Establishment of stable cell lines for personalized melanoma cell vaccine

Abstract

Personalized vaccine, recognized after the failure of allogenic melanoma whole cell and lysate vaccine phase III trials, involves culturing cells from a patient's own tumor within a short duration and with less passages but with optimized expression of tumor-associated antigens (TAAs). Its feasibility is established by comparing pure cell lines generated from fresh and cryopreserved tissues (n=164) of patients with lymph node (LN) and distant metastases. Stable cell lines (from 67% of specimens) are subcultured after cryopreserving them. Pure cell lines established after eliminating fibroblasts (from 96% of the cell lines) include those from LN (69%), soft tissues including cutaneous (60%), liver (64%), lung (75%), bone (80%), brain (75%), and other sites (73%). Within 3.5 months, stable cell lines (> or =50 million cells) are established from initiating the cell culture. For LN metastases, the duration differs significantly (P2<0.05) between fresh (1.4-3.4 months) and cryopreserved (2.4-4.7 months) tissues. The expression of TAAs varies as follows: Tyrosinase (81%) >Melan-A (80%) >HMB45/gp-100 (75%) >Mel-5/TRP-1 (65%) >MAGE-1 (47%) > S-100 (28%). The number of TAAs per cell line differs between early (<7) and late (>7) passages. Among late passage cell lines, lesser percentage of cell lines express three to six antigens pointing out that early passage (<7) cell lines may be needed for antigen-targeted immunotherapy. This study provides a protocol for establishing cell lines within 2-5 months for personalized vaccine therapy for nodal and organ metastatic melanoma patients.