Background Chimeric antigen receptor (CAR) T-cell therapy in solid tumors has been largely disappointing. A key obstacle is the pre-existing antigenic heterogeneity, where not all tumor cells express the antigen targeted by the CAR. Antigen spreading (AS) is the induction and amplification of immune responses to secondary antigens distinct from the original therapeutic target. AS can play an important role in the expansion and broadening of the anti-tumor peripheral T cell repertoire. We recently developed a synthetic vaccine (amph-vax) to enhance CAR T cell activity against solid tumors through direct modulation of CAR T cells and engage host immunity via AS^{1 2} (figure 1A).

Methods The amph-vax was generated by linking a CAR-ligand (i.e., pepvIII in this proposal) to albumin-binding poly (ethylene glycol)-phospholipid and then formulated with cyclic-di-GMP (STING agonist). We used a CT-2A murine glioblastoma model expressing a tumor-specific surface antigen EGFRvIII.

Results Amph-vax boosted CAR T (CAR T-vax) therapy but not CAR T therapy alone, elicited pronounced antigen spreading (AS) in both CD4+ and CD8+ T-cell compartments (figure 1B). Single-cell RNA-seq of 21,835 tumor infiltrating-endogenous T cells confirmed a significant increase of cytotoxic CD8 T cells and induction of Th1 CD4 T cells. Transcriptomic analysis demonstrated that vaccine boosting significantly enhanced CAR T cell metabolism, including oxidative phosphorylation (OXPHOS). AS was reduced by ~50% using PGC-1α-deficient CAR T-vax therapy. IFNγ blockade abolished AS(figure 1C,D). Blockade of IL12 signaling using anti-IL12 (p75) antibody or IL12rb2 knockout mice phenocopied IFNγ blockade, resulting in negligible AS (figure 1E). We further observed a significant reduction of AS in BatF3-deficient mice or mice with deficient IFNγ signaling in CD11c+ DCs (figure 1F). Finally, we showed that IFNGR1- or IL12rb2-deficient CAR T-vax therapy failed to induce AS. Using a heterogeneous tumor model with EGFRvIII+ and EGFRvIII- CT-2A cells mixed at pre-defined ratios, we found that CAR-T vax therapy cured ~50% animals bearing tumors with up to 20% EGFRvIII^- cells, and further elevating CAR T-intrinsic IFNγ expression increased the cure rate to ~80% (figure 1G). And the therapeutic response was completely lost in Rag1-/- mice which have endogenous T cells and therefore no antigen spreading (figure 1G).

Conclusions CAR T-derived IFN-γ were propagated via a positive feedback regulation through crosstalk with DC-derived IL-12 to initiate AS (figure 1H). This finding provides guidance for engineering CAR T therapies to more effectively treat solid tumors with pre-existing antigenic heterogeneity.

References

1. L Ma, T Dichwalkar, JYH Chang, B Cossette, D Garafola, AQ Zhang, M Fichter, C Wang, S Liang, M Silva, S Kumari, NK Mehta, W Abraham, N Thai, N Li, KD Wittrup, DJ Irvine, Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor,Science 2019;365:162–168.

2. L Ma^#, A Hostetler, D Morgan, L Maiorino, I Sulkaj, CA Whittaker, A Neeser, I Pires, P Yousefpour, J Gregory, K Qureshi, J Dye, W Abraham, H Suh, N Li, C Love, DJ Irvine^#. Vaccine-boosted CAR T crosstalk with host immunity to reject tumors with antigen heterogeneity. Cell. 2023 [In press, ^#co-corresponding author]

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Abstract 286 Figure 1

Vaccine boosting of CAR T therapy and antigen spreading. (A) Illustration of amphiphile vaccine design and vaccine boosting of CAR T cells. (B) CAR T-vax therapy induced antigen spreading (AS) to endogenous CD8 and CD4 T cell compartments, AS was monitored using IFNy ELISPOT by co-culturing splenic T cells with WT CT-2A cells. (C-D) Impact of TNFα or IFNy blockade or (E) IL12 (p75) blockade on CAR T-vax induced AS. Arrows indicate administration of neutralizing antibody. (F) Contribution of integral IFNy signaling of CDI lc+ DCs to CAR T-vax induced AS. (G) Growth of heterogeneous tumors (20% EGF-RvIII- CT2A + 80% EGF-RvIII+ CT-2A) in WT mice receiving CAR T, CAR T-vax or NFAT-IFNy CAR T-vax therapy (higher IFNy expression upon activation) or Rag1 -/- mice receiving CAR T-vax therapy. (H) A five-step model detailing how vaccine boosting of CAR T therapy promotes antigen spreading. Error bars show mean ± 95% Cl. **, p<001; *, p<0.05; n.s., not significant by one-way ANOVA with Tukey’s post-test.