Qiangzhong Ma

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 Hepatic Immunotherapy for Metastases study of intra-arterial chimeric antigen receptor modified T cell therapy for CEA+ liver metastases

Purpose: Chimeric antigen receptor–modified T cells (CAR-T) have demonstrated encouraging results in early-phase clinical trials. Successful adaptation of CAR-T technology for CEA-expressing adenocarcinoma liver metastases, a major cause of death in patients with gastrointestinal cancers, has yet to be achieved. We sought to test intrahepatic delivery of anti-CEA CAR-T through percutaneous hepatic artery infusions (HAIs). Experimental Design: We conducted a phase I trial to test HAI of CAR-T in patients with CEA+ liver metastases. Six patients completed the protocol, and 3 received anti-CEA CAR-T HAIs alone in dose-escalation fashion (108, 109, and 1010 cells). We treated an additional 3 patients with the maximum planned CAR-T HAI dose (1010 cells × 3) along with systemic IL2 support. Results: Four patients had more than 10 liver metastases, and patients received a mean of 2.5 lines of conventional systemic therapy before enrollment. No patient suffered a grade 3 or 4 adverse event related to the CAR-T HAIs. One patient remains alive with stable disease at 23 months following CAR-T HAI, and 5 patients died of progressive disease. Among the patients in the cohort that received systemic IL2 support, CEA levels decreased 37% (range, 19%–48%) from baseline. Biopsies demonstrated an increase in liver metastasis necrosis or fibrosis in 4 of 6 patients. Elevated serum IFNγ levels correlated with IL2 administration and CEA decreases. Conclusions: We have demonstrated the safety of anti-CEA CAR-T HAIs with encouraging signals of clinical activity in a heavily pretreated population with large tumor burdens. Further clinical testing of CAR-T HAIs for liver metastases is warranted. Clin Cancer Res; 21(14); 3149–59. ©2015 AACR.

Anti-GD3 Chimeric sFv-CD28/T-Cell Receptor ζ Designer T Cells for Treatment of Metastatic Melanoma and Other Neuroectodermal Tumors

Purpose: The aims of this study are to compare antitumor activities of two generations of GD3-specific chimeric antigen receptors (CAR) in human primary T lymphocytes in vitro and to evaluate the antitumor efficacy of using a combination of systemic infusion of interleukin-2 (IL2) and designer T cells to eradicate subcutaneous established GD3+ melanoma in nude mice. Experimental Design: Antitumor activities were compared for two generations of designer T cells, the progenitor first-generation with immunoglobulin T-cell receptor (TCR) with Signal 1 and the second-generation designer T cells with Signal 1+2. Osmotic IL2 pumps were used to deliver the maximum tolerated dose of IL2 to enhance the antitumor effects of designer T cells on subcutaneous established melanoma in nude mice. Results: Melanoma is associated with high expression of ganglioside GD3, which has been targeted with modest effect in antibody therapies. We previously showed that an anti-GD3 CAR (sFv-TCRζ) will recruit T cells to target this non–T-dependent antigen, with potent killing of melanoma cells. Here, we report the addition of a CD28 costimulation domain to create a second-generation CAR, called Tandem for two signals. We show that this Tandem sFv-CD28/TCRζ receptor on T cells confers advantages of improved cytokine secretion, cytotoxicity, proliferation, and clonal expansion on tumor contact versus the same CAR without costimulation. In an adoptive transfer model using established melanoma tumors, designer T cells with CD28 showed a 50% rate of complete remissions but only where IL2 was supplemented. Conclusions: As a reagent for clinical development, the second-generation product is shown to have superior properties to warrant its preference for clinical designer T-cell immunotherapy for melanoma and other tumors. Systemic IL2 was required for optimal activity in an established tumor model. Clin Cancer Res; 16(10); 2769–80. ©2010 AACR.

Coupling CD28 co-stimulation to immunoglobulin T-cell receptor molecules: the dynamics of T-cell proliferation and death.

Immunoglobulin T-cell receptor (IgTCR) molecules are potentially potent immune response modifiers because they allow T cells to bypass tolerance. Tolerance to self antigens has been one of the major barriers to the development of effective adoptive immunotherapies for treating cancer. In vitro studies in several laboratories have shown that cross-linking IgTCR molecules with the target antigen leads to cytolytic activity, cytokine release, and T-cell proliferation in model systems. However, many of these studies have used established T-cell lines rather than normal T cells or indirect assays of cytotoxicity, proliferation, and cytokine release. We have sought to establish the validity of these model systems while developing more effective adoptive immunotherapies using normal human T cells. In the present study the activation of T-cell proliferation after IgTCR cross-linking was evaluated. The results show that, in addition to IgTCR signals, CD28 costimulation is required to induce expansions of normal peripheral blood mononuclear cell–derived T cells. Signals from IgTCR alone can induce transient cell division, but they do not induce the prolonged polyclonal expansions that are characteristic of native immune responses. Very strong IgTCR signals could circumvent the CD28 requirement, but only at levels that are unlikely to be physiologically relevant. CD28 costimulation also suppressed the deletion of tumor-reactive subclones by activation-induced cell death. These studies confirm the importance of CD28 costimulation to the proliferation of IgTCR-modified human T cells, a key feature of an effective, reconstructed antitumor response.

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.

T cell exhaustion and Interleukin 2 downregulation

T cells reactive to tumor antigens and viral antigens lose their reactivity when exposed to the antigen-rich environment of a larger tumor bed or viral load. Such non-responsive T cells are termed exhausted. T cell exhaustion affects both CD8+ and CD4+ T cells. T cell exhaustion is attributed to the functional impairment of T cells to produce cytokines, of which the most important may be Interleukin 2 (IL2). IL2 performs functions critical for the elimination of cancer cells and virus infected cells. In one such function, IL2 promotes CD8+ T cell and natural killer (NK) cell cytolytic activities. Other functions include regulating naïve T cell differentiation into Th1 and Th2 subsets upon exposure to antigens. Thus, the signaling pathways contributing to T cell exhaustion could be linked to the signaling pathways contributing to IL2 loss. This review will discuss the process of T cell exhaustion and the signaling pathways that could be contributing to T cell exhaustion.

Carcinoembryonic antigen-immunoglobulin Fc fusion protein (CEA-Fc) for identification and activation of anti-CEA immunoglobulin-T-cell receptor-modified T cells, representative of a new class of Ig fusion proteins

Chimeric immunoglobulin-T-cell receptor (IgTCR)-modified T cells (“designer T cells”) kill tumor cells based on antibody-redirected recognition of tumor-associated antigen. Anti-carcinoembryonic antigen (CEA) designer T cells have been prepared and applied in adoptive cellular immunotherapy regimens for CEA-positive cancers. A CEA-immunoglobulin Fc (CEA-Fc) fusion protein was created from the A3B3 region of CEA and the Fc portion of human IgG for the purposes of activation and detection of anti-CEA designer T cells. CEA-Fc was expressed at high yield in CHO cells and purified to homogeneity in a single step on a protein A affinity column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that CEA-Fc formed disulfide-linked dimers with a molecular weight of about 170 kDa and a monomer size of 85kDa. The A3B3 CEA component of the CEA-Fc bound to anti-CEA monoclonal antibody MN-14, as well as to the single-chain Fv (sFv) derived from this antibody that was expressed in the IgTCR on the surface of designer T cells. The Fc portion of CEA-Fc was recognized by anti-human IgG Fc antibody and bound by human monocyte Fc receptors. CEA-Fc activated the anti-CEA designer T cells as plate-bound or monocyte-bound form but not as soluble form, as measured by CD69 expression and T-cell proliferation. Our results indicate that the CEA-Fc fusion protein can be used to detect the expression of the anti-CEA IgTCR chimeric receptors on the modified T cells, as well as to serve as an antigen to activate the anti-CEA IgTCR modified T cells. CEA-Fc is the prototype for a new class of antigen-Fc molecules that may significantly augment the analytic and therapeutic goals of adoptive designer T-cell immunotherapies.

Cell Density Plays a Critical Role in Ex Vivo Expansion of T Cells for Adoptive Immunotherapy

The successful ex vivo expansion of a large numbers of T cells is a prerequisite for adoptive immunotherapy. In this study, we found that cell density had important effects on the process of expansion of T cells in vitro. Resting T cells were activated to expand at high cell density but failed to be activated at low cell density. Activated T cells (ATCs) expanded rapidly at high cell density but underwent apoptosis at low cell density. Our studies indicated that low-cell-density related ATC death is mediated by oxidative stress. Antioxidants N-acetylcysteine, catalase, and albumin suppressed elevated reactive oxygen species (ROS) levels in low-density cultures and protected ATCs from apoptosis. The viability of ATCs at low density was preserved by conditioned medium from high-density cultures of ATCs in which the autocrine survival factor was identified as catalase. We also found that costimulatory signal CD28 increases T cell activation at lower cell density, paralleled by an increase in catalase secretion. Our findings highlight the importance of cell density in T cell activation, proliferation, survival and apoptosis and support the importance of maintaining T cells at high density for their successful expansion in vitro.

Advanced generation anti-prostate specific membrane antigen designer T Cells for prostate cancer immunotherapy

Adoptive immunotherapy by infusion of designer T cells (dTc) engineered with chimeric antigen receptors (CARs) for tumoricidal activity represents a potentially highly specific modality for the treatment of cancer. In this study, 2nd generation (gen) anti‐prostate specific membrane antigen (PSMA) dTc were developed for improving the efficacy of previously developed 1st gen dTc for prostate cancer immunotherapy. The 1st gen dTc are modified with chimeric immunoglobulin‐T cell receptor (IgTCR) while the 2nd gen dTc are engineered with an immunoglobulin‐CD28‐T cell receptor (IgCD28TCR), which incorporates a CD28 costimulatory signal for optimal T cell activation.

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.