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Extracellular Vesicle-Internalizing Receptors (EVIRs) for Cancer ImmunoGeneTherapy

Periodic Reporting for period 4 - EVOLVE (Extracellular Vesicle-Internalizing Receptors (EVIRs) for Cancer ImmunoGeneTherapy)

Reporting period: 2022-01-01 to 2022-12-31

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), namely dendritic cells (DCs), 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) was to propel the development of effective DC therapies based on an innovative strategy that overcomes several key hurdles associated with traditional platforms. EVOLVE has used a novel DC engineering platform whereby chimeric receptors are used to both enable the specific and efficient uptake of tumor-derived extracellular vesicles (EVs) into DCs and to promote the presentation of EV-associated tumor antigens to the host’s immune system. EVOLVE also envisioned the implementation of additional interventions primarily based on the use of DC progenitors (DCPs) with improved engraftment and differentiation potential, engineering of the DCs for the expression of immunostimulatory cytokines, and combination with strategies to reprogram the tumor microenvironment, to facilitate the deployment of more effective anti-tumor immunity. Further to preclinical trials in mouse cancer models, EVOLVE advanced the preclinical development of engineered human DCs for clinical translation of our cell therapy platform. The key results of the project are described below.
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.

We have published a first study describing the EVIR platform in 2018. This work was initiated before the beginning of the project, but during the course of EVOLVE we undertook studies of the mechanism of antigen presentation by EVIR-engineered DCs (DC-EVIR), as described in Aim 1. We obtained exciting results indicating that the EVIR captures tumor-derived EVs and promotes “cross-dressing” of DCs with pre-formed MHC-peptide tumor antigens. This unusual mode of antigen presentation by DCs has been found recently to play important roles in anti-tumor immunity.

As planned in Aim 1, we have also designed new EVIRs with engineered signaling domains. After a laborious process of design, screening and validation, we have selected a new EVIR that induces the activation of transduced DCs in an EV-dependent manner, i.e. only upon binding of the EVIR to tumor-derived EVs. The new EVIR induces expression of co-stimulatory molecules on the DC, 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 EVIR for DC vaccination of mice with melanoma and obtained proof that its activity is superior to that of the parental EVIR. Furthermore, we have engineered the DCs to express a cytokine payload (IL-2, IL-12, and/or FLT3L). DCs engineered to express the EVIR and/or a cytokine payload have been studied extensively in the context of preclinical trials in various tumor models, as described in Aim 2 below.

As planned in Aim 2, we have performed preclinical trials in mouse models of cancer. Although not originally planned, we have made efforts to obtain more efficient DCs. We developed protocols that generate a new population of mouse DC progenitors (DCPs) that efficiently produce cDC1 and cDC2 (i.e. professional DCs) in tumor-bearing mice. Murine DCPs were obtained by culturing bone marrow cells in a cocktail of cytokines enabling (i) expansion of hematopoietic progenitors and (ii) commitment to the cDC lineage. We first demonstrated that traditional monocyte-derived DCs (moDCs) could not contribute to the cDC pool of either naïve or tumor-bearing mice; conversely, transfer of DCPs enabled the engraftment of substantial amounts of cDC1 in both lymphoid organs and tumors. We then found that DCPs transduced to express IL-12 and FLT3L (and, in some experiments, the EVIR as third transgene) boosted anti-tumor immunity in various mouse tumor models. Overall, our results in mouse models of melanoma, liver cancer, lung cancer, and glioblastoma, demonstrated that engineered DCPs robustly activate anti-tumor immunity and clearly outperform traditional vaccination with antigen-loaded moDCs.

As planned in Aim 4, we have developed engineered human DCs. We have shown that human DC-EVIR efficiently and specifically uptake human melanoma EVs and activate T cells toward unrelated melanoma antigens. Furthermore, we could generate engineered human DCPs from CD34+ progenitor cells. The successful generation of engineered human DCPs will facilitate translation of the pre-clinical studies described in Aim 2 to the clinic.
When we conceived EVOLVE, planned experiments involved the use of traditional monocyte-derived DCs. An unforeseen breakthrough of the project has been the successful generation of mouse and human DC progenitors (DCPs) that efficiently produce cDC1 and cDC2 (i.e. professional DCs) in cell culture and in tumor-bearing mice. Upon engineering, these cells induce dramatic immune responses in mouse cancer models. Our results in mouse models of melanoma, liver cancer, lung cancer, and glioblastoma, demonstrate that engineered DCPs robustly activate anti-tumor immunity and clearly outperform traditional vaccination with antigen-loaded monocyte-derived DCs.

Our protocols for the generation of mouse and human DCPs will soon become available to the scientific community and will certainly help to advance both basic and clinically-oriented research in DCs.

One unexpected finding of EVOLVE was that tumor response to engineered DCPs was partly independent of the adaptive immune system (e.g. T cells). In fact, tumor inhibition was largely dependent on DCP-induced interferon-gamma (IFNG) production by a variety of immune cells (including NK cells), which prompted pervasive “immunostimulatory” reprogramming of the tumor microenvironment. These unexpected results have prompted new studies to better understand the biological underpinnings of T-cell independent immune responses in cancer.

In one application, engineered mouse DCPs synergized with CAR-T cells to eradicate glioma in mice. Remarkably, neither DCPs nor CAR-T cells had efficacy as monotherapy, but their combination was highly effective. These unexpected results have motivated further studies to advance the pre-clinical design of combined DCP/CAR-T cell therapy for clinical translation.

A start-up company, EVIR Therapeutics (https://evirtherapeutics.com) has been created in October 2020 to translate our ERC-funded program toward clinical applications. 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