Anthony Bais

Suppression of human glioma xenografts with second-generation IL13R-specific chimeric antigen receptor-modified T cells.

Purpose: Glioblastoma multiforme (GBM) remains highly incurable, with frequent recurrences after standard therapies of maximal surgical resection, radiation, and chemotherapy. To address the need for new treatments, we have undertaken a chimeric antigen receptor (CAR) “designer T cell” (dTc) immunotherapeutic strategy by exploiting interleukin (IL)13 receptor α-2 (IL13Rα2) as a GBM-selective target. Experimental Design: We tested a second-generation IL13 “zetakine” CAR composed of a mutated IL13 extracellular domain linked to intracellular signaling elements of the CD28 costimulatory molecule and CD3ζ. The aim of the mutation (IL13.E13K.R109K) was to enhance selectivity of the CAR for recognition and killing of IL13Rα2+ GBMs while sparing normal cells bearing the composite IL13Rα1/IL4Rα receptor. Results: Our aim was partially realized with improved recognition of tumor and reduced but persisting activity against normal tissue IL13Rα1+ cells by the IL13.E13K.R109K CAR. We show that these IL13 dTcs were efficient in killing IL13Rα2+ glioma cell targets with abundant secretion of cytokines IL2 and IFNγ, and they displayed enhanced tumor-induced expansion versus control unmodified T cells in vitro. In an in vivo test with a human glioma xenograft model, single intracranial injections of IL13 dTc into tumor sites resulted in marked increases in animal survivals. Conclusions: These data raise the possibility of immune targeting of diffusely invasive GBM cells either via dTc infusion into resection cavities to prevent GBM recurrence or via direct stereotactic injection of dTcs to suppress inoperable or recurrent tumors. Systemic administration of these IL13 dTc could be complicated by reaction against normal tissues expressing IL13Ra1. Clin Cancer Res; 18(21); 5949–60. ©2012 AACR.

Phase I trial of anti‐PSMA designer CAR‐T cells in prostate cancer: Possible role for interacting interleukin 2‐T cell pharmacodynamics as a determinant of clinical response

Chimeric antigen receptor (CAR)‐modified “designer” T cells (dTc, CAR‐T) against PSMA selectively target antigen‐expressing cells in vitro and eliminate tumors in vivo. Interleukin 2 (IL2), widely used in adoptive therapies, was proven essential in animal models for dTc to eradicate established solid tumors.

Sorting vector producer cells for high transgene expression increases retroviral titer.

Vector producer cells are derived from helper cell lines expressing viral proteins that have been transduced to express a transgene-carrying retroviral genome. Vector producing cells express two relevant forms of RNA in their cytoplasm: vector RNA (vRNA) that is packaged as the actual gene transfer agent, and messenger RNA (mRNA) from which transgene is translated. Two premises underlie this study: (1) vRNA is limiting for virus production and (2) mRNA is proportional to vRNA. Together, these premises predict that transgene expression in the vector producing cells will be predictive of the viral titer from those cells. In this case, sorting the vector producing cells for high transgene expression should select for more virus production in vector producing cell supernatants. This prediction was supported, with a greater than fivefold benefit in viral titer. This demonstrates a rapid and simple method by which to obtain significantly increased viral titers from the same vector producing cell preparation.

Abstract C13: Phase I trial of anti-PSMA designer T cells in advanced prostate cancer

Introduction: We created chimeric antigen receptors (CAR) specific for prostate specific membrane antigen (PSMA). When expressed in patient T cells, these “designer T cells” (dTc) specifically kill prostate cancer cells in vitro and in vivo in animal models (Ma et al. Prostate 2004:61:12-25). A Phase I clinical trial was approved by the FDA under BB-IND 12084. With the recent publicity surrounding use of this technology to suppress and potentially cure CLL [Porter et al. NEJM 2011:365:725-33], the current aim to apply dTc in metastatic HRPC gains a heightened impetus and significance. Methods: Patient T cells are retrovirally transduced and expanded ex vivo to span the dose range of 10⁁9 to 10⁁11 T cells. Patients undergo prior non-myeloablative (NMA) conditioning to create a “hematologic space” into which dTc are infused for stable engraftment and improved in vivo efficacy. Patients are co-administered continuous infusion IL2. Patients are monitored for safety and response. Outcomes include Phase Ia goals of safety and toxicity and Phase Ib goals of establishing an optimal biologic dose in terms of dTc engraftment and tumor response. Results: To date five subjects have been treated, three at the 10⁁9 cell dose and two at the 10⁁10 cell dose. Excellent T cell modifications of 30-60% were obtained. After NMA conditioning, T cells were infused and stable engraft—ments of 1-20% were observed one-month post recovery, thus affirming one of the study end-points, even at the lowest 10⁁9 dose level, reflecting near 100-fold expansions of dTc in vivo. Two of five patients (40%) had PSA reductions of 50 and 70% in the two months following treatment, whereas four patients (80%) had delays in PSA progression of 2-5 months. Patients experienced neutropenia and lymphopenia after conditioning, but no designer T cell related toxicities. Patient treatments will continue under the dose escalation plan. Conclusion: A new approach to adoptive immune therapy in metastatic prostate cancer has been devised with exciting early results. We postulate that the greater potency of higher dTc doses under adequate IL2 support will induce PSA reductions of 100%, potentially with durable remissions of metastatic prostate cancer that is refractory to all other treatments. Patients are being actively recruited. For referrals, contact by phone: (401) 456-2507 or email: RDavies{at}rwmc.org. This clinical trial received partial funding from US Army/DOD. Preclinical work was supported by the Prostate Cancer Foundation. Note: This abstract was not presented at the conference because the presenter was unable to attend. Citation Format: Richard P. Junghans, Qiangzhong Ma, Ritesh Rathore, Robin Davies, Anthony Bais, Erica Gomes, Ryan Harvey, Steven I. Cohen. Phase I trial of anti-PSMA designer T cells in advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C13.

Recombination–deletion between homologous cassettes in retrovirus is suppressed via a strategy of degenerate codon substitution

Transduction and expression procedures in gene therapy protocols may optimally transfer more than a single gene to correct a defect and/or transmit new functions to recipient cells or organisms. This may be accomplished by transduction with two (or more) vectors, or, more efficiently, in a single vector. Occasionally, it may be useful to coexpress homologous genes or chimeric proteins with regions of shared homology. Retroviridae include the dominant vector systems for gene transfer (e.g., gamma-retro and lentiviruses) and are capable of such multigene expression. However, these same viruses are known for efficient recombination–deletion when domains are duplicated within the viral genome. This problem can be averted by resorting to two-vector strategies (two-chain two-vector), but at a penalty to cost, convenience, and efficiency. Employing a chimeric antigen receptor system as an example, we confirm that coexpression of two genes with homologous domains in a single gamma-retroviral vector (two-chain single-vector) leads to recombination–deletion between repeated sequences, excising the equivalent of one of the chimeric antigen receptors. Here, we show that a degenerate codon substitution strategy in the two-chain single-vector format efficiently suppressed intravector deletional loss with rescue of balanced gene coexpression by minimizing sequence homology between repeated domains and preserving the final protein sequence.