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Host Protective Engineering of Cancer Immunity by Targeting the Intracellular Immune Checkpoint NR2F6

Periodic Reporting for period 2 - HOPE (Host Protective Engineering of Cancer Immunity by Targeting the Intracellular Immune Checkpoint NR2F6)

Reporting period: 2020-04-01 to 2021-09-30

Cancer immunotherapy that targets surface receptors like CTLA4 and/or PD1 with recombinant antibodies (a treatment regimen named immune checkpoint blockade; ICB) or personalized chimeric antigen receptor-engineered T cell immunotherapy (CAR-T) have emerged as innovative therapeutic pillars in oncology. Although it has definitively been a game changer for cancer treatment, a sizeable subset of patients still fails to respond to and even fewer patients are cured by these current clinical strategies.

Importantly, therefore, additional therapeutic target protein candidates suitable for boosting anti-tumor effector responses have been found inside effector CD4+ and CD8+ T (Teff) cells. In my ERC project “HOPE”, we investigate the unique potential of the intracellular orphan receptor NR2F6 (nuclear receptor subfamily 2 group F member 6) and its effector pathway in T cells as emerging alternative target for cancer immunotherapy to significantly increase response rates of i.e. NSCLC lung cancer patients. Utilizing germline gene ablation as well as CRISPR/Cas9-mediated acute gene mutagenesis, NR2F6 has been firmly characterized as immune checkpoint. Targeting this nuclear receptor appears to be a strategy for improving antitumor immunotherapy responses especially in combination with ICB and, potentially, with CAR-T therapy regimens. Taken together, our current preclinical experimental knowledge firmly validates the immune checkpoint function of NR2F6 in murine tumor models, which provides a promising perspective for immunotherapy regimens in humans to strengthen immune system responses in the near future.

Why does the alternative and druggable NR2F6 immune checkpoint appear to be important?

NR2F6 is a recently discovered cancer immune checkpoint protein found at particularly high levels in the effector T cells that have infiltrated solid tumors. In agreement with this observation, genetic NR2F6 inhibition experiments have shown encouragingly strong improvements in T cell responses, employing both human PBMC in vitro and pre-clinical cancer therapy models in vivo. Investigation of the inhibition of Nr2f6 in mice that used an ex vivo CRISPR/Cas9-mediated gene ablation of Nr2f6 in T cells prior to therapeutic cell therapy in conjunction with an approved ICB therapy improved this therapeutic anti-cancer activity. Because the future of immuno-oncology therapy concept is positioned in combination therapies, NR2F6 might be an emerging next generation target that combines intracellular as well as surface receptor pathways, thus improving T cell efficacy and therapeutic outcome, thereby increasing the percentage of cancer patients who positively react to treatment. In deed NR2F6 targeting regimens are now being investigated in R&D laboratories around the world. Such an alternative therapeutic approach, if demonstrated to be successful, could supplement the existing therapeutic models and significantly increase response rates of cancer patients and/or expand the reach of immune therapy regimens to broaden the range of cancer types.

Our current knowledge of NR2F6 is summarized, with special emphasis on the unique potential of NR2F6 and its critical and non-redundant role in T cells. As a note of caution, however, the current target validation of NR2F6 is based on evidence obtained from pre-clinical models only. Thus, the fate of NR2F6-based immuno-oncology therapy envisioned here ultimately depends on functional antagonists for NR2F6, once established, and their outcome in human clinical trials. Nevertheless, continued research on the orphan nuclear receptor NR2F6 may very well represent a suitable path for drug discovery and might offer an innovative, mechanism-based, therapeutic strategy to augment the sensitivity of tumor-infiltrating T cells to tumor antigens in solid cancer patients. Therefore, the critical objective for the second half of my ERC project work is to firmly validate this alternative cancer immune checkpoint NR2F6 as a unique cancer therapeutic candidate target of the T effector cell compartment for next-generation immune-oncology regimens.

Gottfried Baier, home page:
According to the approved project plan, the course of the vast majority of our research project to date, and in spite of Covid-19 pandemics, is on schedule.

@Objective 1: NR2F6 pathway as off switch of tumor T cell responses - status: on time

@Objective 2: Link biological and clinical properties of NR2F6 (M25-48) – status: initiated

@Objective 3: Human NR2F6 as a cancer therapeutic candidate target (M37-60) – status: in preparation phase
Outlook of expected results until the end of "HOPE":

Our detailed insight of NR2F6 mechanism of action (MoA) using biochemical and cell-based assays has yield improved NR2F6 target validation knowledge for laying the groundwork for a streamlined NR2F6 inhibition-based therapy concept. Our discovery that inactivation of NR2F6 as a single regulator of immune suppression provides a unique cancer therapeutic strategy of selectively manipulating the tumor antigen-specific CD4+ and CD8+ effector T cells to elicit host-protective cancer immunity in cancer patients appear particularly relevant. Reversing the newly defined NR2F6-mediated NFATc1 antagonism is essential for effector T cell activation and differentiation, a process critically involved in host protection during malignant disease and, potentially, also other immune disorders.Our long-term research in Innsbruck will continue to explore forward translation of our basic knowledge of NR2F6 immune checkpoint function. Exploiting this newly discovered NR2F6 signaling hub, the purpose of the translational research continues to decisively contribute to the development of strategies that selectively interfere with NR2F6 pathway function(s) for cancer treatment.

First, within this initiative, we will be able to thereby set the stage for validation and characterization of e.g. low molecular weight (LMW) compounds with respect to their future therapeutic potential to treat malignant diseases in the clinic. Most relevant and robust functional HTS as well as secondary assays as basis for the hit-to-lead phase of this drug finding program can now be developed for orthogonal validation of candidates in intact human T cells. Next to the high academic value of our research (that likely leads to high-impact papers in the near future), this represents a pivotal support for the identification of NR2F6 inhibitor candidates.

Second and as the project progresses, we also considering to take the direction of personalized chimeric antigen receptor-engineered (CAR) T cell immunotherapy for solid NSCLC tumors that proves to be particularly promising. Extension of CAR T cell therapy success by NR2F6 pathway blockade may allow to develop more effective treatment regimens for the treatment of advanced/metastatic NSCLC lung cancer. Such pilot experiments, albeit speculative and risky at the moment, as a first proof-of-concept of the anti-tumoral enhancement potential to increase human CAR-T cell functionality and persistence will be pursued within objective 3. At the end of the day, this will likely allow us the time-efficient validation of distinct strategies targeting NR2F6 in vivo using our experimental tumor model systems.