Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning

Abstract

Adoptive cell therapy with tumor-targeted T cells is a promising approach to cancer therapy. Enhanced clinical outcome using this approach requires conditioning regimens with total body irradiation, lymphodepleting chemotherapy, and/or additional cytokine support. However, the need for prior conditioning precludes optimal application of this approach to a significant number of cancer patients intolerant to these regimens. Herein, we present preclinical studies demonstrating that treatment with CD19-specific, chimeric antigen receptor (CAR)-modified T cells that are further modified to constitutively secrete IL-12 are able to safely eradicate established disease in the absence of prior conditioning. We demonstrate in a novel syngeneic tumor model that tumor elimination requires both CD4(+) and CD8(+) T-cell subsets, autocrine IL-12 stimulation, and subsequent IFNγ secretion by the CAR(+) T cells. Importantly, IL-12-secreting, tumor-targeted T cells acquire intrinsic resistance to T regulatory cell-mediated inhibition. Based on these preclinical data, we anticipate that adoptive therapy using CAR-targeted T cells modified to secrete IL-12 will obviate or reduce the need for potentially hazardous conditioning regimens to achieve optimal antitumor responses in cancer patients.

Figure 1

Prior cyclophosphamide conditioning results in 19mz⁺ T cell–mediated B-cell aplasias and eradication of systemic EL4(hCD19) tumors. (A) Schematic of retroviral construct encoding the 19mz and Pmz CARs. CD8 indicates CD8 leader sequence; scFv, single chain variable fragment; V_H and V_L, variable heavy and light chains; TM, transmembrane domain; LTR, long terminal repeat; SD, SA, splice donor and acceptor; and ψ, packaging element. (B) Persistent B-cell aplasias in C57BL6(mCD19^−/− hCD19^+/−) mice pretreated with cyclophosphamide followed by 19mz⁺ but not control Pmz⁺ T-cell therapy. 19mz⁺ T-cell therapy alone or cyclophosphamide alone failed to induce persistent B-cell aplasias. (C) Representative flow cytometry demonstrating B-cell aplasias in mice conditioned with cyclophosphamide followed by 19mz⁺ T-cell infusion at 4 weeks after T-cell infusion. (D) Cyclophosphamide conditioned EL4(hCD19) tumor-bearing C57BL6(mCD19^−/− hCD19^+/−) mice treated with 19mz⁺ T cells have enhanced survival compared with mice treated with 19mz⁺ T cells alone or control Pmz⁺ T cell–treated mice. All results are representative of at least 2 experiments.

Figure 2

Cyclophosphamide conditioning decreases Treg numbers, and induces an increase in serum IL-12 and IFNγ. (A) EL4(hCD19) tumor-bearing C57BL6(mCD19^−/− hCD19^+/−) mice pretreated with the B cell depleting antibody, MB2011 induced B-cell aplasias but failed to mediate tumor eradication on subsequent infusion with 19mz⁺ T-cell infusion (B). (C) Cyclophos-phamide conditioning mediated a significant decrease in peripheral blood Tregs as assessed by flow cytometry. (D) Serum cytokine analyses of cyclophosphamide conditioned C57BL6(mCD19^−/−hCD19^+/−) mice demonstrates transient increases in IL-12p70 and IFNγ as assessed by Luminex IS100 multiplex studies. All results are representative of at least 2 independent experiments.

Figure 3

T cells (19mz/IL-12⁺) induce B-cell aplasias and tumor eradication in the absence of prior cyclophosphamide conditioning. (A) Schematic of CAR/IL-12 retroviral constructs. IRES indicates internal ribosome entry site. (B) 19mz and 19mz/IL-12 expression after retroviral gene transfer as assessed by flow cytometry demonstrated similar gene transfer. (C) Supernatant from 19mz/IL-12⁺ but not 19mz⁺ T cells demonstrates enhanced levels of IL-12p70. (D) B-cell aplasias in EL4(hCD19) tumor-bearing C57BL6(mCD19^−/−hCD19^+/−) mice treated with 19mz/IL-12, but not Pmz/IL-12 or 19mz⁺ T cells, as assessed by flow cytometric analysis of peripheral blood samples at 2 weeks postmodified T-cell infusion. (E) 19mz/IL-12⁺ T cells eradicated disease and enhanced the survival of EL4(hCD19) tumor-bearing mice. In contrast, Pmz/IL-12 T cells significantly enhanced the survival of EL4(hCD19) tumor-bearing mice compared with 19mz⁺ T cell treated mice but similarly failed to eradicate disease. (F) Tregs in the BM of 19mz/IL-12⁺ and 19mz⁺ T cell treated mice as assessed by flow cytometry. All results are representative of at least 2 independent experiments.

Figure 4

In vitro, 19mz/IL-12–modified T cells, compared with 19mz⁺ T cells, exhibit enhanced cytotoxicity, resistance to Treg-mediated inhibition, a proinflammatory cytokine profile and enhanced CD25 expression. (A) 19mz/IL-12 and 19mz transduced T-cell populations were composed of equivalent CD4 and CD8 T cells, as detected by flow cytometry with the former exhibiting enhanced but not statistically significant increase in expression of memory T-cell markers (B). (C) 19mz/IL-12⁺ T cells have significantly higher expression of perforin and granzyme B compared with 19mz⁺ T cells. (D) As determined by standard ⁵¹Cr release assays 19mz/IL-12⁺ T cells have significantly increased ability to lyse EL4(hCD19) tumor cells compared with 19mz⁺ T cells. (E) 19mz/IL-12⁺ T cells, but not 19mz⁺ T cells retain capacity to lyse EL4(hCD19) tumor after preincubation with Tregs. (F) 19mz/IL-12⁺ T cells secrete increased levels of IFNγ, GM-CSF but fail to secrete IL-2 compared with 19mz⁺ T cells after coculture with EL4(hCD19) tumor cells at an E:T of 1:10 for 24 hours. (G) 19mz/IL-12⁺ T cells (thick lines) express higher levels of CD25 compared with 19mz⁺ T cells (thin lines), as detected by flow cytometry. Results are representative of at least 2 independent experiments.

Figure 5

In vivo antitumor efficacy of 19mz⁺ T cells is dependent on continued IL-12 activation in vivo, and requires the presence of both CD4 and CD8 T cells subsets. (A) Similar to 19mz⁺ and Pmz/IL-12⁺ T-cell therapy, treatment of C57BL6(mCD19^−/−hCD19^+/−) mice bearing systemic EL4(hCD19) tumors with 19mz⁺ T cells cultured ex vivo with exogenous IL-12, in contrast to 19mz/IL-12⁺ T-cell treated mice failed to induce either B-cell aplasias or mediate tumor eradication (B). (C) Sorted CD4⁺ 19mz/IL-12⁺ T cells failed to induce B-cell aplasias in EL4(hCD19mCD80) tumor-bearing mice, in contrast to CD8⁺ 19mz/IL-12⁺ T cells, which induced a partial but significant B-cell aplasia and bulk 19mz/IL-12⁺ T cells, which induced significant B-cell aplasias. (D) In addition, in contrast to bulk 19mz/IL-12⁺ T cells, treatment with either sorted CD4⁺ or CD8⁺ 19mz/IL-12⁺ T cells alone failed to eradicate systemic EL4(hCD19⁺) tumors. Results shown are representative of 2 independent experiments with similar results.

Figure 6

In vivo antitumor efficacy of IL-12–secreting targeted T cells is dependent on autocrine IL-12 stimulation and IFNγ secretion. (A) 19mz/IL-12⁺ T cells derived from C57BL6 IL-12Rβ₂^−/− mice, compared with 19mz/IL-12⁺ T cells derived from syngeneic C57BL6(mCD19^−/− hCD19^+/−) mice failed to induce either B-cell aplasias or eradicate systemic EL4(hCD19) tumors in C57BL6(mCD19^−/− hCD19^+/−) mice (B). (C) Similarly, 19mz/IL-12⁺ T cells derived from C57BL6 IFNγ^−/− mice, compared with 19mz/IL-12⁺ T cells derived from syngeneic C57BL6(mCD19^−/− hCD19^+/−) mice were unable to induce B-cell aplasias, or eradicate systemic EL4(hCD19) tumors in C57BL6(mCD19^−/− hCD19^+/−) mice (D). Results shown are representative of 2 independent experiments.