Preview

PDF, English Download (9MB) | Terms of use

Abstract

Bispecific agents are a promising approach for cancer immunotherapy as they enable the redirection of cytotoxic lymphocytes towards tumor cells by targeting different structures on the tumor cell surface and triggering cytotoxic lymphocytes through agonistic binding to activating receptors. Targeted tumor antigens can be roughly divided into peptide antigens presented by major histocompatibility complex (MHC) class I molecules to antigen-specific T cell receptors and cell surface antigens recognized by antibodies. Peptides presented in the context of an MHC-I molecule mostly result from intracellular proteins, which make up the majority of the proteome, and thus serve as an highly attractive target for immunotherapeutic strategies. In contrast to TCR-transgenic T cells that express novel TCR specificities in the natural cellular context, the development of soluble TCR-based therapies is hampered due to the poor stability of recombinant TCRs and a generally significantly lower affinity in comparison to therapeutic antibodies. In the past years progress has been made to address these problems, which include different modifications improving construct stability and methods for TCR affinity maturation enabling the development and clinical application of first TCR-based bispecific recombinant fusion proteins that retarget T cells to tumors displaying intracellular antigens through MHC molecules. This study aimed to develop novel soluble bispecific TCR-based agents for the redirection of NK and T cells. To achieve potent bispecific mediators a bivalent immunoglobulin G (IgG)-like TCR-Fc fusion format was adapted. The ectodomain of the TCR V alpha/C beta chain was fused to the hinge/Fc part of human IgG1 and the ectodomain of the TCR V alpha/C beta chain was expressed as a second soluble protein in cis using a ribosomal skipping sequence. Efficient assembly of TCR alpha and beta chains was facilitated by an additional artificial intermolecular disulfide bridge in the TCR constant domains. The natural intermolecular disulfide bonds of the human IgG1 hinge region enabled assembly of TCR-Fc fusion proteins to stable homodimers which could be successfully expressed by transient transfection of CHO-S producer cells. To enable NK cell redirection, specific mutations known to enhance Fc gamma RIIIa binding were introduced in the Fc fragment. Other investigated formats made use of single chain variable fragments (scFv) recognizing CD16 or NKp46 for NK cell redirection or binding CD3 epsilon for the redirection and activation of T cells. NK- and T-cell-binding scFv antibodies were analyzed after insertion at C-terminal end of TCR C alpha or C beta, respectively. A cytomegalovirus (CMV) pp65 peptide/HLA-A*02:01-specific TCR sequence was used as a model system. To analyze the influence of TCR affinity, the wildtype TCR was compared to an affinity-maturated variant. The soluble IgG1-like TCRs showed a target-specific, affinity- and concentration-dependent binding as well as NK and T cell redirection and activation upon co-culture with peptide-pulsed or transfected cells. In particular, the TCRalpha-alphaNKp46-Fc enhanced and TCRalpha-alphaCD3epsilon-Fc aglycan constructs were found to be highly potent in redirecting NK and T cells, respectively. TCR-scFv-Ig fusion proteins efficiently elicited peptide antigen-specific activation of purified NK cells and T cells and induced cytotoxicity against different tumor targets. These results were further confirmed using low- and and high-affinity variants of three TCRs recognizing HLA-A*02:01-restricted gp100, MART-1 and NY-ESO-1 peptides, respectively, for the redirection of NK cells. TCRhigh aff. -alphaNKp46-Fc enh constructs, however, failed to facilitate NK cytotoxicity against HLA-A2+ melanoma cell lines expressing the antigens of choice unless the cell lines were incubated with an excess of the cognate HLA-A2-binding peptide. Thus, the NK cell-engaging TCR-Fc fusion proteins constructs apparently were not sensitive enough to redirect and activate NK cells against melanoma cells presenting low quantities of naturally processed specific peptide/MHC-I complexes. In sum, in this work a panel of novel recombinant bispecific TCR-Fc and TCR-scFv-Fc fusion proteins were genetically engineered, produced and demonstrated to facilitate the activation and cytotoxicity of NK and T cells towards tumor cells expressing a model peptide-MHC-I complex in sufficient quantities. Further investigations are required to investigate if the avidity of soluble TCR-Fc constructs can be sufficiently increased by multimerization approaches in order to overcome the sensitivity issues resulting from low abundance of peptide-MHC-I complexes and low-affinity TCRs.