Chimeric antigen receptor (CAR) T cell therapy represents a significant advancement in cancer therapy. Larger studies have shown ∼90% complete remission rates against chemoresistant and/or refractory CD19+ leukemia or lymphoma. Effective CAR T cell therapy is highly dependent on lymphodepleting preconditioning, which is achieved through chemotherapy or radiotherapy that carries with it significant toxicities. These can exclude patients of low performance status. In order to overcome the need for preconditioning, we constructed fully mouse first and second generation anti-murine CD19 CARs with or without interleukin-12 (IL-12) secretion. To test these CARs, we established a mouse model to reflect the human situation without preconditioning. Murine second generation CAR T cells expressing IL-12 were capable of eradicating established B cell lymphoma with a long-term survival rate of ∼25%. We believe this to be the first study in a truly lymphoreplete model. We provide evidence that IL-12-expressing CAR T cells not only directly kill target CD19+ cells, but also recruit host immune cells to an anti-cancer immune response. This finding is critical because lymphodepletion regimens required for the success of current CAR T cell technology eliminate host immune cells whose anti-cancer activity could otherwise be harnessed by strategies such as IL-12-secreting CAR T cells.

Chimeric antigen receptor (CAR) T cells form part of a broad wave of immunotherapies that are showing promise in early phase cancer clinical trials. This clinical delivery has been based upon preclinical efficacy testing that confirmed the proof of principle of the therapy. However, CAR T-cell therapy does not exist alone as T cells are generally given in combination with patient preconditioning, most commonly in the form of chemotherapy, and may also include systemic cytokine support, both of which are associated with toxicity. Consequently, complete CAR T-cell therapy includes elements where the toxicity profile is well known, but also includes the CAR T cell itself, for which toxicity profiles are largely unknown. With recent reports of adverse events associated with CAR T-cell therapy, there is now concern that current preclinical models may not be fit for purpose with respect to CAR T-cell toxicity profiling. Here, we explore the preclinical models used to validate CAR T-cell function and examine their potential to predict CAR T-cell driven toxicities for the future.

Enadenotucirev (EnAd) is a chimeric group B adenovirus isolated by bioselection from a library of adenovirus serotypes. It replicates selectively in and kills a diverse range of carcinoma cells, shows effective anticancer activity in preclinical systems, and is currently undergoing phase I/II clinical trials. EnAd kills cells more quickly than type 5 adenovirus, and speed of cytotoxicity is dose dependent. The EnAd death pathway does not involve p53, is predominantly caspase independent, and appears to involve a rapid fall in cellular ATP. Infected cells show early loss of membrane integrity; increased exposure of calreticulin; extracellular release of ATP, HSP70, and HMGB1; and influx of calcium. The virus also causes an obvious single membrane blister reminiscent of ischemic cell death by oncosis. In human tumor biopsies maintained in ex vivo culture, EnAd mediated release of pro-inflammatory mediators such as TNF-α, IL-6, and HMGB1. In accordance with this, EnAd-infected tumor cells showed potent stimulation of dendritic cells and CD4+ T cells in a mixed tumor-leukocyte reaction in vitro. Whereas many viruses have evolved for efficient propagation with minimal inflammation, bioselection of EnAd for rapid killing has yielded a virus with a short life cycle that combines potent cytotoxicity with a proinflammatory mechanism of cell death.

Small molecule Toll-like receptor-7 and -8 agonists (TLR-7/8a) can be used as vaccine adjuvants to induce CD8 T cell immunity but require formulations that prevent systemic toxicity and focus adjuvant activity in lymphoid tissues. Here, we covalently attached TLR-7/8a to polymers of varying composition, chain architecture and hydrodynamic behavior (∼300 nm submicrometer particles, ∼10 nm micelles and ∼4 nm flexible random coils) and evaluated how these parameters of polymer-TLR-7/8a conjugates impact adjuvant activity in vivo. Attachment of TLR-7/8a to any of the polymer compositions resulted in a nearly 10-fold reduction in systemic cytokines (toxicity). Moreover, both lymph node cytokine production and the magnitude of CD8 T cells induced against protein antigen increased with increasing polymer-TLR-7/8a hydrodynamic radius, with the submicrometer particle inducing the highest magnitude responses. Notably, CD8 T cell responses induced by polymer-TLR-7/8a were dependent on CCR2+ monocytes and IL-12, whereas responses by a small molecule TLR-7/8a that unexpectedly persisted in vaccine-site draining lymph nodes (T1/2 = 15 h) had less dependence on monocytes and IL-12 but required Type I IFNs. This study shows how modular properties of synthetic adjuvants can be chemically programmed to alter immunity in vivo through distinct immunological mechanisms.