Haidong Dong - US grants

DESCRIPTION (provided by applicant): Renal cell carcinoma (RCC) encompasses 90% of kidney cancers and 3% of adult cancers. In the U.S. alone, more than 30,000 new cases of RCC are diagnosed and nearly 13,000 patients die from RCC annually. RCC is categorized into three distinct histologic subtypes of which clear cell (ccRCC) is the most common and lethal form, accounting for nearly 90% of RCC-related deaths. The majority of patients now diagnosed with ccRCC will present with organ-confined tumors seemingly amenable to surgical extirpation. Yet, almost half of these patients will experience metastatic relapse after surgery. Treatment options for metastatic ccRCC are limited both in terms of scope and efficacy. Thus, key advances to improve ccRCC patient care will necessarily include: 1) better methods to identify patients at high risk for metastatic relapse following surgery;2) a better understanding of molecular mechanisms that drive ccRCC metastatic progression;and 3) rational, mechanism- targeted therapies to treat metastatic disease in ccRCC patients. Such improvements could emanate from the introduction of newer, more effective drugs or optimization of combination therapies. In this proposal, emphasis will be placed on the latter topic to gain a better understanding of key issues related to the immunobiology and immunotherapeutic treatment of advanced ccRCC. We have recently reported that ccRCC tumors can aberrantly express several relatively novel immune cell coregulatory ligands, namely B7-H1, B7-H3 and B7-H4. We have further shown that enhanced tumor expression of these B7-H ligands can predict aggressive ccRCC behavior including enhanced risk for cancer progression and cancer-related death. Collectively, these observations raise the distinct possibility that human tumors might employ B7-H ligands to disarm host antitumoral immunity in order to promote malignant progression. Thus, tumor-associated B7-H ligands will likely prove useful to refine prognostic algorithms to pinpoint high-risk patients who are most prone to ccRCC progression and death. However, whether tumor- associated B7-H ligands actually function as clinical inhibitors of cell-mediated antitumoral immunity to promote malignant progression remains to be determined. This proposal is singularly devoted to studies that, we believe, will directly improve clinical ccRCC patient care. Specifically, our studies will rigorously test the prognostic and immunotherapeutic potential of B7-H ligands aberrantly expressed by ccRCC. Specifically, we will address a most vital question: "Do B7-H ligands within clinical tumors truly function to impair antitumoral immunity in cancer patients?" Such findings will help to establish the merits (and limitations) of in vivo B7-H ligand blockade as a clinical immunotherapeutic approach to treat human forms of malignancy. Unifying Hypothesis for Specific Aims 1- 4: The overall hypothesis for this proposal is that the aggressive behavior of ccRCC tumors stems, at least in part, from the ability of tumors to elaborate an array of homologous but spatially distributed B7-H ligands that act in concert to undermine cell-mediated immunity in cancer patients. We further postulate that the most aggressive of ccRCC tumors concurrently express increased levels of other proteins that promote tumor cell proliferation and survival and, may also serve as antigenic moieties to lure immune cells into a perilous intratumoral environment. Thus, each aim of our proposal has been crafted to be free-standing, each addressing key clinical and scientific issues pertaining to B7-H ligands and moving from a macroscopic to microscopic understanding of B7-H ligands as immune- regulators and immunotherapeutic targets for clinical ccRCC. Specific Aim 1. Test if putative immunosuppressive, tumor-associated B7-H ligands (B7-H1, B7-H3, B7-H4, and PD-1) can collaborate with each other, as well as with other molecular markers that assist in fostering tumor cell proliferation and survival (IMP3, CAIX, Ki-67, and survivin) to improve outcome prediction for ccRCC patients. Specific Aim 2. Test whether ccRCC metastases are enriched for prognostic B7-H related molecules (B7-H1, B7-H3, B7-H4, and PD-1) as well as IMP3, Ki-67, and survivin when compared against patient-matched primary tumors from which metastases arise. Specific Aim 3. Test if soluble B7-H (sB7-H) ligands can be detected in the sera of ccRCC patients to improve strategies to monitor and, perhaps, immunotherapeutically treat ccRCC patients. Specific Aim 4. Test if tumor-associated B7-H ligands inhibit T and NK cell-mediated immune responses within clinical ccRCC specimens using in situ IHC analysis in combination with in vitro assays of immune cell function. PUBLIC HEALTH RELEVANCE: Clear cell renal cell carcinoma (ccRCC) is the most lethal form of kidney malignancy, killing nearly 13,000 patients annually. Current treatment options for metastatic ccRCC are limited as evident in 2 year survival rates of 10-20%. Advancing ccRCC patient care centers around several areas including better methods for identifying patients at high risk for metastatic relapse following surgery, more thorough understanding of molecular mechanisms that drive ccRCC metastatic progression, and developing rational mechanism-targeted therapies to treat metastatic ccRCC disease. We have determined that ccRCC tumors can aberrantly express several relatively novel immune cell coregulatory ligands, namely B7-H1, B7-H3, and B7-H4, the expression of which predict aggressive ccRCC behavior and increased cancer-related death. These observations raise the distinct possibility that human tumors, especially ccRCC, might employ B7-H ligands to blunt patients'anti-tumor immune responses, thereby promoting malignant progression. Tumor- associated B7-H ligands may likely prove useful to refine prognostic algorithms to pinpoint high-risk patients who are most prone to ccRCC progression and death. However, whether tumor-associated B7-H ligands actually function as clinical inhibitors of cell-mediated antitumoral immunity to promote malignant progression remains to be determined, as does the rationale for B7-H blockade as immunotherapeutic means to improve treatment of advanced ccRCC (or any other malignancy for that matter). This proposal is singularly devoted to studies that, we believe, will directly improve clinical ccRCC patient care. Specifically, our studies will rigorously test the prognostic and immunotherapeutic potential of B7-H ligands aberrantly expressed by ccRCC. Specifically, we will address a most vital question: "Do B7-H ligands within clinical tumors truly function to impair antitumoral immunity in cancer patients?" Such findings will help to establish the merits (and limitations) of in vivo B7-H ligand blockade as a clinical immunotherapeutic approach to treat human forms of malignancy.

DESCRIPTION (provided by applicant): B7-H1 (PD-L1) and its receptor PD-1 are checkpoint molecules in immune system. Elevated B7-H1 expression on tumor cells has been correlated with poor prognosis in several human cancer diseases. Preliminary clinical observations show promising therapeutic effects of B7-H1 blockade in treating advanced human solid tumors (lung cancer, melanoma and kidney cancers). However, only a small portion of patients have long lasting objective responses, although prolonged stabilization of diseases is observed in 12-41% of treated patients. Our compelling preliminary studies suggest that unanticipated disrupting of a previously unknown pro-survival function of B7-H1 expressed by T cells could be the major impediment to effective blockade therapy. The objective of this application is to define T cell intrinsic function of B7-H1 evaluate the impact of B7-H1 antibody capable of disrupting B7-H1's intrinsic function. The central hypothesis of this proposal is that B7-H1 has an intrinsic pro-survival function in activated CD8 T cells and is required for establishing protective immunity, B7-H1 antibody capable of disrupting this function compromises CD8 T cell-mediated antitumor immunity. The clinical impact of this proposal is that it may provide new knowledge and methods for maximizing B7-H1 blockade therapy by selection of optimal antibody. The rationale of this proposal is that the functional role of B7-H1 expressed by T cells is far from complete. Given that T cells are major effectors of immunity and B7-H1 expression on T cells is not static and varies with activation statuses warrants investigations into the significance of T cell-associated B7-H1 in greater detail. This issue becomes even more urgent since systemic antibody- mediated blockade of B7-H1 is being used in a multiple center phase I/II cancer immunotherapy trials. Thus, the proposed research is relevant to the mission of the NIH to develop new approaches in tumor immunotherapies. Supported by solid preliminary data, our hypothesis will be tested by pursuing three specific aims: (1) To define the role of T cell intrinsic B7-H1 in T cel differentiation; (2) To evaluate the impact of B7-H1 antibody on intrinsic function of B7-H1 in T cells; (3) To define the intrinsic signaling pathway of B7-H1 in T cell apoptosis. The proposed research is significant because our studies will provide new knowledge about regulation of T cell survival and provide new approaches in advancing the fields of B7 family checkpoint molecules and tumor immunotherapy.

? DESCRIPTION (provided by applicant): Given the variability in response to the novel immunotherapeutic agents and the desire to extend their long- term benefits to more patients, there is an increased need for the development of biomarkers that can predict treatment outcomes, thereby ensure that these expensive new treatments, which may have significant toxicities, are offered to the patients who are more likely to benefit. The FDA approved anti-PD1 agent (pembrolizumab/MK3475) typifies this phenomenon of dramatic therapeutic responses in a subset of patients who cannot be pre-identified, necessitating broad treatment application in an unselected patient population. The objective in this application is to evaluate and validate Bim (a PD-1 downstream signaling molecule) levels in tumor-reactive PD-1+ T cells, as well as soluble B7-H1(PD-L1), as monitoring or predictive biomarkers for response to anti-PD-1 therapy in patients with metastatic melanoma (MM), an excellent model, as PD-1/PD-L1 pathway is an increasingly exploited therapeutic target in this disease and a variety of other cancers, with extremely promising results in clinical trials. Our central hypothesis is that Bim levels in tumor-reactive CD11a high D-1+CD8+ peripheral blood T cells can objectively monitor responses to anti-PD-1 therapy and that excessive release of soluble B7-H1 by the tumor leads to Bim upregulation and treatment resistance in melanoma. This hypothesis will be tested in MM patients undergoing treatment with an anti-PD-1 monoclonal antibody (pembrolizumab) at our Institution. Guided by strong preliminary data, we propose two Specific Aims to test our central hypothesis: 1) Establish the role of Bim for monitoring disease status during anti-PD-1 therapy; and 2) Identify the mechanisms of resistance to anti-PD-1 blockade. In aim 1, an already proven PD-1 downstream signaling molecule Bim (the pro-apoptotic BH3-only protein), which has been established as a feasible marker for the status of PD-1 engagement in the applicants' hands, will be utilized to monitor and predict the T cell responses to anti-PD-1 therapy, as well as identify patients who may achieve late clinical benefit despite radiologic pseudoprogression. Under aim 2, we will explore that soluble B7-H1 (PD-L1) could influence the sensitivity to anti-PD-1 therapy and be a new therapeutic target of dual blocker therapy (anti-PD-1 and anti-PD-L). Our approach is innovative because it utilizes novel markers to assess the reversibility of exhausted anti-tumor PD-1+ T cells and the efficiency of anti-PD-1 blockade, which would directly influence the therapeutic outcome with anti-PD-1 therapy. Our proposed research is significant, because our results could help identify patients with melanoma (and possibly other malignancies) who are most likely to benefit from anti- PD-1 therapy, therefore increasing drug efficacy and decreasing toxicity through a more personalized approach to cancer treatment.

? DESCRIPTION (provided by applicant): Metastatic castration-refractory prostate cancer (CR-PC) patients have poor prognosis with median survival of approximately 14 months. 10-20% of all prostate cancer patients develop metastatic CR-PC within 5 years of initial diagnosis, and therefore, improved therapeutic strategies are needed. Oligometastasis describes a clinical state where metastases are limited in number, and metastatectomies for various oligometastatic solid tumors can lead to long-term survival. Stereotactic Ablative Radiotherapy (SABR) is a noninvasive therapy with local control (LC) similar to surgery. In our experience, oligometastatic prostate cancer patients (65% were CR-PC) experienced 100% LC at 6 months with SABR with corresponding decline in their PSA. 11C-Choline PET/CT was FDA-approved in recurrent metastatic prostate cancer patients, and prior restaging studies have validated the sensitivity, specificity, positive predictive value, and negative predictive value of Choline PET at 85-100%, 76-96%, 76-91%, and 81-100%, respectively. Therefore, 11C- Choline PET/CT may help to identify true oligometastatic CR-PC patients. Objectives/Hypothesis: The combination of 11C-Choline PET/CT and SABR may improve true oligometastatic (CR-PC) patient selection, alter their clinical management (i.e., from noncurative to curative intent with the addition of SABR), and impact natural disease progression (improve LC, PSA progression-free survival and overall survival [OS]). SABR may also induce anti-prostate cancer immunity, which could be amplified into long-term protective immunity with immune-regulation inhibitors (e.g., ipilimumab, anti-PD1/PD-L1). Specific Aims/Study Design: We propose a prospective single arm phase II clinical trial to assess the role of 11C-Choline PET/CT and SABR in metastatic CR-PC patients with the goal of improving clinical outcomes (Aim 1). We also propose a translational study in Aim 2 to explore the induction anti-prostate cancer immunity elicited by SABR treatments. The confirmation of the ability of SABR to induce anti-prostate cancer immunity would provide a robust rationale to combine SABR and immunomodulating agents (i.e., checkpoint inhibitors) in widespread castration- and chemotherapy- resistant metastatic prostate cancer. The proposed biomarker (CD11ahighPD-1high CD8+ T cells) may improve identification of potential responders to the combined SABR and anti-PD1 therapy. Impact: The incorporation of 11C-Choline PET/CT in the selection of oligometastatic CR-PC patients whose limited metastases are amenable to SABR may improve OS. If our hypothesis is proven successful, there would be a paradigm shift in the clinical management of these patients (i.e., from noncurative to curative intent). The in situ induction of anti-prostate immunity by SABR, if confirmed, would form a robust rationale for the combination of SABR and immunomodulating agents in future clinical trials.

Abstract Targeting immune checkpoint signaling with blocking antibodies has reached a limitation in the treatment of advanced cancers. Although antibodies that bind programmed death ligand 1 (PD-L1) are effective in blocking PD-L1's extracellular interaction with PD-1 receptor on T cells, the potential adaptive upregulation of PD-L1 and its recycling from intracellular compartment to the cell surface may compromise their efficacy. Importantly, the discovery of PD-L1's intracellular functions in cancer cells to promote thier survival and metabolism also highlight a mechanism by which tumor cells can gain resistance to cytotoxic therapy and call for a new strategy to target PD-L1. There is therefore a critical need to design, test, and translate new agents that can simultaneously inhibit PD-L1's extracellular and intracellular functions. This application will utilize a new PD-L1 antibody (clone H1A) that can reduce the expression of PD-L1 in tumor cells through disrupting the association of PD-L1 with CMTM6 (a molecule that can stabilize PD-L1 recycling and expression) and subsequently directing PD-L1 for degradation. H1A-induced degradation of PD-L1 may not only disrupt PD-1/PD-L1 interactions due to the loss of PD-L1, thus removing PD-1's suppressive signals in T cells, but also disrupt PD-L1's cell-intrinsic functions within tumor cells and myeloid cells, thereby decreasing tumor resistance to chemotherapy and releasing the immune-stimulatory function of myeloid cells. Thus, H1A antibody may be a good candidate for targeting the dual functions of PD-L1 for cancer therapy. Based on preliminary data, the central hypothesis of this proposal is that intracellular signaling through PD-L1 results in tumor resistance to cytotoxic chemotherapy and limits the immune-stimulatory function of myeloid cells. Thus, targeted agents that result in degradation of PD-L1 and elimination of its intracellular signaling ability represent a novel therapeutic strategy that will both synergize with chemotherapy and improve the immune response. This hypothesis will be tested by pursuing two specific aims: (1) Determine how H1A antibody synergizes with chemotherapy to overcome tumor resistance; (2) Determine how H1A antibody promotes an enhanced T cell response to attack tumors. To further assess the future clinical use of H1A antibody, a fully humanized version of H1A and humanized PD-1 and PD-L1 mice have been produced, which will allow for evaluation of the therapeutic effects of H1A either alone or in combination with chemotherapy and exploration of H1A's new mechanism of action in vivo. The overall impact of the proposed research is high because it will provide a new therapeutic agent that is capable of targeting the dual functions of PD-L1, resulting in improved efficacy of cytotoxic chemotherapy and an enhanced immune response. This strategy represents a significant paradigm shift within the field in terms of how to target immune checkpoint molecules like PD-L1 for future clinical applications.