Antitumor effects of chimeric receptor engineered human T cells directed to tumor stroma

Abstract

Cancer-associated fibroblasts (CAFs), the principle component of the tumor-associated stroma, form a highly protumorigenic and immunosuppressive microenvironment that mediates therapeutic resistance. Co-targeting CAFs in addition to cancer cells may therefore augment the antitumor response. Fibroblast activation protein-α (FAP), a type 2 dipeptidyl peptidase, is expressed on CAFs in a majority of solid tumors making it an attractive immunotherapeutic target. To target FAP-positive CAFs in the tumor-associated stroma, we genetically modified T cells to express a FAP-specific chimeric antigen receptor (CAR). The resulting FAP-specific T cells recognized and killed FAP-positive target cells as determined by proinflammatory cytokine release and target cell lysis. In an established A549 lung cancer model, adoptive transfer of FAP-specific T cells significantly reduced FAP-positive stromal cells, with a concomitant decrease in tumor growth. Combining these FAP-specific T cells with T cells that targeted the EphA2 antigen on the A549 cancer cells themselves significantly enhanced overall antitumor activity and conferred a survival advantage compared to either alone. Our study underscores the value of co-targeting both CAFs and cancer cells to increase the benefits of T-cell immunotherapy for solid tumors.

Figure 1

Generation of FAP-specific T cells. (a) Schematic of the FAP-specific CAR retroviral vector. (b) Representative data from one donor showing CAR expression and T-cell subsets.

Figure 2

FAP-specific T cells recognize and kill human or murine FAP-positive cells. (a) qRT-PCR. (b) FACS analysis on a panel of cancer cell lines and cancer-associated fibroblast cell lines using human FAP primers. (c) NT- or FAP-T cells were co-cultured with FAP⁺ target cells (A549.mFAP, A549.hFAP) and FAP^- targets (A549) at a 2:1 effector: target (E:T) ratio. The production of proinflammatory cytokines IFN-γ and TNF-α by T cells was determined by Milliplex MAP after 24 hr (n = 4 independent replicates; means + SD). (d,e) FAP-, PSCA-, and NT-T cells were tested in standard 6-hr chromium release assays at 10:1 E:T ratio (n = 5; mean + SD). *P < 0.05; **P < 0.005.

Figure 3

FAP-specific T cells recognize and kill cells expressing FAP from its endogenous promoter. (a) NT- or FAP-T cells were co-cultured with FAP⁺ target cells (U87, SENMA, HPS-19I, and Hs894) and FAP^- (LCL and A549) at a 2:1 effector: target (E:T) ratio. (b) The production of IFN-γ and TNF-α by T cells was determined by Milliplex MAP after 24 hr (n = 3 independent replicates; mean ± SD). (c) FAP- and NT-T cells were tested in standard 6-hr chromium release assays at 10:1 E:T ratio (n = 3 independent replicates; mean ± SD). *P < 0.05.

Figure 4

FAP-specific T cells suppress LCL tumor growth. (a) Induction of murine FAP expression in vivo. Relative fold expression (2^-ΔΔC_t) was quantified in comparison to normal skin using murine FAP primers. Each dot represents a single tumor sample from different mice at each time point. (b) An admixture of LCL.eGFP.FFLuc cells and NT-T cells. (c) FAP-T cells were injected subcutaneously along with 1,500 U of IL-2 administered i.p. (n = 10/group) on day 0. Bioluminescence signal (radiance = photons/sec/cm²/sr) was monitored over time. *P < 0.05; **P < 0.005; ***P < 0.0005.

Figure 5

FAP-specific T cells decrease tumor growth and improve survival in an A549 lung cancer model. (a) A549.eGFP.FFLuc cells were injected i.v. Lungs were excised and qRT-PCR was used to determine relative levels of murine FAP expression in the lungs using murine FAP primers. Relative fold expression (2^-ΔΔCt) was quantified in comparison to normal lungs. Each dot represents a single lung sample from different mice at each time point. (b) A549.eGFP.FFLuc cells were injected i.v. into mice on day 0 and treated i.v. with FAP-T cells and 1, 500 U IL-2 administered i.p. on day 4. Treatment was repeated two more times a week apart. Untreated mice and mice treated with NT-T cells served as controls. Tumor progression was followed by in vivo bioluminescence imaging. Images of representative animals are shown. (c–e) Solid lines represent each individual mouse. (f) Kaplan–Meier survival curve of untreated mice (n = 9) or those treated with FAP (n = 8) or NT-T cells (n = 9).

Figure 6

FAP-specific T cells recognize and kill murine FAP-positive stromal cells. (a) A549.eGFP.FFLuc were injected i.v. on day 0. On day 4 mice were treated with NT- or FAP-T cells administered i.v. along with 1,500 U IL-2 administered i.p. Lungs were excised 48 hr post treatment and relative fold expression (2^-ΔΔCt) was quantified in comparison to normal lungs by qRT-PCR using murine FAP primers. Data are means obtained from six lung specimens in each treatment group. Four normal lung specimens were used as control **P < 0.005. (b) A549.eGFP.FFLuc cells were injected i.v. into SCID-Beige mice on day 0. Lungs were excised on day 4, dissociated to form single cell suspensions and co-cultured with NT- or FAP-T cells at 2:1 E:T ratio or left untreated. 72 hr later the presence of FAP-positive cells and GFP-positive tumor cells was determined by FACS analysis. Representative data for one such single cell suspension is shown. (c) Percentage FAP+ cells were determined for 25,000-gated events. Each symbol represents one lung specimen.*P < 0.05.

Figure 7

Co-targeting CAFs and cancer cells results in an enhanced antitumor response and improved survival. (a) A549.eGFP.FFLuc cells were injected i.v. on day 0. On day 7, mice received an i.v. administration of FAP-T cells (or) EphA2-T cells, (or) FAP-T cells and EphA2-T cells. All groups received an i.p. injection of 1,500 U IL-2. Treatment was repeated two more times a week apart. Untreated mice served as controls. Tumor progression was followed by in vivo bioluminescence imaging. Images of representative animals are shown. (b–e) Solid lines represent each individual mouse (f) Kaplan–Meier survival curve of untreated mice (n = 4) or those treated with FAP-T cells (n = 5) or EphA2-T cells (n = 5), or FAP and EphA2-T cells (n = 5).