(M1030-05-28) Genetically Engineered Multifunctional Immune-Modulating Exosomes (GEMINI-Exos) for Colorectal Cancer Treatment

Purpose: Biotherapeutic strategies have shown promise in the treatment of advanced and metastatic colorectal cancer (CRC), yet significant challenges remain. First-line treatment for advanced CRC is the combination between chemotherapy and targeted therapy and often leads to severe adverse effects in patients with median progression-free duration under a year. Targeted therapy also frequently encounters resistance due to constitutive activation of downstream signaling pathways. On the other hand, immune checkpoint inhibitors such as anti-programmed death 1 (PD1) and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibodies have demonstrated clinical efficacy. However, their benefits are largely restricted to patients with microsatellite instability-high (MSI-H) phenotypes, which are characterized by high mutational burden and pre-existing T cell infiltration. Hence, we hypothesized that an integrated combination of current biotherapeutic strategies could be an approach to tackle the aforementioned challenges in CRC treatment. This study aims to establish a unified platform that combines targeted therapy and immune checkpoint modulation through genetically engineered exosomes to expand immunotherapy application beyond MSI-H colorectal cancer phenotype and overcome tumor-inherent resistance mechanisms.

Methods: We previously developed an integrated exosome-based platform, termed genetically engineered multifunctional immune-modulating exosomes (GEMINI-Exos), that displayed both targeted and immune-modulatory proteins through genetic fusion with exosomal membrane proteins (Figure 1A). Targeting moieties included anti-epidermal growth factor receptor (EGFR) (αEGFR) and anti-CD3 (αCD3) antibodies, designed to redirect T cells toward EGFR-positive tumors and initiate immune-mediated cytotoxicity. PD1 and OX40 ligand (OX40L) were immunomodulatory agents simultaneously expressed for dual immune checkpoint modulation. PD1 expression on exosomes was expected to competitively inhibit PD1–PD1 ligands interactions in the tumor microenvironment, while OX40L–OX40 engagement would promote co-stimulatory signals. The plasmids encoded for the functional proteins were co-transfected to Expi293F cell lines and exosomes were isolated from the culture medium using differential centrifugation method. Surface protein expression was confirmed by immunoblot analysis. Flow cytometry analysis and cell-based assays were performed to evaluate the interaction among colorectal cancer cells, exosomes and human peripheral blood mononuclear cells (PBMCs), as well as the synergic immune activation induced by co-expressed targeting and immunomodulatory domains. SW480 was the selected microsatellite stable (MSS) CRC cell line. In vivo study was conducted in PBMC-humanized NSG mice bearing SW480 xenografts. At the end of the study, the animals were euthanized, and tumors were collected at the end point to assess immune cell infiltration.

Results: The results showed that both αCD3-αEGFR Exos and αCD3-αEGFR-PD1-OX40L GEMINI-Exos could activate immune cells to kill SW480 cells in vitro. The IC₅₀ values were 2.7 ± 1.58 ng/mL and 16.96 ± 6.87 for αCD3-αEGFR Exos and GEMINI-Exos respectively (Figure 1B). IL-2 levels in culture medium when cancer cells were co-cultured with native PBMCs in the presence of exosomes were found to be significantly higher in GEMINI-Exos treatment compared with αCD3-αEGFR Exos treatment. This showed that GEMINI-Exos could augment and sustain the immune activation more efficiently than αCD3-αEGFR Exos, illustrated by higher secreted IL-2 levels throughout 96 hour (Figure 1B). Interestingly, only treatment with αCD3-αEGFR-PD1-OX40L GEMINI-Exos, and not αCD3-αEGFR Exos, resulted in significant tumor suppression in mouse xenograft models. In the GEMINI-Exos–treated group, average tumor volumes remained stable at approximately 400 mm³, whereas average tumor sizes in the vehicle- and αCD3-αEGFR Exos-treated groups by the study endpoint reached around 1560 mm³ and 1325 mm³, respectively (Figure 2A). Immunophenotyping analysis demonstrated increased CD8⁺ T cell infiltration and a reduced Treg population at tumor sites, observed exclusively in GEMINI-Exos group (Figure 2B).

Conclusion: This study highlights the need for integrated combination strategies that concurrently target multiple signaling pathways involved in the immune–cancer crosstalk to overcome resistance to immunotherapy. Inspired by the three clinically validated targets for CRC – EGFR, PD1, and CTLA4 – GEMINI-Exos co-expressing surfaced αCD3, αEGFR, PD1 and OX40L represents a versatile and potent platform for immunotherapy in colorectal cancer.

References: Cheng Q, Dai Z, Smbatyan G, Epstein AL, Lenz HJ, Zhang Y. Eliciting anti-cancer immunity by genetically engineered multifunctional exosomes. Molecular Therapy. 2022;30(9):3066-3077.

Acknowledgements: National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health (NIH) grant R01EB031830 and National Cancer Institute (NCI) of NIH grant R01CA276240.


Figure 1. In vitro characterization of αCD3-αEGFR-PD1-OX40L GEMINI-Exos. (A) Schematic illustration of immunological synapse initiated by GEMINI-Exos. (B) Immune-mediated cytotoxicity and time-dependent activation of human PBMCs by exosomes.


Figure 2. In vivo evaluation of αCD3-αEGFR-PD1-OX40L GEMINI-Exos. (A) Tumor growth inhibition of αCD3-αEGFR-PD1-OX40L GEMINI-Exos in the SW480-xenografted mouse model. (B) Immunophenotype analysis in the tumor at the endpoint of in vivo study.