2022 Awardees

North Carolina State University, Raleigh, NC, United States.

Invasive bladder cancer is a lethal disease that often requires life-altering surgical removal of the bladder to prevent or limit metastasis. Recent approvals of five checkpoint inhibitors for advanced or refractory bladder cancers demonstrate its responsiveness to immunotherapy. However, less than half of advanced bladder cancer patients benefit from checkpoint immunotherapy and antitumor responses are often transient. Our previous preclinical studies in mice demonstrated that intravesical immunotherapy with interleukin- 12 (IL-12) formulated with the mucoadhesive biopolymer chitosan (CS), i.e., CS/IL-12, can eliminate nearly all established orthotopic bladder tumors in a T cell-dependent manner. These studies also demonstrated that intravesical CS/IL-12 can induce robust abscopal responses with the elimination of distant, untreated bladder tumors in most mice. Although these data are promising, several limitations of implanted murine bladder tumor models have hindered clinical translation. Thus, this application proposes to evaluate the safety and activity of intravesical CS/canine IL-12 (caIL-12) immunotherapy in pet dogs with spontaneous invasive urothelial carcinoma (UC) of the bladder. There are numerous similarities between canine and human bladder cancers including mechanisms of tumorigenesis, rates and sites of metastasis, and distinguishable molecular subtypes. Given these similarities, treatments found to be successful in dogs are more likely to be successful in people. The objectives of this project are: 1) to demonstrate that intravesical CS/caIL-12 immunotherapy can safely induce antitumor immunity against canine invasive UC; and 2) to determine if canine bladder cancer is a useful model for the evaluation of this and other novel immunotherapies. The first objective will help prepare intravesical CS/IL-12 for translation into human clinical trials, while the second objective will help bladder cancer researchers overcome the limitations of rodent models and provide a more faithful representation of human bladder cancer. To accomplish these objectives, 2 specific aims have been designed. Aim 1 is focused on safety, as CS/caIL-12 has never been evaluated in dogs. After synthesizing and validating recombinant caIL-12 in vitro, we will perform a dose-escalation study of intravesical CS/caIL-12 in pet dogs with spontaneous invasive UC of the bladder. Aim 1 will establish a recommended dose (RD) based on safety readouts that utilize a combination of clinical examinations and laboratory tests. Proposed pharmacokinetic and immunophenotyping studies will investigate the possible systemic uptake and dissemination of intravesical immunotherapy and the resulting immune impacts. Aim 2 will assess antitumor and immunological responses to the RD of intravesical CS/caIL-12 in an expanded cohort of dogs with bladder cancer. Correlative studies will determine if intravesical CS/caIL-12 influences T cell infiltration, the tumor- immune microenvironment, neoantigen reactivity and/or T cell clonality. The influence of molecular subtype and tumor mutational burden on antitumor and/or immune responsiveness will be assessed.

Purdue University, West Lafayette, IN, United States

Invasive urinary bladder cancer (invasive urothelial carcinoma, InvUC) is lethal in 50% of patients. Immune checkpoint inhibitors (ICIs) can cause dramatic remission of advanced InvUC, but only ~20% of patients have this level of benefit. Pre-clinical animal models are critical for research to improve ICI outcomes, but experimental models lack many of the hallmark features of human cancer and are poor predictors of outcomes in humans. To address the gap in relevant animal cancer models for immunotherapy research, we will study dogs with naturally-occurring InvUC as canine InvUC closely mimics the human condition in pathology, molecular features including luminal and basal subtypes, clinical presentation, local invasion, and frequent metastasis. The proposed work will strengthen the canine InvUC model by defining immune cell responsiveness and ICI outcomes, with comparison to human studies. Our long range goal is to improve the outlook for people with InvUC. The objective of this proposal is to address the gap by determining the suitability of canine InvUC to serve as a model to improve ICI therapy in humans. The central hypothesis is marked similarities will exist between dogs and humans in the immune cell responses in InvUC, and ICI therapy effects including immune adverse events, antitumor activity, immunological responses, and predictors of treatment success and failure. Some differences between dogs and humans are expected, with these also being informative. The hypothesis is formulated and based on strong evidence in the literature and preliminary data. The rationale is that demonstrating the shared immune cell responses and ICI effects between dogs and humans with InvUC will allow the canine model to be fully employed to improve ICI therapy for humans. The objective will be accomplished through two specific aims: (1) determine similarities and differences between dogs and humans in the immune cell responsiveness to InvUC, and (2) determine the safety and antitumor activity of a canine PD-L1 antibody and predictors of success and failure in dogs with InvUC with comparison to findings in humans. The approach will be to: (1) perform dog-human comparison of InvUC through analyses of RNA-seq, scRNA-seq, WGS, and CITE-seq data, and (2) conduct a clinical trial of an ICI, our canine PD-L1 antibody, in dogs with InvUC to assess antitumor activity, pharmacokinetics, adverse event profile, and correlative sequencing and clinical data to predict outcomes, and to compare results to those from human PD- L1 antibody trials. The expected results will define shared immune cell responses and ICI effects in dogs and humans, expand the understanding of predictors of ICI therapy, and justify use of the canine model to improve ICI and other immunotherapies in humans. Samples from dogs in the ICI trial will also be made available for other immune, microRNA, and microbiome research by our collaborators and beyond. We look forward to contributing to the Canine Cancer Immunotherapy Network and depositing our study data in the NCI’s Integrated Canine Data Commons, to complement two InvUC data sets that we have previously deposited.

Tufts University, Boston, MA, United States

Despite substantial improvements in therapeutic strategies, generating robust anti-tumor immune responses in human cancers with a lower somatic mutation burden remains a substantial challenge. Recent data indicate that a critical player in this process, dendritic cells (DCs), fail to effectively elicit efficient and durable T cell responses unless they have entered a unique state of hyperactivation. In this setting, DCs exhibit enhanced migration to local lymph nodes (LNs) and sustained secretion of IL-1β, a cytokine critical for memory T cell formation. In mouse tumor models, vaccination with whole tumor lysate plus an adjuvant consisting of the TLR 7/8 agonist R848 (resiquimod) in combination with a unique isolated lysophosphatidylcholine (22:0 Lyso PC) promotes DC hyperactivation, expansion of antigen specific CD8+ T cells, and robust rejection of tumors. While these findings are encouraging and suggest that identification of specific neoantigens is not necessary to prime and expand a pool of cytotoxic T cells (CTLs), validation and optimization of this approach necessitates the use of a model system that more closely recapitulates human cancers with respect to immune landscape. As such, the purpose of this proposal is to use spontaneous canine cancer, specifically osteosarcoma (OS), as a bridging animal model to validate the utility of DC hyperactivation as a foundational element for generation of robust anti-tumor immunity. The central hypothesis to be tested in this application is that combining hyperactivation of DCs with WTL derived neoantigen will expand a diverse and tumor-specific population of CTLs capable of eliminating residual microscopic metastatic OS tumor cells in dogs following primary tumor removal (amputation). We further predict, that combining DC hyperactivation/WTL with a novel tumor microenvironment (TME) conditioning regimen consisting of toceranib/losartan/ladarixin will enhance the objective response rate in dogs that develop macroscopic lung metastasis. To accomplish this, we will conduct a prospective randomized clinical trial in dogs with OS combining amputation and standard of care carboplatin chemotherapy with adjuvant alone or adjuvant+WTL. Dogs that develop lung metastasis will then be treated with the TME conditioning regimen in combination with adjuvant+WTL. A biobank of tissue samples and blood will be collected from dogs enrolled in these trials including matched primary/metastatic tumors and associated LNs, whole blood, plasma, PBMCs, cell-free DNA, and samples from the vaccine draining LNs. These will be used to perform a set of complementary assays designed to characterize the immune microenvironment and tumor genome over the course of relapse/resistance, credential a novel neoantigen prediction pipeline, and evaluate antigen specific T cell responses. An outstanding team with complementary sets of expertise across clinical trials, translational oncology, comparative genomics, and immuno-oncology has been assembled to ensure stated milestones are achieved. This is bolstered by a dynamic collaboration with our industry partner, Corner Therapeutics, which is committed to supporting this work to facilitate optimization and successful translation into human patients.

University of Pennsylvania, Philadelphia, PA, United States

Chimeric Antigen receptor T (CAR-T) cells have produced unprecedented results in blood cancers but clinical responses in solid tumors are rare. This is due in part to the detrimental effects of the hostile, tumor microenvironment (TME) and the use of patient derived, dysfunctional T cells which are adversely affected by advanced disease states and previous chemotherapy. Invariant Natural Killer T ( iNKTs) are a distinct lineage of CD1d-restricted T lymphocytes with natural tissue (and tumor) tropism, cytolytic effect on CD1d+ cancer cells and tumor associated macrophages and adjuvant effects on anti-tumor immunity. Unlike conventional T cells, allogeneic iNKTs mediate a r obust graft-versus- effect without nducing graft-versus-host disease, thus eliminating the need for gene editing to maintain . Moreover, preclinical studies in mouse models suggest that CAR-enhanced iNKTs can eradicate solid solid-like hematological tumors where CAR-T cells fail. We hypothesize t hat allogeneic CAR-engineered will overcome the barriers to successful CAR-therapy and provide a powerful off-the-shelf universal with curative potential for solid tumors . Yet, unsolved questions related to allo CAR-iNKT safety, optimal regimens to promote persistence, cell dose and therapeutic efficacy in solid tumors remain. while cells from different donors have different it is currently unknown which products are “best” for adoptive cell therapy ( ACT). These cannot be adequately addressed in mice due to the dissimilarity between murine and human iNKT In contrast, Chimeric a i healthy human donors for allogeneic iNKT clinical trials are randomly selected, immunomodulatory capacities that may affect their engraftment our preliminary studies demonstrate that canine and human iNKT cells share remarkable phenotypic and functional similarities and can be CAR engineered and expanded to clinical scale for trial use. Here similar will CAR-iNKT 3 dose and CAR-iNKT immunome the we will use immunocompetent pet dogs with spontaneous steosarcoma (OSA), which is remarkably to pediatric OSA, to advance allogeneic IL-13R  2-targeting CAR-iNKT cells into the human clinic. We first characterize CAR-iNKT cells from different donor dogs and generate a master cell bank of canine allo- products with different immunomodulatory capacities. Next, we will address the safety and effects of strategically designed pre-conditioning regimens, including a combination of cytotoxic chemotherapies, low total body irradiation and a clinical grade iNKT glycolipid agonist, RGI-2001, on allo-CAR-iNKT engraftment persistence in dogs with metastatic OSA. Finally, we will determine the maximum tolerated dose of allo- cells using an accelerated dose escalation trial design and evaluate their effects on the TME, systemic and disease free interval in dogs with appendicular OSA. This work addresses pivotal questions for advancement of allogeneic-CAR-iNKT cells o in a clinically relevant canine cancer “model” to accelerate clinical use of this promising “off the shelf” approach in human patients with solid tumors.