Summary T cell engineering is a powerful means to rapidly generate anti-tumor T cells. The costimulatory properties of second-generation chimeric antigen receptors (CARs) determine the overall potency of adoptively transferred T cells. Utilizing an in vivo “stress test” to challenge CD19-targeted T cells, we studied the functionality and persistence imparted by 7 different CAR structures providing CD28 and/or 4-1BB costimulation. One configuration, which utilizes two signaling domains (CD28 and CD3ζ) and the 4-1BB ligand, provided the highest therapeutic efficacy, showing balanced tumoricidal function and increased T cell persistence accompanied by an elevated CD8/CD4 ratio and decreased exhaustion. Remarkably, induction of the IRF7/IFNβ pathway was required for optimal anti-tumor activity. Thus, 1928z-41BBL T cells possess strikingly potent intrinsic and immunomodulatory qualities.

Translating the recent success of chimeric antigen receptor (CAR) T cell therapy for hematological malignancies to solid tumors will necessitate overcoming several obstacles, including inefficient T cell tumor infiltration and insufficient functional persistence. Taking advantage of an orthotopic model that faithfully mimics human pleural malignancy, we evaluated two routes of administration of mesothelin-targeted T cells using the M28z CAR. We found that intra-pleurally administered CAR T cells vastly out-performed systemically infused T cells, requiring 30-fold fewer M28z T cells to induce long-term complete remissions. Following intrapleural T cell administration, prompt in vivo antigen-induced T cell activation allowed robust CAR T cell expansion and effector differentiation, resulting in enhanced anti-tumor efficacy and functional T cell persistence for 200 days. Regional T cell administration also promoted efficient elimination of extrathoracic tumor sites. This therapeutic efficacy was dependent on early CD4+ T cell activation associated with a higher intra-tumoral CD4/CD8 cell ratios and CD28-dependent CD4+ T cell-mediated cytotoxicity. In contrast, intravenously delivered CAR T cells, even when accumulated at equivalent numbers in the pleural tumor, did not achieve comparable activation, tumor eradication or persistence. The remarkable ability of intrapleurally administered T cells to circulate and persist supports the concept of delivering optimal CAR T cell therapy through “regional distribution centers.” Based on these results, we are opening a phase I clinical trial to evaluate the safety of intrapleural administration of mesothelin-targeted CAR T cells in patients with primary or secondary pleural malignancies.

To enhance the strength of activation afforded by tumor antigen-specific receptors, we investigated the effect of adding combined CD28 and 4-1BB costimulatory signaling domains to a chimeric antigen receptor (CAR) specific for prostate-specific membrane antigen (PSMA). Having transferred receptors encompassing the CD28, 4-1BB, and/or CD3zeta cytoplasmic domains in primary human CD8(+) T cells, we find that the P28BBz receptor, which includes all three signaling domains, is superior to receptors that only include one or two of these domains in promoting cytokine release, in vivo T-cell survival and tumor elimination following intravenous T-cell administration to tumor-bearing severe combined immunodeficient (SCID)/beige mice. Upon in vitro exposure to PSMA, the P28BBZ receptor-induced the strongest PI(3)Kinase/Akt activation and Bcl-X(L) expression, and the least apoptosis in transduced peripheral blood CD8(+) T cells. These findings further support the concept of integrating optimized costimulatory properties into recombinant antigen receptors to augment the survival and function of genetically targeted T cells within the tumor microenvironment.

Radiation-induced inhibition of rapamycin-sensitive pathway and its effect on the cellular response to radiation were studied in the human breast cancer cell line MCF-7. Both radiation and rapamycin shared molecular targets and induced similar physiologic responses. Each of these treatments increased immunostaining of mammalian target of rapamycin (mTOR) in the nucleus, and radiation led to decreased phosphorylation of its autophosphorylation site Ser 2481 . In addition to dephosphorylation of established mTOR downstream effectors 4E-binding protein 1 and p70 ribosomal S6 kinase, both treatments decreased the level of eukaryotic initiation factor 4G. Experiments with the potentiometric dye, JC-1, revealed an oligomycin-dependent increase in mitochondrial membrane potential following radiation or rapamycin treatment, suggesting that both lead to reversal of F 0 F 1 ATPase activity. Both radiation and rapamycin induced sequestration of cytoplasmic material in autophagic vacuoles. In both cases, appearance of autophagic vacuoles involved the participation of microtubule-associated protein 1 light chain 3 (LC3). Transient cotransfection of green fluorescent protein-LC3 with either wild-type or dominant-negative mTOR further showed that inactivation of mTOR pathway is sufficient to induce autophagy in these cells. Finally, administration of rapamycin in combination with radiation led to enhanced mitochondria hyperpolarization, p53 phosphorylation, and increased cell death. Taken together, these experiments show that radiation-induced inhibition of rapamycin-sensitive pathway in MCF-7 cells causes changes in mitochondria metabolism, development of autophagy, and an overall decrease in cell survival. (Cancer Res 2005; 65(23): 11061-70)