Marrow-Infiltrating Lymphocytes (MILs) Provide a Robust Platform for CAR-T Cell Therapy

Adoptive cellular immunotherapy (ACT) using chimeric-antigen receptor (CAR)-modified T cells has shown unprecedented initial response rates in patients with advanced B cell malignancies and is emerging as a powerful tool for cancer treatment. However, there remains room for improvement in initial response rates, and relapse occurs in a significant fraction of those patients that do respond. Reasons for failure to respond are being elucidated. At least two mechanism of relapse - lack of persistence of infused CAR-T cells and the loss of antigen-expression by tumor cells - are well documented.

We have developed a novel platform for ACT called Marrow-infiltrating Lymphocytes (MILs). MILs are a population of polyclonal T cells expanded from the bone marrow (BM) with distinct properties that set them apart from T cells expanded from peripheral blood lymphocytes (PBLs). MILs are enriched with memory T cells and contain tumor antigen-specific T cells that are typically not detected in PBLs. MILs are being developed for several tumor types, including both hematological and solid tumors.

Distinguishing features of MILs compared to PBLs include their memory phenotype, inherent tumor antigen-specificity, and ability to persist long-term. As such, we hypothesized that MILs would provide a robust platform for CAR-T cell therapy with the potential to boost initial response rates and reduce the risk of relapse. Herein, data is presented demonstrating superior anti-tumor activity of CAR-modified MILs (CAR-MILs) compared to CAR-T cells generated from patient-matched PBLs (CAR-PBLs).

We designed a 2nd generation 4-1BB-CD3z CD38 CAR using an scFv derived from Daratumumab. The CD38 CAR was linked to GFP by a T2a cleavage peptide so that GFP could be used as a marker of CAR expression. The CD38 CAR was expressed in a lentiviral construct and demonstrated equal transduction efficiencies and CAR expression levels in MILs and PBLs. The CAR modified MILs demonstrated superior antigen specific killing compared to PBLs against the 8226 myeloma (MM) cell line but not its isogenic CD38 knocked out line. More importantly, CAR-MILs retained their inherent tumor antigen-specificity and capacity to respond through their endogenous tumor antigen-specific T cell receptors - a property that PBL-CARs did not appear to possess. Tumor antigen-specific T cells were quantified by co-culturing MILs with autologous antigen-presenting cells pulsed with allogeneic multiple myeloma cell line lysates as a source of antigen and measuring IFNγ-production as a read-out. An equal or greater number of tumor antigen-specific IFNγ-producing T cells were measured in CD38 CAR-MILs compared to matched unmodified MILs.

Finally, we compared the in vitro cytolytic potential of CD38 CAR-MILs to matched CD38 CAR-PBLs. Co-culture killing assays were performed at decreasing CAR-T effector: target (E:T) ratios. At high E:T ratios, CAR-MILs and CAR-PBLs lysed comparable percentages of CD38⁺ targets with similar kinetics. However, at lower E:T ratios, CAR-MILs killed a greater percentage of CD38⁺ targets with faster kinetics compared to CAR-PBLs.

In conclusion, we have demonstrated 1) the feasibility of producing CAR-modified MILs, 2) that CAR-MILs retain their inherent tumor antigen-specificity and functional capacity - something that was lacking with CAR-PBLs, and 3) that CAR-MILs kill more efficiently in vitro than matched CAR-PBLs. These data suggest that CAR-MILs may provide both better antigen specific killing but more importantly by targeting the endogenous antigenic repertoire, they could also prevent or minimize the risk of relapse via antigen escape variants and thus increase the overall efficacy of adoptive CAR T cell therapy.