"CD8+ Enriched “Young” Tumor Infiltrating Lymphocytes Can Mediate Regression of Metastatic Melanoma".

Mark E. Dudley ¹, Colin A. Gross ¹, Michelle M. Langhan ¹, Marcos R. Garcia ¹, Richard M. Sherry ¹, James C. Yang ¹, Giao Q. Phan ¹, Udai S. Kammula ¹, Marybeth S. Hughes ¹, Deborah E. Citrin ², Nicholas P. Restifo ¹, John R. Wunderlich ¹, Peter A. Prieto ¹, Jenny J. Hong ¹, Russell C. Langan ¹, Daniel A. Zlott ³, Kathleen E. Morton ¹, Donald E. White ¹, Carolyn M. Laurencot ¹, and Steven A. Rosenberg ¹

¹ Surgery Branch and ² Radiation Oncology Branch, National Cancer Institute, Bethesda, Maryland; and
³ Pharmacy Department, Clinical Research Center; National Institutes of Health, Bethesda, Maryland

Corresponding Author:
Mark E. Dudley, National Surgery Branch, National Cancer Institute, CRC 3W-5752, 10 Center Drive, Bethesda, MD 20892-1201.     Phone: 301-496-1436;     Fax: 301-451-6949;     E-mail: med@nih.gov

Received May 13, 2010.   Revision received July 1, 2010.   Accepted July 10, 2010.

NetworkEditors' Perspectives: "Re-infusion of Activated Autologous Lymphocytes Regresses Melanoma".
Abstract:
Translational Relevance:
Introduction:
Methods:
Results:
   Table 1: Demographics of patients and characteristics of treatments administered.
   Fig. 1: CD8+ enriched young TIL caused regression of bulky melanoma lesions at multiple sites.
   Table 2: Responses and toxicities to CD8+ enriched young TIL therapy.
   Fig. 2: Process improvements for the young TIL protocol resulted in predictable, simpler TIL generation.
   Fig. 3: CD8+ enriched young TIL administered to patients were highly tumor reactive.
   Fig. 4: Young TIL impacted lymphocyte reconstitution in the peripheral blood of reconstituting patients.
Discussion:
Potential Conflict of Interests:
Footnotes:
Supplementary Data:
References:
Additional References:
Conclusions from: Euchromatin, Embryomas, Entropy, Enhancers, and EMT.
Further Topics:

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.

Note: Supplementary data for this article are available at Clinical Cancer Research Online

(http://clincancerres.aacrjournals.org/).

This article is featured in Highlights of This Issue,

Translational Relevance:

Cell-based immunotherapy using autologous, tumor-reactive lymphocytes can mediate the regression of bulky, established cancer in animal models and human patients. Tumor-infiltrating lymphocytes (TIL) selected for tumor recognition and highly expanded in vitro have been especially effective for treating melanoma patients, but there are logistic and technical difficulties associated with generating an individualized tumor-reactive therapy for each patient. In this report we use simple, reliable methods of generating individual, autologous TIL products and test them for the first time in a 2-arm, phase II clinical trial. Our results support a wider application of individualized cell therapies based on TIL and define correlates of clinical treatment and response for improvement of future therapies.

Introduction:

Patients with metastatic melanoma have limited treatment options, and only 2 of these treatments have received Food and Drug Administration approval. Interleukin (IL)-2 can mediate durable complete responses in 3% to 5% of treated patients, with an overall response rate of about 13% to 16% (1); decarbazine results in a 15% response rate with rare durable responses (2). Other promising experimental treatments being evaluated in randomized trials for efficacy and toxicity include inhibitors of the dominant BRAF^V600E mutation (3) and an anti-CTLA-4 antibody (4, 5). Despite these recent advances, a pressing need remains for effective treatments.

Adoptive cell therapy (ACT) combines lymphodepletion with a patient's own tumor reactive tumor-infiltrating lymphocytes (TIL) to generate an individualized therapy (6). A total of 93 refractory melanoma patients were treated at our institution with TIL selected for tumor recognition following 1 of 3 different lymphodepleting regimens (7). Forty-three patients received nonmyeloablative chemotherapy (NMA) alone, 25 patients received NMA with 2 Gy of total body irradiation (TBI), and 25 patients received NMA plus 12 Gy TBI. The objective response rates [Response Evaluation Criteria in Solid Tumors (RECIST)] for these sequential trials were 48%, 52%, and 72%, respectively, including 15 patients who had complete responses, all but 1 of which are ongoing at 31 to 89 months. Despite these promising clinical results, the extensive effort, cost, and time required to generate the individual TIL cultures limited the application of this promising therapy to only a few institutions.

Tumor-reactive TIL used in prior studies required an extended duration of multiple microcultures and an individualized assay to identify tumor recognition. Analysis of demographic and biological factors that correlated with TIL function indicated that the size of the lesion and the degree of lymphocyte infiltration impacted TIL generation (8). However, many patients underwent procurement of metastatic tissue for TIL generation but were not ultimately eligible for treatment because their TIL failed the test for tumor recognition, or rapid disease progression occurred during TIL establishment (8). Thus, the complex TIL production process was a substantial limitation to patient therapy. We have now simplified and standardized the TIL production process by generating “young” TIL for therapy (9). These minimally manipulated young TIL cultures consisted of bulk lymphocytes rather than microcultures, and the batch-specific assay for tumor recognition was eliminated. Young TIL have attributes associated with improved persistence and response in vivo including long telomeres and high expression of CD27 and CD28 (10–13). In a small clinical trial, young TIL were shown to be capable of mediating objective clinical responses in some patients (14, 15). We have now developed a simplified procedure to enrich for tumor-reactive CD8+ cells, eliminate nonspecific CD4+ T cells, and deplete T regulatory cells (16).

We report here the first clinical trial with CD8+ enriched young TIL administered to patients following lymphodepletion. CD8+ enriched young TIL were effective for the treatment of some patients with metastatic melanoma. The simplified methods of TIL production allowed rapid accrual to this clinical trial using methods more easily adaptable to laboratories in multiple centers. Here we analyze the clinical and immunologic features of this individualized patient therapy.

Methods:

Patients, clinical samples, and trial design

Patients eligible for this study were aged 18 years or older with measurable metastatic melanoma had at least 1 lesion resectable for TIL; had good clinical performance, adequate liver and kidney function tests, and blood counts near the normal range; were free from active systemic infections, coagulation disorders, cardiovascular disease, or immunodeficiency; were negative for HIV antibody and hepatitis B and C; and had a life expectancy of more than 3 months. All patients signed an informed consent approved by the Institutional Review Board of the National Cancer Institute.

One group of 33 patients received NMA consisting of 60 mg/kg/day cyclophosphamide for 2 days followed by 5 days of 25 mg/m2/day fludarabine. A second cohort of 23 patients received 2 days of 60 mg/kg of cyclophosphamide overlapping the first 2 of 5 days of 25 mg/m²/day fludarabine. On the final day of fludarabine, patients received 2 fractions of 2-Gy TBI separated by at least 6 hours, and the following day they received 1 fraction of 2-Gy TBI. On the day following chemotherapy or radiation all patients received a bolus intravenous infusion of CD8+ enriched young TIL and started high-dose IL-2 therapy (720,000 IU/kg intravenously every 8 hours to tolerance). One day after TIL infusion, patients who received 6-Gy TBI received a minimum of 2 × 10⁶/kg autologous purified (Miltenyi) CD34+ hematopoietic stem cells from a granulocyte colony-stimulating factor ± plerixafor mobilized pheresis.

Patients received trimethoprim, sulfamethoxazole, and fungal prophylaxis following therapy; herpes virus seropositive patients also received valacyclovir. Platelets and packed red blood cells were administered as needed during hematopoietic recovery, and empiric antibiotics were initiated for neutropenic fevers (38.3°C once or for 2 temperatures of 38.0°C at least 1 hour apart and absolute neutrophil count < 500). Patient response was assessed using standard radiographic studies and physical examination at approximately 4 weeks following TIL administration and at regular intervals thereafter. The RECIST guidelines were followed and patients were categorized into complete, partial, or nonresponding categories. Complete blood counts (CBC) were obtained at least once per day while patients were in the hospital and differential counts were obtained when CBC was over 200 cells/mL.

Generation and characterization of CD8+ enriched young TIL

Patients with metastatic melanoma underwent biopsy and as much of the sample as possible was processed to a single cell suspension for generation of young TIL as previously described (9, 17) and detailed in Supplementary Methods. The single cell suspension was evaluated on a hemacytometer with lymphocytes and tumor cells determined on the basis of size and morphology and viability determined by Trypan blue staining. After minimum time in culture, successfully initiated “bulk” young TIL were CD8+ enriched (Miltenyi CliniMACS; ref. 16) and rapidly expanded to clinical cell numbers (18, 19). Aliquots of infused samples were evaluated by fluorescence-activated cell sorting (FACS) analysis and cytokine release assays to determine lymphocyte phenotype and antigen specificity respectively using standard techniques (18).

Statistical analysis

Standard statistical methods are cited in the text, and all P values are 2-tailed assuming unequal variance with P < 0.05 considered significant.

Results:

CD8+ enriched young TIL mediated antitumor responses

The safety and efficacy of therapy using CD8+ enriched young TIL following lymphodepletion was investigated in 2 cohorts of patients with metastatic melanoma. Thirty-three patients received CD8+ enriched TIL with NMA, and 23 patients received CD8+ enriched young TIL following NMA plus 6 Gy of TBI. The demographic characteristics of these patients and the treatments administered are shown in Table 1. Sixty-four percent of patients had received prior IL-2. Median follow-up as of April 1, 2010 in the NMA and 6-Gy TBI cohorts was 14 months and 7 months, respectively.

 Table 1.

Demographics of patients and characteristics of treatments administered

Nineteen of the 33 patients (58%) in the NMA cohort exhibited an objective tumor regression by RECIST criteria, including 16 partial responders (48%) and 3 complete responders (9%). Eleven of 23 patients (48%) in the 6-Gy TBI cohort achieved an objective response, including 2 complete responders (9%). Illustrative examples of clinical tumor regression are shown in Figure 1. Fifteen of 24 patients with M1a or M1b melanoma and 15 of 32 patients with M1c disease responded to therapy. As reported previously (7, 18), all patients experienced transient hematologic toxicities from the lymphodepleting conditioning and received platelet and red blood cell transfusions as medically indicated. Patients were also treated for symptoms associated with high-dose IL-2 therapy. All toxicities typically returned to baseline within a few days. All nonhematologic grade 3 and 4 toxicities not attributable to IL-2 are listed in Table 2. There were no grade 3 or 4 toxicities directly attributable to the infused cells. There were 2 treatment-related mortalities, 1 in each cohort, that resulted from acute sepsis during the neutropenic period associated with lymphodepletion about 5 days after TIL infusion.

Fig. 1. CD8+ enriched young TIL caused regression of bulky melanoma lesions at multiple sites.

Fig. 1. CD8+ enriched young TIL caused regression of bulky melanoma lesions at multiple sites.

A, computed tomography (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 2 months (right) with visible signs of recalcification of prior metastatic sites in the sacrum and ilium.

B, subcutaneous melanoma around the ear and in the auditory canal caused complete hearing loss (left); 11 days after CD8+ enriched young TIL infusion, gross necrosis of the melanoma was visible (middle); at 76 days after treatment the patient experienced a partial response at all sites including liver and subcutaneous lesions (right). 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 1 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. 

Generation of young TIL for treatment was reliable and rapid

During the period of this study (August 2008 through September 2009), 176 tumors from 122 patients were processed to establish young TIL cultures. TIL were successfully grown (>50 × 10⁶ cells within 5 weeks) from 124 of the 176 lesions (70%), comprising 101 of the 122 patients (83%). A striking correlation was observed between the success of establishing TIL and the initial proportion of lymphocytes in the single cell suspension (Fig. 2A). Tumors that successfully yielded TIL had an initial median of 52% lymphocytes whereas tumors that failed to grow TIL cultures in vitro had a median of 8% lymphocytes (P = 5 × 10^-8). Among these 122 patients, 53 were treated (3 additional patients received cryopreserved TIL from prior resections), 21 had samples that failed to grow TIL cultures, 20 developed rapidly progressive disease that prevented treatment, 13 were resected free of evaluable disease (although 9 patients recurred and received TIL treatment subsequently), 9 received other treatments including 1 complete responder to high dose IL-2 therapy, and 6 experienced individual laboratory or clinical issues, including 1 patient whose CD8+ TIL failed to expand during the rapid expansion protocol.

Fig. 2. Process improvements for the young TIL protocol resulted in predictable, simpler TIL generation.

 Fig. 2. Process improvements for the young TIL protocol resulted in predictable, simpler TIL generation.

A, the percentage 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 morphologic criteria after Trypan blue staining. The samples were plotted on the basis of whether sufficient TIL grew to use for treatment (>5 × 10⁷cells in 28 days, R x 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 nonresponding 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 nonresponding patients (n = 26) or responders (n = 30). Standard error bars are shown. 

The CD8+ enrichment was highly effective for reducing the fraction of CD4+ cells in the infused TIL (Table 1 and Supplementary Table S1). The CD4 cell component comprised an average of 22% of the cells in bulk young TIL prior to CD8 enrichment, and natural killer cells comprised 21% (Supplementary Table S1). TIL from prior protocols administered without CD8 enrichment after NMA conditioning also contained about 21% CD4+ cells. Following CD8+ enrichment and expansion, CD4+ cells were reduced to an average of 2% of the infused young TIL.

The age of TIL was compared for patients who received CD8+ enriched TIL and patients on prior TIL protocols at our institution (Fig. 2B). Microculture generated, tumor-selected TIL administered to responding patients were significantly younger than TIL given to patients who did not respond. In the cohorts treated in this report, there was no difference between the age of CD8+ enriched young TIL cultures for responding and nonresponding patients but these cultures were significantly younger than TIL cultures administered on prior protocols (P = 3 × 10^-7).

CD8+ enriched young TIL frequently exhibited tumor recognition

We retrospectively evaluated the ability of the administered CD8+ enriched TIL to recognize tumor and looked for any correlation with clinical efficacy. Recognition of autologous or HLA-matched tumor was evaluated by cytokine release assay (Fig. 3). Twenty-three of the 33 NMA patients had autologous tumor available (18 with cryopreserved tumor digest and 5 with a tumor cell line). Eight of 10 nonresponding patients and 8 of 13 objective responders demonstrated autologous tumor recognition. Four additional objective responding patients recognized HLA-A–matched tumor cell lines. Nineteen of 23 patients treated with 6-Gy TBI had autologous tumor available (15 with fresh frozen tumor and 4 with a cell line). Eight of 11 nonresponding patients and 5 of 8 responding patients demonstrated specific tumor recognition. In addition, 3 patients' TIL recognized HLA-matched tumor cell lines, including 1 nonresponding patient (patient SA, Fig. 6B), whose TIL failed to recognize autologous tumor. In total, 29 of 42 evaluable CD8+ enriched young TIL samples (69%) demonstrated specific autologous tumor recognition and 36 of 56 (64%) patients demonstrated specific recognition in all. Strikingly, 11 of 30 objective responses were mediated by CD8+ enriched young TIL with no evidence of specific tumor recognition as defined in prior TIL clinical protocols.

Fig. 3. CD8+ enriched young TIL administered to patients were highly tumor reactive.

 Fig. 3. CD8+ enriched young TIL administered to patients were highly tumor reactive.

TIL from cryopreserved aliquots of each infused treatment were thawed and rested overnight in IL-2, then washed and incubated at a 1:1 ratio with autologous, HLA-matched, or HLA-mismatched tumors. IFN-g secreted in the coculture supernatant was quantified by ELISA. The data from each separate coculture assay were aggregated and plotted. Patients with specific tumor recognition (greater than 200 pg/mL IFN-g and 2 × HLA-mismatched tumor) are boxed. Off scale > 5,000 pg/mL.

A, CD8+ enriched young TIL administered to patients after NMA conditioning.

B, CD8+ enriched young TIL administered to patients after 6-Gy TBI conditioning. 

CD8+ enriched young TIL influenced lymphocyte reconstitution of the host

The average absolute lymphocyte count (ALC) for all patients who received CD8+ enriched young TIL after NMA or 6-Gy TBI lymphodepletion is plotted in Figs. 4A and B, respectively. For comparison, lymphocyte reconstitution from patients who received antigen-selected TIL derived from microculture expansions after NMA or 12-Gy TBI (7) lymphodepletion are also shown. Interestingly, patients who received CD8+ enriched young TIL demonstrated higher peak ALCs suggesting that CD8+ enriched young TIL have increased capacity for in vivo expansion compared with selected TIL.

Fig. 4. CD8+ enriched young TIL impacted lymphocyte reconstitution in the peripheral blood of reconstituting patients.

 Fig. 4. CD8+ enriched young TIL impacted lymphocyte reconstitution in the peripheral blood of reconstituting patients.

A, CD8+ enriched young TIL quickly repopulated patient peripheral blood to high levels after NMA conditioning. Patient ALCs were determined daily and average ALC is plotted. Arrows, IL-2 therapy. 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 6-Gy TBI conditioning. Arrows, IL-2 therapy; Filled triangles, average of all patients who received CD8+ enriched young TIL with 6?Gy TBI (n = 23). Open triangles, (historic control) average of all patients who received extensively expanded, tumor selected TIL with 12-Gy TBI (n = 25). Vertical bars, standard error.

C and D, CD8+ ALC 1 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 1 month after TIL infusion. C, CD8+ ALC plotted for each patient.

D, CD4+ ALC plotted for each patient. NR, nonresponder; OR, objective responder. Black bars, mean ALC for the population. 

Peripheral blood lymphocytes (PBL) from 47 of the 56 patients who received CD8+ enriched young TIL were sampled at approximately 1 month after cell infusion. ALC and absolute CD3+CD8+ and CD3+CD4+ cell numbers were assessed by FACS analysis in a blinded manner. The treatment resulted in low CD4+ counts at 1 month in most patients (average: 109 CD4+ cells/mL), with no difference between responding and nonresponding patients (P = 0.5; Fig. 4D). Patients in prior clinical trials who received TIL that contained CD4+ lymphocytes had higher peripheral CD4+ cell counts at 1 month after TIL infusion (average: 187 cells/mL). In contrast to CD4+ cells, CD8+ ALC for most patients treated with CD8+ enriched young TIL was in the normal range at 1 month, and there was a significant increase in CD8+ ALC in responding patient compared with nonresponders (P = 0.002; Fig. 4C). Patients who responded had CD8+ ALC about 2-fold higher than nonresponders (1,504 versus 696 cells/mL). In prior clinical trials the average CD8+ cell count was 652 CD8+ cells/mL at 1 month after TIL infusion. Infused TIL were examined by FACS for expression of additional markers including CD27, CD28, CD62L, and CCR7, but none correlated with clinical response.

Discussion:

Adoptive cell therapy (ACT) can mediate durable objective regression (including durable complete responses) in patients with metastatic melanoma refractory to other treatments. The complexity of preparation of cell products unique for each patient has limited the widespread use of this method. In this study we developed a simplified method for cell preparation that may allow wider application of this effective treatment.

Previously, 93 patients were treated over 84 months using highly expanded TIL selected for specific tumor recognition (7). This corresponds to about 1 treatment per month and about 27% of resected patients who finally received a TIL product (8). In the current report 56 additional patients were treated with CD8+ enriched young TIL over 14 months, corresponding to about 3 to 4 patients treated per month, with 53% of eligible patients who underwent resection able to receive TIL therapy. With both methods, clinical response rates were about 55%, but strikingly 11 of 30 objective responders in this study had TIL that would have been ineligible for the prior study. Although comparisons of sequential clinical trials should be made with caution, the changes in the CD8+ enriched young TIL methods were apparently effective for improving delivery of individualized TIL therapy to eligible patients without compromising therapeutic efficacy.

Prior clinical trials with TIL in our institution (7) and preclinical mouse models (20–22) suggested that increased lymphodepletion improves ACT. This study comparing 2 cohorts of patients treated with different conditioning regimens found similar objective response rates and toxicity profiles. This outcome could result from relatively small patient numbers, sequential cohort enrollment, and short follow-up. Alternately, 6 Gy of TBI delivered in fractionated doses may only minimally impact host mechanisms that support the transferred CD8+ cells compared with 12 Gy of TBI (23).

Despite methodologic improvements, 21 of the 123 patients (17%) had TIL that failed to grow in culture. On average, these melanoma lesions started with a median of only 8% lymphocytes (Fig. 2A). In the future, changing the lymphocyte/tumor ratio in vitro could improve the generation of TIL for some tumors. Additionally, some studies have noted a correlation between good prognosis and T-cell infiltration for nonmelanoma cancers including colon cancer (24), breast cancer (25), ovarian cancer (26), and non–small cell lung cancer (27), suggesting that CD8+ enriched young TIL from metastatic lesions from nonmelanoma histologies may be therapeutically active in ACT treatments.

The enrichment of CD8+ cells prior to rapid expansion has practical benefits for TIL generation (16) but the enrichment process is expensive, and depletion of CD4+ cells raises questions about lymphocyte persistence, tumor regression, and treatment toxicity. CD4+ T helper cells are involved in generating and maintaining CD8+ T memory cells in mice (28, 29) and a CD4+ clone was associated with melanoma regression in a patient (30). However, CD4+ T regulatory cells may block CD8+ cell function in tumor immunity (20, 31). The impact of CD4+ cells on the persistence and function of CD8 TIL in vivo is not known. In this study, the CD8+ enriched young TIL repopulated peripheral blood and persisted at high levels for over a month. Furthermore, the objective response rate of 54% reported here is comparable to the 56% objective response rate observed in prior trials with CD4+ replete TIL. These data suggest that elimination of the CD4+ cells was beneficial to the therapy. More study is needed to understand the role of CD4+, CD25+ and FoxP3+ cells in the regulation of TIL responses after infusion and CD4+ T helper cells in the duration of responses. To directly address the impact of CD4+ cells in young TIL therapy, we have initiated a randomized clinical trial where one arm receives unselected young TIL that contain both CD4+ and CD8+ cells, whereas a second arm receives CD8+ enriched young TIL.

This study presents data from 2 cohorts of patients treated with autologous TIL following lymphodepletion for metastatic melanoma. The production process for CD8+ young TIL was relatively rapid and reliable, and the manufactured product was capable of generating tumor regression and objective responses in patients. The minimally manipulated CD8+ cell product had a high frequency of autologous tumor reactivity, and repopulated the host PBL compartment rapidly. Persistently high CD8+ cell numbers were correlated with tumor regression in treated patients. This method for the generation of CD8+ enriched young TIL represents a major simplification in the application of therapeutically effective TIL because of the ease of cell production (one culture versus many microcultures) and the elimination of testing for antitumor specificity. These simplifications can serve to extend the ability to apply this treatment approach to additional institutions.

Disclosure of Potential Conflicts of Interest:

No potential conflicts of interest were disclosed.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes:

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

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26. Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G, et al.Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med 2003;348:203–13.CrossRefMedline

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This progressive study by Mark Dudley, Colin Gross, Michelle Langhan, Marcos Garcia, Richard Sherry , James Yang, Giao Phan, Udai Kammula, Marybeth Hughes, Deborah Citrin, Nicholas Restifo, John Wunderlich, Peter Prieto, Jenny Hong, Russell Langan, Daniel Zlott, Kathleen Morton, Donald White, Carolyn Laurencot, and Steven Rosenberg, reveals new modifications and improvments of autologous lymphocyte immunotherapy of human malignant melanoma, with accompanying improvment in responses and longevity for patients.
Over 40 years of  lymphocyte immunotherapy research may now be reaching a decisive  new phase for cancer patients and their physicians.

Additional References:

1. Frenster JH, and Rogoway, WM, "In-Vitro Activation and Re-Infusion of Autologous Human Lymphocytes", Lancet vol. 2, pp. 979-980, (November 2, 1968).

2. Stanley DA, Frenster JH, and Rigas DA, "Localization of ³H-Phytohemagglutinin within Human Lymphocytes and Monocytes", in: "Proceedings of the Fourth Annual Leukocyte Culture Conference, 1969", (McIntyre OR, ed.), pp. 1-11, (1971), New York: Appleton-Century-Crofts.

3a. Frenster JH, and Rogoway, WM, "Immunotherapy of Human Neoplasms with Autologous Lymphocytes Activated In-Vitro", in: "Proc. Fifth Annual Leukocyte Culture Conf.", (Harris J, ed.), (1970), pp. 359-373, Academic Press, New York.

3b. Keshgegian AA, Meisner LF, and Frenster JH, (1971)
"Thymidine Reversal of Ribothymidine Inhibition of Lymphocyte Mitosis".

3c. Archibald RB, and Frenster JH, "Quantitative Ultrastructural Analysis of In-Vivo Lymphocyte - Reed-Sternberg Cell Interactions in Hodgkin's Disease", Natl. Cancer Inst. Monogr. 36: 239-245 (1973).

4. Kern DH, and Pilch YH,  Immune cytolysis of murine tumor cells mediated by xenogeneic "immune" RNA.
Int. J. Cancer, vol. 13, no. 5, pp. 679-688 (May 15, 1974).

5a. Frenster JH, "Phytohemagglutinin-Activated Autochthonous Lymphocytes for Systemic Immunotherapy of Human Neoplasms", Ann. N.Y. Acad. Sci. 277: 45-51 (1976).

5b. Frenster JH, Papalian MM, Masek MA and Frenster JA,  (1979)
"Electron Microscopic Analysis of Lymph Node Cellular Activity in Hodgkin's Disease".
 J. Natl. Cancer Inst. 63: 331-335.

6. Hellstrom I, Ledbetter JA, Scholler N, Yang Y, Ye Z, Goodman G, Pullman J, Hayden-Ledbetter M, and Hellström KE, "CD3-Mediated Activation of Tumor-Reactive Lymphocytes from Patients with Advanced Cancer", Proc. Natl. Acad. Sci. USA, 98: 6783-6788 (2001).

7. Coughlin CM, Vance BA, Grupp SA, and Vonderheide RH, "RNA-transfected CD40-activated B cells induce functional T-cell responses against viral and tumor antigen targets: implications for pediatric immunotherapy", Blood First Edition Paper, prepublished online November 20, 2003;
DOI 10.1182/blood-2003-07-2379.

8. Liao X, Li Y, Bonini C, Nair S, Gilboa E, Greenberg PD, and Yee C, "Transfection of RNA Encoding Tumor Antigens Following Maturation of Dendritic Cells Leads to Prolonged Presentation of Antigen and the Generation of High-Affinity Tumor-Reactive Cytotoxic T Lymphocytes"., Molecular Therapy, vol. 9, no. 5, pp. 757-764 (May, 2004).

9. Rosenberg SA, and Dudley ME, "Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes", Proc. Natl. Acad. Sci. USA, vol. 101, Suppl. 2, pp. 14639-14645 (October 5, 2004).

10. Bollard CM, Aguilar L, Straathof KC, Gahn B, Huls MH, Rousseau A, Sixbey J, Gresik MV, Carrum G, Hudson M, Dilloo D, Gee A, Brenner MK, Rooney CM, and Heslop HE, "Cytotoxic T Lymphocyte Therapy for Epstein-Barr Virus⁺ Hodgkin's Disease",  J. Exp. Med. 2004 200: 1623-1633.

11. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, and Rosenberg SA.
"Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes".

1. Each cell retains all of its embryonic genes for a lifetime.

2. Controls for embryonic genes are often absent in adults.

3. Uncontrolled embryonic genes can replicate wildly.

4.  Replicating genes participate in  intra-cellular competition.

5.  The basis for gene competition is selective transcription.

6.  MicroRNAs can reprogram embryomic transcription.

7.  Gene reprogramming can produce normal phenotypes.

8.  Normal phenotypes can by-pass chromosomal lesions.

9.  MicroRNA therapy may need to be permanent.

10. Transplantation of microRNAs could be preferred.

http://www.embryomas.net/

1. Pathways within cell genomes involve a flow of information.

2. Information can flow by direct contact or by third parties.

3. Direct contact within whole genomes is difficult to regulate.

4. DNA-DNA direct contects are influenced by agents.

5. Nuclear agents include hydrophilic ionic and hydrophobic conforming ligands.

6. Third parties within genomes involve RNAs and proteins.

7.  RNAs and proteins are easy to regulate or reverse.

8.  Information can be shared, lost, or transformed.

9. System information can be hidden during system isolation.

10.  Local information can be permanently lost during system entropy.

http://www.cancerbiophysics.net/

Links to Current Research in Euchromatin:
Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

A Brief History of Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".
(PowerPoint Presentation).

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Jeannette A. Hovsepian, M.D.
E-mail: frensasc@ix.netcom.com
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