Stephen J. Forman, M.D., F.A.C.P. Research

Adoptive Immunotherapy
Advances in surgery, radiation therapy and chemotherapy over the last decade have increased the cure rates of a variety of malignancies. For patients whose tumors are not eradicated, however, the impediment most frequently encountered is the inability to fully eliminate the minimal residual disease that often has acquired resistance to conventional treatment modalities. An attractive strategy for targeting minimal residual disease is the manipulation of immunologic effector cells to specifically recognize tumor targets. Animal models and an increasing number of clinical trials have implicated the T lymphocyte as a pivotal immunologic effector cell in anti-tumor immunity. This has led to our overall interest in using adoptive immunotherapy of T-cells to target cancer, specifically leukemia/lymphoma, brain tumors and neuroblastoma. Areas of focus in our research program are outlined below.
 
Glioma tumor cells are killed by therapeutic T-cells
Scanning of zetakine redirected cytolytic T-cell lysis of a glioma tumor cell.
 
Genetic Modification of T-cells for Redirected Tumor Recognition
Technologies are now available for identifying immunogenic T-cell target epitopes expressed by human tumors, isolating antigen-specific T-cells and expanding them ex vivo to large numbers for reinfusion. However, to overcome the difficulties of isolating tumor-reactive T-cells from cancer patients, we have developed technology to take T-cells from a cancer patient and reprogram them to target a patient’s cancer through genetic engineering strategies using DNA electroporation and/or lentiviral transduction. Previous studies by our laboratory have provided proof of principle that CTL can be engineered to specifically target lymphoma, neuroblastoma, and glioma tumors via expression of tumor-specific chimeric immunoreceptors resulting in MHC-independent killing of target tumor cells (Cooper et al, Blood 2003; Park et al, Mol Therapy 2007; Kahlon et al, Cancer Res 2004). More recently, we have completed pre-clinical studies of cytotoxic T-cells (CTLs) genetically modified to express an IL13-zetakine chimeric immunoreceptor to target cell-surface IL13Rα2 on medulloblastoma/primitive neuroectodermal tumors, the most common type of brain tumor in children (Stastny et al, J Ped Hematol Oncol 2007). Overall, we are currently involved in studying the potential clinical utility of adoptively transferring T-cells that have been genetically redirected to recognize CD19 (on leukemia and lymphoma); IL13Ra2 (on glioblastoma and medulloblastoma); L1-CAM (on neuroblastoma, lung cancer, and renal cell carcinoma); HER2 (on breast cancer, brain metastases and medulloblastoma); or alpha-3 integrin (on medulloblastoma).
 
 
Murine Xenograft ModelsSurgical suite
To monitor the therapeutic objectives of adoptive T-cell transfer before entering the clinical setting, our lab has developed multiple murine xenograft models. For example, we are now routinely performing stereotactic surgeries in mice for the intracranial injection of human brain tumors to mimic the situation of our patients. In addition, we have also developed lymphoma, neuroblastoma, and medulloblastoma murine xenograft models. Bioluminescent strategies have also been developed by our group for the in vivo imaging of both T-cells and tumors. Our utilization of the firefly and renilla luciferase genes (ffLuc and rLuc, respectively) along with the Xenogen system for serial imaging of anesthetized mice has been critically important for evaluating our animal studies. Specifically, these animal model systems are being used for studying the efficacy of immunotherapy, T-cell trafficking from tail vein injections, contralateral homing of T-cells to tumors within the parenchyma of the brain, and studies of T-cell persistence and proliferation.
 
Trafficking of Adoptively Transferred T-cells
To exert a therapeutic effect, adoptively transferred tumor-specific cytotoxic T-cells (CTLs) must traffic to sites of tumor burden, exit the circulation, and infiltrate the tumor microenvironment. T-cells are able to respond to migration directing chemicals, called chemokines, that are produced by tumor cells. Thus our group has begun to examine the ability of adoptively transferred human CTLs to traffic to tumors. Using a combination of in vivo tumor tropism studies, and in vitro biophotonic chemotaxis assays, we observed that tumors that produce CCL2/MCP1 (>10ng/ml), such as cell lines derived from glioma and medulloblastoma, efficiently chemoattract ex vivo expanded primary human T-cells. These studies suggest that the capacity of adoptively transferred T-cells to home to tumors may be influenced in part by the species and amounts of tumor-derived chemokines, in particular MCP-1 (Brown et al, J Immunol 2007).
 
Targeting Tumor Progenit or Cells with Genetically Engineered Effector T-Cells
Recent hypotheses that tumors arise from cancer stem cells which appear to be resistant to radiation treatment and chemotherapy have also led us to begin to isolate and expand stem cells from human glioma to assess the vulnerability of these cells to T-cell mediated killing in our adoptive immunotherapy models. Our initial studies demonstrate that chimeric immunoreceptor redirected IL13Rα2-specific CTL, currently being evaluated in an FDA-approved pilot Phase I trial for the treatment of recurrent/refractory malignant glioma, can kill cancer stem/progenitor cells in vitro, and reduce engrafted potential of the glioma stem/progenitor population in an orthotopic murine tumor model. Current models now predict that curative therapies for many cancers might require the elimination of the stem/progenitor population, and these studies lay the foundation for an immunotherapy approach to achieve this goal.
 
Development of Strategies to Optimize T-cell Anti-Tumor Activity in Vivo
Our group has also begun to investigate the expression of additional gene products in T-cells that may serve to overcome constraints on T-cell effector function and survival in vivo. For example, we are currently examining the efficacy of providing co-stimulatory signaling to T-cells through integration of costimulatory signaling domains within a tumor targeting chimeric antigen receptor.  Our recent studies in this area demonstrate that integrating costimulation with activation signaling events is important for fully activating CD4+ anti-tumor effector cells, resulting in sustained function in the tumor microenvironment.
 
Another strategy we are currently examining is the utility of genetically modifying T-cells which have first been selected for specificity to viruses such as CMV or EBV, which would allow for endogenous stimulation of these T-cells upon their transfer to patients infected with these viruses that are frequently reactivated in infected hosts. We would also predict that such a combinatorial bispecific T-cell strategy would permit the selective isolation of virus specific memory T-cells as recipients of chimeric antigen receptor genes for improved in vivo persistence following adoptive transfer. In fact, studies on the unique engraftment potential of memory T-cells are also being carried out in primates in collaboration with Dr. Berger at the Fred Hutchison Cancer Research Center in Seattle, WA. Because tumors can avoid immune-mediated elimination by production of immunosuppressive cytokines (e.g., TGF-b), our group is also evaluating strategies by which T-cells may be rendered resistant to such immunosupression. One project is the development of interference RNA sequences that could decrease this susceptibility of T-cells to tumor-mediated suppression. In collaboration with Sangamo Biosciences in Richmond, CA, we are also interested in studying the effects of genetically blocking glucocorticoid sensitivity of the tumor specific T-cells because post-operative brain cancer patients are routinely treated with immunosuppressive steroids (i.e., glucocorticoids) to reduce clinical symptoms of edema.
 
Lastly, another group of projects underway in the lab are examining ways of generating gene products that influence the expression of cytokines such as IL-2 and IL-7 which help promote the survival of T-cells and are critical for generating T-cell memory responses. A new collaboration with Dr. Smolke in the Chemical Engineering Department at CalTech involves the development of RNA regulating systems that are inducible by drugs such as tetracycline to control the expression of cytokines in T-cells which will promote their proliferation and survival.
 
Overall, the recent progress and current directions of Dr. Forman’s research group have generated much enthusiasm and support at City of Hope. The laboratory and office space in the Arnold and Mabel Beckman Center for Cancer Immunotherapeutics & Tumor Immunology is located in the heart of City of Hope. This state-of-the-art center is dedicated to immunotherapy and facilitates collaboration between lab researchers and clinicians, and rapidly moves scientific discoveries from the lab to patients.