EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss

Abstract

Purpose: Chimeric antigen receptor (CAR) transduced T cells represent a promising immune therapy that has been shown to successfully treat cancers in mice and humans. However, CARs targeting antigens expressed in both tumors and normal tissues have led to significant toxicity. Preclinical studies have been limited by the use of xenograft models that do not adequately recapitulate the immune system of a clinically relevant host. A constitutively activated mutant of the naturally occurring epidermal growth factor receptor (EGFRvIII) is antigenically identical in both human and mouse glioma, but is also completely absent from any normal tissues.

Experimental design: We developed a third-generation, EGFRvIII-specific murine CAR (mCAR), and performed tests to determine its efficacy in a fully immunocompetent mouse model of malignant glioma.

Results: At elevated doses, infusion with EGFRvIII mCAR T cells led to cures in all mice with brain tumors. In addition, antitumor efficacy was found to be dependent on lymphodepletive host conditioning. Selective blockade with EGFRvIII soluble peptide significantly abrogated the activity of EGFRvIII mCAR T cells in vitro and in vivo, and may offer a novel strategy to enhance the safety profile for CAR-based therapy. Finally, mCAR-treated, cured mice were resistant to rechallenge with EGFRvIII(NEG) tumors, suggesting generation of host immunity against additional tumor antigens.

Conclusion: All together, these data support that third-generation, EGFRvIII-specific mCARs are effective against gliomas in the brain and highlight the importance of syngeneic, immunocompetent models in the preclinical evaluation of tumor immunotherapies.

Figure 1. EGFRvIII mCAR retroviral gene delivery to murine T cells confers antigen-specific activity

(A) VM/Dk-derived glioma SMA560vIII shows EGFRvIII expression compared with SMA560 by surface antibody staining (L8A4) and flow cytometry; grey represents negative staining control. (B) The EGFRvIII mCAR MSGV1 retroviral vector contains the human anti-EGFRvIII scFv 139, in tandem with murine CD8TM, CD28, 4-1BB, and CD3ζ intracellular regions. EGFRvIII mCAR MSGV1 retrovirus containing supernatant was generated by transient transfection of HEK 293T cells with the corresponding retroviral vectors and the helper pCL-Eco plasmid. Supernatants harvested 48h after transfection were used to transduce previously ConA activated murine VM/Dk splenocytes. (C) Retroviral mCAR gene expression was evaluated by flow cyometric analysis 3 days after transduction; grey represents negative staining control. (D) Antigen specificity of EGFRvIII mCAR transduced and vector control (MSGV1-GFP) T cells was evaluated against EGFRvIII-expressing SMA560 cells (SMA560vIII) or the parental EGFRvIII^NEG line (SMA560) by IFNγ ELISpot assay. Statistical analysis was performed by two-way ANOVA. Horizontal bar represents a statistical significance of P < 0.05 (E) Soluble EGFRvIII peptide (PEPvIII) at 50 μg/mL was used to coat tissue culture plates prior to addition of EGFRvIII mCAR or GFP control T cells. Cells were cultured 18h in the presence of increasing dosages of soluble PEPvIII or irrelevant peptide (IDH) and subjected to IFNγ ELISpot. Statistical analysis was performed by unpaired t test comparing groups defined by EGFRvIII target expression. Horizontal bar represents a statistical significance of P < 0.05. Experiments are representative of two independent repeats with similar results.

Figure 2. EGFRvIII mCAR T-cell therapy against intracerebral tumors requires lymphodepletive host-conditioning for efficacy, and can be abrogated by soluble peptide administration

To evaluate the impact of lymphopenia on the antitumor efficacy of EGFRvIII mCAR T cells, VM/Dk mice (8 per group) with 3-day established intracerebral SMA560vIII tumors were left untreated (A, no TBI) or irradiated (B, 5Gy TBI) prior to intravenous infusion with 3.5×10⁶ vector control (MSGV1-GFP) or EGFRvIII mCAR T cells. (C) To evaluate whether a short peptide corresponding to the antigenic epitope expressed on target cells could be applied to competitively inhibit and reduce the functional activity of EGFRvIII mCAR T cells in vivo, mice with 3 day implanted SMA560vIII treated as in (B) above, were injected with 100 μg PEPvIII or saline 4h following mCAR T cell infusion intravenously, followed by a second injection 10 days later. Mice were monitored for morbidity end points approved by the Duke University IACUC and sacrificed when end points were met. Survival analysis was performed using the Log-Rank (Mantel Cox) test. Statistical significance was determined at P < 0.05.

Figure 3. Adoptive T-cell therapy with EGFRvIII mCAR T cells leads to regression and eradication of subcutaneous and intracerebral syngeneic tumors in a dose-dependent fashion

VM/Dk mice (8 mice per group) with 3-day established (A) intracerebral, or (B) subcutaneous SMA560vIII tumors were irradiated (5Gy TBI) prior to intravenous infusion with various doses of vector control (MSGV1-GFP) or EGFRvIII mCAR T cells as indicated. Mice were monitored for morbidity end points approved by the Duke University IACUC and sacrificed when end points were met. Survival analysis was performed using the Log-Rank (Mantel Cox) test. Tumor growth was monitored in two dimensions. Statistical significance was determined at P < 0.05 using two-way ANOVA. Experiments are representative of three independent repeats with similar results.

Figure 4. Constant numbers of EGFRvIII mCAR T cells persisted in peripheral blood lymphocytes over 5 weeks while endogenous lymphocytes recovered

Constant numbers of EGFRvIII mCAR T cells persisted in peripheral blood lymphocytes (PBL) over 4 weeks. 5Gy irradiated VM/Dk mice (N = 7) had PBL sampled 1, 2, 4 and 5 weeks after intravenous EGFRvIII mCAR T-cell transfer. PBL were stained with mAb against CD3 and PEPvIII-PE multimer was used to identify EGFRvIII mCAR T cells. Absolute numbers per microliter of blood were evaluated by flow cytometry using Flowcount® beads from Beckman Coulter© according to manufacturer instructions. (A) Percentages, and (B) absolute numbers of EGFRvIII mCAR T cells in blood were calculated.

Figure 5. Treatment of mice bearing EGFRvIII^POS tumors with mCARs provides long-term protection against EGFRvIII^NEG tumor

(A) To evaluate whether treatment with EGFRvIII mCAR T cells could elicit de novo priming against antigen-loss variants, long-term survivors (greater than 60 days after treatment) of subcutaneous SMA560vIII tumors by EGFRvIII mCAR therapy were rechallenged with 5×10⁵ EGFRvIII^NEG SMA560 cells on the contralateral flank and their survival was monitored. (B) SMA560 (EGFRvIII^NEG) gliomas were maintained in vitro or passaged in a mouse subcutaneously for 14 days, then harvested and both were stained for EGFRvIII expression using L8A4 mAb and analyzed by flow cytometry. Total numbers and percentages of mCAR T cells in blood was evaluated in (C) tumor-naïve mice, and (D) SMA 560vIII (EGFRvIII^POS) tumor-cured mice before and 7 days after subcutaneous challenge with SMA560 (EGFRvIII^NEG) tumors. Statistical significance was determined using a paired t test. Horizontal bar represents a statistical significance of P < 0.05 (C-E) Tumor naïve VM/Dk mice were either left untreated, received vector control, or EGFRvIII mCAR T cells 1 day after receiving a lymphodepletive conditioning regimen by 5Gy TBI. All mice were challenged subcutaneously with SMA560 (EGFRvIII^NEG) tumors 28 days after adoptive-T-cell transfer. Additionally, VM/Dk mice that were previously inoculated with 5×10⁵ SMA560vIII (EGFRvIII^POS) tumor cells and were cured of tumor after receiving 5Gy TBI and EGFRvIII mCAR T cells (tumor cured) were rechallenged subcutaneously with 5×10⁵ SMA560 (EGFRvIII^NEG) tumor cells at the same time. (E) Tumor growth was monitored in two dimensions. Mice were examined for morbidity end points approved by the Duke University IACUC and sacrificed when end points were met. Experiment is representative of two independent repeats with similar results. Statistical significance of P < 0.0001was determined using two-way ANOVA.