Off the Shelf, GMP Grade Artificial APC Efficiently Generates Large Numbers of Antigen Specific CTLs Sufficient for the Treatment of Cancer and Infectious Disease.
Authors: Butler Marcus O. , Hirano Naoto , Lee Jeng-Shin , Beecham E. Jeffrey , Chung Mirra , Ripley Robin , Murray Andrew P. , Mulligan Richard C. , Nadler Lee M. 
Published: April 2020

Abstract
Generating antigen specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy requires the use of antigen presenting cells (APCs) such as dendritic cells, activated B cells, and/or artificial APC engineered to express immunoaccessory molecules. Although clearly useful, each approach presents certain drawbacks preventing their widespread application to the treatment of minimal residual disease in cancer and the treatment and prophylaxis of infectious disease. For instance, generating autologous APC for each patient is costly and is dependent on a limited supply of patient material. Most artificial APC approaches, while bypassing the individualized preparation of APC, are limited by the requirement to produce clinical grade HLA/peptide tetramers, monoclonal antibodies, polystyrene beads, and/or non-human cell lines. To circumvent all these issues, we have developed an immortalized APC line that can uniquely support the priming and prolonged expansion of antigen specific T cells through the engagement of TCR, CD28, and CD83 ligand. Co-engagement of CD28 and CD83 ligand preferentially enriches and significantly amplifies the number of antigen specific T cells, obviating the necessity to perform any tetramer sorting procedures. Furthermore, we are consistently successful in generating immunity to a wide array of peptide epitopes derived from antigens such as MART1, NY-ESO-1, Her-2/neu, influenza virus, telomerase, and HIV. Since this strategy can provide an unlimited APC source that can be used to prime, expand and select for antigen specific CTLs, we have generated a GMP grade APC for the treatment of patients. Our CASE (clinical grade antigen specific expansion) line was produced by simultaneously transfecting K562 with linearized plasmids encoding the HLA-A*0201 (A2), CD80, CD83, and puromycin resistance genes using a lipofection method. Following drug selection and cloning by limiting dilution, 5 CASE clones were then studied for their ability to induce expansion of antigenic peptide specific CTL, using MART1 as a model antigen. HLA-A2+ CD8 T cells were stimulated with irradiated, peptide pulsed CASE clones, and the percentage of tetramer staining T cells was determined by flow cytometry. The stable CASE clone, #33, was found to consistently generate the highest number of antigen specific T cells. Using the CASE33 clone, we successfully generated several CTL lines, succeeding in 8/8 cases. The generated CTL lines achieved several logs of antigen specific expansion, with tetramer staining up to 57%, and function has been demonstrated by high antigen specific killing and gamma-interferon secretion. Both master and working cell banks for CASE33 have been established and have successfully passed extensive testing including in vitro cultures, testing for bovine adventitious agents, in vivo testing and PCR for viral pathogens, karyotype analysis, and analysis of cellular morphology and growth. The highly effective APC, CASE33, was generated in a relatively short timeframe, and the production of additional clinical grade APCs expressing HLA molecules such as A11, A24, and A30, would be technically straightforward. With such an APC bank, most patients encountered in the clinic could be treated using one or more of these APCs and the widespread testing of adoptive immunotherapy in cancer and infectious diseases will commence.