Oncolytic adeno-immunotherapy modulates the immune system enabling CAR T-cells to cure pancreatic tumors

Abstract

High expression levels of human epidermal growth factor receptor 2 (HER2) have been associated with poor prognosis in patients with pancreatic adenocarcinoma (PDAC). However, HER2-targeting immunotherapies have been unsuccessful to date. Here we increase the breadth, potency, and duration of anti-PDAC HER2-specific CAR T-cell (HER2.CART) activity with an oncolytic adeno-immunotherapy that produces cytokine, immune checkpoint blockade, and a safety switch (CAdTrio). Combination treatment with CAdTrio and HER2.CARTs cured tumors in two PDAC xenograft models and produced durable tumor responses in humanized mice. Modifications to the tumor immune microenvironment contributed to the antitumor activity of our combination immunotherapy, as intratumoral CAdTrio treatment induced chemotaxis to enable HER2.CART migration to the tumor site. Using an advanced PDAC model in humanized mice, we found that local CAdTrio treatment of primary tumor stimulated systemic host immune responses that repolarized distant tumor microenvironments, improving HER2.CART anti-tumor activity. Overall, our data demonstrate that CAdTrio and HER2.CARTs provide complementary activities to eradicate metastatic PDAC and may represent a promising co-operative therapy for PDAC patients.

Fig. 1. HER2.CART and CAdTrio kill human PDAC lines in vitro.

a HER2 expression was analyzed by flow cytometry on PANC-1, CAPAN-1, and CFPAC-1. Tumor cells expressing ffLuc were cultured with NTs or HER2.CARTs (E:T = 1:10). Cells were harvested 0, 1, and 5 days post coculture, and viable cancer cells were analyzed by luciferase assay (n = 4 biologically independent samples, each time point). Data are presented as means ± SD. b Schematic structure of HDAd encoding human IL-12p70, HSVtk safety switch and PDL1 blocking antibody expression cassettes (HDAdTrio). PANC-1, CAPAN-1, and CFPAC-1 were infected with increasing doses of HDAdTrio, OAd, or CAdTrio (OAd: HDAd=1:1) (n = 6 biologically independent samples). We analyzed viable cells at 96 h by MTS assay. Data are presented as means ± SD. c PANC-1, CAPAN-1, and CFPAC-1 were infected with total 10 vp/cell of HDAdTrio, OAd, or CAdTrio (OAd:HDAd=1:1) (n = 4 biologically independent samples). We sampled media 48 h post infection and quantified levels of IL-12p70 and PD-L1 mini-antibody by IL-12p70 ELISA assay and Western blotting for PD-L1 mini-antibody, respectively. Data are presented as means ± SD, p < 0.0001 determined by two-tailed t test (t = 32.93, dF = 6). Statistical significance set at p < 0.05, ns > 0.05.

Fig. 2. HER2.CARTs primarily control CFPAC-1 tumor growth in xenograft mouse model.

a CFPAC-1 cells were transplanted into the right flank of NSG mice (n = 5 animals). A total of 1 × 10⁷ vp of CAdTrio (OAd:HD = 1:1) were injected i.t.. A total of 1 × 10⁶ HER2.CARTs expressing ffLuc were administered i.v. 3 days post injection of CAdTrio. Tumor volumes were monitored at different time points. b Bioluminescence of HER2.CARTs was monitored at the indicated time points. Data are presented as means ± SD. c We collected serum samples from mice at 0, 3, 7, 21, 42, and 63 days post injection of CAdTrio, and measured IFNγ and IL-12p70 levels by ELISA. Data are presented as means ± SD. d Kaplan–Meier survival curve after CAdTrio administration in mice (n = 5 animals) p = 0.0003. P-values were determined using the log-rank Mantel–Cox test (dF=3). Statistical significance set at p < 0.05, ns > 0.05. Abbreviations: s.c. subcutaneous, i.t. intratumoral, i.v. intravenous.

Fig. 3. HER2.CARTs primarily control CAPAN-1 tumor growth but require CAdTrio to cure PDAC in xenograft mouse model.

a CAPAN-1 cells were transplanted into the right flank of NSG mice (n = 5 animals). A total of 1 × 10⁷ vp of CAdTrio (OAd:HD = 1:1) were injected into the tumor. A total of 1 × 10⁶ HER2.CARTs expressing ffLuc were systemically administered 3 days post injection of CAdTrio. We monitored tumor volumes at different time points. b We monitored HER2.CART bioluminescence at different time points. Data are presented as means ± SD, p < 0.005. P-values were determined by two-tailed t test (t = 4.32, dF = 8). c We collected serum samples from mice at 0, 3, 10, 24, 45, and 66 days post injection of CAdTrio, and measured levels of IFNγ and IL-12p70 by ELISA. Data are presented as means ± SD, p = 0.004. P-values were determined by two-tailed t test (t = 5.769, dF = 8). d Kaplan–Meier survival curve after CAdTrio administration in mice (n = 5 animals), p < 0.0001. P-values were determined using the log-rank Mantel–Cox test (dF = 3). Statistical significance set at p < 0.05, ns > 0.05. Abbreviations: s.c. subcutaneous, i.t. intratumoral, i.v. intravenous.

Fig. 4. Combination immunotherapy controls CFPAC-1 tumor growth in humanized mouse model.

a CFPAC-1 cells were transplanted into the right flank of humanized mice (control, CART alone, CAdTrio alone: n = 5 animals, CAdTrio+CART: n = 6 animals). A total of 1 × 10⁷ vp of CAdTrio (OAd:HD = 1:1) were injected into the tumor. A total of 1 × 10⁶ HER2.CARTs expressing ffLuc were systemically administered 3 days post injection of CAdTrio. Tumor volumes were monitored at different time points. b We collected serum samples from mice at 0, 3, 10, 24, 45, and 66 days post injection of CAdTrio, and measured human Th1 and Th2 cytokine levels by Multiplex. Data are presented as means ± SD. c Bioluminescence of HER2.CARTs was monitored at different time points. Data are presented as means ± SD, p = 0.003. P-values were determined using two-tailed t test (t ratio = 4.213, dF = 8). d CFPAC-1 tumors were harvested from non-humanized and humanized mice (non-humanized mice: n = 4 animals, humanized mice: n = 5 animals) at 3 days post injection of CAdTrio, and total RNA was extracted from whole tumors. Pro-inflammatory genes were quantified and normalized with human β-Actin. Data are presented as means ± SD. P-values were determined using two-tailed t test; p = 0.0093 (t ratio = 3.553, dF = 7), p = 0.0004 (t ratio = 6.352, dF = 7), p = 0.0005 (t ratio = 6.084, dF = 7), p = 0.0045 (t ratio = 4.101, dF = 7), p = 0.014 (t ratio = 3.226, dF = 7), p = 0.027 (t ratio = 2.782, dF = 7). Statistical significance set at p < 0.05, ns > 0.05. e CFPAC-1 tumors were harvested from humanized mice (non-humanized mice: n = 4 animals, humanized mice: n = 5 animals) at 3 days post injection of CAdTrio, and tumor infiltrating human immune cells were analyzed with flow cytometry. Box plot elements: central line, median; box limit, upper and lower quartiles; whisker, 1.5x inter-quartile range; points, outliers. Abbreviations: s.c. subcutaneous, i.t. intratumoral, i.v. intravenous, ND not detectable.

Fig. 5. Combination immunotherapy controls tumor growth in humanized mice with multiple PDAC tumors.

a CFPAC-1 cells were transplanted into the right and left flanks of humanized mice (n = 5 animals). A total of 1 × 10⁷ vp of CAdTrio (OAd:HD = 1:1) were injected into the right tumor. A total of 1 × 10⁶ HER2.CARTs expressing ffLuc were systemically administered 3 days post injection of CAdTrio. Tumor volumes were monitored at different time points. b Bioluminescence of HER2.CARTs was monitored at different time points. Data are presented as means ± SD. c CFPAC-1 tumors were harvested from humanized mice at 31 days post injection of CAdTrio, and tumor infiltrating human immune cells were analyzed by flow cytometry. Box plot elements: central line, median; box limit, upper and lower quartiles; whisker, 1.5x inter-quartile range; points, outliers. P-values were determined using ordinary one-way ANOVA with Tukey multiple comparisons, p = 0.0009 (F(3,16) = 9.252), p < 0.01 (F(3,16) = 6.426). Statistical significance set at p < 0.05, ns > 0.05. Total RNA was extracted from whole tumor at 31 days post injection of CAdTrio. Gene expression was profiled with Nanostring, differential gene expression compared to control tumors (d) and compared to single agents (e) are shown. Abbreviations: s.c. subcutaneous, i.t. intratumoral, i.v. intravenous.

Fig. 6. NK cells enhance anti-PDAC activity in the presence of type I IFN and immunomodulatory molecules from CAdTrio.

a PANC-1, CAPAN-1, and CFPAC-1 expressing ffLuc were cultured with NK cells with increasing doses of rIFNα. We also cultured NK cells expressing ffLuc with these cancer cells (E:T = 1:10) with increasing doses of rIFNα. Cells were harvested 0 and 72 h post coculture, and viable cells were analyzed by luciferase assay (n = 4 biologically independent samples, each timepoint). Data are presented as means ± SD. P-values were determined by ordinary one-way ANOVA with Tukey multiple comparisons. CFPAC (F(4,15) = 351.8) NK (F(4,15) = 346.8), Panc1 (F(4,15) = 468.7) NK (F(4,15) = 106.3), CAPAN1 (F(4,15) = 1121) NK (F(4,15) = 318.4. b PANC-1, CAPAN-1, and CFPAC-1 expressing ffLuc (or not expressing) were infected with 100 vp/cell of HDAdTrio. Cells expressing ffLuc were cultured with NK cells (E:T = 1:10). We also cultured NK cells expressing ffLuc with these cancer cells (E:T = 1:10) at 24 h post infection of HDAdTrio in the presence or absence of 1 ng/mL rIFNα. Cells were harvested 72 h post coculture, and viable cells were analyzed by luciferase assay (n = 4 biologically independent samples). Data are presented as means ± SD. P-values were determined by ordinary one-way ANOVA with Tukey multiple comparisons. CFPAC (F(3,12) = 1114) NK (F(3,12) = 458.3), Panc1 (F(3,12) = 356.6) NK (F(3,12) = 410.2), CAPAN1 (F(3,12) = 459.6) NK (F(3,12) = 131.4). c We sampled media 72 h post coculture, and measured granulysin using ELISA assay (n = 4 biologically independent samples). Data are presented as means ± SD. P-values were determined by ordinary one-way ANOVA with Tukey multiple comparisons, p < 0.001 (F(3,12) = 41.07), p < 0.0001 (F(3,12) = 74.40), p < 0.0001 (F(3,12) = 49.49). Statistical significance set at p < 0.05, ns > 0.05.