(E-188) Metabolically activated CAR-Macrophages based therapy as a treatment for lymphoma

Introduction:: Lymphoma is a type of cancer that is quite common in the United States, accounting for about 4% of all cancer cases. To address this issue, it is essential to develop effective treatments for lymphoma. Our immune system works hard to fight off abnormal cells, but cancer has found ways to hide from this system. One promising approach is the CAR (Chimeric Antigen Receptor) strategy, which involves creating a receptor that can target specific cancer antigens and prevent them from escaping. While CAR-T therapy has been popular in recent years, it does have some drawbacks, such as causing an excessive immune response and not being able to infiltrate solid tumors. Recent studies suggest that macrophages might be a better option for CAR therapies because they can naturally get recruited to the tumor microenvironment without causing adverse reactions. However, the tumor microenvironment is often hypoxic, making it difficult for immune cells to combat cancer effectively. To overcome this challenge, a strategy must be developed to enhance glycolysis in the therapeutic agent so that they can survive in the hypoxic region. One way to do this is to introduce a metabolite involved in the glycolysis pathway to the immune cells, which can accelerate their glycolytic pathway and keep them metabolically active even after reaching the tumor region. This study proposes using CAR-Macrophages, known for their tumor-killing properties, treated with Fructose 1,6-Bisphosphate (Poly I:C) MPs as a cell-based therapy against lymphoma.

Materials and Methods:: For this study, we used human macrophages that were isolated from blood using lymphoprep and cultured in RPMI media supplemented with Fetal Bovine Serum and MCSF for 5 days. We then generated Fructose 1,6- Bisphosphate Poly (I:C) MPs using a previously established protocol and treated the human macrophages with F16BP MPs for 2 hours to observe the activation profiles of these cells following treatment. Further in-vitro studies with CFSE labelled Ramos cell line were conducted to examine the efficacy of tumour cell phagocytosing abilities of the F16BP MPs treated CAR-Macrophages. Moving forward, in-vivo studies will be conducted in mice, and we will observe and measure parameters such as survival, weight, size of tumour, and immune responses after tumour injection and administration of formulation.


Results, Conclusions, and Discussions:: We observed a transfection rate of more than 70% in human macrophages after they were transfected with CD19 Tandem plasmid on day 5 after isolation. We also observed an increase death of Ramos cells by more than 50% when they were incubated with transfected macrophages versus non-transfected macrophages. Additionally, when F16BP MPs treated CAR-MACs were incubated with the Ramos cell line, the activation as measured by CD86+ cells were significantly higher compared to untreated. These results are very promising, and we plan to perform the same studies in a nutrient deficient medium to mimic the Tumor microenvironment. Following this, we will conduct further in-vivo studies to fully understand the impact of CAR-MACs on the immune system and TME.

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References (Optional): : Klichinsky M, Ruella M, Shestova O, Lu XM, Best A, Zeeman M, Schmierer M, Gabrusiewicz K, Anderson NR, Petty NE, Cummins KD, Shen F, Shan X, Veliz K, Blouch K, Yashiro-Ohtani Y, Kenderian SS, Kim MY, O'Connor RS, Wallace SR, Kozlowski MS, Marchione DM, Shestov M, Garcia BA, June CH, Gill S. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol. 2020 Aug;38(8):947-953. doi: 10.1038/s41587-020-0462-y. Epub 2020 Mar 23. PMID: 32361713; PMCID: PMC7883632.