Aruna Mahendravada

T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model.

For the adoptive transfer of tumor-directed T lymphocytes to prove effective, there will probably need to be a match between the chemokines the tumor produces and the chemokine receptors the effector T cells express. The Reed-Stemberg cells of Hodgkin lymphoma (HL) predominantly produce thymus- and activation-regulated chemokine/CC chemokine ligand 17 (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), which preferentially attract type 2 T helper (Th2) cells and regulatory T cells (Tregs) that express the TARC/MDC-specific chemokine receptor CCR4, thus generating an immunosuppressed tumor environment. By contrast, effector CD8(+) T cells lack CCR4, are nonresponsive to these chemokines and are rarely detected at the tumor site. We now show that forced expression of CCR4 by effector T cells enhances their migration to HL cells. Furthermore, T lymphocytes expressing both CCR4 and a chimeric antigen receptor directed to the HL associated antigen CD30 sustain their cytotoxic function and cytokine secretion in vitro, and produce enhanced tumor control when infused intravenously in mice engrafted with human HL. This approach may be of value in patients affected by HL.

Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety

T lymphocytes expressing a chimeric antigen receptor (CAR) targeting the CD19 antigen (CAR.19) may be of value for the therapy of B-cell malignancies. Because the in vivo survival, expansion and anti-lymphoma activity of CAR.19+ T cells remain suboptimal even when the CAR contains a CD28 costimulatory endodomain, we generated a novel construct that also incorporates the interleukin-15 (IL-15) gene and an inducible caspase-9-based suicide gene (iC9/CAR.19/IL-15). We found that compared with CAR.19+ T cells, iC9/CAR.19/IL-15+ T cells had: (1) greater numeric expansion upon antigen stimulation (10-fold greater expansion in vitro, and 3- to 15-fold greater expansion in vivo) and reduced cell death rate (Annexin-V+/7-AAD+ cells 10±6% for iC9/CAR.19/IL-15+ T cells and 32±19% for CAR.19+ T cells); (2) reduced expression of the programmed death 1 (PD-1) receptor upon antigen stimulation (PD-1+ cells <15% for iC9/CAR.19/IL-15+ T cells versus >40% for CAR.19+ T cells); and (3) improved antitumor effects in vivo (from 4.7- to 5.4-fold reduced tumor growth). In addition, iC9/CAR.19/IL-15+ T cells were efficiently eliminated upon pharmacologic activation of the suicide gene. In summary, this strategy safely increases the anti-lymphoma/leukemia effects of CAR.19-redirected T lymphocytes and may be a useful approach for treatment of patients with B-cell malignancies.

Interleukin-7 Mediates Selective Expansion of Tumor-redirected Cytotoxic T Lymphocytes (CTLs) without Enhancement of Regulatory T-cell Inhibition

Purpose: The antitumor activity of chimeric antigen receptor (CAR)–redirected CTLs should be enhanced if it were possible to increase their proliferation and function after adoptive transfer without concomitantly increasing the proliferation and function of regulatory T cells (Treg). Here, we explored whether the lack of IL-7Rα in Treg can be exploited by the targeted manipulation of the interleukin-7 (IL-7) cytokine–cytokine receptor axis in CAR-engrafted Epstein–Barr Virus–specific CTLs (EBV-CTLs) to selectively augment their growth and antitumor activity even in the presence of Treg. Experimental Design: We generated a bicistronic retroviral vector encoding a GD2-specific CAR and the IL-7Rα subunit, expressed the genes in EBV-CTLs, and assessed their capacity to control tumor growth in the presence of Treg in vitro and in vivo when exposed to either interleukin-2 (IL-2) or IL-7 in a neuroblastoma xenograft. Results: We found that IL-7, in sharp contrast with IL-2, supports the proliferation and antitumor activity of IL-7Rα.CAR-GD2+ EBV-CTLs both in vitro and in vivo even in the presence of fully functional Treg. Conclusions: IL-7 selectively favors the survival, proliferation, and effector function of IL-7Rα-transgenic/CAR-redirected EBV-CTLs in the presence of Treg both in vitro and in vivo. Thus, IL-7 can have a significant impact in sustaining expansion and persistence of adoptively CAR-redirected CTLs. Clin Cancer Res; 20(1); 131–9. ©2013 AACR.

Interleukin 15 Provides Relief to CTLs from Regulatory T Cell–Mediated Inhibition: Implications for Adoptive T Cell–Based Therapies for Lymphoma

Purpose: Systemic administration of recombinant interleukin (IL)-2 is used to support the expansion and persistence of adoptively transferred antigen-specific CTLs in patients with cancer. However, IL-2 also expands regulatory T cells (Treg) that in turn impair the antitumor activity of CTLs. As recombinant IL-15 is approaching clinical applications, we assessed the effects of this cytokine on the proliferation and antitumor activity of CTLs in the presence of Tregs. We used the model of adoptive transfer of Epstein–Barr virus (EBV)-CTLs, as these cells induce responses in patients with EBV-associated Hodgkin lymphoma, and Tregs are frequently abundant in these patients. Experimental Design: Tregs were isolated from the peripheral blood of healthy donors and patients with Hodgkin lymphoma or from Hodgkin lymphoma tumors and assessed for their ability to inhibit the proliferation and antitumor activity of EBV-CTLs in the presence of IL-15 or IL-2. Specific molecular pathways activated by IL-15 were also explored. Results: We found that in the presence of Tregs, IL-15, but not IL-2, promoted the proliferation, effector function, and resistance to apoptosis of effectors T cells and EBV-CTLs. IL-15 did not reverse or block Tregs but instead preferentially supported the proliferation of CTLs and effector T cells as compared with Tregs. Conclusions: IL-15 selectively favors the survival, proliferation, and effector function of antigen-specific CTLs in the presence of Tregs, and thus IL-15, unlike IL-2, would have a significant impact in sustaining expansion and persistence of adoptively transferred CTLs in patients with cancer, including those infused with EBV-CTLs for treatment of EBV-associated malignancies. Clin Cancer Res; 19(1); 106–17. ©2012 AACR.

Robust and cost effective expansion of human regulatory T cells highly functional in a xenograft model of graft versus host disease

The low frequency of naturally occurring regulatory T cells (nTregs) in peripheral blood and the suboptimal protocols available for their ex vivo expansion limit the development of clinical trials based on the adoptive transfer of these cells. We have, therefore, generated a simplified, robust and cost-effective platform for the large-scale expansion of nTregs using a gas permeable static culture flask (G-Rex) in compliance with Good Manufacturing Practice. More than 109 putative Tregs co-expressing CD25 and CD4 molecules (92±5%) and FoxP3 (69±19%) were obtained within 21 days of culture. Expanded Tregs showed potent regulatory activity in vitro (80±13% inhibition of CD8+ cell division) and in vivo (suppression or delay of graft-versus-host disease in a xenograft mouse model) indicating that the cost-effective and simplified production of nTregs we propose will facilitate the implementation of clinical trials based on their adoptive transfer.