Chimeric antigen receptors (CARs) are bespoke fusion molecules that couple the binding of a tumor-associated cell surface target to the delivery of a tailored T-cell activating signal. CAR T-cells targeting CD19 have demonstrated unprecedented efficacy in the treatment of patients with refractory B-cell malignancy although solid tumors present several additional hurdles to the development of CAR T-cell immunotherapy. One such hurdle is identifying suitable target antigens to maximise tumor targeting and minimise toxicity to healthy tissue. A highly promising candidate is the epithelial-specific integrin, αvβ6. Here, we demonstrate that CAR T-cells targeting αvβ6 have potent anti-tumor activity across a wide range of solid malignancies in pre-clinical models.
The αvβ6 integrin is over-expressed in solid tumors derived from pancreas, head and neck, skin, uterine cervix, lung, colon, breast and fallopian tube/ovary and is generally associated with worsened prognosis. It exerts several pro-tumorigenic activities including activation of TGF-β1, epithelial to mesenchymal transition, cellular migration and matrix metalloproteinase activity. By contrast, αvβ6 is minimally expressed in normal tissue and expression is largely restricted to wound healing.
We have evaluated two peptide-targeting moieties to direct specificity of an αvβ6-specific CD28+CD3ζ-based second generation CAR (A20-28z). We show that a 20mer peptide derived from viral protein 1 of foot and mouth disease virus achieves highly effective targeting. This 20mer peptide (termed A20) contains two αvβ6-binding motifs (RGD and DLXXL) and binds with >1000-fold more specificity to this integrin than other family members such as αvβ3, αvβ5 and α5β1. To evaluate anti-tumor activity we compared A20-28z with two control CAR constructs. An αvβ6 non-binding peptide was generated in which the RGDL motif within A20FMDV2 was substituted with AAAA (termed C20) and a non-signaling CAR constructed whereby A20 was fused to a truncated CD28 endodomain (termed A20-Tr).
A20-28z T-cells destroy and undergo activation by a range of pancreatic, breast and ovarian tumor cell lines in-vitro. By contrast, cells that express low but detectable levels of this integrin are ignored. To expand CAR T-cells preferentially during ex-vivo culture, an IL-4-responsive fusion gene (4βα) was co-expressed which delivers a potent and selective mitogenic signal only to the genetically modified T-cells. In-vivo efficacy of αvβ6 re-targeted human CAR T-cells was demonstrated in SCID Beige mice bearing established Panc04.03 pancreatic and SKOV-3 ovarian tumor xenografts, that express high and intermediate levels of αvβ6 respectively. Ovarian tumor response was significant, but was ultimately limited by transient in-vivo CAR T-cell expansion followed by progressive loss thereafter. In contrast, pancreatic tumours with high αvβ6 expression demonstrated significant tumor regression in response to A20-28z T-cells and exhibited durable responses.
Although the CAR targeting moiety engages murine αvβ6, minimal toxicity was observed in these mouse models, which can fully recapitulate lethal cytokine release syndrome in response to human CAR T-cells. Taken together, these data provide strong support for the clinical evaluation of αvβ6 re-targeted CAR T-cell immunotherapy in solid tumors that express this integrin.
Citation Format: Lynsey May Whilding, Ana C. Parente-Pereira, Tomasz Zabinski, David M. Davies, Roseanna Petrovic, Shelia Violette, Sadaf Ghaem-Maghami, Sabari Vallath, John Marshall, John Maher. Chimeric antigen receptor T-cells targeting the αvβ6 integrin demonstrate potent antitumor activity in multiple solid tumors. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B129.