Background As immune-therapies for solid tumors progress, the tumor microenviroment (TME) presents several hurdles that CAR T-cells need to overcome for efficient tumor control. To limit T-cell effectiveness against the tumor, the TME employs a variety of mechanisms including tumor evasion, secretion of immunosuppressive factors or cellular resistance, among others. Despite being the most aggressive breast cancer subtype with a dismal prognosis, therapeutic options for triple negative breast cancer (TNBC) remain limited to date. Tumor-associated MUC1 antigen is hypoglycosylated in cancer and overexpressed in a large number of TNBC patients, thus providing a useful target for CAR T-cell therapies.

Method We previously presented how TALEN®-mediated gene editing allows programming of various functions addressing both safety and potency aspects of allogenic CAR T-cell therapy. TRAC-KO prevents GvHD; the disruptive insertion of HLA-E at B2M locus mediates resistance to both alloreactive T-cells and NK cells; TGFBR2 KO confers resistance to the immunosuppressive effect of TGFB1 and IL12 knock-in at PD1 locus (ΔPD1-IL12) provides the benefit of IL12 pro-inflammatory properties at the tumor site while avoiding the side effects associated with systemic IL12 accumulation. Here, we present synergistic the benefits of ΔPD1-IL12 and TGFBR2-KO attributes in preclinical models of triple negative breast cancer.

Results Upon intratumoral infusion, CAR T-cells programmed with ΔPD1-IL12 attribute showed complete tumor control at 5 times lower doses than their CAR-only counterpart, despite a tumor burden at least 8 times greater. CAR T-cells engineered with ΔPD1-IL12 infused intratumorally not only demonstrated tumor control locally but also against a distal lesion, potentially recapitulating control of metastatic sites. Finally, we demonstrate that the IL12 and TGFBR2 pathway interact: when combining the ΔPD1-IL12 with TGFBR2 KO, CAR T-cells preserved their activity while mitigating potential accumulation outside the tumor and, thereby, limiting the risks of off-tumor toxicity.

Conclusions Altogether, our pre-clinical data highlight the capability of multi-armored allogeneic CAR T-cells to preserve their activity despite the immunosuppressive microenvironment, while mitigating potential safety concerns and offering a potential therapeutic option for patients with TNBC.

Ethics Approval All procedures involving animals were performed in accordance with regulations and established guidelines of the Animal Ethical Committee and were reviewed and approved by the Institutional Animal Care and Use Committee at Mispro-Biotech (NY).