CD28 single domain antibodies and their multivalent and multispecific constructs

Abstract

Provided herein is a binding polypeptide that specifically binds to CD28. More specifically, provided herein is a fusion protein that comprises a multivalent and / or multispecific construct that binds at least to CD28. Also provided are pharmaceutical compositions that contain these polypeptides, nucleic acid molecules encoding the polypeptides, and vectors thereof, as well as methods for using the provided CD28 binding polypeptides and the use thereof for treating diseases and conditions such as cancer. TIFF2023513003000071.tif67142

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 967,533, filed January 29, 2020, entitled "CD28 SINGLE DOMAIN ANTIBODIES AND MULTIVALENT AND MULTISPECIFIC CONSTRUCTS THEREOF," the contents of which are incorporated by reference in their entirety for all purposes.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application has been submitted with an electronic Sequence Listing. The Sequence Listing is provided as a file entitled 744952001540SeqList.TXT, created on January 28, 2021, and is 327,680 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure generally provides binding polypeptides that specifically bind to cluster of differentiation 28 (CD28). More specifically, the present disclosure relates to fusion proteins, including multivalent and / or multispecific constructs that bind to at least CD28. The present disclosure also provides nucleic acid molecules encoding these polypeptides and vectors, as well as methods and uses of the provided CD28-binding polypeptides to treat diseases and conditions, such as cancer. [Background technology]

[0004] background Cluster of differentiation 28 (CD28) is a homodimeric transmembrane glycoprotein of the immunoglobulin (Ig) superfamily that is primarily expressed by T-lineage cells. CD28 provides costimulatory signals involved in T-cell activation and survival. The role of T cells in various cancers in humans makes CD28 a desirable therapeutic target. However, some CD28 agonists cause substantial toxicity in vivo. Therefore, improved therapeutic molecules that target CD28 are needed. Embodiments that meet this need are provided herein. Summary of the Invention

[0005] overview Provided herein is a CD28-binding polypeptide having at least one single domain antibody (sdAb) that binds to CD28, wherein the at least one CD28 sdAb contains a complementarity determining region 1 (CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 189, 192, 195, 198, 199, 200, and 201; a complementarity determining region 2 (CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 193, 196, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, and 212; and a complementarity determining region 3 (CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 194, and 197.

[0006] In some embodiments, at least one CD28 sdAb comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 191; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 192, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 193, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 194; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 199, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 200, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197;CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 207, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 208, and CDR CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 210, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 211, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; or CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197;

[0007] Also disclosed are CD28 binding polypeptides having at least one single domain antibody (sdAb) that binds to CD28, wherein the at least one CD28 sdAb comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 189, 192, 195, 198, 199, 200, and 201; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 193, 196, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, and 212, 386, 387, 388, 389, 390, 391, 392, 393, 394, and 395; and Also provided herein are CD28 binding polypeptides comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of NOs: 191, 194, and 197, 396, 397, and 398.

[0008] In some embodiments, at least one CD28 sdAb comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 191; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 192, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 193, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 194; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 199, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 200, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197;CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 207, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 208, and CDR CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 210, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 211, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 213, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197 CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197;CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:386, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:387, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:388, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:389, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:389 CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397;CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:390, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:391, and CDR CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396 CDR2 comprising the amino acid sequence set forth in SEQ ID NO:392, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197;CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:393, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:394, and CDR CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 395, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 396 CDR2 comprising the amino acid sequence set forth in SEQ ID NO:388, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197;CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 389, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 397; or CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 398.

[0009] In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 191. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 192, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 193, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 194. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 200, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 207, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:208, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 210, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 211, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:386, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:387, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:388, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:389, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:390, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:391, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:392, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:393, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:394, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:395, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 388, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 389, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 397; or a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 398.

[0010] Also provided herein is a CD28-binding polypeptide having at least one single domain antibody (sdAb) that binds to CD28, wherein the at least one CD28 sdAb contains a complementarity determining region 1 (CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 189, 192, 195, 198, 199, 200, and 201; a complementarity determining region 2 (CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 193, 196, 202, 204, 205, 206, 207, 208, 209, 210, 211, and 212; and a complementarity determining region 3 (CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 194, and 197.

[0011] In some embodiments, at least one CD28 sdAb comprises a CDR1 having the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 191; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 192, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 193, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 194; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 198, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 199 CDR1 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 200, CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 201, CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197;CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 205, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 207, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 208, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 209, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197 CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 210, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 211, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; or CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 212, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197;

[0012] Also disclosed is a CD28 binding polypeptide comprising at least one single domain antibody (sdAb) that binds to CD28, wherein the at least one CD28 sdAb comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 189, 192, 195, 198, 199, 200, and 201; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 193, 196, 202, 204, 205, 206, 207, 208, 209, 210, 211, and 212, 386, 387, 388, 389, 390, 391, 392, 393, 394, and 395; and Also provided herein are CD28 binding polypeptides comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of NOs: 191, 194, and 197, 396, 397, and 398.

[0013] In some embodiments, at least one CD28 sdAb comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 191; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 192, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 193, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 194; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 199, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 200, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197;CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 207, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 208, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209, and CDR CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 210, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 211, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202 CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396;CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:386, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:387, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:388, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:389, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:398 CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398;CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:390, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:391, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:392, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:396 CDR2 comprising the amino acid sequence set forth in SEQ ID NO:393, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197;CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:394, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:395, and CDR CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 388, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 388, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 388 CDR2 comprising the amino acid sequence set forth in SEQ ID NO:389, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197;CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 397; or CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 398.

[0014] Also provided is a CD28 binding polypeptide comprising at least one single domain antibody (sdAb) that binds to CD28, wherein the CD28 VHH domain comprises CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 186; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 187; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 188; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 213; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 214; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 215; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 216; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:217; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:218; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:219; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:220; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:221; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:222; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:223; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:224; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:225; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:226; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:227; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:228; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:229;CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 230; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 231; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 232; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 233; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 234; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 235; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 236; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 237; Also provided herein are CD28-binding polypeptides containing the CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 238; the CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 239; or the CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 280.

[0015] Also disclosed is a CD28-binding polypeptide comprising at least one single domain antibody (sdAb) that binds to CD28, wherein the at least one CD28 sdAb (CD28 VHH) domain comprises CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 186; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 187; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 188; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 213; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 214; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 215; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:216; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:217; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:218; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:219; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:220; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:221; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:222; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:223; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:224; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:225; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:226; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:227;CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:228; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:229; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:230; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:231; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:232; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:233; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:234; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:235; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:236; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:237; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:238; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:239; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:280; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:342; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:343; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:344; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:345; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:346; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:347; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:348;CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 349; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 350; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 351; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 352; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 353; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 354; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 355; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 356; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 357; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 358; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 359; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 360; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 361; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 362; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 363; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 364; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:365; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:366; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:367; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:368;CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 369; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 370; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 371; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 372; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 373; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 374; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 375; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 376; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 377; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 378; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 379; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 380; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 381; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 382; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 383; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 384; or SEQ ID Also provided herein is a CD28-binding polypeptide comprising the CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in NO:385.

[0016] In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 186. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 187. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 188. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 213. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 214. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:215. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:216. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:217. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:218. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:219. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:220.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:221. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:222. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:223. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:224. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:225. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:226. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:227. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:228. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:229. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:230. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:231.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:232. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:233. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:234. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:235. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:236. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:237. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:238. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:239. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:280. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:342. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:343.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:344. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:345. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:346. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:347. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:348. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:349. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:350. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:351. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:352. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:353. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:354.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:355. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:356. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:357. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:358. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:359. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:360. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:361. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:362. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:363. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:364. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:365.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:366. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:367. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:368. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:369. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:370. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:371. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:372. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:373. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:374. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:375. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:376.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:377. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:378. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:379. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:380. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:381. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 382. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 383. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 384; or the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 385.

[0017] Also disclosed is a CD28-binding polypeptide comprising at least one single domain antibody (sdAb) (CD28 VHH) that binds to CD28, wherein the CD28 VHH domain comprises CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 186; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 187; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 188; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 213; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 214; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 215; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:216; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:217; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:218; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:219; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:221; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:222; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:223; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:224; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:225; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:226; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:227; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:228; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:229; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:230;CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 231; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 232; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 233; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 234; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 235; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 236; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 237; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 238; Also provided herein is a CD28-binding polypeptide containing the CDR1, CDR2, and CDR3 contained in the VHH set forth in SEQ ID NO: 239; or the CDR1, CDR2, and CDR3 contained in the VHH set forth in SEQ ID NO: 280.

[0018] Also disclosed is a CD28-binding polypeptide comprising at least one single domain antibody (sdAb) that binds to CD28, wherein the at least one CD28 sdAb (CD28 VHH) domain comprises CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 186; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 187; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 188; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 213; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 214; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 215; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:216; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:217; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:218; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:219; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:221; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:222; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:223; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:224; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:225; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:226; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:227; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:228;CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 229; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 230; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 231; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 232; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 233; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 234; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 235; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 236; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 237; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 238; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 239; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 280; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 342; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 343; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 344; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 345; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:346; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:347; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:348; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:349;CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 350; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 351; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 352; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 353; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 354; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 355; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 356; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 357; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 358; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 359; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 360; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 361; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 362; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 363; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 364; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 365; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:366; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:367; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:368; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:369;CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 370; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 371; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 372; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 373; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 374; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 375; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 376; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 377; Also provided herein is a CD28-binding polypeptide comprising CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 378; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 379; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 380; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 381; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 382; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 383; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 384; or CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 385.

[0019] In some embodiments, at least one CD28 sdAb has an amino acid sequence set forth in any of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 280, or a sequence similar to SEQ ID and NOS: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 280.

[0020] In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 280, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 40 51, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, and 385, or NO:186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234 , 235, 236, 237, 238, 239, 280, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 3 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, and 385.

[0021] In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 186. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 187. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 188. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 213. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 214. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 215. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 216. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 217. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:218. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:219. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:220. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:221. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:222. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:223. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:224. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:225.In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:226. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:227. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:228. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:229. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:230. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:231. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:232. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:233. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:234. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:235. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:236. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:237. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:238. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:239. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:280. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:342.In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:343. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:344. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:345. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:346. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:347. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:348. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:349. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:350. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:351. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:352. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:353. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:354. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:355. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:356. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:357. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:358.In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:359. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:360. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:361. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:362. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:363. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:364. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:365. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:366. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:367. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:368. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:369. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:370. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:371. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:372. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:373. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:374.In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:375. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:376. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:377. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:378. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:379. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:380. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:381. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:382. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 383. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 384. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 385.

[0022] In some embodiments, at least one CD28 sdAb has an amino acid sequence set forth in any of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 280, or a sequence similar to SEQ ID and NOS: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 280.

[0023] In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 280, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, and 385, or the amino acid sequence set forth in any of SEQ ID NOs. NO:186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 23 5, 236, 237, 238, 239, 280, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, and 385.

[0024] In some embodiments, at least one CD28 sdAb contains a sequence set forth in (i) SEQ ID NO:186, (ii) a humanized variant of SEQ ID NO:186, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:186. In some embodiments, at least one CD28 sdAb contains a sequence set forth in SEQ ID NO:186. In some embodiments, at least one CD28 sdAb contains a humanized variant of SEQ ID NO:186. In some embodiments, at least one CD28 sdAb contains a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 186. In some embodiments, at least one CD28 sdAb contains a CDR1 having the amino acid sequence set forth in SEQ ID NO: 189; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 190; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 191.

[0025] In some embodiments, at least one CD28 sdAb contains a sequence set forth in (i) SEQ ID NO:187, (ii) a humanized variant of SEQ ID NO:187, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:187. In some embodiments, at least one CD28 sdAb contains a sequence set forth in SEQ ID NO:187. In some embodiments, at least one CD28 sdAb contains a humanized variant of SEQ ID NO:187. In some embodiments, at least one CD28 sdAb contains a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 187. In some embodiments, at least one CD28 sdAb contains a CDR1 having the amino acid sequence set forth in SEQ ID NO: 192; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 193; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 194.

[0026] In some embodiments, at least one CD28 sdAb contains a sequence set forth in (i) SEQ ID NO:188, (ii) a humanized variant of SEQ ID NO:188, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:188. In some embodiments, at least one CD28 sdAb contains a sequence set forth in SEQ ID NO:188. In some embodiments, at least one CD28 sdAb contains a humanized variant of SEQ ID NO:188. In some embodiments, at least one CD28 sdAb contains a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:188.

[0027] In some embodiments, at least one CD28 sdAb contains a CDR1 having the amino acid sequence set forth in any of SEQ ID NOs: 195, 198, 199, 200, and 201; a CDR2 having the amino acid sequence set forth in any of SEQ ID NOs: 196, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, and 212; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in any of SEQ ID NOs: 195, 198, 199, 200, and 201; a CDR2 containing the amino acid sequence set forth in any of SEQ ID NOs: 196, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 386, 387, 388, 389, 390, 391, 392, 393, 394, and 395; and a CDR3 containing the amino acid sequence set forth in any of SEQ ID NOs: 197, 396, 397, and 398.

[0028] In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 195, 196, and 197, respectively; SEQ ID NOs: 198, 196, and 197, respectively; SEQ ID NOs: 199, 196, and 197, respectively; SEQ ID NOs: 200, 196, and 197, respectively; SEQ ID NOs: 201, 196, and 197, respectively; SEQ ID NOs: 195, 202, and 197, respectively; SEQ ID NOs: 195, 203, and 197, respectively; SEQ ID NOs: 195, 204, and 197, respectively; SEQ ID NOs: 195, 205, and 197, respectively; SEQ ID NOs: 195, 206, and 197, respectively; SEQ ID NOs: 195, 207, and 197, respectively; SEQ ID NOs: 195, 208, and 197; SEQ ID NOs: 195, 209, and 197, respectively; SEQ ID NOs: 195, 210, and 197; SEQ ID NOs: 195, 211, and 197, respectively; or containing CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 195, 212, and 197, respectively. In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 195, 196, and 197, respectively; SEQ ID NOs: 198, 196, and 197, respectively; SEQ ID NOs: 199, 196, and 197, respectively; SEQ ID NOs: 200, 196, and 197, respectively; SEQ ID NOs: 201, 196, and 197, respectively; SEQ ID NOs: 195, 202, and 197, respectively; SEQ ID NOs: 195, 203, and 197, respectively; SEQ ID NOs: 195, 204, and 197, respectively; SEQ ID NOs: 195, 205, and 197, respectively; SEQ ID NOs: 195, 206, and 197, respectively; SEQ ID NOs: 195, 207, and 197, respectively; NO: 195, 208, and 197; SEQ ID NO: 195, 209, and 197, respectively; SEQ ID NO: 195, 210, and 197, respectively; SEQ ID NO: 195, 211, and 197, respectively;SEQ ID NOs: 195, 212, and 197, respectively; SEQ ID NOs: 201, 202, and 197, respectively; SEQ ID NOs: 201, 202, and 396, respectively; SEQ ID NOs: 201, 386, and 197, respectively; SEQ ID NOs: 201, 387, and 197, respectively; SEQ ID NOs: 201, 202, and 397, respectively; SEQ ID NOs: 201, 202, and 398, respectively; SEQ ID NOs: 201, 206, and 197, respectively; SEQ ID NOs: 201, 206, and 398, respectively; SEQ ID NOs: 201, 388, and 197, respectively; SEQ ID NOs: 201, 389, and 197, respectively; SEQ ID NOs: 201, 206, and 397, respectively; SEQ ID SEQ ID NOs:201, 206, and 398; SEQ ID NOs:201, 203, and 197, respectively; SEQ ID NOs:201, 203, and 396, respectively; SEQ ID NOs:201, 390, and 197, respectively; SEQ ID NOs:201, 391, and 197, respectively; SEQ ID NOs:201, 203, and 397, respectively; SEQ ID NOs:201, 203, and 398, respectively; SEQ ID NOs:201, 204, and 197, respectively; SEQ ID NOs:201, 204, and 396, respectively; SEQ ID NOs:201, 392, and 197, respectively; SEQ ID NOs:201, 393, and 197, respectively; SEQ ID NOs:201, 204, and 397, respectively; SEQ ID NOs:201, 204, and 398, respectively; SEQ ID NOs: 201, 196, and 396; SEQ ID NOs: 201, 394, and 197, respectively; SEQ ID NOs: 201, 395, and 197, respectively; SEQ ID NOs: 201, 196, and 397, respectively; SEQ ID NOs: 201, 196, and 398, respectively; SEQ ID NOs: 195, 206, and 396, respectively; SEQ ID NOs: 195, 388, and 197, respectively; SEQ ID NOs: 195, 389, and 197, respectively; SEQ ID NOs: 195, 206, and 397, respectively;or containing CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs: 195, 206, and 398, respectively;

[0029] In some embodiments, at least one CD28 sdAb contains a sequence of amino acids set forth in any one of SEQ ID NOs:213-239 and 280, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:213-239 and 280. In some embodiments, at least one CD28 sdAb contains a sequence of amino acids set forth in any one of SEQ ID NOs:213-239 and 280. In some embodiments, at least one CD28 sdAb contains an amino acid sequence set forth in any of SEQ ID NOs: 213-239, 280, and 342-385, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 213-239, 280, and 342-385. In some embodiments, at least one CD28 sdAb contains an amino acid sequence set forth in any of SEQ ID NOs: 213-239, 280, and 342-385.

[0030] In some embodiments, at least one CD28 sdAb contains a CDR1 having the amino acid sequence set forth in any of SEQ ID NOs: 195, 198, 199, 200, and 201; a CDR2 having the amino acid sequence set forth in any of SEQ ID NOs: 196, 202, 204, 205, 206, 207, 208, 209, 210, 211, and 212; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in any of SEQ ID NOs: 195, 198, 199, 200, and 201; a CDR2 comprising the amino acid sequence set forth in any of SEQ ID NOs: 196, 202, 204, 205, 206, 207, 208, 209, 210, 211, 212, 386, 387, 388, 389, 390, 391, 392, 393, 394, and 395; and a CDR3 comprising the amino acid sequence set forth in any of SEQ ID NOs: 197, 396, 397, and 398.

[0031] In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 195, 196, and 197, respectively; SEQ ID NOs: 198, 196, and 197, respectively; SEQ ID NOs: 199, 196, and 197, respectively; SEQ ID NOs: 200, 196, and 197, respectively; SEQ ID NOs: 201, 196, and 197, respectively; SEQ ID NOs: 195, 202, and 197, respectively; SEQ ID NOs: 195, 204, and 197, respectively; SEQ ID NOs: 195, 205, and 197, respectively; SEQ ID NOs: 195, 206, and 197, respectively; SEQ ID NOs: 195, 207, and 197, respectively; SEQ ID NOs: 195, 208, and 197, respectively; SEQ ID NOs: 195, 209, and 197; SEQ ID NOs: 195, 210, and 197, respectively; SEQ ID NOs: 195, 211, and 197, respectively; or containing the CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 195, 212, and 197, respectively. In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 195, 196, and 197, respectively; SEQ ID NOs: 198, 196, and 197, respectively; SEQ ID NOs: 199, 196, and 197, respectively; SEQ ID NOs: 200, 196, and 197, respectively; SEQ ID NOs: 201, 196, and 197, respectively; SEQ ID NOs: 195, 202, and 197, respectively; SEQ ID NOs: 195, 204, and 197, respectively; SEQ ID NOs: 195, 205, and 197, respectively; SEQ ID NOs: 195, 206, and 197, respectively; SEQ ID NOs: 195, 207, and 197, respectively; SEQ ID NOs: 195, 208, and 197, respectively; NO: 195, 209, and 197; SEQ ID NO: 195, 210, and 197, respectively; SEQ ID NO: 195, 211, and 197, respectively; SEQ ID NO: 195, 212, and 197, respectively; SEQ ID NO: 201, 202, and 197, respectively;SEQ ID NOs: 201, 202, and 396, respectively; SEQ ID NOs: 201, 386, and 197, respectively; SEQ ID NOs: 201, 387, and 197, respectively; SEQ ID NOs: 201, 202, and 397, respectively; SEQ ID NOs: 201, 202, and 398, respectively; SEQ ID NOs: 201, 206, and 197, respectively; SEQ ID NOs: 201, 206, and 398, respectively; SEQ ID NOs: 201, 388, and 197, respectively; SEQ ID NOs: 201, 389, and 197, respectively; SEQ ID NOs: 201, 206, and 397, respectively; SEQ ID NOs: 201, 206, and 398, respectively; SEQ ID NOs: 201, 203, and 197, respectively; SEQ ID SEQ ID NOs:201, 203, and 396; SEQ ID NOs:201, 390, and 197, respectively; SEQ ID NOs:201, 391, and 197, respectively; SEQ ID NOs:201, 203, and 397, respectively; SEQ ID NOs:201, 203, and 398, respectively; SEQ ID NOs:201, 204, and 197, respectively; SEQ ID NOs:201, 204, and 396, respectively; SEQ ID NOs:201, 392, and 197, respectively; SEQ ID NOs:201, 393, and 197, respectively; SEQ ID NOs:201, 204, and 397, respectively; SEQ ID NOs:201, 204, and 398, respectively; SEQ ID NOs:201, 196, and 396, respectively; SEQ ID NOs:201, 394, and 197, respectively; SEQ SEQ ID NOs: 201, 395, and 197; SEQ ID NOs: 201, 196, and 397, respectively; SEQ ID NOs: 201, 196, and 398, respectively; SEQ ID NOs: 195, 206, and 396, respectively; SEQ ID NOs: 195, 388, and 197, respectively; SEQ ID NOs: 195, 389, and 197, respectively; SEQ ID NOs: 195, 206, and 397, respectively; or containing the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 195, 206, and 398, respectively;

[0032] In some embodiments, at least one CD28 sdAb contains a sequence of amino acids set forth in any one of SEQ ID NOs: 213-219, 221-239, and 280, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 213-219, 221-239, and 280. In some embodiments, at least one CD28 sdAb contains a sequence of amino acids set forth in any one of SEQ ID NOs: 213-219, 221-239, and 280.

[0033] In some embodiments, at least one CD28 sdAb contains an amino acid sequence set forth in any of SEQ ID NOs: 213-219, 221-239, 280, and 342-385, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 213-219, 221-239, 280, and 342-385. In some embodiments, at least one CD28 sdAb contains an amino acid sequence set forth in any of SEQ ID NOs: 213-239, 280, and 342-385.

[0034] In some embodiments, at least one sdAb is a CD28 sdAb and / or the CD28 binding polypeptide is monovalent for CD28. In some embodiments, at least one sdAb is a CD28 sdAb. In some embodiments, the CD28 binding polypeptide is monovalent for CD28. In some embodiments, at least one sdAb is a CD28 sdAb and the CD28 binding polypeptide is monovalent for CD28.

[0035] In some embodiments, at least one sdAb is two, three, or four CD28 sdAbs, and / or the CD28 binding polypeptide is multivalent with respect to CD28. In some embodiments, at least one sdAb is two CD28 sdAbs, and / or the CD28 binding polypeptide is bivalent with respect to CD28. In some embodiments, at least one sdAb is two CD28 sdAbs. In some embodiments, the CD28 binding polypeptide is bivalent with respect to CD28. In some embodiments, at least one sdAb is two CD28 sdAbs, and the CD28 binding polypeptide is bivalent with respect to CD28. In some embodiments, the two CD28 sdAbs are the same CD28 sdAb. In some embodiments, both of the CD28 sdAbs contain the amino acid sequence set forth in SEQ ID NO:188. In some embodiments, each of the two CD28 sdAbs contains the amino acid sequence set forth in SEQ ID NO:188. In some embodiments, both of the CD28 sdAbs contain the amino acid sequence set forth in SEQ ID NO:220. In some embodiments, each of the two CD28 sdAbs contains the amino acid sequence set forth in SEQ ID NO:220. In some embodiments, both of the CD28 sdAbs contain the amino acid sequence set forth in SEQ ID NO:280. In some embodiments, each of the two CD28 sdAbs contains the amino acid sequence set forth in SEQ ID NO:280. In some embodiments, the two CD28 sdAbs are different CD28 sdAbs. In some embodiments, one of the two CD28 sdAbs contains the amino acid sequence set forth in SEQ ID NO:188 and the other of the two CD28 sdAbs contains the amino acid sequence set forth in SEQ ID NO:220. In some embodiments, one of the two CD28 sdAbs contains the amino acid sequence shown in SEQ ID NO:188, and the other of the two CD28 sdAbs contains the amino acid sequence shown in SEQ ID NO:280.In some embodiments, one of the two CD28 sdAbs contains the amino acid sequence shown in SEQ ID NO:220, and the other of the two CD28 sdAbs contains the amino acid sequence shown in SEQ ID NO:280.

[0036] In some embodiments, the CD28 binding polypeptide comprises a moiety that binds to Protein A. In some embodiments, at least one CD28 sdAb contains an amino acid modification that reduces binding to Protein A. In some embodiments, an amino acid modification of G65D according to Kabat in framework region 3 (FR3).

[0037] In some embodiments, the CD28 binding polypeptide comprises an immunoglobulin Fc region.

[0038] Also provided herein are anti-CD28 sdAb-Fc fusion proteins containing any of the CD28 binding polypeptides described herein and an immunoglobulin Fc region.

[0039] In some embodiments, the Fc region is linked to at least one of the at least one CD28 sdAb by a connecting peptide (LP). In some embodiments, at least one CD28 sdAb is linked to the Fc region at the N-terminus of the Fc region by a connecting peptide (LP). In some embodiments, the LP is a non-cleavable linker. In some embodiments, the LP is a peptide about 1 to 20 amino acids in length. In some embodiments, the CD28 binding polypeptide contains, from N-terminus to C-terminus: (CD28 sdAb)-LP-Fc. In some embodiments, the CD28 binding polypeptide contains, from N-terminus to C-terminus: (CD28 sdAb)-LP-(CD28 sdAb)-LP-Fc.

[0040] In some embodiments, the CD28 binding polypeptide is a dimer.

[0041] In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the Fc region contains a sequence of amino acids selected from the group consisting of SEQ ID NOs:8, 10, 11, 12, and 13, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs:8, 10, 11, 12, and 13. In some embodiments, the Fc region consists of a sequence of amino acids selected from the group consisting of SEQ ID NOs:8, 10, 11, 12, and 13. In some embodiments, the Fc region is human IgG1. In some embodiments, the Fc region is human IgG1. In some embodiments, the Fc region contains the sequence of amino acids set forth in SEQ ID NO:8, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8. In some embodiments, the Fc region consists of the sequence of amino acids set forth in SEQ ID NO:8.

[0042] In some embodiments, the Fc region is a heterodimeric Fc region.

[0043] In some embodiments, the Fc region exhibits effector function.

[0044] In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce effector function. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to an effector molecule. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to an effector molecule selected from an Fc gamma receptor and C1q. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to an Fc gamma receptor. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to C1q. In some embodiments, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. In some embodiments, the one or more amino acid modifications are deletions of Glu233. In some embodiments, the one or more amino acid modifications are deletions of Leu234. In some embodiments, the one or more amino acid modifications are deletions of Leu 235. In some embodiments, the Fc region contains the sequence of amino acids set forth in SEQ ID NO:9, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9. In some embodiments, the Fc region consists of the sequence of amino acids set forth in SEQ ID NO:9.

[0045] Provided herein are homodimeric Fc fusion proteins containing two identical copies of any of the anti-CD28 sdAb-Fc fusion proteins described herein.

[0046] Also provided herein are heterodimeric Fc fusion proteins containing any two of the anti-CD28 sdAb-Fc fusion proteins described herein.

[0047] In some embodiments, the CD28-binding polypeptide contains one or more binding domains that bind to a target antigen other than CD28. In some embodiments, the one or more binding domains are one or more single-domain antibodies (sdAbs) that bind to a tumor antigen (TAA).

[0048] In some embodiments, the CD28 binding polypeptide comprises a moiety that binds to Protein A.

[0049] Also provided herein are multispecific binding polypeptides comprising any of the provided anti-CD28 sdAb-Fc fusion proteins and one or more binding domains (BDs) that bind to a target antigen other than CD28.

[0050] In some embodiments, the one or more BDs are 1 BD. In some embodiments, the one or more BDs are 2, 3, 4, or more BDs. In some embodiments, the one or more BDs are 2 BDs. In some embodiments, the one or more BDs are 3 BDs. In some embodiments, the one or more BDs are 4 BDs.

[0051] In some embodiments, one or more binding domains (BDs) bind to a tumor-associated antigen (TAA). In some embodiments, one or more binding domains (BDs) bind to a T cell activation marker. In some embodiments, one or more binding domains (BDs) bind to a T cell exhaustion marker. In some embodiments, one or more binding domains (BDs) bind to a tumor microenvironment (TME) marker. In some embodiments, one or more binding domains (BDs) are single domain antibodies. In some embodiments, one or more binding domains (BDs) are single domain antibodies that bind to a TAA.

[0052] In some embodiments, the multispecific binding polypeptide is an Fc fusion protein in which at least one of the one or more BDs and / or at least one CD28 sdAb is linked to an immunoglobulin Fc region.

[0053] Provided herein are multispecific Fc fusion proteins comprising any of the provided multispecific binding polypeptides and an immunoglobulin Fc region. In some embodiments, the multispecific binding polypeptide is an Fc fusion protein in which at least one of one or more BDs and / or at least one CD28 sdAb is linked to the immunoglobulin Fc region.

[0054] In some embodiments, the Fc fusion protein contains, from N-terminus to C-terminus: one or more BDs that bind to a target antigen other than CD28; at least one CD28-binding domain comprising an sdAb that binds to CD28; and an Fc region. In some embodiments, at least one of the one or more BDs is joined to at least one of the at least one CD28 sdAb by a connecting peptide (LP). In some embodiments, the CD28-binding polypeptide contains, from N-terminus to C-terminus: (BD)-LP-(CD28 sdAb)-LP-Fc.

[0055] In some embodiments, the multispecific binding polypeptide is a dimer.

[0056] In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the Fc region contains an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 10, 11, 12, and 13, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:8, 10, 11, 12, and 13. In some embodiments, the Fc region is human IgG1. In some embodiments, the Fc region is human IgG1. In some embodiments, the Fc region contains the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the Fc region contains a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8.

[0057] In some embodiments, the Fc region is a heterodimeric Fc region.

[0058] In some embodiments, the Fc region exhibits effector function.

[0059] In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce effector function. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to an effector molecule selected from an Fc gamma receptor or C1q. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to an Fc gamma receptor. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to C1q. In some embodiments, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. In some embodiments, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. In some embodiments, the one or more amino acid modifications are deletions of Glu233. In some embodiments, the one or more amino acid modifications are deletions of Leu234. In some embodiments, the one or more amino acid modifications are deletions of Leu235. In some embodiments, the Fc region contains the sequence of amino acids set forth in SEQ ID NO:9, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9. In some embodiments, the Fc region consists of the sequence of amino acids set forth in SEQ ID NO:9.

[0060] In any of the provided embodiments, the binding domain (BD) is selected from the group consisting of 1-92-LFA-3, 2B4, 5T4, alpha-4 integrin, alpha-V integrin, alpha4beta1 integrin, alpha4beta7 integrin, alpha-SMA, AGR2, anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD 80, CD81, CD86, CD95, CD107a, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD1 60, CD166, CD172A, CD248, CDH6, CEACAM5(CEA), CEACAM6(NCA-90), CLAUDIN-3, CLAUDIN-4, cMe t, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FR alpha), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFN alpha, IFN beta, IFN gamma, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL- 12R, IL-12R beta 1, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFR alpha, PDGFR beta, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, Siglec15, sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGF beta, TIGIT, TIM-3, TLR2, TLR4, TLR6, The antibody binds to an antigen selected from the group consisting of TLR7, TLR8, TLR9, TMEM31, TNF alpha, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

[0061] In some embodiments, the T cell activation marker is selected from CD25, CD44, CD69, CD71, CD107a, CD137, HLA-DR, and / or KLRG1. In some embodiments, the T cell exhaustion marker is selected from 2B4, CD160, LAG3, PD-1, and / or TIGIT. In some embodiments, the tumor microenvironment marker is selected from alpha-SMA, EDB, FAP, FSP-1, PDGFR alpha, and / or PDGFR beta.

[0062] In some embodiments, one or more TAA BDs are one TAA BD, and / or the multispecific binding polypeptide is monovalent to one TAA. In some embodiments, one or more TAA BDs are two, three, or four TAA BDs, and / or the multispecific binding polypeptide is multivalent to one or more TAA. In some embodiments, one or more TAA BDs are two TAA BDs, and / or the multispecific binding polypeptide is bivalent to one or more TAA. In some embodiments, two TAA BDs are single domain antibodies (sdAbs), and the two sdAbs are the same TAA sdAb. In some embodiments, two TAA BDs are single domain antibodies (sdAbs), and the two sdAbs are different TAA sdAbs that bind to the same TAA. In some embodiments, different TAA sdAbs bind to different epitopes of the same TAA. In some embodiments, the two TAA BDs are single domain antibodies (sdAbs), and the two sdAbs are different TAA sdAbs that bind to different TAAs.

[0063] Also provided herein are homodimeric multispecific Fc-fusion proteins containing two identical copies of any of the provided multispecific polypeptides.

[0064] Also provided herein are heterodimeric multispecific Fc-fusion proteins containing any two of the provided multispecific polypeptides.

[0065] In any of the provided embodiments, the Fc fusion protein contains a connecting peptide between at least one of the at least one CD28 sdAb and the Fc region. In some embodiments, the Fc fusion protein contains a connecting peptide (LP) between at least one of the one or more BDs and the Fc region. In some embodiments, the linker is not cleavable. In some embodiments, the linker is a peptide of about 1-20 amino acids in length. In some embodiments, it contains the amino acid sequence set forth in any of SEQ ID NOs: 1-7, 89, 90, 123-129, 244, and 249. In some embodiments, it consists of the amino acid sequence set forth in any of SEQ ID NOs: 1-7, 89, 90, 123-129, 244, and 249.

[0066] In some embodiments, at least one CD28 sdAb is unable to bind or engage, or is unable to substantially bind or engage, CD28 unless at least one of the one or more BDs is bound to its antigen. In some embodiments, at least one CD28 sdAb is unable to bind or engage, or is unable to substantially bind or engage, CD28 unless each of the one or more BDs is bound to its antigen.

[0067] Also provided herein are CD28-binding polypeptides that contain, from N-terminus to C-terminus: one or more antigen-binding domains that contain a single domain antibody (sdAb) that binds to a tumor-associated antigen (TAA); at least one CD28-binding domain that contains an sdAb that binds to CD28; and an Fc region.

[0068] In any of the provided embodiments, at least one of the one or more TAA sdAbs is joined to at least one of the at least one CD28 sdAb by a connecting peptide (LP). In some embodiments, the CD28 binding polypeptide contains, from N-terminus to C-terminus: (TAA sdAb)-LP-(CD28 sdAb)-LP-Fc.

[0069] In some embodiments, the CD28 binding polypeptide is a dimer. In some embodiments, the Fc region is a homodimeric Fc region.

[0070] Also provided herein is a CD28 binding polypeptide that is a dimer containing, from N-terminus to C-terminus: one or more antigen-binding domains having a single domain antibody (sdAb) that binds to a tumor-associated antigen (TAA); at least one CD28-binding domain having an sdAb that binds to CD28; and an Fc region, wherein the CD28-binding polypeptide is a homodimeric Fc region.

[0071] In some embodiments, the Fc region contains a sequence of amino acids selected from the group consisting of SEQ ID NOs:8, 10, 11, 12, and 13, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs:8, 10, 11, 12, and 13. In some embodiments, the Fc region consists of a sequence of amino acids selected from the group consisting of SEQ ID NOs:8, 10, 11, 12, and 13. In some embodiments, the Fc region is human IgG1. In some embodiments, the Fc region is human IgG1. In some embodiments, the Fc region contains the sequence of amino acids set forth in SEQ ID NO:8, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8. In some embodiments, the Fc region consists of the sequence of amino acids set forth in SEQ ID NO:8.

[0072] In some embodiments, the Fc region is a heterodimeric Fc region.

[0073] In some embodiments, the Fc region exhibits effector function.

[0074] In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce effector function. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to an effector molecule selected from an Fc gamma receptor or C1q. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to an Fc gamma receptor. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to C1q. In some embodiments, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. In some embodiments, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. In some embodiments, the one or more amino acid modifications are deletions of Glu233. In some embodiments, the one or more amino acid modifications are deletions of Leu234. In some embodiments, the one or more amino acid modifications are deletions of Leu235. In some embodiments, the Fc region contains the sequence of amino acids set forth in SEQ ID NO:9, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9. In some embodiments, the Fc region consists of the sequence of amino acids set forth in SEQ ID NO:9.

[0075] In any of the provided embodiments, the TAA is selected from the group consisting of 1-92-LFA-3, 5T4, alpha-4 integrin, alpha-V integrin, alpha4beta1 integrin, alpha4beta7 integrin, AGR2, anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD3 3, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, C D80, CD81, CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A, CD248, CDH6, CEACAM5(CEA), CEACAM6(NCA-90), CLAUDIN-3, CLAUDIN-4, cMet, Collage Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FR alpha), GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HVEM, hyaluronidase, ICOS, IFN alpha, IFN beta, IFN gamma, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R, IL-12Rbeta1, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFR alpha, PDGFR beta, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, Siglec15, sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGF beta, TIGIT, TIM-3, TLR2, TLR4, TL R6, TLR7, TLR8, TLR9, TMEM31, TNF alpha, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

[0076] In some embodiments, the one or more TAA sdAbs are one TAA sdAb. In some embodiments, the CD28 binding polypeptide is monovalent with respect to a TAA. In some embodiments, the one or more TAA sdAbs are two, three, or four TAA sdAbs. In some embodiments, the one or more TAA sdAbs are two TAA sdAbs. In some embodiments, the one or more TAA sdAbs are three TAA sdAbs. In some embodiments, the one or more TAA sdAbs are four TAA sdAbs. In some embodiments, the CD28 binding polypeptide is multivalent with respect to one or more TAAs. In some embodiments, the one or more TAA sdAbs are two TAA sdAbs. In some embodiments, the CD28 binding polypeptide is bivalent with respect to a TAA. In some embodiments, the CD28 binding polypeptide is bivalent with respect to one or more TAAs. In some embodiments, the two TAA sdAbs are the same TAA sdAb. In some embodiments, the two TAA sdAbs are different TAA sdAbs that bind to the same TAA. In some embodiments, the TAA sdAbs bind to different epitopes of the same TAA. In some embodiments, the two TAA sdAbs are different TAA sdAbs that bind to different TAAs.

[0077] In some embodiments, the CD28 binding polypeptide contains a connecting peptide (LP) between at least one of the at least one CD28 sdAb and the Fc region. In some embodiments, the CD28 binding polypeptide contains a connecting peptide (LP) between at least one of the one or more TAA sdAbs and the Fc region. In some embodiments, the LP is a non-cleavable linker. In some embodiments, the LP is a peptide of about 1-20 amino acids in length. In some embodiments, the LP is or contains a sequence set forth in any of SEQ ID NOs: 1-7, 89, 90, 123-129, 244, and 249.

[0078] In any of the embodiments provided, at least one CD28 sdAb is unable to bind or engage with CD28, or is unable to substantially bind or engage with CD28, unless at least one of the one or more TAA sdAbs is bound to that TAA. In any of the embodiments provided, at least one CD28 sdAb is unable to bind or engage with CD28 unless at least one of the one or more TAA sdAbs is bound to that TAA. In any of the embodiments provided, at least one CD28 sdAb is unable to substantially bind or engage with CD28 unless at least one of the one or more TAA sdAbs is bound to that TAA. In some embodiments, at least one CD28 sdAb is unable to bind or engage with CD28, or is unable to substantially bind or engage with CD28, unless each of the one or more TAA sdAbs is bound to its TAA. In some embodiments, at least one CD28 sdAb is unable to bind to or engage CD28 unless each of the one or more TAA sdAbs is bound to that TAA, hi some embodiments, at least one CD28 sdAb is unable to substantially bind to or engage CD28 unless each of the one or more TAA sdAbs is bound to that TAA.

[0079] In any of the provided embodiments, the CD-28 binding polypeptide does not contain a CD3 binding region. In some embodiments, the anti-CD28 sdAb-Fc fusion protein does not contain a CD3 binding region. In some embodiments, the homodimeric Fc fusion protein does not contain a CD3 binding region. In some embodiments, the heterodimeric Fc fusion protein does not contain a CD3 binding region. In some embodiments, the multispecific binding polypeptide does not contain a CD3 binding region. In some embodiments, the homodimeric multispecific binding polypeptide does not contain a CD3 binding region. In some embodiments, the heterodimeric multispecific binding polypeptide does not contain a CD3 binding region. In some embodiments, the fusion protein does not contain a CD3 binding region.

[0080] In some embodiments, the CD28 binding polypeptide comprises a CD3 binding region.

[0081] Provided herein are CD28 binding polypeptides containing at least one CD28 sdAb and a CD3 binding region.

[0082] In some embodiments, at least one CD28 sdAb contains a complementarity determining region 1 (CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 189, 192, 195, 198, 199, 200, and 201; a complementarity determining region 2 (CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 193, 196, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, and 212; and a complementarity determining region 3 (CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 194, and 197. In some embodiments, at least one CD28 sdAb contains a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 189, 192, 195, 198, 199, 200, and 201; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 193, 196, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 386, 387, 388, 389, 390, 391, 392, 393, 394, and 395; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 194, 197, 396, 397, and 398.

[0083] In some embodiments, at least one CD28 sdAb comprises a CDR1 having the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 191; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 192, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 193, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 194; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 198, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 199 CDR1 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 200, CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 201, CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 203, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197;CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 205, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 207, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 208, and CDR CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 209, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 210, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 211, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; or CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 212, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197;

[0084] In some embodiments, at least one CD28 sdAb comprises a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 191; a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 192, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 193, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 194; a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 199, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 200; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 200, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 203, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197;CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 205, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 207, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 208, and CDR CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 209, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 210, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 211, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 212, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197;CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, CDR2 containing the amino acid sequence set forth in SEQ ID NO:202, and CDR3 containing the amino acid sequence set forth in SEQ ID NO:197; CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, CDR2 containing the amino acid sequence set forth in SEQ ID NO:202, and CDR3 containing the amino acid sequence set forth in SEQ ID NO:396; CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, CDR2 containing the amino acid sequence set forth in SEQ ID NO:386, and CDR3 containing the amino acid sequence set forth in SEQ ID NO:197; CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, CDR2 containing the amino acid sequence set forth in SEQ ID NO:387, and CDR3 containing the amino acid sequence set forth in SEQ ID NO:197; CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, CDR2 containing the amino acid sequence set forth in SEQ ID NO:202, and CDR CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 398; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 396; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 388, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197;CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 389, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 397; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 398; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 203, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 203, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 390, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 391, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 203, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 397; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 203, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 398;CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, CDR2 containing the amino acid sequence set forth in SEQ ID NO:204, and CDR3 containing the amino acid sequence set forth in SEQ ID NO:197; CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, CDR2 containing the amino acid sequence set forth in SEQ ID NO:204, and CDR3 containing the amino acid sequence set forth in SEQ ID NO:396; CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, CDR2 containing the amino acid sequence set forth in SEQ ID NO:392, and CDR3 containing the amino acid sequence set forth in SEQ ID NO:197; CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, CDR2 containing the amino acid sequence set forth in SEQ ID NO:393, and CDR3 containing the amino acid sequence set forth in SEQ ID NO:197; CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, CDR2 containing the amino acid sequence set forth in SEQ ID NO:204, and CDR CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 398; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 396; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 394, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 395, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197;CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 397; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 398; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 396; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 388, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197; CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 389, and CDR CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 397; or CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, CDR2 containing the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 containing the amino acid sequence set forth in SEQ ID NO: 398.

[0085] In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 191. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 192, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 193, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 194. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 200, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 207, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:208, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 210, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 211, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:386, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:387, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:388, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:389, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:390, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:391, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:392, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:393, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398.In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:394, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:395, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 388, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 389, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 397; or a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 398.

[0086] In some embodiments, at least one CD28 sdAb contains a complementarity determining region 1 (CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 189, 192, 195, 198, 199, 200, and 201; a complementarity determining region 2 (CDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 193, 196, 202, 204, 205, 206, 207, 208, 209, 210, 211, and 212; and a complementarity determining region 3 (CDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 194, and 197. In some embodiments, at least one CD28 sdAb contains a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 189, 192, 195, 198, 199, 200, and 201; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 193, 196, 202, 204, 205, 206, 207, 208, 209, 210, 211, 212, 386, 387, 388, 389, 390, 391, 392, 393, 394, and 395; and a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 194, 197, 396, 397, and 398.

[0087] In some embodiments, at least one CD28 sdAb comprises a CDR1 having the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 191; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 192, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 193, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 194; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 198, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 199 CDR1 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 200, CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 201, CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197;CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 205, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 207, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 208, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 209, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197 CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 210, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 211, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 212, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197;CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:386, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:387, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:388, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:389, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:389 CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397;CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:390, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:391, and CDR CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396 CDR2 comprising the amino acid sequence set forth in SEQ ID NO:392, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197;CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:393, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO:201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:394, and CDR CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 395, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 397; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 398; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 396; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 396 CDR2 comprising the amino acid sequence set forth in SEQ ID NO:388, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:197;CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 389, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 397; or CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 398.

[0088] In some embodiments, at least one CD28 sdAb comprises a CDR1 having the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 191; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 192, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 193, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 194; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; a CDR1 having the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 198, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 199 CDR1 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 200, CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 201, CDR2 having the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197;CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 205, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 207, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 208, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 209, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197 CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 210, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 211, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197; or CDR1 having the amino acid sequence set forth in SEQ ID NO: 195, CDR2 having the amino acid sequence set forth in SEQ ID NO: 212, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 197;

[0089] In some embodiments, at least one CD28 sdAb (CD28 VHH) comprises CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 186; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 187; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 188; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 213; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 214; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 215; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 216; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:217; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:218; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:219; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:220; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:221; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:222; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:223; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:224; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:225; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:226; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:227; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:228; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:229; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:230;CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 231; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 232; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 233; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 234; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 235; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 236; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 237; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 238; Contains the CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 239; or contains the CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 280;

[0090] In some embodiments, at least one CD28 sdAb (CD28 VHH) comprises CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 186; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 187; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 188; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 213; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 214; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 215; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 216; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:217; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:218; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:219; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:220; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:221; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:222; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:223; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:224; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:225; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:226; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:227; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:228; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:229; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:230;CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 231; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 232; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 233; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 234; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 235; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 236; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 237; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 238; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 239; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 280; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 342; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 343; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 344; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 345; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 346; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 347; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:348; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:349; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:350; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:351; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:352;CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 353; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 354; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 355; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 356; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 357; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 358; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 359; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 360; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 361; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 362; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 363; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 364; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 365; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 366; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 367; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 368; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:369; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:370; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:371; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:372;CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 373; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 374; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 375; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 376; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 377; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 378; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 379; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 380; Contains CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 381; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 382; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 383; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 384; or CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 385;

[0091] In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 186. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 187. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 188. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 213. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 214. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:215. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:216. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:217. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:218. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:219. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:220.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:221. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:222. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:223. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:224. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:225. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:226. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:227. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:228. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:229. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:230. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:231.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:232. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:233. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:234. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:235. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:236. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:237. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:238. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:239. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:280. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:342. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:343.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:344. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:345. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:346. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:347. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:348. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:349. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:350. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:351. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:352. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:353. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:354.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:355. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:356. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:357. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:358. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:359. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:360. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:361. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:362. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:363. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:364. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:365.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:366. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:367. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:368. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:369. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:370. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:371. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:372. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:373. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:374. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:375. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence shown in SEQ ID NO:376.In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:377. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:378. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:379. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:380. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO:381. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 382. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 383. In some embodiments, at least one CD28 sdAb domain comprises the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 384; or the CDR1, CDR2 and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 385.

[0092] In some embodiments, at least one CD28 sdAb (CD28 VHH) comprises CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 186; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 187; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 188; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 213; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 214; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 215; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 216; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:217; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:218; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:219; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:221; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:222; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:223; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:224; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:225; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:226; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:227; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:228; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:229; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:230; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:231;CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 232; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 233; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 234; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 235; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 236; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 237; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 238; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 239; or SEQ ID Contains the CDR1, CDR2, and CDR3 contained in the VHH shown in NO: 280.

[0093] In some embodiments, at least one CD28 sdAb (CD28 VHH) comprises CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 186; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 187; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 188; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 213; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 214; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 215; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 216; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:217; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:218; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:219; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:221; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:222; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:223; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:224; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:225; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:226; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:227; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:228; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:229; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:230; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO:231;CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 232; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 233; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 234; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 235; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 236; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 237; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 238; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 239; CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 280; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 342; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 343; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 344; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 345; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 346; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 347; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 348; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:349; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:350; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:351; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:352; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:353;CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 354; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 355; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 356; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 357; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 358; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 359; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 360; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 361; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 362; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 363; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 364; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 365; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 366; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 367; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 368; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 369; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:370; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:371; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:372; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO:373;CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 374; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 375; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 376; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 377; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 378; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 379; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 380; CDR1, CDR2, and CDR3 contained in the amino acid sequence shown in SEQ ID NO: 381; Contains CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 382; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 383; CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 384; or CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 385;

[0094] In some embodiments, at least one CD28 sdAb has an amino acid sequence set forth in any of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 280, or a sequence similar to SEQ ID and NOS: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 280. In some embodiments, at least one CD28 sdAb contains the amino acid sequence set forth in any of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 280.In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 280, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 40 51, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, and 385, or NO:186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234 , 235, 236, 237, 238, 239, 280, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 36 1, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, and 385.In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 280, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, and 385.

[0095] In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 186. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 187. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 188. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 213. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 214. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 215. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 216. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 217. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:218. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:219. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:220. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:221. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:222. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:223. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:224. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:225.In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:226. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:227. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:228. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:229. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:230. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:231. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:232. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:233. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:234. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:235. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:236. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:237. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:238. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:239. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:280. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:342.In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:343. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:344. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:345. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:346. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:347. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:348. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:349. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:350. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:351. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:352. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:353. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:354. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:355. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:356. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:357. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:358.In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:359. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:360. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:361. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:362. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:363. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:364. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:365. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:366. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:367. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:368. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:369. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:370. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:371. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:372. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:373. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:374.In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:375. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:376. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:377. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:378. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:379. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:380. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:381. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO:382. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 383. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 384. In some embodiments, at least one CD28 sdAb comprises the amino acid sequence set forth in SEQ ID NO: 385.

[0096] In some embodiments, at least one CD28 sdAb has an amino acid sequence set forth in any of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 280, or a sequence similar to SEQ ID and NOS: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 280. In some embodiments, at least one CD28 sdAb contains the amino acid sequence set forth in any of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, and 280.In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 280, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, and 385, or the amino acid sequence set forth in any of SEQ ID NOs. NO:186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 23 5, 236, 237, 238, 239, 280, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, and 385.In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 280, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 40 1, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, and 385.

[0097] In some embodiments, at least one CD28 sdAb contains a sequence set forth in (i) SEQ ID NO:186, (ii) a humanized variant of SEQ ID NO:186, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:186. In some embodiments, at least one CD28 sdAb contains a sequence set forth in SEQ ID NO:186. In some embodiments, at least one CD28 sdAb contains a humanized variant of SEQ ID NO:186. In some embodiments, at least one CD28 sdAb contains a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 186. In some embodiments, at least one CD28 sdAb contains a CDR1 having the amino acid sequence set forth in SEQ ID NO: 189; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 190; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 191.

[0098] In some embodiments, at least one CD28 sdAb contains a sequence set forth in (i) SEQ ID NO:187, (ii) a humanized variant of SEQ ID NO:187, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:187. In some embodiments, at least one CD28 sdAb contains a sequence set forth in SEQ ID NO:187. In some embodiments, at least one CD28 sdAb contains a humanized variant of SEQ ID NO:187. In some embodiments, at least one CD28 sdAb contains a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 187. In some embodiments, at least one CD28 sdAb contains a CDR1 having the amino acid sequence set forth in SEQ ID NO: 192; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 193; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 194.

[0099] In some embodiments, at least one CD28 sdAb contains a sequence set forth in (i) SEQ ID NO:188, (ii) a humanized variant of SEQ ID NO:188, or (iii) a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:188. In some embodiments, at least one CD28 sdAb contains a sequence set forth in SEQ ID NO:188. In some embodiments, at least one CD28 sdAb contains a humanized variant of SEQ ID NO:188. In some embodiments, at least one CD28 sdAb contains a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:188.

[0100] In some embodiments, at least one CD28 sdAb contains a CDR1 having the amino acid sequence set forth in any of SEQ ID NOs: 195, 198, 199, 200, and 201; a CDR2 having the amino acid sequence set forth in any of SEQ ID NOs: 196, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, and 212; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in any of SEQ ID NOs: 195, 198, 199, 200, and 201; a CDR2 comprising the amino acid sequence set forth in any of SEQ ID NOs: 196, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 386, 387, 388, 389, 390, 391, 392, 393, 394, and 395; and a CDR3 comprising the amino acid sequence set forth in any of SEQ ID NOs: 197, 396, 397, and 398.

[0101] In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 195, 196, and 197, respectively; SEQ ID NOs: 198, 196, and 197, respectively; SEQ ID NOs: 199, 196, and 197, respectively; SEQ ID NOs: 200, 196, and 197, respectively; SEQ ID NOs: 201, 196, and 197, respectively; SEQ ID NOs: 195, 202, and 197, respectively; SEQ ID NOs: 195, 203, and 197, respectively; SEQ ID NOs: 195, 204, and 197, respectively; SEQ ID NOs: 195, 205, and 197, respectively; SEQ ID NOs: 195, 206, and 197, respectively; SEQ ID NOs: 195, 207, and 197, respectively; SEQ ID NOs: 195, 208, and 197; SEQ ID NOs: 195, 209, and 197, respectively; SEQ ID NOs: 195, 210, and 197; SEQ ID NOs: 195, 211, and 197, respectively; or containing CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 195, 212, and 197, respectively. In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 195, 196, and 197, respectively; SEQ ID NOs: 198, 196, and 197, respectively; SEQ ID NOs: 199, 196, and 197, respectively; SEQ ID NOs: 200, 196, and 197, respectively; SEQ ID NOs: 201, 196, and 197, respectively; SEQ ID NOs: 195, 202, and 197, respectively; SEQ ID NOs: 195, 203, and 197, respectively; SEQ ID NOs: 195, 204, and 197, respectively; SEQ ID NOs: 195, 205, and 197, respectively; SEQ ID NOs: 195, 206, and 197, respectively; SEQ ID NOs: 195, 207, and 197, respectively; NO: 195, 208, and 197; SEQ ID NO: 195, 209, and 197, respectively; SEQ ID NO: 195, 210, and 197, respectively; SEQ ID NO: 195, 211, and 197, respectively;SEQ ID NOs: 195, 212, and 197, respectively; SEQ ID NOs: 201, 202, and 197, respectively; SEQ ID NOs: 201, 202, and 396, respectively; SEQ ID NOs: 201, 386, and 197, respectively; SEQ ID NOs: 201, 387, and 197, respectively; SEQ ID NOs: 201, 202, and 397, respectively; SEQ ID NOs: 201, 202, and 398, respectively; SEQ ID NOs: 201, 206, and 197, respectively; SEQ ID NOs: 201, 206, and 398, respectively; SEQ ID NOs: 201, 388, and 197, respectively; SEQ ID NOs: 201, 389, and 197, respectively; SEQ ID NOs: 201, 206, and 397, respectively; SEQ ID SEQ ID NOs:201, 206, and 398; SEQ ID NOs:201, 203, and 197, respectively; SEQ ID NOs:201, 203, and 396, respectively; SEQ ID NOs:201, 390, and 197, respectively; SEQ ID NOs:201, 391, and 197, respectively; SEQ ID NOs:201, 203, and 397, respectively; SEQ ID NOs:201, 203, and 398, respectively; SEQ ID NOs:201, 204, and 197, respectively; SEQ ID NOs:201, 204, and 396, respectively; SEQ ID NOs:201, 392, and 197, respectively; SEQ ID NOs:201, 393, and 197, respectively; SEQ ID NOs:201, 204, and 397, respectively; SEQ ID NOs:201, 204, and 398, respectively; SEQ ID NOs: 201, 196, and 396; SEQ ID NOs: 201, 394, and 197, respectively; SEQ ID NOs: 201, 395, and 197, respectively; SEQ ID NOs: 201, 196, and 397, respectively; SEQ ID NOs: 201, 196, and 398, respectively; SEQ ID NOs: 195, 206, and 396, respectively; SEQ ID NOs: 195, 388, and 197, respectively; SEQ ID NOs: 195, 389, and 197, respectively; SEQ ID NOs: 195, 206, and 397, respectively;or containing CDR1, CDR2, and CDR3 as set forth in SEQ ID NOs: 195, 206, and 398, respectively;

[0102] In some embodiments, at least one CD28 sdAb contains a sequence of amino acids set forth in any one of SEQ ID NOs:213-239 and 280, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:213-239 and 280. In some embodiments, at least one CD28 sdAb contains a sequence of amino acids set forth in any one of SEQ ID NOs:213-239 and 280.

[0103] In some embodiments, at least one CD28 sdAb contains an amino acid sequence set forth in any of SEQ ID NOs: 213-239, 280, and 342-385, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 213-239, 280, and 342-385. In some embodiments, at least one CD28 sdAb contains an amino acid sequence set forth in any of SEQ ID NOs: 213-239, 280, and 342-385.

[0104] In some embodiments, at least one CD28 sdAb contains a CDR1 having the amino acid sequence set forth in any of SEQ ID NOs: 195, 198, 199, 200, and 201; a CDR2 having the amino acid sequence set forth in any of SEQ ID NOs: 196, 202, 204, 205, 206, 207, 208, 209, 210, 211, and 212; and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, at least one CD28 sdAb contains a CDR1 comprising the amino acid sequence set forth in any of SEQ ID NOs: 195, 198, 199, 200, and 201; a CDR2 comprising the amino acid sequence set forth in any of SEQ ID NOs: 196, 202, 204, 205, 206, 207, 208, 209, 210, 211, 212, 386, 387, 388, 389, 390, 391, 392, 393, 394, and 395; and a CDR3 comprising the amino acid sequence set forth in any of SEQ ID NOs: 197, 396, 397, and 398.

[0105] In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 195, 196, and 197, respectively; SEQ ID NOs: 198, 196, and 197, respectively; SEQ ID NOs: 199, 196, and 197, respectively; SEQ ID NOs: 200, 196, and 197, respectively; SEQ ID NOs: 201, 196, and 197, respectively; SEQ ID NOs: 195, 202, and 197, respectively; SEQ ID NOs: 195, 204, and 197, respectively; SEQ ID NOs: 195, 205, and 197, respectively; SEQ ID NOs: 195, 206, and 197, respectively; SEQ ID NOs: 195, 207, and 197, respectively; SEQ ID NOs: 195, 208, and 197, respectively; SEQ ID NOs: 195, 209, and 197; SEQ ID NOs: 195, 210, and 197, respectively; SEQ ID NOs: 195, 211, and 197, respectively; or containing the CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 195, 212, and 197, respectively. In some embodiments, at least one CD28 sdAb is selected from the group consisting of SEQ ID NOs: 195, 196, and 197, respectively; SEQ ID NOs: 198, 196, and 197, respectively; SEQ ID NOs: 199, 196, and 197, respectively; SEQ ID NOs: 200, 196, and 197, respectively; SEQ ID NOs: 201, 196, and 197, respectively; SEQ ID NOs: 195, 202, and 197, respectively; SEQ ID NOs: 195, 204, and 197, respectively; SEQ ID NOs: 195, 205, and 197, respectively; SEQ ID NOs: 195, 206, and 197, respectively; SEQ ID NOs: 195, 207, and 197, respectively; SEQ ID NOs: 195, 208, and 197, respectively; NO: 195, 209, and 197; SEQ ID NO: 195, 210, and 197, respectively; SEQ ID NO: 195, 211, and 197, respectively; SEQ ID NO: 195, 212, and 197, respectively; SEQ ID NO: 201, 202, and 197, respectively;SEQ ID NOs: 201, 202, and 396, respectively; SEQ ID NOs: 201, 386, and 197, respectively; SEQ ID NOs: 201, 387, and 197, respectively; SEQ ID NOs: 201, 202, and 397, respectively; SEQ ID NOs: 201, 202, and 398, respectively; SEQ ID NOs: 201, 206, and 197, respectively; SEQ ID NOs: 201, 206, and 398, respectively; SEQ ID NOs: 201, 388, and 197, respectively; SEQ ID NOs: 201, 389, and 197, respectively; SEQ ID NOs: 201, 206, and 397, respectively; SEQ ID NOs: 201, 206, and 398, respectively; SEQ ID NOs: 201, 203, and 197, respectively; SEQ ID SEQ ID NOs:201, 203, and 396; SEQ ID NOs:201, 390, and 197, respectively; SEQ ID NOs:201, 391, and 197, respectively; SEQ ID NOs:201, 203, and 397, respectively; SEQ ID NOs:201, 203, and 398, respectively; SEQ ID NOs:201, 204, and 197, respectively; SEQ ID NOs:201, 204, and 396, respectively; SEQ ID NOs:201, 392, and 197, respectively; SEQ ID NOs:201, 393, and 197, respectively; SEQ ID NOs:201, 204, and 397, respectively; SEQ ID NOs:201, 204, and 398, respectively; SEQ ID NOs:201, 196, and 396, respectively; SEQ ID NOs:201, 394, and 197, respectively; SEQ SEQ ID NOs: 201, 395, and 197; SEQ ID NOs: 201, 196, and 397, respectively; SEQ ID NOs: 201, 196, and 398, respectively; SEQ ID NOs: 195, 206, and 396, respectively; SEQ ID NOs: 195, 388, and 197, respectively; SEQ ID NOs: 195, 389, and 197, respectively; SEQ ID NOs: 195, 206, and 397, respectively; or containing the CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 195, 206, and 398, respectively;

[0106] In some embodiments, at least one CD28 sdAb contains a sequence of amino acids set forth in any one of SEQ ID NOs: 213-219, 221-239, and 280, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 213-219, 221-239, and 280. In some embodiments, at least one CD28 sdAb contains a sequence of amino acids set forth in any one of SEQ ID NOs: 213-219, 221-239, and 280. In some embodiments, at least one CD28 sdAb contains an amino acid sequence set forth in any of SEQ ID NOs: 213-239, 280, and 342-385, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 213-239, 280, and 342-385. In some embodiments, at least one CD28 sdAb comprises an amino acid sequence set forth in any of SEQ ID NOs: 213-239, 280, and 342-385.

[0107] In any of the provided embodiments, the CD28 binding polypeptide contains a CD3 binding region. In some embodiments, the anti-CD28 sdAb-Fc fusion protein comprises a CD3 binding region. In some embodiments, the homodimeric Fc fusion protein comprises a CD3 binding region. In some embodiments, the heterodimeric Fc fusion protein comprises a CD3 binding region. In some embodiments, the multispecific binding polypeptide comprises a CD3 binding region. In some embodiments, the homodimeric multispecific binding polypeptide comprises a CD3 binding region. In some embodiments, the heterodimeric multispecific binding polypeptide comprises a CD3 binding region. In some embodiments, the fusion protein comprises a CD3 binding region.

[0108] Provided herein is a multispecific construct containing at least one of the provided sdAbs, one or more binding domains (BDs) that bind to antigens other than CD28, and a CD3 binding region. In some embodiments, the BD binds to a tumor-associated antigen. In some embodiments, the BD is a single domain antibody.

[0109] In some embodiments, the CD3 binding region binds to CD3 (CD3ε). In some embodiments, the CD3 binding region is an anti-CD3 antibody or antigen-binding fragment. In some embodiments, the CD3 binding region is an anti-CD3 antibody. In some embodiments, the CD3 binding region is an anti-CD3 antigen-binding fragment. In some embodiments, the anti-CD3 antibody or antigen-binding fragment contains a variable heavy chain region (VH) and a variable light chain region (VL). In some embodiments, the CD3 binding region is monovalent. In some embodiments, the CD3 binding region is a variable fragment (Fv) comprising a variable heavy chain region (VH) and a variable light chain region (VL). In some embodiments, the anti-CD3 antibody or antigen-binding fragment is not a single-chain antibody. In some embodiments, the anti-CD3 antibody or antigen-binding fragment is not a single-chain variable fragment (scFv).

[0110] In any of the provided embodiments, the CD28-binding polypeptide contains an immunoglobulin Fc region. In some embodiments, the Fc region is a homodimeric Fc region. In some embodiments, the Fc region contains a sequence of amino acids selected from the group consisting of SEQ ID NOs:8, 10, 11, 12, and 13, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs:8, 10, 11, 12, and 13. In some embodiments, the Fc region consists of a sequence of amino acids selected from the group consisting of SEQ ID NOs:8, 10, 11, 12, and 13. In some embodiments, the Fc region is human IgG1. In some embodiments, the Fc region is human IgG1. In some embodiments, the Fc region contains the sequence of amino acids set forth in SEQ ID NO:8, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8. In some embodiments, the Fc region consists of the sequence of amino acids set forth in SEQ ID NO:8.

[0111] In some embodiments, the Fc region is a heterodimeric Fc region.

[0112] In some embodiments, the Fc region exhibits effector function.

[0113] In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce effector function. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to an effector molecule. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to an effector molecule selected from an Fc gamma receptor and C1q. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to an Fc gamma receptor. In some embodiments, the Fc region contains a polypeptide having one or more amino acid modifications that reduce binding to C1q. In some embodiments, the one or more amino acid modifications are deletions of one or more of Glu233, Leu234, or Leu235. In some embodiments, the one or more amino acid modifications are deletions of Glu233. In some embodiments, the one or more amino acid modifications are deletions of Leu234. In some embodiments, the one or more amino acid modifications are deletions of Leu 235. In some embodiments, the Fc region contains the sequence of amino acids set forth in SEQ ID NO:9, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9. In some embodiments, the Fc region consists of the sequence of amino acids set forth in SEQ ID NO:9.

[0114] In some embodiments, the CD28-binding polypeptide is a dimer. In some embodiments, the Fc is a heterodimeric Fc, and the VH and VL comprising the anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc.

[0115] In some embodiments, the CD3 binding region is unable to bind or engage with CD3, or is unable to substantially bind or engage with CD3, unless at least one of the at least one CD28 sdAb is bound to CD28. In some embodiments, the CD3 binding region is unable to bind or engage with CD3 unless at least one of the at least one CD28 sdAb is bound to CD28. In some embodiments, the CD3 binding region is unable to bind or engage with CD3, or is unable to substantially bind or engage with CD3, unless at least one of the one or more TAA sdAbs is bound to that TAA. In some embodiments, the CD3 binding region is unable to bind or engage with CD3 unless at least one of the one or more TAA sdAbs is bound to that TAA. In some embodiments, the CD3 binding region is unable to substantially bind or engage CD3 unless at least one of the one or more TAA sdAbs is bound to that TAA.

[0116] In some embodiments, the CD28 binding polypeptide comprises a moiety that binds to Protein A.

[0117] In some embodiments, at least one CD28 sdAb comprises an amino acid modification that reduces binding to Protein A. In some embodiments, at least one CD28 sdAb comprises the amino acid modification G65D according to Kabat in framework region 3 (FR3).

[0118] Also provided herein are anti-CD28 single domain antibodies containing a complementarity determining region 1 (CDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 189, 192, 195, 198, 199, 200, and 201; a complementarity determining region 2 (CDR2) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 193, 196, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, and 212, 386, 387, 388, 389, 390, 391, 392, 393, 394, and 395; and a complementarity determining region 3 (CDR3) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 194, and 197, 396, 397, and 398.

[0119] Also provided herein are anti-CD28 sdAbs containing a complementarity determining region 1 (CDR1) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 189, 192, 195, 198, 199, 200, and 201; a complementarity determining region 2 (CDR2) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 193, 196, 202, 204, 205, 206, 207, 208, 209, 210, 211, and 212, 386, 387, 388, 389, 390, 391, 392, 393, 394, and 395; and a complementarity determining region 3 (CDR3) containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 194, and 197, 396, 397, and 398.

[0120] In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 191. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 192, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 193, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 194. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 200, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 202, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 203, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 204, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 205, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 207, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:208, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 209, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 210, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 211, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 212, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 202, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 202, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 396. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:386, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:387, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:202, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:202, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:388, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:389, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197.In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:390, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:391, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:397.In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:392, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:393, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:204, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:398.In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:394, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:395, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:397. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:201, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:196, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:398. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:396. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO:195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO:388, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO:197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 389, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 197. In some embodiments, the CD28 sdAb contains a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 397; or a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 398.

[0121] In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:186. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:187. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:188. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:213. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:214. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:215. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:216. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:217. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:218. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:219. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:220. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:221. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:222. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:223. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:224. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:225. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:226. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:227.In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:228. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:229. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:230. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:231. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:232. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:233. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:234. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:235. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:236. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:237. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:238. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:239. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:280. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:342. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:343. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:344. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:345. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:346.In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:347. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:348. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:349. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:350. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:351. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:352. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:353. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:354. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:355. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:356. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:357. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:358. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:359. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:360. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:361. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:362. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:363. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:364.In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:365. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:366. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:367. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:368. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:369. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:370. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:371. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:372. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:373. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:374. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:375. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:376. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:377. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:378. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:379. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:380. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:381. In some embodiments, the CD28 sdAb contains the amino acid sequence set forth in SEQ ID NO:382.In some embodiments, the CD28 sdAb contains the amino acid sequence shown in SEQ ID NO: 383. In some embodiments, the CD28 sdAb contains the amino acid sequence shown in SEQ ID NO: 384. In some embodiments, the CD28 sdAb contains the amino acid sequence shown in SEQ ID NO: 385.

[0122] In some embodiments, the anti-CD28 sdAb is isolated. In some embodiments, the anti-CD28 sdAb is purified.

[0123] In some embodiments, the CD28 sdAb comprises an amino acid modification that reduces binding to Protein A. In some embodiments, at least one CD28 sdAb comprises an amino acid modification that reduces binding to Protein A. In some embodiments, at least one CD28 sdAb comprises the amino acid modification G65D according to Kabat in framework region 3 (FR3).

[0124] Provided herein are polynucleotides encoding any of the anti-CD28 sdAbs, CD28 binding polypeptides, fusion proteins, and multispecific binding polypeptides, as well as the multispecific constructs described herein.

[0125] Also provided herein is a polynucleotide comprising a first nucleic acid sequence encoding a first polypeptide of any of the CD28 binding polypeptides provided herein and a second nucleic acid sequence encoding a second polypeptide of the CD28 binding polypeptide.In some embodiments, the first and second nucleic acid sequences are separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping.Also provided herein is a polynucleotide comprising a first nucleic acid sequence encoding a first polypeptide of any of the CD28 binding polypeptides provided herein and a second nucleic acid sequence encoding a second polypeptide of a multispecific construct, wherein the first and second nucleic acid sequences are separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping.

[0126] Also provided herein is a polynucleotide containing a first nucleic acid sequence encoding a first polypeptide of any of the fusion proteins provided herein and a second nucleic acid sequence encoding a second polypeptide of the fusion protein. Also provided herein is a polynucleotide containing a first nucleic acid sequence encoding a first polypeptide of any of the multispecific binding polypeptides provided herein and a second nucleic acid sequence encoding a second polypeptide of the multispecific binding polypeptide. Also provided herein is a polynucleotide containing a first nucleic acid sequence encoding a first polypeptide of any of the multispecific constructs provided herein and a second nucleic acid sequence encoding a second polypeptide of the multispecific construct.

[0127] In some embodiments, the first and second nucleic acid sequences are separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping.In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter.In some embodiments, the nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping is selected from T2A, P2A, E2A, or F2A.

[0128] Provided herein is a vector comprising any of the polynucleotides described herein.In some embodiments, the vector is an expression vector.In some embodiments, the vector is a viral vector or a eukaryotic vector.In some embodiments, the eukaryotic vector is a mammalian vector.

[0129] Provided herein is a cell that contains one or more polynucleotides as described herein.In some embodiments, the cell is lymphocyte.In some embodiments, the cell is T cell or natural killer (NK) cell.

[0130] Provided herein is a method for producing polypeptide, comprising: introducing any one or more polynucleotides provided herein or any one or more vectors provided herein into cells, and culturing the cells under conditions to produce anti-CD28 sdAb, CD28 binding polypeptide, fusion protein, multispecific binding polypeptide or multispecific construct.In some embodiments, the method comprises isolating or purifying the polypeptide from cells.Also provided herein is the polypeptide produced by any of the methods described herein.

[0131] Pharmaceutical compositions comprising any of the anti-CD28 sdAbs, CD28 binding polypeptides, fusion proteins, multispecific binding polypeptides, and multispecific constructs described herein are provided.In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.In some embodiments, the pharmaceutical composition is sterile.

[0132] Provided herein is a method for stimulating an immune response in a subject, comprising administering to a subject in need thereof a CD28 sdAb, a CD28 binding polypeptide, a fusion protein, a multispecific binding polypeptide, and a multispecific construct, or a pharmaceutical composition described herein. In some embodiments, the immune response is increased against tumors or cancer. In some embodiments, the method treats a disease or condition in the subject.

[0133] Also provided herein is a method of treating a disease or condition in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of any of the anti-CD28 sdAb, CD28 binding polypeptide, fusion protein, multispecific binding polypeptide, and multispecific construct, or pharmaceutical composition described herein.

[0134] Also provided herein are uses of any of the anti-CD28 sdAbs, CD28 binding polypeptides, fusion proteins, multispecific binding polypeptides, and multispecific constructs, or pharmaceutical compositions described herein. Provided herein are uses of any of the provided multispecific binding polypeptides. Provided herein are uses of any of the provided multispecific constructs. Provided herein are uses of any of the provided pharmaceutical compositions.

[0135] Also provided are compositions comprising any of the anti-CD28 sdAbs, CD28 binding polypeptides, fusion proteins, multispecific binding polypeptides, and multispecific constructs, or pharmaceutical compositions described herein, for use in treating a disease or condition in a subject. Provided herein are compositions comprising multispecific binding polypeptides for use in treating a disease or condition in a subject. Provided herein are compositions comprising multispecific conjugates for use in treating a disease or condition in a subject.

[0136] Also provided are any of the anti-CD28 sdAbs, CD28 binding polypeptides, fusion proteins, multispecific binding polypeptides, and multispecific constructs, or pharmaceutical compositions described herein, for use in the manufacture of a medicament for treating a disease or condition in a subject. Provided herein are compositions comprising multispecific binding polypeptides for use in the manufacture of a medicament for treating a disease or condition in a subject. Provided herein are compositions comprising multispecific conjugates for use in the manufacture of a medicament for treating a disease or condition in a subject.

[0137] In some embodiments, the disease or condition is a tumor or cancer. In some embodiments, the disease or condition is a tumor. In some embodiments, the disease or condition is cancer. In some embodiments, the subject is a human. [Brief explanation of the drawings]

[0138] [Figure 1A]Figure 1 illustrates the ability of CD28-targeting single-domain antibodies formatted as sdAb-IgG1 to bind to cell surface CD28. Binding was assessed by flow cytometry using HEK293FS (293FS) cells transiently transfected with full-length human (hCD28) or cynomolgus monkey (cyCD28) antigens (Figures 1A and 1B, respectively). As a negative control, binding to CD28-untransfected 293FS cells was also tested (Figure 1C). The single-domain antibody 1C9 was formatted with an effector function-reduced Fc domain as sdAb-xELL and tested for binding to Jurkat cells or primary T cells enriched from PBMCs isolated from normal donor whole blood, both of which endogenously express CD28 (Figures 1D and 1E, respectively). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 2A] Figures 2A-I illustrate the ability of 1C9 and its humanized variants to bind to cell surface CD28. Binding was assessed by flow cytometry on HEK293FS (293FS) cells transiently transfected with full-length human (hCD28) or cynomolgus monkey (cyCD28) antigens (Figures 2A, B, D, E), non-transfected HEK293FS cells (Figures 2C and 2F), Jurkat cells that endogenously express CD28 (Figures 2G-H), or primary T cells enriched from PBMCs isolated from normal donor whole blood (Figure 2I). [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Figure 2G] See legend to Figure 2A. [Figure 2H] See legend to Figure 2A. [Figure 2I] See legend to Figure 2A. [Figure 3A] Figures 3A and 3B illustrate exemplary CD28-binding polypeptides with and without an Fc domain, respectively. Figures 3C-3H illustrate the ability of exemplary engineered constructs to bind to cell surface CD28, PDL1, and / or 5T4. Binding was assessed by flow cytometry on HEK293FS (293FS) cells transiently transfected with PDL1 (Figure 3C), T47D cells endogenously expressing 5T4 (Figure 3F), Jurkat cells endogenously expressing CD28 (Figures 3D and 3G), or Raji cells that do not express PDL1, 5T4, or CD28 (Figures 3E and 3H). [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 3G] See legend to Figure 3A. [Figure 3H] See legend to Figure 3A. [Figure 4A]Figure 4A illustrates the ability of CD28-targeting single-domain antibodies 1C12 and 1F10 (but not 2F11, 1G7, and 1C9) formatted as bivalent sdAb-IgG1 to enhance activation of a Jurkat-based reporter cell line by an activating anti-CD3 antibody, where luciferase production is driven by the IL-2 promoter. Figure 4B illustrates the ability of cross-linked (XL)1C9-IgG1, representing a multimer of 1C9 with more than two valencies, to stimulate reporter cells. Figures 4C and 4D illustrate the ability of TAAxCD28 bispecific proteins to agonize CD28 on CD3(IL-2) Jurkat reporter cells in a TAA-dependent manner. Figure 4C illustrates the ability of cx694 (a bispecific construct targeting PDL1 and CD28) to agonize CD28 on CD3(IL-2) Jurkat reporter cells in a PDL1-dependent manner, as evidenced by increased reporter activation in the presence but not in the absence of PDL1-positive CHO-K1 cells (Promega™). Monospecific constructs individually targeting PDL1 (cx1204) or CD28 (cx698) did not significantly enhance reporter cell line activation compared to untreated controls in the presence or absence of PDL1-expressing target cell lines. Figure 4D illustrates the ability of cx8390 (a bispecific construct targeting 5T4 and CD28) to agonize CD28 on CD3(IL-2) Jurkat reporter cells in a 5T4-dependent manner, as evidenced by increased reporter activation in the presence but not in the absence of 5T4-positive T47D cells. Treatment of reporter cells alone or cocultures of T47D and reporter cells with a monospecific protein targeting CD28 (cx8394) did not enhance activation of the reporter cell line compared to untreated controls. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5A]Figure 5 illustrates the ability of cx694 (a bispecific construct targeting PDL1 and CD28) to induce T cell-mediated cytotoxicity in the PDL1+ cell line, A549 (Figures 5A and 5C), but not in the PDL1- cell line, HEK293FS (293FS) (Figures 5B and 5D), as assessed by caspase-3 / 7 activation using a cell imaging system (Figures 5A and 5B) and cell viability using the CellTiter-Glo assay (Figures 5C and 5D). Monospecific constructs targeting PDL1 (cx1204) or CD28 (cx698) individually lacked cytotoxic activity against both A549 and 293FS cells (Figures 5A-5D). [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A] Figures 6A-D illustrate the ability of cx694 (a bispecific protein targeting PDL1 and CD28) to induce antigen-specific activation of CD4+ (Figures 6A-6C) and CD8+ (Figure 6D) T cells, as assessed by analyzing the activation markers CD25 (Figure 6A), CD69 (Figures 6B and 6D), and CD71 (Figure 6C) by flow cytometry. Monospecific constructs targeting PDL1 (cx1204) or CD28 (cx698) individually did not activate either T cell subset in the presence of either target cell line. A549 and 293FS cells were used as PDL1+ and PDL1- cells, respectively. Figure 6E illustrates the lack of IFNγ production by A549 / T cell cocultures treated with cx694. cx5185 is a CD3-stimulatory protein that served as a positive control for the experiment. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 7A]Figures 7A-7C illustrate the ability of bispecific constructs targeting PDL1 and CD28 to costimulate primary T cells. Figure 7A illustrates the ability of cx8370 (a bispecific construct targeting PDL1 and CD28) to enhance IFNγ production by PBMCs treated with a CEF (cytomegalovirus, Epstein-Barr virus, and Flu virus) peptide pool, as assessed by FluoroSpot. Monospecific constructs targeting PDL1 (cx1204) or CD28 (cx984) individually did not affect the levels of cytokines produced by stimulated PBMCs. Figures 7B and 7C illustrate the ability of cx694 (a bispecific construct targeting PDL1 and CD28) to induce TNFα production by cocultures of enriched T cells with autologous immature dendritic cells (iDCs). Figures 7B and 7C illustrate responses from two different effector cell donors, Leuko Pack. Monospecific constructs targeting PDL1 (cx1204) or CD28 (cx698) individually did not induce robust cytokine responses from either donor. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 8] Figures 8A and 8B illustrate the ability of immobilized positive control anti-CD3 (OKT3) and anti-CD28 (TGN1412) antibodies to induce TNFα production by CD4+ T cells in PBMCs pre-cultured at high density, whereas immobilized protein containing exemplary sdAb 1C9 (cx694, cx8370, and cx984) did not induce TNFα production in this cell population at levels above those of untreated control samples. Figures 8A and 8B illustrate responses by two different PBMC donors, Leuko Pack. DETAILED DESCRIPTION OF THE INVENTION

[0139] Detailed Description Provided herein are polypeptides that specifically bind to CD28, hereinafter also referred to as CD28-binding polypeptides. In some embodiments, the provided binding polypeptides contain at least one single-domain antibody (sdAb; e.g., a VHH domain) that binds to CD28. The CD28-binding polypeptides provided herein include monovalent and multivalent (e.g., bivalent) constructs. In some embodiments, the CD28-binding polypeptides provided herein contain one, two, three, four, five, six, seven, or eight VHH domains, each of which individually binds to CD28. In some embodiments, the CD28-binding polypeptides provided herein contain one, two, three, or four VHH domains that bind to CD28. In some embodiments, the CD28-binding polypeptides provided herein contain two VHH domains that bind to CD28. In some embodiments, the CD28-binding polypeptides provided herein contain one VHH domain that binds to CD28. In some embodiments, the CD28-binding polypeptides provided herein contain a single VHH domain that binds to CD28.

[0140] In some embodiments, the CD28-binding polypeptide is monospecific. In some embodiments, the CD28-binding polypeptide is multispecific. For example, the provided CD28-binding polypeptide includes a polypeptide that may contain at least one VHH domain that binds to CD28 and one or more additional binding domains, such as one or more additional VHH domains, that bind to one or more target antigens other than CD28. In a specific embodiment, the target antigen is a tumor-associated antigen (TAA).

[0141] In some embodiments, the CD28-binding polypeptide is provided as an Fc fusion protein. In some embodiments, the CD28-binding polypeptide contains at least one VHH domain that binds to CD28 and an Fc domain. In some embodiments, the CD28-binding polypeptide contains at least one VHH domain that binds to CD28, at least one other binding domain (e.g., VHH) that binds to another target antigen (e.g., TAA), and an Fc domain. In some embodiments, the Fc domain mediates dimerization of the CD28-binding polypeptide under physiological conditions, such that a dimer is formed that doubles the number of CD28-binding sites and, if present, can also double the number of other antigen-binding domains. For example, a CD28-binding polypeptide comprising one VHH domain that binds to CD28 and an Fc region is monovalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization such that the CD28-binding polypeptide exists as a bivalent dimer under such conditions.

[0142] CD28 is a homodimeric transmembrane glycoprotein of the immunoglobulin (Ig) superfamily constitutively expressed by T cells. Its primary binding partners are CD80 (B7-1) and CD86 (B7-2), which can further bind to multiple receptors, including CTLA4. (Esensten et al., Immunity (2019) 44(5):973-88). CD28 provides costimulatory and activation signals for T cells, which are important for T cell proliferation and effector function. (Ibid.) CD28 costimulatory signals provide a secondary signal to enhance primary signaling by the antigen-receptor complex (TCR / CD3) to activate CD8+ cytotoxic T cells (CTLs), which provide adaptive immune responses against cancer and execute tumor-specific immune responses. (Huff et al., International Journal of Molecular Sciences (2019) 20(11):2810). Furthermore, CD8+ T cells with low CD28 expression have been identified in settings where CTL-mediated tumor killing is ineffective. Id.

[0143] An exemplary sequence of standard human CD28 is shown below. (SEQ ID NO:86, e.g., Uniprot No. P10747): TIFF0007789002000001.tif19160

[0144] CD28 agonism has been shown to promote T cell activation, potentially making it an effective immunotherapy for activating an efficient immune response against tumors to treat cancer. However, a problem with certain CD28 agonists is that they may possess superagonist activity, resulting in CD28 agonism independent of TCR signaling. This can sometimes lead to T cell expansion and measurable proinflammatory responses even in the absence of additional stimulation from the T cell receptor. For example, the superagonist monoclonal antibody TGN1412 caused a severe cytokine storm in healthy human patients treated with it (Suntharalingam et al. N Engl J Med 2006; 355:1018-1028). Therefore, improved CD28 agonists with improved safety are needed.

[0145] Provided herein is a VHH domain that binds to CD28 but does not show CD28 agonist activity in monospecific / bivalent format.This CD28 binding VHH domain can be incorporated into many binding formats to show desired immune activity.Various CD28 polypeptide binding formats are provided.See, for example, Figure 3A and 3B.

[0146] Among the provided CD28-binding polypeptides are polypeptides that can bind to and mediate CD28-dependent signaling. In some cases, the provided CD28-binding polypeptides directly engage CD28 and / or agonize the activity of CD28, which in some aspects can be used as a therapy to enhance the anti-tumor activity of T cells.

[0147] In specific embodiments, provided herein are multispecific conditional CD28-binding polypeptides that contain at least one VHH domain that binds to CD28 and at least one binding domain that binds to another antigen, such as a tumor-associated antigen (TAA; e.g., PD-L1 or 5T4) or another antigen present in the tumor microenvironment (TME). In some cases, the binding polypeptide comprises a polypeptide that exhibits dual affinity for CD28 and a tumor-associated antigen (TAA), such as PD-L1 or 5T4. In some cases, the binding polypeptide comprises a polypeptide that exhibits dual affinity for CD28 and another antigen present in the microenvironment, such as a T cell exhaustion marker, a T cell activation marker, or a TME marker. The provided multispecific conditional CD28-binding polypeptides exhibit CD28 agonist activity only when bound to another antigen present on the surface of a cell (e.g., a tumor cell), and T cell activity is independent of CD3 engagement. In some aspects, such dual affinity molecules can engage or activate T cells at the site of the tumor upon binding of a TAA (e.g., PD-L1 or 5T4) expressed on the tumor. In some aspects, such dual affinity molecules can engage or activate T cells at the site of the tumor upon binding of an activation or exhaustion marker expressed on the T cell. In some aspects, such dual affinity molecules can engage or activate T cells at the site of the tumor upon binding of an antigen expressed by cells in the TME. Notably, among such molecules provided herein are those that exhibit conditional CD28 binding and / or engagement. In some embodiments, the CD28-binding polypeptide is multispecific, and its ability to costimulate T cells is blocked or reduced in the absence of binding of a TAA to the multispecific polypeptide. In some embodiments, the multispecific CD28-binding polypeptide can costimulate T cells only when the multispecific polypeptide is bound to a TAA.In some embodiments, the multispecific CD28 binding polypeptide is only capable of costimulating a T cell when the multispecific polypeptide binds to an antigen in the TME.

[0148] In some embodiments, the multispecific conditional CD28-binding molecules comprise formats that are monovalent with respect to CD28 and either monovalent or multivalent (e.g., bivalent) with respect to other antigens (e.g., TAAs). In other embodiments, the multispecific conditional CD28-binding molecules comprise formats that are multivalent (e.g., bivalent) with respect to CD28 and either monovalent or multivalent (e.g., bivalent) with respect to other antigens (e.g., TAAs). In a specific embodiment, the provided multispecific conditional CD28-binding polypeptides are provided as fusion proteins with Fc, resulting in a dimer of the two polypeptide chains.

[0149] Also provided herein are T cell-engaging fusion proteins in the form of multispecific polypeptide constructs containing at least one (and typically only one) VHH that binds to CD28, a CD3-binding region, and at least one other antigen (e.g., TAA). In specific embodiments, the provided T cell-engaging fusion proteins are provided in a format with an Fc region N-terminal to the CD3-binding region. The provided multispecific polypeptide constructs exhibit constrained T cell-engaging activity because such constructs substantially bind to CD3 only when the antigen is bound via the antigen-binding domain. This unique property allows the constrained CD3-engaging protein to distribute to sites where another antigen (e.g., TAA) is present. This format differs from other CD3-engaging multispecific constructs in that constitutive CD3 binding is disabled or eliminated, providing a significant advantage by avoiding peripheral T cell binding and allowing preferential distribution to sites where the antigen is present when recognized by the antigen-binding domain. The constrained T cell-engaging activity of the provided multispecific polypeptide constructs results, in some aspects, from positioning an Fc region N-terminal to the CD3-binding region. In some embodiments, such positioning reduces, attenuates, weakens, and / or prevents CD3 binding by the CD3-binding region. In the absence of antigen binding by the antigen-binding domain, the multispecific polypeptide constructs provided herein exhibit reduced or eliminated CD3 binding and T cell activation capacity. The presence of a VHH that binds CD28 in the provided multispecific polypeptide constructs increases or enhances T cell activity upon co-engagement of CD3 by the CD3-binding region and binding to other antigens (e.g., TAAs).

[0150] In any of the provided embodiments, the Fc is one that exhibits reduced immune effector activity, e.g., via one or more mutations that reduce one or more effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). Exemplary inactive or effector-less Fcs are described herein.

[0151] In some embodiments, the provided CD28-binding polypeptides can be used to stimulate an immune response in a subject, thereby, in some aspects, treating a disease or disorder, such as cancer, in the subject. In some aspects, the CD28-binding polypeptides provided herein, e.g., multispecific conditional CD28-binding polypeptides, can bind to CD28-expressing T cells and stimulate the T cells to induce anti-tumor activity. In some cases, CD28 agonist stimulation and the resulting anti-tumor activity can cause the death of cancerous cells (e.g., costimulated T cells kill cancer cells).

[0152] Also provided herein are engineered cells, such as engineered T cells, that express any of the provided CD28 binding polypeptides. In some embodiments, the engineered cells produce and secrete the CD28 binding polypeptide.

[0153] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that the definitions set forth herein conflict or are otherwise inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein take precedence over the definitions incorporated herein by reference.

[0154] The techniques and procedures described or referenced herein are generally well understood and can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel, et al. eds., (2003)); METHODS IN ENZYMOLOGY (Academic Press, Inc.) series: PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984);Methods in Molecular Biology, Humana Press;Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press;Animal Cell Culture (RI Freshney), ed., 1987);Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press;Cell and Tissue Culture Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CC Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993); and the latest editions thereof, using conventional methodologies commonly used by those skilled in the art.

[0155] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0156] I. Definition Unless otherwise defined, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated, singular terms shall include pluralities and plural terms shall include the singular. In the event of any discrepancy in definitions among various sources or references, the definitions provided herein shall control.

[0157] It is understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments. As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. The use of the term "or" herein is not intended to imply that alternatives are mutually exclusive.

[0158] In this application, unless expressly stated or understood by a person skilled in the art, the use of "or" means "and / or." In the context of multiple dependent claims, the use of "or" refers back to more than one such independent or dependent claim.

[0159] The term "about" as used herein refers to a normal error range for each value, which is readily apparent to those skilled in the art. Reference to a value or parameter with "about" herein includes (and describes) aspects directed to the value or parameter itself. For example, a description that refers to "about X" includes the description of "X".

[0160] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" may be used interchangeably and refer to a polymer of nucleotides. Such a polymer of nucleotides may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0161] As used herein, the term "isolated polynucleotide" is intended to mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, that, by reason of its origin, (1) is not associated with all or a portion of polynucleotides found in nature, (2) is operably linked to polynucleotides to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[0162] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to proteins containing modifications, such as deletions, additions, and substitutions (generally conservative in nature), relative to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or accidental, such as through mutations of the host producing the protein or errors due to PCR amplification.

[0163] The term "isolated protein" as referred to herein means that the subject protein (1) is free from at least some other proteins with which it is typically found in nature, (2) is essentially free from other proteins from the same source, e.g., from the same species, (3) is expressed by cells from a different species, (4) is separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) the "isolated protein" is not associated (by covalent or non-covalent interactions) with portions of proteins with which it is naturally associated, (6) is functionally associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated, or (7) is not naturally occurring. Such isolated proteins can be encoded by genomic DNA, cDNA, mRNA, or other RNA, can be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially pure or substantially free from proteins, polypeptides, or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or other).

[0164] As used herein, "substantially pure" means that the species of interest is the predominant species present (i.e., on a molar basis, it is more abundant than any other individual species in the composition), and a substantially purified fraction is a composition in which the species of interest constitutes at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will constitute greater than about 80% of all macromolecular species present in the composition, e.g., in some embodiments, greater than about 85%, 90%, 95%, and 99%. In some embodiments, the species of interest is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods), such that the composition consists essentially of a single macromolecular species.

[0165] As used herein, the term "operably linked" refers to the positioning of the components so described, in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0166] The term "specifically binds" to an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with higher affinity than with alternative cells or substances. A single domain antibody (sdAb) or VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to the CD28 epitope is an sdAb or VHH-containing polypeptide that binds to this epitope with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other CD28 epitopes or non-CD28 epitopes. For example, it is understood by reading this definition that an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding refers to preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0167] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an sdAb or VHH-containing polypeptide) binds. Epitopes often comprise chemically active surface arrangements of molecules, such as amino acids, polypeptides, or sugar side chains, and possess specific three-dimensional structural and charge characteristics. Epitopes can be formed from both contiguous and / or juxtaposed noncontiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3, at least 5, or 8-10 residues (e.g., amino acids or nucleotides). In some embodiments, an epitope is less than 20 residues (e.g., amino acids or nucleotides) in length, less than 15 residues, or less than 12 residues. Two antibodies may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope can be identified by a certain minimum distance to the CDR residues on the antigen-binding molecule. In some embodiments, an epitope can be identified by the above distance and further limited to those residues involved in binding (e.g., hydrogen bonding) between the antigen-binding molecule residues and the antigen residues. Epitopes can also be identified by various scans; for example, an alanine scan or an arginine scan can reveal one or more residues with which the antigen-binding molecule can interact. Unless explicitly stated, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antigen-binding molecule. Rather, the existence of such a set indicates a minimum series (or species set) of epitopes. Thus, in some embodiments, the set of residues identified as an epitope represents the minimal epitope associated with the antigen, rather than an exclusive list of residues for the epitope on the antigen.

[0168] A "nonlinear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids, and / or sugars within an antigen protein to which an epitope-specific antigen-binding molecule binds. In some embodiments, at least one of the residues will be non-contiguous with other referenced residues of the epitope; however, one or more of the residues may also be contiguous with other residues.

[0169] A "linear" epitope comprises adjacent polypeptides, amino acids, and / or sugars in an antigen protein to which an epitope-specific antigen-binding molecule binds. It is noted that in some embodiments, not all of the residues in a linear epitope need to directly bind (or participate in binding) to an antigen-binding molecule. In some embodiments, a linear epitope can be derived from immunization with a peptide that effectively consists of the sequence of the linear epitope, or from a structural segment of the protein that has exactly that sequence segment and is relatively isolated from the rest of the protein (so that the antigen-binding molecule can at least primarily interact).

[0170] The terms "antibody" and "antigen-binding molecule" are used interchangeably in the broadest sense and encompass a variety of polypeptides containing antibody-like antigen-binding domains, including, but not limited to, conventional antibodies (typically comprising at least one heavy chain and at least one light chain), single-domain antibodies (sdAbs, typically comprising only one chain similar to a heavy chain), VHH-containing polypeptides (polypeptides comprising at least one heavy-chain-only antibody variable domain, i.e., VHH), and fragments of any of the foregoing, so long as they exhibit the desired antigen-binding activity. In some embodiments, an antibody comprises a dimerization domain. Such dimerization domains include, but are not limited to, a heavy-chain constant domain (comprising a CH1, hinge, CH2, and CH3; CH1 typically pairs with a light-chain constant domain, CL, while the hinge mediates dimerization), and an Fc domain (comprising a hinge, CH2, and CH3; the hinge mediates dimerization).

[0171] The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as camelids (including llamas), sharks, mice, humans, cynomolgus monkeys, etc.

[0172] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of a native antibody (V H and V L ) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V H or V L A single domain may be sufficient to confer antigen-binding specificity to a single domain antibody, such as a VHH. Furthermore, antibodies that bind to a particular antigen can be identified by combining VHHs derived from antibodies that bind to the antigen. H or V Ldomains to form complementary V L or V H Libraries of domains may be screened and isolated (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0173] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than a conventional or intact antibody that contains a portion of a conventional or intact antibody that contains at least the variable region that binds to an antigen. Examples of antibody fragments include Fv, single-chain Fv (sdFv), Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; V H This includes, but is not limited to, single domain antibodies (VHH) which contain only the region.

[0174] As used herein, "monovalent" with respect to a binding molecule refers to a binding molecule that has a single antigen recognition site that is specific to a target antigen. Examples of monovalent binding molecules include, for example, monovalent antibody fragments, proteinaceous binding molecules with antibody-like binding properties, or MHC molecules. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single-chain Fv fragments (scFv).

[0175] As used herein, "monovalent" with respect to a binding molecule refers to a binding molecule that has multiple (more than one) antigen recognition sites that are specific for a target antigen.

[0176] The terms "single domain antibody," "sdAb," and "VHH" are used interchangeably herein to refer to an antibody having an antigen-binding / recognition domain that is a single monomer domain. Such antibodies include camelid antibodies or shark antibodies. In some embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N- or C-terminus to comprise only partial FR1 and / or FR4, or to lack one or both of these framework regions, so long as the VHH substantially maintains antigen binding and specificity.

[0177] The term "VHH-containing polypeptide" refers to a polypeptide comprising at least one VHH domain. In some embodiments, a VHH polypeptide comprises two, three, four, or more VHH domains, and each VHH domain may be the same or different. In some embodiments, a VHH-containing polypeptide comprises an Fc domain. In some such embodiments, a VHH polypeptide may form a dimer. Non-limiting structures of a VHH-containing polypeptide include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of such structures, one VHH may be connected to another VHH by a linker, or one VHH may be connected to an Fc by a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids composed primarily of glycine, and optionally serine. In some embodiments, when a VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, the structure VHH1-VHH2-Fc is considered tetravalent when it forms a dimer (i.e., the dimer has four VHH domains). Similarly, the structure VHH1-VHH2-VHH3-Fc is considered hexavalent when it forms a dimer (i.e., the dimer has six VHH domains).

[0178] As used herein, a CD28-binding polypeptide is a polypeptide or protein that specifically binds to CD28. Typically, the CD28-binding polypeptide herein is a VHH-containing polypeptide that contains at least one VHH domain that binds to CD28. The CD28-binding polypeptide includes a conjugate, including a fusion protein. The CD28-binding polypeptide includes a fusion protein, including a fusion protein that contains an Fc domain. In some embodiments, the CD28-binding polypeptide contains two, three, four, or more VHH domains, each of which specifically binds to CD28, and each VHH domain may be the same or different. In some embodiments, the CD28-binding polypeptide is multivalent. In some embodiments, the CD28-binding polypeptide is multispecific. In some cases, the CD28-binding polypeptide may contain one or more additional domains that bind to one or more additional or additional antigens other than CD28.

[0179] The term "monoclonal antibody" refers to an antibody (including an sdAb or VHH-containing polypeptide) from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a sample of monoclonal antibodies may bind to the same epitope on the antigen. The modifier "monoc...

Claims

1. A CD28-binding polypeptide comprising at least one VHH domain that binds to CD28, the at least one VHH domain comprises: a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 195, 198, 199, 200, and 201; a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 206, 196, 202, 204, 205, 207, 208, 209, 210, 211, and 212; and a complementarity determining region 3 (CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 191; or CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 192, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 193, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

194. Including, CD28-binding polypeptide.

2. At least one VHH domain comprises: CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 191; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 192, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 193, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 194; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 200, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 207, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 208, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 210, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 211, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; or CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197 2. The CD28-binding polypeptide of claim 1, comprising:

3. 3. A CD28-binding polypeptide according to claim 1 or 2, wherein at least one CD28 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

197.

4. A CD28-binding polypeptide according to claim 1 or 2, wherein at least one CD28 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

197.

5. A CD28-binding polypeptide according to claim 1 or 2, wherein at least one CD28 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 198, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

197.

6. A CD28-binding polypeptide according to claim 1 or 2, wherein at least one CD28 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 198, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

197.

7. 3. A CD28-binding polypeptide according to claim 1 or 2, wherein at least one CD28 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 199, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

197.

8. 3. A CD28-binding polypeptide according to claim 1 or 2, wherein at least one CD28 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 199, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

197.

9. 3. A CD28-binding polypeptide according to claim 1 or 2, wherein at least one CD28 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 201, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

197.

10. 3. A CD28-binding polypeptide according to claim 1 or 2, wherein at least one CD28 VHH domain comprises a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 201, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

197.

11. 1. A CD28 binding polypeptide comprising at least one VHH domain, the at least one VHH domain comprises: (a) CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 342; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 188; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 213; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 214; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 215; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 216; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 217; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 218; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 219; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 221; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 222; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 223; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 224; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 225; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 226; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 227; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 228; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 229; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 230; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 231; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 232; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 233; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 234; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 235; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 236; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 237; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 238; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 239; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 344; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 345; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 351; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 353; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 358; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 360; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 365; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 367; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 379; or CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 381; (b) CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 186; or (c) CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 187 A CD28-binding polypeptide comprising:

12. At least one VHH domain comprises: an amino acid sequence set forth in any of SEQ ID NOs: 342, 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 344, 345, 351, 353, 358, 360, 365, 367, 379, and 381; or A sequence of amino acids exhibiting at least 90% sequence identity to any of SEQ ID NOs: 342, 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 344, 345, 351, 353, 358, 360, 365, 367, 379, and 381.

12. The CD28-binding polypeptide of any one of claims 1 to 2 or 11, comprising:

13. At least one CD28 VHH domain is an amino acid sequence set forth in any of SEQ ID NOs: 342, 213-218, 223, 225, 228, 229, 234-239, 344, 345, 351, 353, 365, and 367; or A sequence of amino acids that exhibits at least 90% sequence identity to any of SEQ ID NOs: 342, 213-218, 223, 225, 228, 229, 234-239, 344, 345, 351, 353, 365, and 367. Including, 13. A CD28-binding polypeptide according to any one of claims 1, 2, 11, or 12.

14. 14. The CD28-binding polypeptide of any one of claims 1 to 13, wherein the CD28-binding polypeptide is monovalent with respect to CD28.

15. 14. The CD28-binding polypeptide of any one of claims 1 to 13, wherein the CD28-binding polypeptide is multivalent with respect to CD28.

16. 14. The CD28-binding polypeptide of any one of claims 1 to 13, wherein the CD28-binding polypeptide is bivalent with respect to CD28.

17. 17. The CD28-binding polypeptide of claim 16, comprising two identical VHH domains, each of the two VHH domains comprising a sequence of amino acids that exhibits at least 90% sequence identity to any of SEQ ID NOs: 342, 213-219, 221-239, 344, 345, 351, 353, 358, 360, 365, 367, 379, and 381.

18. 17. The CD28-binding polypeptide of claim 16, comprising two different VHH domains, each of the two VHH domains comprising an amino acid sequence that exhibits at least 90% sequence identity to any of SEQ ID NOs: 342, 213-219, 221-239, 344, 345, 351, 353, 358, 360, 365, 367, 379, and 381.

19. the CD28-binding polypeptide comprises a moiety that binds to Protein A; and / or the CD28-binding polypeptide comprises an amino acid modification that reduces binding to Protein A; or the CD28-binding polypeptide comprises an amino acid modification that reduces binding to Protein A, the amino acid modification being G65D according to Kabat in framework region 3 (FR3); A CD28-binding polypeptide according to any one of claims 1 to 18.

20. 20. An anti-CD28 sdAb-Fc fusion protein comprising the CD28-binding polypeptide of any one of claims 1 to 19 and an immunoglobulin Fc region.

21. The Fc region one or more amino acid modifications that reduce effector function and / or reduce binding to an effector molecule selected from an Fc gamma receptor and C1q a polypeptide comprising 21. The anti-CD28 sdAb-Fc fusion protein of claim 20, comprising:

22. 22. A homodimeric Fc fusion protein comprising two identical copies of the anti-CD28 sdAb-Fc fusion protein of claim 20 or 21.

23. A heterodimeric fusion protein comprising two anti-CD28 sdAb-Fc fusion proteins according to claim 20 or 21.

24. 22. An anti-CD28 sdAb-Fc fusion protein according to claim 20 or 21, comprising one or more binding domains (BDs) that bind to a target antigen other than CD28, one or more BDs bind to a tumor-associated antigen (TAA), or one or more BDs bind to a T cell surface marker that is a marker of T cell activation or T cell exhaustion; or One or more BDs bind to cell surface markers that are tumor microenvironment markers; Multispecific binding polypeptides.

25. one or more BDs or each one or more BDs is a VHH domain; and / or the multispecific binding polypeptide is an Fc fusion protein, wherein at least one of the one or more BDs and / or at least one CD28 VHH domain is linked to an immunoglobulin Fc region; 25. The multispecific binding polypeptide of claim 24.

26. 26. The multispecific binding polypeptide of claim 24 or 25, which is a dimer.

27. Antigens that BD binds include 1-92-LFA-3, 2B4, 5T4, alpha-4 integrin, alpha-V integrin, alpha4beta1 integrin, alpha4beta7 integrin, alpha-SMA, AGR2, anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD51, CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD123, CD125 , CD132, CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), C LAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FR alpha), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIIb / IIIa receptor, Gp130, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFN alpha, IFN beta, IFN gamma, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL- 12R, IL-12Rbeta1, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R (wsx1), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFR alpha, PDGFR beta, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4, Siglec15, sphingosine 1 phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGF beta, TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR 9, TMEM31, TNFalpha, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

28. 28. A homodimeric multispecific Fc-fusion protein comprising two identical copies of the multispecific binding polypeptide of any one of claims 24 to 27.

29. A heterodimeric multispecific Fc-fusion protein comprising two multispecific binding polypeptides according to any one of claims 24 to 27.

30. (a) a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 195, 198, 199, 200, and 201, a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 206, 196, 202, 204, 205, 207, 208, 209, 210, 211, and 212, and a complementarity determining region 3 (CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 197; (b) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 191; or (c) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 192, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 193, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

194. Including, Anti-CD28 VHH domain.

31. CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 191; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 192, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 193, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 194; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 200, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 205, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 207, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 208, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 210, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 211, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 202, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197; or CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 197 31. The anti-CD28 VHH domain of claim 30, comprising:

32. An anti-CD28 VHH domain according to claim 30 or 31, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

197.

33. An anti-CD28 VHH domain according to claim 30 or 31, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 195, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

197.

34. An anti-CD28 VHH domain according to claim 30 or 31, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

197.

35. An anti-CD28 VHH domain according to claim 30 or 31, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

197.

36. An anti-CD28 VHH domain according to claim 30 or 31, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

197.

37. An anti-CD28 VHH domain according to claim 30 or 31, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

197.

38. An anti-CD28 VHH domain according to claim 30 or 31, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 196, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

197.

39. An anti-CD28 VHH domain according to claim 30 or 31, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 201, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

197.

40. (a) CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 342; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 188; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 213; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 214; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 215; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 216; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 217; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 218; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 219; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 221; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 222; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 223; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 224; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 225; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 226; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 227; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 228; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 229; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 230; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 231; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 232; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 233; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 234; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 235; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 236; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 237; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 238; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 239; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 344; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 345; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 351; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 353; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 358; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 360; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 365; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 367; CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 379; or CDR1, CDR2, and CDR3 comprised in the amino acid sequence set forth in SEQ ID NO: 381; (b) CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 186; or (c) CDR1, CDR2, and CDR3 contained in the amino acid sequence set forth in SEQ ID NO: 187 an anti-CD28 VHH domain comprising:

41. an amino acid sequence set forth in any of SEQ ID NOs: 342, 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 344, 345, 351, 353, 358, 360, 365, 367, 379, and 381; or A sequence of amino acids exhibiting at least 90% sequence identity to any of SEQ ID NOs: 342, 186, 187, 188, 213, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 344, 345, 351, 353, 358, 360, 365, 367, 379, and 381.

41. The anti-CD28 VHH domain of any one of claims 30, 31, or 40, comprising:

42. below: SEQ ID NOs: 195, 206, and 197, respectively; SEQ ID NOs: 195, 196, and 197, respectively; SEQ ID NOs: 198, 196, and 197, respectively; SEQ ID NOs: 198, 206, and 197, respectively; SEQ ID NOs: 199, 196, and 197, respectively; SEQ ID NOs: 199, 206, and 197, respectively; SEQ ID NOs: 200, 196, and 197, respectively; SEQ ID NOs: 201, 196, and 197, respectively; SEQ ID NOs: 195, 202, and 197, respectively; SEQ ID NOs: 195, 204, and 197, respectively; SEQ ID NOs: 195, 205, and 197, respectively; SEQ ID NOs: 195, 207, and 197, respectively; SEQ ID NOs: 195, 208, and 197, respectively; SEQ ID NOs: 195, 209, and 197, respectively; SEQ ID NOs: 195, 210, and 197, respectively; SEQ ID NOs: 195, 211, and 197, respectively; SEQ ID NOs: 195, 212, and 197, respectively; SEQ ID NOs: 201, 202, and 197, respectively; SEQ ID NOs: 201, 206, and 197, respectively; or SEQ ID NO: 201, 204, and 197, respectively CDR1, CDR2, and CDR3 shown in 42. The anti-CD28 VHH domain of any one of claims 30, 31, 40, or 41, comprising:

43. an amino acid sequence set forth in any of SEQ ID NOs: 342, 213-219, 221-239, 344, 345, 351, 353, 358, 360, 365, 367, 379, and 381; or A sequence of amino acids that exhibits at least 90% sequence identity to any of SEQ ID NOs: 342, 213-219, 221-239, 344, 345, 351, 353, 358, 360, 365, 367, 379, and 381.

43. The anti-CD28 VHH domain of any one of claims 30, 31 or 40-42, comprising:

44. the anti-CD28 VHH domain comprises an amino acid modification that reduces binding to Protein A, or the anti-CD28 VHH domain comprises an amino acid modification that reduces binding to Protein A, the amino acid modification being G65D according to Kabat in framework region 3 (FR3); 44. An anti-CD28 VHH domain according to any one of claims 30 to 43.

45. comprising at least one anti-CD28 VHH domain according to any one of claims 30 to 44, one or more binding domains (BDs) that bind to an antigen other than CD28, and a CD3 binding region; or 45. A method for the treatment of a leukemia comprising administering to a patient a therapeutic agent comprising administering to said patient at least one anti-CD28 VHH domain according to any one of claims 30 to 44, one or more binding domains (BDs) that bind to an antigen other than CD28, and a CD3 binding region, wherein said BDs bind to a TAA and / or said BDs are VHH domains. Multispecific constructs.

46. (i) the CD3 binding region binds to CD3 (CD3ε); (ii) the CD3 binding region is an anti-CD3 antibody or antigen-binding fragment; (iii) the CD3 binding region is monovalent; and / or (iv) the CD3 binding region is a variable fragment (Fv) comprising a variable heavy chain region (VH) and a variable light chain region (VL); 46. ​​The multispecific construct of claim 45.

47. 47. The multispecific construct of claim 45 or 46, wherein the CD28 binding polypeptide comprises an immunoglobulin Fc region.

48. the Fc is a heterodimeric Fc; VH and VL comprising an anti-CD3 antibody or antigen-binding fragment are linked to opposing polypeptides of the heterodimeric Fc; 48. The multispecific construct of claim 47.

49. A CD28-binding polypeptide according to any one of claims 1 to 19; a fusion protein according to any one of claims 20 to 23, 28 or 29. a multispecific binding polypeptide according to any one of claims 24 to 27; a multispecific construct according to any one of claims 45 to 48; or an anti-CD28 VHH domain according to any one of claims 30 to 44 A polynucleotide encoding

50. 50. A vector comprising the polynucleotide of claim 49, the vector is an expression vector, and / or a viral or eukaryotic vector, or a mammalian vector, vector.

51. 51. A cell comprising one or more polynucleotides of claim 49 or a vector of claim 50.

52. 1. A method for producing a polypeptide, comprising: introducing one or more polynucleotides of claim 49 or a vector of claim 50 into a cell; and Culturing the cells under conditions to produce the polypeptide. Including, method.

53. 53. The method of claim 52, further comprising isolating or purifying said polypeptide from said cells.

54. 54. A polypeptide produced by the method of claim 52 or 53.

55. A CD28-binding polypeptide according to any one of claims 1 to 19; a fusion protein according to any one of claims 20 to 23, 28 or 29. a multispecific binding polypeptide according to any one of claims 24 to 27; a multispecific construct according to any one of claims 45 to 48; or an anti-CD28 VHH domain according to any one of claims 30 to 44 Including, Pharmaceutical compositions.

56. 56. The pharmaceutical composition of claim 55, further comprising a pharmaceutically acceptable carrier.

57. for treating a disease or condition in a subject, A CD28-binding polypeptide according to any one of claims 1 to 19; a fusion protein according to any one of claims 20 to 23, 28 or 29. a multispecific binding polypeptide according to any one of claims 24 to 27; an anti-CD28 VHH domain according to any one of claims 30 to 44; a multispecific construct according to any one of claims 45 to 48; or a pharmaceutical composition according to claim 55 or 56. A composition comprising:

58. 58. The composition of claim 57, wherein the disease or condition is a tumor or cancer.

59. For the manufacture of a medicament for treating a disease or condition in a subject, A CD28-binding polypeptide according to any one of claims 1 to 19; a fusion protein according to any one of claims 20 to 23, 28 or 29. a multispecific binding polypeptide according to any one of claims 24 to 27; an anti-CD28 VHH domain according to any one of claims 30 to 44; a multispecific construct according to any one of claims 45 to 48; or a pharmaceutical composition according to claim 55 or 56. Use of a composition comprising:

60. 60. The use of claim 59, wherein the disease or condition is a tumor or cancer.

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