Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients

Abstract

The generation of T lymphocytes with specific reactivity against tumor antigens is a prerequisite for effective adoptive transfer therapies. Melanoma-specific lymphocyte cultures can be established from tumor infiltrating lymphocytes (TILs) by in vitro culture in high levels of IL-2. We have optimized methods for generating melanoma-reactive TIL cultures from small resected tumor specimens. We report a retrospective analysis of 860 attempted TIL cultures from 90 sequential melanoma biopsy specimens from 62 HLA-A2+ patients. Multiple independent TIL derived from a single tumor often exhibited substantial functional and phenotypic variation. Tumor specific activity was detected in TIL from 29 (81%) of 36 patients screened. TIL cultures selected for high activity were generally capable of large numerical expansion using a single round of a rapid expansion protocol. Limited clonal T-cell populations in an oligoclonal TIL culture could confer specific tumor recognition in these highly selected, highly expanded TIL cultures. These methods were efficient at generating TILs suitable for adoptive transfer therapy.

FIGURE 1

Two independent TIL cultures from tumor 1931-2 have similar functional activities but comprised different clones. Top: TIL F4 (left) and F5 (right) exhibit low background staining. Middle: TIL F4 (left) and F5 (right) comprised predominantly cells that are doubled labeled by HLA-A2/MART-1:26-35(27L) tetramer and anti-CD8 antibody. Bottom: TIL F4 comprises predominantly T cells expressing Vβ12, whereas TIL F5 has no Vβ12⁺ cells.

FIGURE 2

Typical TCR β-chain transcript CDR3 size patterns from normal PBMC (upper) and a highly selected, highly in vitro expanded TIL (lower). Fluorescent TRBV-BC PCR products were analyzed on an automated sequencer using Immunoscope software. The fluorescence intensity is expressed in arbitrary units. BV nomenclature is as previously described and also as used by the anti Vβ antibody manufacturer. TRBV3 in this system corresponds to TRBV28 in the IMGT nomenclature. Peripheral blood lymphocytes generate a characteristically wide distribution for each BV family, representing a diversity of expressed TCR. The in vitro selected, REPed TIL exhibits a highly reduced repertoire with 0, 1, or 2 dominant clones in each BV family.