Engineered T Regulatory Cells for Malignancy Associated Vascular Inflammation

Globally, the foremost causes of mortality, cancer and cardiovascular disease (CVD), often co-exist within the same individuals. However, CVD in oncologic patients is not necessarily linked to conventional risk factors like diabetes or dyslipidemia but is a result of inflammatory dysregulation induced by their cancer or previous cancer treatments. This phenomenon is particularly prevalent among patients with hematologic malignancies, such as leukemia, and even those in premalignant states like clonal hematopoiesis of indeterminate potential (CHIP). Patients with CHIP, especially those with DNMT3A, TET2, or TP53 mutations, have a 2-fold increased risk of coronary artery disease (CAD). The mechanism of increased atherosclerosis in these states is due to the continuous activation of inflammatory pathways, leading to oxidative stress and higher levels of oxidized low-density lipoproteins (OxLDL), which promote inflammation, foam cell formation, and plaque deposition. Plaques with increased inflammatory components have a higher risk of rupture. Currently, the medical management of CAD in patients with cancer is more complex than in non-cancer patients due to the rate of thrombocytopenia in this population, making standard management with dual anti-platelet therapy a higher-risk intervention. Similarly, hypercoagulability of cancer patients increases the rate of stent thrombosis after percutaneous intervention. In theory, a targeted anti-inflammatory cellular treatment may result in reduced atherosclerotic burden in cancer patients without increasing the risk of thrombosis or bleeding. Our research proposes using T regulatory cells (Tregs) expressing a chimeric antigen receptor (CAR) specific for OxLDL.

An anti-OxLDL CAR was developed using the variable domains of an OxLDL antibody that targets the MDA-ApoB100 epitope on OxLDL. This epitope is highly expressed in patients with symptomatic coronary artery disease and has previously been shown to reduce atherosclerotic plaque formation by 50% when infused into ApoE^-/- mice. We developed CAR Tregs by transducing naive human CD4 T cells with a bicistronic lentiviral CAR vector that co-expresses FOXP3. The FOXP3-CAR⁺ CD4 T cells were phenotypically CD25^hi and CD127^low, exhibiting similar levels of CTLA-4 compared to natural Tregs. Anti-OxLDL FOXP3 CAR Tregs (OxCAR Tregs) demonstrated specificity and anti-inflammatory reactivity by producing type II cytokines in the presence of MDA-ApoB100 and not native ApoB100. OxCAR Tregs maintained an immunosuppressive phenotype after activation through CAR and TCR and suppressed proliferating T cells. When incubated with macrophages, OxCAR Tregs reduced OxLDL uptake, decreased foam cell formation, and skewed macrophages toward an M2 phenotype.

Murine versions of OxCAR Tregs (muOxCAR Tregs) were then developed and demonstrated similar phenotypes and immunosuppressive capacities as human OxCAR Tregs. The muOxCAR Tregs were evaluated in vivo in a syngeneic model using 8-12 week-old male and female LDLR^-/-; Apobec^-/-; human ApoBTg (LAHB100) mice, which exhibit human-like levels of ApoB100 on LDL particles. LAHB100 mice were treated with non-transduced murine CD4 T cells, murine control CAR Tregs, or muOxCAR Tregs 24 hours after conditioning with cyclophosphamide. At the time of the T cell injection, we initiated the mice on a high-fat diet for two and a half months. The mice received a second injection of cells with conditioning six weeks after the first T-cell injection based on prior data indicating that the longevity of murine CAR T cells is around six weeks. After two and half months, the mice were euthanized, and the descending aortas of each mouse were harvested and analyzed for atherosclerotic lesions via en-face staining. The mice receiving the muOxCAR Tregs had 80% less plaque in the descending aorta than those receiving control T cells. These results were observed in independent experiments with LAHB100 and LDLR^-/- mice. Serum analysis of experimental mice at the time of euthanasia showed no significant change in systemic inflammatory markers. These initial studies demonstrate the feasibility of developing OxCAR Tregs to reduce the inflammation associated with OxLDL, potentially providing a new therapeutic option for atherosclerosis. Studies are ongoing to determine whether OxCAR Tregs can reduce inflammation related to clonal disorders such as CHIP and occult malignancy.