Nanobody-based CAR T cells that target the tumor microenvironment inhibit the growth of solid tumors in immunocompetent mice

Abstract

Chimeric antigen receptor (CAR) T cell therapy has been successful in clinical trials against hematological cancers, but has experienced challenges in the treatment of solid tumors. One of the main difficulties lies in a paucity of tumor-specific targets that can serve as CAR recognition domains. We therefore focused on developing VHH-based, single-domain antibody (nanobody) CAR T cells that target aspects of the tumor microenvironment conserved across multiple cancer types. Many solid tumors evade immune recognition through expression of checkpoint molecules, such as PD-L1, that down-regulate the immune response. We therefore targeted CAR T cells to the tumor microenvironment via the checkpoint inhibitor PD-L1 and observed a reduction in tumor growth, resulting in improved survival. CAR T cells that target the tumor stroma and vasculature through the EIIIB⁺ fibronectin splice variant, which is expressed by multiple tumor types and on neovasculature, are likewise effective in delaying tumor growth. VHH-based CAR T cells can thus function as antitumor agents for multiple targets in syngeneic, immunocompetent animal models. Our results demonstrate the flexibility of VHH-based CAR T cells and the potential of CAR T cells to target the tumor microenvironment and treat solid tumors.

Keywords: chimeric antigen receptor; immunotherapy; tumor microenvironment.

Fig. 1.

VHH-based CAR T cells expressed with retention of antigen specificity. (A) Retroviral construct of VHH-based CAR T cells and their introduction into mouse T cells. (B) Production process for generation of CAR T cells. (C) Immunoblot on T cells transduced with Enh CAR construct. Lysates from transduced and untransduced T cells were blotted against using anti-Enh serum generated from immunization of mice with the Enh VHH. Polypeptides corresponding to the Enh CAR and soluble Enh were seen. (D) Schematic of assay to test for Enh CAR display. T cells were transduced with Enh CAR and probed for binding to GFP by flow cytometry. Nonspecific binding was measured by incubation with an irrelevant protein, TIM3−Fc fusion, probed for with an anti-mouse IgG conjugated to APC. (E) T cells were transduced with A12 CAR targeted to PD-L1. Successful display is probed by binding to recombinant PD-L1−Fc fusion and detected by an anti-mouse IgG conjugated to APC.

Fig. 2.

In vitro activity of CAR T cells: cytokine production and cytotoxicity. T cells were transduced with Enh CAR. (A) IL-2 and (B) IFNγ levels in the supernatant of CAR T cells cultured for 24 h with GFP or an irrelevant protein (TIM3−Fc). (C–J) T cells were transduced with A12 CAR targeted to PD-L1. (C and D) A12 CAR T cells recognized and killed B16 tumors. Coculture of anti−PD-L1 A12 CAR and a nonspecific control 1B7, recognizing a T. gondii calcium-dependent protein kinase, with B16 cells. Cells were cultured for 48 h at various effector:target (E:T) ratios. (C) A Cell Titer Glo assay was performed to measure cytotoxicity. (D) Supernatants were collected and IFNγ levels were measured. (E and F) A12 PD-L1−targeted cells were also effective in killing C3.43 HPV-transformed cancer cell lines. C3.43 cells were cultured with A12 CAR T cells at various E:T ratios. (E) C3.43 killing was measured by Cell Titer Glo, and (F) CAR activation was measured by IFNγ secretion. (G and H) A12 CAR T cells were cytotoxic against MC38 colon adenocarcinoma cells. A12 CAR T cells were cocultured with MC38 cells at various E:T ratios, and (G) MC38 killing and (H) A12 CAR T cell activation and cytokine secretion were measured. (I and J) Blocking experiments were performed using the B16 coculture setup. Cytotoxicity assay mixtures were incubated with varying concentrations soluble A12 VHH, B3 VHH, or an irrelevant 96G3M VHH (14). B3 binds PD-L1 with higher affinity than does A12. Higher levels of target antigen blockade lead to (I) better B16 survival and (J) less IFNγ secretion, indicating specificity. ****P ≤ 0.0001.

Fig. 3.

Anti−PD-L1 CAR T cells are generated more effectively in a PD-L1−deficient background. (A) WT T cells were transduced with the A12 CAR and cultured with B16 cells for 48 h. Supernatants from the B16 coculture experiments were probed for levels of IFNγ. (B) A12 PD-L1−targeted CAR T cells were generated in WT T cells and PD-L1 KO T cells. A12 CAR T cells generated in WT T cells showed increased levels of PD1, TIM3, and LAG3 expression. (C) RAG^−/− mice were injected with B16 tumors s.c., and, 2 d later, A12 CAR T cells generated in WT T cells and PD-L1KO T cells were adoptively transferred. On day 15, tumors, spleens, and lymph nodes were harvested to determine the relative numbers of persisting CAR T cells. (D) Splenocytes were analyzed for the presence of GFP-labeled A12 CAR T cells. More CD4 and CD8 CAR T cells made in the PD-L1 KO background persisted. (E) Greater levels of CD4 CAR T cells made in the PD-L1 KO background were found in the spleen and draining lymph node (for 1B7 PD-L1^−/− vs. A12 PD-L1^−/−: spleen CD4, P = 0.0014; spleen CD8, P = 0.0023; LN CD4, P < 0.0001; LN CD8, P = 0.0757; tumor CD4, P = 0.0238; tumor CD8, P = 0.0162; for A12 PD-L1^−/− vs. A12 WT: LN CD4, P = 0.0007). (F) More CD8 CAR T cells made in the PD-L1 KO background were present in the spleen, draining lymph nodes, and tumor. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 4.

In vivo application of anti−PD-L1 CAR T cells slows growth of solid tumors. (A) PD-L1 KO mice were inoculated with B16 tumor cells. On days 2, 7, and 14, mice were treated with A12 CAR T cells (n = 10) or 1B7-irrelevant CAR T cells (n = 5) or left untreated (n = 5). All mice were given an anti-TRP1 antibody, TA99, in combination with CAR T cell treatment. (B) Kaplan−Meier curves showing survival of each treatment condition (P < 0.0001, Mantel−Cox log-rank test). (C) The average tumor area with SEM and (D) individual tumor area of each mouse was measured. Treatment with the A12 CAR T cells delayed tumor growth (none/A12 P = 0.0296, 1B7/A12 P = 0.04). (E) PD-L1 KO mice were inoculated with B16 tumor cells engineered to express high levels of PD-L1 under the control of a CMV promoter (n = 5). (F) Kaplan−Meier curve showing survival of each group (P = 0.0233, Mantel−Cox log rank). Mice treated with A12 CAR T cells showed improved survival. (G) Average tumor area (none/A12 P = 0.0029, 1B7/A12 P = 0.0422, unpaired t test with Bonferroni correction) and individual tumor area for each group were measured. SEM is shown. (H) PD-L1 KO mice were inoculated with MC38 colon adenocarcinoma. Mice were either left untreated (n = 5), treated with irrelevant CAR T cells (n = 5), or treated with PD-L1−targeted CAR T cells (n = 8). (I) Survival was measured and plotted on a Kaplan−Meier curve, showing that A12 CAR treatment improved survival (P = 0.003). (J) The tumor area average for each group was monitored (none/A12 P = 0.003, 1B7/A12 P = 0.009, unpaired t test with Bonferroni correction).

Fig. 5.

Exhaustion of CAR T cells due to persistent activation overcome by PD-L1 blockade in culture. (A) Chronic antigen exposure and exhaustion of A12 CAR T cells made in a WT background can be blocked by incubation in the course of culture with soluble anti−PD-L1 VHH to mask endogenous PD-L1. A12 CAR T cells were generated in the presence of soluble B3 VHH, which binds PD-L1 with higher affinity than A12. Expression of common exhaustion markers was analyzed using flow cytometry. (B) WT mice were inoculated with B16 tumors, and A12 CAR T cells made in a WT background with and without inclusion of soluble B3 were introduced and compared with A12 CAR T cells made in the PD-L1 KO background. A12 CAR T cells made in a WT background in the presence of soluble B3 showed better persistence than A12 CAR T cells made without inclusion of B3 (CD4: A12 WT vs. A12 WT+ B3, P = 0.0283; CD8: A12 WT vs. A12 WT+ B3, P = 0.1346). (C) Mice were inoculated with B16 overexpressing PD-L1 tumors on day 0, and either A12 or B3 CAR T cells generated in the presence of soluble B3 were introduced on days 3, 10, and 17 (n = 5). (D) Kaplan−Meier curve showing survival of each group. Mice treated with the A12 and B3 CAR T cells showed a slight increase in survival (P = 0.0058, Mantel−Cox log-rank test). (E) Individual tumor area for each group was measured. The A12 CAR T cells generated in the presence of soluble B3 slightly delayed tumor growth (P = 0.0483). SEM is shown. *P ≤ 0.05, **P ≤ 0.01.

Fig. 6.

Anti-EIIIB fibronectin-targeted CAR T cells slow B16 melanoma growth in vivo. (A) T cells were transduced with the EIIIB-specific B2 CAR construct, and transduction efficiency was monitored by mCherry expression. Cells were then incubated with recombinant EIIIB-GST and probed with rabbit anti-GST and anti-rabbit A647 to determine ligand binding. (B) B2 CAR T cells show cytotoxicity in response to ligand recognition. B2 CAR T cells were cocultured with aortic endothelial cells (AEC) that either express the EIIIB fibronectin domain (AEC FN^+/+) or lack it (AEC FN^−/−). (C) Mice were inoculated with B16 tumors on day 0, and B2 CAR T cells (n = 10) were introduced on days 4, 15, and 20. (D) Tumor area was measured for individual mice. Kaplan−Meier curve showing survival of each group (P = 0.0001, Mantel−Cox log-rank test with the Bonferroni correction for multiple comparisons). Mice treated with the B2 CAR T cells showed improved survival. SEM is shown. (E) RAG^−/− mice were inoculated with B16 tumors and treated with B2 (n = 4) or 1B7 CAR T cells (n = 3) on day 4. RAG^−/− mice treated with B2 CAR T cells do not show improved survival increase or delayed tumor growth. SEM is shown. (F) MC38 expresses lower levels of EIIIB (SI Appendix, Fig. S8). MC38 survival curves (P = 0.1895, ns, Mantel−Cox log-rank test) and MC38 individual tumor areas were not significantly affected by treatment with B2 CAR T cells (n = 7). ***P ≤ 0.001, ****P ≤ 0.0001; ns, nonsignificant.

Fig. 7.

Treatment with anti-EIIIB fibronectin-targeted CAR T cells leads to tumor immune infiltration and necrosis. (A) WT mice were inoculated with tumors on day 0 and either left untreated (N = 2) or treated with B2 CAR T cells (n = 3) twice, on days 4 and 11. On day 16, tumors were harvested, fixed, and embedded for IHC and stained for EIIIB, CD31, CD3, CD4, and CD8. (B) The tumor area average measurements and values for individual mice are plotted. (C) Tumor samples were stained with PBS and secondary only (control), NJB2 VHH, anti-CD31, anti-CD3, anti-CD4, and anti-CD8. One representative image is shown. A 20× magnification of the edge (E), capsular region (Top) of the tumor is shown. A similar magnification of a core (C) (Bottom) regions of the tumor is shown. EIIIB is present in the tumor capsule, tumor stroma, and surrounding the tumor vasculature, as inferred from colocalization with CD31 staining. In untreated samples, tumors appeared healthy and live, with intact matrix throughout the tissue. Little T cell and immune infiltration was apparent. (D) Necrotic B2 CAR T cell-treated tumors. Two of the three smaller treated tumors were highly necrotic, with a disintegrated matrix. CD31 staining shows a lack of tumor vasculature with little immune infiltration. (E) One treated tumor appeared to be heterogeneous and showed both (Bottom) necrotic [dead (D)] and (Top) live (L) sectors. The live tissue showed CD31 staining and was heavily infiltrated by CD3-, CD4-, and CD8-positive cells. (F) The number of CD3-, CD4-, and CD8-positive cells was quantified for both treated and untreated tumors. (G) The number of CD3-, CD4-, and CD8-positive cells was quantified for both the live and dead sections of the treated and untreated tumors.