The doubling potential of T lymphocytes allows clinical-grade production of a bank of genetically modified monoclonal T-cell populations.

BACKGROUND AIMS: To produce an anti-leukemic effect after hematopoietic stem cell transplantation we have long considered the theoretical possibility of using banks of HLA-DP specific T-cell clones transduced with a suicide gene. For that application as for any others, a clonal strategy is constrained by the population doubling (PD) potential of T cells, which has been rarely explored or exploited. METHODS: We used clinical-grade conditions and two donors who were homozygous and identical for all HLA-alleles except HLA-DP. After mixed lymphocyte culture and transduction, we obtained 14 HLA-DP-specific T-cell clones transduced with the HSV-TK suicide gene. Clones were then selected on the basis of their specificity and functional characteristics and evaluated for their doubling potential. RESULTS: After these steps of selection the clone NAT-DP4(TK), specific for HLA-DPB1*04:01/04:02, which produced high levels of interferon-γ (IFNγ), tumor necrosis factor (TNF), interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), was fully sequenced. It has two copies of the HSV-TK suicide transgene whose localizations were determined. Four billion NAT-DP4(TK) cells were frozen after 50 PDs. Thawed NAT-DP4(TK) cells retain the potential to undergo 50 additional PDs, a potential very far beyond that required to produce a biological effect. This PD potential was confirmed on 6/16 additional different T-cell clones. This type of well-defined clone can also support a second genetic modification with CAR constructs. CONCLUSION: The possibility of choosing rare donors and exploiting the natural proliferative potential of T lymphocytes may dramatically reduce the clinical and immunologic complexity of adoptive transfer protocols that rely on the use of third-party T-cell populations.

Long-term follow-up of patients treated by adoptive transfer of melanoma tumor-infiltrating lymphocytes as adjuvant therapy for stage III melanoma.

The first analysis of our clinical trial on interest of using tumor-infiltrating lymphocytes (TIL) as adjuvant therapy for stage III (regional lymph nodes) melanoma was published in 2002 [5]. The aim of this paper is to update clinical results of 7 years of follow-up after the last treated patient. In the trial conducted between December 1993 and January 1999, patients without any detectable metastases after lymph node excision were randomly assigned to receive either TIL plus interleukin-2 (IL-2) for 2 months, or IL-2 only. The duration of the relapse-free interval was the primary objective. Eighty-eight patients were enrolled in the study. Currently, the last analysis performed in June 2006, after a median follow-up of 114.8 months, did not show change of non-significant extension of the relapse-free interval or overall survival. However, this second analysis strengthens our first hypothesis about the relationship between number of invaded lymph nodes and TIL treatment effectiveness. In the group with only one invaded lymph node, the estimated relapse rate was significantly lower (P (adjusted) = 0.0219) and the overall survival was increased (P (adjusted) = 0.0125) in the TIL+IL-2 arm compared with the IL-2 only arm. No differences between the two arms, either with regard to the duration of disease-free survival (P (adjusted) = 0.38) or overall survival (P (adjusted) = 0.43), were noted in the group with more than one invaded lymph node, whatever the number of invaded lymph nodes. Treatment was compatible with normal daily activity. This study, with a very long follow up (median of almost 10 years), postulates for the first time relationship between TIL efficiency in stage III melanoma (AJCC) and number of invaded lymph nodes, indicating that tumor burden might be a crucial factor in the production of an effective in vitro expansion of T cells specific for autologous tumor antigen, a finding which could be of value in future vaccine development for the treatment of melanoma.

Randomized trial of adoptive transfer of melanoma tumor-infiltrating lymphocytes as adjuvant therapy for stage III melanoma.

The aim of this study was to demonstrate the interest of using tumor-infiltrating lymphocytes (TIL) as adjuvant therapy for stage III (regional lymph nodes) melanoma. After lymph node excision, patients without any detectable metastases were randomly assigned to receive either TIL plus interleukin-2 (IL-2) for 2 months, or IL-2 only. The primary endpoint was determination of the duration of the relapse-free interval. Eighty-eight patients determined as eligible for treatment were enrolled in the study. After a median follow-up of 46.9 months, for the study population the analysis did not show a significant extension of the relapse-free interval or overall survival. However, a significant interaction ( P<0.001) was found between the treatment and the number of invaded lymph nodes. In the group with only one invaded lymph node, the estimated relapse rate was significantly lower ( P(adjusted)=0.0285) and the overall survival was increased ( P(adjusted)=0.039) in the TIL+IL-2 arm compared with the IL-2 only arm. No differences between the two arms, either as regards the duration of disease-free survival or overall survival, were noted in the group with more than one invaded lymph node whatever the number of invaded lymph nodes. Treatment was compatible with normal daily activity. This study demonstrates for the first time that the efficiency of TIL in stage III melanoma (AJCC) is directly related to the number of invaded lymph nodes, indicating that tumor burden might be a crucial factor in the efficacy and/or in vitro expansion of T cells specific for autologous tumor antigen, a finding which could be of value in future vaccine development for the treatment of melanoma.