Cancer cachexia is a wasting syndrome experienced by 80% of advanced-stage cancer patients that induces significant muscle and fat loss and directly causes approximately 30% of cancer deaths¹. Our project tackles cachexia through engineered macrophages that modulate the levels of interleukin-6 (IL-6), a cytokine whose overabundance in cancer patients causes systemic immune dysfunction and is largely responsible for the wasting associated with cachexia².

Currently, no approved treatments exist beyond healthy diet and exercise, which are inadequate and do not significantly improve patient outcomes¹. Anti-IL-6 antibodies have been developed to treat autoimmune disorders like rheumatoid arthritis, but they are not approved for treating cachexia because they are ineffective at ameliorating cachexia. The lack of available treatment reflects the unfortunate fact that cachexia is largely understudied despite its immense impact on cancer patient outcomes. Our project will be one of the first to directly address cachexia.

Our primary goal is to engineer macrophages to phagocytose IL-6. Inspired by Morrissey et al.³, we designed a chimeric antigen receptor (CAR) construct that, when embedded in the cell membrane of a macrophage, binds IL-6 via an extracellular receptor which then induces phagocytosis via an intracellular signaling domain.

Macrophages typically phagocytose molecules larger than 1 µm that present multiple antigens, since binding multiple of the macrophage's receptors allows for sufficient aggregation of signal to induce phagocytosis. We hypothesize that when individual IL-6 molecules bind to macrophage receptors, they activate a relatively small signal that is not significant enough to induce phagocytosis. Thus, the second aim of our project involves engineering macrophages to secrete an aggregation protein that binds multiple IL-6 molecules to produce large clusters resembling naturally-occurring phagocytic targets.

Macrophages secrete aggregation protein

Aggregation protein binds multiple IL-6

IL-6 aggregates bind CAR

IL-6 binding induces phagocytosis

IL-6 aggregate is degraded in phagolysosome

After reviewing successful CAR constructs in the literature and from past iGEM teams, we designed two novel four-part CARs drawing from previously created parts. We are transfecting these CAR constructs into human embryonic kidney (HEK293) cells as a proxy for the engineered human macrophages in our ideal real therapy.

• Extracellular receptor:
Anti-IL-6 antibody single chain variable fragment, to bind IL-6⁴
• Transmembrane signal transduction and stability domain:
CD8, known to transduce signal and increase CAR stability⁵
• Intracellular signaling domain:
Either Fcγ or Megf10, known to induce phagocytosis^3,6
• Fluorescent tagging:
eGFP, for visualization and for easy folding of the fusion protein

1. “Treating Cancer Cachexia: Progress Looks Possible.” National Cancer Institute, www.cancer.gov/about-cancer/treatment/research/cachexia. Accessed 29 June 2023.
2. Narsale, Aditi A., and James A. Carson. “Role of Interleukin-6 in Cachexia.” Current Opinion in Supportive & Palliative Care, vol. 8, no. 4, 2014, pp. 321–327, https://doi.org/10.1097/spc.0000000000000091.
3. Morrissey, Meghan A, et al. “Chimeric Antigen Receptors That Trigger Phagocytosis.” eLife, vol. 7, 2018, https://doi.org/10.7554/elife.36688.
4. iGEM Part BBa_K4175000
5. Zhang, Cheng, et al. “Engineering Car-T Cells.” Biomarker Research, vol. 5, no. 1, 2017, https://doi.org/10.1186/s40364-017-0102-y.
6. Maverakis, Emanual, et al. “Glycans in the Immune System and the Altered Glycan Theory of Autoimmunity: A Critical Review.” Journal of Autoimmunity, vol. 57, 2015, pp. 1–13, https://doi.org/10.1016/j.jaut.2014.12.002.