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Clinical Trial
. 2010 Dec 15;16(24):6122-31.
doi: 10.1158/1078-0432.CCR-10-1297. Epub 2010 Jul 28.

CD8+ enriched "young" tumor infiltrating lymphocytes can mediate regression of metastatic melanoma

Mark E Dudley  1 , Colin A Gross, Michelle M Langhan, Marcos R Garcia, Richard M Sherry, James C Yang, Giao Q Phan, Udai S Kammula, Marybeth S Hughes, Deborah E Citrin, Nicholas P Restifo, John R Wunderlich, Peter A Prieto, Jenny J Hong, Russell C Langan, Daniel A Zlott, Kathleen E Morton, Donald E White, Carolyn M Laurencot, Steven A Rosenberg
Affiliations
Clinical Trial

CD8+ enriched "young" tumor infiltrating lymphocytes can mediate regression of metastatic melanoma

Mark E Dudley et al. Clin Cancer Res. .

Abstract

Purpose: Tumor-infiltrating lymphocytes (TIL) and interleukin (IL)-2 administered following lymphodepletion can cause the durable complete regression of bulky metastatic melanoma in patients refractory to approved treatments. However, the generation of a unique tumor-reactive TIL culture for each patient may be prohibitively difficult. We therefore investigated the clinical and immunologic impact of unscreened, CD8+ enriched "young" TIL.

Experimental design: Methods were developed for generating TIL that minimized the time in culture and eliminated the individualized tumor-reactivity screening step. Thirty-three patients were treated with these CD8+ enriched young TIL and IL-2 following nonmyeloablative lymphodepletion (NMA). Twenty-three additional patients were treated with CD8+ enriched young TIL and IL-2 after lymphodepletion with NMA and 6 Gy of total body irradiation.

Results: Young TIL cultures for therapy were successfully established from 83% of 122 consecutive melanoma patients. Nineteen of 33 patients (58%) treated with CD8+ enriched young TIL and NMA had an objective response (Response Evaluation Criteria in Solid Tumors) including 3 complete responders. Eleven of 23 patients (48%) treated with TIL and 6 Gy total body irradiation had an objective response including 2 complete responders. At 1 month after TIL infusion the absolute CD8+ cell numbers in the periphery were highly correlated with response.

Conclusions: This study shows that a rapid and simplified method can be used to reliably generate CD8+ enriched young TIL for administration as an individualized therapy for advanced melanoma, and may allow this potentially effective treatment to be applied at other institutions and to reach additional patients.

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Figures

Figure 1
Figure 1
CD8+ enriched young TIL caused regression of bulky melanoma lesions at multiple sites. A) CT scans of metastatic melanoma lesions (arrows) before CD8+ enriched young TIL therapy in the sacrum and ilium (top left), lung (middle left) and spleen (lower left). All lesions showed regression at two months (right) with visible signs of recalcification of prior metastatic sites in the sacrum and ilium. B) (Left) Subcutaneous melanoma around the ear and in the auditory canal caused complete hearing loss. (Middle) Eleven days after CD8+ enriched young TIL infusion, gross necrosis of the melanoma was visible. (Right) At 76 days after treatment the patient experienced a partial response at all sites including liver and subcutaneous lesions. Tumor was absent from the auditory canal and the patient's hearing returned to normal. C) CT scans show mediastinal, lung, nodal and subcutaneous metastatic deposits before (Left) and one month after (Right) treatment with CD8+ enriched young TIL, demonstrating the rapid initial pace of tumor regression in a patient who eventually achieved a complete response.
Figure 2
Figure 2
Process improvements for the young TIL protocol resulted in predictable, simpler TIL generation. A) The percent of lymphocytes in the initial single cell suspension correlated with TIL growth. Specimens from 122 sequential patients who were eligible for TIL therapy were processed to single cell suspensions. The fraction of lymphocytes among viable cells was determined by morphological criteria after trypan blue staining. The samples were plotted based on whether sufficient TIL grew to use for treatment (>5×107 cells in 28 days, Rx TIL) or whether growth was insufficient for treatment (No growth). Black bars indicate median values of the populations. B) Process improvements in TIL generation led to significantly younger cells administered to patients. Prior protocols required all TIL to undergo individualized testing for tumor reactivity (Specific TIL, n=92). TIL from non-responding patients (n=40, NR) spent significantly longer time in culture prior to administration than TIL from objective responders (n=52, OR). There was no difference (NS) between the time spent in culture of CD8+ enriched young TIL (Young TIL) administered to non-responding patients (n=26) or responders (n=30). Standard error bars are shown.
Figure 3
Figure 3
CD8+ enriched young TIL administered to patients were highly tumor reactive. TIL from cryopreserved aliquots of each infused treatment was thawed and rested overnight in IL-2, then washed and incubated at a 1:1 ratio with autologous, HLA-matched, or HLA-mismatched tumors. Interferon (IFN)-gamma secreted in the coculture supernatant was quantified by ELISA. The data from each separate coculture assay was aggregated and plotted. Patients with specific tumor recognition (greater than 200 pg/ml IFN-gamma and 2X HLA-mis-matched tumor) are boxed. Offscale >5000 pg/ml. A) CD8+ enriched young TIL administered to patients after NMA conditioning. B) CD8+ enriched young TIL administered to patients after 6Gy TBI conditioning
Figure 4
Figure 4
CD8+ enriched young TIL impacted lymphocyte reconstitution in the peripheral blood of reconstituting patients. Arrows: IL-2 therapy A) CD8+ enriched young TIL quickly repopulated patient peripheral blood to high levels after NMA conditioning. Patient absolute lymphocyte counts (ALC) were determined daily and average ALC is plotted. Filled square: average of all patients who received CD8+ enriched young TIL with NMA (n=33). Open square: (historic control) average of all patients who received extensively expanded, tumor selected TIL with NMA as their first treatment (n=33). Not all patients had ALC determined every day. B) CD8+ enriched young TIL quickly repopulated patient peripheral blood to high levels after 6Gy TBI conditioning. Filled triangles: average of all patients who received CD8+ enriched young TIL with 6Gy TBI (n=23). Open triangles: (historic control) average of all patients who received extensively expanded, tumor selected TIL with 12Gy TBI (n=25). Vertical bars: standard error. C and D) CD8+ ALC one month after TIL infusion correlated with response. CD4+ and CD8+ ALC was determined in a blinded manner by the Clinical Center Core Immunology Laboratory for 47 of 56 patients treated with CD8+ enriched young TIL at approximately one month after TIL infusion. C: CD8+ ALC plotted for each patient. D: CD4+ ALC plotted for each patient. NR: non-responder, OR: objective responder. Black bars: mean ALC for the population.
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