Following are public and technical abstracts for the New Immune Checkpoint project funded by the Department of Defense Kidney Cancer Research Program (KCRP) for 2017.
Principal Investigator: Chun-Hau Chen
Institution: Beth Israel Deaconess Medical Center, Boston
Funding Mechanism: Concept Award
Award Amount: $131,250
Public Abstract
Renal cell carcinoma (RCC) can lead to significant morbidity and mortality in our patients. Despite the fact that approved therapies, such as pazopanib and sunitinib, which target the vascular endothelial growth factor (VEGF) pathway, and immune-targeted therapies help patients with metastatic RCC, we still see patients develop disease progression. Agents that target VEGF can slow disease progression in a significant number of patients. However, only about one-third of patients who receive these agents respond to treatment. Moreover, the majority of patients develop resistance within about one year. Recently, new advances such as the approval of immune checkpoint inhibitors have come into use. And although the results are generally favorable when patients respond to this type of therapy, only one-quarter of all patients respond, and those that do respond have a limited duration of effect. As a result, there is an urgent need to find novel treatment options for patients with RCC. Our lab has identified a molecule that plays a key role in the regulation of the immune response to cancer. This molecule, HHLA2, is expressed in low levels on normal cells but is found to be highly expressed on cancer cells. T cells are cells in the body that have the ability to destroy harmful cells (i.e., cancer cells); however, it is not known whether HHLA2 inhibits or activates T cells. We believe that HHLA2 plays a key role in regulating T-cell function and that, as a result, HHLA2 may serve as a novel therapeutic target in the treatment of RCC. We will begin to investigate the role of HHLA2 by creating humanized mice or human xenograft models of RCC. Our lab has already created cell lines that express high levels of HHLA2; with these tools we will be able to assess this molecule’s function by using a panel of antibodies to block its function. We will use several different cell lines, from human renal cell cancers, to rigorously test our hypothesis. We are very optimistic that the results of our studies will provide enough rationale to justify clinical trials in the near future. If, by the end of our grant, we are successful in achieving the goals of this research, we hope to learn (1) whether HHLA2 inhibits or activates the immune system; (2) whether any antibodies at our disposal block HHLA2’s function; and, finally, (3) how HHLA2 functions in an organism as examined through our mouse models.
Technical Abstract
Background: Metastatic renal cell carcinoma (mRCC) is a lethal disease. Although recent advances have led to several treatment options for this cancer, patients eventually become resistant to therapy. RCC was long considered a treatment-refractory cancer; however, multiple therapies now exist for patients. A new advance is the approval of inhibitors of the PD-1 pathway with immune checkpoint inhibitors coming into use. One of these agents, Nivolumab, has been approved by the U.S. Food and Drug Administration to treat RCC patients previously treated with the tyrosine kinase inhibitors. Although patients treated with immune checkpoint inhibitors generally respond to therapy, complete responses are rare, and only 25% of patients respond to nivolumab, with 40% showing progressive disease within 4 months of starting treatment. As a result, it is urgent to identify a new therapeutic strategy for treating RCC. HHLA2 is one of several new molecules in the B7/CD28 family of immune checkpoints. HHLA2 is expressed on few normal tissues, but it is expressed in various human cancers with very high levels of expression in RCC. It has been described as both inhibiting and activating T-cell proliferation and cytokine production, but its exact role on T-cell function in RCC is unknown and HHLA2 has not been studied in an in-vivo context. Moreover, there is no mouse orthologue of HHLA2, so mouse studies must be done with humanized mice or human xenografts. We have generated good available tools to study HHLA2 in RCC.
Hypothesis: Inhibition of HHLA2 will prevent T-cell inactivation and ultimately lead to tumor growth stasis or regression. Increased levels of HHLA2 on tumor cells in vivo will correlate with more aggressive and metastatic disease. Objective: We will identify whether or not HHLA2 activates or inhibits T-cell function. We will screen a panel of antibodies to assess their ability to inhibit HHLA2 function both in vitro and in vivo. We will examine HHLA2’s function in vivo by developing mouse models to investigate HHLA2’s role in RCC.
Specific Aims and Study Design
Aim 1: Does HHLA2 inhibit T-cell function and response in co-cultures of human RCC lines and T cells? Although HHLA2 has been shown to inhibit T-cell function, it is not known whether HHLA2 has a stimulatory or inhibitory function in RCC. To further examine the role of HHLA2 in T-cell responses in primary and secondary stages of T-cell activation, the overexpression of human RCC cell lines overexpressing HHLA2 (786-O or A498) will be co-cultured with CFSE-labeled naïve or differentiated T cells, and we will use the following parameters to evaluate the effect of HHLA2 overexpression: (1) on T-cell proliferation using the analysis of CFSE-dilution pattern; (2) on T-cell apoptosis using cytotoxicity assay and, (3) on cytokine production using a multiplex cytokine array and assay T cell responses by RNA Sequencing.
Aim 2: Test an available panel of antibodies to identify a potential HHLA2 immunotherapeutic agent and identify its downstream signaling pathways.
To elucidate the modulation of T-cell function by HHLA2, a panel of antibodies that blocks or activates HHLA2 or its receptor TMIGD2 will be used to test the efficacy of blockade using the same assays from Aim 1. The antibodies we will use will depend on whether we find in Aim 1 that HHLA2 is inhibitory or stimulatory in RCC. To identify HHLA2 downstream signaling pathways, a putative phospholipid-binding motif of HHLA2 will be tested for its effects on T-cell function. We will (1) use phospholipid-binding assays to test the binding ability of the basic-rich domain in the HHLA2 cytoplasmic trail; (2) use the recently generated HHLA2 deleted tail mutant to test its activity in T-cell responses.
Aim 3. Test the in vivo function of HHLA2 in a humanized mouse model to examine the relationship between HHLA2 and regulation of T-cell responses during RCC formation.
To examine the relationship between HHLA2 and its regulation of T-cell responses during RCC formation, we will (1) design a therapeutic mouse trial in a humanized RCC xenograft model with a blocking antibody selected from our results in Aim 2. The HHLA2-overexpressing or vector control RCC cell lines will be implanted in mice, followed by the injection of educated T cells. When the tumor is measureable, mice will be treated with control or anti-HHLA2 monoclonal antibodies (mAbs). The endpoints will include (1) the size and weight of primary tumors and (2) assessing downstream effects of HHLA2 pathway inhibition on the immune environment in treated tumors.
Innovation: In the field of RCC, we are fortunate to have agents such as VEGFR inhibitors and immune checkpoint inhibitors that have clear activity. However, patients still develop disease progression and die. Innovative strategies to improve these treatments could add to the efficacy of these agents and extend life and also improve quality of life. We propose that HHLA2 is a novel therapeutic target in RCC. The main innovation in this project is the idea that HHLA2 interacts with T cells and has dramatic impact on tumor growth. We believe that the data from these studies, taken together, will support our hypothesis that HHLA2 is a novel target in RCC and that it can be blocked effectively to provide clinical benefit to patients.
Impact: Identification of a new target could translate to development of new therapies for patients who have failed traditional therapy or have progressed on standard of care treatments. Our proposal will lay the groundwork for the confirmation that HHLA2 plays a pivotal role in T-cell function and response to tumor cells, ultimately providing rationale for the development of clinical HHLA2 blockers and future clinical trials. Our hope is that the study outlined above will provide the bedrock upon which more studies will rest, eventually leading to therapies that will increase responses and the overall survival of patients with metastatic renal cell carcinoma.