Establishing CD8+ T Cell Immunity by Adoptive Transfer of Autologous, IL-15 Expanded, Anti-Tumor CTL with a Central/Effector Memory Phenotype Can Induce Objective Clinical Responses.
Authors: Butler Marcus , Friedlander Philip , Mooney Mary , Drury Linda , Metzler Martha , Milstein Matthew , Berezovskaya Alla , Flavin Marisa , Imataki Osamu , Tanaka Makito , Daley Heather , Hodi F. Stephen , Nadler Lee , Hirano Naoto 
Published: October 2019

Abstract
Abstract 782
The goal of cellular immunotherapy is to build long-lasting anti-tumor immunologic “memory” in patients and reject tumors for a lifetime. Previously, we and others demonstrated that IL-15 promotes the generation of T cells with a central memory (CM) phenotype which have the capacity to persist and establish effective anti-tumor memory in vivo. Furthermore, it has been shown that CD83 delivers a CD80-dependent T cell stimulatory signal that allows T cells to be long-lived. Based on these findings, we developed a system to generate large numbers of long-lived antigen-specific CD8+ T cells with a memory phenotype. This in vitro culture system utilizes IL-15 and a standardized, renewable artificial antigen presenting cell (aAPC) which was produced by transducing CD80, CD83, and HLA-A*0201 to the human cell line, K562. This aAPC can uniquely support the priming and prolonged expansion of large numbers of antigen-specific CD8+ CTL which display a central/effector memory (CM/EM) phenotype, possess potent effector function, and can be maintained in vitro for >1 year without any feeder cells or cloning.
We hypothesized that adoptive transfer of these CTL with a CM/EM phenotype should result in anti-tumor memory in humans even without lymphodepletion or high dose IL-2. For our “first-in-human” clinical study, we chose the melanoma antigen MART1 as a target antigen, since MART1-specific HLA-A*0201+-restricted precursor CTL are detectable in some melanoma patients and can be immunophenotyped pre-infusion. Autologous CD8+ T cells were stimulated weekly with peptide-pulsed human cell-based aAPC and expanded with low dose IL-2 and IL-15. After three weeks, polyclonal MART1 CTL were reinfused without additional lymphodepletion, chemotherapy, IL-2, or vaccination. Eight study participants have enrolled and received a total of 15 MART1 CTL infusions (31% MART1 multimer positivity, median). All but one subject received two reinfusions where the 2nd graft was produced from CD8+ T cells harvested two weeks after the 1st reinfusion. To date, ≥2×109 CTL with potent effector function and a CM/EM phenotype were successfully generated for all subjects. No dose limiting toxicities were observed at either Dose Level 1 (2×108/m2) or Dose Level 2 (2×109/m2). Clinical activity was observed with a response by RECIST criteria in 1 subject, which was confirmed by a negative PET/CT 100 days following the last CTL infusion. In addition, 1 patient experienced a mixed response, 1 had stable disease, 3 had progression, and 2 are currently on active therapy.
Multimer staining showed that, immediately post infusion, the percentage of CD8+ T cells specific for MART1 temporarily increased in all subjects, with the highest (6.5%) observed in subject #7. In 4 subjects, sustained increases in the frequency of MART1 specific T cells by more than two-fold (range 2.0-10x) for ≥21 days were observed despite the fact that no exogenous cytokines or vaccination was administered. Moreover, an increase of detectable MART1 specific T cells which display a CM phenotype was observed in all evaluable subjects and was observed for ≥35 days in 6 of 8 subjects. In subject #2, the conversion of MART1 CTL immunophenotype from a naïve to a mixture of naïve/memory phenotypes was observed for more than 6 months. We identified 10 individual MART1 T cell clonotypes from peripheral CD45RA- memory T cells on day 21. Clonotypic TCR Vbeta CDR3 analysis revealed that CTL grafts contained 7 out of 10 of these clonotypes. Furthermore, 6 clonotypes persisted in the peripheral CD45RA- memory fraction on days 39, 67 and/or 132. In Subject #3, who showed a mixed clinical response, 5 individual MART1 T cell clonotypes were isolated from lung metastases. 4 out of 5 clones were included in the CTL grafts. This finding supports the possibility that infused CTL can traffic and localize to sites of disease. Intriguingly, in both subjects, we were able to identify MART1 CTL clonotypes that were not detectable in the CTL grafts but possibly emerged after CTL infusion, indicating that adoptive transfer of MART1-specific CTL may provoke a de novo antitumor response.
Taken together, these results suggest that CM/EM MART1 CTL generated ex vivo using our cell-based artificial APC in the presence of IL-15 may persist in vivo and induce de novo anti-tumor responses. Further enhancement of anti-tumor activity may be achieved through vaccination, cytokine administration, and/or removal of cytokine sinks and inhibitory factors following appropriate lymphodepletion.

Disclosures:
No relevant conflicts of interest to declare.