LNs draining a site of vaccine inoculation are an enriched source of T lymphocytes that have been sensitized to tumor antigens. Preclinical kinetic studies have clearly demonstrated that the optimal number of preeffector T cells is present between 6 and 14 d with subsequent waning of activity. These findings recommend a strategy of placing a tumor vaccine in a region that is uninvolved with tumor so that newly sensitized T cells can be obtained by removal of the vaccine draining LNs at the peak of the reaction.
In an initial study of VDLN cells, subjects with metastatic melanoma or RCC were treated. Irradiated autologous tumor cells admixed with bacille Calmette-Guerin (BCG) was used as a vaccine and 7 d later draining LNs were removed. The T cells were stimulated with immobilized anti-CD3 (OKT3) for 2 d followed by culture in IL-2 to reach a mean treatment dose of 8.4 x 1010 cells. These activation conditions generated a mixture of CD4 and CD8 that demonstrated specific release of GM-CSF and IFN-y against autologous tumor targets. Eleven melanoma patients and 12 RCC patients were treated with T-cell infusion and concomitant IL-2. One patient with melanoma experienced a partial response (PR); two patients with RCC had a CR and two had a PR. Immune monitoring studies indicated a trend correlating development of a positive delayed-type hypersensitivity (DTH) response to autologous tumor with clinical response (100).
The feasibility of adoptive immunotherapy using VDLN cells was also tested in subjects with malignant gliomas. Two sequential trials were performed based on preclinical studies demonstrating that adoptive transfer of ex vivo-activated LN T cells could mediate regression of intracranial tumors including glioma (36,101). In a major departure from previous clinical studies of T-cell adoptive transfer, T cells alone were used for therapy without concomitant IL-2. This modification was based on findings that systemic IL-2 actually diminished the trafficking of adoptively transferred T cells into brain tumors and inhibited their therapeutic efficacy (38). In the first study, nine patients with recurrent glioblastoma multiforme (GBM) and one patient with anaplastic astrocytoma were vaccinated with irradiated autologous tumor cells mixed with GM-CSF (Sargamostim) as an adjuvant. Treatment with adjuvant GM-CSF was tolerated without toxicity and promoted local inflammation at the vaccine site but without skin ulceration that had occasionally been observed previously when BCG was used as an adjuvant. Draining LNs were removed 7 d later and stimulated with the bacterial superantigen Staphylococcus aureus enterotoxin A (SEA) and IL-2. The combination of SEA and a low concentration of IL-2 stimulated vigorous proliferation of both CD4+ and CD8+ T cells. The cell dose ranged from 9 x 108 to 1.5 x 1011 cells and the treatment was tolerated without significant toxicity, leading to partial regression in two subjects (102).
A second cohort of patients with newly diagnosed gliomas was treated to assess whether adoptive transfer of sensitized T cells induced any neurologic toxicity over a longer period of time. Patients were vaccinated with irradiated autologous tumor cells mixed with GM-CSF approx 1 mo after completion of standard radiation treatment. The yield of LN cells for these patients ranged from 9 x 107 to 1.1 x 109 and was approximately four times greater than what was previously obtained in the cohort of patients with recurrent GBM. Minor modifications to the procedure of ex vivo stimulation but with retention of a low concentration of IL-2 (10 U/mL) resulted in a median of 42-fold proliferation of cells over 6-8 d. These culture conditions permitted delivery of a median of 1.1 x 1010 cells that were predominantly CD4+ and nearly uniformly CD25+. The T cells were administered as outpatient therapy, without concomitant IL-2, and there was no immediate toxicity. Interestingly, four patients had partial regression of measurable residual enhancing tumor masses without development of immediate or delayed neurologic toxicity. In addition, three patients with minimal residual tumor have been free of tumor progression for greater than 2 yr (103).
Similarly designed phase I clinical studies were performed in patients with metastatic RCC or head and neck squamous cell carcinoma (104,105). These studies used T cells alone without concomitant IL-2 and were performed entirely as outpatient therapy. The extent of numerical amplification of T cells and retention of a predominance of CD4+ T cells was similar to the previous experience with glioma patients, indicating that there was not a significant restriction of this approach to a single disease. There was no significant toxicity from the T-cell transfer. Among 20 patients with progressive metastatic RCC, 1 had a partial response, 1 a mixed response, and 8 had stable disease lasting greater than 5 mo. These results indicated that further modifications to improve the function or to sustain the T cells were needed before more extensive clinical testing was warranted.
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