38P - Feasibility of expanding tumor-infiltrating lymphocytes from cryopreserved tumor specimens after long-term storage

Background

Tumor-infiltrating lymphocyte (TIL) therapy has emerged as a promising treatment for late-stage solid cancer patients. Most studies use fresh tumor fragments to expand TIL. Limited research exists on cryopreserving and long-term storing of tumors as TIL starting material. The aim of this study was to expand and characterize tumor-infiltrating lymphocytes (TILs) from tumor specimens stored in liquid nitrogen for over 12 months.

Methods

Tumors, resected from 20 patients with colorectal cancer, were immediately cut into small pieces and cryopreserved in liquid nitrogen for 14-20 months until use. Upon thawing tumor fragments were either placed in a TIL expansion media or were first subjected to enzymatic digestion. Serum-free culture media containing 500 units of IL2 was used for TIL expansion. CD3/CD28 antibodies were added at the start of the cultivation and every 7 days to activate TIL. After 10-22 days in culture, expanded TILs were characterized by phenotype. The ability of the expanded TIL to secrete interferon-ɣ (IFNɣ) was measured in the ELISPOT assay. T cell receptor (TCR) repertoire was analyzed on the starting tumor material and expanded TILs.

Results

The average storage time of the tumor starting material was 16 months. The success rate of TIL expansion was 50% and was the same when TILs were grown directly from small fragments or from single-cell tissue suspensions after enzymatic digestion. The mean percentage of CD3+ cells in TIL cultures was 85.31%. The mean percentage of CD4+ cells and CD8+ cells was 81.01% and 10.29% correspondingly. Regardless of the isolation method, 9 TIL cultures exhibited the high production of IFNgamma in the ELISPOT assay upon reactivation following cocultivation with phytohemagglutinin. Extended phenotype characterization and TCR repertoire analysis is ongoing.

Conclusions

Our study demonstrates the feasibility of using long-term cryopreserved tumor fragments for TIL expansion with further assessment of clonality, phenotyping and neoantigen specificity.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Federal Medical Biological Agency funding for the «T-kletki» project, 123032900030-7.

Disclosure

All authors have declared no conflicts of interest.