Children s Hospital of Philadelphia Annual Progress Report: 2011 Formula Grant

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1 Children s Hospital of Philadelphia Annual Progress Report: 2011 Formula Grant Reporting Period July 1, 2012 June 30, 2013 Formula Grant Overview The Children s Hospital of Philadelphia received $3,521,179 in formula funds for the grant award period January 1, 2012 through December 31, Accomplishments for the reporting period are described below. Research Project 1: Project Title and Purpose Highly Active Cell Therapy of Cancer Our purpose is to develop engineered T cell therapies for B cell malignancies, leukemias, and certain specific solid tumors such as neuroblastoma and synovial sarcoma. Using chimeric antigen receptors (CARs) which target tumors and activate T cells, and an efficacious clinical-grade (GMP) ex vivo cell manufacturing system, we will continue our highly promising use of CAR-engineered T cells. This grant will support preclinical studies to optimize CARs in mouse xenograft models, as well as early phase clinical trials testing a variety of CAR-mediated T cell therapy approaches. Anticipated Duration of Project 1/1/ /31/2015 Project Overview The overall goal of this Pennsylvania Department of Health Formula Project is to develop clinically efficacious methods of treating high-risk and relapsed leukemia, lymphoma and some solid tumors with chimeric antigen receptor (CAR)-armed T cells. The long-term goal of our cell therapy group is to establish improved treatments for hematologic malignancies and other tumors by engineering, optimizing, and clinically testing these highly active anti-cancer T cells. This treatment approach could potentially obviate the need for allogeneic stem cell transplant for some patients. Our preliminary clinical data showing cures of patients with high burdens of refractory tumor provide the first clear proof of concept and proof of mechanism for an anticancer cell therapy, and are potentially paradigm-shifting. They suggest that the central problems of expansion and persistence of therapeutic cells in the patient after infusion are solvable using the right cell manufacturing system and the right CAR design. With successful expansion and long-term persistence in the patient, even small doses of T cells can lyse very large tumor burdens. In order to leverage the dramatic results we have seen and continue to develop these approaches for pediatric cancer patients, we are proposing a 4 year combined basic/translational Children s Hospital of Philadelphia 2011 Formula Grant Page 1

2 cell therapy program for CHOP. We propose 3 Aims to build on these results and establish cell therapy infrastructure at CHOP: Aim 1. Develop novel CARs targeting antigens other than CD19, and develop RNA transfection as an alternative approach to lentiviral transduction to temporarily express CARs on T cells. Aim 2. Perform 3 cell therapy trials testing CD19-targeted CAR+ T cell approaches in patients with B cell malignancies such as acute lymphoblastic leukemia (ALL), CLL and non-hodgkin lymphoma (NHL). Aim 3. Using CARs and approaches developed in Aim 1, take engineered T cell-based therapy into trials enrolling non-b cell cancers, including sarcoma (target NY-ESO-1), acute myelogenous leukemia (AML; target mesothelin), and neuroblastoma (target GD2). Principal Investigator Stephan A. Grupp, MD, PhD Professor of Pediatrics The Children s Hospital of Philadelphia 3501 Civic Center Blvd. Philadelphia, PA Other Participating Researchers David Barrett, MD PhD employed by The Children s Hospital of Philadelphia Carl H. June, MD; Bruce L. Levine, PhD; Yangbing Zhao, MD PhD employed by the University of Pennsylvania Expected Research Outcomes and Benefits Expected outcomes and benefits include the following: 1. Initiate a trial to test CAR-T cell therapy in children with CD19+ malignancies. CARs are chimeric antigen receptors that redirect T cells to cancer cells and activate the T cells so they kill the tumor. Any cancer target or tumor-associated antigen which is recognized by an antibody can in principle be made into a CAR. 2. Develop mrna-based CARs to supplement permanent modification of T cell by lentivirus. We anticipate gaining a better understanding of the performance of mrna CAR+ T cells in animal models that will guide our use of them in the clinic. 3. A better understanding of the nature and phenotype of long-term engrafting T cells and their impact on disease control in animal models. 4. Extend the CD19 CAR concept to mrna CARs and to the allogeneic transplant setting. 5. Develop CARs that can target non B cell tumor antigens, to test CARs against AML and solid tumors. 6. Further develop clinically relevant xenograft models of pediatric cancer, translating data from these models to clinical trial design. Children s Hospital of Philadelphia 2011 Formula Grant Page 2

3 7. The overall goal is improve treatment options for patients with high-risk and relapsed cancers. Summary of Research Completed Project milestone(s) for the period 7/1/2012-6/30/2013: 1. Have 2 cell therapy trials open and accruing 2. Accrue patients to 2 cell therapy trials 3. Collect biology samples on each treated/infused cell therapy patient We completed milestones 1-3. The long-term goal of our the CHOP Cell Therapy Group (CCTG) is to establish improved treatments for leukemias and other tumors by engineering, optimizing, and clinically testing highly active anti-cancer T cells. This treatment approach could potentially replace expensive and risky bone marrow transplants for some patients. CCTG has made significant progress over the past year in both clinical trials and lab work exploring T cells engineered with chimeric antigen receptors (CARs; Figure 1A). Our work, supported by the CURE grant, has been directed toward testing the hypothesis that pediatric cancers can be targets for CAR-engineered T cells. Aim 1. Develop novel CARs targeting antigens other than CD19, and develop RNA transfection as an alternative approach to lentiviral transduction to temporarily express CARs on T cells. Aim 1 Progress: The researchers continue to work with engineered T cells that target neuroblastoma (NB), focusing on 2 targets proposed in the grant, GD2 and NY-ESO. We have seen excellent anti-tumor effects with an anti-gd2 CAR, and have continued to optimize the RNA transfection system. Using the CD19 CAR system as the gold standard, recent experiments by the CHOP Cell Therapy Group (CCTG) show that multiple infusions of RNAmodified T cells, which temporarily express CARs, can approximate the efficacy of permanently modified lentiviral CARs. These data are now published (Barrett, DM, X Liu, S Jiang, CH June, SA Grupp *, and Y Zhao* Regimen specific effects of RNA-modified chimeric antigen receptor T cells in mice with advanced leukemia. Human Gene Therapy), so they will not be summarized in detail here. The important point of these studies is that 3 infusions of RNA CARs, given with interval lymphodepletion with Cytoxan (CTX; this is a key requirement), can cure xenografted mice of human ALL (Figure 2). This result is immediately translatable and will drive the design of upcoming RNA CAR trials. One of the patients (see Aim 2) CCTG has treated went into a complete remission (CR) but then relapsed with CD19 negative disease. Analysis of the blasts of this patient showed a small CD19- negative population which then provided the seeds of a CD19-negative recurrence (Figure 1C). Importantly, CART19 (CAR T cells directed against CD19 for B cell malignancies) therapy can be modeled in NSG mice using human ALL and human T cells, both of which are available from the patients (milestone 3). The animals typically receive by tail vein injection 10 6 B-ALL cells expressing firefly luciferase, which results in 100% engraftment in the bone marrow ( Day 0 time point in Figure 1B). Mice are then given 7x10 6 CART19 cells, which eliminates the B- ALL-derived luminescent signal after 72 hours ( Day 3 time point in Figure 1B, compare to Children s Hospital of Philadelphia 2011 Formula Grant Page 3

4 tumor load in saline-treated mice). There was also statistically significant prolongation in survival and cures in most of the mice. CCTG is using this animal model to study relapse of human disease by reconstituting mice with pre-and post-cart19 leukemia specimens. Specifically, the experiment used the CD19-positive pre-cart19 leukemia with a small CD19- negative fraction ( shoulder in Figure 1C left) and the relapsed CD19-negative leukemia from the same patient (Figure 1C). These specimens were successfully expanded in NSG mice (Figure 1D) and the loss of CD19 expression was confirmed by Western blotting (Figure 1E). Of note, while phosphorylation of Lyn (a component of CD19-mediated signaling) was somewhat reduced, it was still maintained in an apparently CD19-independent fashion. CCTG hypothesizes that this residual activation of Lyn might be responsible for the relapse and could be eliminated with dasatinib (Figure 1F), and this is under exploration, facilitated by the discoveries made in and the samples available from the clinical trials. The CCTG have also moved forward with NY-ESO-1 as a target. In addition to opening a clinical trial of NY-ESO-1 targeted T cells (see Aim 2), the group has begun to validate NY-ESO as a potential target in NB. Human T cells transfected with a high affinity anti-ny-eso TCR were injected, showing good activity in these early experiments (Figure 3). Adding NB patients to the synovial sarcoma trial testing NY-ESO as an immunotherapy target is under discussion. Aim 2. Perform 3 cell therapy trials testing CD19-targeted CAR+ T cell approaches in patients with B cell malignancies such as acute lymphoblastic leukemia (ALL), CLL and non-hodgkin lymphoma (NHL). Over the past year, CCTG has treated the first children on our CART19 trial. Accrual has greatly exceeded expectations and the results are dramatic. The CCTG initiated the trial to treat children with relapsed/refractory ALL, children who were ineligible for Bone Marrow Transplant (BMT) because they could not achieve a remission. Four such patients have been treated (milestone 2). The results from the 1 st two patients demonstrated rapid complete remissions in patients with chemorefractory disease and no other treatment options. These results were recently published in the New England Journal of Medicine (Grupp, SA, M Kalos, D Barrett, R Aplenc, DL Porter, SR Rheingold, DT Teachey, BL Levine, and CH June Induction of complete remissions of ALL by chimeric antigen receptor-expressing T cells. New England Journal of Medicine. 368(16): ). One of these patients remains in CR 15 months after treatment. Given these results, the CCTG proposed to the FDA that we open a second trial, this time to treat ALL patients who had relapsed after allogeneic bone marrow transplant (BMT). This is a group with high unmet medical need, because there is no established, effective therapy for patients who experience such a relapse. In addition, there is the added complication that the T cells collected from the patients after a BMT are of donor origin and might potentially cause graft vs. host disease (GVHD). CCTG hypothesized that allogeneic T cells collected from the patient, rather than the donor, might be well-tolerized and might have less potential to cause GVHD. The FDA accepted this proposal, but requested that we not do this as a second, stand-alone trial, but instead add this new treatment group to the ongoing trial as a second cohort. Their rationale was that this would allow unified toxicity reporting and allow them to see the full picture across all pediatric ALL cell therapy patients at CHOP. This was done, and rather than the planned two Children s Hospital of Philadelphia 2011 Formula Grant Page 4

5 trials, we now have a trial with two cohorts (milestone 1). Cohort 1 is no prior allo BMT, and Cohort 2 is s/p allo BMT. At the same time, the researchers have now opened the proposed synovial sarcoma trial, A Pilot Study of Genetically Engineered NY-ESO-1 Specific (c259) T cells in HLA-A2+ Patients with Synovial Sarcoma, using NY-ESO-1 as a target (milestone 1). This trial is pending accrual of the first patient as of the writing of this report. Given the NY-ESO-1 results reported above in Aim 1, CCTG has proposed to the Sponsor to include neuroblastoma as another tumor to be tested, which would potentially enhance accrual. Status of the clinical trials: Researchers are now accruing children to both allo and no-allo cohorts on CHP-959: PILOT STUDY OF REDIRECTED AUTOLOGOUS T CELLS ENGINEERED TO CONTAIN ANTI- CD19 ATTACHED TO TCRζ AND 4-1BB SIGNALING DOMAINS IN PATIENTS WITH CHEMOTHERAPY RESISTANT OR REFRACTORY CD19+ LEUKEMIA AND LYMPHOMA. As indicated above, the second cell therapy trial proposed in last year s report, using allogeneic CART19 cells, is now accruing as Cohort 2 in the above trial at the FDA s suggestion (milestone 1). We have accrued 4 patients to Cohort 1 (no prior allo BMT) and 10 patients to Cohort 2 (s/p allo BMT; milestone 2). This accrual is well in excess of our anticipated accrual rate, and fortunately the Cell and Vaccine Production Facility at U Penn has been able to ramp up to the point where they can now manufacture up to 3 cell therapy products per month for the CHOP Cell Therapy Group. In these 14 initial patients, the trial results demonstrate a CR rate of 80%. This shows astonishing short-term efficacy in this group of relapsed/refractory pediatric ALL patients. Over the next year, the results will mature from this initial group and it will be possible to assess the ability of a single dose of CART19 T cells to sustain remissions. Importantly, 0 of the 10 allo Cohort 2 patients have developed GVHD, supporting the hypothesis that these tolerized cells have a low risk of this post-bmt complication. Our research sample compliance has been 100% to date (milestone 3). Aim 3 proposes using CARs and approaches developed in Aim 1 to take engineered T cell-based therapy into trials enrolling non-b cell cancers, including sarcoma (target NY-ESO-1), acute myelogenous leukemia (AML; possible target CD123), and neuroblastoma (targets GD2 and NY-ESO-1). Further progress in Aim 1 over the next year will direct the approaches for Aim 3. Children s Hospital of Philadelphia 2011 Formula Grant Page 5

6 Figure 1. Pre-clinical modeling of primary patient leukemias. A. Structure of the CAR targeting the B cell antigen CD19. B. NSG mice with bioluminescent leukemia immediately before and 72 hours after infusion of CART19 cells. C. Primary bone marrow flow cytometry profiles gated on CD45+ blasts from pre- and post- CART19 therapy, demonstrating the emergence of a CD19 negative population. D. Both samples from panel C were successfully engrafted in NSG mice. The neoplastic cells were rendered bioluminescent via lentiviral transfer of the luciferase gene. E. Western blot analysis of CD19 and Lyn expression and phosphorylation in samples from panel D (IR82 and IR243). F. Kaplan-Meier survival curves of NSG mice engrafted with a primary patient sample and treated with vehicle (ctrl) or dasatinib by oral gavage. Children s Hospital of Philadelphia 2011 Formula Grant Page 6

7 Figure 2. Dose splitting impacts efficacy of mrna CAR T cells. A) This is a composite survival of 4 independent experiments (with 3 different donors, n=20) using 10 6 Nalm-6 ALL cells on Day 0 and either a single dose 2.5x10 7 on Day 7, or split infusions on Days 7,14 and 28 with interval CTX lymphodepletion at 60mg/kg IP x1 24 hours prior to RNA CART19 doses 2 and 3. Survival is significantly improved by the (x10 6 RNA CART19 cells) schedule despite the total dose of T cells being equivalent (p= ). The only long term survivors are observed with this dose schedule. B) Compressed T cell infusion schedule further improves efficacy. This composite of 2 experiments with 2 donors demonstrates the significant effect of a compressed infusion schedule of on Days (n=15, p<0.0001). The compressed infusion and CTX schedule did result in some meso-41bbz (irrelevant CAR) treated mice surviving beyond CTX only controls, perhaps reflecting a slight allogeneic effect of this donor against Nalm-6 ALL. The difference approached significance for control vs. meso-41bbz (p=0.09), while the comparison of control vs. anti-cd19 targeted RNA CAR T cells from the same donor was highly significant (p<0.001). Figure 3. Validation of NY-ESO-1 as a target in NB % of NB tumors express NY-ESO-1. The group has developed a high affinity TCR in collaboration with Adaptimmune which recognizes NY-ESO. The NY-ESO+ human NB line NB16 was established as both flank tumors and in a disseminated model. Human T cells lentivirally transfected with NY-ESO hatcr were injected after tumor establishment and the animals followed for tumor size (flank model, left panel) and survival (disseminated model, right panel). Papers published and abstracts presented with CURE grant support. (Papers newly reported this year) 1. Hale, GA, M Arora, KW Ahn, W He, B Camitta, MR Bishop, M Bitan, MS Cairo, K Chan, RW Childs, E Copelan, SM Davies, MA Diaz Perez, JJ Doyle, RP Gale, M Gonzalez Vicent, BN Horn, AA Hussein, S Jodele, NR Kamani, KA Kasow, M Kletzel, HM Lazarus, VA Lewis, KC Myers, R Olsson, M Pulsipher, M Qayed, JE Sanders, PJ Shaw, S Soni, PJ Stiff, EA Stadtmauer, NT Ueno, DA Wall, and SA Grupp Allogeneic hematopoietic cell transplantation for neuroblastoma. Bone Marrow Transplant. Feb 18. doi: /bmt Epub ahead of print. PMID: NIHMS: Feb. 18, Barrett, DM, X Liu, S Jiang, CH June, SA Grupp *, and Y Zhao* Regimen specific effects of RNA-modified chimeric antigen receptor T cells in mice with advanced leukemia. Human Gene Therapy, in press. Accepted June 28, 2013 *equal contribution Children s Hospital of Philadelphia 2011 Formula Grant Page 7