Introduction:: Cancer is the second most deadly disease in the US, with over 50,000 solid cancer deaths projected for 2023, including from melanoma and breast cancer. Cancer immunotherapy refers to treatments that utilize the immune system to eradicate cancerous cells. Despite this field’s progress, reducing the severe systemic toxicity experienced by solid cancer patients (heavy fevers, organ damage, etc.) remains a significant unmet need in clinical settings. Moreover, there are inherent limitations in current therapeutic options to treat “immunologically cold” tumors unresponsive to immunotherapies. To provide an emerging synthetic biology approach that addresses these issues, we utilize a genetic circuit that “hijacks” tumor cells, forcing them to release therapeutics that promote a localized immune response against the tumor itself, without harming healthy tissues. This project’s objective was to promote cancer-specific allorejection in mouse strains with solid tumors. We developed a genetic circuit that “hijacked” tumors of the C57BL/6 (BL/6) mouse strain, forcing them to express MHC Class I alloantigens derived from a different mouse strain, BALB/c. Alloantigens are allogeneic antigens exclusively native to certain individuals and immunologically “foreign” to other individuals of a species. Since BALB/c alloantigens have a different immunological haplotype to BL/6, the genetic circuit selectively turns BL/6 tumors into “foreign” organs; this process would induce tumor eradication via CD8+ T-cell mediated allorejection, without negatively impacting the surrounding healthy tissues.
To characterize our circuit, we achieved 3 experimental sub-objectives: 1. Validated circuit’s tumor specificity 2. Synthesized BALB/c alloantigens for tumor-specific expression 3. Evaluated circuit’s in vivo anti-tumor efficacy
Materials and Methods:: The methods used to achieve each sub-objective are as follows: Sub-objective 1: The circuit comprises 3 modules: Modules 1 and 2 are TF-sensor modules, which harness tumor-specific activity of transcription factors (TFs) and promoters (sensors). Together, both TF-sensor modules act as a AND gate, requiring tumor-specific activity of two different TFs; this combinatorial logic increases tumor specificity and safety of the circuit, preventing off-tumor effects in healthy tissues. To validate TF-sensor modules’ tumor specificity, cancerous (YUMM1.7, OVCAR-8) and non-cancerous (keratinocytes, IOSE) cells were lentivirally infected with synthetic sensors associated with cancerous TFs. To quantify tumor specificity, flow cytometry elucidated the ratio of sensors’ fluorescent intensities between both cell types. Sub-objective 2: Driven by the TF-sensor modules, Module 3 is the output module that enables tumor cells to secrete any genetically encodable output; to construct the output module, 3 MHC Class I BALB/c alloantigen sequences (H2Dd, H2Ld, H2Kd) were reverse-transcribed (RT-PCR) and amplified (PCR, HiFi assembly) from murine spleen mRNA and sequence-validated with Sanger sequencing. The 3-module genetic circuit would thus use tumor-specific transcriptional activity to force BL/6 tumor cells to express BALB/c alloantigens. Sub-objective 3: To assemble the 3-module circuit, tumor cell lines, BPPNM (breast cancer) and YUMM1.7 (melanoma), were lentivirally infected with TF-sensor modules and the alloantigens output module for integration into the cells’ genome to induce tumor-specific alloantigen expression. These host cells were injected intratumorally into BL/6 mice to assess in vivo anti-tumor efficacy, with tumor burdens measured volumetrically and with bioluminescent imaging over time.
Results, Conclusions, and Discussions:: In vitroresults: Modules 1 & 2: From lentiviral and flow cytometry experiments, the most tumor-specific sensors were selected for normalization of experimental TF-sensor modules. The two sensors for driving output module expression had normalized tumor specificities of 20x and 6x, between cancerous (YUMM1.7, OVCAR-8) and non-cancerous (keratinocytes, IOSE) cells. Module 3: We successfully amplified 3 BALB/c alloantigens (H2Dd, H2Ld, H2Kd) based on high DNA concentrations, 1.2 kb bands from gel electrophoresis, and Sanger sequencing.
In vivo efficacy results: Breast cancer models: 5 days post-tumor inoculation, mice injected with the alloantigen-genetic circuit had tumor burdens that were 51.1% less than negative control tumors (no therapeutic introduced). Melanoma models: 6 days post-tumor inoculation, there was a statistically significant decrease in mean tumor burden in alloantigen-treated mice (relative to negative control). At 10 days, alloantigen-treated burdens increased non-significantly (potentially attributed to tumor-mediated immunosuppression directed against alloantigens), decreasing at 14 days. However, tumors grew more slowly in alloantigen-treated mice, persisting 19 days post-tumor inoculation. Negative control tumors grew aggressively and ulcerated, prompting euthanization after 10 days.
The circuit’s TF-sensor modules displayed remarkable tumor specificity, with high activity in cancerous cells and minimal leakage in non-cancerous cells. By selectively targeting tumor cells, the circuit promotes tumor-localized therapeutic delivery to overcome systemic toxicity in healthy tissues. The alloantigen-genetic circuit demonstrated in vivo anti-tumor efficacy, curtailing tumor growth during the therapy’s preliminary stages. Longer survival times of alloantigen-treated mice signify lower toxicity via the circuit’s heightened functionality in solid tumors. These results demonstrate alloantigens’ unique capacity to directly convert tumors into immunological targets for cytotoxic T-cells. To mitigate tumor-mediated immunosuppression, we can introduce CRISPR-Cas9 knockouts of immunosuppressive genes together with the circuit to maximize the tumor allorejection mechanism's potency.
We established proof-of-concept for a genetic circuit that “hijacked” tumors to express alloantigens and promoted solid tumor allorejection. Although we demonstrated the circuit’s functionality in 3 cancer types (melanoma, ovarian, and breast cancer), the individual modules can be further engineered to match any tissue-specific state. Thus, the genetic circuit has potential to tackle multiple solid cancers and address the systemic toxicity issue in cancer immunotherapy.
Acknowledgements (Optional): : I would like to thank Dr. Ming-Ru Wu, the Wu lab, and Dana-Farber Cancer Institute for supporting this work.
References (Optional): : Nissim, L. et al. (2017) “Synthetic RNA-based immunomodulatory gene circuits for cancer immunotherapy,” Cell, 171(5). Available at: https://doi.org/10.1016/j.cell.2017.09.049. Immunobiology, 5th edition: The immune system in health and disease (2001). Garland Publishing.