'Final common pathway' of human cancer immunotherapy: targeting random somatic mutations

Abstract

Effective clinical cancer immunotherapies, such as administration of the cytokine IL-2, adoptive cell transfer (ACT) and the recent success of blockade of the checkpoint modulators CTLA-4 and PD-1, have been developed without clear identification of the immunogenic targets expressed by human cancers in vivo. Immunotherapy of patients with cancer through the use of ACT with autologous lymphocytes has provided an opportunity to directly investigate the antigen recognition of lymphocytes that mediate cancer regression in humans. High-throughput immunological testing of such lymphocytes in combination with improvements in deep sequencing of the autologous cancer have provided new insight into the molecular characterization and incidence of anti-tumor lymphocytes present in patients with cancer. Here we highlight evidence suggesting that T cells that target tumor neoantigens arising from cancer mutations are the main mediators of many effective cancer immunotherapies in humans.

Figure 1

Identification of neoantigen-reactive T cells from patients with cancer. Next-generation sequencing (whole exome and whole transcriptome) is performed on tumor and matched normal cells to identify non-synonymous somatic mutations expressed by the cancer (left). Next, two approaches that do not rely on predictions of HLA–peptide binding can be used to investigate the reactivity of T cells to neoantigens encoded by the identified mutations. In the first approach (middle), minigenes encoding the mutation flanked by nucleotides encoding 12 amino acids from the wild-type gene can be synthesized in tandem to create TMG constructs, which are then cloned into an appropriate expression vector. Linking multiple minigenes in tandem allows a relatively large number of mutations to be evaluated at once. Plasmids encoding TMGs or TMG RNAs transcribed in vitro are then introduced into the appropriate antigen-presenting cells (APCs), such as autologous dendritic cells or B cells, through techniques such as electroporation or lipid-based transfection, to allow processing and presentation of the neoantigens in the context of the patient’s own HLA class I and II molecules. T cells derived from tumor (TILs) or from the blood (right) are then co-cultured with the antigen-presenting cells expressing the TMGs, and T cell reactivity is evaluated by immunological methods such as cytokine ELISPOT or ELISA or the analysis of T cell–activation molecules such as CD137 (4–1BB) or CD134 (OX40) by flow cytometry (far right). The second approach (bottom) is identical to the first approach, except that instead of genetic constructs encoding the mutations, long peptides containing the mutant amino acid flanked by 12 amino acids from the wild-type protein are synthesized and then pulsed onto antigen-presenting cells, which process and present the mutant peptides to T cells. Similar to the minigenes and TMG concept, in this approach, a variable number of individual long peptides can be combined to generate peptide pools, which increases the throughput of neoantigen screening.