Drew M. Pardoll - US grants

Antigen-specific, major histocompatibility restricted recognition by classical T cells is mediated by a T cell receptor (TCR) consisting of a disulfide-linked alpha beta heterodimer. Recently, a second heterodimeric T cell receptor, termed TCR gamma delta, was identified. TCY gamma delta appears to be expressed by a separate T cell lineage distinct from TCR alpha beta-bearing cells. Despite the rapidly accumulating information based on the biochemical nature of the gamma delta receptor and the structure of the gamma and delta genes, there is at present no firm understanding of the function of T cells which bear gamma delta receptors. The goal of this project is to gain insight into the role of TCR gamma delta+ T cells in the immune system. The first step in determining the function of the new lymphocyte population is the identification of an immune response in which that population participates. We have recently found that TCR gamma delta+ cells proliferate in vivo during the primary immune response to certain foreign antigens. For example, the primary immune response to heat-killed mycobacterium tuberculosis is characterized by a 30-fold expansion in TCR gamma delta cells. Evidence that this expansion is associated with specific activation comes from the finding that greater than 30% of TCR gamma delta+ cells in the MT-primed lymph node express high densities of functional IL-2 receptors. Using this primary immune response as a model system, this proposal describes experimental approaches aimed at addressing the following specific questions: 1) what specific ligand(s) are recognized by the population of activated TCR gamma delta cells? 2) which specific gene segments of the gamma and delta loci are utilized in the response to foreign antigen? 3) what is the physiologic effector or regulatory role of TCR gamma delta+ cells in the antigen-specific immune response? The analyses of ligand recognition in the TCR gamma delta repertoire will involve the in vitro cloning of TCR gamma delta-bearing T cells followed by specific antigen activation studies and sequence analysis of the gamma and delta gene segments utilized in these clones. Evaluation of the effector functions of these TCR gamma delta T cells will involve an analysis of lymphokine production both in vitro and in vivo as well as a series of adoptive transfer experiments utilizing purified lymphocyte populations. Finally, attempts will be made to raise specific monoclonal antibodies which recognize gamma delta receptors that are expressed by clones grown out of the MT primed lymph node. By comparing the normal immune response with the immune response generated during in vivo treatment with these anti-gamma delta antibodies, it should be possible to gain yet further understanding of the role of TCR gamma delta+ T cells.

One of the most critical questions in cancer immunology is why the immune system fails to eliminate tumors that arise de novo. Concentrating on the T cell-mediated immune response, we sought to determine whether this was due to an absence of CTL precursors capable of recognizing tumor-specific neoantigens or rather, a failure of the helper arm to produce the lymphokines that act as critical second signals for CTL activation. We devised a novel strategy to answer this question that involved engineering tumor cells by gene transfection to produce helper lymphokines known to be important for CTL priming. The critical feature of this approach is that the helper lymphokine is produced only where the antigens are. We reasoned that these engineered tumors might effectively prime "latent" tumor specific CTL by bypassing a defective T helper arm. Indeed, we found that animals injected with poorly immunogenic tumors engineered to secrete IL-2, IL-4 or GMGSF developed systemic immunity against challenge with the parental tumor. The broad objective of this proposal is to dissect the antitumor immune response generated by immunization with lymphokine gene-transfected tumors at the cellular and molecular level. In particular, the following aims will be pursued: 1) To characterize the mechanisms by which tumor antigen-specific CTL are primed. We propose a set of experiments to determine the role of each of the infiltrating cells in the priming of CTL in vivo and to distinguish whether MHC class I-restricted tumor antigens are presented by the engineered tumor itself or by bone marrow-derived antigen-presenting cells 2) We plan to use T cell receptor transgenic mice expressing identifiable T cell receptors reactive with tumor-specific helper antigens presented on MHC class II molecules to determine why the helper arm of the immune system apparently fails to function in the antitumor response 3) We plan to further explore the immune response to tumors that have turned off their MHC class I antigens 4) We plan to use tumor-specific CTL to identify tumor specific peptide-epitopes which will ultimately be purified and sequenced.

Human papillomaviruses have been implicated as etiologic agents in cervical cancer. The transforming protein, E6 and E7 of oncogenic HPVs are consistently expressed in tumor cells and therefore represent examples of tumor specific antigens. Vaccines targeted to these proteins may provide a means to prevent and treat HPV associated malignancies. In order to design and characterize the efficacy of various vaccine strategies and other immunotherapeutic strategies, it will be important to characterize antigen specific T cell responses to the HPV E6 and E7 proteins. Over the past few years, our group has developed novel genetic approaches to cancer immunotherapy which result in the induction or enhancement of both CTL and T helper responses to tumor specific antigens. As HPV associated malignancies represent one of the best models for human cancer in which there are identified tumor specific antigen, we propose to study these immune responses and characterize specific E6 and E7 epitopes that are expressed in patients bearing the most common MHC class I allele - HLA A2. Specifically, we propose to: 1) develop and utilize assays to measure CTL and Th responses to HPV 16 E6 and E7 proteins. A variety of strategies will be considered to prepare stimulator and target cells for CTL and Th assays. 2) identify epitopes on HPV 16 E6 and E7 proteins which bind to commonly expressed human HLA class I alleles and generate CTL responses. Naturally processed HPV 16 E6 and E7 peptides will be characterized by immunoprecipitation of MHC class I-peptide complexes from HPV-associated cervical carcinoma. Acid dissociated peptides will be analyzed by HPLC and compared with the elution profiles of synthetic peptides. 3) measure and characterize CTL and Th responses to HPV 16 E6 and E7 proteins/peptides in peripheral blood of patients treated with cytokine gene transduced tumor cell vaccines or with recombinant vaccinia-E6/E7 vaccines.

The currently proposed project represents a phase I study in humans with metastatic kidney (renal) cancer to evaluate a novel form of gene modified tumor cell vaccine. This study is based on extensive preclinical experiments in animal tumor models for gene targeted immunotherapy. The animal tumor experiments have shown that introducing specific genes into tumor cells that encode stimulating growth factors (termed cytokines) for the immune system enhance the ability of these gene modified tumor cells to activate an immune response in the animal against the original tumor. Specifically, we have found that a particular cytokine gene, GM-CSF, when introduced into the tumor, results in the most effective generation of clinically relevant systemic antitumor immune responses. Using highly efficient and safety tested viruses as a vehicle to transfer genes into the tumor cells, we have been able to genetically modify human cancer cells to produce equivalent levels of the immunostimulatory GM-GSF as has been accomplished in the animal tumor cells. The current human protocol seeks to accomplish specific clinical and laboratory objectives. First, as a phase I protocol, it seeks to determine the maximum tolerated dose of irradiated autologous renal tumor cells, unmodified and after transfer of the human GM-CSF gene. Second, we seek to quantitate the toxicities, if any, of introducing increasing amounts of GM-CSF gene transduced tumor cells. Thirdly, we seek to quantitate the specific antitumor immune responses induced by these vaccination approaches. Fourth, we will seek preliminary evidence of therapeutic activity of the vaccine preparation. This trial is designed with two arms: one arm evaluates escalating doses of GM-CSF transduced tumor cells and the second parallel arm evaluates escalating doses of nontransduced tumor cells. While the primary objective of the two arm trial is to determine whether observed toxicity is due to cell administration of the GM-CSF gene transfer, we will also seek preliminary comparative evidence to determine whether the introduction of the GM-CSF gene into the tumor cells modifies their vaccination potential relative to untransduced tumor cells with regards to either immunologic or clinical antitumor responses.

One of the most critical questions in cancer immunology is why the immune system fails to eliminate tumors that arise de novo. Concentrating on the T cell-mediated immune response, we sought to determine whether this was due to an absence of CTL precursors capable of recognizing tumor-specific neoantigens or rather, a failure of the helper arm to produce the lymphokines that act as critical second signals for CTL activation. We devised a novel strategy to answer this question that involved engineering tumor cells by gene transfection to produce helper lymphokines known to be important for CTL priming. The critical feature of this approach is that the helper lymphokine is produced only where the antigens are. We reasoned that these engineered tumors might effectively prime "latent" tumor specific CTL by bypassing a defective T helper arm. Indeed, we found that animals injected with poorly immunogenic tumors engineered to secrete IL-2, IL-4 or GMGSF developed systemic immunity against challenge with the parental tumor. The broad objective of this proposal is to dissect the antitumor immune response generated by immunization with lymphokine gene-transfected tumors at the cellular and molecular level. In particular, the following aims will be pursued: 1) To characterize the mechanisms by which tumor antigen-specific CTL are primed. We propose a set of experiments to determine the role of each of the infiltrating cells in the priming of CTL in vivo and to distinguish whether MHC class I-restricted tumor antigens are presented by the engineered tumor itself or by bone marrow-derived antigen-presenting cells 2) We plan to use T cell receptor transgenic mice expressing identifiable T cell receptors reactive with tumor-specific helper antigens presented on MHC class II molecules to determine why the helper arm of the immune system apparently fails to function in the antitumor response 3) We plan to further explore the immune response to tumors that have turned off their MHC class I antigens 4) We plan to use tumor-specific CTL to identify tumor specific peptide-epitopes which will ultimately be purified and sequenced.

Antigen-specific, major histocompatibility restricted recognition by classical T cells is mediated by a T cell receptor (TCR) consisting of a disulfide-linked alpha beta heterodimer. Recently, a second heterodimeric T cell receptor, termed TCR gamma delta, was identified. TCY gamma delta appears to be expressed by a separate T cell lineage distinct from TCR alpha beta-bearing cells. Despite the rapidly accumulating information based on the biochemical nature of the gamma delta receptor and the structure of the gamma and delta genes, there is at present no firm understanding of the function of T cells which bear gamma delta receptors. The goal of this project is to gain insight into the role of TCR gamma delta+ T cells in the immune system. The first step in determining the function of the new lymphocyte population is the identification of an immune response in which that population participates. We have recently found that TCR gamma delta+ cells proliferate in vivo during the primary immune response to certain foreign antigens. For example, the primary immune response to heat-killed mycobacterium tuberculosis is characterized by a 30-fold expansion in TCR gamma delta cells. Evidence that this expansion is associated with specific activation comes from the finding that greater than 30% of TCR gamma delta+ cells in the MT-primed lymph node express high densities of functional IL-2 receptors. Using this primary immune response as a model system, this proposal describes experimental approaches aimed at addressing the following specific questions: 1) what specific ligand(s) are recognized by the population of activated TCR gamma delta cells? 2) which specific gene segments of the gamma and delta loci are utilized in the response to foreign antigen? 3) what is the physiologic effector or regulatory role of TCR gamma delta+ cells in the antigen-specific immune response? The analyses of ligand recognition in the TCR gamma delta repertoire will involve the in vitro cloning of TCR gamma delta-bearing T cells followed by specific antigen activation studies and sequence analysis of the gamma and delta gene segments utilized in these clones. Evaluation of the effector functions of these TCR gamma delta T cells will involve an analysis of lymphokine production both in vitro and in vivo as well as a series of adoptive transfer experiments utilizing purified lymphocyte populations. Finally, attempts will be made to raise specific monoclonal antibodies which recognize gamma delta receptors that are expressed by clones grown out of the MT primed lymph node. By comparing the normal immune response with the immune response generated during in vivo treatment with these anti-gamma delta antibodies, it should be possible to gain yet further understanding of the role of TCR gamma delta+ T cells.

Advances in understanding of T cell immunobiology have engendered a rapidly expanding interest in molecular engineering of antitumor immune responses. In particular, the identification and cloning of genes encoding cytokines provides a potent set of reagents for activating immunologic effector responses in vivo. One of the major concepts in cytokine biology is that their activity is most potent when they are expressed in a paracrine fashion, that is, local to the site of antigen. We have developed two major strategies for the paracrine expression of cytokines in vivo. One approach involves the transduction of tumor cells with genes encoding cytokines. Two distinct phenomena are observed when these cytokine secreting tumors are injected in vivo. Local sustained release of some cytokines such as IL-2, IL-4 and TNF-alpha result in inflammatory responses that mediate destruction of the transduced tumors. Additionally, certain cytokine producing tumors result in the activation of potent systemic T cell dependent antitumor responses. GM-CSF producing tumors appear to generate the most potent vaccines. Recently, we have developed an alternate approach to sustained local cytokine release using biodegradable polymer microspheres. Mixture of irradiated nontransduced tumor cells with biopolymer microspheres containing GM-CSF produce equivalent immunization to GM-CSF gene transduced tumor cells. This approach is simpler and less labor intensive for clinical applications than direct gene transfer because it eliminated the necessity for culturing and transducing human tumor explants. The overall objective of this project, is to explore these strategies of cytokine-enhanced immunotherapy to treat tumors in the brain. To assess the feasibility of this approach we have developed an intracranial tumor model using the B16F10 melanoma, a well characterized variant of a spontaneous melanoma originally derived from C57BL/6 mice. Because it is poorly immunogenic, it does not incite an effective local or systemic immune response, and hence provides an ideal model to examine how cytokines enhance the immune response to tumor. We will use two complementary strategies: B16F10 cells, transduced with the gene for GM-CSF, as a systemic tumor vaccine to protect against challenge with tumor in the CNS; and local delivery of irradiated tumor cells genetically programmed to produce specific cytokines IL-2, IL-4, and TNF-alpha directly to the site of a brain tumor. Additionally, we will develop polymer mediated delivery of cytokines as a prelude to the translation of these approaches to human brain cancer therapy.

Cytokines play a central role in the induction and regulation of immune response. Physiologically, they act in a paracrine rather than endocrine fashion. Experiments with cytokine gene transduced tumor vaccines have demonstrated that the paracrine release of certain cytokines at the site of the tumor results in priming of immune responses against certain tumor antigens. A broad comparative analysis of multiple cytokine genes demonstrated that GM-CSF transduced tumor vaccines produced a particularly potent systemic immune response. Paracrine GM-CSF vaccines enhance the presentation of tumor-specific antigens to the immune system and induce expression of co-stimulatory signals for T cell activation. A major limitation in the translation of this strategy to clinical practice is the labor intensity and technical difficulty of reliably generating genetically-altered tumor vaccines for the majority of patients. Recently, Dr. Pardoll and Leong's laboratories have developed an alternative to gene transfer by providing the local and sustained delivery of cytokines through biodegradable polymer microspheres. The hypothesis was that synthetic controlled release microspheres containing cytokines, mixed with irradiated autologous tumor cells, can replace the requirement for gene transfer and in vitro tumor expansion, while maintaining the ability to generate an effective, systemic antitumor immune response. This project proposes to apply this polymer based paracrine cytokine adjuvant approach to E7 and HER-2/neu specific vaccines by incorporating recombinant protein and/or peptides into timed release biodegradable polymer microspheres mixed with GM-CSF containing polymer microspheres. Specifically, we plan to: 1. Optimize functional GM-CSF-containing microspheres. 2. Produce recombinant E7 and HER-2/neu protein and peptides and incorporate them into polymer microspheres. 3. Optimize E7 and HER-2/neu vaccines by testing different combinations and formulations of microspheres containing recombinant protein and/or peptide mixed with GM-CSF containing microspheres. 4. Characterize the types of E7 and HER-2/neu specific cellular (helper and cytotoxic) and humoral immune responses elected by these polymer microsphere based vaccines. 5. Evaluate potential synergies between these polymer microsphere based vaccines and other vaccine approaches being developed in this NCDDG.

The ectopic expression of tumor antigens in animals models has been shown in induce tumor-specific immunity and is an approach that may prove useful for human cancer treatment. We propose to study adenovirus-mediated expression of tumor antigens for inducing anti-tumor immunity in order to better understand the nature of this process and explore its potential for human use. Adenoviruses can be oncogenic in animals but they can also induce a strong immune response that is protective against tumors that express adenoviral antigens. This suggests that under the right conditions, ectopic antigens expressed by the virus could generate a potent cellular immune response that can destroy cells expressing those antigens. During infection, adenovirus vectors can achieve high levels of antigen expression and the host anti-viral response may play an "adjuvant" role in stimulating an immune response tot he antigen by recruiting needed immune effector cells or by inducing production of immunostimulatory cytokines in the local environment. Much knowledge has been accumulated relating to the biology of adenoviral infections in the last 20 years. We will use that knowledge to systematically develop a vector to generate strong antitumor immunity. Specifically, we propose to: 1) Optimize several basic variables relating to dose, route and scheduling using a adenoviral vector that expresses either the human papilloma virus 16 E7 protein or the rat HER-2/neu protein. The vaccines will be tested against a challenge of increasing numbers of tumor cells that express the same proteins. The tumor models have been developed as part of Projects 1 and 2. The vaccine will also be tested in a therapeutic mode to see if it can cause regression of established tumors. If the vaccine is not maximally protective, we will determine if variation of the virus that affect viral replication and immunogenicity will improve the response against ectopic antigens. 2) Develop combinations of the virus that can deliver additional genes that can influence the immune response including B7.1 GM-CSF, IL-2 and IFNgamma. 3) Perform immunological studies to understand the mechanisms of adenovirally induced anti-tumor response. 4) Use that information to design better vaccine strategies that may combine components of all of the projects within this program.

DESCRIPTION (Adapted from the Investigator's Abstract): In murine colon cancer, the investigator has identified an immunodominant T-cell epitope recognized by CD8+ CTL. This peptide epitope, AH1, is derived from a normally silent endogenous retroviral gp70 gene that has been transcriptionally activated in the tumor cells. He now plans to study in detail the kinetics of interactions between this peptide and its MHC class I restricting element, Ld, as well as the T-cell receptor specific for the peptide MHC complex. By comparing these values with other Ld-restricted murine tumor antigens and display distinct biological properties, he hopes to define specific parameters of immunogenicity and immunodominance of tumor antigens. Specifically, he proposes to: 1) measure association and dissociation kinetics of tumor specific peptide antigens with their MHC class I restricting elements, and with T-cell receptors specific for the peptide MHC complex; 2) analyze the effects of specific amino acid modifications of the tumor antigens to further correlate MHC and TCR binding kinetics with in vivo immunogenicity; 3) develop and utilize multivalent soluble Ig-Ld molecules to directly visualize the cellular dynamics of peptide + MHC specific T cells in vivo; and 4) analyze the mechanisms responsible for lack of presentation of AH1 in certain BALB/c tumors that are gp70+.

Antigen-specific T cell responses are an essential component in antitumor immunity. Definition of immunodominant tumor antigens recognized by T cells has been a very important endeavor in cancer immunology for two reasons. First, direct molecular identification of T cell recognized antigens is a critical first step in understanding and further delineating the relationship between the immune system and the endogenously arising cancers. Second, the identification of shared immunodominant tumor antigens will provide the basis for development of antigen-specific vaccines as well as other forms of T cell mediated cancer immunotherapy. While much emphasis has been placed on the study of MHC class I restricted CD8 antitumor responses and antigens recognized by CD8+ CTL, relatively little is known about the MHC class II restricted CD4 antitumor response and tumor antigens recognized by CD4 cells. GM-CSF transduced tumor cell vaccines represent an effective means of activating both CD4+ and CD8+ T cells. In order to further define CD4+ recognized antigens and CD4 responses in human cancer, we have analyzed renal cancer patients vaccinated with autologous GM-CSF transduced tumor vaccines that have generated strong DTH responses against their tumor post-vaccination as well as clinical responses. We have successfully cultured tumor specific CD4+ T cell clones from two patients. Some of the clones within the panel recognize antigens uniquely expressed by the autologous tumor while others recognize antigens shared by multiple renal tumors. We propose to identify the antigens recognized by these CD4 cells and study immune responses against them. Specifically, we propose to: 1) Utilize biochemical approaches to identify and characterize both unique and shared renal cancer antigens recognized by clones within our panel. 2) Study responses specific for the identified CD4 recognized renal cancer antigens within PBL from renal cancer patients pre- and post-vaccination and 3) Construct IgG-MHC class II-peptide chimeras as a means of directly staining antigen specific CD4 T cells and utilize these constructs to follow antigen specific CD4 responses.

DESCRIPTION: (Applicant's Abstract) This application seeks to evaluate a novel vaccine approach involving targeting the HPV-16 E7 antigen to the MHC class II processing pathway. One of the most important endeavors in evaluating therapeutic vaccines is the measurement of induced in vivo immune responses relevant to anti-tumor efficacy. Recent studies call into question the sensitivity of standard bulk and limiting dilution CTL analyses in estimating the true frequency and functional status of antigen specific T cells. An exciting new approach to directly visualize antigen specific T cells has been the use of multimeric MHC+ peptide complexes that bind stably and specifically to antigen specific T cells. The applicant has developed a simple and versatile approach to MHC-peptide multimerization by linking MHC molecules genetically to a dimeric IgG scaffold. These peptide-MHC-IgG chimeras have been shown to bind stably and specifically to antigen-specific CD8+ and CD4+ T cells. He proposes to utilize these reagents to evaluate the in vivo dynamics and functional status of E7 specific T cells in patients receiving the LAMP targeted E7 vaccines. Specifically, he proposes to: 1) Develop stable CD8+ T cell lines and clones specific for immunodominant HLA-A2 restricted E7 peptides. 2) Develop and test E7-HLA-A2-IgG dimers for three identified immunodominant HLA-A2 restricted E7 peptides. 3) Evaluate the in vivo dynamics of E7+A2 specific CD8+ T cell responses in vaccinated HLA-A2+ patients utilizing the E7-HLA-A2-IgG chimeras developed in Specific Aim 2. 4) Develop analogous E7-MHC II-IgG chimeras capable of detecting CD4+ T cells specific for E7 peptides presented by the common HLA Class II alleles DR1 and DR4. Ultimately, these analyses will test the hypothesis that vaccination of patients expressing HPV 16 E7+ SIL or cervical cancer with LAMP-targeted E7 vaccines increases the numbers of activated E7 specific T cells in peripheral blood and at the site of disease.

DESCRIPTION: (Adapted from the applicant's abstract) The aim of this proposal is the generation of a murine model of Human Papilloma Virus associated cancer that will allow us to specifically test the efficacy of a new class of heat shock protein based vaccines using HLA-A2 restricted HPV-16 E7 epitopes. HPV associated malignancies are the second most common cause of cancer death in women worldwide. Such malignancies are an ideal target for immunotherapy due to the uniform expression of E6 and E7. Furthermore, the capacity of the human immune system to recognize the E7 antigen has been well documented based on the detection of both antibody responses as well as CTL responses. Specifically, they propose to: Introduce a chimeric HLA-A2.1/Kb and a human beta2 microglobulin gene into the established E6/E7+ murine TC-1 tumor (TC-1/A2). Validate that TC-1/A2 can indeed be recognized by HLA-A2 restricted T cells. Produce HSP70 vaccines using a proprietary peptide technology. Assessing the growth kinetics and immunogenicity of TC-1/A2 in HLA-A2/Kb transgenic mice determines whether immunization with the HSP70:HPV16E7-70BP peptide vaccines delay outgrowth of TC-1/A2 tumors. If the HSP70 vaccines demonstrate antitumor efficacy in this model for HPV associated malignancy, they will be developed for clinical trials. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE

neoplasm /cancer; biomedical equipment resource; biomedical facility;

neoplasm /cancer; biohazard control; antiseptic sterilization; biomedical equipment resource; biomedical facility;

DESCRIPTION (provided by the applicant): In addition to providing ligands for T cell receptor engagement, antigen presenting cells (APCs) direct the activation and differentiation of T cells by providing costimulatory signals. One of the most important families of costimulatory molecules is the B7 family, which currently consists of 4 members, each of which appears to be important in mediating various aspects of T cell dependent immune responses. Dendritic cells (DCs), unique APCs with potent T cell stimulatory capacity, are critical modulators of both natural and vaccine induced immune responses. We have cloned a new B7 family member, termed B7-DC, whose expression is highly restricted to DCs. B7-DC was identified among a library of genes differentially expressed between DCs and activated macrophages. B7-DC costimulates T cell proliferation more efficiently than B7-1 and induces a distinct pattern of lymphokine secretion, characterized by high levels of the proinflammatory lymphokines, gamma-interferon (g-IFN) and interleukin-6 (IL-6). These properties of B7-DC may account for some of the unique activity of DCs relative to other APCs. The current application seeks to define the biologic role of B7-DC in mediating immune responses. Specifically, we plan to: 1) Quantitatively determine the expression pattern of B7-DC and which signals induce its expression, 2) Perform a detailed analysis of B7-DC's costimulatory activity in vivo, 3) Analyze its in vivo role in mediating immune responses, 4) Identify receptor(s) for B7-DC and 5) Determine whether human B7-DC functions similarly to murine B7-DC.

[unreadable] DESCRIPTION (provided by applicant): The goal of this project is to investigate the utility of bone marrow transplant (BMT) with gene modified BM to induce potent anti-tumor immunity. A major focus of cancer immunotherapy is to develop strategies to induce T cell responses through efficient presentation of tumor antigens by dendritic cells (DCs). Current vaccination strategies are limited in their ability to efficiently load DCs in vivo. Ex vivo generated DCs can be efficiently loaded, but after re-injection, few DCs traffic to secondary lymphoid organs, which are the critical sites for antigen presentation to T cells. To enhance the efficiency and durability of antigen presentation by DCs, we transduced hematopoietic stem cells (HSC) with a lentiviral vector encoding a model tumor antigen, then transplanted the modified cells. Our results showed high-level expression of a transgene by DCs in lymphoid organs. The combination of bone marrow-transplant using antigen expressing HSC and systemic agents that activate DCs along with mature donor lymphocyte infusions resulted in dramatic expansion and activation of antigen specific T cells. This tripartite therapy provided potent antigen specific immunotherapy of an aggressive established murine lymphoma. In this application, we are proposing to investigate the mechanism and kinetics of the immune response to the tumor, selective expression of the tumor Ag by regulatable vectors, and optimization of the treatment strategy. Specifically, we will: (1) examine the T cell immune responses in mice receiving Ag-transduced bone marrow stem/progenitor cells (HSCs), using HA as a model tumor Ag; (2) investigate combinations of molecules for in vivo activation of DCs and T cell priming subsequent to HSC transduction/transplantation, and develop DC specific and pharmacologically regulatable vectors to enhance immune responsiveness to antigen; (3) determine the efficacy of tumor Ag expression in the BM for myeloablative and nonmyeloablative allogeneic BMT; and (4) determine whether transplantation of Ag transduced BM will be effective against solid tumors. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Regulatory T cells (Treg) have emerged as a major mechanism in the maintenance of immunologic self-tolerance. Treg cells, which can emerge directly from the thymus (so called natural Treg cells) or be induced in the periphery (induced Treg cells) are usually CD4+CD25+ and function by inhibiting effector T cells. Elucidation of the mechanisms of Treg generation would be greatly facilitated by the identification of specific markers for Treg cells and molecules that mediate their function. We have been studying a self-tolerance system in which TCR transgenic CD4 T cells adoptively transferred into transgenic animals expressing the target antigen in multiple epithelial tissues become anergic and simultaneously develop into Treg cells capable of suppressing autoimmunity. Gene expression profiling of in vivo anergic/Treg cells compared with effector/memory T cells identified the LAG-3 gene as highly upregulated on anergic/Treg cells relative to effector/memory T cells. LAG-3hi Treg cells mediated suppression of T cell proliferative responses in an in vitro suppression system and anti-LAG-3 antibodies inhibited this suppression. These results identify LAG-3 as the first known specific cell surface marker for Treg cells that is potentially directly involved in mediating Treg function. This proposal seeks to further elucidate the mechanisms of suppression by LAG-3hi Treg cells and the specific role of the LAG-3 molecule in their activity. Specifically, we propose to: 1) Study the capacity and mechanisms of suppression mediated by Treg cells defined by expression of LAG-3 and CD25. 2) Elucidate the direct role of LAG-3 in modulating the activity of Treg cells and 3) Analyze the effect of blockade or elimination of LAG-3+ Treg cells in enhancing the activity of immunotherapies for established cancer.

Applications of functional genomics to the identification of relevant human tumor antigens such as mesothelin in pancreatic and ovarian cancer provide the basis for antigen specific vaccines. However, the ultimate potency of vaccine induced antitumor immune responses is limited by preexisting tolerance, even when the targeted tumor antigen displays limited expression by normal tissues. We and others have demonstrated that amplification of immune responses in the setting of preexisting tolerance can be achieved by a) increasing costimulation and b) inhibiting the activity of Treg cells. Therefore, the major aim of this NCDDG element is to develop a costimulatory agonist and a Treg antagonist, based on gene discovery efforts within our laboratory, which will be used to amplify mesothelin-based vaccines. Recently, we discovered a new B7 family member, termed B7-DC, that is selectively expressed by dendritic cells. B7-DC can act as a strong costimulator for Th1 responses in synergy with B7.1 or B7.2. This costimulatory activity is independent of PD-1, an inhibitory receptor to which B7-DC can bind. Aim#1 of this section will be the development and characterization of both murine and human mutant B7-DC-Fc molecules (termed B7-DC*-Fc) that have lost PD-1 binding activity but retain costimulatory activity. As part of a gene profiling project to identify Treg specific genes, we identified LAG-3 as a Treg specific cell surface molecule. In the mouse, both natural and induced Treg express increased levels of LAG-3 and antibodies to murine LAG-3 block Treg activity both in vivo and in vitro. Anti-LAG-3 monoclonal antibodies will be developed as Treg antagonists. Based on our findings that anti-LAG-3 antibodies block Treg activity in murine systems, together with the finding that tolerant T cells in various murine tumor systems express increased LAG-3, Aim#2 of this section will be to produce both murine and human anti-LAG-3 antibodies and test them for their ability to inhibit Treg cells. These agents will be combined with mesothelin-based vaccines in Sections 1 and 2 of the NCDDG.

The Common Equipment Core resource provides support to Cancer Center Investigators for the maintenance and operation of equipment that is shared among multiple groups or programs with an emphasis to ensure young investigators access to equipment necessary to their laboratories. The services provided by this resource are repair and/or preventative maintenance contracts for shared equipment, annual inspections of freezers, cryo units and balances, water filters for film processors and ice machines and maintenance and chemicals for the film processors. Shared equipment covered are three film processors, eight ice machines, four autoclaves (1 in CRBII), two Coulter counters, two real time PCR instruments, two liquid scintillation/luminescence counters, two stills, and A/V equipment in four shared conference rooms. Equipment such as ultra centrifuges, mid-range centrifuges, scintillation counters, and imaging equipment are included to make sure young investigators have access to necessary equipment crucial in developing their laboratory programs. All equipment is currently located in the Bunting-Blaustein Building and some will be located in CRBII when it opens in January 2006. Service monitoring and oversight are provided by the Laboratory Facilities and Services Manager and the Laboratory Services Coordinator. User logs are required and kept for each piece of equipment where appropriate. Exceptions are autoclaves, film processors, ice machines and A/V equipment. There is no charge back system for these services. There is a chargeback for the use of the real time PCR units, the liquid scintillation/luminescence counters (as of January 2005) and cryo storage.

The Glassware Washing Facility (a CCSG Core since 1976) is a Cancer Center managed resource which provides cleaning and sterilization of reusable laboratory glassware, instruments and equipment and provides sterilization of biohazardous tissue culture waste from Center research laboratories. Routine services are washing, material wrapping/preparation for sterilization, pipette plugging, accomodating individual investigators' customized washing and preparation needs, centralized autoclaving for media and liquids and biohazardous material. The facility is located in the Bunting-Blaustein Cancer Research Building (CRB I) and services are provided by glassware washers supervised by the Laboratory Facilities and Services Manager. Service logs are maintained and each investigator is charged for these services. The newly upgraded facility is equiped with three washers, three autoclaves, two dryers and other assorted support equipment. Currently three glassware washing staff members process glassware, media and waste. In January 2006, this facility will provide support for Cancer Research Building II (CRB II), which will double the number of research investigators to whom the Core provides services. The existing facility has been recently renovated and has added one washer, one autoclave and one dryer. Casework has been added to increase and improve the efficiency of the prep/plugging area. There will also be one additional autoclave in CRBII to handle autoclaving of biohazardous waste. Two additional staff member will be added to handle the increased volumes from CRB II.

[unreadable] DESCRIPTION (provided by applicant): The innate and adaptive immune systems represent our two arms of defense against infection and cancer. Natural killer (NK) cells are a central component of innate immunity, recognizing infected cells, stressed cells and tumor cells via specific nonpolymorphic receptors. As the critical presenters of antigen to naive T cells, dendritic cells (DCs) are the critical initiators of adaptive immunity. Coordination of immune responses requires efficient communication between innate and adaptive immunity. Recently, we discovered a unique hybrid cell type that expresses both NK molecules (activating and inhibitory) and DC molecules, termed NKDC. Murine NKDCs are related to, but distinct from classic plasmacytoid DC (PDCs). Upon activation with CpG oligonucleotide ligands for toll-like receptor (TLR)-9, NKDC become transiently activated to kill target cells. The killing is, at least in part, NKG2D-dependent. Their NK activity subsequently diminishes associated with loss of NKG2D and its adaptor, DAP-12. As NK activity is lost, DC-like antigen presenting activity is gained, associated with upregulation of surface MHC class II and costimulatory molecules. In vivo, splenic NKDCs preferentially display NK activity while lymph node NKDCs preferentially display ARC activity. By virtue of their capacity to mediate natural killing activity, followed by antigen presenting activity, NKDC may represent a direct link between innate and adaptive immunity. We propose to characterize the development and functions of this cell type in detail. The following specific aims will be pursued. Specific Aim #1: Define the developmental biology of NKDC relative to NK cells and classic DC.Specific Aim #2: Define the activation, recognition and effector molecules involved in NKDC killing. Specific Aim #3: Define the capacity of NKDC to process and present antigens to T cells. Specific Aim #4: Define the role of NKDC in response to tumors. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): As tumors develop and progress, they disrupt surrounding tissues through direct invasion and distant tissues through metastases. These processes are intrinsically proinflammatory and thus have the potential to induce anti-tumor immune responses directed against tumor specific antigens. In this fashion, the immune system can act as an extrinsic tumor suppressor. We have hypothesized that tumors must actively develop mechanisms to suppress the release and sensing of these immunologic danger signals in order to survive and propagate without inciting anti-tumor immunity. Along these lines, we have demonstrated that the oncogenic Stat3 signaling pathway, which is constitutively activated in over 50% of diverse cancers, may serve such a role. In transplantable tumor models with constitutively activated Stat3, inhibition of Stat3 signaling results in the release of proinflammatory cytokines and chemokines as well as factors, such as VEGF and IL10, which inhibit DC maturation. Recently, we have further derived evidence that tumor induced Stat3 signaling in multiple hematopoietic cell types restrains tumor immune surveillance, defining a potential second level through which Stat3 signaling inhibits the generation of antitumor immunity. These findings suggest that Stat3 may be an interesting target for inhibition to enhance antitumor immunity. The role of Stat3 signaling has not yet been studied in endogenously arising cancer models. In the current proposal, we will use both genetic and pharmacologic approaches to evaluate the role of Stat3 signaling in blocking antitumor immunity in endogenously arising tumors from our autochthonous transgenic models of prostate and breast cancer as well as the potential for inhibition of Stat3 alone and in combination with vaccines and other agents to enhance therapeutic anti-tumor immunity. Specifically, we propose to: 1)Evaluate the role of tumor Stat3 signaling on prostate/prostate cancer specific immunity and tolerance in the ProHAxTRAMP autochthonous prostate cancer model, 2)Evaluate the role of hematopoietic Stat3 signaling on prostate/prostate cancer specific immunity and tolerance in the ProHAxTRAMP autochthonous prostate cancer model and breast cancer specific immunity and tolerance in the MMTV-Her2 autochthonous breast cancer model, 3)Evaluate the role of Stat3 signaling on the expression of B7-H1 and B7-H4 on tumor and other cellular elements of the tumor microenvironment and 4) Evaluate the capacity of a small molecule Stat3 inhibitor, CPA-7, to inhibit Stat3 signaling in tumor and infiltrating hematopoietic cells and subsequent enhancement of therapeutic anti-tumor immunity. [unreadable] [unreadable] [unreadable]

Our long term goal is to develop immune therapies for disease caused by Human Papillomavirus (HPV). While the field of cancer immunotherapy has shown proof of principle in humans, namely, that established malignancies can be recognized and eradicated by a tumor-specific adaptive T cell response, reproducibly achieving this effect has been difficult. One reason may be that therapeutic vaccines tested to date have not been immunogenic enough to eliminate established disease. Another reason may be immunologic suppression directly mediated by developing and progressing tumors, which are operative in the lesion microenvironment. This project will test the hypothesis that HPV-specific adaptive T cell responses can be elicited by therapeutic vaccination in subjects with high grade cervical dysplasia (CIN3), and that the application of a topical TLR7 agonist, imiquimod, directly on the lesion, will enhance access of CD8+ T cells to the lesions. CIN3 lesions are relatively accessible, and some of them do regress within our 15-week study window. The disease provides rational, non-self antigenic targets for therapeutic vaccination. This project will test a heterologous DNA prime, recombinant vaccinia-based boost regimen to enhance the immune response against HPV16 E6 and E7, which are expressed in a functionally obligate manner in cervical cancer and its precursor lesion, high grade cervical dysplasia (CIN3). This project also provides the opportunity to analyze target lesions directly to determine if we can identify evidence of mechanisms that would mitigate the function of immune effector responses, namely, impairment of homing and function of immune cell subsets. In fact, we found that CD8+ T cells accumulate at CIN3 lesions, but in the case of persistent disease, fail to access lesional epithelium. While patterns of immune cell infiltration have been identified to predict clinical outcome in the clinical setting of invasive disease, to our knowledge, they have not been identified early in disease, before development of an overtly invasive phenotype. This work is an opportunity to study immune responses that are localized to incipient neoplasia, and immune homeostatic mechanisms which could be locally uncoupled to enhance both access and function of effector immune cells to eliminate disease.

The Developmental Research Program is an important component of the SPORE and critical to the long-term fight against cervical cancer. It provides an avenue for soliciting new. research ideas and for developing innovative high-risk, but high-impact projects to stimulate cervical cancer research in the context of the SPORE. Pilot studies provide investigators with the resources to conduct translational research consistent with the SPORE'S objectives. This program will encourage participation from a broad range of investigators at Johns Hopkins and University of Alabama Birmingham (UAB) by providing support for pilot projects with the potential to develop into more fully developed translational projects. It will also encourage and facilitate the development of new research directions, methodologies, and collaborations. In addition, the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins also provides $75,000 annually as match funds for the Developmental Research Program. UAB will also contribute $40,000 annually to be used in the Career Development or Developmental Research program.

DESCRIPTION (provided by applicant): Chronic inflammation and, specifically, infection-associated inflammatory processes, enhance carcinogenesis in the affected organs. Chronic innate immune responses are known to contribute to these processes whereas the contributions of adaptive immunity to carcinogenesis are less clear. We have identified a human colon anaerobic bacterium, enterotoxigenic Bacteroides fragilis (ETBF), that induces a rapid and dramatic increase in colon tumors in multiple intestinal neoplasia mice (MinApc). This model replicates features of human colorectal cancer and our data demonstrate that colon tumorigenesis in this system is dependent, in part, on a novel Stat3/Th17 pathway, thereby defining a distinct role for adaptive immunity in colon cancer pathogenesis. Herein we seek to further define the mechanisms by which Stat3 activation in distinct cellular compartments and the resultant components of the Th17 response crosstalk with the colonic epithelium, inducing genetic and/or epigenetic epithelial cell changes that result in colon tumorigenesis. Our studies will begin to identify links between specific inflammatory mediators and the genetic changes critical to colon carcinogenesis. This work has direct relevance to the design of studies to investigate the pathogenesis of human colorectal cancer and may have implications for novel approaches to colorectal cancer therapy. Colorectal cancer is a major public health problem being the second leading cause of cancer death in the United States in women and men. PUBLIC HEALTH RELEVANCE: Colon cancer is the second leading cause of cancer death for women and men. The microbial and immunologic mechanisms contributing to colon cancer are unknown. This project will study the immune and genetic mechanisms by which a newly recognized common human stool bacterium called enterotoxigenic Bacteroides fragilis (ETBF) triggers colon tumors in mice, providing new insights into how colon cancer developes and potentially new approaches to colon cancer therapy.

DESCRIPTION (provided by applicant): We have identified a human colon anaerobic bacterium, enterotoxigenic Bacteroides fragilis (ETBF), as a candidate etiologic agent for colon cancer and our murine models of ETBF colonization delineate a potential procarcinogenic immune pathway that ETBF induce. Just as the understanding of peptic ulcer disease and ensuing stomach cancer was transformed by the discovery of H. pylori, we hypothesize that ETBF, by secreting the potent B. fragilis toxin (BFT), precipitate procarcinogenic mucosal immune responses, thereby promoting formation of colon cancer. Our model does not propose to alter existing mutational paradigms of colon cancer but rather proposes that ETBF colonization is an integral mechanism accounting for the accumulation of genetic mutations necessary for colon carcinogenesis. This proposal will study the relationship between colon colonization by ETBF in humans, specific colon immune responses and, ultimately, colon cancer. Defining a microbial etiology for colon cancer has key implications as colon cancer is a major public health problem being the second leading cause of cancer death in the United States in women and men. A significant correlation between any two of the studied variables - ETBF colonization, colon immune responses and colon cancer -- will substantively alter current paradigms for the pathogenesis, prevention and therapy of colon cancer.

DESCRIPTION (provided by applicant): Naturally occurring as well as adaptively developed Foxp3+CD4+ regulatory T cells (Treg) are central to the maintenance of immunological self-tolerance and immune homeostasis by suppressing aberrant or excessive immune responses. Treg specifically express the transcription factor Foxp3, which mediates the coordinate activation of genes such as CTLA-4 and GITR along with silencing of cytokines such as interleukin-2, IL-17 and interferon-?, that are normally expressed by effector T cells (Teff). The functional boundary between Treg and the Teff they are meant to suppress is not absolute. In fact, the flexibility between Treg and Teff may be an important component to both physiologic and pathophysiologic immune responses. Understanding this functional interchange requires an understanding of how immune effector genes are normally silenced in Treg. Despite progress in understanding mechanisms of Foxp3-dependent gene activation, the molecular mechanism of Foxp3-dependent gene repression has not been elucidated. Recently, we identified Eos, a zinc-finger transcription factor of the Ikaros family, as a critical mediator of Foxp3-dependent gene silencing in Treg. Eos interacts directly with Foxp3 and is necessary for gene silencing without affecting expression of Foxp3 activated genes. We further demonstrated that Eos and its corepressor C-terminal binding protein 1 (CtBP1) are necessary for histone modifications and ultimately promoter methylation involved in selective gene silencing in Treg. Knockdown of Eos in Treg abrogates their ability to suppress immune responses in vitro and in vivo. We hypothesize that Eos-CtBP-1 gene silencing is a highly regulated process in Treg that impacts on their function as suppressors vs. effectors of immunity. In the current proposal, we will further explore metabolic and microRNA dependent mechanisms of regulation of Eos-CtBP1 gene silencing in Treg as well as the in vivo consequences of ablation of this pathway for Treg development, homeostasis and modulation of adaptive immune responses. Specifically, we will 1) Elucidate the role of metabolic stimuli that affect NADH/NAD balance in Eos mediated gene silencing via the CtBP1 complex, 2) Study the regulation of Eos expression in Treg cells by microRNAs and 3) Study the consequences of Eos deletion to Treg cell homeostasis, differentiation and adaptive Treg cell development. PUBLIC HEALTH RELEVANCE: Foxp3 is a critical transcription factor that mediates both gene activation and gene repression in regulatory T cells. We have discovered a specific molecule, term Eos, that is selectively responsible for gene silencing in regulatory T cell. This proposal explore mechanism of Eos dependent Gene silencing and test the hypothesis that regulation of Eos is an important mechanism to control the regulatory T cell/ effector T cell balance in immunity.

DESCRIPTION (provided by applicant): The promise of cancer immunotherapy is based upon the exquisite specificity of the immune system, through which a potent machinery can eliminate targeted cells. However, despite some notable examples of success, progress in developing this form of cancer therapy has fallen short of expectations. Major insights explaining the limitations of T cell-based cancer immunotherapies have come from the discovery of inhibitory co-receptors or pathways termed immune checkpoints, which restrain T cell functions in normal physiologic settings as well as in the context of neoplastic disease. Recent evidence suggests that tumors may "usurp" immunological checkpoint mechanisms to create a barrier against antitumor immune responses - including endogenous responses and those induced by immunotherapies such as cancer vaccines. Animal cancer models demonstrate that blocking the interaction of inhibitory molecules on tumor cells with their co-receptors on tumor-specific T cells can "release the brakes" on antitumor immunity and cause tumor regression. Thus, checkpoint inhibition, applied alone or in combination with vaccines, represents an important new therapeutic approach for enhancing antitumor immunity. One of the most interesting inhibitory co-receptors is PD-1, that is induced on activated T cells and down-modulates critical functions in both CD4+ ("helper") and CD8+ ("killer") subsets. The major ligand for PD-1 is B7-H1 (PDL1), a B7 family member normally expressed by several leukocyte subsets upon activation, and aberrantly expressed in many human cancers. These findings, highlighting multiple mechanisms by which PD-1/B7-H1 interactions may inhibit antitumor immunity, have provided a rationale for clinical trials in cancer patients using fully human antibodies blocking PD-1 or B7-H1. Notably, objective tumor regressions were observed in the first phase I trial of PD-1 blockade in patients with advanced treatment-refractory metastatic solid tumors. It is now critically important to better understand the regulation and function of PD-1 and B7-H1, and to discern the effects of PD-1/B7-H1 blockade on antitumor immunity. In current proposal, we will test hypothesis that modulation of B7-H1/PD-1 inhibitory pathway could vastly enhance efficacy of cancer immunotherapy by improving tumor microenvironment and protecting ongoing T cell activity. The current proposal integrates basic and clinical science, and will use animal models and human in vitro systems to achieve the following aims: 1) To define mechanisms regulating B7-H1 expression by tumor cells and other cell types in the tumor microenvironment;2) To characterize factors influencing PD-1 expression by T cells, particularly in the context of vaccine-induced stimulation;and 3) To characterize immunological mechanisms underlying the clinical effects of B7-H1/PD-1 blockade in cancer therapy. Taken together, results from these studies will enable the rational clinical development of PD-1/B7-H1 blockade, alone or in combinatorial regimens, in cancer therapy. PUBLIC HEALTH RELEVANCE: Although remarkable progress has been made on a scientific level in understanding regulatory processes governing the activity of the immune system against cancer, the clinical application of these findings to develop effective cancer therapies will require a more detailed knowledge of the molecules and pathways involved. Our objectives are 1) to elucidate how interactions of the immune regulatory molecules B7-H1, expressed by cancer cells, and PD-1, expressed by activated antitumor T lymphocytes, support cancer progression;and 2) to use this knowledge to develop effective cancer immunotherapies based on blockade of B7-H1/PD-1 ligation.

? DESCRIPTION (provided by applicant): Over the past 10 years, studies of the immune microenvironment of cancer in both murine models and in humans has identified intracellular signaling pathways and expression of membrane ligands and receptors that locally inhibit antitumor immune responses. Among the most important are the ligands PD-L1 and PD-L2 that interact with the co-inhibitory receptor PD-1 on activated T cells. PD-1 pathway-blocking antibodies have so far had significant impact in melanoma, renal, lung, bladder, head and neck, and ovarian cancers, and Hodgkin's lymphoma. During the initial R01 funding period, we have made major discoveries regarding regulation of the expression of PD-1 and its ligands, that have important implications for identifying biomarkers and developing combinatorial approaches to cancer immunotherapy. Specifically, we showed that PD-L1 expression in tumors predicts response to PD-1 blockade. This finding has led to the development of PD-L1 IHC tests by several major pharmaceutical companies developing PD-1 pathway blocking drugs. We also demonstrated coordinate expression of PD-L1 and other checkpoint molecules, supporting the clinical development of the anti-PD-1/LAG-3 combination. Finally, we developed major biological and molecular concepts for understanding regulation of PD-1 and its ligands in the tumor microenvironment (TME), namely that expression represents a dynamic cross- talk between tumor cells and TILs. We showed that a dominant mechanism for PD-L1 up-regulation on certain tumors is not constitutive induction but rather adaptive resistance, whereby tumors respond to sensing of immune threat through IFN-g. The adaptive resistance concept now guides thinking in the field. Conversely, we also showed that a major cytokine produced by tumor cells, TGF-b, can enhance TCR-driven PD-1 promoter activity and thus PD-1 expression on T cells. These discoveries from our previous grant period raise a number of additional questions that bear on advances in immunotherapy: Why do some tumor types express PD-L1 adaptively and others constitutively? What are the relative functional roles of PD-L1 expression by tumor cells vs. infiltrating immune cells? Is the expression of PD-1 on TILs regulated in vivo by levels of TGF-b and TGF-b dependent signaling molecules? How heterogeneous is expression of PD-1, PD-L1 and other coordinately regulated checkpoint molecules among metastases in a given patient? Why do many patients with PD-L1+ tumors NOT respond to PD-1 pathway blockers? To answer these questions, we propose three Aims in this competing renewal: 1) Define mechanisms regulating PD-L1 expression by tumor cells and other cell types in the TME; 2) Characterize factors influencing PD-1 expression by T cells; and 3) Characterize immunological mechanisms underlying the clinical effects of PD-L1/PD-1 blockade in cancer therapy, including the co-expression of multiple checkpoint pathways that might provide resistance pathways to therapy.

DESCRIPTION (provided by applicant): The long-term objective of these studies is to determine whether a unique set of CD8 T cells can play a role in cancer therapy using the immune system. Specifically, we and others found that CD8 (killer) T cells that secrete a chemical substance (cytokine) known as interleukin-17 (IL-17) appear to last longer and function better than those that secrete other cytokines. We plan to determine how these cells (called Tc17) kill tumors, and whether they absolutely need IL-17 to work. Next, we plan to figure out the best way to make these cells, focusing on whether the target they recognize (antigen) plays a major role in that process. Finally, we found that Tc17 cells show an extremely interesting property that might be important for their superior anti-tumor function: in animals these cells convert, or flip the cytokines they produce. This conversion process is fascinating, and a better understanding of it might be important in understanding how other tumor treatments that use the immune system work. These experiments will not be undertaken in a vacuum; several groups, including ours have already showed that CD8 T cells that make IL-17 have superior anti-tumor activity in animals. Thus, these studies are designed to follow up on those intriguing results in an effort to engineer and understand a superior method for cancer treatment.

ABSTRACT The mission of the Cancer Immunology (CI) Program is to understand the basic biology of the immune response to cancer and to use those data to optimize immunotherapy for cancer?first in preclinical models, and subsequently through the design and execution of cutting-edge translational clinical trials. The Program, formerly led by Drew Pardoll, M.D., Ph.D., and Elizabeth Jaffee, M.D., is now led by Dr. Pardoll and Charles Drake, M.D., Ph.D., as Dr. Jaffee assumed the role of Deputy Director of the Sidney Kimmel Comprehensive Cancer Center (SKCCC). The Program includes 21 members from seven departments within the Johns Hopkins University School of Medicine. National Cancer Institute (NCI) and other peer-reviewed support of Program members totals $8.9 million total costs annually, and the Program receives an additional $26.1 million annually in nonpeer-reviewed funding. Sixteen members have peer-reviewed funding. The total number of publications by Program members is 434, of which 108 (25%) were Intra-Programmatic, 188 (43%) were Inter- Programmatic and 142 (33%) were multi-institutional collaborations. Building on basic, translational and clinical trials successes over the past five years, the Program seeks to harvest the untapped capacity of the immune system's power to provide further durable cancer remission and even cure. The Programs aims are to: Aim 1: Continue to unravel basic mechanisms of immune regulation and cancer immunity that will fuel the next generation of cancer immunotherapies. Aim 2: Utilize appropriate preclinical models to understand and optimize antitumor immunity, including identification of the most potent combinatorial approaches, such as vaccines together with checkpoint inhibitors. Aim 3: Initiate and complete cutting-edge clinical trials, including the incorporation of appropriate translational biomarker studies designed to guide, with precision, current and next-generation immunotherapies. In particular, the Program will build on studies over the past five years identifying ligands in the tumor microenvironment (TME) and on tumor genetics and viral association as predictors of response to checkpoint blockade. These aims are facilitated by partnerships among laboratory-focused investigators, translational investigators in the CI Program and multiple other SKCCC Programs to jointly develop specific preclinical immunotherapy strategies and then translate them into the most suitable clinical settings. Notable advances over the past five years include 1) the preclinical testing and foundational clinical development of the first anti-PD-1 antibodies in cancer therapy, 2) development of the PD-L1 biomarker, 3) demonstration that mismatch repair deficiency (MMRd) cancers and virus-associated cancers have a very high response to anti-PD-1, 3) demonstration of patient benefit with novel prime-boost vaccine strategies in pancreas cancer, 4) development of marrow- infiltrating lymphocytes for adoptive cell therapy, 5) discovery of four novel immune checkpoint pathways, 6) development of a novel vaccine platform (STINGVAX), and 7) discovery of metabolic pathways that modulate T cell function and enhance antitumor efficacy.

PROJECT SUMMARY We previously identified tumor cell PD-L1 membranous (cell surface) expression in pretreatment biopsies as a correlate of the likelihood of response to anti-PD-1 therapy. This general finding of an association of PD-L1 expression with tumor response to anti-PD-1 therapy has now been substantiated across thousands of patients with certain tumor types treated with both anti-PD-1 and anti-PD-L1. However, while PD-L1 expression enriches for response to anti-PD-1/PD-L1, it is not sufficient. Other related features which have been suggested to improve on the PD-L1 biomarker includes whether PD-L1 is expressed on a tumor cell or immune cell, the CD8 cell density at the tumor's leading edge, CD8:FoxP3+ cell ratio, the distance between PD-1 and PD-L1 protein expression, and PD-1 expression levels (low vs. high). We have developed a quantitative multiplex immunofluorescence assay which captures all of these features and includes PD-L1, PD-1, CD8, FoxP3, CD68, a tumor marker (e.g. Sox10/S100 for melanoma), and DAPI. Through this assay, it is possible to define which cell type is expressing PD-1 and PD-L1 and to enumerate specific cellular subsets. It is also possible to include spatial parameters, including the distance between PD-1 and PD-L1, which have not previously been included in predictive or prognostic surgical pathology specimen-based assays. As a part of the parent R01, we have shown that our assay has increased sensitivity and specificity for response when compared to the assessment of PD-L1 expression alone in multiple tumor types, including melanoma, non-small cell lung carcinoma, Merkel cell carcinoma, head and neck squamous cell carcinoma, amongst others. We have completed formal assay validation studies at a single site, and in some instances have completed comparisons between two sites. The purpose of this proposal is to perform inter-site validation of the assay amongst four major academic sites (Johns Hopkins, Yale University, MD Anderson, Providence Portland Medical Center). This proposal includes two main Aims: 1) validation of the staining and scoring reproducibility across all four academic sites on tonsil and archival melanoma specimens, and 2) the establishment of final assay thresholds using ~100 pre- treatment specimens from patients with melanoma treated with anti-PD-1. For Aim 2, staining for each case will also be performed across three academic sites to provide additional information regarding assay reproducibility. Discovery and Validation cohorts will be used to establish final assay parameters linked to clinical outcomes. The deliverable of the study is a refined, multiplex biomarker assay for response/resistance to anti-PD-1 that has been validated across multiple academic sites. The result will be a multiplex IF assay that is suitably staged for advanced development aimed at clinical implementation. Such an assay will facilitate more precise therapeutic guidance in patients receiving anti-PD-1. While melanoma is the focus of the current grant proposal, our preliminary results suggest that this assay will also have great value in numerous other solid tumor types.

Project Summary Tumor cells exploit mechanisms of immune regulation to evade detection and eradication by host defenses. Foxp3+CD4+CD25+ regulatory T cell (Treg)-mediated immune suppression is crucial for immune evasion by tumor cells and an obstacle for successful tumor immunotherapy. Hence, the ability to disrupt Treg function is of major therapeutic significance. Recent work by us and others revealed that the key Treg transcription factor, Foxp3 is subject to polyubiquitination-dependent posttranslational regulation. Particularly, we found the E3 ubiquitin ligase Stub1, which is induced in response to a range of stress signals, facilitates the degradation of Foxp3 providing a potential target for dynamic modulation of Treg suppression. In the current proposal, we are seeking to: 1) Dissect molecular signaling pathways involved in Stub1 expression and its post-translational modification; 2) Understand the consequences of physiological Stub1 induction and genetic deletion for Treg cell homeostasis, differentiation and function; and 3) Test pharmacological activators of Foxp3 ubquitination as novel immunotherapic strategies to undermine immune suppression in the cancer setting. These studies will expand our understanding of the mechanisms behind posttranslational Foxp3 regulation. Specifically, we will further explore pathways determining Stub1 activity and expression, including the previously unappreciated phosphorylation of the ligase by the kinase GS3K?. To this end, we will utilize biochemical approaches and well-characterized models of in vitro and in vivo Treg function to establish the consequences of ablating these pathways. Furthermore, pharmacological modifiers of the Stub1/Ubiquitin-dependent pathway for Foxp3 degradation (identified in a drug screen and previous studies) will be tested for efficacy as breakers of immune suppression - a major obstacle for anti-cancer immunotherapy. This vetting will be carried out in an aggressive murine melanoma model (in vivo) as well as in ex vivo studies of human leukocytes obtained from healthy donors and advanced cancer patients. In so doing we will determine the potential therapeutic application of modulating Stub1 activity to boost anti-tumor immunity. Our experiments may reveal novel modes of regulating Stub1 activity and Foxp3 protein downregulation. Detailed assessment of physiological Stub1 induction and its impact on Foxp3 and Treg function is predicted to demonstrate a potent therapeutic application. Use of Stub1- activators in combination with proven checkpoint targeting agents may yield even better anti-tumor efficacy.

Background: Foxp3+CD4+CD25+ regulatory T cells (Treg)-mediated immune suppression is crucial for immune evasion by tumor cells and an obstacle for successful tumor immunotherapy. Hence, the ability to disrupt Treg function is of major therapeutic significance. Although Foxp3 is a master regulator of Treg, Foxp3 expression is not sufficient to account for the suppressive capacity of Tregs. It has been suggested that Foxp3 needs to associate with other co-factors in order to suppress non-Treg (T effector) genes and enforce Treg associated gene expression and function. Recently, we found that Yes-associated protein (YAP), a downstream co-activator of the Hippo pathway, is highly expressed by Tregs and is critical for Foxp3-mediated suppressive activity. Furthermore, T cell specific YAP knockout mice mount superior immune responses to implanted B16 melanomas. We hypothesize that YAP is an attractive target for immunotherapeutic strategies aimed at breaking tolerance and enhancing anti-tumor immunity in the cancer setting. Specific Aims: In the current proposal, we are seeking to: 1) Further dissect the molecular mechanisms by which YAP facilitates Foxp3+ Treg function; 2) Understand the consequences of YAP deletion for Treg cell differentiation and function; and 3) Explore the anti-tumor efficacy of YAP inhibitor(s) alone or in combination with immune checkpoint blockade. Objectives & Significance: These studies will expand our understanding of the mechanisms behind YAP facilitates Foxp3 regulation and Treg function. In so doing we will further dissect the molecular mechanism by which YAP facilitates Foxp3-mediated Treg function. Furthermore, we will explore Yap as a potential novel therapeutic target by pharmacologically manipulating YAP activity, and testing various inhibitors for efficacy as breakers of immune tolerance, which is a major obstacle for anti-cancer immunotherapy. The use of such Yap inhibitors in combination with other immune modulators such as anti-PD-1 is expected to improve the effectiveness of immunotherapy, and boost anti-tumor immunity and patient survival. Methodology: In these studies we will deploy biochemical, molecular biology, genetic and bioinformatic approaches to further dissect the role of YAP in Treg cell biology, and attempt to discover known and novel inhibitors of Yap that are effective alone and synergistic with immunotherapies in reducing tumor burden. Both novel and known YAP inhibitors will be tested for their capacity to undermine Treg function and break immune tolerance in vitro and in vivo. Expected Results & Implications: Our experiments will reveal a novel role of YAP in Treg cell function. We predict that a detailed understanding of the physiological role of YAP induction and its impact on Foxp3 and Treg function will provide insight into therapeutic targeting of this pathway. The use of YAP inhibitors in combination with proven checkpoint targeting agents may yield even better anti-tumor efficacy.