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Extracellular Vesicle-Internalizing Receptors (EVIRs) for Cancer ImmunoGeneTherapy

Periodic Reporting for period 3 - EVOLVE (Extracellular Vesicle-Internalizing Receptors (EVIRs) for Cancer ImmunoGeneTherapy)

Reporting period: 2020-07-01 to 2021-12-31

In the last decade we have witnessed transformative results in the clinical application of both cancer immunotherapies and gene transfer technologies. Tumor vaccines are a specific modality of cancer immunotherapy. Similar to vaccination against pathogens, tumor vaccines are designed to elicit a specific immune response against cancer. They are based on the administration of inactivated cancer cells or tumor antigens, or the inoculation of antigen-presenting cells (APCs) previously exposed to tumor antigens. In spite of significant development and testing, tumor vaccines have largely delivered unsatisfactory clinical results. Indeed, while some patients show dramatic and durable cancer regressions, many do not respond, highlighting both the potential and the shortcomings of current vaccination strategies. Hence, identifying and abating the barriers to effective cancer vaccines is key to broadening their therapeutic reach.

The goal of EVOLVE (EVirs to Optimize and Leverage Vaccines for cancer Eradication) is to propel the development of effective APC-based tumor vaccines using an innovative strategy that overcomes several key hurdles associated with available treatments. EVOLVE puts forward a novel APC engineering platform whereby chimeric receptors are used to both enable the specific and efficient uptake of cancer-derived extracellular vesicles (EVs) into APCs, and to promote the cross-presentation of EV-associated tumor antigens for stimulating anti-tumor immunity. EVOLVE also envisions a combination of ancillary ‘outside of the box’ interventions, primarily based on further APC engineering combined with innovative pre-conditioning of the tumor microenvironment, to facilitate the deployment of effective APC-driven, T-cell-mediated anti-tumor immunity. Further to preclinical trials in mouse models of breast cancer and melanoma, our APC platform will be used to prospectively identify novel human melanoma antigens and reactive T cell clones for broader immunotherapy applications.
EVOLVE is focused on the development of dendritic cells (DCs) that are transduced with extracellular vesicle-internalizing receptors (EVIRs). These chimeric receptors promote (i) the uptake of tumor-derived EVs by the engineered DCs, and (ii) the presentation of tumor-associated antigens to T cells. In the first 3 reporting periods we focused on Aims 1, 2 and 4 of the project and achieved most of the milestones of EVOLVE. A majority of the studies have used an EVIR that targets GD2 as bait antigen for EV uptake.

As planned in Aim 1, we have designed new EVIRs with engineered signaling domains. After a lengthy process of design, screening and validation, we have selected two structurally unrelated EVIRs that induce the activation of transduced DCs in an EV-dependent manner, i.e. only upon binding of the EVIR to tumor-derived EVs. The new EVIRs induce expression of MHC and co-stimulatory molecules on the DCs, and may thus help to overcome some of the inherent limitations of early-generation EVIRs lacking intracellular signaling domains, such as antigen-induced tolerance. We have tested the new EVIRs in the context of DC vaccination studies and obtained promising results (tumor growth inhibition; T cell recruitment and activation; etc.), which will be followed up during the final reporting period.

As planned in Aim 2, we have made substantial progress with establishing more suitable DCs for EVIR transduction and vaccination. We established protocols to generate a new population of mouse DC progenitors (DCPs) that efficiently produce cDC1 and cDC2 (i.e. professional DCs) in tumor-bearing mice. Our results in mouse models of melanoma, lung cancer and colorectal cancer demonstrate that EVIR-engineered DCPs robustly activate antigen-specific T cells and outperform traditional vaccination with antigen-loaded DCs. We are currently implementing the new EVIRs (see Aim 1 above) into the DCP platform.

As planned in Aim 4, we have developed protocols for the validation of our DC platform in the human system. We set up immunoassays involving patient-derived melanoma cells/EVs, HLA-matched DCs transduced with an EVIR that targets GD2, and T cells. These assays have shown that human DC-EVIRs efficiently and specifically uptake GD2+ melanoma EVs and activate T cells toward unrelated melanoma antigens. Furthermore, we have developed protocols to generate DCPs from human blood CD34+ cells, which should enable translating the pre-clinical studies described in Aim 2 to the clinic.

During the 3 reporting periods we have also performed studies aimed to establish and characterize a genetically engineered mouse model of lung cancer that is resistant to immune checkpoint blockade (ICB, a clinically approved albeit most often inefficacious form of immunotherapy). Indeed, we aim to clinically test our DC vaccination platform in both melanoma and lung cancer patients who are refractory to ICB, and we are currently performing DC vaccination studies in the lung cancer model. Thus, work planned for the final period will especially focus on DC vaccination studies in mouse models of melanoma and lung cancer; this will leverage on the know-how (new EVIRs, new DC protocols) developed during the last reporting period.
Although not originally planned, we have made substantial progress with establishing a novel population of DCs for EVIR transduction and vaccination; such DCs may overcome some of the limitations that are inherent to clinically used monocyte-derived DCs. We generated mouse DC progenitors (DCPs) that efficiently produce cDC1 and cDC2 (i.e. professional DCs) in tumor-bearing mice. Our results in mouse models of melanoma, lung cancer and colorectal cancer demonstrate that EVIR-engineered DCPs robustly activate antigen-specific T cells and outperform traditional vaccination with antigen-loaded DCs.

In addition to DC vaccination protocols, our EVIR platform may be exploited to identify EV-associated tumor antigens and cognate T cell receptors (TCR) through ex vivo cells assays. This approach may help to select or identify tumor antigen-specific CD8+ T cells for adoptive T cell therapy applications.

A start-up company, EVIR Therapeutics (https://evirtherapeutics.com) has been created in October 2020 to translate our ERC-funded program toward a clinical application. The company is primarily focused on addressing manufacturing issues and other translational aspects of the new vaccination platform.
EVIR-engineered DCs uptake tumor-drived EVs