, rat, etc.

[0199] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additionalAttorney Ref.62571.00009WO01 (RND-0002-WO) components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. “Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.

[0200] A “sterile” formulation is aseptic or free or essentially free from all living microorganisms and their spores. A “frozen” formulation is one at a temperature below 0 ºC.

[0201] A “stable” formulation is one in which the protein therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones. A. Adv. Drug Delivery Rev. 10: 29-90) (1993), for example. Stability can be measured at a selected temperature for a selected time period. Stability can be evaluated qualitatively and / or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.

[0202] A “benchmark molecule” means a molecule having known properties, such as target cell binding properties. Benchmark molecules are used herein as comparator molecules to measure or characterize one or more attributes of a non-benchmark antibody molecule described herein. II. Detailed Description

[0203] The present disclosure provides several families of closely related antibodies that bind to the binding targets described herein, namely, CD28 and Nectin-4. Aspects of the present disclosure include multi-specific antibodies containing one arm with a first binding unit that binds to CD28 (the “CD28- binding arm”), and another arm with a second binding unit that binds to a DAA (e.g., Nectin-4) on a target cell (the “DAA-binding arm”). In some embodiments, one or more arms of a multi-specific antibody can comprise multiple binding units that bind to the same binding target or to different binding targets. Aspects of the present disclosure also include multi-specific antibodies containing one arm with a first binding unit that binds to Nectin-4 (the “Nectin-4-binding arm”), and another arm with a secondAttorney Ref.62571.00009WO01 (RND-0002-WO) binding unit that binds to an EAA (e.g., CD28) on an effector cell (the “EAA-binding arm”). Each arm of the multi-specific antibodies herein comprises a VH domain comprising heavy chain CDR1, CDR2 and CDR3 sequences in a human VH framework. The CDR sequences may be situated, as an example, in the region of around amino acid residues 26-33; 51-58; and 97-116 for CDR1, CDR2 and CDR3, respectively, of the provided exemplary variable region sequences set forth in SEQ ID NOs: 93-102, 107, 121-237, and 333-334. Each arm of the multi-specific antibodies herein also comprises a VL domain comprising light chain CDR1, CDR2 and CDR3 sequences in a human VL framework. The CDR sequences may be situated, as an example, in the region of around amino acid residues 26-33; 51- 58; and 97-116 for CDR1, CDR2 and CDR3, respectively, of the provided exemplary variable region sequence set forth in SEQ ID NO: 110. Antibodies in accordance with embodiments of the present disclosure can utilize common light chain sequences, such that the same light chain CDRs can be combined with any of the heavy chain CDRs described herein to generate a binding unit that binds to the indicated target (e.g., CD28, or Nectin-4).

[0204] It will be understood by one of ordinary skill in the art that the CDR sequences may be in different positions if a different framework sequence is selected, although generally the order of the sequences will remain the same.

[0205] In some embodiments, the heavy chain CDR sequences for a CD28 binding unit belong to family 1 and have heavy chain CDR sequences corresponding to the following formulae. An X indicates a variable amino acid, which may be specific amino acids as indicated below. CDR1 G1G2X3F4X5X6X7Y8(SEQ ID NO: 320) where: X3is S or T. X5is S, T, or N. X6is G or F. X7is Y or N.

[0206] In some embodiments, a heavy chain CDR1 sequence of a family 1 antibody comprises the sequence set forth in any one of SEQ ID NOs: 1, or 57-61. CDR2 X1X2X3X4G5X6X7where: X1is I, A, or V. X2is H, K, Q, N, S or T. X3is H or D. X4is S or T.Attorney Ref.62571.00009WO01 (RND-0002-WO) X6is S, R, I, or N. X7is T or A.

[0207] In some embodiments, a heavy chain CDR2 sequence of a family 1 antibody comprises the sequence set forth in any one of SEQ ID NOs: 9, 11, 13, or 68-78. CDR3 A1R2X3X4X5G6A7X8X9D10X11(SEQ ID NO: 322) where: X3is V, A, or S. X4 is G or P. X5is S, or no amino acid (e.g., position does not exist). X8is T or L. X9is V, P, L, or F. X11is Y, H, or F.

[0208] In some embodiments, a heavy chain CDR3 sequence of a family 1 antibody comprises the sequence set forth in any one of SEQ ID NOs: 18, 20, 21, or 86-90.

[0209] In some embodiments, the heavy chain CDR sequences for an EAA binding unit that binds to CD28 belong to family 2 and have CDR sequences corresponding to the following formulae. An X indicates a variable amino acid, which may be specific amino acids as indicated below. CDR1 G1G2S3X4X5S6X7X8X9X10(SEQ ID NO: 323) where: X4is I or S. X5is S or R. X7is G or S. X8is G or S. X9is Y or F. X10is Y or N.

[0210] In some embodiments, a heavy chain CDR1 sequence of a family 2 antibody comprises the sequence set forth in any one of SEQ ID NOs: 2-3, or 62-67. CDR2Attorney Ref.62571.00009WO01 (RND-0002-WO) I1X2X3X4G5X6T7where: X2is Y, H, or N. X3is Y, R, or H. X4is S or F. X6is R, N, S, or T.

[0211] In some embodiments, a heavy chain CDR2 sequence of a family 2 antibody comprises the sequence set forth in any one of SEQ ID NO: 10, 73, or 79-85. CDR3 X1R2G3D4S5X6W7P8F9D10Y11(SEQ ID NO: 325) where: X1is A or T. X6is S or A.

[0212] In some embodiments, a heavy chain CDR3 sequence of a family 2 antibody comprises the sequence set forth in any one of SEQ ID NOs: 19, 22, or 91-92.

[0213] The antibodies of both family 1 and family 2 comprise a set of heavy chain CDR sequences as defined herein and shown in Tables 1A, 1B, 4A and 4B, and are exemplified by the provided heavy chain CDR1, CDR2 and CDR3 sequences set forth in Tables 2 and 5, the heavy chain variable region (VH) sequences of SEQ ID NOs: 93-102, 107, 121-237, and 333-334 set forth in Tables 3A and 6A, and the light chain variable region (VL) sequence of SEQ ID NO: 110 set forth in Tables 3B and 6B. The family 1 and family 2 antibodies provide a number of benefits that contribute to utility as clinically therapeutic agent(s). The family 1 and family 2 antibodies include members with a range of binding affinities, allowing the selection of a specific sequence with a desired binding affinity. Table 1A: Anti-CD28 unique heavy chain CDR amino acid sequences. SEQ_aa_CDR1 SEQ_aa_CDR2 SEQ_aa_CDR3 0)Attorney Ref.62571.00009WO01 (RND-0002-WO) GGSFTFYY (SEQ ID NO: IHHSGRT (SEQ ID NO: 68) ARAGGATLDY (SEQ ID NO: 59) 86) GGSFSGNY (SEQ ID NO: IKHSGST (SEQ ID NO: 69) ARVGGATPDH (SEQ ID NO: 8) 9)SEQ_aa_CDR1 SEQ_aa_CDR2 SEQ_aa_CDR3 QSVSSN (SEQ ID NO: 28) GAS QQYNNWPWT (SEQ ID NO: 44)Table 2: Anti-CD28 heavy chain CDR1, CDR2, CDR3 amino acid sequences. Antibody SEQ_aa_CDR1 SEQ_aa_CDR2 SEQ_aa_CDR3 Construct O:Attorney Ref.62571.00009WO01 (RND-0002-WO) R007 GGSIRSSSYY (SEQ IYYSGST (SEQ ID NO: ARGDSSWPFDY (SEQ ID ID NO: 3) 10) NO: 22) R008 GGSFSGYY (SEQ ID INHSGNT (SEQ ID NO: ARVGGATPDY (SEQ ID O: O: O: O: O:Attorney Ref.62571.00009WO01 (RND-0002-WO) GGSFSGYY (SEQ ID INHSGST (SEQ ID NO: ARVGGATPDY (SEQ ID R050 NO: 1) 73) NO: 21) GGSFSGYY (SEQ ID INHSGST (SEQ ID NO: ARAGGATLDY (SEQ ID O: O: O: O: O: O: O: O:Attorney Ref.62571.00009WO01 (RND-0002-WO) GGSFSFYY (SEQ ID ITHSGST (SEQ ID NO: ARAPGATFDY (SEQ ID NO: R076 NO: 57) 77) 89) GGSFSGYY (SEQ ID INHSGST (SEQ ID NO: ARVGGATVDY (SEQ ID O: O: O:Attorney Ref.62571.00009WO01 (RND-0002-WO) GGSISSGSYY (SEQ IYYSGRT (SEQ ID NO: ARGDSSWPFDY (SEQ ID R103 ID NO: 62) 79) NO: 22) GGSISSSSYY (SEQ ID IYYSGST (SEQ ID NO: ARGDSAWPFDY (SEQ IDAttorney Ref.62571.00009WO01 (RND-0002-WO) GGSISSGSYY (SEQ INHSGST (SEQ ID NO: TRGDSAWPFDY (SEQ ID R129 ID NO: 62) 73) NO: 19) GGSSSSSSYY (SEQ IYYSGST (SEQ ID NO: ARGDSAWPFDY (SEQ IDTable 3A: Anti-CD28 heavy chain variable region amino acid sequences. Antibody SEQ_aa_FR1_FR4 SEQ IDAttorney Ref.62571.00009WO01 (RND-0002-WO) Reference No. R001 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQPP 93Attorney Ref.62571.00009WO01 (RND-0002-WO) R034 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPP 122 GKGLEWIGEIHHSGRTNYNPSLKSRVTISVDTSKHQFSLRLTS VTAADTAVYYCARVGGATPDYWGQGTLVTVSSAttorney Ref.62571.00009WO01 (RND-0002-WO) R052 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQSP 140 GKGLEWIGEINHSGITNYNPSLKSRVTISVDTSKNQFSLNLSS VTAADTAVYYCARVGGATPDYWGQGTLVTVSSAttorney Ref.62571.00009WO01 (RND-0002-WO) R069 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSFYYWTWIRQSP 157 GKGLEWIGEINHSGSTNYNPSLKSRLTISVDTSKNQFSLKLSS VTAADTAVYYCARVGGATVDYWGQGSLVTVSSAttorney Ref.62571.00009WO01 (RND-0002-WO) R087 QVQLQQWGAGLLKPSETLSLTCAVYGGSFNFYYWTWIRQPP 175 GKGLEWIGEIKHSGSTYYNPSLKSRVIISVDTPKNQFSLRLTS VTAADTAVYYCARVGGATVDYWGQGSLVTVSSAttorney Ref.62571.00009WO01 (RND-0002-WO) R105 QLLLQEPGPRLVKPSETLSLTCTVSGGSISSGSYYWGWIRQPP 193 GKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKKQFSLKLTS VTAADTAVYYCARGDSAWPFDYWGQGTLVTVSSAttorney Ref.62571.00009WO01 (RND-0002-WO) R122 QLLLQEPGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPP 210 GKGLEWIGTIYYSGTTYSNPSLKSRVTISVDTSKSQFSLKLTS VTAADTAVYYCARGDSSWPFDYWGQGTLVTVSSAttorney Ref.62571.00009WO01 (RND-0002-WO) R140 QLLLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPP 228 GKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKKQFSLKLNS VTAADTAVYYCTRGDSSWPFDYWGQGTLVTVSSain sequence). Antibody SEQ_aa_FR1_FR4 SEQ ID Construct NOAttorney Ref.62571.00009WO01 (RND-0002-WO)

[0214] In some embodiments, the heavy chain CDR sequences for a DAA binding unit that binds to Nectin-4 belong to family 3 and have CDR sequences corresponding to the following formulae. An X indicates a variable amino acid, which may be specific amino acids as indicated below. CDR1 G1X2S3X4X5X6G7X8X9Y10(SEQ ID NO: 326) where: X2is G or V. X4is L or I. X5is R, S, or T. X6 is S or I. X8is I, P, G, or A. X9is S or Y.

[0215] In some embodiments, a heavy chain CDR1 sequence of a family 3 antibody comprises the sequence set forth in any one of SEQ ID NOs: 4, or 286-291. CDR2 X1Y2X3S4G5X6X7where: X1is I or L. X3is Y or F. X6is T or S. X7is T or A.

[0216] In some embodiments, a heavy chain CDR2 sequence of a family 3 antibody comprises the sequence set forth in any one of SEQ ID NOs: 10, 83, or 292-294. CDR3 A1X2E3X4X5X6X7H8Y9F10X11X12(SEQ ID NO: 328) where: X2is R or K. X4is V, T, Q, I, or N. X5is N, H, W, or Q. X6is W or G. X7is N, A, K, H, E, or L. X11is D or E.Attorney Ref.62571.00009WO01 (RND-0002-WO) X12is Y or F.

[0217] In some embodiments, a heavy chain CDR3 sequence of a family 3 antibody comprises the sequence set forth in any one of SEQ ID NOs: 23, or 295-300.

[0218] The antibodies of family 3 comprise a set of heavy chain CDR sequences as defined herein and shown in Tables 4A and 4B, and are exemplified by the provided heavy chain CDR1, CDR2 and CDR3 sequences set forth in Table 5, the heavy chain variable region (VH) sequences of SEQ ID NOs: 101- 102, 301-317 set forth in Table 6A, and the light chain variable region (VL) sequences of SEQ ID NO: 110 set forth in Table 6B. The family 3 antibodies provide a number of benefits that contribute to utility as clinically therapeutic agent(s). The family 3 antibodies include members with a range of binding affinities, allowing the selection of a specific sequence with a desired binding affinity. Table 4A: Anti-Nectin-4 unique heavy chain CDR amino acid sequences. SEQ_aa_CDR1 SEQ_aa_CDR2 SEQ_aa_CDR3 GGSISSGISY (SEQ ID NO: 4) IYYSGST (SEQ ID NO: 10) AKENHWKHYFDY (SEQ IDTable 4B: Anti-Nectin-4 light chain CDR amino acid sequences (common light chain sequences). SEQ_aa_CDR1 SEQ_aa_CDR2 SEQ_aa_CDR3 SVSSN (SE ID NO 28) GAS YNNWPWT (SE ID NO 44)Antibody SEQ_aa_CDR1 SEQ_aa_CDR2 SEQ_aa_CDR3 Construct D DAttorney Ref.62571.00009WO01 (RND-0002-WO) R156 GGSLRSGISY (SEQ IYYSGTT (SEQ ID NO: AREVNWNHYFDY (SEQ ID ID NO: 286) 83) NO: 295) R157 GGSISSGISY (SEQ ID IYYSGSA (SEQ ID NO: ARETHWAHYFDY (SEQ ID D D D D D D D D DTable 6A: Anti-Nectin-4 heavy chain variable region amino acid sequences. Antibody SEQ_aa_FR1_FR4 SEQ ID NO. ConstructAttorney Ref.62571.00009WO01 (RND-0002-WO) R158 QVQLQESGPGLVKPSQTLSLTCTVSGVSITSGISYWSWIRQHP 303 GKGLEWLGYIYYSGTTYYNPSLKSRLAISVDTSKNQFSLKLSS LTAADTAVYFCAKEQNWKHYFDYWGQGTLVTVSSain sequence). Antibody SEQ_aa_FR1_FR4 SEQ ID NO. t tAttorney Ref.62571.00009WO01 (RND-0002-WO) R011, EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQ 110 R015 APRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYY CQQYNNWPWTFGQGTKVEIK

[0219] A suitable antibody may be selected from those provided herein for development and therapeutic or other use, as shown, for example, in FIG.1.

[0220] Aspects of the present disclosure include antibodies comprising means for binding to the binding targets described herein, namely, CD28 and Nectin-4. In some embodiments, an antibody comprises means for binding to CD28, such as, for example a binding unit comprising means for binding to CD28. In some embodiments, an antibody comprises means for binding to Nectin-4, such as, for example a binding unit comprising means for binding to Nectin-4. Antibodies comprising means for binding to a binding target include equivalents of the structural features of the antibodies described herein, for example, equivalents of the CD28 binding domains and / or Nectin-4 binding domains described herein.

[0221] Determination of affinity for a candidate protein can be performed using methods known in the art, such as surface plasmon resonance (SPR). Members of the antibody family may have an affinity for an EAA (e.g., CD28) or a DAA (e.g., Nectin-4) with a Kd of from about 10-6molar (M) to around about 10-11M, including without limitation: from about 10-6M to around about 10-10M; from about 10-6M to around about 10-9M; from about 10-6M to around about 10-8M; from about 10-8M to around about 10-11M; from about 10-8M to around about 10-10M; from about 10-8M to around about 10-9M; from about 10-9M to around about 10-11M; from about 10-9M to around about 10-10M; or any value within these ranges. The affinity selection may be confirmed with a biological assessment for modulating, e.g., increasing, a CD28 biological activity, including in vitro assays, pre-clinical models, and clinical trials, as well as assessment of potential toxicity. Cynomolgus monkey (cyno) cross- reactivity is important for evaluation of potential toxicity of therapeutic molecules. The extracellular domain of cynomolgus CD28 is identical in sequence to that of human CD28, resulting in no differences in cross-reactivity, making cyno a potentially relevant species for toxicity studies of the CD28 antibodies described herein. The anti-human Nectin-4 antibodies described herein have an affinity for cyno Nectin-4 within the range of 2 - 3 fold, which is considered similar and therefore makes cyno a potentially relevant species for toxicity studies of the anti-Nectin-4 antibodies described herein as well. CD28 Binding Domains

[0222] In some embodiments, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising a CDR1 sequence of any one of SEQ ID NOs: 1, 2, 3, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 1. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 2. In a particular embodiment, a multi-specific anti-CD28Attorney Ref.62571.00009WO01 (RND-0002-WO) antibody comprises a CDR1 sequence of SEQ ID NO: 3. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 57. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 58. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 59. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 60. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 61. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 62. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 63. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 64. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 65. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 66. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR1 sequence of SEQ ID NO: 67.

[0223] In some embodiments, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising a CDR2 sequence of any one of SEQ ID NOs: 9, 10, 11, 12, 13, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 9. In a particular embodiment, a multi- specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 10. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 11. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 12. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 13. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 68. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 69. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 70. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 71. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 72. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 73. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 74. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 75. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 76. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 77. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 78. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 79. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 80. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 81. InAttorney Ref.62571.00009WO01 (RND-0002-WO) a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 82. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 83. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 84. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR2 sequence of SEQ ID NO: 85.

[0224] In some embodiments, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising a CDR3 sequence of any one of SEQ ID NOs: 18, 19, 20, 21, 22, 86, 87, 88, 89, 90, 91, or 92. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 18. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 19. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 20. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 21. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 22. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 86. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 87. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 88. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 89. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 90. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 91. In a particular embodiment, a multi-specific anti-CD28 antibody comprises a CDR3 sequence of SEQ ID NO: 92. (Table 1A).

[0225] In some embodiments, a multi-specific anti-CD28 antibody comprises a light chain variable region sequence comprising a CDR1 sequence of SEQ ID NO: 28. In some embodiments, a multi- specific anti-CD28 antibody comprises a light chain variable region sequence comprising a CDR2 sequence of SEQ ID NO: 36. In some embodiments, a multi-specific anti-CD28 antibody comprises a light chain variable region sequence comprising a CDR3 sequence of SEQ ID NO: 44. (Table 1B).

[0226] In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 9, and the CDR3 sequence of SEQ ID NO: 18. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 19. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 11, and the CDR3 sequence of SEQ ID NO: 20. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 13, and the CDR3 sequence of SEQ ID NO: 21. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequenceAttorney Ref.62571.00009WO01 (RND-0002-WO) comprising the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 12, and the CDR3 sequence of SEQ ID NO: 21. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 68, and the CDR3 sequence of SEQ ID NO: 21. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 57, the CDR2 sequence of SEQ ID NO: 69, and the CDR3 sequence of SEQ ID NO: 86. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 57, the CDR2 sequence of SEQ ID NO: 9, and the CDR3 sequence of SEQ ID NO: 21. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 57, the CDR2 sequence of SEQ ID NO: 70, and the CDR3 sequence of SEQ ID NO: 87. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 57, the CDR2 sequence of SEQ ID NO: 9, and the CDR3 sequence of SEQ ID NO: 88. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO:58, the CDR2 sequence of SEQ ID NO: 9, and the CDR3 sequence of SEQ ID NO: 89. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 71, and the CDR3 sequence of SEQ ID NO: 90. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 72, and the CDR3 sequence of SEQ ID NO: 87. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 68, and the CDR3 sequence of SEQ ID NO: 87. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 69, and the CDR3 sequence of SEQ ID NO: 87. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 73, and the CDR3 sequence of SEQ ID NO: 86. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 73, and the CDR3 sequence of SEQ ID NO: 18. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 57, the CDR2 sequence of SEQ ID NO: 73, and the CDR3 sequence of SEQ ID NO: 87. In a further embodiment, a multi-specific anti-CD28 antibodyAttorney Ref.62571.00009WO01 (RND-0002-WO) comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 73, and the CDR3 sequence of SEQ ID NO: 87. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 73, and the CDR3 sequence of SEQ ID NO: 21. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 74, and the CDR3 sequence of SEQ ID NO: 21. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 73, and the CDR3 sequence of SEQ ID NO: 88. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 57, the CDR2 sequence of SEQ ID NO: 73, and the CDR3 sequence of SEQ ID NO: 21. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 9, and the CDR3 sequence of SEQ ID NO: 88. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 59, the CDR2 sequence of SEQ ID NO: 68, and the CDR3 sequence of SEQ ID NO: 21. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 75, and the CDR3 sequence of SEQ ID NO: 20. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 57, the CDR2 sequence of SEQ ID NO: 70, and the CDR3 sequence of SEQ ID NO: 89. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 73, and the CDR3 sequence of SEQ ID NO: 89. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 57, the CDR2 sequence of SEQ ID NO: 73, and the CDR3 sequence of SEQ ID NO: 18. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 76, and the CDR3 sequence of SEQ ID NO: 90. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 60, the CDR2 sequence of SEQ ID NO: 69, and the CDR3 sequence of SEQ ID NO: 18. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 57, the CDR2 sequence of SEQ ID NO: 70, and the CDR3 sequence of SEQ ID NO: 18. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 57, the CDR2 sequence of SEQ ID NO: 77, and the CDR3 sequence of SEQ ID NO: 89. In a further embodiment, a multi-specific anti-CD28 antibodyAttorney Ref.62571.00009WO01 (RND-0002-WO) comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 69, and the CDR3 sequence of SEQ ID NO: 89. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 78, and the CDR3 sequence of SEQ ID NO: 18. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 77, and the CDR3 sequence of SEQ ID NO: 18. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 71, and the CDR3 sequence of SEQ ID NO: 18. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 61, the CDR2 sequence of SEQ ID NO: 69, and the CDR3 sequence of SEQ ID NO: 18. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 79, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 80, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 19. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 80, and the CDR3 sequence of SEQ ID NO: 19. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 80, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 91. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 81, and the CDR3 sequence of SEQ ID NO: 19. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 91. In a furtherAttorney Ref.62571.00009WO01 (RND-0002-WO) embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 82, and the CDR3 sequence of SEQ ID NO: 91. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 80, and the CDR3 sequence of SEQ ID NO: 19. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 79, and the CDR3 sequence of SEQ ID NO: 19. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 79, and the CDR3 sequence of SEQ ID NO: 91. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 80, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 19. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 73, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 79, and the CDR3 sequence of SEQ ID NO: 19. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 83, and the CDR3 sequence of SEQ ID NO: 91. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 83, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 63, the CDR2 sequence of SEQ ID NO: 80, and the CDR3 sequence of SEQ ID NO: 91. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 73, and the CDR3 sequence of SEQ ID NO: 19. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 64, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 91. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 84, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 10, and theAttorney Ref.62571.00009WO01 (RND-0002-WO) CDR3 sequence of SEQ ID NO: 92. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 62, the CDR2 sequence of SEQ ID NO: 80, and the CDR3 sequence of SEQ ID NO: 92. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 65, the CDR2 sequence of SEQ ID NO: 80, and the CDR3 sequence of SEQ ID NO: 19. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 66, the CDR2 sequence of SEQ ID NO: 85, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 67, the CDR2 sequence of SEQ ID NO: 83, and the CDR3 sequence of SEQ ID NO: 22. In a further embodiment, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 91. (Table 2).

[0227] In a further embodiment, a multi-specific anti-CD28 antibody comprises a light chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 28, the CDR2 sequence of SEQ ID NO: 36, and the CDR3 sequence of SEQ ID NO: 44.

[0228] In a further embodiment, a multi-specific anti-CD28 antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 93-100, 107, 121-237, or 333-334. (Table 3A).

[0229] In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 93. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 94. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 95. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 96. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 97. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 98. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 99. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 100. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 107. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 121. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 122. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 123. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 124. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable regionAttorney Ref.62571.00009WO01 (RND-0002-WO) sequence of SEQ ID NO: 125. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 126. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 127. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 128. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 129. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 130. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 131. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 132. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 133. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 134. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 135. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 136. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 137. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 138. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 139. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 140. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 141. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 142. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 143. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 144. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 145. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 146. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 147. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 148. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 149. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 150. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 151. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chainAttorney Ref.62571.00009WO01 (RND-0002-WO) variable region sequence of SEQ ID NO: 152. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 153. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 154. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 155. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 156. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 157. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 158. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 159. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 160. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 161. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 162. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 163. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 164. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 165. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 166. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 167. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 168. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 169. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 170. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 171. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 172. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 173. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 174. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 175. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 176. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 177. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 178. In a still further embodiment, a multi-specific anti-CD28 antibody comprises theAttorney Ref.62571.00009WO01 (RND-0002-WO) heavy chain variable region sequence of SEQ ID NO: 179. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 180. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 181. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 182. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 183. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 184. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 185. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 186. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 187. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 188. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 189. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 190. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 191. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 192. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 193. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 194. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 195. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 196. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 197. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 198. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 199. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 200. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 201. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 202. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 203. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 204. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 205. In a still further embodiment, a multi-specific anti-CD28 antibodyAttorney Ref.62571.00009WO01 (RND-0002-WO) comprises the heavy chain variable region sequence of SEQ ID NO: 206. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 207. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 208. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 209. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 210. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 211. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 212. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 213. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 214. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 215. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 216. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 217. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 218. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 219. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 220. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 221. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 222. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 223. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 224. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 225. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 226. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 227. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 228. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 229. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 230. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 231. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 232. In a still further embodiment, a multi-specific anti-CD28Attorney Ref.62571.00009WO01 (RND-0002-WO) antibody comprises the heavy chain variable region sequence of SEQ ID NO: 233. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 234. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 235. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 236. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 237. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 333. In a still further embodiment, a multi-specific anti-CD28 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 334.

[0230] In some embodiments, a CDR sequence in a multi-specific anti-CD28 antibody of the present disclosure comprises one or two amino acid substitutions relative to a CDR1, CDR2 and / or CDR3 sequence or set of CDR1, CDR2 and CDR3 sequences in any one of SEQ ID NOs: 1-92 (Table 1A; Table 1B; Table 2).

[0231] In some embodiments, a multi-specific anti-CD28 antibody preferably comprises a heavy chain variable domain (VH) in which the CDR3 sequence has greater than or equal to 80%, such as at least 85%, at least 90%, at least 95%, or at least 99% sequence identity at the amino acid level to a CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in Table 1A or Table 2, and binds to CD28.

[0232] In some embodiments, a multi-specific anti-CD28 antibody preferably comprises a heavy chain variable domain (VH) in which the full set of CDRs 1, 2, and 3 (combined) has greater than or equal to eighty-five percent (85%) sequence identity at the amino acid level to the CDRs 1, 2, and 3 (combined) of the antibodies whose CDR sequences are provided in Table 1A or Table 2, and binds to CD28.

[0233] In some embodiments, a multi-specific anti-CD28 antibody comprises a heavy chain variable region sequence with at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any of the heavy chain variable region sequences of SEQ ID NOs: 93-100, 107, 121-237, or 333-334 (shown in Table 3A), and binds to CD28.

[0234] In some embodiments, a multi-specific anti-CD28 antibody preferably comprises a light chain variable domain (VL) in which the CDR3 sequence has greater than or equal to 80%, such as at least 85%, at least 90%, at least 95%, or at least 99% sequence identity at the amino acid level to a CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in Table 1B and binds to CD28.

[0235] In some embodiments, a multi-specific anti-CD28 antibody preferably comprises a light chain variable domain (VL) in which the full set of CDRs 1, 2, and 3 (combined) has greater than or equal to eighty-five percent (85%) sequence identity at the amino acid level to the CDRs 1, 2, and 3 (combined) of the antibodies whose CDR sequences are provided in Table 1B and binds to CD28.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0236] In some embodiments, a multi-specific anti-CD28 antibody comprises a light chain variable region sequence with at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to the light chain variable region sequence of SEQ ID NO: 110 (shown in Table 3B), and binds to CD28. Nectin-4 Binding Domains

[0237] In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR1 sequence of SEQ ID NO: 4. In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR1 sequence of SEQ ID NO: 286. In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR1 sequence of SEQ ID NO: 287. In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR1 sequence of SEQ ID NO: 288. In some embodiments, a multi-specific anti-Nectin- 4 antibody comprises a heavy chain variable region sequence comprising a CDR1 sequence of SEQ ID NO: 289. In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR1 sequence of SEQ ID NO: 290. In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR1 sequence of SEQ ID NO: 291.

[0238] In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR2 sequence of SEQ ID NO: 10. In some embodiments, a multi- specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR2 sequence of SEQ ID NO: 83. In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR2 sequence of SEQ ID NO: 292. In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR2 sequence of SEQ ID NO: 293. In some embodiments, a multi-specific anti-Nectin- 4 antibody comprises a heavy chain variable region sequence comprising a CDR2 sequence of SEQ ID NO: 294.

[0239] In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR3 sequence of SEQ ID NO: 23. In some embodiments, a multi- specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR3 sequence of SEQ ID NO: 295. In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR3 sequence of SEQ ID NO: 296. In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR3 sequence of SEQ ID NO: 297. In some embodiments, a multi-specific anti-Nectin- 4 antibody comprises a heavy chain variable region sequence comprising a CDR3 sequence of SEQ ID NO: 298. In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR3 sequence of SEQ ID NO: 299. In some embodiments, aAttorney Ref.62571.00009WO01 (RND-0002-WO) multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising a CDR3 sequence of SEQ ID NO: 300. (Table 4A).

[0240] In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a light chain variable region sequence comprising a CDR1 sequence of SEQ ID NO: 28. In some embodiments, a multi- specific anti- Nectin-4 antibody comprises a light chain variable region sequence comprising a CDR2 sequence of SEQ ID NO: 36. In some embodiments, a multi-specific anti- Nectin-4 antibody comprises a light chain variable region sequence comprising a CDR3 sequence of SEQ ID NO: 44. (Table 4B).

[0241] In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 23. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 286, the CDR2 sequence of SEQ ID NO: 83, and the CDR3 sequence of SEQ ID NO: 295. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 292, and the CDR3 sequence of SEQ ID NO: 296. In a further embodiment, a multi- specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 287, the CDR2 sequence of SEQ ID NO: 83, and the CDR3 sequence of SEQ ID NO: 297. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 288, the CDR2 sequence of SEQ ID NO: 83, and the CDR3 sequence of SEQ ID NO: 298. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 289, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 299. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 289, the CDR2 sequence of SEQ ID NO: 83, and the CDR3 sequence of SEQ ID NO: 295. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 23. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 293, and the CDR3 sequence of SEQ ID NO: 23. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 286, the CDR2 sequence of SEQ ID NO: 83, and the CDR3 sequence of SEQ ID NO: 296. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 289, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQ ID NO: 23. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 289, the CDR2 sequence of SEQ ID NO: 10, and the CDR3 sequence of SEQAttorney Ref.62571.00009WO01 (RND-0002-WO) ID NO: 296. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 286, the CDR2 sequence of SEQ ID NO: 83, and the CDR3 sequence of SEQ ID NO: 23. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 290, the CDR2 sequence of SEQ ID NO: 83, and the CDR3 sequence of SEQ ID NO: 300. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 286, the CDR2 sequence of SEQ ID NO: 83, and the CDR3 sequence of SEQ ID NO: 300. In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 291, the CDR2 sequence of SEQ ID NO: 294, and the CDR3 sequence of SEQ ID NO: 300. (Table 5).

[0242] In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises a light chain variable region sequence comprising the CDR1 sequence of SEQ ID NO: 28, the CDR2 sequence of SEQ ID NO: 36, and the CDR3 sequence of SEQ ID NO: 44.

[0243] In a further embodiment, a multi-specific anti-Nectin-4 antibody comprises any of the heavy chain variable region amino acid sequences of SEQ ID NOs: 101-102, or 301-317. (Table 6A).

[0244] In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 101. In a still further embodiment, a multi-specific anti- Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 102. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 301. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 302. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 303. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 304. In a still further embodiment, a multi-specific anti- Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 305. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 306. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 307. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 308. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 309. In a still further embodiment, a multi-specific anti- Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 310. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 311. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 312. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ IDAttorney Ref.62571.00009WO01 (RND-0002-WO) NO: 313. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 314. In a still further embodiment, a multi-specific anti- Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 315. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 316. In a still further embodiment, a multi-specific anti-Nectin-4 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 317.

[0245] In some embodiments, a CDR sequence in a multi-specific anti-Nectin-4 antibody of the present disclosure comprises one or two amino acid substitutions relative to a CDR1, CDR2 and / or CDR3 sequence or set of CDR1, CDR2 and CDR3 sequences in any one of SEQ ID NOs: 4, 10, 23, 28, 36, 44, 83, or 286-300. (Table 4A; Table 4B; Table 5).

[0246] In some embodiments, a multi-specific anti-Nectin-4 antibody preferably comprises a heavy chain variable domain (VH) in which the CDR3 sequence has greater than or equal to 80%, such as at least 85%, at least 90%, at least 95%, or at least 99% sequence identity at the amino acid level to a CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in Table 4A or Table 5, and binds to Nectin-4.

[0247] In some embodiments, a multi-specific anti-Nectin-4 antibody preferably comprises a heavy chain variable domain (VH) in which the full set of CDRs 1, 2, and 3 (combined) has greater than or equal to eighty-five percent (85%) sequence identity at the amino acid level to the CDRs 1, 2, and 3 (combined) of the antibodies whose CDR sequences are provided in Table 4A or Table 5, and binds to Nectin-4.

[0248] In some embodiments, a multi-specific anti-Nectin-4 antibody comprises a heavy chain variable region sequence with at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any of the heavy chain variable region sequences of SEQ ID NOs: 101-102 or 300-317 (shown in Table 6A), and binds to Nectin-4.

[0249] In some embodiments, a multi-specific anti-Nectin-4 antibody preferably comprises a light chain variable domain (VL) in which the CDR3 sequence has greater than or equal to 80%, such as at least 85%, at least 90%, at least 95%, or at least 99% sequence identity at the amino acid level to a CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in Table 4B and binds to Nectin-4.

[0250] In some embodiments, a multi-specific anti-CD28 antibody preferably comprises a light chain variable domain (VL) in which the full set of CDRs 1, 2, and 3 (combined) has greater than or equal to eighty-five percent (85%) sequence identity at the amino acid level to the CDRs 1, 2, and 3 (combined) of the antibodies whose CDR sequences are provided in Table 4B and binds to Nectin-4.

[0251] In some embodiments, a multi-specific anti-CD28 antibody comprises a light chain variable region sequence with at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to the light chain variable region sequence of SEQ ID NO: 110 (shown in Table 6B), and binds to Nectin-4.Attorney Ref.62571.00009WO01 (RND-0002-WO) Multi-specific Antibody Constructs

[0252] In some embodiments, multi-specific antibodies are provided (e.g., bispecific antibodies), which can have any of the configurations discussed herein, including, without limitation, a bispecific antibody comprising two heavy chain polypeptide subunits and two light chain polypeptide subunits.

[0253] In some embodiments, a multi-specific antibody can comprise a first arm comprising a binding unit that binds to an EAA and a second arm comprising a binding unit that binds to a DAA. In some embodiments, a multi-specific antibody can comprise a first arm comprising a binding unit that binds an EAA and a second arm comprising a binding unit that binds to Nectin-4. In some embodiments, a multi-specific antibody can comprise a first arm comprising a binding unit that binds to CD28 and a second arm comprising a binding unit that binds to a DAA. In some embodiments, the DAA can be Nectin-4. In some embodiments, a bispecific antibody can comprise a first arm comprising a binding unit that binds to CD28 and a second arm comprising a binding unit that binds to Nectin-4. In some embodiments, a bispecific antibody can comprise a first arm comprising a binding unit that binds to CD3 and a second arm comprising a binding unit that binds to Nectin-4.

[0254] In some embodiments, a multi-specific antibody can comprise a first arm comprising a binding unit that binds to Nectin-4 and a second arm comprising a binding unit that binds to an EAA. In some embodiments, the EAA can be CD28. In some embodiments, a bispecific antibody can comprise a first arm comprising a binding unit that binds to Nectin-4 and a second arm comprising a binding unit that binds to CD28.

[0255] Non-limiting examples of EAAs include cell surface proteins expressed by any immune effector cell, such as, for example, CD28 expressed by T-cells, or CD3 expressed by T-cells. Non- limiting examples of DAAs include tumor associated antigens (TAAs) and tumor specific antigens (TSAs). TAAs are relatively restricted to tumor cells, whereas TSAs are unique to tumor cells. In some embodiments, the DAA is a TAA such as, for example, Nectin-4.

[0256] In some embodiments, a multi-specific antibody can comprise at least one binding unit that binds to CD28, and at least one binding unit that binds to a DAA (e.g., Nectin-4).

[0257] In some embodiments, a multi-specific antibody can comprise at least one binding unit that binds to Nectin-4, and at least one binding unit that binds to an EAA (e.g., CD28).

[0258] Any of the binding domains described herein can be combined with additional sequences, such as constant region and Fc region sequences described herein (e.g., Tables 7 and 8), to generate multi- specific antibodies that bind to an EAA and / or a DAA. In addition, any of the variable region sequences herein can be combined with constant region sequences of any known antibody isotype (IgG, IgM, IgD, IgA, and IgE) as well as any of their subtypes (e.g., IgG1, IgG2, IgG3, IgG4) to generate desired full- length antibody molecules.

[0259] Aspects of the present disclosure include multi-specific antibodies comprising the binding units discussed herein, in addition to heavy chain and light chain constant region sequences. In someAttorney Ref.62571.00009WO01 (RND-0002-WO) embodiments, a heavy chain constant region sequence comprises a CH1 domain, a hinge region, a CH2 domain, and / or a CH3 domain.

[0260] In some embodiments, an antibody comprises a human IgG1 wild type constant region sequence. In some embodiments, an antibody comprises a human IgG1 variant constant region sequence comprising one or more amino acid mutations, in comparison to the wild type sequence. In some embodiments, an antibody comprises a human IgG1 region sequence comprising a silencing mutation, such as, for example, an L234A mutation, an L235A mutation, a P329G mutation, or any combination thereof. In some embodiments, an antibody comprises a human IgG1 region sequence comprising a T366W mutation (knob). In some embodiments, an antibody comprises a human IgG1 region sequence comprising a T366S mutation, an L368A mutation, a Y407V mutation, or any combination thereof (hole). In some embodiments, an antibody comprises a human IgG1 region sequence comprising a disulfide mutation, such as, for example, an S354C mutation, a Y349C mutation, or both. These and other sequences are provided in Tables 7, 8A, and 8B.

[0261] In some embodiments, an antibody comprises a human IgG4 wild type constant region sequence. In some embodiments, an antibody comprises a human IgG4 variant constant region sequence comprising one or more amino acid mutations, in comparison to the wild type sequence. In some embodiments, an antibody comprises a human IgG4 hinge region sequence comprising an S228P mutation. In some embodiments, an antibody comprises a human IgG4 region sequence comprising a silencing mutation, such as, for example, an F234A mutation, an L235A mutation, or both. In some embodiments, an antibody comprises a human IgG4 region sequence comprising a T366W mutation (knob). In some embodiments, an antibody comprises a human IgG4 region sequence comprising a T366S mutation, an L368A mutation, a Y407V mutation, or any combination thereof (hole). In some embodiments, an antibody comprises a human IgG4 region sequence comprising a disulfide mutation, such as, for example, an S354C mutation, a Y349C mutation, or both. In some embodiments, an antibody comprises a human IgG4 region sequence comprising a heavy chain constant region sequence comprising a L445P mutation. These and other sequences are provided in Tables 7, 8A, and 8B. Table 7: Human IgG1, IgG4, Fc Region sequences. Sequence Name Amino Acid SequenceAttorney Ref.62571.00009WO01 (RND-0002-WO) Human IgG4 ASTKGPSVFP LAPCSRSTSESTAALGCLVKDYFPEPVTVS (UniProt WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV S K V V E S K D Q T YTable 8A: Summary of benchmark antibody molecules with functional descriptions. Antibody Construct Reference No. Functional Description of Antibody it e n it n it n sAttorney Ref.62571.00009WO01 (RND-0002-WO) R020 R020 is a bispecific antibody and includes the same VH and VL domains of the CD28 binding unit of R017 and the same Fc as R005, R006, and R007. The tumor binding unit is formatted as a scFv that binds to the 8 s n s s s n n-Attorney Ref.62571.00009WO01 (RND-0002-WO) 4 binding unit as R005, R006 and R007. As provided in FIG.25A, the affinity for human CD3 delta epsilon heterodimer protein is measured in the single digit nM range by BLI. 6 s AsAntibody Name Amino Acid Sequence Construct N N K N K L N N K N K L L N N K N K L LAttorney Ref.62571.00009WO01 (RND-0002-WO) R002, pIG_hIgH4- ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN R004 PAA-Hole SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPK N K L N N K N K L N L E S F P N L E S L G H

[0262] Aspects of the present disclosure include antibodies comprising the full-length polypeptide subunits described in Table 9A. In some embodiments, an antibody can include a full length common light chain sequence that comprises a common light chain variable region sequence (VL) and a light chain constant region sequence (CL). In some embodiments, an antibody can include two full lengthAttorney Ref.62571.00009WO01 (RND-0002-WO) common light chain sequences, each comprising a common light chain variable region sequence (VL) and a light chain constant region sequence (CL). Table 9A: Full Length Heavy Chain Sequences. Antibody Subunit Sequence Construct Reference Q S N P E Q S Q L S W T L K S K Q S N P E Q S Q L S W T LAttorney Ref.62571.00009WO01 (RND-0002-WO) FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK S K Q A N C F N K Q L S W T L K S K Q A N C F N K Q L S W T L K S KAttorney Ref.62571.00009WO01 (RND-0002-WO) First heavy QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQ chain PPGKGLEWIGEIQHSGSTNYNPSLKSRVTISVDTSKNQFSL polypeptide KLSSVTAADTAVYYCARVGGATPDFWGQGTLVTVSSAST N P E Q S Q L S W T L K S K Q L T N P E Q S Q L S W T L K S K Q S S N PAttorney Ref.62571.00009WO01 (RND-0002-WO) PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQ S Q L S W T L K S K Q L T N P E Q G L Q T N P E Q G L Q S S N P E Q G LAttorney Ref.62571.00009WO01 (RND-0002-WO) First and QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGISYWSWIRQ second heavy HPGKGLEWIGYIYYSGSTYYNPSLKSRVTILVDTSKNQFSL chain ELSSVTAADTAVYYCAKENHWKHYFDYWGQGTLVTVSS W T L K S K Q L T N P E Q G L Q T N P E Q G L Q S S N P E Q G L Q L S W T LAttorney Ref.62571.00009WO01 (RND-0002-WO) FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK S K Q L T S N F G L T Q L S G N E N Q S S N P E Q S Q L S W T L K S KAttorney Ref.62571.00009WO01 (RND-0002-WO) First heavy QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQ chain PPGKGLEWIGEIQHSGSTNYNPSLKSRVTISVDTSKNQFSL polypeptide KLSSVTAADTAVYYCARVGGATPDFWGQGTLVTVSSAST S N F G L T Q L S G N E N Q L S N P E Q G L Q L T N P E Q G L Q S N PAttorney Ref.62571.00009WO01 (RND-0002-WO) PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ G L Q S S N P E Q G LAntibody Subunit Amino Acid Sequence Construct G L E T [02a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 240, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 241.

[0264] In some embodiments, a multi-specific antibody that binds to CD28 and Nectin-4 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 243, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 244.

[0265] In some embodiments, a multi-specific antibody that binds to CD28 and Nectin-4 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 245, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 246.

[0266] In some embodiments, a multi-specific antibody that binds to CD28 and Nectin-4 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 247, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 248.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0267] In some embodiments, a multi-specific antibody that binds to CD28 and Nectin-4 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 249, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 250.

[0268] In some embodiments, a multi-specific antibody that binds to CD28 and Nectin-4 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 251, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 252.

[0269] In some embodiments, a multi-specific antibody that binds to CD28 and Nectin-4 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 253, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 254.

[0270] In some embodiments, a multi-specific antibody that binds to CD28 and Nectin-4 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 263, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 264.

[0271] In some embodiments, a multi-specific antibody that binds to CD28 and Nectin-4 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 265, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 266.

[0272] In some embodiments, a multi-specific antibody that binds to CD28 and Nectin-4 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 267, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 268.

[0273] In some embodiments, a monospecific antibody that binds to CD28 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 255, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 255.

[0274] In some embodiments, a monospecific antibody that binds to CD28 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 256, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 256.

[0275] In some embodiments, a monospecific antibody that binds to CD28 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 257, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 257.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0276] In some embodiments, a monospecific antibody that binds to CD28 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 259, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 259.

[0277] In some embodiments, a monospecific antibody that binds to CD28 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 260, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 260.

[0278] In some embodiments, a monospecific antibody that binds to CD28 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 261, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 261.

[0279] In some embodiments, a monospecific antibody that binds to Nectin-4 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 258, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 258.

[0280] In some embodiments, a monospecific antibody that binds to Nectin-4 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 262, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 262.

[0281] In some embodiments, a monospecific antibody that binds to CD28 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 329, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 329.

[0282] In some embodiments, a monospecific antibody that binds to CD28 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 335, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 335.

[0283] In some embodiments, a monospecific antibody that binds to CD28 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 336, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 336.

[0284] In some embodiments, a monospecific antibody that binds to CD28 comprises a first light chain polypeptide subunit comprising SEQ ID NO: 242, a second light chain polypeptide subunit comprising SEQ ID NO: 242, a first heavy chain polypeptide subunit comprising SEQ ID NO: 337, and a second heavy chain polypeptide subunit comprising SEQ ID NO: 337.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0285] Aspects of the present disclosure include use of the variable regions described herein in CARs. In some embodiments, the variable regions of the antibodies described herein can be used as a binding (targeting) domain of a chimeric antigen receptor (CAR). Such antibodies or CARs can induce potent T-cell mediated killing of cells expressing a disease associated antigen (e.g., Nectin-4).

[0286] Aspects of the present disclosure include immunoconjugates, or antibody-drug conjugates (ADCs) that comprise an antibody as described herein (Ab), a linker (L), and a drug moiety (D). In some embodiments, an antibody described herein is conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In another aspect, the present disclosure further provides methods of using the immunoconjugates. In one aspect, an immunoconjugate comprises any of the antibodies described herein covalently attached to a cytotoxic agent or a detectable agent. ADCs are described, for example, in U.S. Patent No.8,362,213, the disclosure of which is incorporated by reference herein in its entirety.

[0287] Aspects of the present disclosure include multispecific antibodies comprising means for binding to the binding targets described herein, namely, CD28 and Nectin-4. In some embodiments, a multispecific antibody comprises means for binding to CD28, such as, for example a first binding unit comprising means for binding to CD28, and a second binding unit that binds to a DAA (e.g., Nectin-4). In some embodiments, a multispecific antibody comprises means for binding to Nectin-4, such as, for example, a first binding unit comprising means for binding to Nectin-4, and a second binding unit that binds to an EAA (e.g., CD28). In some embodiments, a multispecific antibody comprises a first binding unit comprising means for binding to CD28, and a second binding unit comprising means for binding to Nectin-4. In some embodiments, a bispecific antibody comprises a first binding unit comprising means for binding to CD28, and a second binding unit comprising means for binding to Nectin-4. Multispecific antibodies (e.g., bispecific antibodies) comprising means for binding to a binding target include equivalents of the structural features of the antibodies described herein, for example, equivalents of the CD28 binding domains and / or Nectin-4 binding domains described herein. Preparation of Antibodies

[0288] The antibodies of the present disclosure can be prepared by methods known in the art. Recombinant technology using, for example, phagemid technology, allows for preparation of antibodies having a desired specificity from recombinant genes encoding a range of antibodies. Certain recombinant techniques involve isolation of antibody genes by immunological screening of combinatorial immunoglobulin phage expression libraries prepared from RNA isolated from an immunized animal. For such methods, combinatorial immunoglobulin phagemid libraries can be prepared from RNA isolated from an immunized animal, and phagemids expressing appropriate antibodies can be selected by panning using cells expressing antigen and control cells.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0289] In addition to the combinatorial immunoglobulin phage expression libraries disclosed above, one molecular cloning approach is to prepare antibodies from transgenic animals containing human antibody transgenes. Such transgenic animals can be employed to produce human antibodies of a single isotype, more specifically an isotype that is essential for B cell maturation, such as IgM and possibly IgD.

[0290] In a preferred embodiment, the antibodies herein are produced by transgenic animals, including transgenic mice and rats, in which the endogenous immunoglobulin genes are knocked out or disabled. Endogenous immunoglobulin loci in rodents can be knocked out or silenced using a variety of technologies known in the art.

[0291] Multi-specific antibodies, including their functional fragments, can also be produced by recombinant DNA technology through expression of the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (e.g., CHO cells), E. coli or yeast. Methods of recombinant DNA technology may also include co-expression of all the chains making up the protein in a single recombinant host cell, or co-expression of a heavy chain polypeptide and an antibody, e.g., a human antibody. Recombinant DNA methods are described, for example, in U.S. Patent No.4,816,567, the disclosure of which is incorporated by reference herein in its entirety.

[0292] Antibodies binding to non-overlapping epitopes on a target protein (e.g., CD28 or Nectin-4) can be identified by competition binding assays, such as enzyme-linked immunoassays (ELISA assays) or flow cytometric competitive binding assays. For example, one can use competition between known antibodies binding to the target antigen and the antibody of interest. By using this approach, one can divide a set of antibodies into those that compete with the reference antibody and those that do not. The non-competing antibodies are identified as binding to a distinct epitope that does not overlap with the epitope bound by the reference antibody. Often, one antibody is immobilized, the antigen is bound, and a second, labeled (e.g., biotinylated) antibody is tested in an ELISA assay for ability to bind the captured antigen. This can be performed also by using surface plasmon resonance (SPR) platforms, including PROTEONTMXPR36 (BioRad, Inc), BIACORE® 2000 and BIACORE® T200 (GE Healthcare Life Sciences), and MX96 SPR imager (Ibis technologies B.V.), as well as on biolayer interferometry platforms, such as OCTET® Red384 and OCTET® HTX (ForteBio, Pall Inc). For further details, see the examples herein.

[0293] Typically, an antibody “competes” with a reference antibody if it causes about 15-100% reduction in the binding of the reference antibody to the target antigen, as determined by standard techniques, such as by the competition binding assays described above. In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or higher.Attorney Ref.62571.00009WO01 (RND-0002-WO) Pharmaceutical Compositions, Uses and Methods of Treatment

[0294] It is another aspect of the present disclosure to provide pharmaceutical compositions comprising one or more antibodies of the present disclosure in admixture with a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers as used herein are exemplified, but not limited to, adjuvants, solid carriers, water, buffers, or other carriers used in the art to hold therapeutic components, or combinations thereof.

[0295] In one embodiment, a pharmaceutical composition comprises an antibody (e.g., a monospecific or multi-specific antibody) that comprises a first binding unit that binds to CD28. In another embodiment, a pharmaceutical composition comprises a multi-specific antibody (e.g., a bispecific antibody) that binds to two or more non-overlapping epitopes on CD28. In another embodiment, a pharmaceutical composition comprises a multi-specific antibody (e.g., a bispecific antibody) that comprises a first binding unit that binds to an CD28 and a second binding unit that binds to a DAA (e.g., Nectin-4).

[0296] In some embodiments, a pharmaceutical composition comprises an antibody (e.g., a monospecific or multi-specific antibody) that comprises a first binding unit that binds Nectin-4. In another embodiment, a pharmaceutical composition comprises a multi-specific antibody (e.g., a bispecific antibody) that binds to two or more non-overlapping epitopes on Nectin-4. In some embodiments, a pharmaceutical composition comprises a multi-specific antibody (e.g., a bispecific antibody) that comprises a first binding unit that binds to Nectin-4 and a second binding unit that binds to an EAA (e.g., CD28).

[0297] Pharmaceutical compositions of the antibodies used in accordance with the present disclosure are prepared for storage by mixing proteins having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (see, e.g. Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), such as in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt- forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0298] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). The formulation depends on the route of administration chosen. The antibodies herein can be administered by intravenous injection or infusion or subcutaneously. For injection administration, the antibodies herein can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection. The solution can contain carriers, excipients, or stabilizers as discussed above. Alternatively, antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0299] Antibody formulations are disclosed, for example, in U.S. Patent No. 9,034,324. Similar formulations can be used for the antibodies of the present disclosure. Subcutaneous antibody formulations are described, for example, in US20160355591 and US20160166689. Methods of Use – Monotherapies and Combination Therapies

[0300] Aspects of the present disclosure involve the treatment of diseases and conditions using the antibodies and pharmaceutical compositions described herein, either as monotherapies or as combination therapies.

[0301] In some embodiments, the methods involve monotherapies, such as administering a multi- specific antibody or pharmaceutical composition as described herein, comprising a first binding unit that binds to CD28. In some embodiments, the methods involve monotherapies, such as administering a multi-specific antibody (e.g., a bispecific antibody) or pharmaceutical composition as described herein, comprising a first binding unit that binds to CD28 and a second binding unit that binds to a DAA (e.g., Nectin-4).

[0302] In some embodiments, the methods involve monotherapies, such as administering a multi- specific antibody or pharmaceutical composition as described herein, comprising a first binding unit that binds to Nectin-4. In some embodiments, the methods involve monotherapies, such as administering a multi-specific antibody (e.g., a bispecific antibody) or pharmaceutical composition as described herein, comprising a first binding unit that binds Nectin-4 and a second binding unit that binds to an EAA (e.g., CD28).

[0303] In some embodiments, the methods involve combination therapies, which can involve administering one of the antibodies or pharmaceutical compositions described herein as a Signal 2 molecule, to provide Signal 2 activation in addition to a Signal 1 activating signal. In some embodiments, the Signal 2 molecule can amplify the activating signal (Signal 1) provided to effector cells (e.g., T- cells) in response to interaction with a target cell. In some embodiments, the methods involve administering a Signal 2 molecule as described herein and also administering a cosignaling molecule to modulate the Signal 2. In some embodiments, the cosignaling molecule can be costimulatory in orderAttorney Ref.62571.00009WO01 (RND-0002-WO) to amplify the Signal 2 signal. In some embodiments, the cosignaling molecule may be coinhibitory in order to decrease the Signal 2 signal.

[0304] In some embodiments, a combination therapy method involves administering a Signal 1 molecule first, such as, for example, administering a first multi-specific antibody in order to trigger cytotoxicity of a T-cell towards a target cell (i.e., Signal 1 activation), followed by administering a second multi-specific antibody in order to trigger further cytotoxicity of a T-cell towards a target cell (i.e., Signal 2 activation). Signal 2 activation can be necessary in instances where Signal 1 activation alone is insufficient to eliminate target cells (e.g., tumor cells).

[0305] In some embodiments, a combination therapy method involves administering a Signal 1 molecule first, such as, for example, administering a first multi-specific antibody in order to trigger cytotoxicity of a T-cell towards a target cell (i.e., Signal 1 activation), followed by administering a second multi-specific antibody in order to trigger further cytotoxicity of a T-cell towards a target cell (i.e., Signal 2 activation), and then administering a cosignaling molecule to modulate the Signal 2 signal by further increasing cytotoxicity of the T-cell towards the target cell (e.g., costimulation). Costimulation of Signal 2 activation using a cosignaling molecule can be necessary in instances where the provided Signal 1 and Signal 2 activation signals alone are insufficient to eliminate target cells (e.g., tumor cells).

[0306] In some embodiments, a combination therapy method involves administering a Signal 1 molecule first, such as, for example, administering a first multi-specific antibody in order to trigger cytotoxicity of a T-cell towards a target cell (i.e., Signal 1 activation), followed by administering a second multi-specific antibody in order to trigger further cytotoxicity of a T-cell towards a target cell (i.e., Signal 2 activation), and then administering a cosignaling molecule to modulate the Signal 2 signal by decreasing cytotoxicity of the T-cell towards the target cell (e.g., coinhibition). Coinhibition of Signal 2 activation using a cosignaling molecule can be necessary in instances where Signal 1 activation alone is insufficient to eliminate target cells (e.g., tumor cells), but Signal 2 activation causes excess cytotoxicity of the T-cell towards the target cell.

[0307] Non-limiting examples of a Signal 1 molecule triggering Signal 1 activation can include, for example, a multi-specific antibody that binds to an EAA on an effector cell and a DAA on a target cell. In some embodiments, the EAA that the Signal 1 multi-specific antibody binds to is a CD3 protein. In some embodiments, the EAA that the Signal 1 multi-specific antibody binds to is a peptide MHC complex. In some embodiments, the DAA that the Signal 1 multi-specific antibody binds to is a TAA or a TSA. In some embodiments, the TAA that the Signal 1 multi-specific antibody binds to is Nectin- 4. In some embodiments, the multi-specific antibody can include, for example without limitation, a multi-specific antibody as described herein (e.g., a multi-specific antibody that contains a first binding unit that binds to Nectin-4 and a second binding unit that binds to an EAA (e.g., CD3). Additional non- limiting examples of Signal 1 activation antibodies include those described in WO2018052503 and WO2021222578, both of which are hereby incorporated by reference in their entirety.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0308] Non-limiting examples of a Signal 2 molecule triggering Signal 2 activation can include, for example, a multi-specific antibody as described herein (e.g., a multi-specific antibody that contains a first binding unit that binds to CD28 and a second binding unit that binds to a DAA (e.g., Nectin-4) on a target cell.

[0309] In some embodiments, combination therapies may include administration of an additional therapeutic agent before, after, or concurrently with administration of an antibody or pharmaceutical composition described herein. In some embodiments, combination therapies may include administration of an additional therapeutic agent before, after, or concurrently with administration of an antibody or pharmaceutical composition described herein as a Signal 2 molecule. In some embodiments, combination therapies may include administration of an additional therapeutic agent before, after, or concurrently with administration of an antibody or pharmaceutical composition described herein as a Signal 2 molecule and a cosignaling molecule.

[0310] In some embodiments, the methods comprise administering a checkpoint inhibitor antibody as an additional therapeutic agent. In some embodiments, the methods comprise administering an antibody-drug conjugate (ADC) as an additional therapeutic agent. In one or more embodiments, the methods comprise administering a chemotherapeutic drug as an additional therapeutic agent. In further embodiments, the methods comprise administering an oncolytic virus as an additional therapeutic agent.

[0311] Aspects of the present disclosure include using the monotherapies and / or combination therapies as described above in the treatment of various diseases and disorders.

[0312] Aspects of the present disclosure include treatment of disorders, including, without limitation, cancers, such as solid tumor cancers and liquid tumor cancers. Non-limiting examples of cancers include carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), skin cancer, melanoma, lung cancer, including small-cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, ovarian cancer (e.g., high grade serous ovarian carcinoma), liver cancer (e.g., hepatocellular carcinoma (HCC)), bladder cancer (e.g., urothelial bladder cancer), testicular (germ cell tumor) cancer, hepatoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer (e.g., renal cell carcinoma, nephroblastoma or Wilms' tumor), prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. Additional examples of cancer include, without limitation, retinoblastoma, thecomas, arrhenoblastomas, hepatoma, hematologic malignancies including non- Hodgkin's lymphoma (NHL), multiple myeloma and acute hematologic malignancies, endometrial or uterine carcinoma, endometriosis, fibrosarcomas, choriocarcinoma, vulval cancer, thyroid cancer, esophageal carcinomas, nasopharyngeal carcinoma, laryngeal carcinomas, Kaposi's sarcoma,Attorney Ref.62571.00009WO01 (RND-0002-WO) melanoma, skin carcinomas, Schwannoma, oligodendroglioma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, and urinary tract carcinomas. Further examples of cancer include, without limitation, metastatic cancer, which refers to the state of cancer where the cancer cells of a tissue of origin are transmitted from the original site to one or more sites elsewhere in the body, by the blood vessels or lymphatics, to form one or more secondary tumors in one or more organs besides the tissue of origin. Non-limiting examples of metastatic cancer include metastatic breast cancer.

[0313] Aspects of the present disclosure include treatment of disorders characterized by expression of Nectin-4. The treatment methods may comprise treatment of disorders characterized by expression of Nectin-4, including, without limitation, cancers, such as solid tumor cancers, including bladder cancer, breast cancer, kidney cancer, pancreatic cancer, penile cancer, prostate cancer, lung cancer, head and neck cancer, gastric cancer, esophageal cancer, genitourinary cancer, ovarian cancer, and endometrial cancer.

[0314] In one embodiment, the multi-specific anti-CD28 x anti-Nectin-4 antibodies and pharmaceutical compositions described herein can be used to treat disorders characterized by the expression of Nectin-4, including, without limitation, the diseases and disorders described herein.

[0315] Nectin-4, also known as nectin cell adhesion molecule 4 (UniProt KB No. Q96NY8). Nectin-4 is a tumor-associated antigen with a molecular weight of 55.5 kDa. Nectin-4 may be expressed by various types of cancer cells, such as for example, bladder cancer cells, breast cancer cells, and pancreatic cancer cells. Associated with low survival rates in patients, it has been designated as a cancer- specific biomarker, as Nectin-4 expression by non-cancerous, normal human cells is very limited.

[0316] Effective doses of the compositions of the present disclosure for the treatment of disease vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but nonhuman mammals may also be treated, e.g., companion animals such as dogs, cats, horses, etc., laboratory mammals such as rabbits, mice, rats, etc., and the like. Treatment dosages can be titrated to optimize safety and efficacy.

[0317] Dosage levels can be readily determined by the ordinarily skilled clinician, and can be modified as required, e.g., as required to modify a subject's response to therapy. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient.

[0318] In some embodiments, the therapeutic dosage the agent may range from about 0.0001 to 100 mg / kg, and more usually 0.01 to 5 mg / kg, of the host body weight. For example, dosages can be 1 mg / kg body weight or 10 mg / kg body weight or within the range of 1-10 mg / kg. An exemplary treatment regime entails administration once every two weeks or once a month or once every 3 to 6 months. Therapeutic entities of the present disclosure are usually administered on multiple occasions.Attorney Ref.62571.00009WO01 (RND-0002-WO) Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the therapeutic entity in the patient. Alternatively, therapeutic entities of the present disclosure can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0319] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The pharmaceutical compositions herein are suitable for intravenous or subcutaneous administration, directly or after reconstitution of solid (e.g., lyophilized) compositions. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of the present disclosure can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0320] Toxicity of the antibodies and antibody structures described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in humans. The dosage of the antibodies described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0321] The compositions for administration will commonly comprise an antibody or other ablative agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs (e.g.,Attorney Ref.62571.00009WO01 (RND-0002-WO) Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0322] Also within the scope of the present disclosure are kits comprising the active agents and formulations thereof, of the present disclosure and instructions for use. The kit can further contain at least one additional reagent, e.g. a chemotherapeutic drug, etc. Kits typically include a label indicating the intended use of the contents of the kit. The term “label” as used herein includes any writing, or recorded material supplied on or with a kit, or which otherwise accompanies a kit.

[0323] The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. III. Examples Example 1: Mechanism of action between an immune effector cell and a target cell

[0324] Activation on an immune effector cell (e.g., a T-cell) can trigger cytotoxicity against a target cell of interest, such as a tumor cell. One mechanism for activating cytotoxicity of an immune effector cell is by crosslinking the immune effector cell to a target cell with a multispecific antibody. FIG. 1, Panels A and B, provide two examples of this process.

[0325] FIG.1, Panel A, shows a T-cell as an example of an immune effector cell, and a tumor cell as an example of a target cell.

[0326] In the upper portion of Panel A, a CD3xTAA-1 (Tumor associated antigen 1) bispecific antibody is shown crosslinking a CD3 protein on the surface of the T-cell with a TAA-1 protein on the surface of the tumor cell. This interaction can provide a signal that, if sufficient, results in triggering cytotoxicity of the T-cell towards the tumor cell (referred to in the figure as “Signal 1 activation”). CD3 is provided as an example of an effector associated antigen (EAA), and TAA-1 is provided as an example of a disease associated antigen (DAA).

[0327] In the lower portion of Panel A, a CD28xTAA-2 (Tumor associated antigen 2) bispecific antibody is shown crosslinking a CD28 protein on the surface of the T-cell with a TAA-2 protein on the surface of the tumor cell. This interaction can provide an additional signal that, again, if sufficient, can result in triggering cytotoxicity of the T-cell towards the tumor cell (referred to in the figure as “Signal 2 activation”). CD28 is provided as an example of an effector associated antigen (EAA), and TAA-2 is provided as an example of a disease associated antigen (DAA).

[0328] FIG.1, Panel B, shows a T-cell as an example of an immune effector cell, and a tumor cell as an example of a target cell.

[0329] In the upper portion of Panel B, a T-cell receptor (TCR) is shown recognizing, or binding to, its cognate peptide major histocompatibility complex (pMHC), present on the surface of the tumor cell. This interaction can provide a signal that, if sufficient, results in triggering cytotoxicity of the T-cell towards the tumor cell (referred to in the figure as “Signal 1 activation”). Here, the T-cell receptorAttorney Ref.62571.00009WO01 (RND-0002-WO) (TCR) is provided as an example of an effector associated antigen (EAA), and the pMHC is provided as an example of a disease associated antigen (DAA).

[0330] In the lower portion of Panel B, a CD28xTAA-2 (Tumor associated antigen 2) bispecific antibody is shown crosslinking a CD28 protein on the surface of the T-cell with a TAA-2 protein on the surface of the tumor cell. This interaction can provide an additional signal that, again, if sufficient, can result in triggering cytotoxicity of the T-cell towards the tumor cell (referred to in the figure as “Signal 2 activation”). CD28 is provided as an example of an effector associated antigen (EAA), and TAA-2 is provided as an example of a disease associated antigen (DAA).

[0331] In some cases, Signal 1 alone is insufficient to eliminate target cells, such as tumor cells. In such cases, the addition of Signal 2 can increase the activity of the immune effector cell. As shown in FIG. 1, adding a second antibody, such as a CD28 x TAA bispecific antibody, can enhance T-cell activity in the presence of Signal 1, thereby improving T-cell activation and function, and leading to higher levels of T-cell-mediated cytotoxicity toward the tumor cells. Example 2: Linking KiH mutant Fc with an extra disulfide bond in the CH3 domain increases thermal stability of bispecific antibodies

[0332] Melting (Tm) and aggregation (Tagg) behavior of identical bispecific antibodies, with and without an extra disulfide bond in the Fc domain, were investigated. Two bispecific antibodies, named R001 and R003, were purified with additional disulfide bonds in their Fc domains. These antibodies contained mutated residues S354C and Y349C, in accordance with the teachings of Zhu et. al.; Nat Biotechnol. 1998 Jul;16(7):677-81. Melting temperatures (Tm) as well as onset-of-aggregation temperatures (Tagg) were measured using the Uncle protein stability screening platform from Unchained Labs (California, USA). Direct comparison to the same antibodies lacking the additional disulfide bonds (R002, R004) revealed that melting- as well as onset-temperatures for aggregation were significantly increased for the versions of these antibodies containing the extra disulfide bonds in their Fc domains. The results are provided in FIG. 2, and demonstrate that in each case, the version of the antibody with the additional disulfide bonds had a higher melting temperature and higher onset-of- aggregation temperature. Tm1 indicates the melting temperature of the VH region, whereas Tm2 indicates the melting temperature of the Fc region.

[0333] R001 is identical to R002, but R001 contains the S354C and Y349C Fc mutations that form the extra disulfide bonds, while R002 does not contain these mutations. R003 is identical to R004, but R003 contains the S354C and Y349C Fc mutations that form the extra disulfide bonds, while R004 does not contain these mutations. Example 3: Impact of framework sequence mutations on CD28 protein binding

[0334] Binding kinetics of the indicated monospecific anti-CD28 antibodies were assessed using OCTET® bio-layer interferometry (BLI) by loading the respective antibodies at a concentration of 2.5Attorney Ref.62571.00009WO01 (RND-0002-WO) μg / mL onto anti-hIgG Fc capture (AHC) sensors and associating to dimeric human CD28 extracellular domain (ECD) antigen (which is identical between humans and cynomolgus monkeys), with a 7-point, 2-fold dilution series starting at 300 nM.

[0335] The results for antibodies R042 and R173 binding to human CD28 are shown in FIG. 3A. Antibodies R042 and R173 contain the same CDR sequences, but differ in their framework region sequences. The results for antibodies R009 and R013 binding to human CD28 are shown in FIG.3B. Antibodies R009 and R013 contain the same CDR sequences, but differ in their framework region sequences. The results for antibodies R174 and R175 binding to human CD28 are shown in FIG.3C. Antibodies R174 and R175 contain the same CDR sequences, but differ in their framework region sequences.

[0336] KD, Ka and Kdis for the indicated antibodies are shown in the table in FIG. 3D. These data show that the indicated anti-CD28 antibodies bind with similar affinity to human CD28 protein. Example 4: Impact of framework sequence mutations on Nectin-4 protein binding

[0337] Binding kinetics of the indicated anti-Nectin-4 antibodies were assessed using OCTET® bio- layer interferometry (BLI) by loading the respective antibodies at a concentration of 2.5 μg / mL onto anti-hIgG Fc capture (AHC) sensors and associating to Nectin-4 cynomolgus and human antigen with a 7-point, 2-fold dilution series starting at 100 nM. The results for antibodies R011 and R015 binding to human Nectin-4 are shown in FIG. 4A. The results for antibodies R011 and R015 binding to cynomolgus Nectin-4 are shown in FIG. 4B. Antibodies R011 and R015 contain the same CDR sequences, but differ in their framework region sequences. KD, Ka and Kdis for the indicated antibodies are shown in the table in FIG. 4C. These data show that the indicated antibodies bind with similar affinity to both human and cyno Nectin-4 protein, and that the affinity is not impacted by framework sequence mutations. Example 5: CD28 protein binding affinity comparison

[0338] CD28 binding kinetics of the indicated antibodies were assessed using OCTET® bio-layer interferometry (BLI) by loading CD28 dimeric antigen onto streptavidin (SA) sensors at a concentration of 2.5 μg / mL and associating with the indicated bispecific antibodies, using a 7-point, 2-fold dilution series starting at 300 nM. Kinetic graphs were fitted with a 1:1 global binding model. The dissociation fit was reduced to 120s to allow a better R2 fit and lower RSS value.

[0339] The results for antibodies R005-R007 are shown in FIG.5A. The results for antibodies R019- R021 are shown in FIG.5B. KD, Ka and Kdis are shown in the table in FIG.5C.

[0340] These results demonstrate that R005, R006 and R007 exhibit CD28 binding kinetics that are unique compared to each other as well as in comparison to the three CD28 benchmark molecules, R019, R020 and R021. In the case of R006, the calculated affinity is similar to the benchmark R020; however,Attorney Ref.62571.00009WO01 (RND-0002-WO) both the ka and kdis are approximately two-fold faster, suggesting that this antibody may exhibit different activity in the context of an in vitro or in vivo system. Example 6: Cell binding of bispecific antibodies to CD28+ and Nectin-4+ cell lines

[0341] Cell binding of the listed antibodies was assessed using the CD28-positive Jurkat cell line, the Nectin-4-positive T47D cell line, and the CD28-negative / Nectin-4-low TCCSUP cell line. Binding studies were conducted in triplicate using the indicated antibodies, titrated from 4 μg / mL. Goat anti- human AF488 labeled secondary antibody was used for detection of primary antibody binding. Binding was analyzed by flow cytometry and plotted as the mean fluorescence intensity (MFI) vs. antibody concentration. The results are shown in FIG.6, Panels A, B, and C. Cell binding EC50 values for the indicated antibodies and cell lines are summarized in the table in FIG. 6, Panel D. Panel A shows the expected range of cell binding on CD28+ Jurkat cells, which correlate with the CD28 protein affinities reported in example 5. From this data it is clear that R006 binds distinctly from the three CD28 benchmarks, R016, R017 and R018. Panel B shows the expected binding pattern to Nectin- 4+ T47D cells, since each bispecific antibody contains the same Nectin-4 binding arm the binding curves are overlapping. Panel C shows no appreciable binding to the TCCSUP cell line which does not express CD28 and is extremely low for Nectin-4 expression (90 copies per cell + / - 130 as shown in FIG. 8). Example 7: CD28 x Nectin-4 bispecific antibodies show specific target binding by ELISA

[0342] Binding of the indicated bispecific antibodies to the indicated recombinant protein targets was assessed via enzyme linked immunoabsorbant assay (ELISA). Target antigens were coated at 2 µg / mL in coating buffer onto 384-well plates and incubated at 4°C overnight. The next day, plates were washed, followed by a blocking step, followed by washing and incubation with serially diluted test articles (starting at 16 µg / mL for 12-point dilution in 4-fold series in replicates of 3). Bound antibodies were detected using a secondary anti-huIgG-Fc-HRP antibody, followed by addition of TMB substrate. The reaction was stopped by addition of HCl and absorbance at 450 nm was measured using a plate reader. Specific binding was observed against human and cynomolgus target proteins CD28 and Nectin-4. No binding was observed to off target proteins: Nectin-1, Nectin-3, CTLA4 or human serum albumin. The graphical results are shown in FIG.7A. FIG.7B shows the EC50 results in a tabular format. Example 8: Nectin-4 antigen density on tumor cell lines, primary cells and dissociated tumor cells

[0343] Nectin-4 antigen density was assessed on human tumor cell lines (sourced from ATCC), primary human cells and dissociated tumor cells (DTCs) using a QUANTIBRITETMPE quantitation kit (BD Biosciences) according to the manufacturer’s protocol. Primary human keratinocytes were tested pre- and post-differentiation using the differentiation protocol described in Xie et al., 2005 Mol BiolAttorney Ref.62571.00009WO01 (RND-0002-WO) Cell.2005 Jul; 16(7): 3236–3246. The results are shown in FIG.8. These results show that the tumor cell lines HT-1197 and SCaBER express similar levels of Nectin-4 as differentiated primary keratinocytes, the tumor cell lines TCCSUP and 5637 express similar levels of Nectin-4 as primary keratinocytes and the tumor cell lines RT-112 and RT4 express similar Nectin-4 levels as dissociated tumor cells from patient bladder tumors. Example 9: Assessment of CD28 super-agonist activity

[0344] The indicated antibodies were tested for CD28 super-agonist activity by wet coating using the methods described by Eastwood, D. et al., Br J Pharmacol.2010 Oct; 161(3):512-26 and Stebbings, R. et al., J Immunotoxicol.2013 Jan-Mar; 10(1):75-82. Briefly, antibodies were diluted to 8 μg / mL in PBS and coated for 2 hours at 37°C in wells of untreated polystyrene cell culture plates. Excess antibody was then removed and wells were washed with PBS. PBMCs from two healthy human donors were added to antibody-coated wells and incubated for 3 days in complete media. Interferon gamma production and T cell proliferation were analyzed at 24 hours and 72 hours, respectively. The results are shown in FIG.9. Individual data points were plotted with a horizontal line indicating the mean of each group. A surrogate for the known super-agonist antibody, TGN1412, showed T-cell proliferation and interferon gamma production, while antibodies R005, R006 and R007, and a negative control non- CD28 targeting antibody (R015) did not. CD28 super-agonist activity is a known risk of CD28 engagers and can cause serious side effects in humans, as evidenced by the theralizumab (TGN1412) clinical trial failure. See, e.g., Suntharalingam G, et al., N Engl J Med. 2006 Sep 7;355(10):1018-28. These data demonstrate that the anti-CD28 antibodies of the present disclosure do not exhibit super-agonist activity, as measured either by cytokine production (IFNγ) or T-cell proliferation. Example 10: Reduced cytokine production from lower affinity anti-CD28 antibodies

[0345] Cytokine production in response to effector cell activation was assessed using a variety of cell lines expressing various levels of target antigen.10,000 Nectin-4 positive, 5T4 positive cells (SCaBER, HT1197 and 5637) were combined with primary human pan T-cells at an effector : target cell ratio of 2:1. CD28 x Nectin-4 bispecific targeting antibodies (Signal 2) were titrated from 4 μg / mL + / - a CD3 x 5T4 bispecific antibody (R028) for delivery of Signal 1. A different concentration of the Signal 1 antibody was used for each cell line at EC10: 2ng / mL, 4ng / mL and 3ng / mL for SCaBER, HT1197 and 5637 respectively. IL-2 production was analyzed in assay supernatants after 24 hours of incubation. IL- 2 production stimulated from CD3 x 5T4 treatment alone was subtracted from all wells. The results are shown in FIG.10, and demonstrate that antibody R006 resulted in reduced cytokine production on cell lines that express Nectin-4 at levels comparable to normal, healthy tissues (see FIG.8 and Example 8), as compared to the results obtained with the comparator antibody R017. Antibody R007 showed reduced cytokine production as compared to the comparator antibody R018. Reduced cytokine production at levels of Nectin-4 expression expected to be found on healthy patient tissues may improveAttorney Ref.62571.00009WO01 (RND-0002-WO) patient safety in comparison to other CD28 engaging antibodies such as the clinical benchmark R017. Importantly, R006 can induce high levels of cytotoxicity of tumor cell lines expressing Nectin-4 levels similar to those found in patient tumors (for example FIG. 11, panel B, FIG. 14, panel A) as well as enhance cytokine production comparable to higher affinity CD28 x Nectin-4 bispecifics when target cell Nectin-4 expression is high (FIG. 13, panel A). In contrast, the benchmark R018 has reduced cytokine production as shown in FIG. 10 but also reduced efficacy as shown in FIG. 11, panel B. Therefore, it is possible that R006 has the ideal balance between safety on normal tissues and efficacy against cancerous tissues. Example 11: Analysis of cytotoxicity activity of anti-CD28 bispecifics in bladder cancer target cells

[0346] Bladder cancer target cells, RT112 (Nectin-4 positive, 5T4 positive), were combined at either a 5:1 or 1:1 E:T ratio with primary T-cells and incubated for six days with 2 ng / mL CD3 x 5T4 bispecific antibody (R028; Signal 1) and a titration of the indicated CD28 x Nectin-4 bispecific antibodies from 4 μg / mL. Cytotoxicity methods: Target cells were plated at a density of 10,000 cells per well in 96-well plates and allowed to attach overnight. The next day, human pan T-cells isolated from healthy donors were added to each well along with the indicated antibody treatments in a total volume of 200 μL. After incubation for 72 hours at 37C, 5% CO2, the plates were washed with PBS to remove the T-cells and cell titer glo was added to quantify the remaining live target cells. Percent target cell cytotoxicity was calculated by the formula 100*(1 - (untreated target cell wells / treated target cell wells)). The results are shown in FIG.11.

[0347] At a low E:T ratio (1:1), the lower affinity antibodies R018 and R007 did not show appreciable cytotoxicity activity. The E:T ratio of tumor-specific cytotoxic T cells expected within tumors is 1:1 or lower. As such, the data from FIG.11, Panel B are likely more representative of the tumor environment in vivo. These results demonstrate that antibody R006 has an intermediate CD28 potency between comparator antibodies R017 and R018. Under some conditions, such as a lower E:T ratio, antibody R006 outperforms antibody R018 in terms of Emax. Since tumors are generally thought to contain lower E:T ratios, antibody R006 demonstrates ideal properties for therapeutic use. Antibodies with lower CD28 potency may not provide sufficient Signal 2 to show clinical efficacy in patients. Example 12: Enhancement of cytotoxic activity of CD3x5T4 bispecific antibodies (Signal 1) at both high and low E:T ratios with the addition of CD28xNectin-4 bispecific antibodies (Signal 2)

[0348] A bispecific antibody targeting CD3 x 5T4 (Signal 1; R028) was titrated from 1 μg / mL on T47D cells (dual positive for Nectin-4 and 5T4) in the presence of primary human pan T-cells at a 5:1 or 1:1 effector : target cell ratio, + / - 1 μg / mL of antibody R005, R006 or R007. After incubation for 72 hours at 37C, 5% CO2, the plates were washed with PBS to remove the T-cells and cell titer glo was added to quantify the remaining live target cells. Percent target cell cytotoxicity was calculated by theAttorney Ref.62571.00009WO01 (RND-0002-WO) formula 100*(1 - (untreated target cell wells / treated target cell wells)). At a 5:1 E:T ratio, the addition of CD28xNectin-4 bispecific antibody increased the potency of R028 by approximately 4- to 8-fold, and slightly boosted the maximum cytotoxicity. At a 1:1 E:T ratio, the addition of the CD28 x Nectin- 4 bispecific antibodies boosted the cytotoxicity maximum from less than 10% to 50 – 70%. The results are provided in FIG.12, and demonstrate that a CD28 x Nectin-4 bispecific antibody (Signal 2) boosts activity of a CD3x5T4 bispecific antibody (Signal 1) at both a high and a low E:T ratio, where the CD3x5T4 antibody alone (no Signal 2) has a low Emax activity. Solid tumors within patients are expected to have E:T ratios of 1:1 or lower, therefore this data highlights the potential benefits of treatment with these CD28 bispecific antibodies as compared to treatment with a CD3 bispecific antibody. Example 13: CD28 bispecific antibodies enhance T-cell cytokine production in the presence of both Signal 1 and Nectin-4 tumor expression

[0349] 10,000 Nectin-4 positive, 5T4 positive T47D cells, or Nectin-4 negative, 5T4 positive UM-UC- 3 cells, were combined with primary human pan T-cells at an effector : target cell ratio of 5:1. CD28 x Nectin-4 bispecific targeting antibodies (Signal 2) or a CD28 x non-targeting negative control bispecific (R031) were titrated from either 4 μg / mL or 16 μg / mL + / - 15 ng / mL CD3 x 5T4 bispecific antibody (R028) for delivery of Signal 1. IL-2 production was analyzed in assay supernatants after 24 hours of incubation. IL-2 production stimulated from CD3 x 5T4 treatment alone was subtracted from all wells. The results are shown in FIG. 13, and demonstrate that the CD28 x Nectin-4 bispecific antibodies enhance cytokine production by T-cells only if Signal 1 is present (shown by FIG.13, Panel B; no IL- 2 production when Signal 1 is absent) and if the CD28 bispecific can crosslink T-cells and target cells (shown by non-targeting control R031 in FIG.13, Panel A and by the Nectin-4 negative cell line in FIG. 13, Panel C). These results are consistent with the mechanism of action of these bispecific CD28 x Nectin-4 targeting antibodies. Example 14: CD28 x Nectin-4 bispecific antibodies require Nectin-4 expression on target cells to enhance cytotoxicity

[0350] 10,000 Nectin-4 positive (T47D) or negative (UM-UC-3) target cells were incubated with primary human pan T-cells at an effector : target cell ratio of 5:1. Both T47D and UM-UC-3 cells express 5T4. CD28 x Nectin-4 bispecific targeting antibodies, or a CD28 x non-targeting negative control bispecific (R031) were titrated from 1 μg / mL.0.8 ng / mL CD3 x 5T4 bispecific antibody (R028) was added to all wells for delivery of Signal 1. After incubation for 72 hours at 37C, 5% CO2, the plates were washed with PBS to remove the T-cells and cell titer glo was added to quantify the remaining live target cells. Percent target cell cytotoxicity was calculated by the formula 100*(1 - (untreated target cell wells / treated target cell wells)). The results are provided in FIG.14, and demonstrate that target antigenAttorney Ref.62571.00009WO01 (RND-0002-WO) expression is required for cytotoxicity activity of CD28 x Nectin-4 bispecific antibodies. These results are consistent with the mechanism of action of these bispecific CD28 x Nectin-4 targeting antibodies. Example 15: CD28 x Nectin-4 bispecific antibodies require Signal 1 to induce cytotoxicity activity

[0351] Nectin-4 positive (T47D) or negative (UM-UC-3) target cell lines were combined with primary human pan T-cells at an effector : target cell ratio of 5:1. CD28 x Nectin-4 bispecific antibodies or a CD28 x non-targeting negative control bispecific antibody were titrated from 16 μg / mL. To assess cytotoxicity activity, target cells were plated at a density of 10,000 cells per well in 96-well plates and allowed to attach overnight. The next day, 50,000 human pan T-cells isolated from healthy donors were added to each well along with the indicated antibody treatments in a total volume of 200 μL. After incubation for 72 hours at 37C, 5% CO2, the plates were washed with PBS to remove the T-cells and cell titer glo was added to quantify the remaining live target cells. Percent target cell cytotoxicity was calculated by the formula 100*(1 - (untreated target cell wells / treated target cell wells)). The results are provided in FIG.15, and demonstrate that Signal 1 (activation of TCR / CD3) is required for cytotoxicity activity of CD28 x Nectin-4 bispecific antibodies, consistent with the intended mechanism of action of these bispecific antibodies. Example 16: CD28 bispecific antibodies enhance T-cell proliferation activity of a CD3 x 5T4 bispecific antibody

[0352] 10,000 Nectin-4 positive, 5T4 positive (T47D) target cells were incubated with primary human pan T-cells at an effector : target cell ratio of 1:4. A CD3 x 5T4 bispecific antibody was titrated from 1 μg / mL + / - addition of 1 μg / mL of antibody R005, R006 or R007. T-cell proliferation was analyzed after 5 days of incubation using flow cytometry to identify viable, CD3 positive events and results were reported as T-cells per μL. The results are shown in FIG.16, and demonstrate that the indicated CD28 x Nectin-4 bispecific antibodies enhance the activity of a CD3 x 5T4 bispecific antibody in regards to T-cell proliferation from two different T-cell donors. Under the conditions of this experiment, the CD3 x 5T4 antibody treatment alone showed virtually no T-cell proliferation, indicating that Signal 1 alone was insufficient to stimulate substantial T-cell proliferation. Solid tumors within patients are expected to contain E:T ratios of 1:1 or lower. These results highlight the potential of the described CD28 x Nectin-4 bispecific antibodies to show an anti-tumor benefit by enhancing T cell expansion under conditions of low E:T ratio as may be found in patients. Example 17: CD28 bispecific antibodies enhance T-cell activation in the presence of TCR stimulation

[0353] Nectin-4 positive (T47D) target cells were combined with primary human pan T-cells at an effector : target cell ratio of 5:1 and treated with 0.8 ng / mL of CD3 x 5T4 bispecific antibody (R028;Attorney Ref.62571.00009WO01 (RND-0002-WO) Signal 1). CD28 x Nectin-4 bispecific targeting antibodies (Signal 2) or a CD28 x non-targeting negative control bispecific (R031) were titrated from 4 μg / mL. After incubation for 72 hours, T-cells were collected for flow cytometry. T-cell activation was assessed by staining for CD25 expression. The percent CD25 positive population of CD3+ T-cells is reported vs. CD28 bispecific antibody concentration. The results are shown in FIG. 17, and demonstrate that CD28 x Nectin-4 bispecific antibodies enhance expression of the T-cell surface activation marker CD25. A control containing a CD28 targeting arm but lacking the Nectin-4 targeting arm showed a minimal impact on CD25 expression. These results are consistent with the mechanism of action of the CD28 x Nectin-4 bispecific antibodies. Example 18: CD28 bispecific antibodies show similar cytotoxicity activity whether in an IgG4 or IgG1 format

[0354] CD28 x Nectin-4 bispecific antibodies with the same variable domains expressed with either IgG1 or IgG4 Fcs were tested in a cytotoxicity assay against the Nectin-4 positive cell line T47D. R005 and R032 contain identical variable domains and utilize either an IgG4 or IgG1 Fc, while R006 and R029 contain identical variable domains and utilize either an IgG4 or IgG1 Fc. In the presence of 0.8 ng / mL CD3 x 5T4 bispecific antibody (R028) to deliver Signal 1, both IgG1 and IgG4 bispecific antibodies with the same VH regions showed similar activity. These results are shown in FIG.18 and demonstrate that similar cytotoxicity activity was observed with IgG1 and IgG4 Fc region sequences.

[0355] To assess cytotoxicity activity, target cells were plated at a density of 10,000 cells per well in 96-well plates and allowed to attach overnight. The next day, 50,000 human pan T-cells isolated from healthy donors were added to each well along with the indicated antibody treatments in a total volume of 200 µL. After incubation for 72 hours at 37C, 5% CO2, the plates were washed with PBS to remove the T-cells and cell titer glo was added to quantify the remaining live target cells. Percent target cell cytotoxicity was calculated by the formula 100*(1 - (untreated target cell wells / treated target cell wells)). Example 19: Functional activity of CD28 bispecific antibodies containing 5T4-targeting binding units

[0356] CD28 x 5T4 bispecific antibodies were generated and tested in a cytotoxicity assay against the 5T4-positive target cell line T47D. In the presence of 0.8 ng / mL CD3 x 5T4 bispecific antibody (R028) to deliver Signal 1, enhancement of cytotoxicity was observed for antibodies R024, R023 and R022. These results are shown in FIG. 19. R024, R023 and R022 contain the same CD28 targeting arms as R007, R005 and R006 respectively and a different DAA binding unit targeting 5T4 instead of Nectin- 4. The relative activity of R024, R023 and R022 correlates with that observed by R007, R005 and R006, indicating the CD28 targeting arms behave similarly when paired with different DAA targeting arm.Attorney Ref.62571.00009WO01 (RND-0002-WO) Example 20: Functional activity of CD28 bispecific antibodies containing FRα-targeting

[0357] CD28 x FRα bispecific antibodies were generated and tested in a cytotoxicity assay against the FRa-positive target cell line T47D. In the presence of 0.8 ng / mL CD3 x 5T4 bispecific antibody (R028) to deliver Signal 1, a range of cytotoxicity enhancement was observed. These results are shown in FIG. 20. R026, R025 and R027 contain the same CD28 targeting arms as R005, R006 and R007 respectively and a different DAA binding unit targeting Folate receptor alpha (FRα) instead of Nectin-4. The relative activity of R025, R026 and R027 correlates with that observed by R005, R006 and R007, indicating the CD28 targeting arms behave similarly when paired with different DAA targeting arms. Consistent with its activity when used as a bispecific antibody targeting Nectin-4, the CD28 binding domain of R025 achieves similar maximum levels of cytotoxicity as R026, which contains a higher affinity CD28 binding domain.

[0358] To assess cytotoxicity activity, target cells were plated at a density of 10,000 cells per well in 96-well plates and allowed to attach overnight. The next day, 50,000 human pan T-cells isolated from healthy donors were added to each well along with the indicated antibody treatments in a total volume of 200 µL. After incubation for 72 hours at 37C, 5% CO2, the plates were washed with PBS to remove the T-cells and cell titer glo was added to quantify the remaining live target cells. Percent target cell cytotoxicity was calculated by the formula 100*(1 - (untreated target cell wells / treated target cell wells)). Example 21: Cytoxicity activity of a CD3xNectin-4 bispecific antibody

[0359] A bispecific antibody (R154) was constructed using a high affinity anti-CD3 binding domain and a Nectin-4 binding domain from the monoclonal antibody R015. In a cytotoxicity assay with the bladder cancer cell line RT4, this antibody induced dose dependent cytotoxicity of the tumor cells, indicating that antibody was able to activate and T-cells and direct them to lyse the Nectin-4 positive target cells.

[0360] Cytotoxicity methods: Target cells were plated at a density of 10,000 cells per well in 96-well plates and allowed to attach overnight. The next day, 100,000 human pan T-cells isolated from healthy donors were added to each well along with the indicated antibody treatment in a total volume of 200 µL. After incubation for 72 hours at 37C, 5% CO2, the plates were washed with PBS to remove the T-cells and cell titer glo was added to quantify the remaining live target cells. Percent target cell cytotoxicity was calculated by the formula 100*(1 - (untreated target cell wells / treated target cell wells)). The results are shown in FIG.21. Example 22: Binding data for an anti-Nectin-4 antibody

[0361] Binding kinetics of an anti-Nectin-4 antibody were assessed on BLI by loading the antibody at a concentration of 2.5 μg / mL onto AHC sensors and associating to Nectin-4 cynomolgus and human antigen with a 7-point, 2-fold dilution series starting at 100 nM. KD, Ka and Kdis areAttorney Ref.62571.00009WO01 (RND-0002-WO) shown in the table in FIG.22A. The binding traces for antibody R153 are also shown in FIG.22A, and demonstrate that R153 binds tightly to human and cynomolgus Nectin-4 protein.

[0362] Epitope binning studies show that antibody R011 binds to a different epitope on human Nectin-4 as compared to the benchmark molecules R151 and R152 because they exhibit different patterns of competition with a third anti-Nectin-4 antibody, R153. These results are summarized in the table shown in FIG.22B.

[0363] Epitope binning on human Nectin-4 was assessed for a panel of anti-Nectin-4 antibodies by BLI. All samples were diluted in kinetics buffer made of 1x PBS, 300 mM NaCl, 5 mM Imidazole and 0.02% Tween20, pH7.4. Recombinant human Nectin-4 protein was loaded on Ni-NTA sensors at a concentration of 1 µg / mL for 180s to Ni-NTA sensor tips, followed by incubation with 5-fold molar excess of antibody 1 for 300s, followed by 30s baseline step and 200s soaking in wells with antibody 2, at 2.5-fold molar excess of antigen. Sensors are regenerated with 10mM Glycine pH 1.5, neutralized in kinetics buffer and recoated with 10mM Ni-Sulfate.

[0364] R151 is an anti-Nectin-4 benchmark antibody currently used clinically in an ADC format. R152 and R153 are also anti-Nectin-4 benchmark antibodies. Example 23: Binding affinity of anti-Nectin-4 antibodies in comparison to benchmark molecules

[0365] Three anti-Nectin-4 antibodies, including one benchmark antibody, were evaluated to determine their binding kinetics on BLI by loading the antibodies diluted in kinetics buffer (1x PBS, 0.1% BSA, 0.02% Tween20, pH 7.4) at a concentration of 2.5 μg / mL onto AHC sensors. A loading threshold was set to a 0.4 nm shift. Antigen association was performed for 180s to Nectin-4 mouse, cynomolgus and human antigen with a 7-point, 2-fold dilution series starting at 100 nM (human and cyno) and 1000nM (mouse). Dissociation was analyzed for 400s in kinetics buffer. Binding data for antibody R151 are shown in FIG.23A. Binding data for antibody R015 are shown in FIG.23B. Binding data for antibody R152 are shown in FIG.23C. KD, Ka and Kdis are shown in the table provided in FIG.23D.

[0366] These BLI binding data demonstrate that antibody R015 has higher affinity to human Nectin- 4 than the benchmark antibody R151. The binding affinity of antibody R015 is similar to the benchmark antibody R152; however, antibody competition studies indicate it is targeting a different epitope than R152 (see FIG.22B showing epitope binning). A higher Nectin-4 affinity is advantageous for use in an EAA x DAA bispecific antibody format because it can lead to improved targeting of the tumor. A high affinity tumor binding domain can increase accumulation of the antibody in the tumor as opposed to binding on all peripheral immune cells.Attorney Ref.62571.00009WO01 (RND-0002-WO) Example 24: Bispecific CD28x5T4 and CD28xFRα antibodies show tumor target specific cell binding

[0367] Cell binding studies were performed using CD28x5T4 and CD28xFRα bispecific antibodies and 5T4- and FRα-positive cell lines. FIG.24A shows the results of bispecific CD28x5T4 antibodies R024, R022 and R023 binding to the 5T4-positive target cell line T47D. As shown, all antibodies showed 5T4 specific cell binding.

[0368] FIG.24B shows the results of bispecific CD28x FRα antibodies R025, R026 and R027 binding to the FRα -positive target cell line T47D. As shown, all three antibodies showed FRα specific cell binding.

[0369] Cell binding on the TAA-positive T47D cell line was conducted in triplicate with the indicated antibodies titrated from 16 μg / mL. Goat anti-human AF488 labeled secondary antibody was used for detection of primary antibody binding. Binding was analyzed on a flow cytometer and plotted as the mean fluorescence intensity (MFI) vs. antibody concentration. An IgG4 isotype control antibody was used as a negative control. Example 25: Protein affinities of benchmark antibodies

[0370] Protein affinity of R020 to human 5T4 was measured as single digit nM by BLI. Methods: Binding kinetics were assessed by BLI by loading the antibody at a concentration of 2 µg / mL onto AHC sensors and associating to 5T4 human antigen with a 7-point, 2-fold dilution series starting at 250nM.

[0371] Protein affinity of R155 to human FOLR1 was measured as single digit nM by BLI. The antibody was loaded onto AHC sensors at a concentration of 2.5 µg / mL. FOLR1 human protein were prepared in a 7 point 2-fold dilution series with a top concentration of 1000nM.

[0372] Protein affinity of R176 and R177 to human CD3 delta epsilon (CD3δε) protein was measured as single digit nM by BLI.7-point 2-fold dilution series of the antibodies were generated, with a top concentration of 100nM. CD3δε antigen was loaded at a concentration of 5µg / mL onto Ni- NTA sensors for 120s. Baseline was established in kinetics buffer and antibodies were associated.

[0373] The results for CD28 benchmark antibody R020 binding to human 5T4 are shown in FIG.25A. R020 is a bispecific antibody and includes the same VH and VL domains of the CD28 binding unit of R017 and the same Fc as R005, R006, and R007. The tumor binding unit is formatted as an scFv that binds to the tumor antigen 5T4.

[0374] The results for CD28 benchmark antibody R155 binding to human FOLR1 are shown in FIG. 25B. R155 is a bispecific antibody and contains the same Fc and the same binding unit to FOLR1 as R025, R026 and R027.

[0375] The results for antibodies R176 and R177 binding to human CD28 are shown in FIG. 25C. R176 is a bispecific antibody and contains the same Fc and the same CD3δε binding unit as R154. R177 is a bispecific antibody and contains the same Fc and the same CD3δε binding unit as R028.Attorney Ref.62571.00009WO01 (RND-0002-WO)

[0376] KD, Ka and Kdis for the indicated antibodies are shown in the table in FIG.25D. These data provide the binding affinities of the non-CD28 binding unit (e.g., binding affinity for 5T4, FOLR1, or CD3δε) of the indicated bispecific antibodies. Example 26: Anti-tumor activity of a CD28 x Nectin-4 bispecific antibody in an in vivo mouse tumor model and an additive therapeutic effect in combination with an immune checkpoint inhibitor

[0377] Tumor growth inhibition by R006 was evaluated in an immunocompetent mouse model. In this model, the extracellular region of endogenous mouse CD28 (exons 2-3) was replaced with the human CD28 extracellular region in C57BL / 6 mice, (referred to as hCD28 mice).10-15 week old hCD28 mice were implanted subcutaneously with 2 x 106MC38-hNectin4 cells (an MC38 syngeneic mouse cell line where the mouse Nectin4 gene has been replaced with the human Nectin4 gene). When the tumors reached an average volume of 100 mm3, mice were randomized and treated intraperitoneally twice weekly with R006 alone, or in combination with the checkpoint inhibitor aPD1, (an antibody that blocks the interaction between mouse PD-1 and PD-L1, antibody clone RMP1-14). Tumor volumes were measured twice weekly, and the results are shown in FIGS. 26A-B as tumor volume over time represented as the average ± SEM of 7-8 mice per group.

[0378] The MC38 cell line expresses immunogenic neoepitopes and therefore endogenous signal 1 is present in these tumors (Yadav et al.; Nature 2014 Nov;27;515(7525):572-6). As seen in FIG. 26A, compared to a control group, R006 shows significant anti-tumor activity in an in vivo mouse model in a dose-dependent manner. The combination of R006 with aPD1 treatment showed substantially improved anti-tumor efficacy compared to either treatment alone, highlighting the benefit of the combination treatment, as seen in FIG. 26B. p values were calculated by mixed-effects multiple comparison analysis using Tukey’s correction. Significance is shown for day 24 for each treatment group relative to the control group (ns – not significant, * p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).

[0379] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Attorney Ref.62571.00009WO01 (RND-0002-WO) CLAIMS 1. An antibody that binds to CD28, comprising a variable region comprising: (a) a heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 1, 2, 3, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67; and / or (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 9, 10, 11, 12, 13, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85; and / or (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 18, 19, 20, 21, 22, 86, 87, 88, 89, 90, 91, and 92; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO:

44.

2. The antibody of claim 1, wherein the variable region comprises: (a) a heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 1, 2, 3, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67; and (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 9, 10, 11, 12, 13, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85; and (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 18, 19, 20, 21, 22, 86, 87, 88, 89, 90, 91, and 92; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO:

44.

3. The antibody of claim 2, wherein the variable region comprises: (a) a heavy chain variable region sequence comprising: (1) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (2) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (3) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 11; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (4) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 13; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (5) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (6) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 12; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (7) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (8) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 86; or (9) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (10) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or (11) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or (12) a heavy chain CDR1 sequence comprising SEQ ID NO: 58; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or (13) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 71; a heavy chain CDR3 sequence comprising SEQ ID NO: 90; or (14) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 72; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or (15) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (16) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or (17) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 86; or (18) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (19) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or (20) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or (21) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (22) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 74; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (23) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or (24) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (25) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or (26) a heavy chain CDR1 sequence comprising SEQ ID NO: 59; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (27) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 75; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (28) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or (29) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or (30) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (31) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 76; a heavy chain CDR3 sequence comprising SEQ ID NO: 90; or (32) a heavy chain CDR1 sequence comprising SEQ ID NO: 60; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (33) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (34) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 77; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or (35) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or (36) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 78; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (37) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 77; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (38) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 71; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (39) a heavy chain CDR1 sequence comprising SEQ ID NO: 61; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (40) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (41) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (42) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (43) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (44) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (45) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (46) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (47) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 81; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (48) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (49) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (50) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 82; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (51) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (52) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (53) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (54) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (55) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (56) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (57) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (58) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (59) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (60) a heavy chain CDR1 sequence comprising SEQ ID NO: 63; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (61) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (62) a heavy chain CDR1 sequence comprising SEQ ID NO: 64; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (63) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 84; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (64) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 92; or (65) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 92; or (66) a heavy chain CDR1 sequence comprising SEQ ID NO: 65; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (67) a heavy chain CDR1 sequence comprising SEQ ID NO: 66; a heavy chain CDR2 sequence comprising SEQ ID NO: 85; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (68) a heavy chain CDR1 sequence comprising SEQ ID NO: 67; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (69) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; and (b) a light chain variable region sequence comprising: (1) a light chain CDR1 sequence comprising SEQ ID NO: 28, a light chain CDR2 sequence comprising SEQ ID NO: 36, and a light chain CDR3 sequence comprising SEQ ID NO:

44.

4. The antibody of claim 3, wherein the variable region comprises: (a) a heavy chain variable region sequence comprising: (1) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (2) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (3) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 11; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; or (4) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 13; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (5) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (6) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 12; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; and (b) a light chain variable region sequence comprising: (1) a light chain CDR1 sequence comprising SEQ ID NO: 28, a light chain CDR2 sequence comprising SEQ ID NO: 36, and a light chain CDR3 sequence comprising SEQ ID NO:

44.

5. The antibody of any one of claims 1-4, wherein the heavy chain variable region CDR1, CDR2 and CDR3 sequences are present in a human VH framework.

6. The antibody of claim 5, comprising a heavy chain variable region sequence having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 93-100, 107, 121-237, and 333- 334.

7. The antibody of claim 6, wherein the heavy chain variable region sequence is selected from the group consisting of SEQ ID NOs: 93-100, 107, 121-237, and 333-334.

8. The antibody of claim 7, comprising a heavy chain variable region sequence of SEQ ID NO:

93.

9. The antibody of claim 7, comprising a heavy chain variable region sequence of SEQ ID NO:

94.

10. The antibody of claim 7, comprising a heavy chain variable region sequence of SEQ ID NO:

95.

11. The antibody of claim 7, comprising a heavy chain variable region sequence of SEQ ID NO:

96.

12. The antibody of claim 7, comprising a heavy chain variable region sequence of SEQ ID NO: 97.Attorney Ref.62571.00009WO01 (RND-0002-WO) 13. The antibody of claim 7, comprising a heavy chain variable region sequence of SEQ ID NO:

98.

14. The antibody of claim 7, comprising a heavy chain variable region sequence of SEQ ID NO:

99.

15. The antibody of claim 7, comprising a heavy chain variable region sequence of SEQ ID NO:

100.

16. The antibody of any one of claims 1-4, wherein the light chain variable region CDR1, CDR2 and CDR3 sequences are present in a human VL framework.

17. The antibody of any one of claims 1-4, comprising a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO:

110.

18. The antibody of claim 6, wherein the light chain variable region sequence comprises the sequence of SEQ ID NO:

110.

19. An antibody that binds to Nectin-4, comprising a variable region comprising: (a) a heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 4, and 286- 291; and / or (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 10, 83, and 292-294; and / or (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 23, and 295- 300; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO:

44.

20. The antibody of claim 19, wherein the variable region comprises: (a) a heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 4, and 286- 291; and (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 10, 83, and 292-294; andAttorney Ref.62571.00009WO01 (RND-0002-WO) (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 23, and 295- 300; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO:

44.

21. The antibody of claim 19, wherein the variable region comprises: (a) a heavy chain variable region sequence comprising: (1) a heavy chain CDR1 sequence comprising SEQ ID NO: 4, a CDR2 sequence comprising SEQ ID NO: 10, and a CDR3 sequence comprising SEQ ID NO: 23; or (2) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; or (3) a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 292; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; or (4) a heavy chain CDR1 sequence comprising SEQ ID NO: 287; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 297; or (5) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; or (6) a heavy chain CDR1 sequence comprising SEQ ID NO: 288; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 298; or (7) a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 299; or (8) a heavy chain CDR1 sequence comprising SEQ ID NO: 288; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 298; or (9) a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; or (10) a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (11) a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 293; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or (12) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; or (13) a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or (14) a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; or (15) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or (16) a heavy chain CDR1 sequence comprising SEQ ID NO: 290; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or (17) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or (18) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or (19) a heavy chain CDR1 sequence comprising SEQ ID NO: 291; a heavy chain CDR2 sequence comprising SEQ ID NO: 294; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO:

44.

22. The antibody of any one of claims 19-21, wherein the heavy chain variable region CDR1, CDR2 and CDR3 sequences are present in a human VH framework.

23. The antibody of claim 22, comprising a heavy chain variable region sequence having at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 101-102, and 301-317.Attorney Ref.62571.00009WO01 (RND-0002-WO) 24. The antibody of claim 23, wherein the heavy chain variable region sequence is selected from the group consisting of SEQ ID NOs: 101-102, and 301-317.

25. The antibody of claim 24, comprising a heavy chain variable region sequence of SEQ ID NO:

101.

26. The antibody of claim 24, comprising a heavy chain variable region sequence of SEQ ID NO:

102.

27. The antibody of claim 19, wherein the light chain variable region CDR1, CDR2 and CDR3 sequences are present in a human VL framework.

28. The antibody of claim 27, comprising a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO:

110.

29. The antibody of claim 28, wherein the light chain variable region sequence comprises the sequence of SEQ ID NO:

110.

30. A multi-specific antibody comprising: a first binding unit that binds to CD28, comprising: (a) a first heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 1, 2, 3, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67; and / or (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 9, 10, 11, 12, 13, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85; and / or (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 18, 19, 20, 21, 22, 86, 87, 88, 89, 90, 91, and 92; and (b) a first light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44; and a second binding unit that binds to a disease-associated antigen (DAA).

31. The multi-specific antibody of claim 30, wherein the first binding unit comprises: (a) a heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 1, 2, 3, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67; andAttorney Ref.62571.00009WO01 (RND-0002-WO) (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 9, 10, 11, 12, 13, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, and 85; and (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 18, 19, 20, 21, 22, 86, 87, 88, 89, 90, 91, and 92; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO:

44.

32. The multi-specific antibody of claim 31, wherein the first binding unit comprises: (a) a heavy chain variable region sequence comprising: (1) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (2) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (3) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 11; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; or (4) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 13; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (5) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (6) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 12; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (7) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (8) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 86; or (9) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (10) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or (11) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or (12) a heavy chain CDR1 sequence comprising SEQ ID NO: 58; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or (13) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 71; a heavy chain CDR3 sequence comprising SEQ ID NO: 90; or (14) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 72; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or (15) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or (16) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or (17) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 86; or (18) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (19) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or (20) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 87; or (21) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (22) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 74; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (23) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or (24) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (25) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 88; or (26) a heavy chain CDR1 sequence comprising SEQ ID NO: 59; a heavy chain CDR2 sequence comprising SEQ ID NO: 68; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (27) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 75; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; or (28) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or (29) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or (30) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (31) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 76; a heavy chain CDR3 sequence comprising SEQ ID NO: 90; or (32) a heavy chain CDR1 sequence comprising SEQ ID NO: 60; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (33) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 70; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (34) a heavy chain CDR1 sequence comprising SEQ ID NO: 57; a heavy chain CDR2 sequence comprising SEQ ID NO: 77; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or (35) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 89; or (36) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 78; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (37) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 77; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (38) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 71; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (39) a heavy chain CDR1 sequence comprising SEQ ID NO: 61; a heavy chain CDR2 sequence comprising SEQ ID NO: 69; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (40) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (41) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (42) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (43) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (44) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (45) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (46) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (47) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 81; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (48) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (49) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (50) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 82; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (51) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (52) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (53) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (54) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (55) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (56) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (57) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 79; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (58) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (59) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (60) a heavy chain CDR1 sequence comprising SEQ ID NO: 63; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (61) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 73; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (62) a heavy chain CDR1 sequence comprising SEQ ID NO: 64; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; or (63) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 84; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (64) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 92; or (65) a heavy chain CDR1 sequence comprising SEQ ID NO: 62; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 92; or (66) a heavy chain CDR1 sequence comprising SEQ ID NO: 65; a heavy chain CDR2 sequence comprising SEQ ID NO: 80; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (67) a heavy chain CDR1 sequence comprising SEQ ID NO: 66; a heavy chain CDR2 sequence comprising SEQ ID NO: 85; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (68) a heavy chain CDR1 sequence comprising SEQ ID NO: 67; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (69) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 91; and (b) a light chain variable region sequence comprisingAttorney Ref.62571.00009WO01 (RND-0002-WO) (1) a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 sequence comprising SEQ ID NO: 36, and a light chain CDR3 sequence comprising SEQ ID NO:

44.

33. The multi-specific antibody of claim 32, wherein the first binding unit comprises: (a) a heavy chain variable region sequence comprising: (1) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 9; a heavy chain CDR3 sequence comprising SEQ ID NO: 18; or (2) a heavy chain CDR1 sequence comprising SEQ ID NO: 2; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 19; or (3) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 11; a heavy chain CDR3 sequence comprising SEQ ID NO: 20; or (4) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 13; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; or (5) a heavy chain CDR1 sequence comprising SEQ ID NO: 3; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 22; or (6) a heavy chain CDR1 sequence comprising SEQ ID NO: 1; a heavy chain CDR2 sequence comprising SEQ ID NO: 12; a heavy chain CDR3 sequence comprising SEQ ID NO: 21; and (b) a light chain variable region sequence comprising: (1) a light chain CDR1 sequence comprising SEQ ID NO: 28, a light chain CDR2 sequence comprising SEQ ID NO: 36, and a light chain CDR3 sequence comprising SEQ ID NO:

44.

34. The multi-specific antibody of any one of claims 30-33, wherein: the CDR1, CDR2, and CDR3 sequences in the heavy chain variable region of the first binding unit are present in a human VH framework; and the CDR1, CDR2, and CDR3 sequences in the light chain variable region of the first binding unit are present in a human VL framework.

35. The multi-specific antibody of claim 33, wherein:Attorney Ref.62571.00009WO01 (RND-0002-WO) the heavy chain variable region sequence of the first binding unit comprises at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 93-100, 107, 121-237, and 333-334; and the light chain variable region sequence of the first binding unit comprises at least 95% sequence identity to SEQ ID NO:

110.

36. The multi-specific antibody of claim 35, wherein: the heavy chain variable region sequence of the first binding unit is selected from the group consisting of SEQ ID NOs: 93-100, 107, 121-237, and 333-334; and the light chain variable region sequence of the first binding unit comprises the sequence of SEQ ID NO:

110.

37. The multi-specific antibody of claim 36, wherein: the heavy chain variable region sequence of the first binding unit comprises the sequence of SEQ ID NO: 93; or the heavy chain variable region sequence of the first binding unit comprises the sequence of SEQ ID NO: 94; or the heavy chain variable region sequence of the first binding unit comprises the sequence of SEQ ID NO: 95; or the heavy chain variable region sequence of the first binding unit comprises the sequence of SEQ ID NO: 96; or the heavy chain variable region sequence of the first binding unit comprises the sequence of SEQ ID NO: 97; or the heavy chain variable region sequence of the first binding unit comprises the sequence of SEQ ID NO: 98; or the heavy chain variable region sequence of the first binding unit comprises the sequence of SEQ ID NO: 99; or the heavy chain variable region sequence of the first binding unit comprises the sequence of SEQ ID NO: 100; and the light chain variable region sequence of the first binding unit comprises the sequence of SEQ ID NO:

110.

38. The multi-specific antibody of any one of claims 30-37, wherein the DAA is a tumor associated antigen (TAA) or a tumor specific antigen (TSA).

39. The multi-specific antibody of claim 38, wherein the TAA is Nectin-4.Attorney Ref.62571.00009WO01 (RND-0002-WO) 40. A multi-specific antibody comprising: a first binding unit that binds to Nectin-4, comprising: (a) a first heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 4, and 286- 291; and / or (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 10, 83, and 292-294; and / or (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 23, and 295- 300; and (b) a first light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO: 44; and a second binding unit that binds to an effector-associated antigen (EAA).

41. The multi-specific antibody of claim 40, wherein the first binding unit comprises: (a) a heavy chain variable region sequence comprising: (1) a CDR1 sequence selected from the group comprising SEQ ID NOs: 4, and 286- 291; and (2) a CDR2 sequence selected from the group comprising SEQ ID NOs: 10, 83, and 292-294; and (3) a CDR3 sequence selected from the group comprising SEQ ID NOs: 23, and 295- 300; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO:

44.

42. The multi-specific antibody of claim 40, wherein the first binding unit comprises: (a) a heavy chain variable region sequence comprising: (1) a heavy chain CDR1 sequence comprising SEQ ID NO: 4, a CDR2 sequence comprising SEQ ID NO: 10, and a CDR3 sequence comprising SEQ ID NO: 23; or (2) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (3) a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 292; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; or (4) a heavy chain CDR1 sequence comprising SEQ ID NO: 287; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 297; or (5) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; or (6) a heavy chain CDR1 sequence comprising SEQ ID NO: 288; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 298; or (7) a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 299; or (8) a heavy chain CDR1 sequence comprising SEQ ID NO: 288; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 298; or (9) a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 295; or (10) a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or (11) a heavy chain CDR1 sequence comprising SEQ ID NO: 4; a heavy chain CDR2 sequence comprising SEQ ID NO: 293; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or (12) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; or (13) a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or (14) a heavy chain CDR1 sequence comprising SEQ ID NO: 289; a heavy chain CDR2 sequence comprising SEQ ID NO: 10; a heavy chain CDR3 sequence comprising SEQ ID NO: 296; orAttorney Ref.62571.00009WO01 (RND-0002-WO) (15) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 23; or (16) a heavy chain CDR1 sequence comprising SEQ ID NO: 290; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or (17) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or (18) a heavy chain CDR1 sequence comprising SEQ ID NO: 286; a heavy chain CDR2 sequence comprising SEQ ID NO: 83; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; or (19) a heavy chain CDR1 sequence comprising SEQ ID NO: 291; a heavy chain CDR2 sequence comprising SEQ ID NO: 294; a heavy chain CDR3 sequence comprising SEQ ID NO: 300; and (b) a light chain variable region sequence comprising a CDR1 sequence comprising SEQ ID NO: 28, a CDR2 sequence comprising SEQ ID NO: 36, and a CDR3 sequence comprising SEQ ID NO:

44.

43. The multi-specific antibody of any one of claims 40-42, wherein: the CDR1, CDR2, and CDR3 sequences in the heavy chain variable region of the first binding unit are present in a human VH framework; and the CDR1, CDR2, and CDR3 sequences in the light chain variable region of the first binding unit are present in a human VL framework.

44. The multi-specific antibody of claim 43, wherein: the heavy chain variable region sequence of the first binding unit comprises at least 95% sequence identity to any one of the sequences of SEQ ID NOs: 101-102, and 301-317; and the light chain variable region sequence of the first binding unit comprises at least 95% sequence identity to SEQ ID NO:

110.

45. The multi-specific antibody of claim 44, wherein: the heavy chain variable region sequence of the first binding unit is selected from the group consisting of SEQ ID NOs: 101-102, and 301-317; and the light chain variable region sequence of the first binding unit comprises SEQ ID NO: 110.Attorney Ref.62571.00009WO01 (RND-0002-WO) 46. The multi-specific antibody of any one of claims 40-45, wherein the EAA is a cell surface protein on an immune effector cell.

47. The multi-specific antibody of claim 46, wherein the EAA is a T-cell antigen.

48. The multi-specific antibody of claim 47, wherein the T-cell antigen is CD3.

49. The multi-specific antibody of claim 47, wherein the T-cell antigen is CD28.

50. The multi-specific antibody of any one of claims 30-49, wherein the light chain comprises a light chain constant region (CL).

51. The multi-specific antibody of any one of claims 30-50, wherein the light chain is a kappa LC or a lambda LC.

52. The multi-specific antibody of any one of claims 30-51, further comprising a heavy chain constant region sequence.

53. The multi-specific antibody of claim 52, wherein the heavy chain constant region sequence is a wild type human IgG1 constant region sequence.

54. The multi-specific antibody of claim 52, wherein the heavy chain constant region sequence is a variant human IgG1 constant region sequence comprising an L234A mutation, an L235A mutation, a P329G mutation, or any combination thereof.

55. The multi-specific antibody of claim 52, wherein the heavy chain constant region sequence is a variant human IgG1 constant region sequence comprising an S354C mutation or a Y349C mutation.

56. The multi-specific antibody of any one of claims 54-55, wherein the variant human IgG1 constant region sequence comprises a T366W mutation, an S354C mutation, or both a T366W mutation and an S354C mutation.

57. The multi-specific antibody of any one of claims 54-56, wherein the variant human IgG1 constant region sequence comprises a T366S mutation, an L368A mutation, a Y407V mutation, a Y349C mutation, or any combination thereof.Attorney Ref.62571.00009WO01 (RND-0002-WO) 58. The multi-specific antibody of claim 52, wherein the heavy chain constant region sequence is a wild type human IgG4 constant region sequence.

59. The multi-specific antibody of claim 52, wherein the heavy chain constant region sequence comprises a variant human IgG4 constant region sequence comprising an S228P mutation, an F234A mutation, an L235A mutation, or any combination thereof.

60. The multi-specific antibody of claim 52, wherein the heavy chain constant region sequence comprises a variant human IgG4 constant region sequence comprising an S354C mutation or a Y349C mutation.

61. The multi-specific antibody of any one of claims 59-60, wherein the variant human IgG4 constant region sequence comprises a T366W mutation, an S354C mutation, or both a T366W mutation and an S354C mutation.

62. The multi-specific antibody of one of claims 59-60, wherein the variant human IgG4 constant region sequence comprises a T366S mutation, an L368A mutation, a Y407V mutation, a Y349C mutation, or any combination thereof.

63. The multi-specific antibody of one of claims 59-62, wherein the variant human IgG4 constant region sequence comprises an L445P mutation.

64. A multi-specific antibody that binds to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 240; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

241.

65. A multi-specific antibody that binds to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 243; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

244.

66. A multi-specific antibody that binds to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242;Attorney Ref.62571.00009WO01 (RND-0002-WO) (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 245; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

246.

67. A multi-specific antibody that binds to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 247; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

248.

68. A multi-specific antibody that binds to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 249; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

250.

69. A multi-specific antibody that binds to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 251; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

252.

70. A multi-specific antibody that binds to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 253; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

254.

71. A multi-specific antibody that binds to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 263; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

264.

72. A multi-specific antibody that binds to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 265;Attorney Ref.62571.00009WO01 (RND-0002-WO) (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

266.

73. A multi-specific antibody that binds to CD28 and Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 267; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

268.

74. An antibody that binds to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 255; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

255.

75. An antibody that binds to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 256; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

256.

76. An antibody that binds to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 257; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

257.

77. An antibody that binds to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 259; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

259.

78. An antibody that binds to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 260; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 260.Attorney Ref.62571.00009WO01 (RND-0002-WO) 79. An antibody that binds to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 261; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

261.

80. An antibody that binds to Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 258; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

258.

81. An antibody that binds to Nectin-4, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 262; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

262.

82. An antibody that binds to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 329; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

329.

83. An antibody that binds to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 335; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

335.

84. An antibody that binds to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 336; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO: 336.Attorney Ref.62571.00009WO01 (RND-0002-WO) 85. An antibody that binds to CD28, comprising: (a) a first light chain polypeptide subunit comprising SEQ ID NO: 242; (b) a second light chain polypeptide subunit comprising SEQ ID NO: 242; (c) a first heavy chain polypeptide subunit comprising SEQ ID NO: 337; (d) a second heavy chain polypeptide subunit comprising SEQ ID NO:

337.

86. An antibody comprising means for binding to CD28.

87. An antibody comprising means for binding to Nectin-4.

88. A multispecific antibody comprising: a first binding unit comprising means for binding to CD28; and a second binding unit that binds to a disease-associated antigen (DAA).

89. The multispecific antibody of claim 88, wherein the DAA is a TAA or a TSA.

90. The multispecific antibody of claim 89, wherein the TAA is Nectin-4.

91. A multispecific antibody comprising: a first binding unit comprising means for binding to Nectin-4; and a second binding unit that binds to an effector-associated antigen (EAA).

92. The multispecific antibody of claim 91, wherein the EAA is CD28 or CD3.

93. A multispecific antibody comprising: a first binding unit comprising means for binding to CD28; and a second binding unit comprising means for binding to Nectin-4.

94. A bispecific antibody comprising: a first binding unit comprising means for binding to CD28; and a second binding unit comprising means for binding to Nectin-4.

95. A pharmaceutical composition comprising an antibody of any one of claims 1-94.

96. A polynucleotide encoding an antibody of any one of claims 1-95.

97. A vector comprising the polynucleotide of claim 96.Attorney Ref.62571.00009WO01 (RND-0002-WO) 98. A cell comprising the vector of claim 97.

99. A method of producing an antibody of any one of claims 1-94, the method comprising growing a cell according to claim 98 under conditions permissive for expression of the antibody, and isolating the antibody from the cell and / or a cell culture medium in which the cell is grown.

100. A method of making an antibody of any one of claims 1-94, the method comprising immunizing a transgenic animal with CD28 and identifying CD28-binding heavy chain sequences.

101. A method of making an antibody of any one of claims 1-94, the method comprising immunizing a transgenic animal with Nectin-4 and identifying Nectin-4-binding heavy chain sequences.

102. A method of treatment, comprising administering to an individual in need an effective dose of an antibody of any one of claims 1-94, or a pharmaceutical composition of claim 95.

103. Use of an antibody of any one of claims 1-94 in the preparation of a medicament for the treatment of a disease or disorder in an individual in need.

104. The antibody of any one of claims 1-94 or the pharmaceutical composition of claim 95, for use in therapy in an individual in need.

105. A method for the treatment of a disorder, comprising administering to a subject with said disorder an antibody of any one of claims 1-94, or a pharmaceutical composition of claim 95.

106. The method of claim 96, wherein the disorder is cancer.

107. The method of claim 106, wherein the cancer is a solid tumor cancer.

108. The method of claim 107, wherein the solid tumor cancer is bladder cancer, urothelial bladder cancer, breast cancer, kidney cancer, pancreatic cancer, penile cancer, prostate cancer, lung cancer, a head and neck cancer, small-cell lung cancer, non-small cell lung cancer (NSCLC), gastric cancer, cervical cancer, ovarian cancer, esophageal cancer, genitourinary cancer, ovarian cancer, skin cancer, liver cancer, brain cancer, testicular cancer, colon cancer, rectal cancer, colorectal cancer, vulval cancer, thyroid cancer, melanoma, or endometrial cancer.Attorney Ref.62571.00009WO01 (RND-0002-WO) 109. The method of claim 107, wherein the solid tumor cancer is a carcinoma.

110. The method of claim 109, wherein the carcinoma is adenocarcinoma, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), squamous carcinoma of the lung, epithelial squamous cell carcinoma, transitional cell carcinoma, non melanoma skin cancer, renal cell carcinoma (RCC), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), hepatocellular carcinoma (HCC), hepatic carcinoma, skin carcinoma, anal carcinoma, penile carcinoma, esophageal carcinoma, salivary gland carcinoma, nasopharyngeal carcinoma, laryngeal carcinomas, choriocarcinoma, urinary tract carcinoma, endometrial carcinoma, high grade serous ovarian carcinoma, or uterine carcinoma.

111. The method of claim 110, wherein the adenocarcinoma is adenocarcinoma of the lung, or pancreatic ductal adenocarcinoma.

112. The method of claim 107, wherein the solid tumor cancer is a blastoma.

113. The method of claim 112, wherein the blastoma is hepatoblastoma, medulloblastoma, nephroblastoma, Wilms tumor, neuroblastoma, glioblastoma multiforme, pleuropulmonary blastoma, retinoblastoma, arrhenoblastoma, or pancreatoblastoma.

114. The method of claim 107, wherein the solid tumor cancer is a sarcoma.

115. The method of claim 114, wherein the sarcoma is angiosarcoma, chondrosarcoma, clear cell sarcoma, epithelioid sarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumors, Kaposi sarcoma, leiomyosarcoma, liposarcoma, myeloid sarcoma, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, spindle cell sarcoma, synovial sarcoma, or soft tissue sarcoma.

116. The method of claim 114, wherein the sarcoma results from Schwannoma or oligodendroglioma.

117. The method of claim 106, wherein the cancer is a liquid tumor cancer.

118. The method of claim 117, wherein the liquid tumor cancer is a hematologic malignancy.

119. The method of claim 118, wherein the hematologic malignancy is a lymphoma.

120. The method of claim 119, wherein the lymphoma is a Hodgkin’s lymphoma, a non-Hodgkin’s lymphoma, T-cell lymphoma, nonspecific peripheral T-cell lymphoma (PTCL-NOS),Attorney Ref.62571.00009WO01 (RND-0002-WO) angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), adult T-cell leukemia / lymphoma (ATLL), cutaneous T-cell lymphoma (CTCL), natural killer / T-cell lymphoma, or cutaneous lymphoma.

121. The method of claim 118, wherein the hematologic malignancy is a leukemia.

122. The method of claim 121, wherein the leukemia is acute myeloid leukemia, T-cell acute lymphoblastic leukemia (T-ALL), or mixed-phenotype acute leukemia.

123. The method of claim 118, wherein the hematologic malignancy is acute hematologic malignancy, myeloma, or multiple myeloma.

124. The method of claim 106, wherein the cancer is metastatic cancer.

125. A method for the treatment of a disorder characterized by expression of Nectin-4, comprising administering to a subject with said disorder an antibody of any one of claims 1-94, or a pharmaceutical composition of claim 95.

126. The method of claim 125, wherein the disorder is cancer.

127. The method of claim 126, wherein the cancer is a solid tumor cancer.

128. The method of claim 127, wherein the solid tumor cancer is bladder cancer, breast cancer, kidney cancer, pancreatic cancer, penile cancer, prostate cancer, lung cancer, a head and neck cancer, gastric cancer, esophageal cancer, genitourinary cancer, ovarian cancer, or endometrial cancer.

129. The method of claim 127, wherein the solid tumor cancer is bladder cancer.

130. The method of claim 127, wherein the solid tumor cancer is breast cancer.

131. The method of claim 127, wherein the solid tumor cancer is ovarian cancer.

132. The method of claim 127, wherein the solid tumor cancer is esophageal cancer.

133. The method of claim 127, wherein the solid tumor cancer is gastric cancer.Attorney Ref.62571.00009WO01 (RND-0002-WO) 134. The method according to any one of claims 102-133, further comprising administering a Signal 1 molecule.

135. The method of claim 134, wherein the Signal 1 molecule comprises a multi-specific antibody that binds to an EAA on an effector cell and a DAA on a target cell.

136. The method of claim 135, wherein the EAA that the multi-specific antibody binds to is a CD3 protein.

137. The method of claim 135 or claim 136, wherein the EAA that the multi-specific antibody binds to is a peptide MHC complex.

138. The method of claim 135 or claim 136, wherein the DAA that the multi-specific antibody binds to is a TAA or a TSA.

139. The method of claim 135 or claim 136, wherein the TAA that the multi-specific antibody binds to is Nectin-4.

140. The method of claim 135, wherein the multi-specific antibody is a Nectin-4 x CD3 antibody.

141. The method of any one of claims 102-140, further comprising administering a cosignaling molecule.

142. The method of claim 141, wherein the cosignaling molecule is a costimulatory cosignaling molecule.

143. The method of claim 141, wherein the cosignaling molecule is a coinhibitory cosignaling molecule.

144. The method according to any one of claims 102-143, further comprising administering an additional therapeutic agent.

145. The method of claim 144, wherein the additional therapeutic agent comprises a checkpoint inhibitor antibody.

146. The method of claim 144, wherein the additional therapeutic agent comprises an antibody- drug conjugate (ADC).Attorney Ref.62571.00009WO01 (RND-0002-WO) 147. The method of claim 144, wherein the additional therapeutic agent comprises a chemotherapeutic drug.

148. The method of claim 144, wherein the additional therapeutic agent comprises an oncolytic virus.

149. A kit for treating a disease or disorder in an individual in need, comprising an antibody of any one of claims 1-94, or a pharmaceutical composition of claim 95, and instructions for use.

150. The kit of claim 149, further comprising at least one additional reagent.

151. The kit of claim 150, wherein the at least one additional reagent comprises a multi-specific antibody that binds to CD28 and Nectin-4, a checkpoint inhibitor antibody, an antibody-drug conjugate (ADC), a chemotherapeutic drug, a Signal 1 molecule, a cosignaling molecule, an oncolytic virus, or any combination thereof.

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