Cancer Immunotherapy: The 2018 Dr. Paul Janssen Award Symposium

The multiple, non-redundant mechanisms that limit T cell responses offer novel strategies for mobilizing the immune system against cancer. CTLA-4, the best characterized immune checkpoint, inhibits T cell proliferation by blocking the interaction of the costimulatory molecule CD28 with its ligands B7-1 and B7-2 on antigen presenting cells. CTLA-4 antibodies have proven effective against multiple tumor types in pre-clinical and clinical studies. Ipilimumab, an antibody to human CTLA-4, was approved by the FDA in 2011 for late stage melanoma and provides long term survival benefit to ~20% of patients. PD-1, another checkpoint, seems to interfere with T cell antigen receptor mediated signaling. Its two ligands, PD-L1 and PD-L2, are normally expressed on dendritic cells, but many tumor cells express PD-L1. Antibodies to PD-1 and PD-L1 provided objective responses against several tumor types in clinical trials in about 25% of patients. In a phase II trial, combined anti-PD-1 and anti-CTLA-4 provided objective responses in ~50% of late stage melanoma patients. We used high parameter flow cytometry to identify the mostly non-overlapping cellular mechanisms of CTLA-4 and PD-1 blockade, which may partially explain the enhanced effect of their combination. T cell responses to tumor cells appear largely directed toward neoantigens arising from mutational events associated with carcinogenesis. While all tumors with antigens recognizable by the immune system should be targets for checkpoint blockade, tumors with lower mutational burdens (prostate, breast, and kidney cancer) present challenges. Strategies for their treatment will be discussed.

Cellular circuits are dynamic networks which involve many interacting proteins, responding to multiple stimuli before manifesting a functional phenotypic effect.  With rapid developments taking place in cellular immunotherapy, design of synthetic cellular circuit components is becoming more complex and novel techniques are required for assessing how a novel circuit functions, rather then simply if it functions.  Whilst functional analysis of engineered cells can provide some insight into the downstream effects of synthetic constructs such as chimeric antigen receptors (CARs), to date signalling analysis has predominantly relied on bulk techniques which average over a large number of cells and are inadequate for visualising network states.

High-dimensional single-cell technologies such as mass cytometry allow simultaneous analysis of dozens of protein epitopes in millions of individual cells, each providing information on co-occurrence of protein or phospho-proteins to form complex distributions which should not be collapsed to mean or median statistics.  Using techniques from transportation theory, we describe stimulus-induced changes in phospho-protein expression at single-cell resolution.  By exploiting the non-linearity of protein-protein relationships, we describe the strength of signals between proteins and how they change between natural and synthetic circuits under various stimulus conditions.

CARs redirect T-cell immunity against chosen antigens.  We sought to clarify signalling circuit states in CAR-T cells.  Using single-cell resolution data from over 8x10⁶ cells we explore the circuit changes induced by pre-existing and novel designs of chimeric antigen receptor expressed in abT and gdT cells.  CARs exert different influences on T-cell circuits to their native receptor counterparts, in particular through increased signal strength and tonic signalling which is associated with increased exhaustion marker expression.

We also demonstrate that CAR-gdT cells offer an opportunity to overcome on-target off-tumor toxicity which has hampered progress in CAR-abT cell design.  Removal of CD3z from the CAR abrogated reduced exhaustion marker expression to baseline and CD3z-free CARs still enhanced gdT cell cytotoxicity against tumor cells expressing their cognate antigen.  Excitingly, CD3z-free CAR-gdT cells demonstrated no toxicity against cognate-antigen expressing healthy cells.

Regulatory T (Treg) cells represent a specialized lineage of cells of the adaptive immune system, whose biological role is in preserving tissue functions. Treg cells mediate a unique means of negative regulation in-trans of inflammation and, thereby, contain the consequent tissue damage. T cell and interleukin-2 receptor signaling are required for differentiation of Treg cells in the thymus and - in combination with TGF-β signaling - for their extrathymic differentiation. These signals also promote generation of potently suppressive activated “effector” Treg cells in the secondary lymphoid organs and non-lymphoid tissues, which partake in a variety of biological processes ranging from tissue repair and maintenance to suppression of immune responses to “self” and infectious agents to control of metabolite levels and cancer progression. Common and distinct features of Treg cells in diverse physiological and pathological settings and their functions will be discussed.

Immune checkpoint therapies, including anti-CTLA-4, anti-PD-1 and anti-PD-L1, have led to significant clinical responses in cancer patients. To investigate immunologic changes and mechanistic pathways that are elicited by these therapies, we conducted pre-surgical and tissue-based clinical trials, which permit access to tumor tissues for laboratory studies. To compare and contrast data, we chose one tumor type that responds well to immune checkpoint therapy and one that does not. The first pre-surgical trial was conducted with anti-CTLA-4 (ipilimumab) in a cohort of patients with localized bladder cancer. The second pre-surgical trial was conducted with anti-CTLA-4 (ipilimumab) in patients with localized prostate cancer. In addition, we evaluated data from patients with melanoma who received immune checkpoint therapy. These data enabled us to identify the ICOS/ICOSL pathway as relevant for anti-tumor immune responses in the setting of anti-CTLA-4 therapy. We also identified resistance mechanisms including expression of other immune inhibitory pathways, such as VISTA, and loss of the IFN signaling pathway in tumor cells. These data will be discussed in greater details.