Antibodies binding PD-l1, PD-l2 and / or CD28

Abstract

Provided herein are novel antibodies to aPD-L1, aPD-L2, and aCD28. In some embodiments, the antibody is an aPD-L1 x aPD-L2 x aCD28 antibody. Such antibodies enhance anti-tumor activity by providing a co-stimulatory signal against T cell activation of tumor cells while also advantageously blocking inhibitory PD-L1: PD1 and / or PD-L2: PD1 pathway interactions.

Description

[0001] Priority Declaration

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 330,769, filed April 13, 2022, U.S. Provisional Application No. 63 / 379,100, filed October 11, 2022, U.S. Provisional Application No. 63 / 382,592, filed November 7, 2022, U.S. Provisional Application No. 63 / 478,881, filed January 6, 2023, and U.S. Provisional Application No. 63 / 480,478, filed January 18, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is electronically submitted in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on April 11, 2023, is named 067461-5301-WO_SL.xml, and is 3,420,050 bytes in size. Background Art

[0005] The innate immune response against tumors sends immune effector cells (such as natural killer (NK) cells and T cells) to attack and destroy tumor cells. Tumor infiltrating lymphocytes (TILs) typically express a variety of immune checkpoint receptors (e.g., PD-1, CTLA-4) and co-stimulatory receptors (e.g., ICOS, 4-1BB, OX40, GITR, and CD28). Over time, due to the upregulation of inhibitory immune checkpoints, TILs lose their cytotoxicity. Although checkpoint blockade has been shown to increase clinical response rates relative to other treatment options, many patients still cannot achieve a response to checkpoint blockade. The engagement of co-stimulatory receptors on TILs can provide positive signals that can overcome the negative signals of immune checkpoints. Preclinical and clinical studies of agonist co-stimulatory receptor antibodies have indeed demonstrated that the agonism of co-stimulatory receptors can produce impressive anti-tumor responses, thereby activating T cells to attack tumor cells.

[0006] It is also important for cancer treatment to enhance anti-tumor activity by specifically destroying tumor cells while minimizing peripheral toxicity. In this case, it is crucial that, only in the presence of target tumor cells, costimulatory signals are provided to T cells. However, for TIL, peripheral T cells and the autoantigen reactive T cells that cause autoimmune toxicity, the agonism of monospecific full-length antibodies to costimulatory receptors may be indiscriminate. For example, Urelumab (urelumab) (a monospecific indiscriminate pan-4-1BB agonist antibody) shows significant hepatotoxicity (Segal et al., 2016) in early clinical trials. Therefore, there is still a need for novel immune response enhancing compositions for treating cancer. Summary of the Invention

[0007] Provided herein are novel αPD-L1, αPD-L2, and αCD28 antibodies. In some embodiments, the antibodies are trispecific αPD-L1 x αPD-L2 x αCD28 antibodies. Such antibodies enhance anti-tumor activity by providing co-stimulatory signals for T cell activation against tumor cells while also advantageously blocking inhibitory PD-L1: PD1 and / or PD-L2: PD1 pathway interactions (see Figure 94 In some embodiments, such trispecific antibodies can be used in combination with αCD3xα tumor target antigen (TTA) bispecific antibodies to treat cancer.

[0008] In one aspect, provided herein are multispecific antibodies comprising a PD-L1 antigen binding domain, a PD-L2 antigen binding domain, and a CD28 binding domain, wherein each of the binding domains comprises a variable heavy domain and a variable light domain.

[0009] In one aspect, provided herein is a novel 1+1+1 stacked Fab2-Fab-Fc format antibody ( Figure 83D). The antibody comprises a) a first monomer, b) a second monomer, and c) a first common light chain, a second common light chain, and a third common light chain. The first monomer comprises VH1-CH1-hinge-CH2-CH3 from the N-terminus to the C-terminus, wherein VH1 is the first variable heavy domain, and CH2-CH3 is the first Fc domain. The second monomer comprises VH2-CH1-linker-VH3-CH1-hinge-CH2-CH3 from the N-terminus to the C-terminus, wherein VH2 is the second variable heavy domain, VH3 is the third variable heavy domain, and CH2-CH3 is the second Fc domain. Each common light chain comprises VL-CL from the N-terminus to the C-terminus, wherein VL is a common variable light domain, and CL is a constant light domain. In this format, the first variable heavy domain and the common variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the common variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the common variable light domain of the third common light chain form a third antigen binding domain.

[0010] In some embodiments, the antibody is a trispecific antibody that binds PD-L1, PD-L2, and CD28, and each of the first, second, and third antigen binding domains is selected from the group consisting of a PD-L1 antigen binding domain, a PD-L2 antigen binding domain, and a CD28 binding domain.

[0011] In some embodiments, the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain are selected from the group consisting of: i) the first antigen binding domain is a PD-L1 binding domain, the second antigen binding domain is a PD-L2 binding domain, and the third antigen binding domain is a CD28 binding domain, ii) the first antigen binding domain is a PD-L1 binding domain, the second antigen binding domain is a CD28 binding domain, and the third antigen binding domain is a PD-L2 binding domain, iii) the first antigen binding domain is a PD-L2 binding domain, the second antigen binding domain is a PD-L1 binding domain, and the third antigen binding domain is a CD28 binding domain, iv) the first antigen binding domain is a PD-L2 binding domain, the second antigen binding domain is a CD28 binding domain, and the third antigen binding domain is a PD-L1 binding domain, v) the first antigen binding domain is a CD28 binding domain, the second antigen binding domain is a PD-L1 binding domain, and the third antigen binding domain is a PD-L2 binding domain, or vi) the first antigen binding domain is a CD28 binding domain, the second antigen binding domain is a PD-L2 binding domain, and the third antigen binding domain is a PD-L1 binding domain.

[0012] In some embodiments, the CD28 binding domain comprises a variable heavy domain having an amino acid selected from SEQ ID NOs: 3354-3389, or a variant thereof, and a variable light domain having an amino acid sequence of SEQ ID NO: 3239, or a variant thereof.

[0013] In some embodiments, the PD-L1 antigen binding domain comprises a variable heavy domain having an amino acid selected from SEQ ID NOs: 3235 and 3243-3260, or a variant thereof, and a variable light domain having an amino acid sequence of SEQ ID NO: 3239, or a variant thereof.

[0014] In some embodiments, the PD-L2 antigen binding domain comprises a variable heavy domain having amino acids selected from SEQ ID NOs: 3267 and 3275-3347, or a variant thereof, and a variable light domain having an amino acid sequence of SEQ ID NO: 3239, or a variant thereof.

[0015] In some embodiments, the first Fc domain and the second Fc domain are variant Fc domains. In certain embodiments, the first Fc domain and the second Fc domain include a group of heterodimerization (heterodimerization) skew variants selected from the group consisting of: S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V, wherein numbering is according to EU numbering. In some embodiments, the first Fc domain and the second Fc domain include heterodimerization skew variants S364K / E357Q:L368D / K370S.

[0016] In some embodiments, the first Fc domain and the second Fc domain each comprise one or more ablation variants. In some embodiments, the one or more ablation variants are E233P / L234V / L235A / G236del / S267K, wherein numbering is according to EU numbering.

[0017] In certain embodiments, one of the first monomer or the second monomer comprises one or more pi variants. In some embodiments, the CH1-hinge-CH2-CH3 of the first monomer comprises the pi variants N208D / Q295E / N384D / Q418E / N421D, wherein numbering is according to EU numbering.

[0018] In some embodiments, the first Fc domain comprises the amino acid variant L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K and the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variant S364K / E357Q / E233P / L234V / L235A / G236del / S267K, and wherein the numbering is according to EU numbering.

[0019] In some embodiments, the first Fc domain and the second Fc domain each further comprise amino acid variant 428 / 434S.

[0020] In some embodiments, a) the PD-L1 binding domain comprises a variable heavy domain having the amino acid sequence of SEQ ID NO: 3251, and a variable light domain having the amino acid sequence of SEQ ID NO: 3239; b) the PD-L2 binding domain comprises a variable heavy domain having the amino acid sequence of SEQ ID NO: 3319, and a variable light domain having the amino acid sequence of SEQ ID NO: 3239; and c) the CD28 binding domain comprises a variable heavy domain having the amino acid sequence of SEQ ID NO: 3380, and a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0021] In some embodiments, a) the first monomer has the amino acid sequence of SEQ ID NO:3201; b) the second monomer has the amino acid sequence of SEQ ID NO:3202; and c) the first common light chain, the second common light chain, and the third common light chain each have the amino acid sequence of SEQ ID NO:3203.

[0022] In one aspect, provided herein is a novel 1+1+1 stacked Fab2-scFv-Fc format antibody ( Figure 83A). The 1+1+1 stacked Fab2-scFv-Fc format comprises a) a first monomer, b) a second monomer, and c) a first common light chain and a second common light chain. The first monomer comprises an scFv-linker-CH2-CH3 from N-terminus to C-terminus, wherein CH2-CH3 is a first Fc domain. The second monomer comprises a VH1-CH1-linker-VH2-CH1-hinge-CH2-CH3 from N-terminus to C-terminus, wherein VH1 is a first variable heavy domain, VH2 is a second variable heavy domain, and CH2-CH3 is a second Fc domain. The first common light chain and the second common light chain each comprise a VL1-CL from N-terminus to C-terminus, wherein VL1 is a first variable light domain, and CL is a constant light domain. In addition, the scFv comprises a third VH domain (VH3), an scFv linker, and a second variable light domain (VL2). In this format, the first variable heavy domain and the first variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the first variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the second variable light domain form a third antigen binding domain.

[0023] In some embodiments, the antibody is a trispecific antibody that binds PD-L1, PD-L2, and CD28, and each of the first, second, and third antigen binding domains is selected from the group consisting of a PD-L1 antigen binding domain, a PD-L2 antigen binding domain, and a CD28 binding domain.

[0024] In some embodiments, the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain are selected from the group consisting of:

[0025] i) the first antigen binding domain is a PD-L1 binding domain, the second antigen binding domain is a PD-L2 binding domain, and the third antigen binding domain is a CD28 binding domain, ii) the first antigen binding domain is a PD-L1 binding domain, the second antigen binding domain is a CD28 binding domain, and the third antigen binding domain is a PD-L2 binding domain, iii) the first antigen binding domain is a PD-L2 binding domain, the second antigen binding domain is a PD-L1 binding domain, and the third antigen binding domain is a CD28 binding domain, iv) the first antigen binding domain is a PD-L2 binding domain, the second antigen binding domain is a CD28 binding domain, and the third antigen binding domain is a PD-L1 binding domain, v) the first antigen binding domain is a CD28 binding domain, the second antigen binding domain is a PD-L1 binding domain, and the third antigen binding domain is a PD-L2 binding domain, or vi) the first antigen binding domain is a CD28 binding domain, the second antigen binding domain is a PD-L2 binding domain, and the third antigen binding domain is a PD-L1 binding domain.

[0026] In some embodiments, the CD28 binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from Figure 35 、 Figure 36 , a variable heavy domain of any one of the variable heavy domains in FIG. 38 , and a variable heavy domain selected from Figure 35 、 Figure 37 and the variable light domains of any one of the variable light domains in Figure 38, ii) the variable heavy domain and variable light domain of the CD28 binding domain in Figure 38, iii) a variable heavy domain selected from Figures 41 to 74 A variable heavy domain of any one of the variable heavy domains in Figures 41 to 74 a variable light domain of any one of the variable light domains in Figures 41 to 74 The variable heavy domain and variable light domain of the CD28 binding domain in Figures 41 to 74 The variable heavy domain of any one of the variable light domains in , and the variable light domain having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 ), vi) Figure 81 and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 163, and a variable light domain selected from Figure 35 and Figure 37 A variable light domain of any one of the variable light domains in , or a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0027] In some embodiments, the PD-L1 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from Figure 17 and Figure 19 A variable heavy domain of any one of the variable heavy domains in Figure 17 and Figure 20 a variable light domain of any one of the variable light domains in Figure 17 and Figure 21 iii) a variable heavy domain and a variable light domain of the PD-L1 binding domain selected from Figure 25 and a variable heavy domain of any one of the variable heavy domains in Figure 26, and a variable heavy domain selected from Figure 25 and the variable light domain of any one of the variable light domains in Figure 26, iv) Figure 25 and the variable heavy domain and variable light domain of the PD-L1 binding domain in Figure 26, v) is selected from Figure 25 and the variable heavy domain of any one of the variable heavy domains in Figure 26, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 ), vi) Figure 28 and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 161, and a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0028] In some embodiments, the PD-L2 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from Figure 29 to any of the variable heavy domains in Figure 32, and a variable heavy domain selected from Figure 29 to the variable light domain of any one of the variable light domains in Figure 32, ii) Figure 29 to the variable heavy domain and variable light domain of the PD-L2 binding domain in Figure 32, iii) selected from Figure 29 to the variable heavy domains of any one of the variable heavy domains in Figure 32, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 ),iv) Figure 34 162 , and a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0029] In some embodiments, the first Fc domain and the second Fc domain are variant Fc domains. In certain embodiments, the first Fc domain and the second Fc domain comprise a set of heterodimerization skew variants selected from the group consisting of: S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V, wherein numbering is according to EU numbering. In some embodiments, the first Fc domain and the second Fc domain comprise heterodimerization skew variants S364K / E357Q:L368D / K370S.

[0030] In some embodiments, the first Fc domain and the second Fc domain each comprise one or more ablation variants. In some embodiments, the one or more ablation variants are E233P / L234V / L235A / G236del / S267K, wherein numbering is according to EU numbering.

[0031] In some embodiments, one of the first monomer or the second monomer comprises one or more pi variants. In some embodiments, the CH1-hinge-CH2-CH3 of the second monomer comprises the pi variants N208D / Q295E / N384D / Q418E / N421D, wherein numbering is according to EU numbering.

[0032] In some embodiments, the first Fc domain comprises the amino acid variant S364K / E357Q / E233P / L234V / L235A / G236del / S267K, the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variant L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K, and the numbering is according to EU numbering.

[0033] In some embodiments, the scFv linker is a charged scFv linker having the amino acid sequence (GKPGS) 4. In some embodiments, the first Fc domain and the second Fc domain each further comprise the amino acid variant 428 / 434S.

[0034] In one aspect, provided herein is a novel 1+1+1 Fab-(Fab-scFv)-Fc format antibody ( Figure 83B). The 1+1+1 Fab-(Fab-scFv)-Fc format comprises a) a first monomer, b) a second monomer, and c) a first common light chain and a second common light chain. The first monomer comprises VH1-CH1-linker-scFv-linker-CH2-CH3 from N-terminus to C-terminus, wherein VH1 is the first variable heavy domain, and CH2-CH3 is the first Fc domain. The second monomer comprises VH2-CH1-hinge-CH2-CH3 from N-terminus to C-terminus, wherein VH2 is the second variable heavy domain, and CH2-CH3 is the second Fc domain. The first common light chain and the second common light chain each comprise VL1-CL from N-terminus to C-terminus, wherein VL1 is the first variable light domain, and CL is a constant light domain. In addition, the scFv comprises a third VH domain (VH3), an scFv linker, and a second variable light domain (VL2). In this format, the first variable heavy domain and the first variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the first variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the second variable light domain form a third antigen binding domain.

[0035] In some embodiments, the antibody is a trispecific antibody that binds PD-L1, PD-L2, and CD28, and each of the first, second, and third antigen binding domains is selected from the group consisting of a PD-L1 antigen binding domain, a PD-L2 antigen binding domain, and a CD28 binding domain.

[0036] In some embodiments, the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain are selected from the group consisting of:

[0037] i) the first antigen binding domain is a PD-L1 binding domain, the second antigen binding domain is a PD-L2 binding domain, and the third antigen binding domain is a CD28 binding domain, ii) the first antigen binding domain is a PD-L1 binding domain, the second antigen binding domain is a CD28 binding domain, and the third antigen binding domain is a PD-L2 binding domain, iii) the first antigen binding domain is a PD-L2 binding domain, the second antigen binding domain is a PD-L1 binding domain, and the third antigen binding domain is a CD28 binding domain, iv) the first antigen binding domain is a PD-L2 binding domain, the second antigen binding domain is a CD28 binding domain, and the third antigen binding domain is a PD-L1 binding domain, v) the first antigen binding domain is a CD28 binding domain, the second antigen binding domain is a PD-L1 binding domain, and the third antigen binding domain is a PD-L2 binding domain, or vi) the first antigen binding domain is a CD28 binding domain, the second antigen binding domain is a PD-L2 binding domain, and the third antigen binding domain is a PD-L1 binding domain.

[0038] In some embodiments, the CD28 binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from Figure 35 、 Figure 36 , a variable heavy domain of any one of the variable heavy domains in FIG. 38 , and a variable heavy domain selected from Figure 35 、 Figure 37 and the variable light domains of any one of the variable light domains in Figure 38, ii) the variable heavy domain and variable light domain of the CD28 binding domain in Figure 38, iii) a variable heavy domain selected from Figures 41 to 74 A variable heavy domain of any one of the variable heavy domains in Figures 41 to 74 a variable light domain of any one of the variable light domains in Figures 41 to 74 The variable heavy domain and variable light domain of the CD28 binding domain in Figures 41 to 74 The variable heavy domain of any one of the variable light domains in , and the variable light domain having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 ), vi) Figure 81 and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 163, and a variable light domain selected from Figure 35 and Figure 37 A variable light domain of any one of the variable light domains in , or a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0039] In some embodiments, the PD-L1 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from Figure 17 and Figure 19 A variable heavy domain of any one of the variable heavy domains in Figure 17 and Figure 20 a variable light domain of any one of the variable light domains in Figure 17 and Figure 21 iii) a variable heavy domain and a variable light domain of the PD-L1 binding domain selected from Figure 25 and a variable heavy domain of any one of the variable heavy domains in Figure 26, and a variable heavy domain selected from Figure 25 and the variable light domain of any one of the variable light domains in Figure 26, iv) Figure 25 and the variable heavy domain and variable light domain of the PD-L1 binding domain in Figure 26, v) is selected from Figure 25 and the variable heavy domain of any one of the variable heavy domains in Figure 26, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 ), vi) Figure 28 and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 161, and a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0040] In some embodiments, the PD-L2 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from Figure 29 to any of the variable heavy domains in Figure 32, and a variable heavy domain selected from Figure 29 to the variable light domain of any one of the variable light domains in Figure 32, ii) Figure 29 to the variable heavy domain and variable light domain of the PD-L2 binding domain in Figure 32, iii) selected from Figure 29 to the variable heavy domains of any one of the variable heavy domains in Figure 32, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 ),iv) Figure 34 162 , and a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0041] In some embodiments, the first Fc domain and the second Fc domain are variant Fc domains. In certain embodiments, the first Fc domain and the second Fc domain comprise a set of heterodimerization skew variants selected from the group consisting of: S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V, wherein numbering is according to EU numbering. In some embodiments, the first Fc domain and the second Fc domain comprise heterodimerization skew variants S364K / E357Q:L368D / K370S.

[0042] In some embodiments, the first Fc domain and the second Fc domain each comprise one or more ablation variants. In some embodiments, the one or more ablation variants are E233P / L234V / L235A / G236del / S267K, wherein numbering is according to EU numbering.

[0043] In some embodiments, one of the first monomer or the second monomer comprises one or more pi variants. In some embodiments, the CH1-hinge-CH2-CH3 of the second monomer comprises the pi variants N208D / Q295E / N384D / Q418E / N421D, wherein numbering is according to EU numbering.

[0044] In some embodiments, the first Fc domain comprises the amino acid variant S364K / E357Q / E233P / L234V / L235A / G236del / S267K, the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variant L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K, and the numbering is according to EU numbering.

[0045] In some embodiments, the scFv linker is a charged scFv linker having the amino acid sequence (GKPGS) 4. In some embodiments, the first Fc domain and the second Fc domain each further comprise the amino acid variant 428 / 434S.

[0046] In one aspect, provided herein is a novel 1+1+1 mAb-scFv format antibody ( Figure 83C). 1+1+1 mAb-scFv format antibodies generally comprise a) a first monomer, b) a second monomer, and c) a first common light chain and a second common light chain. The first monomer comprises VH1-CH1-hinge-CH2-CH3-domain linker-scFv from N-terminus to C-terminus, wherein VH1 is the first variable heavy domain, and CH2-CH3 is the first Fc domain. The second monomer comprises VH2-CH1-hinge-CH2-CH3 from N-terminus to C-terminus, wherein VH2 is the second variable heavy domain, and CH2-CH3 is the second Fc domain. The first common light chain and the second common light chain each comprise VL1-CL from N-terminus to C-terminus, wherein VL1 is the first variable light domain, and CL is a constant light domain. In addition, the scFv comprises a third VH domain (VH3), an scFv linker, and a second variable light domain (VL2). In this format, the first variable heavy domain and the first variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the first variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the second variable light domain form a third antigen binding domain.

[0047] In some embodiments, the antibody is a trispecific antibody that binds PD-L1, PD-L2, and CD28, and each of the first, second, and third antigen binding domains is selected from the group consisting of a PD-L1 antigen binding domain, a PD-L2 antigen binding domain, and a CD28 binding domain.

[0048] In some embodiments, the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain are selected from the group consisting of:

[0049] i) the first antigen binding domain is a PD-L1 binding domain, the second antigen binding domain is a PD-L2 binding domain, and the third antigen binding domain is a CD28 binding domain, ii) the first antigen binding domain is a PD-L1 binding domain, the second antigen binding domain is a CD28 binding domain, and the third antigen binding domain is a PD-L2 binding domain, iii) the first antigen binding domain is a PD-L2 binding domain, the second antigen binding domain is a PD-L1 binding domain, and the third antigen binding domain is a CD28 binding domain, iv) the first antigen binding domain is a PD-L2 binding domain, the second antigen binding domain is a CD28 binding domain, and the third antigen binding domain is a PD-L1 binding domain, v) the first antigen binding domain is a CD28 binding domain, the second antigen binding domain is a PD-L1 binding domain, and the third antigen binding domain is a PD-L2 binding domain, or vi) the first antigen binding domain is a CD28 binding domain, the second antigen binding domain is a PD-L2 binding domain, and the third antigen binding domain is a PD-L1 binding domain.

[0050] In some embodiments, the CD28 binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from Figure 35 、 Figure 36 , a variable heavy domain of any one of the variable heavy domains in FIG. 38 , and a variable heavy domain selected from Figure 35 、 Figure 37 and the variable light domains of any one of the variable light domains in Figure 38, ii) the variable heavy domain and variable light domain of the CD28 binding domain in Figure 38, iii) a variable heavy domain selected from Figures 41 to 74 A variable heavy domain of any one of the variable heavy domains in Figures 41 to 74 a variable light domain of any one of the variable light domains in Figures 41 to 74 The variable heavy domain and variable light domain of the CD28 binding domain in Figures 41 to 74 The variable heavy domain of any one of the variable light domains in , and the variable light domain having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 ), vi) Figure 81 and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 163, and a variable light domain selected from Figure 35 and Figure 37 A variable light domain of any one of the variable light domains in , or a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0051] In some embodiments, the PD-L1 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from Figure 17 and Figure 19 A variable heavy domain of any one of the variable heavy domains in Figure 17 and Figure 20 a variable light domain of any one of the variable light domains in Figure 17 and Figure 21 iii) a variable heavy domain and a variable light domain of the PD-L1 binding domain selected from Figure 25 and a variable heavy domain of any one of the variable heavy domains in Figure 26, and a variable heavy domain selected from Figure 25 and the variable light domain of any one of the variable light domains in Figure 26, iv) Figure 25 and the variable heavy domain and variable light domain of the PD-L1 binding domain in Figure 26, v) is selected from Figure 25 and the variable heavy domain of any one of the variable heavy domains in Figure 26, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 ), vi) Figure 28 and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 161, and a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0052] In some embodiments, the PD-L2 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from Figure 29 to any of the variable heavy domains in Figure 32, and a variable heavy domain selected from Figure 29 to the variable light domain of any one of the variable light domains in Figure 32, ii) Figure 29 to the variable heavy domain and variable light domain of the PD-L2 binding domain in Figure 32, iii) selected from Figure 29 to the variable heavy domains of any one of the variable heavy domains in Figure 32, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 ),iv) Figure 34 162 , and a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0053] In some embodiments, the first Fc domain and the second Fc domain are variant Fc domains. In certain embodiments, the first Fc domain and the second Fc domain comprise a set of heterodimerization skew variants selected from the group consisting of: S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V, wherein numbering is according to EU numbering. In some embodiments, the first Fc domain and the second Fc domain comprise heterodimerization skew variants S364K / E357Q:L368D / K370S.

[0054] In some embodiments, the first Fc domain and the second Fc domain each comprise one or more ablation variants. In some embodiments, the one or more ablation variants are E233P / L234V / L235A / G236del / S267K, wherein numbering is according to EU numbering.

[0055] In some embodiments, one of the first monomer or the second monomer comprises one or more pi variants. In some embodiments, the CH1-hinge-CH2-CH3 of the second monomer comprises the pi variants N208D / Q295E / N384D / Q418E / N421D, wherein numbering is according to EU numbering.

[0056] In some embodiments, the first Fc domain comprises the amino acid variant S364K / E357Q / E233P / L234V / L235A / G236del / S267K, the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variant L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K, and the numbering is according to EU numbering.

[0057] In some embodiments, the scFv linker is a charged scFv linker having the amino acid sequence (GKPGS) 4. In some embodiments, the first Fc domain and the second Fc domain each further comprise the amino acid variant 428 / 434S.

[0058] In another aspect, provided herein is a method for treating cancer, comprising administering to a patient in need thereof a subject antibody disclosed herein or a pharmaceutical composition comprising the subject antibody and a pharmaceutically acceptable carrier. In some embodiments, the method further comprises administering to the patient an anti-CD3 x tumor target antigen (TTA) bispecific antibody.

[0059] Also provided herein are nucleic acid compositions encoding the compositions and antibodies provided herein, expression vectors comprising the nucleic acids, and host cells comprising the nucleic acids and expression vectors.

[0060] In another aspect, provided herein is a method of treating cancer, comprising administering to a patient in need thereof one of the subject antibodies provided herein. In some embodiments, the method further comprises administering to the patient an anti-CD3 x tumor target antigen bispecific antibody.

[0061] In another aspect, provided herein is a composition comprising a CD28 antigen binding domain, wherein the CD28 binding domain comprises: a) a variable heavy domain having vhCDR1-3 of the variable heavy domain in FIG163; and b) a variable heavy domain having vhCDR1-3 of the variable heavy domain in FIG163; Figure 35 and Figure 37 The variable light domain of vlCDR1-3 of the variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0062] In another aspect, provided herein is a CD28 antigen binding domain, wherein the CD28 binding domain comprises: a) a variable heavy domain having at least 85%, 90%, 95% or 99% sequence identity to the variable heavy domain in Figure 163; and b) a variable heavy domain having at least 85%, 90%, 95% or 99% sequence identity to the variable heavy domain in Figure 163; Figure 35 and Figure 37 In some embodiments, the variable heavy domain has the amino acid sequence of the variable heavy domain in Figure 163; and the variable light domain has Figure 35 and Figure 37 The amino acid sequence of the variable light domain in or the variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0063] In another aspect, provided herein is a composition comprising a PD-L1 antigen binding domain, wherein the PD-L1 binding domain comprises: a) a variable heavy domain having vhCDR1-3 of the variable heavy domain in Figure 161; and b) a variable light domain having vlCDR1-3 of a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0064] In another aspect, provided herein is a composition comprising a PD-L1 antigen binding domain, wherein the PD-L1 binding domain comprises: a) a variable heavy domain having at least 85%, 90%, 95%, or 99% sequence identity to the variable heavy domain in Figure 161; and b) a variable light domain having at least 85%, 90%, 95%, or 99% sequence identity to a variable light domain having the amino acid sequence of SEQ ID NO: 3239. In some embodiments, the variable heavy domain has the amino acid sequence of the variable heavy domain in Figure 161; and the variable light domain has the amino acid sequence of a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0065] In another aspect, provided herein is a composition comprising a PD-L2 antigen binding domain, wherein the PD-L1 binding domain comprises: a) a variable heavy domain having vhCDR1-3 of the variable heavy domain in Figure 162; and b) a variable light domain having vlCDR1-3 of a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

[0066] In another aspect, provided herein is a composition comprising a PD-L2 antigen binding domain, wherein the PD-L1 binding domain comprises: a) a variable heavy domain having at least 85% sequence identity to the variable heavy domain in Figure 162; and b) a variable light domain having at least 85% sequence identity to a variable light domain having the amino acid sequence of SEQ ID NO: 3239. In some embodiments, the variable heavy domain has the amino acid sequence of the variable heavy domain in Figure 162; and the variable light domain has the amino acid sequence of a variable light domain having the amino acid sequence of SEQ ID NO: 3239. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The sequences of human, mouse, and cynomolgus monkey PDL1 are depicted. This PDL1 can be used to develop cross-reactive PDL1 antigen-binding domains to facilitate clinical development.

[0068] Figure 2 The sequences of human, mouse, and cynomolgus monkey PDL2 are depicted. This PDL2 can be used to develop cross-reactive PDL2 antigen-binding domains to facilitate clinical development.

[0069] Figures 3A to 3B The sequences of human, mouse, and cynomolgus monkey CD28 are depicted. This CD28 can be used to develop cross-reactive CD28 antigen-binding domains to facilitate clinical development.

[0070] Figures 4A to 4FPairs of useful sets of heterodimerization variants are depicted (including skewed and pi variants). Figure 4F There are variants for which there are no corresponding "monomer 2" variants. Such variants are pI variants that can be used alone on either monomer of a bispecific antibody (e.g., αPD-L1 x αCD28 bsAb) or trispecific antibody (e.g., PDL1 x PDL2 x CD28 triAb), or, for example, included on the non-scFv side of a format utilizing an scFv with an appropriately charged scFv linker as a component on the second monomer (suitable charged linkers are shown in FIG. Figure 7 The heterodimer yield (%) and CH3T of the preferred Fc heterodimerization variants were previously described. m (°C) (see, for example, FIG. 8 of U.S. Patent Application No. 2019 / 0248898).

[0071] Figure 5 Depicted is a list of isomeric variant antibody constant regions and their corresponding substitutions. pi_(-) indicates a lower pi variant, while pi_(+) indicates a higher pi variant. These variants can be optionally and independently combined with other variants (including heterodimerization variants) outlined herein.

[0072] Figure 6 Useful ablation variants (also referred to as "knockout" or "KO" variants) that ablate FcγR binding are described. In some embodiments, such ablation variants are included in the Fc domains of two monomers of the subject antibodies described herein. In other embodiments, the ablation variant is included on only one variant Fc domain.

[0073] Figure 7 Described are multiple charged scFv linkers that can be used to increase or decrease the pI of the subject multimeric bispecific and trispecific antibodies (e.g., PDL1x CD28 bsAb) utilizing one or more scFv as components as described herein. (+H) positively charged linkers are particularly useful herein, particularly for use with the anti-CD28 VL and VH sequences shown herein. According to Whitlow et al., Protein Engineering 6(8):989-995 (1993), a single prior art scFv linker with a single charge is referred to as "Whitlow". It should be noted that such linkers are used to reduce aggregation in scFv and enhance proteolytic stability in scFv. Such charged scFv linkers can be used in any of the subject antibody formats disclosed herein (e.g., 1+1 Fab-scFv-Fc, 2+1 Fab2-scFv-Fc formats, etc.) comprising scFv.

[0074] Figures 8A to 8BDepict a plurality of exemplary domain linkers.In some embodiments, these linkers can be used to connect single-chain Fv to Fc chain.In some embodiments, these linkers can be combined in any orientation.For example, GGGGS linker can be combined with "lower half hinge" linker at N-terminus or C-terminus.

[0075] Figure 9 Particularly useful embodiments are shown for the heterodimeric Fc domains (ie, CH2-CH3 in this embodiment) of the PDL1 x CD28 and PDL2 x CD28 bsAbs and PDL1 x PDL2 x CD28 triAbs of the invention.

[0076] Figure 10The sequences of several useful human IgG1-based multimeric PDL1 x CD28 and PDL2 x CD28 bispecific antibodies (bsAbs) or PDL1 x PDL2 x CD28 trispecific antibody (triAb) backbones are shown, without cytokine sequences. The heterodimeric Fc backbone 1 is based on human IgG1 (356E / 358M allotype) and comprises an L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, an S364K / E357Q skew variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant on both chains. Heterodimeric Fc backbone 2 is based on human IgG1 (356E / 358M allotype) and comprises an L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, an S364K skew variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant on both chains. Heterodimeric Fc backbone 3 is based on human IgG1 (356E / 358M allotype) and comprises an L368E / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, an S364K skew variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant on both chains. Heterodimeric Fc backbone 4 is based on human IgG1 (356E / 358M allotype) and comprises a K360E / Q362E / T411E skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, a D401K skew variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant on both chains. The heterodimeric Fc backbone 5 is based on human IgG1 (356D / 358L allotype) and comprises an L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, an S364K / E357Q skew variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant on both chains.Heterodimeric Fc backbone 6 is based on human IgG1 (356E / 358M allotype) and comprises an L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, an S364K / E357Q skew variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant and a deglycosylated N297A variant on both chains. The heterodimeric Fc backbone 7 is based on human IgG1 (356E / 358M allotype) and comprises an L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, an S364K / E357Q skew variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant and a deglycosylated N297S variant on both chains. Heterodimeric Fc backbone 8 is based on human IgG4 and comprises an L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, an S364K / E357Q skew variant on the second heterodimeric Fc chain, and an S228P (S241P in Kabat according to EU numbering) variant that ablate Fab arm exchange (as known in the art) on both chains. Heterodimeric Fc backbone 9 is based on human IgG2 and comprises an L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, and an S364K / E357Q skew variant on the second heterodimeric Fc chain. The heterodimeric Fc backbone 10 is based on human IgG2 and comprises a L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, a S364K / E357Q skew variant on the second heterodimeric Fc chain, and an S267K ablation variant on both chains. The heterodimeric Fc backbone 11 is based on human IgG1 (356E / 358M allotype) and comprises an L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, an S364K / E357Q skew variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant and an M428L / N434S Xtend variant on both chains.The heterodimeric Fc backbone 12 is based on human IgG1 (356E / 358M allotype) and comprises an L368D / K370S skew variant on the first heterodimeric Fc chain, an S364K / E357Q skew variant and a P217R / P229R / N276K pI variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant on both chains. The heterodimeric Fc backbone 13 is based on human IgG1 (356D / 358L allotype) and comprises an L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, an S364K / E357Q skew variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant and an M428L / N434S Xtend variant on both chains. The heterodimeric Fc backbone 14 is based on human IgG1 (356E / 358M allotype) and comprises an L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, an S364K / E357Q skew variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant and an M428L / N434AXtend variant on both chains. The heterodimeric Fc backbone 15 is based on human IgG1 (356D / 358L allotype) and comprises an L368D / K370S skew variant and a Q295E / N384D / Q418E / N421D pI variant on the first heterodimeric Fc chain, an S364K / E357Q skew variant on the second heterodimeric Fc chain, and an E233P / L234V / L235A / G236del / S267K ablation variant and an M428L / N434AXtend variant on both chains.

[0077] Within each of these backbones are sequences that are 90%, 95%, 98% and 99% identical (as defined herein) to the recited sequences and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions (compared to the "parent" of the figures, as will be understood by those skilled in the art, already contain multiple amino acid modifications compared to the parent human IgG1 (or IgG2 or IgG4, depending on the backbone). That is, the recited backbones may contain additional amino acid modifications (typically amino acid substitutions) in addition to or as an alternative to the skew variants, pI variants and ablation variants contained in the backbone of this figure. In addition, the backbones depicted herein may contain deletions of C-terminal glycine (K446_) and / or lysine (K447_). C-terminal glycine and / or lysine deletions may be intentionally engineered to reduce heterogeneity or in the context of certain bispecific formats (such as mAb-scFv formats). Additionally, C-terminal glycine and / or lysine deletions may occur naturally, for example during production and storage. Additionally, these sequences may include the H435R / Y436F variant in either monomer 1 or monomer 2 to facilitate purification.

[0078] Figure 11 Depicted are exemplary sequences of backbones used in 2+1 mAb-scFv and 1+1+1 mAb-scFv formats. The formats depicted here are based on Figure X The depicted heterodimeric Fc backbone 1 also includes G446_ on monomer 1 (-) and G446_ / K447_ on monomer 2 (+). It should be noted that Figure X Any of the additional backbones depicted can be adapted for use in a 2+1 mAb-scFv format, with or without K447_ on one or both chains. It should be noted that these sequences can also include the M428L / N434S variant. Additionally, these sequences can include the H435R / Y436F variant in either monomer 1 or monomer 2 to facilitate purification.

[0079] Figure 12 Depicted are sequences of "CH1" useful in embodiments of bsAbs and triAbs of the invention.

[0080] Figure 13 Depicted are sequences of the "hinge" useful in embodiments of the bsAb and triAb of the invention.

[0081] Figure 14 Depicted are the constant domains of the cognate light chains useful in the subject PDL1 x CD28 or PDL2 x CD28 bsAbs and PDL1 x PDL2 x CD28 triAbs utilizing Fab binding domains.

[0082] Figures 15A to 15F The sequences of exemplary anti-CD3 binding domains suitable for use in CD3 bispecific antibodies that can be combined with the CD28 bispecific or trispecific antibodies of the invention are depicted. The CDRs are underlined and the scFv linker is double-underlined (in the sequence, the scFv linker is the positively charged scFv(GKPGS)4 linker (SEQ ID NO: 892), but as will be appreciated by one skilled in the art, this linker can be replaced by other linkers, including uncharged linkers or negatively charged linkers, some of which are depicted in FIG). Figure 6 (in the middle), and the slashes indicate one or more boundaries of the variable domain. In addition, the naming convention describes the orientation of the scFv from the N-terminus to the C-terminus. As indicated herein and for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv format or Fab format.

[0083] Figure 16 Depicted are sequences of exemplary αPSMAxαCD3 bsAbs in a 2+1 Fab2-scFv-Fc format comprising H1.30_L1.47 anti-CD3 scFv (also known as CD3 High [VHVL]) or L1.47_H1.32 anti-CD3 scFv (also known as CD3 High-Intermediate (High-Int) #1 [VLVH]), respectively. The CDRs are underlined, and slashes indicate one or more boundaries between the variable region and other chain components (e.g., constant region and domain linker). It should be noted that αPSMAxαCD3 bsAbs can utilize variable region, Fc region, and constant domain sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. Additionally, each of the sequences outlined herein may or may not include the M428L / N434S variant in one, or preferably both, Fc domains, which results in a longer serum half-life.

[0084] Figure 17Depicted are the variable heavy chain sequence and variable light chain sequence of 2G4 (an exemplary humanized hybridoma-derived PDL1 binding domain), as well as the sequences of XENP25859 (an anti-PDL1 mAb based on 2G4 and an IgG1 backbone with E233P / L234V / L235A / G236del / S267K ablation variants) and XENP36627 (a monovalent anti-PDL1 mAb based on 2G4). The CDRs are underlined, and the slashes indicate one or more boundaries between the variable region and the constant domain. As indicated herein and for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may be slightly different depending on the numbering used, and therefore not only the underlined CDRs but also the CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figure, these VH and VL sequences can be used in either scFv or Fab formats.

[0085] Figure 18 Depicted are PD1:PDL1 blockade (binding of a PDL1-mFc fusion to PD1-transfected HEK293T cells) of anti-PDL1 clone 2G4 (XENP25859), partially blocking anti-PDL1 (XENP25853), non-blocking anti-PDL1 (XENP25858), and XENP24118, a benchmark avelumab-based anti-PDL1 mAb.

[0086] Figure 19 Depicted is the sequence of the affinity-optimized variable heavy domain from anti-PDL1 clone 2G4. It should be noted that the variable heavy domain can be paired with any other 2G4 variable light domain depicted herein, including SEQ ID NOs: 1467-1528 (e.g., 2G4_H1.12_L1.14 as utilized in XENP40706).

[0087] Figure 20 Depicted is the sequence of the affinity-optimized variable light domain from anti-PDL1 clone 2G4. It should be noted that the variable heavy domain can be paired with any other 2G4 variable heavy domain depicted herein, including SEQ ID NOs: 1529-1599 (e.g., 2G4_H1.12_L1.14 as utilized in XENP40706).

[0088] Figure 21 Depicted are sequences of exemplary affinity-optimized 2G4 VH / VL pairs. Note that these pairs can be formatted as Fab or scFv.

[0089] Figure 22The consensus framework regions (FRs) and complementarity determining regions (CDRs) (as in Kabat) of the anti-PDL1 clone 2G4 variable heavy domain variant and variable light domain variant are depicted.

[0090] Figure 23 Depicted are exemplary affinity-engineered 2G4 VH / VL pairs and their binding affinities in the context of scFv (in the context of a 1+1 Fab-scFv-Fc bsAb format).

[0091] Figure 24 Depicted is the sequence of the common variable light domain (called 6B1_L1) used in humanized mice that was the basis of the PDL1, PDL2, and CD28 common light chain research campaign. This variable light domain sequence can be used with Figure 25 Figure 26 Figure 29 、 Figure 30 、 Figure 31 Figure 32 Figure 41 、 Figure 42 、 Figure 43 、 Figure 44 、 Figure 45 、 Figure 46 、 Figure 47 、 Figure 48 、 Figure 49 、 Figure 50 、 Figure 51 、 Figure 52 、 Figure 53 、 Figure 54 、 Figure 55 、 Figure 56 、 Figure 57 、 Figure 58 、 Figure 59 、 Figure 60 、 Figure 61 、 Figure 62 、 Figure 63 Figure 64 Figure 65 、 Figure 66 、 Figure 67 、 Figure 68 、 Figure 69 、 Figure 70 、 Figure 71 、 Figure 72 、 Figure 73 and Figure 74 Variable heavy domain pairings depicted.

[0092] Figure 25The novel PDL1 binding domain clone 13G1 is depicted. The CDRs are underlined, and slashes indicate one or more boundaries between the variable region and the constant domain. As noted herein and for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore, not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0093] FIG. 26A to FIG. 26B Depicted are the novel PDL1 binding domain clone 13G7, alternative VH 13G7_H2, alternative VL 13G7_L2, and exemplary affinity engineered VH variants (in addition, it should be noted that the CDRs in the affinity engineered VH can be grafted onto alternative parental frameworks as described in Example 1B). It should be noted that 13G7_H1 and 13G7_H2, as well as affinity engineered 13G7 VH variants, can additionally be combined with Figure 24 13G7 VH is paired with the 6B1_L1 described. In addition, the common framework regions (FRs) and complementary determining regions (CDRs) of 13G7 VH are depicted. The CDRs are underlined, and the slashes indicate one or more boundaries between the variable region and the constant domain. As indicated herein and for each sequence containing CDRs herein, as shown in Table 2, the precise identification of the CDR positions may be slightly different depending on the numbering used, and therefore not only the underlined CDRs but also the CDRs included in the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figure, these VH and VL sequences can be used in scFv format or Fab format.

[0094] Figure 27 Depicted are the dissociation constant (KD), association rate (ka), and dissociation rate (kd) of PDL1 antibodies produced by single-cell technology in mice genetically engineered to have a fully human heavy chain variable domain combined with human common light chain substitutions.

[0095] Figure 28 A to Figure 28T depicts the variable heavy chain sequence and variable light chain sequence of additional PDL1 binding domains that can be used in the PDL1 x CD28 bsAb and PDL1 x PDL2 x CD28 triAb of the present invention. As indicated herein and for each sequence containing CDRs herein, as shown in Table 2, the exact designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figure, these VH and VL sequences can be used in either scFv or Fab format.

[0096] Figure 29 Depicted are the novel PDL2 binding domain clone 5C11, and the alternative VH 5C11_H2. It should be noted that both 5C11_H1 and 5C11_H2 can additionally be used as Figure 24 6B1_L1 pairing depicted. As indicated herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0097] Figure 30 Depicted are the novel PDL2 binding domain clone 8G2, and the alternative VH 8G2_H2. It should be noted that both 8G2_H1 and 8G2_H2 can additionally be used as Figure 24 6B1_L1 pairing depicted. As indicated herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0098] Figure 31 Depicted are the novel PDL2 binding domain clone 8G5, and the alternative VH 8G5_H3. It should be noted that both 8G5_H1 and 8G5_H3 can additionally be used as Figure 246B1_L1 pairing depicted. As indicated herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0099] Figures 32A to 32D Depicted are the novel PDL2 binding domain clone 16G11, as well as alternative VHs 16G11_H2 and 16G11_H3 and exemplary affinity engineered VH variants (in addition, it should be noted that the CDRs in the affinity engineered VHs can be grafted onto alternative parental frameworks as described in Example 1B). It should be noted that 16G11_H1, 16G11_H2 and 16G11_H3 and affinity engineered 16G11 VH variants can additionally be combined with Figure 24 16G11 VH is depicted. In addition, the consensus framework regions (FRs) and complementarity determining regions (CDRs) of the 16G11 VH are depicted. As indicated herein and for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0100] Figure 33 Depicted are the dissociation constant (KD), association rate (ka), and dissociation rate (kd) of PDL2 antibodies produced by single-cell technology in mice genetically engineered to have a fully human heavy chain variable domain combined with human common light chain substitutions.

[0101] Figure 34 Depicted are the variable heavy and light chain sequences of additional PDL2 binding domains that can be used in the PDL2 x CD28 bsAb and PDL1 x PDL2 x CD28 triAb of the present invention. As noted herein and for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore, not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0102] Figure 35 Depicted are the variable heavy and light chain sequences of 1A7, an exemplary non-superagonist phage-derived CD28 binding domain. As noted herein and for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0103] Figure 36 The sequence of the affinity optimized variable heavy domain from anti-CD28 clone 1A7 is depicted. It should be noted that the variable heavy domain can be used with Figure 35 and Figure 37 Any other variable light domain pairing depicted, including SEQ ID NOs: 2456-2524.

[0104] Figure 37 The sequence of the affinity-optimized variable light domain from anti-CD28 clone 1A7 is depicted. It should be noted that the variable light domain can be used with Figure 35 and Figure 36 Any other variable light domain pairing depicted, including SEQ ID NOs: 2525-2630.

[0105] Figures 38A to 38C Depicted are sequences of exemplary affinity-optimized 1A7 VH / VL pairs. It should be noted that these pairs can be formatted as Fab or scFv. Additionally, in the scFv format, these pairs can be formatted in either a VHVL orientation or a VLVH orientation.

[0106] Figures 39A to 39B The consensus framework regions (FRs) and complementarity determining regions (CDRs) (as in Kabat) of the anti-CD28 clone 1A7 variable heavy domain variants and variable light domain variants are depicted.

[0107] Figure 40 Depicted are exemplary affinity-engineered 1A7 VH / VL pairs and their binding affinities in the context of scFv (in the context of a 1+1 Fab-scFv-Fc bsAb format).

[0108] Figure 41Depicted are the sequences of the novel CD28 binding domain clone 1B1-5.88488, as well as XENP41791 (an anti-CD28 mAb based on 1B1-5.88488) and XENP42418 (an anti-CD28 mAb utilizing the 1B1-5.88488 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0109] Figure 42 Depicted are the sequences of the novel CD28 binding domain clone 1B1-4.88488, as well as XENP41792 (an anti-CD28 mAb based on 1B1-4.88488) and XENP42419 (an anti-CD28 mAb utilizing the 1B1-4.88488 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0110] Figure 43 Depicted are the sequences of the novel CD28 binding domain clone 1D8.88474, as well as XENP41834 (an anti-CD28 mAb based on 1D8.88474) and XENP42420 (an anti-CD28 mAb utilizing the 1D8.88474 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0111] Figure 44Depicted are the sequences of the novel CD28 binding domain clone 1G2-2.88474, as well as XENP41846 (an anti-CD28 mAb based on 1G2-2.88474) and XENP42421 (an anti-CD28 mAb utilizing the 1G2-2.88474 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0112] Figure 45 Depicted are the sequences of the novel CD28 binding domain clone 2G5.88497, as well as XENP41864 (an anti-CD28 mAb based on 2G5.88497) and XENP42423 (an anti-CD28 mAb utilizing the 2G5.88497 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0113] Figure 46 Depicted are the sequences of the novel CD28 binding domain clone 2A3.88497, as well as XENP41882 (an anti-CD28 mAb based on 2A3.88497) and XENP42425 (an anti-CD28 mAb utilizing the 2A3.88497 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0114] Figure 47Depicted are the sequences of the novel CD28 binding domain clone 1D9-3.83967, as well as XENP41907 (an anti-CD28 mAb based on 1D9-3.83967) and XENP42426 (an anti-CD28 mAb utilizing the 1D9-3.83967 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0115] Figure 48 Depicted are the sequences of the novel CD28 binding domain clone 1C9-1.83967, as well as XENP41927 (an anti-CD28 mAb based on 1C9-1.83967) and XENP42429 (an anti-CD28 mAb utilizing the 1C9-1.83967 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0116] Figure 49 Depicted are the sequences of the novel CD28 binding domain clone 1D10-4.83967, as well as XENP41936 (an anti-CD28 mAb based on 1D10-4.83967) and XENP42430 (an anti-CD28 mAb utilizing the 1D10-4.83967 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may be slightly different depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0117] Figure 50Depicted are the sequences of the novel CD28 binding domain clone 1A12.83967, as well as XENP41957 (an anti-CD28 mAb based on 1A12.83967). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0118] Figure 51 Depicted are the sequences of the novel CD28 binding domain clone 1B11.83967, and XENP41949 (an anti-CD28 mAb based on 1B11.83967). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0119] Figure 52 Depicted are the sequences of the novel CD28 binding domain clone 1D10-2.83967, and XENP41935 (an anti-CD28 mAb based on 1D10-2.83967). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0120] Figure 53 Depicted are the sequences of the novel CD28 binding domain clone 1D7.83967, and XENP41904 (an anti-CD28 mAb based on 1D7.83967). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0121] Figure 54 Depicted are the sequences of the novel CD28 binding domain clone 1D3.83967, and XENP41901 (an anti-CD28 mAb based on 1D3.83967). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0122] Figure 55 Depicted are the sequences of the novel CD28 binding domain clone 2B10.88497, as well as XENP41891 (an anti-CD28 mAb based on 2B10.88497). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0123] Figure 56 Depicted are the sequences of the novel CD28 binding domain clone 2B9.88497, and XENP41890 (an anti-CD28 mAb based on 2B9.88497). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0124] Figure 57Depicted are the sequences of the novel CD28 binding domain clone 2B8.88497, as well as XENP41889 (an anti-CD28 mAb based on 2B8.88497). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0125] Figure 58 Depicted are the sequences of the novel CD28 binding domain clone 1G6-1.83967, as well as XENP41877 (an anti-CD28 mAb based on 1G6-1.83967). As noted herein, and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0126] Figure 59 Depicted are the sequences of the novel CD28 binding domain clone 1C7.88474, as well as XENP41874 (an anti-CD28 mAb based on 1C7.88474). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0127] Figure 60 Depicted are the sequences of the novel CD28 binding domain clone 1A5-2.88474, as well as XENP41869 (an anti-CD28 mAb based on 1A5-2.88474). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0128] Figure 61 Depicted are the sequences of the novel CD28 binding domain clone 1A2.88474, as well as XENP41868 (an anti-CD28 mAb based on 1A2.88474). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0129] Figure 62 Depicted are the sequences of the novel CD28 binding domain clone 2F5.88497, as well as XENP41860 (an anti-CD28 mAb based on 2F5.88497). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0130] Figure 63 Depicted are the sequences of the novel CD28 binding domain clone 2E9.88497, and XENP41858 (an anti-CD28 mAb based on 2E9.88497). As noted herein, and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0131] Figures 64A to 64CDepicted are the sequences of the novel CD28 binding domain clone 1A3.88474, as well as XENP41849 (an anti-CD28 mAb based on 1A3.88474) and exemplary affinity-engineered VH variants. In addition, the consensus framework regions (FRs) and complementarity determining regions (CDRs) of the 1A3.88474 VH are depicted. As noted herein and for each sequence herein containing CDRs, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore, not only the underlined CDRs but also the CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0132] Figure 65 Depicted are the sequences of the novel CD28 binding domain clone 1H11.88474, as well as XENP41816 (an anti-CD28 mAb based on 1H11.88474). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0133] Figure 66 Depicted are the sequences of the novel CD28 binding domain clone 1G1-1.88474, as well as XENP41807 (an anti-CD28 mAb based on 1G1-1.88474). As noted herein, and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0134] Figure 67Depicted are the sequences of the novel CD28 binding domain clone 1E6-1.88474, as well as XENP41802 (an anti-CD28 mAb based on 1E6-1.88474). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0135] Figure 68 Depicted are the sequences of the novel CD28 binding domain clone 1A1-5.88488, and XENP41797 (an anti-CD28 mAb based on 1A1-5.88488). As noted herein, and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0136] Figure 69 Depicted are the sequences of the novel CD28 binding domain clone 1E2-5.88488, as well as XENP41781 (an anti-CD28 mAb based on 1E2-5.88488). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0137] Figure 70Depicted are the sequences of the novel CD28 binding domain clone 2F6.88497, as well as XENP41861 (an anti-CD28 mAb based on 2F6.88497) and XENP42422 (an anti-CD28 mAb utilizing the 2F6.88497 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0138] Figure 71 Depicted are the sequences of the novel CD28 binding domain clone 1H2.83967, as well as XENP41880 (an anti-CD28 mAb based on 1H2.83967) and XENP42424 (an anti-CD28 mAb utilizing the 1H2.83967 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0139] Figure 72 Depicted are the sequences of the novel CD28 binding domain clone 1E4-3.83967, as well as XENP41909 (an anti-CD28 mAb based on 1E4-3.83967) and XENP42427 (an anti-CD28 mAb utilizing the 1E4-3.83967 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0140] Figure 73Depicted are the sequences of the novel CD28 binding domain clone 1C2-2.83967, as well as XENP41918 (an anti-CD28 mAb based on 1C2-2.83967) and XENP42428 (an anti-CD28 mAb utilizing the 1C2-2.83967 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0141] Figure 74 Depicted are the sequences of the novel CD28 binding domain clone 1A11.83967, as well as XENP41956 (an anti-CD28 mAb based on 1A11.83967) and XENP42431 (an anti-CD28 mAb utilizing the 1A11.83967 VH and 6B1 common VL). As noted herein and indeed for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0142] Figure 75 Depicted are the maximum BLI responses of CD28 mAbs based on single-cell technology-derived binding domains (WT sequence compared to VH paired with 6B1_L1) binding to the CD28 antigen. The data show that most binding domains retain CD28 binding when paired with 6B1_L1 VL; however, VHs from several clones (e.g., 1B1-4.88488, 2G5.88497, and 1D10-4.83967) exhibited diminished CD28 binding when paired with 6B1_L1.

[0143] Figure 76 Depicted are the maximum BLI responses for binding of CD28 mAbs to the CD28 antigen based on additional single cell technology-derived binding domains.

[0144] Figure 77 Binding of CD28 mAbs to Jurkat cells based on single-cell technology-derived binding domains is depicted.

[0145] Figure 78Binding of CD28 mAbs based on single cell technology-derived binding domains (WT sequence compared to VH paired with the 6B1_L1 common light chain) to Jurkat cells is depicted.

[0146] Figure 79 Depicted is the induction of IL2 secretion by purified T cells stimulated with CD3 (100 ng / mL OKT3) by CD28 mAbs based on binding domains derived from single cell technology.

[0147] Figure 80 Depicted is the induction of IL2 secretion by purified T cells stimulated with CD3 (100 ng / mL OKT3) by a CD28 mAb based on a single cell technology-derived binding domain (WT sequence compared to VH paired with the 6B1_L1 common light chain).

[0148] Figure 81 Depicted are the variable heavy and light chain sequences of additional CD28 binding domains useful in the PDL1 x CD28 and PDL2 x CD28 bsAbs and PDL1 x PDL2 x CD28 triAbs of the present invention. As indicated herein and for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore, not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. Furthermore, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0149] Figures 82A to 82M The bispecific formats of the invention are depicted. Figure 82A A "1+1 Fab-scFv-Fc" format is depicted having a first Fab arm that binds a first antigen and a second scFv arm that binds a second antigen. The 1+1 Fab-scFv-Fc format comprises: a first monomer comprising a first heavy chain variable region (VH1) covalently linked (optionally via a linker) to the N-terminus of a first heterodimeric Fc backbone; a second monomer comprising a single-chain Fv covalently linked (optionally via a linker) to the N-terminus of a second corresponding heterodimeric Fc backbone; and a third monomer comprising a light chain variable region covalently linked to a light chain constant domain, wherein the light chain variable region is complementary to VH1. Figure 82BA "2+1 Fab2-scFv-Fc" format is depicted having a first Fab arm and a second Fab-scFv arm, wherein the Fab binds to a first antigen and the scFv binds to a second antigen. The 2+1 Fab2-scFv-Fc format comprises: a first monomer comprising a first heavy chain variable region (VH1) covalently linked (optionally via a linker) to the N-terminus of a first heterodimeric Fc backbone; a second monomer comprising VH1 of a single chain Fv covalently linked (optionally via a linker) to the N-terminus of a second corresponding heterodimeric Fc backbone; and a third monomer comprising a light chain variable region covalently linked to a light chain constant domain, wherein the light chain variable region is complementary to VH1. Figure 82C A "1+1 common light chain" or "1+1 CLC" format is depicted, having a first Fc comprising a first Fab arm that binds a first antigen and a second Fc comprising a second Fab arm that binds a second antigen. The 1+1 CLC format includes a first monomer comprising VH1-CH1-hinge-CH2-CH3, a second monomer comprising VH2-CH1-hinge-CH2-CH3, and a third monomer comprising VL-CL. VL pairs with VH1 to form a binding domain with binding specificity for a first antigen; and VL pairs with VH2 to form a binding domain with binding specificity for a second antigen. Figure 82D A "2+1 common light chain" or "2+1 CLC" format is depicted, having a first Fc comprising two Fab arms, each of which binds to a first antigen, and a second Fc comprising one Fab arm that binds to a second antigen. The 2+1 CLC format includes a first monomer comprising VH1-CH1-hinge-VH1-CH1-hinge-CH2-CH3, a second monomer comprising VH2-CH1-hinge-CH2-CH3, and a third monomer comprising VL-CL. The VL pairs with the first and second VH1s to form a binding domain with binding specificity for the first antigen; and the VL pairs with the VH2 to form a binding domain with binding specificity for the second antigen. Figure 82EDepicted is a "2+1 mAb-scFv" format having a first Fc comprising an N-terminal Fab arm that binds a first antigen and a second Fc comprising an N-terminal Fab arm that binds the first antigen and a C-terminal scFv that binds a second antigen. The 2+1 mAb-scFv format includes a first monomer comprising VH1-CH1-hinge-CH2-CH3, a second monomer comprising VH1-CH1-hinge-CH2-CH3-scFv, and a third monomer comprising VL-CL. VL pairs with the first VH1 and the second VH1 to form a binding domain with binding specificity for the first antigen. Additional bispecific formats include F) dual scFv, G) single-arm scFv-mAb, H) scFv-mAb, I) bispecific mAb, J) single-arm central scFv, K) mAb-Fv, L) central Fv, and M) trident.

[0150] Figures 83A to 83D The trispecific format of the present invention is depicted. Figure 83A A "1+1+1 stacked Fab2-scFv-Fc" format is depicted, comprising: a first monomer, which comprises, from N-terminus to C-terminus, VH1-CH1-linker-VH2-CH1-hinge-CH2-CH3, wherein CH2-CH3 is a first heterodimeric Fc domain; a second monomer, which comprises, from N-terminus to C-terminus, scFv-linker-CH2-CH3, wherein CH2-CH3 is a second heterodimeric Fc domain complementary to the first heterodimeric Fc domain, and wherein the scFv has a first antigen specificity; and a third monomer, which is a common light chain, which comprises, from N-terminus to C-terminus, VL-CL, wherein VL pairs with VH1 to form an antigen binding domain with a second antigen specificity, and pairs with VH2 to form an antigen binding domain with a third antigen specificity. Figure 83D A "1+1+1 Fab-(Fab-scFv)-Fc format" is depicted, comprising: a first monomer, which is a heavy chain, comprising from N-terminus to C-terminus VH1-CH1-hinge-CH2-CH3, wherein CH2-CH3 is a first heterodimeric Fc domain; a second monomer, which comprises from N-terminus to C-terminus VH2-CH1-linker-scFv-linker-CH2-CH3, wherein CH2-CH3 is a second heterodimeric Fc domain complementary to the first heterodimeric Fc domain, and wherein the scFv has a first antigen specificity; and a third monomer, which is a common light chain, comprising from N-terminus to C-terminus VL-CL, wherein VL pairs with VH1 to form an antigen binding domain with a second antigen specificity, and pairs with VH2 to form an antigen binding domain with a third antigen specificity. Figure 83CA "1+1+1 mAb-scFv" format is depicted, comprising: a first monomer, which is a heavy chain, comprising from N-terminus to C-terminus VH1-CH1-hinge-CH2-CH3, wherein CH2-CH3 is a first heterodimeric Fc domain; a second monomer, which comprises from N-terminus to C-terminus VH2-CH1-hinge-CH2-CH3-linker-scFv, wherein CH2-CH3 is a second heterodimeric Fc domain complementary to the first heterodimeric Fc domain, and wherein the scFv has a first antigen specificity; and a third monomer, which is a common light chain, comprising from N-terminus to C-terminus VL-CL, wherein VL pairs with VH1 to form an antigen binding domain with a second antigen specificity, and pairs with VH2 to form an antigen binding domain with a third antigen specificity. Figure 83D A "1+1+1 stacked Fab2-Fab-Fc" format is depicted, comprising: a first monomer, which is a heavy chain, comprising from N-terminus to C-terminus VH1-CH1-hinge-CH2-CH3, wherein CH2-CH3 is a first heterodimeric Fc domain; a second monomer, which comprises from N-terminus to C-terminus VH2-CH1-linker-VH3-CH1-hinge-CH2-CH3, wherein CH2-CH3 is a second heterodimeric Fc domain complementary to the first heterodimeric Fc domain; and a third monomer, which is a common light chain, comprising from N-terminus to C-terminus VL-CL, wherein VL pairs with VH1 to form an antigen binding domain with a first antigen specificity, pairs with VH2 to form an antigen binding domain with a second antigen specificity, and pairs with VH3 to form an antigen binding domain with a third antigen specificity.

[0151] Figures 84A to 84T Depicted is the sequence of an exemplary PDL1 x CD28 bsAb in a 1 + 1 Fab-scFv-Fc format. The CDRs are underlined, and slashes indicate one or more boundaries between the variable region, Fc region, and constant domain. It should be noted that αPD-L1 xαCD28 bsAbs can utilize variable region, Fc region, and constant domain sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In addition, each of the sequences outlined herein may or may not include the M428L / N434S variant in one or preferably both Fc domains, with the inclusion of M428L / N434S resulting in a longer serum half-life.

[0152] Figure 85The sequence of an exemplary αPD-L1 xαCD28 bsAb in a 2+1 Fab2-scFv-Fc format is depicted. The CDRs are underlined, and slashes indicate one or more boundaries between the variable region, Fc region, and constant domain. The scFv domain has a VH-scFv linker-VL orientation (from N-terminus to C-terminus), but this can be reversed. It should be noted that the scFv domain sequence includes the sequence GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: XX) as the scFv linker between the variable heavy region and the variable light region; however, this linker can be used Figure 6 It should also be noted that the chain 2 sequence includes the sequence GGGGSGGGGSKTHTCPPCP (SEQ ID NO: XX) as a domain linker between the C-terminus of the scFv and the N-terminus of the CH2 domain, which is a "flexible half-hinge" domain linker; however, this linker can be replaced with Figure 7 It should be noted that the αPD-L1 x αCD28 bsAb can utilize variable region, Fc region, and constant domain sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In addition, each of the sequences outlined herein may or may not include the M428L / N434S variant in one or preferably both Fc domains, which results in a longer serum half-life.

[0153] Figure 86 The sequence of an exemplary PDL1 x CD28 bsAb in a 2+1 mAb-scFv format is depicted. The CDRs are underlined, and slashes indicate one or more boundaries between the variable region, linker, Fc region, and constant domain. The scFv domain has a V H -scFv linker-V L orientation (from N-terminus to C-terminus), but this can be reversed. It should be noted that the chain 2 sequence includes the sequence GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: XX) as a domain linker; however, this linker can be replaced with any domain linker (including Figure 6 It should be noted that the αPDL1 x αCD28 bsAbs can utilize variable region, Fc region, and constant domain sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In addition, each of the sequences outlined herein may or may not include the M428L / N434S variant in one or preferably both Fc domains, which results in a longer serum half-life.

[0154] Figures 87A to 87B Depicted are sequences of exemplary PDL2 x CD28 bsAbs in a 1+1 Fab-scFv-Fc format. The CDRs are underlined, and slashes indicate one or more boundaries between the variable region, Fc region, and constant domain. It should be noted that PDL2 x CD28 bsAbs can utilize variable region, Fc region, and constant domain sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In addition, each of the sequences outlined herein may or may not include the M428L / N434S variant in one or preferably both Fc domains, with inclusion of M428L / N434S resulting in a longer serum half-life.

[0155] Figures 88A to 88M Depicted are sequences of exemplary PDL1 x PDL2 x CD28 triAbs in a 1+1+1 stacked Fab2-scFv-Fc format. The CDRs are underlined, and slashes indicate one or more boundaries between the variable region and other domains (e.g., constant domains). It should be noted that PDL1 x PDL2 x CD28 triAbs can utilize variable region, Fc region, and constant domain sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In addition, each of the sequences outlined herein may or may not include the M428L / N434S variant in one or preferably both Fc domains, with inclusion of M428L / N434S resulting in a longer serum half-life.

[0156] Figures 89A to 89B Depicted are sequences of exemplary PDL1 x PDL2 x CD28 triAbs in a 1+1+1 Fab-(Fab-scFv)-Fc format. The CDRs are underlined, and slashes indicate one or more boundaries between the variable region and other domains (e.g., constant domains). It should be noted that PDL1 x PDL2 x CD28 triAbs can utilize variable region, Fc region, and constant domain sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In addition, each of the sequences outlined herein may or may not include the M428L / N434S variant in one or, preferably, both Fc domains, with inclusion of M428L / N434S resulting in a longer serum half-life.

[0157] Figure 90Depicted are sequences of exemplary PDL1 x PDL2 x CD28 triAbs in a 1+1+1 mAb-scFv format. The CDRs are underlined, and slashes indicate one or more boundaries between the variable region and other domains (e.g., constant domains). It should be noted that PDL1 x PDL2 x CD28 triAbs can utilize variable region, Fc region, and constant domain sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In addition, each of the sequences outlined herein may or may not include the M428L / N434S variant in one or preferably both Fc domains, with inclusion of M428L / N434S resulting in a longer serum half-life.

[0158] Figures 91A to 91P Depicted are sequences of exemplary PDL1 x PDL2 x CD28 triAbs in a 1+1+1 stacked Fab2-Fab-Fc format. The CDRs are underlined, and slashes indicate one or more boundaries between the variable region and other domains (e.g., constant domains). It should be noted that PDL1 x PDL2 x CD28 triAbs can utilize variable region, Fc region, and constant domain sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In addition, each of the sequences outlined herein may or may not include the M428L / N434S variant in one or preferably both Fc domains, with inclusion of M428L / N434S resulting in a longer serum half-life.

[0159] Figure 92 Depicted are A) classical T cell / APC interactions, and B) by combining CD3 bispecific antibodies with CD28 bispecific antibodies to replicate classical T cell / APC interactions. In classical T cell / APC interactions, there is a first signal (signal 1) provided by the reactivity of TCR and peptide-MHC and a second signal (signal 2) provided by the CD28 cross-linking of CD80 / CD86 expressed on APC, which together fully activate T cells. In contrast, in the treatment using CD3 bispecific antibodies, only the first signal is provided. The CD28 signal can be provided by CD28 bispecifics, with the idea being to promote activation and proliferation by CD28 co-stimulation.

[0160] Figure 93 Depicted are the introduction of CD28 signaling via a CD28 bispecific antibody and the mitigation of any checkpoint-mediated repression of the added CD28 signal by checkpoint blockade (e.g., PD-1 blockade).

[0161] Figure 94 It is depicted that the PDL1 x CD28 bispecific antibody provides Signal 2 while advantageously further enabling blockade of the PDL1:PD1 interaction.Although not depicted, the PDL2 x CD28 bispecific antibody can similarly provide Signal 2 while advantageously further enabling blockade of the PDL2:PD1 interaction.

[0162] Figure 95 Depicted is the sequence of XENP24118, a PDL1 mAb based on avelumab and an IgG1 backbone with E233P / L234V / L235A / G236del / S267K ablation variants. The CDRs are underlined, and slashes indicate one or more boundaries between the variable and constant domains.

[0163] Figure 96 Figure 3: Expansion of CMV+ T cells following incubation of NLV-loaded MDA-MB-231 cancer cells with purified CD3+ T cells from CMV+ donors and either the αPD-L1 antibody XENP24118 or the αPD-L1 x αCD28 bsAb XENP34963. αPD-L1 x αCD28 bsAb XENP34963 significantly enhanced T cell expansion compared to PD-L1 blockade alone.

[0164] Figure 97 Depicted are the induction of A) IL-2 secretion, B) IFNγ secretion, and C) CD3+ T cell expansion by 1 μg / ml αB7H3 x αCD3 bsAb in combination with αPD-L1 mAb XENP24118 or αPD-L1 x αCD28 bsAb XENP34963. αPD-L1 x αCD28 bsAb enhances the activity of the CD3 bsAb T cell engager.

[0165] Figure 98 Depicted are the changes in cell killing over time after incubation of LNCaP cancer cells (PSMA+) with CD3+ T cells at a 1:1 effector:target ratio and an exemplary CD3 bispecific antibody (αPSMA x αCD3 XENP32220) alone or in combination with XENP36233. The data show that XENP32220 enhanced cell killing compared to incubating cancer cells and T cells alone; however, the addition of αPD-L1 x αCD28 overcame the cancer cells' resistance to the CD3 bispecific antibody and further enhanced cell killing.

[0166] Figure 99Depicted are A) group median changes in tumor volume (as determined by caliper measurement; baseline corrected) over time (days after first dose) and B) tumor volume at day 20 (after first dose) in MC38 (engineered to stably express human PD-L1) and huPBMC-engrafted human CD28 knock-in mice dosed with 5 mg / kg αPD-L1 mAb XENP24118, 8.3 mg / kg αPD-L1 xαCD28 bsAb XENP34963, 6 mg / kg αPD-L1 xαCD28 XENP34961, or PBS control.

[0167] Figure 100 Depicted are the induction of A) IL-2 release and B) IFNγ release by anti-PDL1 clone 2G4 (XENP25859), partially blocking anti-PDL1 (XENP25853), non-blocking anti-PDL1 (XENP25858), and XENP24118 (a benchmark anti-PDL1 mAb based on avelumab). Data show that partially blocking and non-blocking anti-PDL1 clones induce less cytokine release compared to anti-PDL1 clone 2G4.

[0168] Figure 101 Depicted are the blockade of PD1:PDL1 interactions during T cell:cancer cell interactions by αPDL1 xαCD28 bsAbs (XENP36233), partially blocking anti-PDL1 (XENP36232), non-blocking anti-PDL1 (XENP26783), and XENP34963 (a benchmark bsAb based on avelumab with an anti-PDL1 arm) with anti-PDL1 clone 2G4 (as modeled by Jurkat-PD1 cells incubated with CHO-PDL1-CD80-αCD3 and CHO-PDL1-αCD3 cells). Data show that bsAbs containing partially blocking and non-blocking anti-PDL1 clones induced lower activity compared to bsAbs containing anti-PDL1 clone 2G4.

[0169] Figure 102 Depicts the interaction between αPDL1 xαCD28 (XENP36233) and parental PDL1 缺失 Binding of MC38 or MC38 cells transfected to express PDL1 with low or medium-high antigen density.

[0170] Figure 103 Depicts the presence of parental PDL1 缺失 Induction of IL-2 release by αPDL1 xαCD28 (XENP36233) in HEK293T cells or HEK293T cells transfected to express PDL1 at medium or high antigen density.

[0171] Figure 104 Depicted are CD3+ T cells and PDL1 in the presence of A) 10:1 E:T ratio and B) 1:1 E:T ratio. 缺失 Induction of cell killing by αPSMA x αCD3 alone or in combination with αPDL1 x αCD28 XENP36233 in the context of the 22Rv1 cell line. The data show that the αPDL1 x αCD28 bsAb does not synergize with the CD3 bsAb on PDL1-negative cell lines such as 22Rv1.

[0172] Figure 105 Figure 3. Serum concentrations of XENP36764 over time in cynomolgus monkeys. The αPDL1 x αCD28 bsAb exhibited favorable pharmacokinetics.

[0173] Figure 106 Depicted is a diagram of the assumptions used in the mechanism-based PK / PD in silico model.

[0174] Figure 107 Predictions from mechanism-based modeling are depicted, demonstrating A) linear PK at dose levels consistent with typical checkpoint inhibitor regimens, B) trimer formation in tumors indicative of costimulation, and C) sustained PDL1 blockade.

[0175] Figure 108 Depicted are the induction of IL-2 release by an αPDL1 x αCD28 bsAb with an affinity-engineered CD28 binding domain in the presence of an αB7H3 x αCD3 bsAb, CD3+ T cells, and A) MDA-MB-231 or B) DU145-NLR cells. The data show that increasing CD28 affinity results in more potent and more effective IL-2 secretion by the αPDL1 x αCD28 bsAb.

[0176] Figure 109 Depicts the presence of αB7H3 xαCD3 bsAb, CD3+ T cells, and PDL1 低 Induction of cell killing by αPDL1 x αCD28 bsAb with affinity-engineered CD28 binding domain in the context of LnCAP cancer cells. Data showed that increasing CD28 affinity increased PDL1 at a low 1:1 E:T. 低 Targeting of cancer cells.

[0177] Figure 110 Depicts the presence of αB7H3 xαCD3 bsAb, CD3+ T cells, and PDL1 中Induction of cell killing by αPDL1 x αCD28 bsAb with affinity-engineered CD28 binding domain in the context of DU145 cells. The data show that increasing CD28 affinity enhances PDL1 binding at even lower E:T of 0.1:1. 中 Targeting of cancer cells.

[0178] Figure 111 Depicted are changes in tumor volume (as determined by caliper measurement; baseline corrected) over time for individual mice in hPDL1-MC38-engrafted hCD28 knock-in mice administered A) PBS control, B) monovalent αPDL1 mAb XENP36627, and C) XENP37261 with enhanced CD28 binding affinity, and D) tumor volume at day 28.

[0179] Figure 112 Figure 3: Induction of IL-2 release by an αPDL1 x αCD28 bsAb with an affinity-engineered PDL1 binding domain in the presence of an αB7H3 x αCD3 bsAb, CD3+ T cells, and DU145-NLR cells. Data show that increasing PDL1 affinity promotes IL-2 secretion.

[0180] Figure 113 Figure 3. Depicted is the induction of A) IL2 release and B) IFNγ release by αPDL1 mAb XENP24118 and the αPDL1 x αCD28 bsAb XENP38514 with enhanced PDL1 binding in the DC:T cell MLR. Data show that αPDL1 x αCD28 enhances T cell / APC interactions.

[0181] Figure 114 Depicts PD1:PDL1 blockade by an αPDL1 x αCD28 bsAb with an affinity-engineered PDL1-binding domain (binding of a PDL1-mFc fusion to PD1-transfected HEK293T cells). The data demonstrate that the αPDL1 x αCD28 bsAb blocks the interaction between PD1 and PDL1.

[0182] Figure 115 Figure 3: Induction of IL-2 release by an αPDL1 x αCD28 bsAb with an affinity-engineered CD28-binding domain and an affinity-engineered PDL1-binding domain in the presence of SEB-stimulated PBMCs. Data show that increasing PDL1 affinity promotes IL-2 secretion.

[0183] Figure 116Depicted is the induction of IL-2 release by αPDL1 xαCD28 bsAbs with affinity-engineered CD28 binding domains and affinity-engineered PDL1 binding domains in the presence of CD3+ enriched T cells, MDA-MB0231 transfected to express αCD3 scFv (to serve as signal 1), and 1 μg / mL exemplary B7H3 x CD3 bsAbs. The data show that XENP40409 (a non-Xtend analog of XENP40706) with 2G4_H1.12_L1.14 most potently induces IL2 production.

[0184] Figure 117 Depicted are CD28 receptor occupancy on cynomolgus monkey T cells (as indicated by reduced binding of the secondary CD28 mAb) following administration of A) XENP36803 (1X dose, 4X dose, and 10X dose) or B) XENP36764 (4X dose, 10X dose, and 20X dose). The data show CD28 receptor occupancy on T cells up to day 14.

[0185] Figure 118 Depicted are PDL1 receptor occupancy (reduction in free receptor, as indicated by reduced binding of the 2G4-based single-armed PDL1 mAb) on cynomolgus monkey T cells following administration of A) XENP36803 (1X dose, 4X dose, and 10X dose) or B) XENP36764 (4X dose, 10X dose, and 20X dose).

[0186] Figures 119A to 119B Depicted are the proliferation of cynomolgus macaque T cells (as indicated by increased Ki67 expression) following administration of A) XENP36803 (1X dose, 4X dose, and 10X dose) or B) XENP36764 (4X dose, 10X dose, and 20X dose). Notably, the PDL1 x CD28 bsAb selectively induced the proliferation of effector CD4+ and CD8+ T cells (i.e., CD45RA-).

[0187] Figure 120 Depicted in the presence of MDA-MB-231PDL1 + PDL2 +Figure 1. Induction of A) IL-2 secretion and B) IFNγ secretion by T cells of an exemplary B7H3 x CD3 bsAb at 1 μg / ml in combination with XENP37261, XENP40409, and additional PDL1 x CD28 bsAbs utilizing additional PDL1 binding domains obtained from the common light chain research campaign in the presence of cancer cells (1:1 E:T). XENP42047 and XENP42048 utilizing the 13G1 and 13G7 PDL1 CLC binding domains, respectively, exhibited similar potency and efficacy to XENP37261, while additional bsAbs utilizing other PDL1 CLC binding domains had lower potency and / or efficacy.

[0188] Figure 121 Depicted in the presence of MDA-MB-231PDL1 + PDL2 + Figure 1. Induction of A) IL-2 secretion and B) IFNγ secretion by T cells of 1 μg / ml of exemplary B7H3 x CD3 bsAbs in combination with XENP37261, XENP40409, and PDL2 x CD28 bsAbs utilizing a PDL2 binding domain derived from a common light chain research campaign in the presence of cancer cells (1:1 E:T). Most PDL2 x CD28 bsAbs (XENP42051, XENP42052, XENP42053, and XENP42054 utilizing the 5C11, 8G2, 8G5, and 16G11 PDL2 CLC binding domains, respectively), but not all (i.e., XENP42050 utilizing another high-affinity PDL2 CLC binding domain), were as potent and / or more effective than the PDL1 x CD28 bsAb in inducing cytokine release.

[0189] Figure 122 Depicts scenarios where tumor cells have varying levels of PDL1 and PDL2 expression and the relative binding capacities of PDL1 x CD28 bsAb, a combination of PDL1 x CD28 and PDL2 x CD28 bsAbs, and a PDL1 x PDL2 x CD28 triAb (in a 1+1+1 Fab-Fab-scFv format with PDL1 and PDL2 CLC-binding domains and a CD28-binding domain), demonstrating that the triAb targets PDL1 - PDL2 + tumors (assuming saturation, i.e., 100% affinity, where possible (no switch to monovalent interactions as might occur at high concentrations)).

[0190] Figure 123Depicted are the interactions of 1 μg / ml of exemplary murine B7H3 x CD3 bsAb with XENP40409, PDL1 x CD28 bsAbs utilizing the CLC PDL1 binding domain (XENP42047 and XENP42049), PDL2 x CD28 bsAbs utilizing the CLC PDL2 binding domain (XENP42051, XENP42052, XENP42053, and XENP42054), PDL1 x PDL2 x CD28 triAbs (in a 1+1+1 stacked Fab2-scFv-Fc format with PDL2 at the bottom) (XENP42324, XENP42325, XENP42326, XENP42327, XENP42328, XENP42329, XENP42330, and XENP42331), PDL1 x PDL2 x CD28 triAbs (in a 1+1+1 stacked Fab2-scFv-Fc format with PDL1 at the bottom) (XENP42332, XENP42333, XENP42334, XENP42335, XENP42336, XENP42337, XENP42338, and XENP42339), and PDL1 x PDL2 x CD28 Induction of T cell IL-2 secretion by combinations of triAbs (in 1+1+1 mAb-scFv format) (XENP42340, XENP42341, XENP42342, XENP42343, XENP42344, XENP42345, XENP42346, and XENP42347).

[0191] Figure 124Depicted are the interactions of 1 μg / ml of exemplary murine B7H3 x CD3 bsAb with XENP40409, PDL1 x CD28 bsAbs utilizing the CLC PDL1 binding domain (XENP42047 and XENP42049), PDL2 x CD28 bsAbs utilizing the CLC PDL2 binding domain (XENP42051, XENP42052, XENP42053, and XENP42054), PDL1 x PDL2 x CD28 triAbs (in a 1+1+1 stacked Fab2-scFv-Fc format with PDL2 at the bottom) (XENP42324, XENP42325, XENP42326, XENP42327, XENP42328, XENP42329, XENP42330, and XENP42331), PDL1 x PDL2 x CD28 triAbs (in a 1+1+1 stacked Fab2-scFv-Fc format with PDL1 at the bottom) (XENP42332, XENP42333, XENP42334, XENP42335, XENP42336, XENP42337, XENP42338, and XENP42339), and PDL1 x PDL2 x CD28 Induction of T cell IL-2 secretion by combinations of triAbs (in 1+1+1 mAb-scFv format) (XENP42340, XENP42341, XENP42342, XENP42343, XENP42344, XENP42345, XENP42346, and XENP42347).

[0192] Figure 125 Depicted are the relative PDL1, PDL2, and B7H3 expression levels on LCLC-103H, SNU-423, and NCI-H460 cancer cells.

[0193] Figure 126Depicted is the induction of T cell IL-2 secretion by 1 μg / ml of an exemplary B7H3 x CD3 bsAb in combination with XENP40409, PDL1 x CD28 bsAbs utilizing a PDL1 binding domain derived from a common light chain research campaign (XENP42047 and XENP42049), and PDL2 x CD28 bsAbs utilizing a PDL2 binding domain derived from a common light chain as well as campaign (XENP42051, XENP42052, XENP42053, and XENP42054) in the presence of A) LCLC-103H, B) SNU-423, and C) NCI-H460 cancer cells (1:1 E:T). Each bsAb was active against LCLC-103H and SNU-423, with XPL1-13G1 being the most potent PDL1 binding domain (as in XENP42047) and 8G2 being the most potent PDL2 binding domain (as in XENP42052).

[0194] Figures 127A to 127C Depicted are 1 μg / ml of exemplary B7H3 x CD3 bsAbs in combination with PDL1 x PDL2 x CD28 triAbs (in a 1+1+1 stacked Fab2-scFv-Fc format with PDL2 on the bottom) (XENP42324, XENP42325, XENP42326, XENP42327, XENP42328, XENP42329, XENP42330, and XENP42331) and PDL1 x PDL2 x CD28 in the presence of A) LCLC-103H, B) SNU-423, and C) NCI-H460 cancer cells (1:1 E:T). Induction of T cell IL-2 secretion by combinations of triAbs (in a 1+1+1 stacked Fab2-scFv-Fc format with PDL1 at the bottom) (XENP42332, XENP42333, XENP42334, XENP42335, XENP42336, XENP42337, XENP42338, and XENP42339). D) Legend is depicted.

[0195] Figure 128 Depicted are blockade of PD-1 by PDL1 x CD28 and PDL2 x CD28 bsAbs based on various PDL1 and PDL2 binding domains on CHO-PDL1 cells.

[0196] Figure 129 Depicted are blockade of PD-1 by PDL1 x CD28 and PDL2 x CD28 bsAbs based on various PDL1 and PDL2 binding domains on CHO-PDL2 cells.

[0197] Figures 130A to 130C Depicted are the induction of T cell IL-2 secretion by exemplary murine B7H3 x CD3 bsAbs in combination with A) XENP42049 PDL1 x CD28, B) XENP42054 PDL2 x CD28, and C) XENP43461 PDL1 x PDL2 x CD28 in the presence of the LCLC103H-NLR cell line and either PDL1 blockade or PDL2 blockade.

[0198] Figure 131 A control antibody utilizing the RSV binding domain is depicted.

[0199] Figure 132 Depicted is the expression of PDL1 on PC3 cells co-cultured with T cells following treatment with exemplary B7H3 x CD3 bsAbs alone or in combination with an IFNγ-neutralizing mAb. The CD3 bsAbs induce PDL1 and PDL2 expression by promoting IFNγ release.

[0200] Figures 133A to 133C Depicted is IFNγ secretion by T cells co-cultured with PC3 cells and then treated with the indicated antibodies (exemplary B7H3 x CD3 bsAb alone or in combination with XENP40409) for A) 1 day, B) 2 days, and C) 5 days. As PDL1 expression increases, the CD3 bsAb synergizes with PDL1 x CD28 over time.

[0201] Figure 134 Depicted are the induction of IFNγ release in mixed lymphocyte reactions (n=14) following incubation with PBS, XENP43456 (PDL1 x PDL2 x RSV), or anti-PD1 mAb (5 days post-treatment). Blockade of PDL1 and PDL2 is functionally equivalent to blockade of PD1.

[0202] Figure 135 Depicted are IFNγ release from PBMCs treated with the indicated air-dried αCD28 bivalent antibodies (TGN1412 or 1A7 derived) (1 day after treatment). The 1A7 derived anti-CD28 epitope lacks superagonist properties.

[0203] Figures 136A to 136B Depicted with A) PDL1 高 MDA-MB-231 cancer cells (1:1 effector:target ratio; approximately 130,000 PDL1 antigens) and B) PDL1 低Figure 3. Induction of IL-2 release by T cells co-cultured with LNCaP (10:1 effector:target ratio; approximately 13,000 PDL1 antigens) and treated with the indicated concentrations of the indicated PDL1 x PDL2 x CD28 triAbs in combination with an exemplary B7H3 x CD3 bsAb. The PDL1 x PDL2 x CD28 triAbs enhanced IL-2 release on both PDL1-high and PDL1-low cancer cells.

[0204] Figures 137A to 137B Depicted with A) PDL1 高 MDA-MB-231 cancer cells (1:50 effector:target ratio; approximately 130,000 PDL1 antigens) and B) PDL1 低 Figure 3. Induction of redirected T cell cytotoxicity (RTCC; as determined by luminescence 5 days after treatment) by T cells co-cultured with LNCaP (10:1 effector:target ratio; approximately 13,000 PDL1 antigens) and treated with the indicated concentrations of exemplary B7H3 x CD3 bsAbs alone or in combination with 1 μg / ml of the indicated PDL1 x PDL2 x CD28 triAbs. The PDL1 x PDL2 x CD28 triAbs enhanced redirected T cell cytotoxicity at low effector to target ratios and on low PDL1 cancer cells.

[0205] Figures 138A to 138B Depicted are the induction of IL2 release by T cells from A) the first and B) the second CMV+ PBMC donors co-cultured with A431-β2M-deficient cells or A431-β2M-deficient cells stably expressing a fusion of HLA-A2, β2M, and NLV peptides and treated with PDL1 x PDL2 x CD28, PDL1 x PDL2 x RSV, or RSV x CD28 (24 hours post-treatment).

[0206] Figure 139A and Figure 139B The sequences of XENP43734 and XENP43735 (mouse surrogates PDL1 x PDL2 x CD28 and PDL1 x PDL2 x RSV triAbs, respectively) are depicted. It should be noted that XENP43735 can be considered a mouse surrogate for XENP43461 (without Xtend).

[0207] Figures 140A to 140DDepicted are changes in tumor volume (as determined by caliper measurement) over time in individual mice in hPDL1(high)-MC38-engrafted hCD28 knock-in mice dosed with A) PBS control, B) anti-mouse PD1 mAb, and C) XENP43735 (mouse surrogate PDL1 x PDL2 x RSV triAb control), and D) XENP43734 (mouse surrogate PDL1 x PDL2 x CD28 triAb, similar to XENP43461 (without Xtend). Arrows indicate days of dosing.

[0208] Figure 141 Depicted are blockade of PD1:PDL1 and PD1:PDL2 on Jurkat-PD1 cells incubated with A431-αCD3 by bivalent PD-1 mAb or XENP43456 PDL1 x PDL2 x RSV triAb.

[0209] Figures 142A to 142E Depicted are the changes in tumor volume (as determined by caliper measurement) over time in individual mice in MDA-MB-231- and huPBMC-engrafted NSG-DKO mice dosed with A) PBS control, B) an exemplary B7H3 x CD3 bsAb (0.5 mg / kg), C) a combination of PDL1 x PDL2 x CD28 XENP44676 and B7H3 x CD3 bsAb (5 mg / kg and 0.5 mg / kg, respectively), D) a combination of control PDL1 x PDL2 x RSV XENP44796 and B7H3 x CD3 bsAb (5 mg / kg and 0.5 mg / kg, respectively), or E) a combination of control RSV x RSV x CD28 XENP44797 and B7H3 x CD3 bsAb (5 mg / kg and 0.5 mg / kg, respectively).

[0210] Figure 143Depicted are tumor volumes (as determined by caliper measurement) on day 39 in single MDA-MB-231 and huPBMC-engrafted NSG-DKO mice following administration of A) PBS control, B) exemplary B7H3 x CD3 bsAb (0.5 mg / kg), C) a combination of PDL1 x PDL2 x CD28 XENP44676 and B7H3 x CD3 bsAb (5 mg / kg and 0.5 mg / kg, respectively), D) a combination of control PDL1 x PDL2 x RSV XENP44796 and B7H3 x CD3 bsAb (5 mg / kg and 0.5 mg / kg, respectively), or E) a combination of control RSV x RSV x CD28 XENP44797 and B7H3 x CD3 bsAb (5 mg / kg and 0.5 mg / kg, respectively).

[0211] Figure 144 Depicted are CD3+ cell counts in the blood of MDA-MB-231 and huPBMC-engrafted NSG-DKO mice dosed with A) PBS control, B) an exemplary B7H3 x CD3 bsAb (0.5 mg / kg), C) a combination of PDL1 x PDL2 x CD28 x ENP44676 and B7H3 x CD3 bsAb (5 mg / kg and 0.5 mg / kg, respectively), D) a combination of control PDL1 x PDL2 x RSV x ENP44796 and B7H3 x CD3 bsAb (5 mg / kg and 0.5 mg / kg, respectively), or E) a combination of control RSV x RSV x CD28 x ENP44797 and B7H3 x CD3 bsAb (5 mg / kg and 0.5 mg / kg, respectively) on day 7 after the first dose.

[0212] Figure 145 The K of affinity-engineered anti-PDL1 clone 13G7 to human PDL1 is depicted. D Binding constant, association constant (k a ) and dissociation constant (k d ).

[0213] Figure 146 The K of affinity-engineered anti-PDL1 clone 13G7 against cynomolgus monkey PDL1 is depicted. D Binding constant, association constant (k a ) and dissociation constant (k d ).

[0214] Figure 147 The K of affinity-engineered anti-PDL2 clone 16G11 to human PDL2 is depicted. DBinding constant, association constant (k a ) and dissociation constant (k d ).

[0215] Figure 148 The K of affinity-engineered anti-PDL2 clone 16G11 against cynomolgus monkey PDL2 is depicted. D Binding constant, association constant (k a ) and dissociation constant (k d ).

[0216] Figure 149 The K of affinity-engineered anti-PDL1 clone 1A3A4.248 to human PDL1 is depicted. D Binding constant, association constant (k a ) and dissociation constant (k d ).

[0217] Figure 150 The K of affinity-engineered anti-PDL1 clone 1A3A4.248 to cynomolgus monkey PDL1 is depicted. D Binding constant, association constant (k a ) and dissociation constant (k d ).

[0218] Figure 151 The K of affinity engineered anti-PDL2 clone 1F12A4.249 (round 1) to human PDL2 is depicted. D Binding constant, association constant (k a ) and dissociation constant (k d ).

[0219] Figure 152 Figure 3 depicts the K affinity of affinity engineered anti-PDL2 clone 1F12A4.249 (round 1) against cynomolgus monkey PDL2. D Binding constant, association constant (k a ) and dissociation constant (k d ).

[0220] Figures 153A to 153B The K of affinity engineered anti-PDL2 clone 1F12A4.249 (round 2) to human PDL2 is depicted. D Binding constant, association constant (k a ) and dissociation constant (k d ).

[0221] Figures 154A to 154B Figure 3 depicts the K affinity of affinity engineered anti-PDL2 clone 1F12A4.249 (round 2) against cynomolgus monkey PDL2. D Binding constant, association constant (k a ) and dissociation constant (kd ).

[0222] Figure 155 Depicts the presence of A) LnCAP(PDL1 低 PDL2 缺失 ), B)DU145(PDL1 中 PDL2 缺失 ) or C)LCLC103H(PDL1 高 PDL2 中 ) tumor cells. Induction of IL-2 secretion by 1 μg / mL of an exemplary B7H3 x CD3 bsAb in combination with PDL1 x PDL2 x CD28 in 1+1+1 Fab-(Fab-scFv)-Fc (with CD28 scFv in either VHVL or VLVH orientation), 1+1+1 stacked Fab2-scFv-Fc, and 1+1+1 mAb-scFv formats.

[0223] Figure 156 Depicts the presence of DU145 (PDL1 中 PDL2 缺失 Figure 2. Induction of IL-2 secretion by 1 μg / mL of an exemplary B7H3 x CD3 bsAb in combination with PDL1 x PDL2 x CD28 in a 1+1+1 stacked Fab2-scFv-Fc with either WT scFv (XENP43461) or stapled scFv (XENP43462) in the presence of WT (XENP43461) ) tumor cells (1:1 effector:target ratio).

[0224] Figure 157 Depicted is the induction of IL-2 secretion by 1 μg / mL of an exemplary B7H3 x CD3 bsAb in combination with PDL1 x PDL2 x CD28 utilizing either the 1A7 or 1A3 CD28 binding domains in various formats and orientations in the presence of DU145 tumor cells. The data show that XENP43461 with the 1A7 binding domain outperformed triAbs with the 1A3 binding domain in all formats (PDL1 and PDL2 binding domains matched).

[0225] Figure 158 Depicted is the induction of IL-2 secretion by 1 μg / mL of an exemplary B7H3 x CD3 bsAb in combination with PDL1 x PDL2 x CD28 of 1A7 utilizing a germline VL and affinity-optimized VH in the presence of DU145 tumor cells. The data show that several full CLC triAbs achieved potency comparable to that of XENP43465.

[0226] Figure 159Depicted are the EC50 for induction of IL-2 secretion by 1 μg / mL of an exemplary B7H3 x CD3 bsAb in combination with PDL1 x PDL2 x CD28 utilizing 1A7 with germline VL and affinity-optimized VH, and the monovalent K for CD28 binding by the affinity-engineered 1A7 binding domain. D The data showed that several full-CLCtriAbs achieved potency comparable to that of XENP43465.

[0227] Figure 160 Depicted is the induction of IL-2 secretion by 1 μg / mL of an exemplary B7H3 x CD3 bsAb in combination with PDL1 x PDL2 x CD28 utilizing a 1A7 or 1A3 derived CD28 binding domain in the presence of Caki-1 tumor cells (1:1 effector:target). The data show that the 1A7-based full CLC triAb is more potent and efficacious than the 1A3-based full CLC triAb.

[0228] Figures 161A to 161C Depicted are novel PDL1 binding domain clone 2A3A4.248, as well as exemplary affinity engineered VH 2A3A4.248_H1.9 (additional variants are depicted as SEQ ID NOs: 3235-3266) that can be paired with L1 (note that L1 is an IGKV1-39 germline sequence). In addition, the consensus framework regions (FRs) and complementarity determining regions (CDRs) of the 1F12A4.249 VH are depicted. As noted herein and indeed for each sequence herein containing CDRs, as shown in Table 2, the precise designation of the CDR positions may vary slightly depending on the numbering used, and therefore not only the underlined CDRs but also CDRs included within VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0229] Figures 162A to 162GDepicted is a novel PDL2 binding domain clone 1F12A4.249, as well as exemplary affinity engineered VH 1F12A4.249_H1.45 (additional variants are depicted as SEQ ID NOs: 3267-3353) that can be paired with L1 (it should be noted that L1 is an IGKV1-39 germline sequence). In addition, the consensus framework regions (FRs) and complementarity determining regions (CDRs) of the 1F12A4.249 VH are depicted. As indicated herein and for each sequence containing CDRs herein, as shown in Table 2, the precise designation of the CDR positions may be slightly different depending on the numbering used, and therefore, not only the underlined CDRs but also the CDRs included within the VH and VL domains using other numbering systems are included herein. In addition, for all sequences in the figures, these VH and VL sequences can be used in either scFv or Fab formats.

[0230] Figures 163A to 163B Depicted are the novel 1A7 VH affinity engineered to pair with the IGKV1-39 germline sequence, along with the consensus framework regions (FRs) and complementarity determining regions (CDRs).

[0231] Figures 164A to 164D Depicted are exemplary 1A7-based scFvs with engineered cysteines for disulfide bond stabilization.

[0232] Figure 165 Depicted are the CD28 affinities of additional 1A7 scFv variants with or without disulfide stabilization (as indicated by [SS]) obtained from different experiments in the context of different CD28 multispecific antibodies.

[0233] Figure 166 Depicted is the increase in thermal stability of the disulfide-stabilized 1A7 scFv. DETAILED DESCRIPTION

[0234] I. Overview

[0235] Provided herein are novel anti-PD-L1, anti-PD-L2, and anti-CD28 antibodies, including novel anti-CD28 x anti-PD-L1 and anti-CD28 x anti-PD-L2 bispecific antibodies, as well as novel anti-CD28 x anti-PD-L1 x anti-PD-L2 trispecific antibodies. Also provided herein are methods for preparing such antibodies and using such antibodies to treat cancer. The subject bispecific and trispecific antibodies are capable of agonistically binding to CD28 costimulatory molecules on T cells and targeting PD-L1 and / or PD-L2 on tumor cells. Thus, such antibodies selectively enhance anti-tumor activity at the tumor site while minimizing peripheral toxicity. The subject antibodies provided herein are particularly useful for enhancing anti-tumor activity when used in combination with other anti-cancer therapies.

[0236] Therefore, on the one hand, provided herein are heterodimeric antibodies that are bound to two or three different antigens. In some embodiments, the antibody is "bispecific" and binds to two different target antigens, typically CD28 and PD-L1 or CD28 and PD-L2 as described below. In some embodiments, the antibody is "trispecific" and binds to three different target antigens, typically CD28, PD-L1 and PD-L2 as described below. These heterodimeric antibodies can be monovalent (e.g., there is a single antigen binding domain, such as a variable heavy domain and a variable light domain pair) or bivalent (there are two antigen binding domains that each independently bind to an antigen) to bind to each of the target antigens. In some embodiments, provided herein are heterodimeric antibodies comprising 1) a CD28 binding domain, and 2) a PD-L1 or a PD-L2 binding domain (e.g., a heterodimeric antibody in a "1+1 Fab-scFv-Fc" format as described herein). In other embodiments, the heterodimeric antibodies provided herein comprise 1) one CD28 binding domain, and 2) two PD-L1 or PD-L2 binding domains (e.g., heterodimeric antibodies in the format of "2+1 Fab2-scFv-Fc" as described herein). In some embodiments, the heterodimeric antibodies provided herein are trispecific antibodies that comprise three different antigen binding domains, each of which binds to a different target antigen, typically PD-L1, PD-L2, and CD28 (e.g., heterodimeric antibodies in the format of "1+1+1 stacked Fab2-scFv-Fc", "1+1+1 Fab-(Fab-scFv)-Fc", "1+1+1 mAb-scFv", and "1+1+1 stacked Fab2-Fab-Fc", Figure 83). The heterodimeric antibodies provided herein are based on the use of different monomers containing amino acid substitutions (i.e., "skew variants") that are biased towards forming heterodimers rather than homodimers, as more fully outlined below. In some embodiments, the heterodimeric antibodies are also coupled to "pI variants" to allow for simple purification of the heterodimers from the homodimers, as similarly outlined below. The heterodimeric bispecific antibodies provided generally rely on the use of engineered or variant Fc domains that can self-assemble in production cells to produce heterodimeric proteins, as well as methods for producing and purifying such heterodimeric proteins.

[0237] II. Nomenclature

[0238] The antibodies provided herein are listed in several different formats. In some cases, each monomer in a particular antibody is given a unique "XENP" number, but as will be understood in the art, longer sequences may contain shorter XENP numbers. For example, the "scFv-Fc" monomer of an antibody in a 1+1 Fab-scFv-Fc format may have a first XENP number, while the scFv domain itself will have a different XENP number. Some molecules have three polypeptides, so the XENP number and component are used as the name. Thus, the molecule XENP34961 in a 2+1 Fab2-scFv-Fc format comprises three sequences (see Figure 85 A): "Fab-Fc heavy chain" monomer ("chain 1"); 2) "Fab-scFv-Fc heavy chain" monomer ("chain 2"); and 3) "light chain" monomer or equivalent, but one skilled in the art will be able to readily identify these sequences through sequence alignment. These XENP numbers are provided in the sequence listings and identifiers and are used in the accompanying drawings. In addition, a molecule comprising three components generates multiple sequence identifiers. For example, the listing for Fab includes the full heavy chain sequence, the variable heavy domain sequence, and the three CDRs of the variable heavy domain sequence, the full light chain sequence, the variable light domain sequence, and the three CDRs of the variable light domain sequence. The Fab-scFv-Fc monomer includes the full-length sequence, the variable heavy domain sequence, the three heavy chain CDR sequences, and the scFv sequence (including the scFv variable heavy domain sequence, the scFv variable light domain sequence, and the scFv linker). It should be noted that some molecules with scFv domains herein use a single charged scFv linker (+H), but other linkers may also be used. Additionally, the nomenclature for specific antigen binding domains (e.g., PD-L1, PD-L2, and CD28 binding domains) uses a format such as "Hx.xx_Ly.yy" or "Hx.xxLy.yy," where the numbering serves as a unique identifier for a specific variable chain sequence. Thus, the variable domain on the Fab side of the PD-L1 binding domain 2G4[PDL1] (e.g., Figure 17 ) is "H1L1", which indicates that the variable heavy domain H1 is combined with the light domain L1. In the case where these sequences are used as scFv, the designation "H1_L1" or "H1L1" indicates that the variable heavy domain H1 is combined with the light domain L1 and is in a VH-linker-VL orientation from N-terminus to C-terminus. Such a molecule with the same heavy variable domain sequence and light variable domain sequence but in reverse order (VL-linker-VH orientation from N-terminus to C-terminus) will be designated as "L1_H1". Similarly, different constructs may "mix and match" heavy and light chains, as will be apparent from the sequence listing and accompanying drawings.

[0239] III. Definitions

[0240] In order that this application may be more fully understood, several definitions are set forth below. Such definitions are intended to encompass grammatical equivalents.

[0241] "CD28", "cluster of differentiation 28" and "Tp44" herein (e.g., Genebank accession numbers NP_001230006 (human), NP_001230007 (human), NP_006130 (human) and NP_031668 (mouse)) refer to the B7 receptor expressed on T cells, which provides the co-stimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation by CD28 also provides a powerful signal for the production of various interleukins. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2) proteins. CD28 contains an intercellular domain with a YMNM motif, which is critical for recruiting SH2 domain-containing proteins, particularly PI3K. CD28 also contains two proline-rich domains, which are capable of binding to SH3-containing proteins. An exemplary CD28 sequence is depicted in Figure 1 middle.

[0242] As used herein, "ablation" refers to a reduction or elimination of an activity. Thus, for example, "ablation of FcγR binding" means that the Fc region amino acid variant has less than 50% of the initial binding compared to an Fc region not containing the particular variant, with a loss of activity exceeding 70%-80%-90%-95%-98% being preferred, and generally, the activity is below the level of detectable binding in a Biacore, SPR or BLI assay. Particularly useful in ablating FcγR binding are Figure 5 Those shown, they are usually added to two monomers.

[0243] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC is associated with binding to FcγRIIIa; increased binding to FcγRIIIa results in increased ADCC activity.

[0244] As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis refers to a cell-mediated reaction in which nonspecific phagocytes expressing FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.

[0245] As used herein, the term "antibody" is used in a generic manner. The antibodies provided herein can take a variety of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments and mimetics as described herein.

[0246] Traditional immunoglobulin (Ig) antibodies are "Y"-shaped tetramers. Each tetramer is typically composed of two pairs of identical polypeptide chains, each pair having one "light chain" monomer (usually with a molecular weight of about 25 kDa) and one "heavy chain" monomer (usually with a molecular weight of about 50-70 kDa).

[0247] Useful bispecific antibody formats include, but are not limited to, the "1+1 Fab-scFv-Fc", "2+1 Fab2-scFv-Fc", "1+1 common light chain", and "2+1 common light chain" formats provided herein (see, e.g., Figure 82). Useful trispecific antibody formats include, but are not limited to, 1+1+1 stacked Fab2-scFv-Fc", "1+1+1 Fab-(Fab-scFv)-Fc", "1+1+1 mAb-scFv", and "1+1+1 stacked Fab2-Fab-Fc" formats (see, e.g., Figure 83). Additional useful antibody formats include, but are not limited to, "mAb-Fv", "mAb-scFv", "central Fv", "single-arm scFv-mAb", "scFv-mAb", "double scFv", and "trident" format antibodies as disclosed in US20180127501A1, which is incorporated herein by reference, particularly with respect to the relevant portions relating to antibody formats (see, e.g., Figure 2 ).

[0248] An antibody heavy chain typically comprises a variable heavy (VH) domain (including vhCDR1-3) and an Fc domain (including a CH2-CH3 monomer). In some embodiments, an antibody heavy chain comprises a hinge and a CH1 domain. Traditional antibody heavy chains are monomers organized from N-terminus to C-terminus as follows: VH-CH1-hinge-CH2-CH3. CH1-hinge-CH2-CH3 are collectively referred to as the heavy chain "constant domain" or "constant region" of an antibody, of which there are five different classes or "isotypes": IgA, IgD, IgG, IgE, and IgM.

[0249] In some embodiments, the antibodies provided herein include IgG isotype constant domains, and the IgG isotype constant domains have several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. In immunoglobulin IgG subclasses, there are several immunoglobulin domains in the heavy chain. "Immunoglobulin (Ig) domain" herein means the region of immunoglobulins with different tertiary structures. The present invention focuses on heavy chain domains, including constant heavy (CH) domains and hinge domains. In the case of IgG antibodies, IgG isotypes each have three CH regions. Therefore, in the case of IgG, "CH" domains are as follows: "CH1" refers to positions 118-215 according to the EU index as in Kabat. "Hinge" refers to positions 216-230 according to the EU index as in Kabat. "CH2" refers to positions 231-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat. As shown in Table 1, the exact numbering and position of the heavy chain domains may be different in different numbering systems. As shown herein and described below, pi variants can be in one or more of the CH regions as well as the hinge region, as discussed below.

[0250] It should be noted that IgG1 has different allotypes, with polymorphisms at 356 (D or E) and 358 (L or M). The sequences depicted herein use the 356E / 358M allotype, however other allotypes are contemplated herein. That is, any sequence comprising an IgG1 Fc domain as contemplated herein may have 356D / 358L, instead of the 356E / 358M allotype. It should be understood that therapeutic antibodies may also comprise hybrids of isotypes and / or subclasses. For example, as shown in U.S. Publication 2009 / 0163699, incorporated by reference, in some embodiments, the antibodies of the present invention comprise human IgG1 / G2 hybrids.

[0251] As used herein, "Fc" or "Fc region" or "Fc domain" means a polypeptide that comprises the constant region of an antibody, in some cases, excluding all or a portion of the first constant region immunoglobulin domain (e.g., CH1), and in some cases, optionally comprising all or a portion of a hinge. For IgG, the Fc domain comprises immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3), and optionally all or a portion of the hinge region between CH1 (Cγ1) and CH2 (Cγ2). Thus, in some cases, the Fc domain comprises CH2-CH3 and hinge-CH2-CH3 from N-terminus to C-terminus. In some embodiments, the Fc domain is an Fc domain from IgG1, IgG2, IgG3, or IgG4, wherein IgG1 hinge-CH2-CH3 and IgG4 hinge-CH2-CH3 have particular uses in many embodiments. Additionally, in the case of a human IgG1 Fc domain, the hinge may comprise a C220S amino acid substitution. Additionally, in the case of a human IgG4 Fc domain, the hinge may comprise an S228P amino acid substitution. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as comprising residues E216, C226, or A231 at its carboxyl terminus, where numbering is according to the EU index as in Kabat. In some embodiments, as described more fully below, amino acid modifications are made to the Fc region, for example, to alter binding to one or more FcγRs or FcRn.

[0252] By "heavy chain constant region" herein is meant the CH1-hinge-CH2-CH3 portion of an antibody (or fragment thereof), excluding the variable heavy domain; in EU numbering of human IgG1, this is amino acids 118-447. By "heavy chain constant region fragment" herein is meant a heavy chain constant region that contains fewer amino acids from either or both the N-terminus or the C-terminus but still retains the ability to form a dimer with another heavy chain constant region.

[0253] The domain of another type of heavy chain is hinge region. " Hinge " or " hinge region " or " antibody hinge region " or " hinge domain " herein means the flexible polypeptide of the amino acid between the first constant domain and the second constant domain that comprise antibody. Structurally, IgG CH1 domain terminates at EU position 215, and IgG CH2 domain starts at residue EU position 231. Therefore, for IgG, antibody hinge is defined herein as comprising position 216 (E216 in IgG1) to 230 (P230 in IgG1), wherein numbering is according to the EU index as in Kabat. In some cases, " hinge fragment " is used, and it contains less amino acid at one or both ends in the N-terminal and C-terminal of hinge domain. As noted herein, pi variants can also be prepared in hinge region. Many antibodies herein have at least one cysteine ​​replaced by serine at position 220 according to EU numbering (hinge region). Generally, this modification is on the "scFv monomer" side of most of the sequences depicted herein, but it can also be on the "Fab monomer" side or on both to reduce disulfide bond formation. Specifically included in the sequences herein are substitutions of one or both of these cysteines (C220S).

[0254] As will be appreciated by those skilled in the art, the exact numbering and position of the heavy chain constant region domains (i.e., CH1, hinge, CH2, and CH3 domains) may vary in different numbering systems. A useful comparison of the heavy constant region numbering according to EU and Kabat is as follows, see Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85 and Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, United States Public Health Service, National Institutes of Health, Bethesda, which are incorporated by reference in their entirety.

[0255] Table 1

[0256] EU Number Kabat numbering CH1 118-215 114-223 hinge 216-230 226-243 CH2 231-340 244-360 CH3 341-447 361-478

[0257] The antibody light chain is generally composed of two domains: a variable light domain (VL), which includes light chain CDR v1CDR1-3; and a constant light chain region (usually referred to as CL or CK). The antibody light chain is generally organized from N-terminus to C-terminus as follows: VL-CL.

[0258] By "antigen binding domain" or "ABD" herein is meant a set of six complementary determining regions (CDRs) that specifically bind to a target antigen as discussed herein (e.g., PD-L1 or CD28) when present as part of a polypeptide sequence. As is known in the art, these CDRs are typically present as a first set of variable heavy CDRs (vhCDRs or VHCDRs) and a second set of variable light CDRs (vlCDRs or VLCDRs), each set of CDRs comprising three CDRs: vhCDR1, vhCDR2, vhCDR3 variable heavy CDRs, and vlCDR1, vlCDR2, and vlCDR3 vhCDR3 variable light CDRs. The CDRs are present in the variable heavy domain (vhCDR1-3) and the variable light domain (vlCDR1-3). The variable heavy domain and the variable light domain are from the Fv region.

[0259] The present invention provides a large number of different CDR groups. In this case, a "full CDR group" comprises three variable light CDRs and three variable heavy CDRs, such as vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2, and vhCDR3. These can be part of a larger variable light domain or a variable heavy domain, respectively. In addition, as more fully outlined herein, when heavy and light chains are used (e.g., when Fab is used), the variable heavy domain and the variable light domain can be on separate polypeptide chains, or on a single polypeptide chain in the case of an scFv sequence.

[0260] As will be appreciated by those skilled in the art, the exact numbering and position of the CDRs may vary in different numbering systems. However, it will be understood that disclosure of a variable heavy sequence and / or variable light sequence includes disclosure of the associated (intrinsic) CDRs. Thus, disclosure of each variable heavy region is disclosure of a vhCDR (e.g., vhCDR1, vhCDR2, and vhCDR3), and disclosure of each variable light region is disclosure of a vlCDR (e.g., vlCDR1, vlCDR2, and vlCDR3). A useful comparison of CDR numbering is as follows, see Lafranc et al., Dev. Comp. Immunol. 27(1): 55-77 (2003):

[0261] Table 2

[0262]

[0263] Throughout this specification, the Kabat numbering system (e.g., Kabat et al., supra (1991)) is generally used when referring to residues in the variable domain (approximately residues 1-107 in the light chain variable region and residues 1-113 in the heavy chain variable region), as well as the EU numbering system for the Fc region.

[0264] CDRs contribute to the formation of the antigen-binding domain and the antigen-binding site of an antibody, or more specifically, the epitope binding site. An "epitope" refers to a determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, called a paratope. An epitope is a group of molecules, such as amino acids or sugar side chains, and typically has specific structural characteristics as well as specific charge characteristics. A single antigen can have more than one epitope.

[0265] An epitope can include amino acid residues that are directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by the specific antigen-binding peptide; in other words, the amino acid residues are within the coverage area of ​​the specific antigen-binding peptide.

[0266] Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. Conformational and nonconformational epitopes can be distinguished by the loss of binding to the former, while the binding to the latter is not lost, in the presence of denaturing solvents.

[0267] In a unique spatial conformation, an epitope typically comprises at least 3 and more usually at least 5 or 8-10 amino acids. Antibodies that recognize the same epitope can be validated using a simple immunoassay that shows the ability of one antibody to block the binding of another antibody to the target antigen, such as "binning." As outlined below, the present invention includes not only the antigen-binding domains and antibodies enumerated herein, but also antigen-binding domains and antibodies that compete for binding to the epitope to which the enumerated antigen-binding domains bind.

[0268] In some embodiments, the six CDRs of the antigen-binding domains are derived from variable heavy domains and variable light domains. In a "Fab" format, one group of six CDRs is derived from two different peptide sequences, variable heavy domains (vh or VH; containing vhCDR1, vhCDR2 and vhCDR3) and variable light domains (vl or VL; containing vlCDR1, vlCDR2 and vlCDR3), the C-terminus of the vh domain is connected to the N-terminus of the CH1 domain of the heavy chain and the C-terminus of the vl domain is connected to the N-terminus of the constant light domain (and thus forming a light chain). In the scFv format, vh and vl domains are typically covalently linked into a single polypeptide sequence by using a joint ("scFv joint") as outlined herein, which can be (from N-terminus) vh-joint-vl or vl-joint-vh, the former being typically preferred (including the optional domain joint on each side, depending on the format used) (e.g., from Figure 26). Generally, the C-terminus of the scFv domain is linked to the N-terminus of the hinge in the second monomer.

[0269] As used herein, "variable region" or "variable domain" means a region of an immunoglobulin that comprises one or more Ig domains that are substantially encoded by any of the Vκ, Vλ and / or VH genes that constitute the kappa, lambda and heavy chain immunoglobulin loci, respectively, and that contains the CDRs that confer antigen specificity. Thus, a "variable heavy domain" is paired with a "variable light domain" to form an antigen binding domain ("ABD"). In addition, each variable domain comprises three hypervariable regions ("complementarity determining regions," "CDRs") arranged in the following order from amino-terminus to carboxyl-terminus (vhCDR1, vhCDR2 and vhCDR3 of the variable heavy domain, and vlCDR1, vlCDR2 and vlCDR3 of the variable light domain) and four framework (FR) regions: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0270] As used herein, "Fab" or "Fab region" refers to the region of an antibody that contains the VH, CH1, VL, and CL immunoglobulin domains, typically on two different polypeptide chains (e.g., VH-CH1 on one chain and VL-CL on another chain). Fab can refer to this region in isolation or in the context of the bispecific antibodies of the present invention. In the case of Fab, in addition to the CH1 and CL domains, Fab also contains an Fv region.

[0271] As used herein, "Fv" or "Fv fragment" or "Fv region" refers to the region of an antibody that comprises the VL domain and the VH domain. The Fv region can be formatted as two Fabs (typically two different polypeptides that also comprise a constant region as outlined above, as discussed above) and single-chain Fvs (scFv), in which the VL and VH domains are contained in a single peptide, typically connected with a linker as discussed herein.

[0272] "Single-chain Fv" or "scFv" herein means a variable heavy domain that is covalently linked to a variable light domain to form an scFv or scFv domain, typically using an scFv linker as discussed herein. The scFv domain can be in either orientation (vh-linker-vl or vl-linker-vh) from N-terminus to C-terminus. In the sequences depicted in the sequence listing and accompanying drawings, the order of the vh and vl domains is indicated by the name, for example, H.X_L.Y means vh-linker-vl from N-terminus to C-terminus, and L.Y_H.X means vl-linker-vh from N-terminus to C-terminus.

[0273] Some embodiments of the subject antibodies provided herein comprise at least one scFv domain that, although not naturally occurring, typically comprises a variable heavy domain and a variable light domain connected together by an scFv linker. As outlined herein, while scFv domains are typically oriented from N-terminus to C-terminus as VH-scFv linker-VL, for any scFv domain (or scFv domain constructed using the vh and vl sequences of a Fab), this can be reversed to VL-scFv linker-VH, with an optional linker at one or both ends depending on the format.

[0274] As used herein, "modification" or "variant" refers to amino acid substitutions, insertions, and / or deletions in a polypeptide sequence, or changes to moieties chemically linked to a protein. For example, a modification can be a change in the carbohydrate or PEG structure linked to a protein. As used herein, "amino acid modification" refers to amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. For clarity, unless otherwise indicated, amino acid modifications are always to amino acids encoded by DNA, e.g., the 20 amino acids having codons in DNA and RNA.

[0275] As used herein, "amino acid substitution" or "substitution" means replacing an amino acid at a specific position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is for an amino acid that does not naturally occur at a specific position, does not naturally occur in an organism, or in any organism. For example, the substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced by tyrosine. For clarity, proteins that have been engineered to change the nucleic acid coding sequence but do not change the starting amino acid (e.g., replacing CGG (encoding arginine) with CGA (still encoding arginine) to increase expression levels in the host organism) are not "amino acid substitutions"; that is, although a new gene encoding the same protein is generated, if the protein has the same amino acid at the specific position where it starts, then the protein is not an amino acid substitution.

[0276] As used herein, "amino acid insertion" or "insertion" means adding an amino acid sequence at a specific position in a parent polypeptide sequence. For example, -233E or 233E indicates the insertion of glutamic acid after position 233 and before position 234. In addition, -233ADE or A233ADE indicates the insertion of AlaAspGlu after position 233 and before position 234.

[0277] As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid sequence at a specific position in a parent polypeptide sequence. For example, E233- or E233#, E233(), or E233del indicates a deletion of glutamic acid at position 233. In addition, EDA233- or EDA233# indicates a deletion of the sequence GluAspAla starting at position 233.

[0278] As used herein, "variant protein" or "protein variant" or "variant" refers to a protein that differs from a parent protein by at least one amino acid modification. A protein variant has at least one amino acid modification compared to the parent protein, but not so many that the variant protein will not align with the parent protein using an alignment program as described below. In general, variant proteins (such as the variant Fc domains outlined herein, etc.) are typically at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the parent protein using an alignment program (such as BLAST) described below.

[0279] As used herein, "variant" also refers to specific amino acid modifications that confer a specific function (eg, "heterodimerization variant," "pi variant," "ablation variant," etc.).

[0280] As described below, in some embodiments, the parent polypeptide, e.g., the Fc parent polypeptide, is a human wild-type sequence, such as a heavy constant domain or Fc region from IgG1, IgG2, IgG3, or IgG4, but human sequences with variants may also be used as "parent polypeptides," for example, including the IgG1 / 2 hybrid of U.S. Publication No. 2006 / 0134105. The protein variant sequences herein will preferably possess at least about 80% identity to the parent protein sequence, and most preferably at least about 90% identity, more preferably at least about 95%-98%-99% identity. Thus, as used herein, "antibody variant" or "variant antibody" means an antibody that differs from a parent antibody by at least one amino acid modification, as used herein, "IgG variant" or "variant IgG" means an antibody that differs from a parent IgG (again, in many cases, from a human IgG sequence) by at least one amino acid modification, and as used herein, "immunoglobulin variant" or "variant immunoglobulin" means an immunoglobulin sequence that differs from a parent immunoglobulin sequence by at least one amino acid modification. As used herein, "Fc variant" or "variant Fc" means a protein comprising amino acid modifications in the Fc domain as compared to the Fc domain of human IgG1, IgG2, or IgG4.

[0281] As used herein, "Fc variant" or "variant Fc" means a protein comprising an amino acid modification in the Fc domain. The modification can be an addition, deletion, or substitution. Fc variants are defined according to the amino acid modifications that constitute them. Thus, for example, N434S or 434S is an Fc variant having a serine substitution at position 434 relative to the parent Fc polypeptide, wherein the numbering is according to the EU index. Similarly, M428L / N434S defines an Fc variant having the substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid may not be specified, in which case the aforementioned variant is referred to as 428L / 434S. It should be noted that the order in which the substitutions are provided is arbitrary, that is, for example, 428L / 434S is the same Fc variant as 434S / 428L, etc. For all positions discussed herein relating to antibodies or their derivatives and fragments (e.g., Fc domains), unless otherwise indicated, the amino acid position numbering is according to the EU index. The "EU index" or "EU index as in Kabat" or "EU numbering" scheme refers to the EU numbering for antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby incorporated by reference in its entirety). A modification may be an addition, deletion or substitution.

[0282] Generally, the variant Fc domain has at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the corresponding parent human IgG Fc domain (using the identity algorithm discussed below, one embodiment of which utilizes the BLAST algorithm as known in the art using default parameters). Alternatively, the variant Fc domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications compared to the parent Fc domain. Alternatively, the variant Fc domain may have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the parent Fc domain. Additionally, as discussed herein, the variant Fc domains described herein still retain the ability to form a dimer with another Fc domain as measured using known techniques such as native gel electrophoresis as described herein.

[0283] As used herein, "protein" means at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides, and peptides. In addition, the polypeptides constituting the antibodies of the present invention may include synthetic derivatization of one or more side chains or termini, glycosylation, PEGylation, cyclic arrangement, cyclization, linkers to other molecules, fusion to proteins or protein domains, and the addition of peptide tags or labels.

[0284] As used herein, "residue" refers to a position in a protein and its associated amino acid identity. For example, asparagine 297 (also known as Asn297 or N297) is the residue at position 297 in the human antibody IgG1.

[0285] As used herein, "IgG subclass modification" or "isotype modification" refers to an amino acid modification that converts an amino acid of one IgG isotype to the corresponding amino acid in a different aligned IgG isotype. For example, since IgG1 contains a tyrosine and IgG2 contains a phenylalanine at EU position 296, the F296Y substitution in IgG2 is considered an IgG subclass modification.

[0286] As used herein, "non-naturally occurring modification" refers to a non-isotypic amino acid modification. For example, since human IgG does not contain a serine at position 434, the substitution 434S of IgG1, IgG2, IgG3, or IgG4 (or a hybrid thereof) is considered a non-naturally occurring modification.

[0287] As used herein, "amino acid" and "amino acid identity" mean one of the 20 naturally occurring amino acids encoded by DNA and RNA.

[0288] As used herein, "effector function" refers to the biochemical events resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0289] As used herein, "IgG Fc ligand" means a molecule, preferably a polypeptide, that is bound to the Fc region of an IgG antibody from any organism to form an Fc / Fc ligand complex. Fc ligands include, but are not limited to, FcγRI, FcγRII, FcγRIII, FcRn, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are Fc receptor families homologous to FcγR (Davis et al., 2002, Immunological Reviews 190: 123-136, which are incorporated by reference in their entirety). Fc ligands may include undiscovered molecules that bind to Fc. Specific IgG Fc ligands are FcRn and Fcγ receptors. As used herein, "Fc ligand" means a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.

[0290] As used herein, "Fcγ receptor," "FcγR," or "FcgammaR" means any member of the family of proteins that bind to the Fc region of an IgG antibody and are encoded by an FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporated by reference), as well as any undiscovered human FcγR or FcγR isoform or allotype. FcγRs can be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγR or FcγR isoform or allotype.

[0291] As used herein, "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by an FcRn gene. FcRn can be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, a functional FcRn protein comprises two polypeptides, commonly referred to as a heavy chain and a light chain. The light chain is beta-2 microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise indicated herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and beta-2-microglobulin. Various FcRn variants are used to increase binding to the FcRn receptor and, in some cases, to increase serum half-life. "FcRn variants" are amino acid modifications that contribute to increased binding to the FcRn receptor, and suitable FcRn variants are shown below.

[0292] As used herein, "parent polypeptide" means the starting polypeptide that is subsequently modified to produce a variant. The parent polypeptide can be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide. Thus, as used herein, "parent immunoglobulin" means the unmodified immunoglobulin polypeptide that is modified to produce a variant, and as used herein, "parent antibody" means the unmodified antibody that is modified to produce a variant antibody. It should be noted that "parent antibody" includes known commercial recombinantly produced antibodies as outlined below. In this case, the "parent Fc domain" will be relative to the variant being listed; thus, a "variant human IgG1 Fc domain" will be compared to the parent Fc domain of human IgG1, a "variant human IgG4 Fc domain" will be compared to the parent Fc domain of human IgG4, etc.

[0293] As used herein, "position" means a position in a protein sequence. Positions can be numbered sequentially or according to an established format, such as using the EU index to number antibody domains (e.g., CH1, CH2, CH3 or hinge domain).

[0294] As used herein, "target antigen" means the molecule that is specifically bound by an antigen binding domain comprising the variable region of a given antibody.

[0295] In this article, in the case of the monomers of the heterodimeric antibodies of the present invention, "strandedness" means that, similar to the two chains of "matching" of DNA, heterodimerization variants are incorporated into each monomer in order to maintain the ability to "match" to form heterodimers. For example, if some pI variants are engineered into monomer A (e.g., to make pI higher), the spatial variants of the "charge pairs" that are also available do not interfere with the pI variants, e.g., the charge variants that make pI higher are placed on the same "chain" or "monomer" to maintain both functions. Similarly, for "skewed" variants that appear in paired groups as more fully outlined below, the skilled person will consider pI to decide which chain or monomer a group in the pair will enter, thereby also using the pI of the skewed variants to maximize pI separation.

[0296] As used herein, "target cell" means a cell that expresses a target antigen.

[0297] Herein, in the context of producing a bispecific antibody according to the present invention, a "host cell" means a cell that contains exogenous nucleic acid encoding the components of the bispecific antibody and is capable of expressing the bispecific antibody under appropriate conditions. Suitable host cells are discussed below.

[0298] "Wild type" or "WT" herein refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0299] Provided herein are various antibody domains (eg, Fc domains) that have sequence identity to human antibody domains. The sequence identity between two similar sequences (e.g., antibody variable domains) can be measured by an algorithm such as Smith, TF and Waterman, MS (1981) "Comparison Of Biosequences," Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, SB and Wunsch, CD. (1970) "A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins," J. Mol. Biol. 48:443 [homology alignment algorithm]; Pearson, WR and Lipman, DJ (1988) "Improved Tools For Biological Sequence Comparison," Proc. Natl. Acad. Sci. (USA) 85:2444 [search for similarity method]; or Altschul, SF et al., (1990) "Basic Local Alignment Search Tool," J. Mol. Biol. 215:403-10, the "BLAST" algorithm, see https: / / blast.ncbi.nlm.nih.gov / Blast.cgi. When using any of the aforementioned algorithms, the default parameters (regarding window length, gap penalties, etc.) are used. In one embodiment, sequence identity is achieved using the BLAST algorithm using the default parameters.

[0300] The antibodies of the present invention are generally isolated or recombinant. When used to describe the various polypeptides disclosed herein, "isolated" means a polypeptide that has been identified and separated and / or recovered from a cell or cell culture expressing the polypeptide. Typically, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. "Recombinant" means that the antibody is produced using recombinant nucleic acid technology in an exogenous host cell, and the antibody can also be isolated.

[0301] "Specific binding" or "specifically binds to" or "specific for" a particular antigen or epitope means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule of a similar target.

[0302] Specific binding for a particular antigen or epitope can be demonstrated, for example, by an antibody having a KD for the antigen or epitope of at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M or greater, where KD refers to the off-rate of a specific antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen has a KD relative to the antigen or epitope that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more greater for a control molecule.

[0303] Likewise, specific binding for a particular antigen or epitope can be manifested, for example, by an antibody having a KA or Ka for the antigen or epitope that is at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more greater than for the epitope relative to a control, where KA or Ka refers to the on-rate of the specific antibody-antigen interaction. Binding affinity is typically measured using Biacore, SPR, or BLI assays.

[0304] IV. PD-L1, PD-L2, and CD28 Antigen Binding Domains

[0305] Provided herein are antigen binding domains (ABDs) and ABD compositions that bind to PD-L1, PD-L2, or CD28. In some embodiments, one or more of the ABDs are included in an antibody format described herein (including any of the bispecific and trispecific formats in Figures 82 and 83).

[0306] A. PD-L1 antigen binding domain

[0307] In one aspect, provided herein are PD-L1 antigen binding domains (ABDs) and compositions comprising such PD-L1 antigen binding domains (ABDs), including anti-PD-L1 antibodies (e.g., anti-PD-L1 x anti-CD28 bispecific antibodies, and anti-PD-L1 x anti-PD-L2 x anti-CD28 trispecific antibodies). Such PD-L1 binding domains and related antibodies (e.g., bispecific antibodies and trispecific antibodies disclosed herein) can be used, for example, to treat PD-L1-related cancers. In some embodiments, the PD-L1 ABD is capable of binding to human and cynomolgus monkey PD-L1 (see Figure 1 and Example 1).

[0308] As will be appreciated by those skilled in the art, a suitable PD-L1 binding domain may comprise a set of six such as Figure 25 and CDRs depicted in Figure 26 and the sequence listing. Suitable PD-L1 ABDs may also comprise sequences such as these and Figure 25 The complete VH and VL sequences depicted in Figure 26 and the Sequence Listing were used as scFv or Fab domains.

[0309] In one embodiment, the PD-L1 antigen binding domain comprises the Figure 25 26 and the sequence listing), the six CDRs of the PD-L1 ABD (i.e., vhCDR1-3 and vlCDR1-3) are either underlined or, where a different numbering scheme is used as described herein and as shown in Table 2, are numbered using Figure 25 26 and those depicted in the sequence listing (see Table 2). Suitable PD-L1 ABDs may also comprise the complete VH and VL sequences as depicted in these sequences and the accompanying figures, used as scFv or Fab domains.

[0310] In one embodiment, the PD-L1 antigen binding domain comprises the Figure 25 26 and the sequence listing). In an exemplary embodiment, the PD-L1 antigen binding domain includes the six CDRs of PD-L1 comprising a variable heavy domain (VH) and a variable light domain (VL), wherein VH and VL are selected from the following:

[0311] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 21; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 25;

[0312] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 33;

[0313] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 33;

[0314] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0315] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 to Figure 2 6).

[0316] In addition to the parent CDR groups forming the ABD of PD-L1 disclosed in the accompanying drawings and sequence listings, variant PD-L1 ABDs having at least one modified CDR comprising the PD-L1 ABD CDRs disclosed herein are also provided herein. In one embodiment, the PD-L1 ABD comprises a set of 6 CDRs, which have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to the 6 CDRs of the PD-L1 ABD described herein (including the accompanying drawings and sequence listings). In an exemplary embodiment, the PD-L1 ABD comprises a set of 6 CDRs, which have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to the 6 CDRs of the PD-L1 ABD comprising a variable heavy domain (VH) and a variable light domain (VL) selected from the following:

[0317] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 21; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 25;

[0318] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 33;

[0319] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 33;

[0320] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0321] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 to Figure 2 6).

[0322] In certain embodiments, the variant PD-L1 ABD is capable of binding to the PD-L1 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay), the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L1 ABD is capable of binding to both human and cynomolgus monkey PD-L1.

[0323] In one embodiment, the anti-PD-L1 ABD comprises a Figure 24 to Figure 2 6 and the sequence listing). In an exemplary embodiment, the anti-PD-L1 ABD comprises six CDRs that are at least 90%, 95%, 97%, 98% or 99% identical to the six CDRs of a PD-L1 ABD comprising a variable heavy domain (VH) and a variable light domain (VL) selected from the following:

[0324] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 21; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 25;

[0325] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 33;

[0326] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 33;

[0327] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0328] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 to Figure 2 6).

[0329] In certain embodiments, the anti-PD-L1 ABD is capable of binding to the PD-L1 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay), the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L1 ABD is capable of binding to both human and cynomolgus monkey PD-L1.

[0330] In another exemplary embodiment, the anti-PD-L1 ABD comprises the Figure 24 to Figure 2 6 and sequence listing) of any of the PD-L1 ABDs described herein. In exemplary embodiments, VH and VL are selected from the following:

[0331] (i) a VH having the amino acid sequence of SEQ ID NO: 21; and (ii) a VL having the amino acid sequence of SEQ ID NO: 25; and

[0332] (i) a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL having the amino acid sequence of SEQ ID NO: 33;

[0333] (i) a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL having the amino acid sequence of SEQ ID NO: 33;

[0334] (i) a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0335] (i) a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 to Figure 2 6).

[0336] In addition to the parent anti-PD-L1 binding domain variable heavy domain and variable light domain disclosed herein, anti-PD-L1 ABDs comprising variable heavy domains and / or variable light domains that are variants of the anti-PD-L1 ABD VH and VL domains disclosed herein are also provided herein. In one embodiment, Figures 28 to 31 The variant VH domain and / or VL domain has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes compared to the VH and / or VL domains of the anti-PD-L1 ABD described in (e.g., SEQ ID NO: 100). In exemplary embodiments, the variant VH domain and / or VL domain has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes compared to a VH and / or VL selected from the group consisting of:

[0337] (i) a VH having the amino acid sequence of SEQ ID NO: 21; and (ii) a VL having the amino acid sequence of SEQ ID NO: 25; and

[0338] (i) a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL having the amino acid sequence of SEQ ID NO: 33;

[0339] (i) a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL having the amino acid sequence of SEQ ID NO: 33;

[0340] (i) a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0341] (i) a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 to Figure 2 6).

[0342] In certain embodiments, the anti-PD-L1 ABD is capable of binding to PD-L1 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay), the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L1 ABD is capable of binding to both human and cynomolgus monkey PD-L1.

[0343] In some embodiments, the PD-L1 ABD comprises a variable heavy domain having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes relative to one of the following PD-L1 ABD variable heavy domains: SEQ ID NOs: 21, 29, and 37. In some embodiments, the PD-L1 ABD comprises any one of the variable light domains provided herein, or a variant thereof.

[0344] In some embodiments, the PD-L1 ABD comprises a variable light domain having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes relative to one of the following PD-L1 ABD variable light domains: SEQ ID NOs: 20, 25, and 33. In some embodiments, the PD-L1 ABD comprises any one of the variable heavy domains provided herein, or a variant thereof.

[0345] In one embodiment, the variant VH and / or VL domains are as described herein (including Figure 25 26 and the sequence listing). In exemplary embodiments, the variant VH and / or VL domains are at least 90%, 95%, 97%, 98% or 99% identical to the VH and / or VL of one of the anti-PD-L1 ABDs comprising a VH and VL selected from:

[0346] (i) a VH having the amino acid sequence of SEQ ID NO: 21; and (ii) a VL having the amino acid sequence of SEQ ID NO: 25; and

[0347] (i) a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL having the amino acid sequence of SEQ ID NO: 33;

[0348] (i) a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL having the amino acid sequence of SEQ ID NO: 33;

[0349] (i) a VH having the amino acid sequence of SEQ ID NO: 29; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0350] (i) a VH having the amino acid sequence of SEQ ID NO: 37; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 to Figure 2 6).

[0351] In certain embodiments, the anti-PD-L1 ABD is capable of binding to PD-L1 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay), the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L1 ABD is capable of binding to both human and cynomolgus monkey PD-L1.

[0352] In some embodiments, the PD-L1 ABD comprises a variable heavy domain that is at least 90%, 95%, 97%, 98%, or 99% identical to one of the following PD-L1 ABD variable heavy domains: SEQ ID NOs: 21, 29, and 37. In some embodiments, the PD-L1 ABD comprises any one of the variable light domains provided herein, or a variant thereof.

[0353] In some embodiments, the PD-L1 ABD comprises a variable light domain that is at least 90%, 95%, 97%, 98%, or 99% identical to one of the following PD-L1 ABD variable light domains: SEQ ID NOs: 20, 25, and 33. In some embodiments, the PD-L1 ABD comprises any one of the variable heavy domains provided herein, or a variant thereof.

[0354] In some embodiments, the PD-L1 ABD has a variable heavy domain and a variable light domain that are 90%, 95%, 97%, 98%, or 99% identical to the VH and / or VL domains of a PD-L1 ABD as described herein, except that the CDRs are identical. In some embodiments, the VH is selected from the group consisting of SEQ ID NOs: 21, 29, and 37. In some embodiments, the VL is selected from the group consisting of SEQ ID NOs: 20, 25, and 33.

[0355] In some embodiments, the PD-L1 ABD has a variable heavy domain and a variable light domain that are 90%, 95%, 97%, 98%, or 99% identical to the VH and / or VL domains of the PD-L1 ABD as described herein, and there may be 0 to 6 amino acid modifications in the CDRs. In some embodiments, the VH is selected from the group consisting of SEQ ID NOs: 21, 29, and 37. In some embodiments, the VL is selected from the group consisting of SEQ ID NOs: 20, 25, and 33.

[0356] In some embodiments, the PD-L1 ABD has a variable heavy domain and a variable light domain that are 90%, 95%, 97%, 98%, or 99% identical to the VH and / or VL domains of the PD-L1 ABD as described herein, and there may be 0 to 6 amino acid modifications in the CDRs, with no CDR having more than 1 amino acid modification. In some embodiments, the VH is selected from the group consisting of SEQ ID NOs: 21, 29, and 37. In some embodiments, the VL is selected from the group consisting of SEQ ID NOs: 20, 25, and 33.

[0357] In certain embodiments, the PD-L1 ABD is capable of binding to PD-L1 as measured by at least one of a Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay) assay, the latter of which is particularly useful in many embodiments. In specific embodiments, the CD28 ABD is capable of binding to human PD-L1 within the detection limits of the assay (see Figure 2 ).

[0358] Such PD-L1 binding domains can be contained in any of the antibodies provided herein (e.g., including the bispecific and trispecific antibody formats provided in Figures 82 and 83).

[0359] 1. Additional PD-L1 binding domains

[0360] In another aspect, provided herein are additional PD-L1 binding domains that can be used in anti-PD-L1 antibodies, including any of the anti-PD-L1 antibodies described herein. In some embodiments, the anti-PD-L1 binding domain comprises a variable heavy domain selected from any one of those in FIG. 161 (SEQ ID NOs: 3335 and 3243-3260), or a variant thereof, and a SEQ ID NO: 3239, wherein the SEQ ID NO: 3240 is a SEQ ID NO: 3251. Figure 161A ) and "1F12A4.249[PDL2]_L1" (SEQ ID NO: 3271, see Figure 162A)) or a common light chain of a variable light domain or a variant thereof, wherein the common light chain can also be used as a light chain for a CD28 and / or PD-L2 binding domain. In some embodiments, the anti-PD-L1 antibodies provided herein (e.g., anti-CD28 x anti-PD-L1 and anti-CD28 x anti-PD-L1 x anti-PDL2 antibodies) comprise a PD-L1 binding domain comprising a common light chain having an IGKV1-39_L1 variable light domain. In some embodiments, the anti-PD-L1 antibody is an anti-CD28 x anti-PD-L1 antibody having a CD28 binding domain and a PD-L1 binding domain, each of which comprises a common light chain having an IGKV1-39_L1 variable light domain (SEQ ID NO: 3239). In some embodiments, the anti-PD-L1 antibody is an anti-CD28 x anti-PD-L1 x anti-PD-L2 antibody having a CD28 binding domain, a PD-L1 binding domain, and a PD-L2 binding domain, each comprising a common light chain having the IGKV1-39_L1 variable light domain (SEQ ID NO: 3239). Such PD-L1 binding domains utilizing a common light chain having the IGKV1-39_L1 variable light domain (SEQ ID NO: 3239) can be used, for example, in any of the antibody formats utilizing a common light chain provided herein (see, e.g., 1+1 CLC, 2+1 CLC, 1+1+1 stacked Fab2-scFv-Fc, 1+1+1 Fab-(Fab-scFv)-Fc, 1+1+1 mAb-scFv, and 1+1+1 stacked Fab2-Fab-Fc formats disclosed herein, Figures 82 and 83).

[0361] In one embodiment, the PD-L1 antigen binding domain comprises the vhCDRs 1-3 of any one of the PD-L1 variable heavy domains depicted in Figure 161 (SEQ ID NOs: 3335 and 3243-3260), and the vlCDRs 1-3 of the IGK1-39_L1 variable light domain (SEQ ID NO: 3239).

[0362] In one embodiment, a PD-L1 ABD of a subject anti-PD-L1 antibody described herein comprises: a) a vhCDR1, vhCDR2, and / or vhCDR3 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to the vhCDR1, vhCDR2, and / or vhCDR3 of one of the PD-L1 variable heavy domains depicted in Figure 161 (SEQ ID NOs: 3335 and 3243-3260), respectively; and / or b) a vlCDR1, vlCDR2, and / or vlCDR3 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to the vlCDR1, vlCDR2, and / or vlCDR3 of the IGK1-39_L1 variable light domain (SEQ ID NO: 3239). In certain embodiments, the PD-L1 ABD of a subject anti-PD-L1 antibody is capable of binding to the PD-L1 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet assay) assays, the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L1 ABD is capable of binding to the human PD-L1 antigen (see Figure 1 ).

[0363] In one embodiment, the PD-L1 ABD of a subject anti-PD-L1 antibody comprises a vhCDR1, vhCDR2, and / or vhCDR3 that are at least 90%, 95%, 97%, 98%, or 99% identical to the vhCDR1, vhCDR2, and / or vhCDR3, respectively, of one of the PD-L1 variable heavy domains depicted in Figure 161 (SEQ ID NOs: 3335 and 3243-3260); and / or a vlCDR1, vlCDR2, and / or vlCDR3 that are at least 90%, 95%, 97%, 98%, or 99% identical to the vlCDR1, vlCDR2, and / or vlCDR3, respectively, of the IGK1-39_L1 variable light domain (SEQ ID NO: 3239). In certain embodiments, the PD-L1 ABD is capable of binding to PD-L1 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet assay) assays, the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L1 ABD is capable of binding to human PD-L1 antigen (see Figure 1 ).

[0364] In another exemplary embodiment, the PD-L1 ABD of a subject anti-PD-L1 antibody comprises the variable heavy (VH) domain of one of the PD-L1 variable heavy domains depicted in FIG. 161 (SEQ ID NOs: 3335 and 3243-3260), and the IGK1-39_L1 variable light domain (SEQ ID NO: 3239).

[0365] In some embodiments, an anti-PD-L1 antibody comprises a PD-L1 ABD comprising a variable heavy domain that is a variant of one of the PD-L1 variable heavy domains depicted in FIG. 161 (SEQ ID NOs: 3335 and 3243-3260) and / or a variable light domain that is a variant of the IGK1-39_L1 variable light domain (SEQ ID NO: 3239). In some embodiments, the variant VH domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to one of the PD-L1 variable heavy domains depicted in Figure 161 (SEQ ID NOs: 3335 and 3243-3260), and / or the variable light domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to the IGK1-39_L1 variable light domain (SEQ ID NO: 3239). In some embodiments, the one or more amino acid changes are in the VH and / or VL framework regions (FR1, FR2, FR3, and / or FR4). In some embodiments, the one or more amino acid changes are in one or more CDRs. In certain embodiments, the PD-L1 ABD is capable of binding to PD-L1 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet assay) assays, the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L1 ABD is capable of binding to human PD-L1 antigen (see Figure 1 ).

[0366] In one embodiment, the variant VH domain is at least 90%, 95%, 97%, 98% or 99% identical to one of the PD-L1 variable heavy domains depicted in Figure 161 (SEQ ID NOs: 3335 and 3243-3260), and / or the variable light domain is at least 90%, 95%, 97%, 98% or 99% identical to the IGK1-39_L1 variable light domain (SEQ ID NO: 3239). In certain embodiments, the PD-L1 ABD is capable of binding to PD-L1 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet assay) assays, the latter being particularly useful in many embodiments. In specific embodiments, the PD-L1 ABD is capable of binding to the human PD-L1 antigen (see Figure 1 ).

[0367] In some embodiments, the PD-L1 binding domain comprises Figure 161C The VH of any of the depicted VHCDR1-3 and / or HFR1-4 sequences and the IGK1-39_L1 variable light domain (SEQ ID NO: 3239), or variants thereof.

[0368] In another aspect, provided herein are PD-L1 binding domains that compete with any of the PD-L1 binding domains disclosed herein for binding to human PD-L1.

[0369] B. PD-L2 antigen binding domain

[0370] In one aspect, provided herein are PD-L2 antigen binding domains (ABDs) and compositions comprising such PD-L2 antigen binding domains (ABDs), including anti-PD-L2 antibodies (e.g., anti-PD-L1 x anti-CD28 bispecific antibodies, and anti-PD-L1 x anti-PD-L2 x anti-CD28 trispecific antibodies). Such PD-L2 binding domains and related antibodies (e.g., bispecific antibodies and trispecific antibodies disclosed herein) can be used, for example, to treat PD-L2-related cancers. In some embodiments, the PD-L2 ABD is capable of binding to human and cynomolgus monkey PD-L2 (see Figure 2 and Example 1).

[0371] As will be appreciated by those skilled in the art, a suitable PD-L2 binding domain may comprise a set of six such as Figure 29 32 and the CDRs depicted in the sequence listing. Suitable PD-L2 ABDs may also include sequences such as these and Figure 29 The complete VH and VL sequences depicted in Figure 32 and the Sequence Listing were used as scFv or Fab domains.

[0372] In one embodiment, the PD-L2 antigen binding domain comprises the Figure 29 32 and the sequence listing), the six CDRs of the PD-L2 ABD (i.e., vhCDR1-3 and vlCDR1-3) are either underlined or, where a different numbering scheme is used as described herein and as shown in Table 2, are numbered using Figure 29 32 and those depicted in the sequence listing (see Table 2). Suitable PD-L2 ABDs may also comprise the complete VH and VL sequences as depicted in these sequences and the accompanying figures, used as scFv or Fab domains.

[0373] In one embodiment, the PD-L2 antigen binding domain comprises the Figure 29 32 and the sequence listing). In an exemplary embodiment, the PD-L2 antigen binding domain comprises the six CDRs of PD-L2 comprising a variable heavy domain (VH) and a variable light domain (VL), wherein VH and VL are selected from the following:

[0374] ((i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 41; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 45;

[0375] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 45;

[0376] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 41; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0377] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0378] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 57;

[0379] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 57;

[0380] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0381] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0382] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 69;

[0383] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 69;

[0384] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0385] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0386] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 81;

[0387] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 81;

[0388] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0389] (i) a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 of the VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 29 to Figure 32). ( Figure 24 as well as Figure 29 to Figure 32).

[0390] In addition to the parent CDR groups that form the ABD of PD-L2 disclosed in the Figures and Sequence Listing, variant PD-L2 ABDs having at least one modified CDR comprising the PD-L2 ABD CDRs disclosed herein are also provided herein. In one embodiment, the PD-L2 ABD comprises a set of 6 CDRs that have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to the 6 CDRs of the PD-L2 ABD described herein (including the Figures and Sequence Listing). In an exemplary embodiment, the PD-L2 ABD comprises a set of 6 CDRs that have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to the 6 CDRs of the PD-L2 ABD comprising a variable heavy domain (VH) and a variable light domain (VL) selected from the following:

[0391] ((i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 41; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 45;

[0392] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 45;

[0393] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 41; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0394] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0395] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 57;

[0396] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 57;

[0397] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0398] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0399] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 69;

[0400] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 69;

[0401] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0402] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0403] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 81;

[0404] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 81;

[0405] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0406] (i) a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 of the VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 29 to Figure 32). ( Figure 24 as well as Figure 29 to Figure 32).

[0407] In certain embodiments, the variant PD-L2 ABD is capable of binding to the PD-L2 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay), the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L2 ABD is capable of binding to both human and cynomolgus monkey PD-L2.

[0408] In one embodiment, the anti-PD-L2 ABD comprises a Figure 24 and Figure 29 32 and the sequence listing). In exemplary embodiments, the anti-PD-L2 ABD comprises six CDRs that are at least 90%, 95%, 97%, 98% or 99% identical to the six CDRs of the anti-PD-L2 ABD described in

[0015] to

[0016] Figure 32 and the sequence listing. In exemplary embodiments, the anti-PD-L2 ABD comprises six CDRs that are at least 90%, 95%, 97%, 98% or 99% identical to the six CDRs of a PD-L2 ABD comprising a variable heavy domain (VH) and a variable light domain (VL) selected from:

[0409] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 41; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 45;

[0410] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 45;

[0411] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 41; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0412] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0413] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 57;

[0414] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 57;

[0415] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0416] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0417] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 69;

[0418] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 69;

[0419] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0420] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0421] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 81;

[0422] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, the VH CDR2, and the VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 81;

[0423] (i) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 respectively have the amino acid sequences of VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL comprising a VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO: 20;

[0424] (i) a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1, VL CDR2, and VL CDR3 respectively have the amino acid sequences of the VL CDR1, VL CDR2, and VL CDR3 of the VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 29 to Figure 32). ( Figure 24 as well as Figure 29 to Figure 32).

[0425] In certain embodiments, the anti-PD-L2 ABD is capable of binding to the PD-L2 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay), the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L2 ABD is capable of binding to both human and cynomolgus monkey PD-L2.

[0426] In another exemplary embodiment, the anti-PD-L2 ABD comprises the Figure 24 and Figure 29 32 and the sequence listing). In exemplary embodiments, VH and VL are selected from the following:

[0427] ((i) VH having the amino acid sequence of SEQ ID NO: 41; and (ii) VL having the amino acid sequence of SEQ ID NO: 45;

[0428] (i) a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL having the amino acid sequence of SEQ ID NO: 45;

[0429] (i) a VH having the amino acid sequence of SEQ ID NO: 41; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0430] (i) a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0431] (i) a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL having the amino acid sequence of SEQ ID NO: 57;

[0432] (i) a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL having the amino acid sequence of SEQ ID NO: 57;

[0433] (i) a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0434] (i) a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0435] (i) a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL having the amino acid sequence of SEQ ID NO: 69;

[0436] (i) a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL having the amino acid sequence of SEQ ID NO: 69;

[0437] (i) a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0438] (i) a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0439] (i) a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL having the amino acid sequence of SEQ ID NO: 81;

[0440] (i) a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL having the amino acid sequence of SEQ ID NO: 81;

[0441] (i) a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0442] (i) a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 and Figure 29 to Figure 32).

[0443] In addition to the parent anti-PD-L2 binding domain variable heavy domain and variable light domain disclosed herein, anti-PD-L2 ABDs are also provided herein comprising variable heavy domains and / or variable light domains that are variants of the anti-PD-L2 ABD VH and VL domains disclosed herein. In one embodiment, the parent anti-PD-L2 binding domain variable heavy domain and variable light domain disclosed herein (including Figures 28 to 31 The variant VH domain and / or VL domain has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes compared to the VH and / or VL domains of the anti-PD-L2 ABD described in (e.g., SEQ ID NO: 100). In exemplary embodiments, the variant VH domain and / or VL domain has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes compared to a VH and / or VL selected from the group consisting of:

[0444] (i) a VH having the amino acid sequence of SEQ ID NO: 41; and (ii) a VL having the amino acid sequence of SEQ ID NO: 45;

[0445] (i) a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL having the amino acid sequence of SEQ ID NO: 45;

[0446] (i) a VH having the amino acid sequence of SEQ ID NO: 41; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0447] (i) a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0448] (i) a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL having the amino acid sequence of SEQ ID NO: 57;

[0449] (i) a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL having the amino acid sequence of SEQ ID NO: 57;

[0450] (i) a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0451] (i) a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0452] (i) a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL having the amino acid sequence of SEQ ID NO: 69;

[0453] (i) a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL having the amino acid sequence of SEQ ID NO: 69;

[0454] (i) a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0455] (i) a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0456] (i) a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL having the amino acid sequence of SEQ ID NO: 81;

[0457] (i) a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL having the amino acid sequence of SEQ ID NO: 81;

[0458] (i) a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0459] (i) a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 and Figure 29 to Figure 32).

[0460] In certain embodiments, the anti-PD-L2 ABD is capable of binding to PD-L2 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay) assays, the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L2 ABD is capable of binding to both human and cynomolgus monkey PD-L2.

[0461] In some embodiments, the PD-L2 ABD comprises a variable heavy domain having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes relative to one of the following PD-L2 ABD variable heavy domains: SEQ ID NOs: 41, 49, 53, 61, 65, 73, 77, and 85. In some embodiments, the PD-L2 ABD comprises any one of the variable light domains provided herein, or a variant thereof.

[0462] In some embodiments, the PD-L2 ABD comprises a variable light domain having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes relative to one of the following PD-L2 ABD variable light domains: SEQ ID NOs: 20, 45, 57, 69, and 81. In some embodiments, the PD-L2 ABD comprises any one of the variable heavy domains provided herein, or a variant thereof.

[0463] In one embodiment, the variant VH and / or VL domains are as described herein (including Figure 29 32 and the sequence listing). In exemplary embodiments, the variant VH and / or VL domains are at least 90%, 95%, 97%, 98% or 99% identical to the VH and / or VL of one of the anti-PD-L2 ABDs comprising a VH and VL selected from:

[0464] ((i) VH having the amino acid sequence of SEQ ID NO: 41; and (ii) VL having the amino acid sequence of SEQ ID NO: 45;

[0465] (i) a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL having the amino acid sequence of SEQ ID NO: 45;

[0466] (i) a VH having the amino acid sequence of SEQ ID NO: 41; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0467] (i) a VH having the amino acid sequence of SEQ ID NO: 49; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0468] (i) a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL having the amino acid sequence of SEQ ID NO: 57;

[0469] (i) a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL having the amino acid sequence of SEQ ID NO: 57;

[0470] (i) a VH having the amino acid sequence of SEQ ID NO: 53; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0471] (i) a VH having the amino acid sequence of SEQ ID NO: 61; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0472] (i) a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL having the amino acid sequence of SEQ ID NO: 69;

[0473] (i) a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL having the amino acid sequence of SEQ ID NO: 69;

[0474] (i) a VH having the amino acid sequence of SEQ ID NO: 65; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0475] (i) a VH having the amino acid sequence of SEQ ID NO: 73; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0476] (i) a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL having the amino acid sequence of SEQ ID NO: 81;

[0477] (i) a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL having the amino acid sequence of SEQ ID NO: 81;

[0478] (i) a VH having the amino acid sequence of SEQ ID NO: 77; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0479] (i) a VH having the amino acid sequence of SEQ ID NO: 85; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20 ( Figure 24 and Figure 29 to Figure 32).

[0480] In certain embodiments, the anti-PD-L2 ABD is capable of binding to PD-L2 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay) assays, the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L2 ABD is capable of binding to both human and cynomolgus monkey PD-L2.

[0481] In some embodiments, the PD-L2 ABD comprises a variable heavy domain that is at least 90%, 95%, 97%, 98%, or 99% identical to one of the following PD-L2 ABD variable heavy domains: SEQ ID NOs: 41, 49, 53, 61, 65, 73, 77, and 85. In some embodiments, the PD-L2 ABD comprises any one of the variable light domains provided herein, or a variant thereof.

[0482] In some embodiments, the PD-L2 ABD comprises a variable light domain that is at least 90%, 95%, 97%, 98%, or 99% identical to one of the following PD-L2 ABD variable light domains: SEQ ID NOs: 20, 45, 57, 69, and 81. In some embodiments, the PD-L2 ABD comprises any one of the variable heavy domains provided herein, or a variant thereof.

[0483] In some embodiments, the PD-L2 ABD has a variable heavy domain and a variable light domain that are 90%, 95%, 97%, 98%, or 99% identical to the VH and / or VL domains of a PD-L2 ABD as described herein, except that the CDRs are identical. In some embodiments, the VH is selected from the group consisting of SEQ ID NOs: 41, 49, 53, 61, 65, 73, 77, and 85. In some embodiments, the VL is selected from the group consisting of SEQ ID NOs: 20, 45, 57, 69, and 81.

[0484] In some embodiments, the PD-L2 ABD has a variable heavy domain and a variable light domain that are 90%, 95%, 97%, 98% or 99% identical to the VH and / or VL domains of the PD-L2 ABD as described herein, and there may be 0 to 6 amino acid modifications in the CDRs. In some embodiments, the VH is selected from the group consisting of SEQ ID NOs: 41, 49, 53, 61, 65, 73, 77, and 85. In some embodiments, the VL is selected from the group consisting of SEQ ID NOs: 20, 45, 57, 69, and 81.

[0485] In some embodiments, the PD-L2 ABD has a variable heavy domain and a variable light domain that are 90%, 95%, 97%, 98% or 99% identical to the VH and / or VL domains of the PD-L2 ABD as described herein, and there may be 0 to 6 amino acid modifications in the CDRs, with no CDR having more than 1 amino acid modification. In some embodiments, the VH is selected from the group consisting of SEQ ID NOs: 41, 49, 53, 61, 65, 73, 77, and 85. In some embodiments, the VL is selected from the group consisting of SEQ ID NOs: 20, 45, 57, 69, and 81.

[0486] In certain embodiments, the PD-L2 ABD is capable of binding to PD-L2 as measured by at least one of a Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay) assay, the latter of which is particularly useful in many embodiments. In specific embodiments, the CD28 ABD is capable of binding to human PD-L2 within the detection limits of the assay (see Figure 2 ).

[0487] Such PD-L2 binding domains can be contained in any of the antibodies provided herein (e.g., including the bispecific and trispecific antibody formats provided in Figures 82 and 83).

[0488] 1. Additional PD-L2 binding domains

[0489] In another aspect, provided herein are additional PD-L1 binding domains that can be used in anti-PD-L2 antibodies, including any of the anti-PD-L2 antibodies described herein. In some embodiments, the anti-PD-L1 binding domain comprises a variable heavy domain selected from any one of those in FIG. 162 (SEQ ID NOs: 3267 and 3275-3347), or a variant thereof, and a SEQ ID NO: 3239, wherein the SEQ ID NO: 3240 is a 2A3A4.248[PDL1]_L1. Figure 161A) and "1F12A4.249[PDL2]_L1" (SEQ ID NO: 3271, see Figure 162A )) or a common light chain of a variable light domain or a variant thereof, wherein the common light chain can also be used as a light chain for a CD28 and / or PD-L1 binding domain. In some embodiments, the anti-PD-L2 antibodies provided herein (e.g., anti-CD28 x anti-PD-L1 and anti-CD28 x anti-PD-L1 x anti-PDL2 antibodies) comprise a PD-L2 binding domain comprising a common light chain having an IGKV1-39_L1 variable light domain. In some embodiments, the anti-PD-L2 antibody is an anti-CD28 x anti-PD-L2 antibody having a CD28 binding domain and a PD-L2 binding domain, each of which comprises a common light chain having an IGKV1-39_L1 variable light domain (SEQ ID NO: 3239). In some embodiments, the anti-PD-L2 antibody is an anti-CD28 x anti-PD-L1 x anti-PD-L2 antibody having a CD28 binding domain, a PD-L1 binding domain, and a PD-L2 binding domain, each comprising a common light chain having the IGKV1-39_L1 variable light domain (SEQ ID NO: 3239). Such PD-L2 binding domains utilizing a common light chain having the IGKV1-39_L1 variable light domain (SEQ ID NO: 3239) can be used, for example, in any of the antibody formats utilizing a common light chain provided herein (see, e.g., 1+1 CLC, 2+1 CLC, 1+1+1 stacked Fab2-scFv-Fc, 1+1+1 Fab-(Fab-scFv)-Fc, 1+1+1 mAb-scFv, and 1+1+1 stacked Fab2-Fab-Fc formats disclosed herein, Figures 82 and 83).

[0490] In one embodiment, the PD-L2 antigen binding domain comprises the vhCDRs 1-3 of any one of the PD-L2 variable heavy domains depicted in Figure 161 (SEQ ID NOs: 3335 and 3243-3260), and the vlCDRs 1-3 of the IGK1-39_L1 variable light domain (SEQ ID NO: 3239).

[0491] In one embodiment, a PD-L2 ABD of a subject anti-PD-L2 antibody described herein comprises: a) a vhCDR1, vhCDR2, and / or vhCDR3 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to the vhCDR1, vhCDR2, and / or vhCDR3 of one of the PD-L2 variable heavy domains depicted in Figure 162 (SEQ ID NOs: 3267 and 3275-3347), respectively; and / or b) a vlCDR1, vlCDR2, and / or vlCDR3 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to the vlCDR1, vlCDR2, and / or vlCDR3 of the IGK1-39_L1 variable light domain (SEQ ID NO: 3239). In certain embodiments, the PD-L2 ABD of a subject anti-PD-L2 antibody is capable of binding to the PD-L2 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet assay) assays, the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L2 ABD is capable of binding to the human PD-L2 antigen (see Figure 1 ).

[0492] In one embodiment, the PD-L2 ABD of a subject anti-PD-L2 antibody comprises: a vhCDR1, vhCDR2, and / or vhCDR3 that are at least 90%, 95%, 97%, 98%, or 99% identical to the vhCDR1, vhCDR2, and / or vhCDR3, respectively, of one of the PD-L2 variable heavy domains depicted in Figure 162 (SEQ ID NOs: 3267 and 3275-3347); and / or a vlCDR1, vlCDR2, and / or vlCDR3 that are at least 90%, 95%, 97%, 98%, or 99% identical to the vlCDR1, vlCDR2, and / or vlCDR3, respectively, of the IGK1-39_L1 variable light domain (SEQ ID NO: 3239). In certain embodiments, the PD-L2 ABD is capable of binding to PD-L2 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet assay) assays, the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L2 ABD is capable of binding to the human PD-L2 antigen (see Figure 1 ).

[0493] In another exemplary embodiment, the PD-L2 ABD of a subject anti-PD-L2 antibody comprises the variable heavy (VH) domain of one of the PD-L2 variable heavy domains depicted in FIG. 162 (SEQ ID NOs: 3267 and 3275-3347), and the IGK1-39_L1 variable light domain (SEQ ID NO: 3239).

[0494] In some embodiments, an anti-PD-L2 antibody comprises a PD-L2 ABD comprising a variable heavy domain that is a variant of one of the PD-L2 variable heavy domains depicted in FIG. 162 (SEQ ID NOs: 3267 and 3275-3347) and / or a variable light domain that is a variant of the IGK1-39_L1 variable light domain (SEQ ID NO: 3239). In some embodiments, the variant VH domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to one of the PD-L2 variable heavy domains depicted in Figure 162 (SEQ ID NOs: 3267 and 3275-3347), and / or the variable light domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to the IGK1-39_L1 variable light domain (SEQ ID NO: 3239). In some embodiments, the one or more amino acid changes are in the VH and / or VL framework regions (FR1, FR2, FR3, and / or FR4). In some embodiments, the one or more amino acid changes are in one or more CDRs. In certain embodiments, the PD-L2 ABD is capable of binding to PD-L2 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet assay) assays, the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L2 ABD is capable of binding to the human PD-L2 antigen (see Figure 1 ).

[0495] In one embodiment, the variant VH domain is at least 90%, 95%, 97%, 98% or 99% identical to one of the PD-L2 variable heavy domains depicted in Figure 162 (SEQ ID NOs: 3267 and 3275-3347), and / or the variable light domain is at least 90%, 95%, 97%, 98% or 99% identical to the IGK1-39_L1 variable light domain (SEQ ID NO: 3239). In certain embodiments, the PD-L2 ABD is capable of binding to PD-L2 as measured by at least one of Biacore, surface plasmon resonance (SPR), flow cytometry, and / or BLI (biolayer interferometry, e.g., Octet assay) assays, the latter of which is particularly useful in many embodiments. In specific embodiments, the PD-L2 ABD is capable of binding to the human PD-L2 antigen (see Figure 1 ).

[0496] In some embodiments, the PD-L2 binding domain comprises Figure 162G The VH of any of the depicted VHCDR1-3 and / or HFR1-4 sequences and the IGK1-39_L1 variable light domain (SEQ ID NO: 3239), or variants thereof.

[0497] In another aspect, provided herein are PD-L2 binding domains that compete with any of the PD-L1 binding domains disclosed herein for binding to human PD-L2.

[0498] C. CD28 antigen binding domain

[0499] In one aspect, provided herein are CD28 antigen binding domains (ABDs) and compositions comprising such CD28 antigen binding domains (ABDs), including anti-CD28 antibodies (e.g., anti-PD-L1 x anti-CD28 bispecific antibodies, and anti-PD-L1 x anti-PD-L2 x anti-CD28 trispecific antibodies). Such CD28 binding domains and related antibodies (e.g., bispecific antibodies and trispecific antibodies disclosed herein) can be used, for example, to treat CD28-related cancers. In some embodiments, the CD28 ABDs are capable of binding to human and cynomolgus monkey CD28 (see Figure 2 and Example 2).

[0500] As will be appreciated by those skilled in the art, a suitable CD28 binding domain may comprise a set of six such as Figures 41 to 74 And the CDRs depicted in the sequence listing. Suitable CD28 ABDs may also include sequences such as these and Figures 41 to 74 and the complete VH and VL sequences depicted in the sequence listing for use as scFv or Fab domains.

[0501] In one embodiment, the CD28 antigen binding domain comprises the Figures 41 to 74 The six CDRs of the CD28 ABD (i.e., vhCDR1-3 and vlCDR1-3) described in the SEQ ID NO: 1 and SEQ ID NO: 2 are either underlined or, where a different numbering scheme is used as described herein and as shown in Table 2, are denoted by the underlined CDRs. Figures 41 to 74 The CDRs identified by other alignments within the variable heavy (VH) domain and variable light domain (VL) sequences of the SEQ ID NO: 1 and those depicted in the sequence listing (see Table 2). Suitable CD28 ABDs may also comprise the complete VH and VL sequences as depicted in these sequences and the accompanying figures, for use as scFv or Fab domains.

[0502] In one embodiment, the CD28 antigen binding domain comprises the Figures 41 to 74 and sequence listing) of the CD28 ABD (i.e., vhCDR1-3 and vlCDR1-3). In an exemplary embodiment, the CD28 antigen binding domain comprises the six CDRs of CD28 comprising a variable heavy domain (VH) and a variable light domain (VL), wherein VH and VL are selected from the following:

[0503] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 93;

[0504] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 101;

[0505] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 109;

[0506] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 117;

[0507] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 125;

[0508] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 133;

[0509] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 141;

[0510] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 149;

[0511] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 157;

[0512] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 165;

[0513] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 173;

[0514] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 181;

[0515] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 189;

[0516] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 197;

[0517] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 205;

[0518] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 213;

[0519] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 221;

[0520] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 229;

[0521] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 237; and

[0522] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 245;

[0523] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 253;

[0524] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 261;

[0525] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 269;

[0526] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 277;

[0527] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 285;

[0528] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 293;

[0529] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 301;

[0530] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 309;

[0531] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 317;

[0532] (i) a VH having the amino acid sequence of SEQ ID NO: 321; and (ii) a VL having the amino acid sequence of SEQ ID NO: 325;

[0533] (i) a VH having the amino acid sequence of SEQ ID NO: 329; and (ii) a VL having the amino acid sequence of SEQ ID NO: 333;

[0534] (i) a VH having the amino acid sequence of SEQ ID NO: 337; and (ii) a VL having the amino acid sequence of SEQ ID NO: 341;

[0535] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0536] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0537] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0538] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0539] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0540] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0541] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0542] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0543] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0544] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0545] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0546] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0547] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0548] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0549] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0550] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0551] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0552] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0553] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0554] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0555] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0556] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0557] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0558] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0559] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0560] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0561] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0562] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0563] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0564] (i) a VH having the amino acid sequence of SEQ ID NO: 321; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0565] (i) a VH having the amino acid sequence of SEQ ID NO: 329; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0566] (i) a VH having the amino acid sequence of SEQ ID NO: 337; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20. Figure 24 and Figures 41 to 74 ).

[0567] In addition to the parent CDR sets that form the ABD of CD28 disclosed in the accompanying drawings and sequence listings, variant CD28 ABDs having at least one modified CDR comprising the CD28 ABD CDRs disclosed herein are also provided herein. In one embodiment, the CD28 ABD comprises a set of 6 CDRs that have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to the 6 CDRs of the CD28 ABD described herein (including the accompanying drawings and sequence listings). In an exemplary embodiment, the CD28 ABD comprises a set of 6 CDRs that have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications compared to the 6 CDRs of a CD28 ABD comprising a variable heavy domain (VH) and a variable light domain (VL) selected from the group consisting of:

[0568] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 93;

[0569] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 101;

[0570] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 109;

[0571] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 117;

[0572] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 125;

[0573] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 133;

[0574] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 141;

[0575] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 149;

[0576] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 157;

[0577] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 165;

[0578] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 173;

[0579] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 181;

[0580] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 189;

[0581] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 197;

[0582] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 205;

[0583] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 213;

[0584] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 221;

[0585] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 229;

[0586] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 237; and

[0587] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 245;

[0588] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 253;

[0589] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 261;

[0590] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 269;

[0591] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 277;

[0592] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 285;

[0593] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 293;

[0594] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 301;

[0595] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 309;

[0596] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 317;

[0597] (i) a VH having the amino acid sequence of SEQ ID NO: 321; and (ii) a VL having the amino acid sequence of SEQ ID NO: 325;

[0598] (i) a VH having the amino acid sequence of SEQ ID NO: 329; and (ii) a VL having the amino acid sequence of SEQ ID NO: 333;

[0599] (i) a VH having the amino acid sequence of SEQ ID NO: 337; and (ii) a VL having the amino acid sequence of SEQ ID NO: 341;

[0600] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0601] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0602] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0603] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0604] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0605] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0606] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0607] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0608] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0609] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0610] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0611] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0612] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0613] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0614] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0615] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0616] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0617] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0618] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0619] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0620] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0621] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0622] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0623] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0624] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0625] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0626] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0627] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0628] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0629] (i) a VH having the amino acid sequence of SEQ ID NO: 321; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0630] (i) a VH having the amino acid sequence of SEQ ID NO: 329; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0631] (i) a VH having the amino acid sequence of SEQ ID NO: 337; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20. Figure 24 as well as Figure 29 to Figure 32).

[0632] In certain embodiments, the variant CD28 ABD is capable of binding to the CD28 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay), the latter of which is particularly useful in many embodiments. In specific embodiments, the CD28 ABD is capable of binding to both human and cynomolgus monkey CD28.

[0633] In one embodiment, the anti-CD28 ABD comprises a Figure 24 and Figures 41 to 74and sequence listing). In exemplary embodiments, the anti-CD28 ABD comprises six CDRs that are at least 90%, 95%, 97%, 98% or 99% identical to the six CDRs of a CD28 ABD comprising a variable heavy domain (VH) and a variable light domain (VL) selected from the group consisting of:

[0634] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 93;

[0635] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 101;

[0636] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 109;

[0637] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 117;

[0638] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 125;

[0639] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 133;

[0640] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 141;

[0641] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 149;

[0642] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 157;

[0643] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 165;

[0644] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 173;

[0645] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 181;

[0646] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 189;

[0647] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 197;

[0648] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 205;

[0649] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 213;

[0650] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 221;

[0651] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 229;

[0652] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 237; and

[0653] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 245;

[0654] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 253;

[0655] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 261;

[0656] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 269;

[0657] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 277;

[0658] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 285;

[0659] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 293;

[0660] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 301;

[0661] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 309;

[0662] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 317;

[0663] (i) a VH having the amino acid sequence of SEQ ID NO: 321; and (ii) a VL having the amino acid sequence of SEQ ID NO: 325;

[0664] (i) a VH having the amino acid sequence of SEQ ID NO: 329; and (ii) a VL having the amino acid sequence of SEQ ID NO: 333;

[0665] (i) a VH having the amino acid sequence of SEQ ID NO: 337; and (ii) a VL having the amino acid sequence of SEQ ID NO: 341;

[0666] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0667] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0668] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0669] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0670] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0671] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0672] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0673] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0674] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0675] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0676] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0677] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0678] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0679] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0680] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0681] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0682] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0683] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0684] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0685] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0686] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0687] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0688] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0689] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0690] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0691] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0692] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0693] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0694] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0695] (i) a VH having the amino acid sequence of SEQ ID NO: 321; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0696] (i) a VH having the amino acid sequence of SEQ ID NO: 329; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0697] (i) a VH having the amino acid sequence of SEQ ID NO: 337; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20. Figure 24 and Figures 41 to 74 ).

[0698] In certain embodiments, the anti-CD28 ABD is capable of binding to the CD28 antigen as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay), the latter of which is particularly useful in many embodiments. In specific embodiments, the CD28 ABD is capable of binding to both human and cynomolgus monkey CD28.

[0699] In another exemplary embodiment, the anti-CD28 ABD comprises the Figure 24 and Figures 41 to 74 and sequence listing) of any of the CD28 ABDs described herein. In exemplary embodiments, VH and VL are selected from the following:

[0700] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 93;

[0701] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 101;

[0702] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 109;

[0703] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 117;

[0704] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 125;

[0705] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 133;

[0706] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 141;

[0707] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 149;

[0708] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 157;

[0709] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 165;

[0710] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 173;

[0711] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 181;

[0712] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 189;

[0713] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 197;

[0714] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 205;

[0715] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 213;

[0716] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 221;

[0717] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 229;

[0718] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 237; and

[0719] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 245;

[0720] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 253;

[0721] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 261;

[0722] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 269;

[0723] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 277;

[0724] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 285;

[0725] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 293;

[0726] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 301;

[0727] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 309;

[0728] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 317;

[0729] (i) a VH having the amino acid sequence of SEQ ID NO: 321; and (ii) a VL having the amino acid sequence of SEQ ID NO: 325;

[0730] (i) a VH having the amino acid sequence of SEQ ID NO: 329; and (ii) a VL having the amino acid sequence of SEQ ID NO: 333;

[0731] (i) a VH having the amino acid sequence of SEQ ID NO: 337; and (ii) a VL having the amino acid sequence of SEQ ID NO: 341;

[0732] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0733] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0734] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0735] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0736] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0737] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0738] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0739] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0740] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0741] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0742] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0743] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0744] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0745] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0746] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0747] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0748] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0749] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0750] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0751] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0752] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0753] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0754] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0755] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0756] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0757] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0758] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0759] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0760] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0761] (i) a VH having the amino acid sequence of SEQ ID NO: 321; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0762] (i) a VH having the amino acid sequence of SEQ ID NO: 329; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0763] (i) a VH having the amino acid sequence of SEQ ID NO: 337; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20. Figure 24 and Figures 41 to 74 ).

[0764] In addition to the parent anti-CD28 binding domain variable heavy domain and variable light domain disclosed herein, anti-CD28 ABDs are also provided herein comprising variable heavy domains and / or variable light domains that are variants of the anti-CD28 ABD VH and VL domains disclosed herein. In one embodiment, the parent anti-CD28 binding domain variable heavy domain and variable light domain disclosed herein (including Figures 28 to 31 The variant VH domain and / or VL domain has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes compared to the VH and / or VL domains of the anti-CD28 ABD described in (e.g., SEQ ID NO: 100). In exemplary embodiments, the variant VH domain and / or VL domain has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes compared to a VH and / or VL selected from:

[0765] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 93;

[0766] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 101;

[0767] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 109;

[0768] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 117;

[0769] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 125;

[0770] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 133;

[0771] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 141;

[0772] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 149;

[0773] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 157;

[0774] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 165;

[0775] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 173;

[0776] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 181;

[0777] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 189;

[0778] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 197;

[0779] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 205;

[0780] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 213;

[0781] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 221;

[0782] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 229;

[0783] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 237; and

[0784] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 245;

[0785] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 253;

[0786] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 261;

[0787] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 269;

[0788] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 277;

[0789] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 285;

[0790] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 293;

[0791] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 301;

[0792] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 309;

[0793] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 317;

[0794] (i) a VH having the amino acid sequence of SEQ ID NO: 321; and (ii) a VL having the amino acid sequence of SEQ ID NO: 325;

[0795] (i) a VH having the amino acid sequence of SEQ ID NO: 329; and (ii) a VL having the amino acid sequence of SEQ ID NO: 333;

[0796] (i) a VH having the amino acid sequence of SEQ ID NO: 337; and (ii) a VL having the amino acid sequence of SEQ ID NO: 341;

[0797] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0798] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0799] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0800] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0801] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0802] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0803] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0804] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0805] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0806] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0807] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0808] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0809] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0810] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0811] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0812] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0813] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0814] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0815] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0816] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0817] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0818] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0819] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0820] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0821] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0822] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0823] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0824] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0825] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0826] (i) a VH having the amino acid sequence of SEQ ID NO: 321; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0827] (i) a VH having the amino acid sequence of SEQ ID NO: 329; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0828] (i) a VH having the amino acid sequence of SEQ ID NO: 337; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20. Figure 24 and Figures 41 to 74 ).

[0829] In certain embodiments, the anti-CD28 ABD is capable of binding to CD28 as measured by at least one of Biacore, surface plasmon resonance (SPR), and / or BLI (biolayer interferometry, e.g., Octet assay), the latter of which is particularly useful in many embodiments. In specific embodiments, the CD28 ABD is capable of binding to both human and cynomolgus monkey CD28.

[0830] In some embodiments, the CD28 ABD comprises a variable heavy domain having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes relative to one of the following CD28 ABD variable heavy domains: SEQ ID NO: 89, 97, 105, 113, 121, 129, 137, 145, 153, 161, 169, 177, 185, 193, 201, 209, 217, 225, 233, 241, 249, 257, 265, 273, 281, 289, 297, 305, 313, 321, 329, and 337. In some embodiments, the CD28 ABD comprises any one of the variable light domains provided herein, or a variant thereof.

[0831] In some embodiments, the CD28 ABD comprises a variable light domain having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes relative to one of the following CD28 ABD variable light domains: SEQ ID NO: 20, 93, 101, 109, 117, 125, 133, 141, 149, 157, 165, 173, 181, 189, 197, 205, 213, 221, 229, 237, 245, 253, 261, 269, 277, 285, 293, 301, 309, 317, 325, 333, and 341. In some embodiments, the CD28 ABD comprises any one of the variable heavy domains provided herein, or a variant thereof.

[0832] In one embodiment, the variant VH and / or VL domains are as described herein (including Figures 41 to 74and Sequence Listing). In exemplary embodiments, the variant VH and / or VL domains are at least 90%, 95%, 97%, 98% or 99% identical to the VH and / or VL of one of the anti-CD28 ABDs comprising a VH and VL selected from:

[0833] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 93;

[0834] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 101;

[0835] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 109;

[0836] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 117;

[0837] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 125;

[0838] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 133;

[0839] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 141;

[0840] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 149;

[0841] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 157;

[0842] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 165;

[0843] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 173;

[0844] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 181;

[0845] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 189;

[0846] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 197;

[0847] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 205;

[0848] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 213;

[0849] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 221;

[0850] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 229;

[0851] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 237; and

[0852] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 245;

[0853] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 253;

[0854] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 261;

[0855] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 269;

[0856] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 277;

[0857] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 285;

[0858] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 293;

[0859] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 301;

[0860] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 309;

[0861] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 317;

[0862] (i) a VH having the amino acid sequence of SEQ ID NO: 321; and (ii) a VL having the amino acid sequence of SEQ ID NO: 325;

[0863] (i) a VH having the amino acid sequence of SEQ ID NO: 329; and (ii) a VL having the amino acid sequence of SEQ ID NO: 333;

[0864] (i) a VH having the amino acid sequence of SEQ ID NO: 337; and (ii) a VL having the amino acid sequence of SEQ ID NO: 341;

[0865] (i) a VH having the amino acid sequence of SEQ ID NO: 89; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0866] (i) a VH having the amino acid sequence of SEQ ID NO: 97; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0867] (i) a VH having the amino acid sequence of SEQ ID NO: 105; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0868] (i) a VH having the amino acid sequence of SEQ ID NO: 113; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0869] (i) a VH having the amino acid sequence of SEQ ID NO: 121; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0870] (i) a VH having the amino acid sequence of SEQ ID NO: 129; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0871] (i) a VH having the amino acid sequence of SEQ ID NO: 137; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0872] (i) a VH having the amino acid sequence of SEQ ID NO: 145; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0873] (i) a VH having the amino acid sequence of SEQ ID NO: 153; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0874] (i) a VH having the amino acid sequence of SEQ ID NO: 161; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0875] (i) a VH having the amino acid sequence of SEQ ID NO: 169; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0876] (i) a VH having the amino acid sequence of SEQ ID NO: 177; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0877] (i) a VH having the amino acid sequence of SEQ ID NO: 185; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0878] (i) a VH having the amino acid sequence of SEQ ID NO: 193; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0879] (i) a VH having the amino acid sequence of SEQ ID NO: 201; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0880] (i) a VH having the amino acid sequence of SEQ ID NO: 209; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0881] (i) a VH having the amino acid sequence of SEQ ID NO: 217; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0882] (i) a VH having the amino acid sequence of SEQ ID NO: 225; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0883] (i) a VH having the amino acid sequence of SEQ ID NO: 233; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20; and

[0884] (i) a VH having the amino acid sequence of SEQ ID NO: 241; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0885] (i) a VH having the amino acid sequence of SEQ ID NO: 249; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0886] (i) a VH having the amino acid sequence of SEQ ID NO: 257; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0887] (i) a VH having the amino acid sequence of SEQ ID NO: 265; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0888] (i) a VH having the amino acid sequence of SEQ ID NO: 273; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0889] (i) a VH having the amino acid sequence of SEQ ID NO: 281; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0890] (i) a VH having the amino acid sequence of SEQ ID NO: 289; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0891] (i) a VH having the amino acid sequence of SEQ ID NO: 297; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0892] (i) a VH having the amino acid sequence of SEQ ID NO: 305; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0893] (i) a VH having the amino acid sequence of SEQ ID NO: 313; and (ii) a VL having the amino acid sequence of SEQ ID NO: 20;

[0894] (...

Claims

1. A multispecific antibody, comprising: a) PD-L1 antigen binding domain; b) a PD-L2 antigen binding domain; and c) CD28 antigen binding domain.

2. A multispecific antibody, comprising: a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3, wherein VH1 is a first variable heavy domain, and CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-linker-VH3-CH1-hinge-CH2-CH3, wherein VH2 is a second variable heavy domain, VH3 is a third variable heavy domain, and CH2-CH3 is a second Fc domain, c) a first common light chain, a second common light chain, and a third common light chain, each of which comprises, from N-terminus to C-terminus, VL-CL, wherein VL is a common variable light domain, and CL is a constant light domain; wherein the first variable heavy domain and the common variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the common variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the common variable light domain of the third common light chain form a third antigen binding domain, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L1 antigen binding domain, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L2 antigen binding domain, and wherein one of the first antigen binding domain, the second antigen binding domain and the third antigen binding domain is a CD28 binding domain.

3. The multispecific antibody of claim 2, wherein the first antigen binding domain, the second antigen binding domain and the third antigen binding domain are selected from the following: i) the first antigen binding domain is the PD-L1 binding domain, the second antigen binding domain is the PD-L2 binding domain, and the third antigen binding domain is the CD28 binding domain, ii) the first antigen binding domain is the PD-L1 binding domain, the second antigen binding domain is the CD28 binding domain, and the third antigen binding domain is the PD-L2 binding domain, iii) the first antigen binding domain is the PD-L2 binding domain, the second antigen binding domain is the PD-L1 binding domain, and the third antigen binding domain is the CD28 binding domain, iv) the first antigen binding domain is the PD-L2 binding domain, the second antigen binding domain is the CD28 binding domain, and the third antigen binding domain is the PD-L1 binding domain, v) the first antigen binding domain is the CD28 binding domain, the second antigen binding domain is the PD-L1 binding domain, and the third antigen binding domain is the PD-L2 binding domain, vi) the first antigen binding domain is the CD28 binding domain, the second antigen binding domain is the PD-L2 binding domain, and the third antigen binding domain is the PD-L1 binding domain.

4. The multispecific antibody of claim 2 or 3, wherein the CD28 binding domain comprises a variable heavy domain having an amino acid selected from SEQ ID NO: 3354-3389, or a variant thereof, and a variable light domain having an amino acid sequence of SEQ ID NO: 3239, or a variant thereof.

5. The multispecific antibody of any one of claims 2 to 4, wherein the PD-L1 antigen binding domain comprises a variable heavy domain having an amino acid selected from SEQ ID NOs: 3235 and 3243-3260, or a variant thereof, and a variable light domain having an amino acid sequence of SEQ ID NO: 3239, or a variant thereof.

6. The multispecific antibody of any one of claims 2 to 5, wherein the PD-L2 antigen binding domain comprises a variable heavy domain having an amino acid selected from SEQ ID NO: 3267 and 3275-3347, or a variant thereof, and a variable light domain having an amino acid sequence of SEQ ID NO: 3239, or a variant thereof.

7. The multispecific antibody according to any one of claims 2 to 6, wherein the first Fc domain and the second Fc domain are variant Fc domains.

8. The multispecific antibody of claim 7, wherein the first Fc domain and the second Fc domain comprise a set of heterodimerization skew variants selected from the group consisting of: S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V, wherein numbering is according to EU numbering.

9. The multispecific antibody of claim 8, wherein the first Fc domain and the second Fc domain comprise the heterodimerization skew variant S364K / E357Q:L368D / K370S.

10. The multispecific antibody according to any one of claims 7 to 9, wherein the first Fc domain and the second Fc domain each comprise one or more ablation variants.

11. The multispecific antibody of claim 10, wherein the one or more ablation variants are E233P / L234V / L235A / G236del / S267K, wherein numbering is according to EU numbering.

12. The multispecific antibody of any one of claims 7 to 11, wherein one of the first monomer or the second monomer comprises one or more pi variants.

13. The multispecific antibody of claim 12, wherein the CH1-hinge-CH2-CH3 of the first monomer comprises the pI variants N208D / Q295E / N384D / Q418E / N421D, wherein the numbering is according to EU numbering.

14. The multispecific antibody of claim 13, wherein the first Fc domain comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K, wherein the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, and wherein the numbering is according to EU numbering.

15. The multispecific antibody of claim 14, wherein the first Fc domain and the second Fc domain each further comprise the amino acid variant 428 / 434S.

16. The multispecific antibody of claim 2, wherein: a) the PD-L1 binding domain comprises a variable heavy domain having an amino acid sequence of SEQ ID NO: 3251, and a variable light domain having an amino acid sequence of SEQ ID NO: 3239; b) the PD-L2 binding domain comprises a variable heavy domain having an amino acid sequence of SEQ ID NO: 3319, and a variable light domain having an amino acid sequence of SEQ ID NO: 3239; and c) the CD28 binding domain comprises a variable heavy domain having an amino acid sequence of SEQ ID NO: 3380, and a variable light domain having an amino acid sequence of SEQ ID NO: 3239.

17. The multispecific antibody of claim 2, wherein: a) the first monomer has an amino acid sequence of SEQ ID NO: 3201; b) the second monomer has the amino acid sequence of SEQ ID NO: 3202; and c) the first common light chain, the second common light chain and the third common light chain each have the amino acid sequence of SEQ ID NO: 3203.

18. A nucleic acid composition, comprising: a) a first polynucleotide encoding a first monomer as described in any one of claims 2 to 17; b) a second polynucleotide encoding the second monomer of any one of claims 2 to 17; and c) a third polynucleotide encoding the first common light chain, the second common light chain and the third common light chain of any one of claims 1 to 17.

19. An expression vector composition, comprising: a) a first expression vector comprising the first polynucleotide according to claim 17, b) a second expression vector comprising the second polynucleotide of claim 17, and c) a third expression vector comprising the third polynucleotide of claim 17.

20. A host cell comprising the nucleic acid composition of claim 18 or the expression vector composition of claim 19.

21. A pharmaceutical composition comprising the antibody of any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

22. A method for treating cancer, the method comprising administering the antibody according to any one of claims 1 to 17 or the pharmaceutical composition according to claim 21 to a patient in need thereof.

23. The method of claim 22, further comprising administering to the patient an anti-CD3 x tumor target antigen (TTA) bispecific antibody.

24. A multispecific antibody, comprising: a) a first monomer comprising, from N-terminus to C-terminus, scFv-linker-CH2-CH3, wherein CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH1-CH1-linker-VH2-CH1-hinge-CH2-CH3, wherein VH1 is a first variable heavy domain, VH2 is a second variable heavy domain, and CH2-CH3 is a second Fc domain, c) a first common light chain and a second common light chain, each of the first common light chain and the second common light chain comprising, from N-terminus to C-terminus, VL1-CL, wherein VL1 is a first variable light domain, and CL is a constant light domain; wherein the scFv comprises a third VH domain (VH3), a scFv linker and a second variable light domain (VL2) wherein the first variable heavy domain and the first variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the first variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the second variable light domain form a third antigen binding domain, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L1 antigen binding domain, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L2 antigen binding domain, and wherein one of the first antigen binding domain, the second antigen binding domain and the third antigen binding domain is a CD28 binding domain.

25. The multispecific antibody of claim 24, wherein the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain are selected from the following: i) the first antigen binding domain is the PD-L1 binding domain, the second antigen binding domain is the PD-L2 binding domain, and the third antigen binding domain is the CD28 binding domain, ii) the first antigen binding domain is the PD-L1 binding domain, the second antigen binding domain is the CD28 binding domain, and the third antigen binding domain is the PD-L2 binding domain, iii) the first antigen binding domain is the PD-L2 binding domain, the second antigen binding domain is the PD-L1 binding domain, and the third antigen binding domain is the CD28 binding domain, iv) the first antigen binding domain is the PD-L2 binding domain, the second antigen binding domain is the CD28 binding domain, and the third antigen binding domain is the PD-L1 binding domain, v) the first antigen binding domain is the CD28 binding domain, the second antigen binding domain is the PD-L1 binding domain, and the third antigen binding domain is the PD-L2 binding domain, vi) the first antigen binding domain is the CD28 binding domain, the second antigen binding domain is the PD-L2 binding domain, and the third antigen binding domain is the PD-L1 binding domain.

26. The multispecific antibody of claim 24 or 25, wherein the CD28 binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from any one of the variable heavy domains in Figures 35, 36, and 38, and a variable light domain selected from any one of the variable light domains in Figures 35, 37, and 38, ii) the variable heavy domain and variable light domain of the CD28 binding domain in Figure 38, iii) a variable heavy domain selected from any one of the variable heavy domains in Figures 41 to 74, and a variable light domain selected from any one of the variable light domains in Figures 41 to 74, iv) the variable heavy domain and variable light domain of the CD28 binding domain in Figures 41 to 74, v) a variable heavy domain selected from any one of the variable light domains in Figures 41 to 74, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 (Figure 24), vi) the variable heavy domain and variable light domain of the CD28 binding domain in Figure 81, and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 163, and a variable light domain selected from any one of the variable light domains in Figures 35 and 37, or a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

27. The multispecific antibody of any one of claims 24-26, wherein the PD-L1 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from any one of the variable heavy domains in Figures 17 and 19, and a variable light domain selected from any one of the variable light domains in Figures 17 and 20, ii) the variable heavy domain and the variable light domain of the PD-L1 binding domain in Figures 17 and 21, iii) a variable heavy domain selected from any one of the variable heavy domains in Figures 25 and 26, and a variable light domain selected from any one of the variable light domains in Figures 25 and 26, iv) the variable heavy domain and the variable light domain of the PD-L1 binding domain in Figures 25 and 26, v) a variable heavy domain selected from any one of the variable heavy domains in Figures 25 and 26, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 (Figure 24), vi) the variable heavy domain and variable light domain of the PD-L1 binding domain in Figure 28; and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 161, and a variable light domain having the amino acid sequence of SEQ ID NO:3239.

28. The multispecific antibody of any one of claims 24-27, wherein the PD-L2 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from any one of the variable heavy domains in Figures 29 to 32, and a variable light domain selected from any one of the variable light domains in Figures 29 to 32, ii) the variable heavy domain and the variable light domain of the PD-L2 binding domain in Figures 29 to 32, iii) a variable heavy domain selected from any one of the variable heavy domains in Figures 29 to 32, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 (Figure 24), iv) the variable heavy domain and the variable light domain of the PD-L2 binding domain in Figure 34, and v) a variable heavy domain selected from any one of the variable heavy domains in Figure 162, and a variable light domain having the amino acid sequence of SEQ ID NO:3239.

29. The multispecific antibody of any one of claims 24-28, wherein the first Fc domain and the second Fc domain are variant Fc domains.

30. The multispecific antibody of claim 29, wherein the first Fc domain and the second Fc domain comprise a set of heterodimerization skew variants selected from the group consisting of: S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V, wherein numbering is according to EU numbering.

31. The multispecific antibody of claim 30, wherein the first Fc domain and the second Fc domain comprise the heterodimerization skew variant S364K / E357Q:L368D / K370S.

32. The multispecific antibody of any one of claims 29 to 31, wherein the first Fc domain and the second Fc domain each comprise one or more ablation variants.

33. The multispecific antibody of claim 32, wherein the one or more ablation variants are E233P / L234V / L235A / G236del / S267K, wherein numbering is according to EU numbering.

34. The multispecific antibody of any one of claims 29 to 33, wherein one of the first monomer or the second monomer comprises one or more pi variants.

35. The multispecific antibody of claim 34, wherein the CH1-hinge-CH2-CH3 of the second monomer comprises the pi variant N208D / Q295E / N384D / Q418E / N421D, wherein the numbering is according to EU numbering.

36. The multispecific antibody of claim 29, wherein the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, wherein the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K, and wherein the numbering is according to EU numbering.

37. The multispecific antibody of claim 36, wherein the scFv linker is a charged scFv linker having the amino acid sequence (GKPGS)4.

38. The multispecific antibody of claim 36, wherein the first Fc domain and the second Fc domain each further comprise the amino acid variant 428 / 434S.

39. A nucleic acid composition comprising: a) a first polynucleotide encoding a first monomer as described in any one of claims 24 to 38; b) a second polynucleotide encoding the second monomer of any one of 24 to 38; and c) a third polynucleotide encoding the first common light chain and the second common light chain of any one of claims 24 to 38.

40. An expression vector composition, comprising: a) a first expression vector comprising the first polynucleotide according to claim 39, b) a second expression vector comprising the second polynucleotide of claim 39, and c) a third expression vector comprising the third polynucleotide of claim 39.

41. A host cell comprising the nucleic acid composition of claim 39 or the expression vector composition of claim 40.

42. A pharmaceutical composition comprising the antibody of any one of claims 24 to 38 and a pharmaceutically acceptable carrier.

43. A method for treating cancer, comprising administering the antibody of any one of claims 24 to 38 or the pharmaceutical composition of claim G20 to a patient in need thereof.

44. The method of claim 43, further comprising administering to the patient an anti-CD3 x tumor target antigen (TTA) bispecific antibody.

45. A multispecific antibody, comprising: a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-linker-scFv-linker-CH2-CH3, wherein VH1 is a first variable heavy domain, and CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variable heavy domain, and CH2-CH3 is a second Fc domain, c) a first common light chain and a second common light chain, each of the first common light chain and the second common light chain comprising, from N-terminus to C-terminus, VL1-CL, wherein VL1 is a first variable light domain, and CL is a constant light domain; wherein the scFv comprises a third VH domain (VH3), a scFv linker and a second variable light domain (VL2) wherein the first variable heavy domain and the first variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the first variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the second variable light domain form a third antigen binding domain, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L1 antigen binding domain, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L2 antigen binding domain, and wherein one of the first antigen binding domain, the second antigen binding domain and the third antigen binding domain is a CD28 binding domain.

46. ​​The multispecific antibody of claim 45, wherein the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain are selected from the following: i) the first antigen binding domain is the PD-L1 binding domain, the second antigen binding domain is the PD-L2 binding domain, and the third antigen binding domain is the CD28 binding domain, ii) the first antigen binding domain is the PD-L1 binding domain, the second antigen binding domain is the CD28 binding domain, and the third antigen binding domain is the PD-L2 binding domain, iii) the first antigen binding domain is the PD-L2 binding domain, the second antigen binding domain is the PD-L1 binding domain, and the third antigen binding domain is the CD28 binding domain, iv) the first antigen binding domain is the PD-L2 binding domain, the second antigen binding domain is the CD28 binding domain, and the third antigen binding domain is the PD-L1 binding domain, v) the first antigen binding domain is the CD28 binding domain, the second antigen binding domain is the PD-L1 binding domain, and the third antigen binding domain is the PD-L2 binding domain, vi) the first antigen binding domain is the CD28 binding domain, the second antigen binding domain is the PD-L2 binding domain, and the third antigen binding domain is the PD-L1 binding domain.

47. The multispecific antibody of claim 45 or 46, wherein the CD28 binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from any one of the variable heavy domains in Figures 35, 36, and 38, and a variable light domain selected from any one of the variable light domains in Figures 35, 37, and 38, ii) the variable heavy domain and variable light domain of the CD28 binding domain in Figure 38, iii) a variable heavy domain selected from any one of the variable heavy domains in Figures 41 to 74, and a variable light domain selected from any one of the variable light domains in Figures 41 to 74, iv) the variable heavy domain and variable light domain of the CD28 binding domain in Figures 41 to 74, v) a variable heavy domain selected from any one of the variable light domains in Figures 41 to 74, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 (Figure 24), vi) the variable heavy domain and variable light domain of the CD28 binding domain in Figure 81, and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 163, and a variable light domain selected from any one of the variable light domains in Figures 35 and 37, or a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

48. The multispecific antibody of any one of claims 45 to 47, wherein the PD-L1 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from any one of the variable heavy domains in Figures 17 and 19, and a variable light domain selected from any one of the variable light domains in Figures 17 and 20, ii) the variable heavy domain and the variable light domain of the PD-L1 binding domain in Figures 17 and 21, iii) a variable heavy domain selected from any one of the variable heavy domains in Figures 25 and 26, and a variable light domain selected from any one of the variable light domains in Figures 25 and 26, iv) the variable heavy domain and the variable light domain of the PD-L1 binding domain in Figures 25 and 26, v) a variable heavy domain selected from any one of the variable heavy domains in Figures 25 and 26, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 (Figure 24), vi) the variable heavy domain and variable light domain of the PD-L1 binding domain in Figure 28; and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 161, and a variable light domain having the amino acid sequence of SEQ ID NO:3239.

49. The multispecific antibody of any one of claims 45 to 48, wherein the PD-L2 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from any one of the variable heavy domains in Figures 29 to 32, and a variable light domain selected from any one of the variable light domains in Figures 29 to 32, ii) the variable heavy domain and the variable light domain of the PD-L2 binding domain in Figures 29 to 32, iii) a variable heavy domain selected from any one of the variable heavy domains in Figures 29 to 32, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 (Figure 24), iv) the variable heavy domain and the variable light domain of the PD-L2 binding domain in Figure 34, and v) a variable heavy domain selected from any one of the variable heavy domains in Figure 162, and a variable light domain having the amino acid sequence of SEQ ID NO:3239.

50. The multispecific antibody of any one of claims 45 to 49, wherein the first Fc domain and the second Fc domain are variant Fc domains.

51. The multispecific antibody of claim 50, wherein the first Fc domain and the second Fc domain comprise a set of heterodimerization skew variants selected from the group consisting of: S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V, wherein numbering is according to EU numbering.

52. The multispecific antibody of claim 51, wherein the first Fc domain and the second Fc domain comprise the heterodimerization skew variant S364K / E357Q:L368D / K370S.

53. The multispecific antibody of any one of claims 50 to 52, wherein the first Fc domain and the second Fc domain each comprise one or more ablation variants.

54. The multispecific antibody of claim 53, wherein the one or more ablation variants are E233P / L234V / L235A / G236del / S267K, wherein numbering is according to EU numbering.

55. The multispecific antibody of any one of claims 50 to 54, wherein one of the first monomer or the second monomer comprises one or more pi variants.

56. The multispecific antibody of claim 55, wherein the CH1-hinge-CH2-CH3 of the second monomer comprises the pi variants N208D / Q295E / N384D / Q418E / N421D, wherein the numbering is according to EU numbering.

57. The multispecific antibody of claim 56, wherein the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, wherein the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K, and wherein the numbering is according to EU numbering.

58. The multispecific antibody of claim 57, wherein the scFv linker is a charged scFv linker having the amino acid sequence (GKPGS)4.

59. The multispecific antibody of claim 57, wherein the first Fc domain and the second Fc domain each further comprise the amino acid variant 428 / 434S.

60. A nucleic acid composition, comprising: a) a first polynucleotide encoding a first monomer as described in any one of claims 45 to 59; b) a second polynucleotide encoding the second monomer of any one of claims 45 to 59; and c) a third polynucleotide encoding the first common light chain and the second common light chain of any one of claims 45 to 59, respectively.

61. An expression vector composition, comprising: a) a first expression vector comprising the first polynucleotide according to claim 60, b) a second expression vector comprising the second polynucleotide of claim 60, and c) a third expression vector comprising the third polynucleotide of claim 60.

62. A host cell comprising the nucleic acid composition of claim 60 or the expression vector composition of claim 61.

63. A pharmaceutical composition comprising the antibody of any one of claims 45 to 59 and a pharmaceutically acceptable carrier.

64. A method for treating cancer, the method comprising administering to a patient in need thereof an antibody according to any one of claims 45 to 59 or a pharmaceutical composition according to claim H20.

65. The method of claim 64, further comprising administering to the patient an anti-CD3 x tumor target antigen (TTA) bispecific antibody.

66. A multispecific antibody, comprising: a) a first monomer, which comprises, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3-domain linker-scFv, wherein VH1 is the first variable heavy domain, and CH2-CH3 is the first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variable heavy domain, and CH2-CH3 is a second Fc domain; and c) a first common light chain and a second common light chain, each of the first common light chain and the second common light chain comprising, from N-terminus to C-terminus, VL1-CL, wherein VL1 is a first variable light domain, and CL is a constant light domain; wherein the scFv comprises a third VH domain (VH3), a scFv linker and a second variable light domain (VL2) wherein the first variable heavy domain and the first variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the first variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the second variable light domain form a third antigen binding domain, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L1 antigen binding domain, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L2 antigen binding domain, and wherein one of the first antigen binding domain, the second antigen binding domain and the third antigen binding domain is a CD28 binding domain.

67. The multispecific antibody of claim 66, wherein the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain are selected from the group consisting of: i) the first antigen binding domain is the PD-L1 binding domain, the second antigen binding domain is the PD-L2 binding domain, and the third antigen binding domain is the CD28 binding domain, ii) the first antigen binding domain is the PD-L1 binding domain, the second antigen binding domain is the CD28 binding domain, and the third antigen binding domain is the PD-L2 binding domain, iii) the first antigen binding domain is the PD-L2 binding domain, the second antigen binding domain is the PD-L1 binding domain, and the third antigen binding domain is the CD28 binding domain, iv) the first antigen binding domain is the PD-L2 binding domain, the second antigen binding domain is the CD28 binding domain, and the third antigen binding domain is the PD-L1 binding domain, v) the first antigen binding domain is the CD28 binding domain, the second antigen binding domain is the PD-L1 binding domain, and the third antigen binding domain is the PD-L2 binding domain, vi) the first antigen binding domain is the CD28 binding domain, the second antigen binding domain is the PD-L2 binding domain, and the third antigen binding domain is the PD-L1 binding domain.

68. The multispecific antibody of claim 66 or 67, wherein the CD28 binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from any one of the variable heavy domains in Figures 35, 36, and 38, and a variable light domain selected from any one of the variable light domains in Figures 35, 37, and 38, ii) the variable heavy domain and variable light domain of the CD28 binding domain in Figure 38, iii) a variable heavy domain selected from any one of the variable heavy domains in Figures 41 to 74, and a variable light domain selected from any one of the variable light domains in Figures 41 to 74, iv) the variable heavy domain and variable light domain of the CD28 binding domain in Figures 41 to 74, v) a variable heavy domain selected from any one of the variable light domains in Figures 41 to 74, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 (Figure 24), vi) the variable heavy domain and variable light domain of the CD28 binding domain in Figure 81, and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 163, and a variable light domain selected from any one of the variable light domains in Figures 35 and 37, or a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

69. The multispecific antibody of any one of claims 66 to 68, wherein the PD-L1 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from any one of the variable heavy domains in Figures 17 and 19, and a variable light domain selected from any one of the variable light domains in Figures 17 and 20, ii) the variable heavy domain and the variable light domain of the PD-L1 binding domain in Figures 17 and 21, iii) a variable heavy domain selected from any one of the variable heavy domains in Figures 25 and 26, and a variable light domain selected from any one of the variable light domains in Figures 25 and 26, iv) the variable heavy domain and the variable light domain of the PD-L1 binding domain in Figures 25 and 26, v) a variable heavy domain selected from any one of the variable heavy domains in Figures 25 and 26, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 (Figure 24), vi) the variable heavy domain and variable light domain of the PD-L1 binding domain in Figure 28; and vii) a variable heavy domain selected from any one of the variable heavy domains in Figure 161, and a variable light domain having the amino acid sequence of SEQ ID NO:3239.

70. The multispecific antibody of any one of claims 66 to 69, wherein the PD-L2 antigen binding domain comprises a variable heavy domain and a variable light domain selected from: i) a variable heavy domain selected from any one of the variable heavy domains in Figures 29 to 32, and a variable light domain selected from any one of the variable light domains in Figures 29 to 32, ii) the variable heavy domain and the variable light domain of the PD-L2 binding domain in Figures 29 to 32, iii) a variable heavy domain selected from any one of the variable heavy domains in Figures 29 to 32, and a variable light domain having the amino acid sequence of SEQ ID NO: 20 (Figure 24), iv) the variable heavy domain and the variable light domain of the PD-L2 binding domain in Figure 34, and v) a variable heavy domain selected from any one of the variable heavy domains in Figure 162, and a variable light domain having the amino acid sequence of SEQ ID NO:3239.

71. The multispecific antibody of any one of claims 66 to 70, wherein the first Fc domain and the second Fc domain are variant Fc domains.

72. The multispecific antibody of claim 71, wherein the first Fc domain and the second Fc domain comprise a set of heterodimerization skew variants selected from the group consisting of: S364K / E357Q:L368D / K370S; S364K:L368D / K370S; S364K:L368E / K370S; D401K:T411E / K360E / Q362E; and T366W:T366S / L368A / Y407V, wherein numbering is according to EU numbering.

73. The multispecific antibody of claim 73, wherein the first Fc domain and the second Fc domain comprise the heterodimerization skew variant S364K / E357Q:L368D / K370S.

74. The multispecific antibody of any one of claims 71 to 73, wherein the first Fc domain and the second Fc domain each comprise one or more ablation variants.

75. The multispecific antibody of claim 74, wherein the one or more ablation variants are E233P / L234V / L235A / G236del / S267K, wherein numbering is according to EU numbering.

76. The multispecific antibody of any one of claims 71 to 75, wherein one of the first monomer or the second monomer comprises one or more pi variants.

77. The multispecific antibody of claim 76, wherein the CH1-hinge-CH2-CH3 of the second monomer comprises the pi variant N208D / Q295E / N384D / Q418E / N421D, wherein the numbering is according to EU numbering.

78. The multispecific antibody of claim 77, wherein the first Fc domain comprises the amino acid variants S364K / E357Q / E233P / L234V / L235A / G236del / S267K, wherein the CH1-hinge-CH2-CH3 of the second monomer comprises the amino acid variants L368D / K370S / N208D / Q295E / N384D / Q418E / N421D / E233P / L234V / L235A / G236del / S267K, and wherein the numbering is according to EU numbering.

79. The multispecific antibody of claim 78, wherein the scFv linker is a charged scFv linker having the amino acid sequence (GKPGS)4.

80. The multispecific antibody of claim 78, wherein the first Fc domain and the second Fc domain each further comprise the amino acid variant 428 / 434S.

81. A nucleic acid composition, comprising: a) a first polynucleotide encoding a first monomer as described in any one of claims 66 to 80; b) a second polynucleotide encoding the second monomer of any one of claims 66 to 80; and c) a third polynucleotide encoding the first common light chain and the second common light chain of any one of claims 66 to 80.

82. An expression vector composition, comprising: a) a first expression vector comprising the first polynucleotide according to claim 60, b) a second expression vector comprising the second polynucleotide of claim 60, and c) a third expression vector comprising the third polynucleotide of claim 60.

83. A host cell comprising the nucleic acid composition of claim 60 or the expression vector composition of claim 61.

84. A pharmaceutical composition comprising the antibody of any one of claims 66 to 80 and a pharmaceutically acceptable carrier.

85. A method for treating cancer, comprising administering to a patient in need thereof an antibody according to any one of claims 66 to 80 or a pharmaceutical composition according to claim H20.

86. The method of claim 85, further comprising administering to the patient an anti-CD3 x tumor target antigen (TTA) bispecific antibody.

87. A composition comprising a CD28 antigen binding domain, wherein the CD28 binding domain comprises: a) a variable heavy domain having vhCDR1-3 of the variable heavy domain in Figure 163; and b) a variable light domain having vlCDR1-3 of the variable light domain in Figures 35 and 37 or a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

88. A composition comprising a CD28 antigen binding domain, wherein the CD28 binding domain comprises: a) a variable heavy domain that has at least 85% sequence identity to the variable heavy domain in Figure 163; and b) a variable light domain that has at least 85% sequence identity with the variable light domains in Figures 35 and 37 or a variable light domain having the amino acid sequence of SEQ ID NO: 3239.

89. A composition as described in claim 88, wherein the variable heavy domain has at least 90% sequence identity with the variable heavy domain in Figure 163; and the variable light domain has at least 90% sequence identity with the variable light domain in Figures 35 and 37 or a variable light domain with the amino acid sequence of SEQ ID NO:3239.

90. A composition as described in claim 88, wherein the variable heavy domain has at least 95% sequence identity with the variable heavy domain in Figure 163; and the variable light domain has at least 95% sequence identity with the variable light domain in Figures 35 and 37 or a variable light domain having the amino acid sequence of SEQ ID NO:3239.

91. A composition as described in claim 88, wherein the variable heavy domain has at least 99% sequence identity with the variable heavy domain in Figure 163; and the variable light domain has at least 99% sequence identity with the variable light domain in Figures 35 and 37 or a variable light domain with the amino acid sequence of SEQ ID NO:3239.

92. compositions as claimed in claim 88, wherein said variable heavy domain has the amino acid sequence of the variable heavy domain in Figure 163; And said variable light domain has the variable light domain in Figure 35 and Figure 37 or the amino acid sequence of the variable light domain with the amino acid sequence of SEQ IDNO:3239.

93. A composition comprising a PD-L1 antigen binding domain, wherein the PD-L1 binding domain comprises: a) a variable heavy domain having vhCDR1-3 of the variable heavy domain in Figure 161; and b) a variable light domain having vlCDR1-3 of a variable light domain having the amino acid sequence of SEQ ID NO:3239.

94. A composition comprising a PD-L1 antigen binding domain, wherein the PD-L1 binding domain comprises: a) a variable heavy domain that has at least 85% sequence identity to the variable heavy domain in Figure 161; and b) a variable light domain that has at least 85% sequence identity to the variable light domain having the amino acid sequence of SEQ ID NO:3239.

95. A composition as described in claim 94, wherein the variable heavy domain has at least 90% sequence identity with the variable heavy domain in Figure 161; and the variable light domain has at least 90% sequence identity with the variable light domain having the amino acid sequence of SEQ ID NO:3239.

96. A composition as described in claim 94, wherein the variable heavy domain has at least 95% sequence identity with the variable heavy domain in Figure 161; and the variable light domain has at least 95% sequence identity with the variable light domain having the amino acid sequence of SEQ ID NO:3239.

97. A composition as described in claim 94, wherein the variable heavy domain has at least 99% sequence identity with the variable heavy domain in Figure 161; and the variable light domain has at least 99% sequence identity with the variable light domain having the amino acid sequence of SEQ ID NO:3239.

98. A composition as described in claim 94, wherein the variable heavy domain has the amino acid sequence of the variable heavy domain in Figure 161; and the variable light domain has the amino acid sequence of a variable light domain having the amino acid sequence of SEQ ID NO:3239.

99. A composition comprising a PD-L2 antigen binding domain, wherein the PD-L1 binding domain comprises: a) a variable heavy domain having vhCDR1-3 of the variable heavy domain in Figure 162; and b) a variable light domain having vlCDR1-3 of a variable light domain having the amino acid sequence of SEQ ID NO:3239.

100. A composition comprising a PD-L2 antigen binding domain, wherein the PD-L1 binding domain comprises: a) a variable heavy domain that has at least 85% sequence identity to the variable heavy domain in Figure 162; and b) a variable light domain that has at least 85% sequence identity to the variable light domain having the amino acid sequence of SEQ ID NO:3239.

101. A composition as described in claim 100, wherein the variable heavy domain has at least 90% sequence identity with the variable heavy domain in Figure 162; and the variable light domain has at least 90% sequence identity with the variable light domain having the amino acid sequence of SEQ ID NO:3239.

102. A composition as described in claim 94, wherein the variable heavy domain has at least 95% sequence identity with the variable heavy domain in Figure 162; and the variable light domain has at least 95% sequence identity with the variable light domain having the amino acid sequence of SEQ ID NO:3239.

103. A composition as described in claim 94, wherein the variable heavy domain has at least 99% sequence identity with the variable heavy domain in Figure 162; and the variable light domain has at least 99% sequence identity with the variable light domain having the amino acid sequence of SEQ ID NO:3239.

104. A composition as described in claim 100, wherein the variable heavy domain has the amino acid sequence of the variable heavy domain in Figure 162; and the variable light domain has the amino acid sequence of a variable light domain having the amino acid sequence of SEQ ID NO:3239.

105. An antibody comprising: a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3, wherein VH1 is a first variable heavy domain, and CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-linker-VH3-CH1-hinge-CH2-CH3, wherein VH2 is a second variable heavy domain, VH3 is a third variable heavy domain, and CH2-CH3 is a second Fc domain, c) a first common light chain, a second common light chain, and a third common light chain, each of which comprises, from N-terminus to C-terminus, VL-CL, wherein VL is a common variable light domain, and CL is a constant light domain; wherein the first variable heavy domain and the common variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the common variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the common variable light domain of the third common light chain form a third antigen binding domain, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L1 antigen binding domain, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L2 antigen binding domain, and wherein one of the first antigen binding domain, the second antigen binding domain and the third antigen binding domain is a CD28 binding domain.

106. An antibody comprising: a) a first monomer comprising, from N-terminus to C-terminus, scFv-linker-CH2-CH3, wherein CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH1-CH1-linker-VH2-CH1-hinge-CH2-CH3, wherein VH1 is a first variable heavy domain, VH2 is a second variable heavy domain, and CH2-CH3 is a second Fc domain, c) a first common light chain and a second common light chain, each of the first common light chain and the second common light chain comprising, from N-terminus to C-terminus, VL1-CL, wherein VL1 is a first variable light domain, and CL is a constant light domain; wherein the scFv comprises a third VH domain (VH3), a scFv linker and a second variable light domain (VL2) wherein the first variable heavy domain and the first variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the first variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the second variable light domain form a third antigen binding domain.

107. An antibody comprising: a) a first monomer comprising, from N-terminus to C-terminus, VH1-CH1-linker-scFv-linker-CH2-CH3, wherein VH1 is a first variable heavy domain, and CH2-CH3 is a first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variable heavy domain, and CH2-CH3 is a second Fc domain, c) a first common light chain and a second common light chain, each of the first common light chain and the second common light chain comprising, from N-terminus to C-terminus, VL1-CL, wherein VL1 is a first variable light domain, and CL is a constant light domain; wherein the scFv comprises a third VH domain (VH3), a scFv linker and a second variable light domain (VL2) wherein the first variable heavy domain and the first variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the first variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the second variable light domain form a third antigen binding domain.

108. An antibody comprising: a) a first monomer, which comprises, from N-terminus to C-terminus, VH1-CH1-hinge-CH2-CH3-domain linker-scFv, wherein VH1 is the first variable heavy domain, and CH2-CH3 is the first Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variable heavy domain, and CH2-CH3 is a second Fc domain; and c) a first common light chain and a second common light chain, each of the first common light chain and the second common light chain comprising, from N-terminus to C-terminus, VL1-CL, wherein VL1 is a first variable light domain, and CL is a constant light domain; wherein the scFv comprises a third VH domain (VH3), a scFv linker and a second variable light domain (VL2) wherein the first variable heavy domain and the first variable light domain of the first common light chain form a first antigen binding domain, the second variable heavy domain and the first variable light domain of the second common light chain form a second antigen binding domain, and the third variable heavy domain and the second variable light domain form a third antigen binding domain.

109. The antibody of any one of claims 105 to 108, wherein one of the first antigen binding domain, the second antigen binding domain, or the third antigen binding domain binds CD28.

110. The antibody of any one of claims 108, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L1 antigen binding domain, wherein one of the first antigen binding domain, the second antigen binding domain, and the third antigen binding domain is a PD-L2 antigen binding domain, and wherein one of the first antigen binding domain, the second antigen binding domain and the third antigen binding domain is a CD28 binding domain.

Images