Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor

Abstract

Chimeric antigen receptor-T cell (CAR-T) therapy has been effective in the treatment of hematologic malignancies, but it has shown limited efficacy against solid tumors. Here we demonstrate an approach to enhancing CAR-T function in solid tumors by directly vaccine-boosting donor cells through their chimeric receptor in vivo. We designed amphiphile CAR-T ligands (amph-ligands) that, upon injection, trafficked to lymph nodes and decorated the surfaces of antigen-presenting cells, thereby priming CAR-Ts in the native lymph node microenvironment. Amph-ligand boosting triggered massive CAR-T expansion, increased donor cell polyfunctionality, and enhanced antitumor efficacy in multiple immunocompetent mouse tumor models. We demonstrate two approaches to generalizing this strategy to any chimeric antigen receptor, enabling this simple non-human leukocyte antigen-restricted approach to enhanced CAR-T functionality to be applied to existing CAR-T designs.

Fig. 1.. Design of an amphiphile-ligand vaccine to boost CAR-T cells.

(A) Schematic of the general chemical structure of amph-ligands (top), and the steps in amph-ligand vaccine boosting in vivo (bottom): Upon injection, amph-ligands associate with albumin at the injection site and are subsequently trafficked to the draining lymph node. The amphiphiles then transfer to the membrane of lymph node-resident cells, including antigen presenting cells (APCs). CAR-T cells that encounter decorated APCs in the LN are stimulated by the surface-displayed amph-ligand as well as costimulatory receptors and cytokines produced by the APC. (B) Structures of amph-FITC and cognate FITC-CAR, and representative flow cytometry analysis of T cell surface expression for FITC-CAR. (C, D) Flow cytometry analysis at 24 hr (C) and confocal imaging after 30 min (D) of amph-FITC insertion into DC2.4 cell membranes, by direct FITC fluorescence or staining with an anti-FITC antibody. (E-F) IFN-γ secretion (pg/ml) (E) and killing (% of target cell death) (F) of amph-FITC-coated DC2.4 cells after 6 hr co-culture with FITC-CAR-T or control untransduced T-cells at a 10:1 E:T ratio. Shown in E and F is a representative experiment with technical triplicates. P-values were determined by unpaired student’s t-test. Error bars represent 95% CI; ***p<0.0001, **p<0.01, *p<0.05.

Fig. 2.. Amph-ligands accumulate on lymph node APCs and prime CAR-T cells in vivo.

(A-E) C57BL/6 mice (n=3 animals/group for A-C, and n=5 animals/group for D-E) were immunized s.c. with amph-FITC and cyclic di-GMP adjuvant (A-C, E) or amph-FITC alone (D, E). Shown are histological images of LNs (A-B); confocal imaging of sorted amph-FITC-coated CD11c⁺ cells isolated from LNs at 24 hours (C); and flow cytometry analysis of the cellular biodistribution of amph-FITC 1 or 3 days after injection (D-E). (F-H) CD45.2⁺ C57BL/6 mice (n=7 animals/group) with (F, G) or without (H) prior lymphodepletion (LD) were adoptively transferred with CD45.1⁺ FITC-CAR-T cells followed by amph-FITC vaccination. Shown are frequencies of peripheral blood CAR-T cells (F, H) and cellular phenotypes at day 30 (G). P-values were determined by unpaired student’s t-test for E and G, and by RM (repeated measures) two-way ANOVA with Tukey’s multiple comparisons test for F and H. Error bars represent 95% CI; ***p<0.001, **p<0.01, *p<0.05. n.s, not significant.

Fig. 3.. Amph-peptide ligands boost CAR-T cells in vivo for enhanced solid tumor immunotherapy in mice.

(A) Structure of amph-pepVIII and surface expression of EGFRvIII CAR. (B) Representative histogram showing EGFRvIII-CAR-T proliferation in LNs 48 hours after amph-pepVIII vaccination (n=3 animals/group). (C-D) Expansion (C) and cytokine polyfunctionality at day 7 (D) of circulating EGFRvIII-CAR-T cells following a single amph-pepVIII immunization (n=5 animals/group). (E) Enumeration, granzyme B levels, and Ki67 levels of tumor-infiltrating EGFRvIII-CAR-T cells (n=4 animals/group) with or without amph-pepVIII boost. (F-J) Tumor growth (F, I), ELISPOT of enriched CD3+ splenocytes cultured with irradiated parental CT-2A tumor cells (H), and survival (G, J) of mEGFRvIII-CT-2A tumor-bearing mice treated with EGFRvIII-CAR-T with or without amph-pepvIII vaccination for animals that were lympho-depleted (F-G, n=5 animals/group, H, n=4 animals/group) or lympho-replete (I-J, n=7 animals/group) prior to adoptive transfer. Black arrow indicates time of CT-2A-EGFRvIII tumor re-challenge. Red arrow indicates time of parental CT-2A tumor re-challenge. P-values were determined by unpaired student’s t-test for D, E and H, by RM (repeated measures) two-way ANOVA with Tukey’s multiple comparisons test for C, F and I, and by log-rank test for G and J. Error bars represent 95% CI; ***p<0.001, **p<0.01, *p<0.05. n.s, not significant.

Fig. 4.. Amph-FITC ligands boost the anti-tumor activity of bispecific CAR-T cells.

(A) Schematic of bispecific CAR design: the FITC-binding scFv 4m5.3 is fused through a short linker to a tumor antigen-specific CAR, enabling the T cell to be triggered by binding to either FITC-decorated cells or tumor cells. (B) Representative T cell surface expression of FITC/TRP1-CAR. (C) Killing of TRP1-expressing B16F10 cells in vitro after a 6 hr co-culture with FITC/TRP1-CAR-T, monospecific TRP1-CAR-T or control untransduced T cells at E:T of 10:1. (D, E) Tumor growth (D) and survival (E) of B16F10 tumor-bearing mice (n=7 animals/group) treated with 10×10⁶ CAR-T alone or CAR-T plus amph-FITC vaccination. P-values were determined by RM (repeated measures) two-way ANOVA with Tukey’s multiple comparisons test for D, and by log-rank test for E. (F) Surface expression of FITC/hCD19 bispecific CAR on human T cells. (G) FITC/TRP1 bispecific CAR-T responding to either hCD19⁺ Raji cells or amph-FITC-coated K562 cells as monitored by IFN-γ secretion. Shown in C and G is a representative experiment with technical triplicates. P-values were determined by unpaired student’s t-test for C and G. Error bars represent 95% CI; ***p<0.0001, **p<0.01, *p<0.05. n.s, not significant.