Introduction Chimeric Antigen Receptor (CAR) T-cell Therapy Engineering Approach References

Project Description

Introduction

We are living in the age of precision medicine; over the past twenty years, we have seen the emergence of a powerful pathway for treating cancer—engineering our own immune system to target and destroy malignant cells. This year, the Duke iGEM team has worked to improve cancer immunotherapy for patient safety and improved efficacy.

Chimeric Antigen Receptor (CAR) T-cell Therapy

CAR T-cell therapy is a type of adoptive cell therapy where a patient’s immune cells are isolated, genetically modified, and then reintroduced to the body to activate the immune system against a specific cancer antigen. Native T-cells are a type of lymphocyte, or white blood cell, that recruit other immune cells to trigger a downstream cytotoxic effect on a foreign cell. This effect relies on specific membrane-bound receptors that recognize the foreign antigen. Using CAR T-cell therapy, these native receptors are replaced with CARs, or receptors specifically designed to activate the T-cell program against a cell expressing a specific cancer antigen.

As cancer incidence rates rise globally with increased prevalence in prostate, breast, and other solid tumors, engineering novel immunotherapy approaches will be essential to combating these diseases. CAR T-cell therapy has been heralded as the next-and-best generation of immunotherapy that allows for de novo T-cell targeting of cancer cells using synthetic tumor-specific receptors.

Adapted from "CAR T Therapy”, by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates

Classically, this consists of a single-chain variable fragment (scFv) antibody that binds to the extracellular domains of upregulated cancer antigens, a conserved transmembrane domain, and co-stimulatory intracellular domains (e.g. CD28, CD3ζ, 4-1BB) from native T-cell receptors (TCRs) that promote adaptive killing of nearby cancer cells. While successful for treating liquid tumors in leukemias and lymphomas, CAR T-cell therapy in solid tumors has been limited due to heterogeneous expression and reduced accessibility of cancer antigens on solid tumors. In addition, the harsh tumor microenvironment (TME) excludes immune cells and restricts tumor penetration.

Engineering approach

CARs detect the cell-surface cancer antigen (input), interpret the signal into T-cell activation mechanisms (processing), and secrete stimulatory cytokines (e.g. IL2, TNF-ɑ), perforins, and granzymes to mediate killing of cancer cells (output).

While a single CAR solution has been successful in hematologic cancers using CD19-targeting, there are problems with specificity, antigen escape, and immunosuppression that cannot be addressed in a one-logic circuit. T-cell gene circuits that modulate cytokine expression to prevent overactivation and T cell exhaustion have been reported.

Our group is particularly interested in rewiring CARs to soluble factors versus receptor-mediated ligands that require a mechanical force to conduct the T-cell activation pathways. This is combined with previous work using the synNotch cell signaling platform to drive cytokine production, CAR expression, and therapeutic antibodies to remodel the TME for improved tumor killing.

Our approach combines systematic analysis of soluble secretion molecules to activate synthetic receptors and computational protein modeling to design extracellular domains for CAR and synNotch activation. These synthetic receptors enable cellular “computational” logic that leads to our integrated CAR-synNotch logic circuit. This signal amplification of soluble secretion molecules that use autocrine and paracrine signaling to promote T-cell activation, proliferation, and killing should improve solid tumor killing effects. When all project aims are combined, we will develop the Intelligent Chimeric Antigen Receptor Upregulation System (ICARUS), which employs an operational amplifier genetic circuit model that allows cells to produce their own secreted “neo-antigen” that can activate its own CAR function and that of nearby CAR T-cells. Through this amplification, we aim to eliminate concerns of antigen escape and accessibility, promote T-cell proliferation at the site of tumors to combat TME exclusion, and augment the killing response through increased signaling and T-cell activation.

References

  1. Allen, G. M., Frankel, N. W., Reddy, N. R., Bhargava, H. K., Yoshida, M. A., Stark, S. R., Purl, M., Lee, J., Yee, J. L., Yu, W., Li, A. W., Garcia, K. C., El-Samad, H., Roybal, K. T., Spitzer, M. H., & Lim, W. A. (2022). Synthetic cytokine circuits that drive T cells into immune-excluded tumors. Science (New York, N.Y.), 378(6625), eaba1624. https://doi.org/10.1126/science.aba1624
  2. Brenner, J., Cho, J. H., Wong, N. M. L., & Wong, W. W. (2018). Synthetic Biology: Immunotherapy by Design. Annual Review of Biomedical Engineering, 20, 95–118. https://doi.org/10.1146/annurev-bioeng-062117-121147
  3. Chang, Z. L., Lorenzini, M. H., Chen, X., Tran, U., Bangayan, N. J., & Chen, Y. Y. (2018). Rewiring T-cell responses to soluble factors with chimeric antigen receptors. Nature Chemical Biology, 14(3), Article 3. https://doi.org/10.1038/nchembio.2565
  4. López-Cantillo, G., Urueña, C., Camacho, B. A., & Ramírez-Segura, C. (2022). CAR-T Cell Performance: How to Improve Their Persistence? Frontiers in Immunology, 13, 878209. https://doi.org/10.3389/fimmu.2022.878209
  5. Murthy, H., Iqbal, M., Chavez, J. C., & Kharfan-Dabaja, M. A. (2019). Cytokine Release Syndrome: Current Perspectives. ImmunoTargets and Therapy, 8, 43–52. https://doi.org/10.2147/ITT.S202015
  6. Safarzadeh Kozani, P., Safarzadeh Kozani, P., Ahmadi Najafabadi, M., Yousefi, F., Mirarefin, S. M. J., & Rahbarizadeh, F. (2022). Recent Advances in Solid Tumor CAR-T Cell Therapy: Driving Tumor Cells From Hero to Zero? Frontiers in Immunology, 13, 795164. https://doi.org/10.3389/fimmu.2022.795164
  7. Shih, R. M., & Chen, Y. Y. (2022). Engineering Principles for Synthetic Biology Circuits in Cancer Immunotherapy. Cancer Immunology Research, 10(1), 6–11. https://doi.org/10.1158/2326-6066.CIR-21-0769
  8. Sterner, R. C., & Sterner, R. M. (2021). CAR-T cell therapy: Current limitations and potential strategies. Blood Cancer Journal, 11(4), 69. https://doi.org/10.1038/s41408-021-00459-7