University of Pennsylvania Annual Progress Report: 2009 Nonformula Grant

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1 University of Pennsylvania Annual Progress Report: 2009 Nonformula Grant Reporting Period July 1, 2012 June 30, 2013 Nonformula Grant Overview The University of Pennsylvania received $4,620,420 in nonformula funds for the grant award period June 1, 2010 through May 31, Accomplishments for the reporting period are described below. Research Project: Project Title and Purpose Novel Adjuvants for Cancer Vaccine Immunotherapy - The purpose is to conduct a series of thematically related projects to test new cancer vaccine approaches. The projects will include a clinical trial in patients with lung and ovarian cancer, and basic, translational and pre-clinical investigation at three institutions to encourage collaboration. A programmatic effort to promote technology transfer in cancer vaccine research in Pennsylvania is included. Finally, there will be an innovative program to develop a pipeline of new scientists and clinicians trained in cancer research in the Commonwealth which will involve outreach in Philadelphia and Lincoln University. Anticipated Duration of Project 6/1/2010-5/31/2014 Project Overview The overall research objectives of this program are: 1. To prolong survival and reduce mortality of patients with ovarian and lung cancer by enhancing T cell activation in the tumor microenvironment. 2. To support cancer vaccine research and training throughout eastern Pennsylvania. 3. To provide a training and mentoring program in translational cancer research for underrepresented minorities. 4. To promote technology transfer and the potential for job growth within the Commonwealth in the biotechnology of cancer vaccines. We propose the following specific research aims to accomplish the general objectives of this program: 1. Establish the safety, antitumor activity and optimum biologic dose of a bispecific antibody that targets CD326 (epithelial cell adhesion molecule (EpCAM)) with the first arm and anti-cd3 (MT110) with the second arm in a phase I/II clinical trial. (Project 1: CD326 (EpCAM) based University of Pennsylvania 2009 Nonformula Grant on Cancer Vaccines Page 1

2 immunotherapy in patients with ovarian and lung cancer ) 2. Determine whether or not the inclusion of specificities for costimulatory molecules or antagonists of inhibitory receptors will enhance the activity of the bispecific T cell engager platform. (Project 2: Engineering Second Generation BiTE antibodies ) 3. Explore the role of tumor endothelial marker-1 (TEM1) vaccination targeting the tumor microvasculature with combination therapies that antagonize vascular endothelial growth factor (VEGF). (Project 3: Enhancing Cancer Immunotherapy by Immune Attack of the Tumor Vasculature ) 4. Determine whether or not the inclusion of adjuvants that augment the Th17 arm of the cellular immune system enhances antitumor effects. (Project 4: ICOSL based tumor vaccines ) 5. Establish an educational program for undergraduate and graduate level training in translational cancer research. (Translational Research Fellowship Program) Principal Investigator Carl H. June, MD Professor University of Pennsylvania 421 Curie Blvd Philadelphia, PA Other Participating Researchers Robert H. Vonderheide, MD, DPhil, Michael Kalos, PhD, George Coukos, MD, PhD, Andrea Facciabene, PhD, Yangbing Zhao, MD, PhD, Nicole Aqui, MD, Richard G. Carroll, PhD, Elizabeth Veloso, RN, JD employed by University of Pennsylvania Hossein Borghaei, DO, Gregory P. Adams, PhD, Matthew K. Robinson, PhD employed by Fox Chase Cancer Center John O. Chikwem, PhD employed by Lincoln University Expected Research Outcomes and Benefits This program in cancer vaccines will produce both short term and long term benefits. In the near term, the program will enable the conduct of a promising clinical trial for patients with lung and ovarian cancer, thereby providing scientific information on the ability of CD326 to serve as a vaccine target using bispecific T cell engager antibody technology. As a result of the interinstitutional collaborative research efforts, long term benefits will continue to accrue for patients in eastern Pennsylvania who will benefit by improved access to state of the art trials in cancer vaccine research. At the same time, the development of new approaches to harness the power of the immune system to attack tumor cells and the tumor microenvironment will be pursued in the laboratory. A world-class group of clinicians and scientists has been assembled to address this vital problem and will synergize to develop new cancer therapies. In summary, this Program will allow outstanding scientists, educators and clinicians to leverage the power and potential of the immune system to develop new cancer vaccines with improved efficacy and reduced toxicity. University of Pennsylvania 2009 Nonformula Grant on Cancer Vaccines Page 2

3 In addition to research outcomes for cancer therapy, this project contains a significant focus on teaching and education in order to produce a durable result for years to come. We will reach out to the City of Philadelphia, and to college students at Lincoln University to enhance the pipeline of new investigators trained in this promising scientific field, while providing role models and unparalleled opportunities for aspiring under-represented minority students. Summary of Research Completed Specific Aim 1. During the last year, our team efforts were focused on opening the clinical study testing safety and feasibility of the cmet CAR RNA eletroporated T cells by intratumoral injection in breast cancer subjects. The study went through several revisions by the University of Pennsylvania (UPenn) regulatory committees which led to a modified study design and addition of a new patient population for an increased safety design. Thus, in addition to testing the safety of the cmet CAR T cells in triple negative breast cancer subjects, we have included testing the safety of this approach in metastatic breast cancer patients. The study was opened for enrollment and a first metastatic breast cancer subject enrolled. Specific Aim 2. The overarching goal of this aim is to evaluate the therapeutic impact of 2 nd generation BiTE (bispecific T-cell engager) molecules that recruit and activate T cells through interactions with one of three different receptors (ICOS, PD-1, CTLA4) present on the surface of T cells. Work in the past year has focused both on the expression/characterization of the T cell binding single chain Fv (scfv) antibodies and on construction/expression of the BiTE molecules. A panel of scfv against PD1 and CTLA4 were expressed and purified from HEK293 cell system and sent to colleagues at UPenn for analysis of biological function (Figure 1 and data not shown). The previously isolated anti-pd1 scfv failed to elicit three biomarkers of T cell activation (IFNγ production, IL-2 production and proliferation) as compared to a control antibody. To overcome lack of activity seen with the anti-pd1 scfv, we generated a scfv version of an anti-pd1 IgG antibody that has demonstrated the appropriate activity in clinical trials. The CTLA4 scfv being used in our work has been provided to our UPenn colleagues and testing is in progress. This scfv is based upon the FDA-approved antibody ipilumumab so these experiments are being performed to confirm, rather than determine if, the scfv has the appropriate activity. The anti-icos scfv have been purified from mammalian culture and await shipment to our UPenn colleagues for testing. As a control, we have begun constructing an anti-cd3 scfv based on the OKT3 antibody that serves as the T cell binding domain for the 1 st generation BiTE molecules. This will allow for a direct comparison of 1 st vs 2 nd generation BiTE activity. Each class of CTLA4 and PD1 scfv have been cloned into the bispecific scfv mammalian expression vector described last year that is designed to target HER2 as a tumor cell marker. Transfected cells have been shown to express the 2 nd generation BiTEs (Figure 2) and selection of high-expressing stable clones is currently ongoing. Work over the past year has demonstrated that generation of 2 nd generation BiTEs targeting cmet as our Specific Aim 1 study is challenging; this is due to the fact that the most promising class of cmet scfv isolated by phagedisplay exhibited moderate binding to cmet but had stability issues that precluded its use in the bispecific format. Work is underway to identify alternative cmet binders with immediate work University of Pennsylvania 2009 Nonformula Grant on Cancer Vaccines Page 3

4 focusing on the construction of a scfv version of MetMAb, a clinically validated anti-cmet antibody with the appropriate activity when used in a monovalent format. Specific Aim 3. In this third year, we further dissected the roles of the tumor associated antigen (TAA) versus the TEM1 immune responses, improved potency of TEM vaccination by regulatory T cells depletion, and expanded the toxicology safety testing of TEM1 vaccine. This work was described in detail in our manuscript under review by JCI (please note that for publication reasons we changed the name of our vaccine from TEM1-DOM to TEM1-TT). In the previous report we demonstrated that vaccination against tumor vasculature/stroma in TC1 as well CT26 models resulted in induction of anti-taa immune responses. This year, in order to dissect the anti-tumor impact of TEM1 versus TAA-specific responses, we performed adoptive cell transfers of CD4, CD8 and total CD3 T cells from tumor bearing and tumor-free TEM1-TT immunized mice into naïve mice injected with tumor CT26 cells. Our results indicate that both TEM1-specific CD8 T cells and combined TEM1 and TAA-specific CD8 T cells improved survival of tumor bearing mice (Figure 3), and demonstrate the effectiveness of TEM1-TT immune response alone and its synergy with the anti-taa responses mounted by epitope spreading. Immunohistochemisty on CT26 tumor sections indicates decreased proliferation (Ki67 staining) and increased apoptosis (tunel staining) in specimens from TEM1-TT vaccinated compared with control treated mice. In an attempt to improve the vaccination potency, we tested the impact of regulatory T cells (Treg) depletion on TEM1-TT vaccination. C57b/6 mice were depleted of Treg by i.p. injection of the PC61 clone of the anti-cd25 monoclonal antibody and 4 days later, they were immunized with TEM1-TT. The response to the immune dominant mouse TEM peptide was assessed from mice splenocytes by ELISpot IFNγ secretion. Our results indicate that Treg depletion potentiated the TEM1-TT immune response and may impact the strategy for transitioning into the clinic. Because antiangiogenic agents may raise safety concerns, we expanded the pregnancy toxicology studies by testing the reproductive endocrine functions following TEM1-TT vaccination. Mice with synchronized estrous cycles were vaccinated, and serum levels of 17β-estradiol, progesterone, luteinizing hormone, and follicle stimulating hormone were measured longitudinally and found to be similar between TEM1-TT and control vaccinated groups. To ensure that pregnancy did not affect the effectiveness of TEM1-TT vaccination, splenocytes of vaccinated pregnant and non-pregnant mice were tested against TEM peptide or control peptide using IFN- ELISpot. We found that there was significant recognition of TEM peptide by TEM1-TT vaccinated splenocytes from both mice groups. Collectively, these data indicate that TEM1-TT vaccination does not affect the estrus cycle, has no untoward effects on physiological processes that depend on de novo angiogenesis, and is unaltered by pregnancy supporting the safety of the approach for advancement into clinical testing. Specific Aim 4. In the previous year, we showed that incorporation of ICOS intracellular domain in a chimeric antigen receptor (CAR) specific for mesothelin (ss1) tumor antigen promote the Th17 phenotype after antigen encounter, enhance the in vivo antitumor activity of redirected T cells, and T cells persistence in a mesothelioma tumor mouse model. A major issue currently facing the field of adoptive therapy is defining the T cells subsets that are most potent in mediating an antitumor effect when redirected with tumor specific CARs. In order to determine University of Pennsylvania 2009 Nonformula Grant on Cancer Vaccines Page 4

5 the optimal T cell subsets for engraftment and efficacy, we analyzed the in vivo antitumor activity of retargeted Th17/Tc17 cells versus non-polarized Th1/Tc1 cells redirected with ss1- specific CARs. NSG mice were injected in the flank with a fast growing tumor expressing the ss1 antigen, the non-small cell lung tumor cell line, L55. After 3 weeks, when the tumors reached a volume of 150 mm 3, mice were treated with 2 i.v. injections of 10 7 Th17 /Tc17 cells (1:1 mixture) redirected with ss1-icosz or control ss1-delz, or 10 7 Th0 /Tc0 cells redirected with ss1-icosz (n=7-10). Non-polarized cells redirected with ss1-icosz mediated a significantly better anti-tumor effect than Th17/Tc17 cells, with most tumors gone 3 days after the administration of the second dose (Figure 4A); however, non-polarized cells did not persist, and T cells were detectable in very low numbers in the blood (Figure 4B) and in the spleens (Figure 4C) of treated mice. By contrast, Th17/Tc17 cells redirected with ss1-icosz were not able to eliminate the tumor, but they showed an enhanced persistence when compared to the other groups. We hypothesize that differences seen in the antitumor activity are due to the effect of the CD8 + T cells, but that under these conditions (fast growing tumors), we were unable to analyze the effect of the CD4 + in tumor rejection, or the effect of T cell persistence. New experiments to evaluate the role of CD4+ T cells in tumor rejection are underway. Specific Aim 5. The objectives of the Minority Training Core are to provide undergraduate and graduate students with an introduction and the basic tools necessary to pursue careers in translational research. Each student participates in an intensive, supervised research experience with mentors involved in some aspect of translational research, from bench work to clinical trials. As part of this goal, we established both a Summer Undergraduate Internship and a monthly seminar series for undergraduate students at Lincoln University during the academic year. The seminar series at Lincoln University has been completed for this academic year. Our undergraduate program received two applications for the Both applicants were excellent, and both were accepted for internships at UPenn. Danielle Mathis from Lincoln University has been placed in the laboratory of Dr. Andrea Facciabene. The goal of her project is to improve the immunogenicity of a DNA vaccine (Tumor Endothelial Marker-1, TEM1) by a gene expression modifying approach. Danielle is now sub-cloning the mouse vaccine into a new vaccine that will be able to target the human form of TEM1. These vaccines will be subsequently tested in humanized HLA-A2 mice to assess efficacy, before being put into clinical trials. Conner Paez from UPenn is working with Dr. Bruce Sachais to study heparin-induced thrombocytopenia (HIT). Dr. Sachais has previously developed, optimized and published a retrospective study on several assays that may have improved diagnostic parameters for HIT. Conner is currently validating these assays with a prospective cohort. Given his interest in medical school and surgery, learning about HIT and about assay validation will be beneficial for his career. Both students are participating in the CTSA Summer Undergraduate Internship Program s workshops and seminars, and are expected to present their research at the CTSA Research Symposium. Ms. Ebanks, our undergraduate student from 2011, is continuing her research in the laboratory of Dr. Karen Baskerville, an Associate Professor at Lincoln University, with funding for supplies provided by this grant. We are still reviewing applications for the graduate program. Ernestina Nyarko completed her independent study project with Dr. Jun Mao, focusing on cancer survivor s perceptions of University of Pennsylvania 2009 Nonformula Grant on Cancer Vaccines Page 5

6 survivorship care from primary care physicians, and immunological markers in acupuncture as treatment of althralgia in breast cancer patients on aromatase inhibitor therapy. She is currently working on a manuscript for publication. Ernestina graduated from the UPenn Perelman School of Medicine in May, and is a resident in family practice at Carolinas Medical Center. She hopes to continue her interest in population studies, particularly women s health. A. B. Figure 1. Impact of anti-pd1 antibodies on receptor function. The ability of anti-pd1 antibodies (C9 and PD-1-33) to block receptor function were measured using induction of IFNγ production (A), and IL-2 production (B) as biomarkers of activity. A human PD-1 antibody from R&D (AF1086) was used as a positive control for blocking PD1 signaling. Lack of IFNγ production suggests the antibodies are not effectively blocking PD1 function. Figure 2. Expression of 2 nd generation BiTEs. Western blot of media from pools of cells transfected with anti-pd1/anti-her2 and anti-ctla4/anti-her2 BiTEs constructed from clinically validated anti-ctla4 and anti-pd1 mabs demonstrating expression of the 2 nd generation BiTEs. Selection of high-expressing clones is underway. University of Pennsylvania 2009 Nonformula Grant on Cancer Vaccines Page 6

7 Survival (%) Survival-Tem1 Survival-Tem1(CT26) CD3 CD4 CD8 CTRL Time after ACT (days) Time after ACT (days) Figure 3. TEM1 and TAA immune responses enhance survival of CT26 tumor bearing mice. C57b/6 mice bearing CT26 tumors were infused with 1x10^7 CD3, CD4 or CD8 T cells from TEM1-TT immunized mice either free of tumor (left panel) or bearing CT26 tumors (right panel). Survival was monitored for 10 animals per group. A B C A Figure 4. Non-polarized T cells redirected with an ICOS based CAR show enhanced antitumor effect but reduced persistence in vivo. Human L55 tumors were established in the flanks of NSG mice. After 3 weeks, when the tumors reached a volume of 150 mm 3, mice were treated with 2 i.v. injections of 10 x 10 6 Th17 /Tc17 cells or Th0/Tc0 cells redirected with ICOS-CAR. (A) Tumor volume was analyzed at indicated time points. The absolute numbers of CD4+ and CD8+ cells was determined in the blood (B) and spleen (C) on weeks 3 and 4, respectively. University of Pennsylvania 2009 Nonformula Grant on Cancer Vaccines Page 7