Superior Efficacy of CAR-T Cells Using Marrow-Infiltrating Lymphocytes (MILsTM) As Compared to Peripheral Blood Lymphocytes (PBLs)
Authors: Lutz Eric R. , Jana Srikanta , Rudraraju Lakshmi , DeOliveira Elizabeth , Zhou Jing , Mackay Sean , Borrello Ivan M. , Noonan Kimberly 
Published: November 2019

Background
The type of T cell used in generating chimeric antigen receptor (CAR) T cells is an important choice. Evidence suggests that T cells that are early in the effector/memory differentiation pathway with more stemness and greater potential to persist are better than more differentiated T cells with less stemness that are more readily exhausted and have less potential to persist.
Marrow-infiltrating Lymphocytes (MILsTM) is a novel form of adoptive T cell therapy composed of patient-autologous, polyclonal CD4 and CD8 T cells that are activated and expanded from the bone marrow. Genetically unmodified MILsTM have demonstrated antitumor activity in patients with multiple myeloma and are being developed for several other tumor types, including non-small cell lung cancer and other solid tumors. Distinguishing features of bone marrow T cells used to produce MILsTM include their memory phenotype, inherent tumor antigen-specificity, higher CD8:CD4 ratio and ability to persist long-term when compared to peripheral blood lymphocytes (PBLs) which is the T cell source used to produce currently approved CAR-T therapies. Based on these differences, we hypothesize that MILsTM provide a more robust and better fit platform for CAR-T therapy compared to PBLs.
Using a CD38-specific, 4-1BB/CD3z-signaling CAR as an initial model, we have demonstrated the feasibility of producing CAR-modified MILsTM (CAR-MILsTM) and showed that CAR-MILsTM demonstrate superior killing in vitro compared to CAR-T cells generated from patient-matched PBLs (CAR-PBLs). Herein, we build on our previous data and add a second BCMA-specific CAR model. We use the two multiple myeloma model systems to compare cytolytic potential, functionality, and expression of phenotypic markers of memory, stemness and exhaustion between patient-matched CAR-MILsTM and CAR-PBLs.
Methods
Matched pairs of CAR-MILsTM and CAR-PBLs were produced from the bone marrow and blood of multiple myeloma patients. Two different in vitro cytotoxicity assays, the RTCA xCelligence real-time impedance and FACS assays, were used to evaluate antigen-specific killing of target tumor cells. Functionality of CD4 and CD8 CAR-T cells, at the single-cell level, was evaluated by measuring the secretion of 32 cytokines and chemokines following in vitro antigen-specific stimulation using IsoPlexis IsoCode chips and analyzed using IsoPeak. Expression of markers of T cell memory (CD45RO & CCR7/CD62L), stemness (CD27) and exhaustion (PD1 & TIM3) on CAR-MILsTM and CAR-PBLs prior to and following antigen-specific stimulation was evaluated by flow-cytometry (FACS).
Results
CAR-MILsTM demonstrated superior killing of tumor target cells in vitro, regardless of the antigen specificity of the CAR, when compared to matched CAR-PBLs and this superiority persisted even upon repeated antigen encounter - a factor that may be critical in guaranteeing better anti-tumor efficacy and persistence. CAR-MILsTM demonstrated increased polyfunctionality (secretion of 2+ cytokines per cell) and an increased polyfunctional strength index (PSI) following antigen-stimulation compared to CAR-PBL in both CD4 and CD8 T cells. The enhanced PSI in CAR-MILsTM was predominately mediated by effector, stimulatory and chemoattractive proteins associated with antitumor activity including Granzyme B, IFNg, IL-8, MIP1a and MIP1b. Coincidentally, increased PSI and enhanced secretion of these same proteins was reported to be associated with improved clinical responses in patients with Non-Hodgkin lymphoma treated with CD19-specific CAR-T therapy. Expression of memory markers on CD4 and CD8 T cells were similar in CAR-MILsTM and CAR-PBLs both prior to and following antigen-stimulation. Although expression of CD27, PD1 and TIM3 were similar at baseline, CAR-MILs maintained higher levels of CD27 and lower levels of PD1 and TIM3 compared to CAR-PBLs following antigen-stimulation in both CD4 and CD8 T cells.
Conclusions
Collectively, our data suggest that CAR-MILsTM have several advantages over CAR-PBLs, including increased cytolytic potential, enhanced polyfunctionality, increased stemness and less exhaustion. Based on these differences and the inherent antitumor properties of MILsTM, we speculate that CAR-MILsTM would be more potent and effective than currently approved CAR-T products derived from PBLs.

Disclosures
Lutz: WindMIL Therapeutics: Employment, Equity Ownership. Jana:WindMIL Therapeutics: Employment, Equity Ownership. Rudraraju:WindMIL Therapeutics: Employment, Equity Ownership. DeOliveira:WindMIL Therapeutics: Employment, Equity Ownership. Zhou:Isoplexis: Employment, Equity Ownership. Mackay:Isoplexis: Employment, Equity Ownership. Borrello:Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX; BMS: Consultancy; WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau. Noonan:WindMIL Therapeutics: Employment, Equity Ownership, Patents & Royalties; Aduro: Patents & Royalties: intellectual property on allogeneic MM GVAX.