We describe an in vitro method for the development of CD4+ T-cell clones with specificity for a tumor antigen and the use of one such clone for the treatment of a patient with metastatic melanoma. Development of the method has been difficult, in part because of the paucity of well-defined immunogenic HLA class II–restricted epitopes of tumor antigens and also because of the difficulty in isolating and expanding CD4+ T-cell clones to obtain numbers sufficient for clinical use. The HLA-DP4–restricted peptide epitope of the melanoma-associated cancer–testis antigen NY-ESO-1 is one of the most immunogenic tumor antigens10,14
and can elicit responses by CD4+ T cells from patients with cancer who have been immunized with NY-ESO-1.15,16
Using the NY-ESO-1 peptide, we succeeded in generating NY-ESO-1–specific CD4+ T cells from the blood of an unimmunized patient with meta-static melanoma. We were able to expand these cells to several billion and use them to treat the patient. No exogenous cytokines were administered because these CD4+ T cells, when stimulated in vitro by the NY-ESO-1 antigen, produce auto-crine-active interleukin-2. After infusion of the cells, influenza-like symptoms and lymphopenia, signs of a cytokine release syndrome, developed. In contrast to transferred CD8+ T cells, which survive only briefly (<20 days) in vivo in the absence of exogenous cytokine,1,3
the CD4+ T-cell clones that we administered without any cytokine treatment were detectable in the patient’s blood for at least 3 months. Therefore, the possibility that in vitro–derived and clonally expanded T cells were functionally exhausted17
was not substantiated by our results.
Our studies and those performed by others have shown that immunotherapy with antigen-specific T cells can eliminate tumor cells that express the corresponding antigen, but it can also allow the outgrowth of antigen-loss tumor variants.1,3
The target antigen in our case, NY-ESO-1, was not uniformly expressed by the cells of the patient’s melanoma, yet the tumor regressed completely. We postulate that the complete regression of the tumor can be attributed in part to responses by the patient’s immune system to other antigens displayed by the tumor. This additional response could have come about through antigens in apoptotic bodies that were released from killed tumor cells. When collected and processed by antigen-presenting cells, these antigens probably activated a variety of T cells, thereby broadening the immune response, a process referred to as antigen spreading.18,19
The delay of days to weeks in the development of these additional responses is consistent with antigen spreading ().
The fact that T cells against glycoprotein 100, an antigen that was not expressed by the melanoma, were undetectable indicates the selectivity of the antitumor immune responses. It is also possible that the reduction in the tumor burden relieved immunosuppression in the tumor microenvironment caused by inhibitory cytokines and regulatory T cells, thereby contributing to the broadened T-cell response. IgG antibodies against NY-ESO-1 were detectable in plasma samples from the patient before infusion of the T-cell clone and remained unchanged in titer after the infusion. The presence of antibodies against NY-ESO-1 is associated with an increased likelihood that NY-ESO-1–specific CD4+ and CD8+ T-cell responses will be generated ex vivo.14,20
Antibodies against MAGE-3 and MART-1 were undetectable before the T-cell infusion and afterward for up to 87 days ( in the Supplementary Appendix
). The lack of an increase in the titers of antibodies against melanoma antigens despite the appearance of CD4+ T cells against these antigens suggests that the production of such antibodies is independent of T cells.
In summary, we showed that the infusion of a clonal population of CD4+ T cells with specificity for a single tumor-associated antigen caused complete regression of a tumor. During regression of the tumor, this clone appears to have induced the patient’s own T cells to respond to other antigens of his tumor. These findings support further clinical studies of antigen-specific CD4+ T cells in the treatment of malignant disease.