Richard E. Royal

Adoptive Cell Transfer Therapy Following Non-Myeloablative but Lymphodepleting Chemotherapy for the Treatment of Patients With Refractory Metastatic Melanoma

PURPOSE We investigated the combination of lymphodepleting chemotherapy followed by the adoptive transfer of autologous tumor reactive lymphocytes for the treatment of patients with refractory metastatic melanoma. PATIENTS AND METHODS Thirty-five patients with metastatic melanoma, all but one with disease refractory to treatment with high-dose interleukin (IL) -2 and many with progressive disease after chemotherapy, underwent lymphodepleting conditioning with two days of cyclophosphamide (60 mg/kg) followed by five days of fludarabine (25 mg/m(2)). On the day following the final dose of fludarabine, all patients received cell infusion with autologous tumor-reactive, rapidly expanded tumor infiltrating lymphocyte cultures and high-dose IL-2 therapy. RESULTS Eighteen (51%) of 35 treated patients experienced objective clinical responses including three ongoing complete responses and 15 partial responses with a mean duration of 11.5 +/- 2.2 months. Sites of regression included metastases to lung, liver, lymph nodes, brain, and cutaneous and subcutaneous tissues. Toxicities of treatment included the expected hematologic toxicities of chemotherapy including neutropenia, thrombocytopenia, and lymphopenia, the transient toxicities of high-dose IL-2 therapy, two patients who developed Pneumocystis pneumonia and one patient who developed an Epstein-Barr virus-related lymphoproliferation. CONCLUSION Lymphodepleting chemotherapy followed by the transfer of highly avid antitumor lymphocytes can mediate significant tumor regression in heavily pretreated patients with IL-2 refractory metastatic melanoma.

Adoptive Cell Therapy for Patients With Metastatic Melanoma: Evaluation of Intensive Myeloablative Chemoradiation Preparative Regimens

PURPOSE The two approved treatments for patients with metastatic melanoma, interleukin (IL)-2 and dacarbazine, mediate objective response rates of 12% to 15%. We previously reported that adoptive cell therapy (ACT) with autologous antitumor lymphocytes in lymphodepleted hosts mediated objective responses in 51% of 35 patients. Here, we update that study and evaluate the safety and efficacy of two increased-intensity myeloablative lymphodepleting regimens. PATIENTS AND METHODS We performed two additional sequential trials of ACT with autologous tumor-infiltrating lymphocytes (TIL) in patients with metastatic melanoma. Increasing intensity of host preparative lymphodepletion consisting of cyclophosphamide and fludarabine with either 2 (25 patients) or 12 Gy (25 patients) of total-body irradiation (TBI) was administered before cell transfer. Objective response rates by Response Evaluation Criteria in Solid Tumors (RECIST) and survival were evaluated. Immunologic correlates of effective treatment were studied. RESULTS Although nonmyeloablative chemotherapy alone showed an objective response rate of 49%, when 2 or 12 Gy of TBI was added, the response rates were 52% and 72% respectively. Responses were seen in all visceral sites including brain. There was one treatment-related death in the 93 patients. Host lymphodepletion was associated with increased serum levels of the lymphocyte homeostatic cytokines IL-7 and IL-15. Objective responses were correlated with the telomere length of the transferred cells. CONCLUSION Host lymphodepletion followed by autologous TIL transfer and IL-2 results in objective response rates of 50% to 70% in patients with metastatic melanoma refractory to standard therapies.

Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen

Gene therapy of human cancer using genetically engineered lymphocytes is dependent on the identification of highly reactive T-cell receptors (TCRs) with antitumor activity. We immunized transgenic mice and also conducted high-throughput screening of human lymphocytes to generate TCRs highly reactive to melanoma/melanocyte antigens. Genes encoding these TCRs were engineered into retroviral vectors and used to transduce autologous peripheral lymphocytes administered to 36 patients with metastatic melanoma. Transduced patient lymphocytes were CD45RA(-) and CD45RO(+) after ex vivo expansion. After infusion, the persisting cells displayed a CD45RA(+) and CD45RO(-) phenotype. Gene-engineered cells persisted at high levels in the blood of all patients 1 month after treatment, responding patients with higher ex vivo antitumor reactivity than nonresponders. Objective cancer regressions were seen in 30% and 19% of patients who received the human or mouse TCR, respectively. However, patients exhibited destruction of normal melanocytes in the skin, eye, and ear, and sometimes required local steroid administration to treat uveitis and hearing loss. Thus, T cells expressing highly reactive TCRs mediate cancer regression in humans and target rare cognate-antigen-containing cells throughout the body, a finding with important implications for the gene therapy of cancer. This trial was registered at www.ClinicalTrials.gov as NCI-07-C-0174 and NCI-07-C-0175.

Autoimmunity Correlates With Tumor Regression in Patients With Metastatic Melanoma Treated With Anti–Cytotoxic T-Lymphocyte Antigen-4

PURPOSE Previously, we reported our experience treating 14 patients with metastatic melanoma using a fully human antibody to cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4) in conjunction with peptide vaccination. We have now treated 56 patients to evaluate two different dose schedules of anti-CTLA-4 and to explore the relationship between autoimmunity and tumor regression. PATIENTS AND METHODS A total of 56 patients with progressive stage IV melanoma were enrolled onto the study. All had Karnofsky performance status > or = 60% with no prior history of autoimmunity. Twenty-nine patients received 3 mg/kg anti-CTLA-4 every 3 weeks, whereas 27 received 3 mg/kg as their initial dose with subsequent doses reduced to 1 mg/kg every 3 weeks. In both cohorts patients received concomitant vaccination with two modified HLA-A*0201-restricted peptides from the gp100 melanoma-associated antigen, gp100:209-217(210M) and gp100:280-288(288V). RESULTS Two patients achieved a complete response (ongoing at 30 and 31 months, respectively) and five patients achieved a partial response (durations of 4, 6, 25+, 26+, and 34+ months, respectively), for an overall objective response rate of 13%. Tumor regression was seen in lung, liver, brain, lymph nodes, and subcutaneous sites. Of 14 patients with grade 3/4 autoimmune toxicity, five (36%) experienced a clinical response compared with only two responses in the 42 patients (5%) with no autoimmune toxicity (P = .008). There were no significant differences in response rate or toxicity between the two dose schedules. CONCLUSION Administration of anti-CTLA-4 monoclonal antibody plus peptide vaccination can cause durable objective responses, which correlate with the induction of autoimmunity, in patients with metastatic melanoma.

Enterocolitis in Patients With Cancer After Antibody Blockade of Cytotoxic T-Lymphocyte–Associated Antigen 4

PURPOSE Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) is an inhibitory receptor on T cells. Knocking out CTLA4 in mice causes lethal lymphoproliferation, and polymorphisms in human CTLA4 are associated with autoimmune disease. Trials of the anti-CTLA4 antibody ipilimumab (MDX-010) have resulted in durable cancer regression and immune-mediated toxicities. A report on the diagnosis, pathology, treatment, clinical outcome, and significance of the immune-mediated enterocolitis seen with ipilimumab is presented. PATIENTS AND METHODS We treated 198 patients with metastatic melanoma (MM) or renal cell carcinoma (RCC) with ipilimumab. RESULTS The overall objective tumor response rate was 14%. We observed several immune mediated toxicities including dermatitis, enterocolitis, hypophysitis, uveitis, hepatitis, and nephritis. Enterocolitis, defined by grade 3/4 clinical presentation and/or biopsy documentation, was the most common major toxicity (21% of patients). It presented with diarrhea, and biopsies showed both neutrophilic and lymphocytic inflammation. Most patients who developed enterocolitis responded to high-dose systemic corticosteroids. There was no evidence that steroid administration affected tumor responses. Five patients developed perforation or required colectomy. Four other patients with steroid-refractory enterocolitis appeared to respond promptly to tumor necrosis factor alpha blockade with infliximab. Objective tumor response rates in patients with enterocolitis were 36% for MM and 35% for RCC, compared with 11% and 2% in patients without enterocolitis, respectively (P = .0065 for MM and P = .0016 for RCC). CONCLUSION CTLA4 seems to be a significant component of tolerance to tumor and in protection against immune mediated enterocolitis and these phenomena are significantly associated in cancer patients.

Ipilimumab (Anti-CTLA4 Antibody) Causes Regression of Metastatic Renal Cell Cancer Associated With Enteritis and Hypophysitis

The inhibitory receptor CTLA4 has a key role in peripheral tolerance of T cells for both normal and tumor-associated antigens. Murine experiments suggested that blockade of CTLA4 might have antitumor activity and a clinical experience with the blocking antibody ipilimumab in patients with metastatic melanoma did show durable tumor regressions in some patients. Therefore, a phase II study of ipilimumab was conducted in patients with metastatic renal cell cancer with a primary end point of response by Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Two sequential cohorts received either 3 mg/kg followed by 1 mg/kg or all doses at 3 mg/kg every 3 weeks (with no intention of comparing cohort response rates). Major toxicities were enteritis and endocrine deficiencies of presumed autoimmune origin. One of 21 patients receiving the lower dose had a partial response. Five of 40 patients at the higher dose had partial responses (95% confidence interval for cohort response rate 4% to 27%) and responses were seen in patients who had previously not responded to IL-2. Thirty-three percent of patients experienced a grade III or IV immune-mediated toxicity. There was a highly significant association between autoimmune events (AEs) and tumor regression (response rate=30% with AE, 0% without AE). CTLA4 blockade with ipilimumab induces cancer regression in some patients with metastatic clear cell renal cancer, even if they have not responded to other immunotherapies. These regressions are highly associated with other immune-mediated events of presumed autoimmune origin by mechanisms as yet undefined.

Tumor Regression and Autoimmunity in Patients Treated With Cytotoxic T Lymphocyte–Associated Antigen 4 Blockade and Interleukin 2: A Phase I/II Study

BackgroundCytotoxic T lymphocyte–associated antigen (CTLA)-4 can inhibit T-cell responses and is involved in tolerance against self antigens. We previously reported autoimmune manifestations and objective cancer regressions in patients with metastatic melanoma treated with CTLA-4 blockade. The possibility of activating tumor-reactive T cells while removing inhibitory activity with CTLA-4 blockade has stimulated interest in using anti–CTLA-4 antibodies in combination with other cancer immunotherapies to improve clinical outcomes. In this study, we assessed the antitumor activity and autoimmune toxicity of CTLA-4 blockade in combination with an immune-activating stimulus, interleukin (IL)-2, in patients with metastatic melanoma.MethodsThirty-six patients received anti–CTLA-4 antibody every 3 weeks. Three patients per cohort received doses of .1, .3, 1.0, and 2.0 mg/kg. Twenty-four patients received 3.0 mg/kg. All patients received IL-2 therapy (720,000 IU/kg every 8 hours to a maximum of 15 doses).ResultsEight patients (22%) experienced objective tumor responses (three complete and five partial), including metastases in the lungs, lymph nodes, mediastinum, and subcutaneous tissues. Six of the eight patients have ongoing objective responses at 11 to 19 months. Five patients (14%) developed grade III/IV autoimmune toxicities secondary to anti–CTLA-4 administration, including four patients with enterocolitis and one with arthritis and uveitis.ConclusionsThere is not evidence to support a synergistic effect of CTLA-4 blockade plus IL-2 administration, because the 22% objective response rate is that expected from the sum of these two agents administered alone. Durable cancer regressions were seen in patients treated with this combination.

Prognostic factors related to clinical response in patients with metastatic melanoma treated by CTL-associated antigen-4 blockade.

Purpose: CTL-associated antigen 4 (CTLA-4) can inhibit T-cell activation and helps maintain peripheral self-tolerance. Previously, we showed immune-related adverse events (IRAE) and objective, durable clinical responses in patients with metastatic melanoma treated with CTLA-4 blockade. We have now treated 139 patients in two trials and have sufficient follow-up to examine factors associated with clinical response. Experimental Design: A total of 139 patients with metastatic melanoma were treated: 54 patients received ipilimumab in conjunction with peptide vaccinations and 85 patients were treated with intra-patient dose escalation of ipilimumab and randomized to receive peptides in accordance with HLA-A*0201 status. Results: Three patients achieved complete responses (CR; ongoing at 29+, 52+, and 53+ months); an additional 20 patients achieved partial responses (PR) for an overall objective response rate of 17%. The majority of patients (62%, 86 of 139) developed some form of IRAE, which was associated with a greater probability of objective antitumor response (P = 0.0004); all patients with CR had more severe IRAEs. Prior therapy with IFNα-2b was a negative prognostic factor, whereas prior high-dose interleukin-2 did not significantly affect the probability of response. There were no significant differences in the rate of clinical response or development of IRAEs between the two trials. The duration of tumor response was not affected by the use of high-dose steroids for abrogation of treatment-related toxicities (P = 0.23). There were no treatment-related deaths. Conclusion: In patients with metastatic melanoma, ipilimumab can induce durable objective clinical responses, which are related to the induction of IRAEs.

Tumor Progression Can Occur despite the Induction of Very High Levels of Self/Tumor Antigen-Specific CD8+ T Cells in Patients with Melanoma

The identification of many tumor-associated epitopes as nonmutated “self” Ags led to the hypothesis that the induction of large numbers of self/tumor Ag-specific T cells would be prevented because of central and peripheral tolerance. We report in this study on vaccination efforts in 95 HLA-A*0201 patients at high risk for recurrence of malignant melanoma who received prolonged immunization with the “anchor-modified” synthetic peptide, gp100209–217(210M). Vaccination using this altered peptide immunogen was highly effective at inducing large numbers of self/tumor-Ag reactive T cells in virtually every patient tested, with levels as high as 42% of all CD8+ T cells assessed by tetramer analysis. From 1 to 10% of all CD8+ cells were tumor-Ag reactive in 44% of patients and levels >10% were generated in 17% of patients. These studies were substantiated using the ELISPOT assay and a bulk cytokine release assay. Although our data regarding “tumor escape” were inconclusive, some patients had growing tumors that expressed Ag and HLA-A*0201 in the presence of high levels of antitumor T cells. There was no difference in the levels of antitumor Ag-specific T cells in patients who recurred compared with those that remained disease-free. Thus, the mere presence of profoundly expanded numbers of vaccine-induced, self/tumor Ag-specific T cells cannot by themselves be used as a “surrogate marker” for vaccine efficacy. Further, the induction of even high levels of antitumor T cells may be insufficient to alter tumor progression.

Phase 2 trial of single agent ipilimumab (Anti-CTLA-4) for locally advanced or metastatic pancreatic adenocarcinoma

New, effective therapies are needed for pancreatic ductal adenocarcinoma. Ipilimumab can mediate an immunologic tumor regression in other histologies. This phase II trial evaluated the efficacy of Ipilimumab for advanced pancreatic cancer. Subjects were adults with locally advanced or metastatic pancreas adenocarcinoma with measurable disease, good performance status, and minimal comorbidities. Ipilimumab was administered intravenously (3.0 mg/kg every 3 wk; 4 doses/course) for a maximum of 2 courses. Response rate by response evaluation criteria in solid tumors criteria and toxicity were measured. Twenty-seven subjects were enrolled (metastatic disease: 20 and locally advanced: 7) with median age of 55 years (27 to 68 y) and good performance status (26 with Eastern Cooperative Oncology Group performance status =0 to 1). Three subjects experienced ≥ grade 3 immune-mediated adverse events (colitis:1, encephalitis:1, hypohysitis:1). There were no responders by response evaluation criteria in solid tumors criteria but a subject experienced a delayed response after initial progressive disease. In this subject, new metastases after 2 doses of Ipilimumab established progressive disease. But continued administration of the agent per protocol resulted in significant delayed regression of the primary lesion and 20 hepatic metastases. This was reflected in tumor markers normalization, and clinically significant improvement of performance status. Single agent Ipilimumab at 3.0 mg/kg/dose is ineffective for the treatment of advanced pancreas cancer. However, a significant delayed response in one subject of this trial suggests that immunotherapeutic approaches to pancreas cancer deserve further exploration.