Robert Somerville

Cancer Immunotherapy Based on Mutation-Specific CD4+ T Cells in a Patient with Epithelial Cancer

T Cells for Epithelial Tumors Malignant tumors harbor genetic alterations. Recently, adoptive T cell therapies have taken advantage of this: T cells specific for mutations in tumors are infused into patients to generate an antitumor immune response. Although therapeutic benefit has been seen for melanomas, effectiveness against more common epithelial tumors is unclear. Using whole-exome sequencing, Tran et al. (p. 641) identified tumor-infiltrating CD4+ T cells specific for a mutated antigen expressed by a tumor from a patient with metastatic cholangiocarcinoma. Infusion of this patient with an expanded-population, mutation-specific T cell resulted in tumor regression and stabilization of disease. T cells specific for a mutation expressed by tumor cells show antitumor activity in a patient with an epithelial cancer. Limited evidence exists that humans mount a mutation-specific T cell response to epithelial cancers. We used a whole-exomic-sequencing-based approach to demonstrate that tumor-infiltrating lymphocytes (TIL) from a patient with metastatic cholangiocarcinoma contained CD4+ T helper 1 (TH1) cells recognizing a mutation in erbb2 interacting protein (ERBB2IP) expressed by the cancer. After adoptive transfer of TIL containing about 25% mutation-specific polyfunctional TH1 cells, the patient achieved a decrease in target lesions with prolonged stabilization of disease. Upon disease progression, the patient was retreated with a >95% pure population of mutation-reactive TH1 cells and again experienced tumor regression. These results provide evidence that a CD4+ T cell response against a mutated antigen can be harnessed to mediate regression of a metastatic epithelial cancer.

Chemotherapy-Refractory Diffuse Large B-Cell Lymphoma and Indolent B-Cell Malignancies Can Be Effectively Treated With Autologous T Cells Expressing an Anti-CD19 Chimeric Antigen Receptor

PURPOSE T cells can be genetically modified to express an anti-CD19 chimeric antigen receptor (CAR). We assessed the safety and efficacy of administering autologous anti-CD19 CAR T cells to patients with advanced CD19(+) B-cell malignancies. PATIENTS AND METHODS We treated 15 patients with advanced B-cell malignancies. Nine patients had diffuse large B-cell lymphoma (DLBCL), two had indolent lymphomas, and four had chronic lymphocytic leukemia. Patients received a conditioning chemotherapy regimen of cyclophosphamide and fludarabine followed by a single infusion of anti-CD19 CAR T cells. RESULTS Of 15 patients, eight achieved complete remissions (CRs), four achieved partial remissions, one had stable lymphoma, and two were not evaluable for response. CRs were obtained by four of seven evaluable patients with chemotherapy-refractory DLBCL; three of these four CRs are ongoing, with durations ranging from 9 to 22 months. Acute toxicities including fever, hypotension, delirium, and other neurologic toxicities occurred in some patients after infusion of anti-CD19 CAR T cells; these toxicities resolved within 3 weeks after cell infusion. One patient died suddenly as a result of an unknown cause 16 days after cell infusion. CAR T cells were detected in the blood of patients at peak levels, ranging from nine to 777 CAR-positive T cells/μL. CONCLUSION This is the first report to our knowledge of successful treatment of DLBCL with anti-CD19 CAR T cells. These results demonstrate the feasibility and effectiveness of treating chemotherapy-refractory B-cell malignancies with anti-CD19 CAR T cells. The numerous remissions obtained provide strong support for further development of this approach.

Immunogenicity of somatic mutations in human gastrointestinal cancers

Low mutation rate okay for T cells Cancers that tend to have high numbers of mutations, such as melanoma and smoking-induced lung cancer, respond well to immunotherapies, whereas those with fewer mutations, such as pancreatic cancer, do not. Tran et al. searched for tumor mutation–reactive T cells in 10 patients with metastatic gastrointestinal cancers, which have relatively low mutation burdens, and discovered that 9 out of 10 harbored such cells. T cells from one patient recognized a mutation common to many types of cancers. Engineering T cells to express this particular mutation-reactive T cell receptor may extend adoptive cell immunotherapy to a larger pool of patients than previously anticipated. Science, this issue p. 1387 Individuals with cancers that have low mutation frequencies often harbor mutation-reactive T cells. It is unknown whether the human immune system frequently mounts a T cell response against mutations expressed by common epithelial cancers. Using a next-generation sequencing approach combined with high-throughput immunologic screening, we demonstrated that tumor-infiltrating lymphocytes (TILs) from 9 out of 10 patients with metastatic gastrointestinal cancers contained CD4+ and/or CD8+ T cells that recognized one to three neo-epitopes derived from somatic mutations expressed by the patient’s own tumor. There were no immunogenic epitopes shared between these patients. However, we identified in one patient a human leukocyte antigen–C*08:02–restricted T cell receptor from CD8+ TILs that targeted the KRASG12D hotspot driver mutation found in many human cancers. Thus, a high frequency of patients with common gastrointestinal cancers harbor immunogenic mutations that can potentially be exploited for the development of highly personalized immunotherapies.

Allogeneic T Cells That Express an Anti-CD19 Chimeric Antigen Receptor Induce Remissions of B-Cell Malignancies That Progress After Allogeneic Hematopoietic Stem-Cell Transplantation Without Causing Graft-Versus-Host Disease

PURPOSE Progressive malignancy is the leading cause of death after allogeneic hematopoietic stem-cell transplantation (alloHSCT). After alloHSCT, B-cell malignancies often are treated with unmanipulated donor lymphocyte infusions (DLIs) from the transplant donor. DLIs frequently are not effective at eradicating malignancy and often cause graft-versus-host disease, a potentially lethal immune response against normal recipient tissues. METHODS We conducted a clinical trial of allogeneic T cells genetically engineered to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. Patients with B-cell malignancies that had progressed after alloHSCT received a single infusion of CAR T cells. No chemotherapy or other therapies were administered. The T cells were obtained from each recipients alloHSCT donor. RESULTS Eight of 20 treated patients obtained remission, which included six complete remissions (CRs) and two partial remissions. The response rate was highest for acute lymphoblastic leukemia, with four of five patients obtaining minimal residual disease-negative CR. Responses also occurred in chronic lymphocytic leukemia and lymphoma. The longest ongoing CR was more than 30 months in a patient with chronic lymphocytic leukemia. New-onset acute graft-versus-host disease after CAR T-cell infusion developed in none of the patients. Toxicities included fever, tachycardia, and hypotension. Peak blood CAR T-cell levels were higher in patients who obtained remissions than in those who did not. Programmed cell death protein-1 expression was significantly elevated on CAR T cells after infusion. Presence of blood B cells before CAR T-cell infusion was associated with higher postinfusion CAR T-cell levels. CONCLUSION Allogeneic anti-CD19 CAR T cells can effectively treat B-cell malignancies that progress after alloHSCT. The findings point toward a future when antigen-specific T-cell therapies will play a central role in alloHSCT.

Randomized Selection Design Trial Evaluating CD8+-Enriched Versus Unselected Tumor-Infiltrating Lymphocytes for Adoptive Cell Therapy for Patients With Melanoma

PURPOSE Adoptive cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) and high-dose interleukin-2 (IL-2) administered to lymphodepleted patients with melanoma can cause durable tumor regressions. The optimal TIL product for ACT is unknown. PATIENTS AND METHODS Patients with metastatic melanoma were prospectively assigned to receive unselected young TILs versus CD8(+)-enriched TILs. All patients received lymphodepleting chemotherapy and high-dose IL-2 therapy and were assessed for response, toxicity, survival, and immunologic end points. RESULTS Thirty-four patients received unselected young TILs with a median of 8.0% CD4(+) lymphocytes, and 35 patients received CD8(+)-enriched TILs with a median of 0.3% CD4(+) lymphocytes. One month after TIL infusion, patients who received CD8(+)-enriched TILs had significantly fewer CD4(+) peripheral blood lymphocytes (P = .01). Twelve patients responded to therapy with unselected young TILs (according to Response Evaluation Criteria in Solid Tumors [RECIST]), and seven patients responded to CD8(+)-enriched TILs (35% v 20%; not significant). Retrospective studies showed a significant association between response to treatment and interferon gamma secretion by the infused TILs in response to autologous tumor (P = .04), and in the subgroup of patients who received TILs from subcutaneous tumors, eight of 15 patients receiving unselected young TILs responded but none of eight patients receiving CD8(+)-enriched TILs responded. CONCLUSION A randomized selection design trial was feasible for improving individualized TIL therapy. Since the evidence indicates that CD8(+)-enriched TILs are not more potent therapeutically and they are more laborious to prepare, future studies should focus on unselected young TILs.

Randomized, Prospective Evaluation Comparing Intensity of Lymphodepletion Before Adoptive Transfer of Tumor-Infiltrating Lymphocytes for Patients With Metastatic Melanoma

PURPOSE Adoptive cell transfer, the infusion of large numbers of activated autologous lymphocytes, can mediate objective tumor regression in a majority of patients with metastatic melanoma (52 of 93; 56%). Addition and intensification of total body irradiation (TBI) to the preparative lymphodepleting chemotherapy regimen in sequential trials improved objective partial and complete response (CR) rates. Here, we evaluated the importance of adding TBI to the adoptive transfer of tumor-infiltrating lymphocytes (TIL) in a randomized fashion. PATIENTS AND METHODS A total of 101 patients with metastatic melanoma, including 76 patients with M1c disease, were randomly assigned to receive nonmyeloablative chemotherapy with or without 1,200 cGy TBI before transfer of tumor-infiltrating lymphcytes. Primary end points were CR rate (as defined by Response Evaluation Criteria in Solid Tumors v1.0) and overall survival (OS). Clinical and laboratory data were analyzed for correlates of response. RESULTS CR rates were 24% in both groups (12 of 50 v 12 of 51), and OS was also similar (median OS, 38.2 v 36.6 months; hazard ratio, 1.11; 95% CI, 0.65 to 1.91; P = .71). Thrombotic microangiopathy was an adverse event unique to the TBI arm and occurred in 13 of 48 treated patients. With a median potential follow-up of 40.9 months, only one of 24 patients who achieved a CR recurred. CONCLUSION Adoptive cell transfer can mediate durable complete regressions in 24% of patients with metastatic melanoma, with median survival > 3 years. Results were similar using chemotherapy preparative regimens with or without addition of TBI.

Lymphoma Remissions Caused by Anti-CD19 Chimeric Antigen Receptor T Cells Are Associated With High Serum Interleukin-15 Levels

Purpose T cells genetically modified to express chimeric antigen receptors (CARs) targeting CD19 (CAR-19) have potent activity against acute lymphoblastic leukemia, but fewer results supporting treatment of lymphoma with CAR-19 T cells have been published. Patients with lymphoma that is chemotherapy refractory or relapsed after autologous stem-cell transplantation have a grim prognosis, and new treatments for these patients are clearly needed. Chemotherapy administered before adoptive T-cell transfer has been shown to enhance the antimalignancy activity of adoptively transferred T cells. Patients and Methods We treated 22 patients with advanced-stage lymphoma in a clinical trial of CAR-19 T cells preceded by low-dose chemotherapy. Nineteen patients had diffuse large B-cell lymphoma, two patients had follicular lymphoma, and one patient had mantle cell lymphoma. Patients received a single dose of CAR-19 T cells 2 days after a low-dose chemotherapy conditioning regimen of cyclophosphamide plus fludarabine. Results The overall remission rate was 73% with 55% complete remissions and 18% partial remissions. Eleven of 12 complete remissions are ongoing. Fifty-five percent of patients had grade 3 or 4 neurologic toxicities that completely resolved. The low-dose chemotherapy conditioning regimen depleted blood lymphocytes and increased serum interleukin-15 (IL-15). Patients who achieved a remission had a median peak blood CAR+ cell level of 98/μL and those who did not achieve a remission had a median peak blood CAR+ cell level of 15/μL ( P = .027). High serum IL-15 levels were associated with high peak blood CAR+ cell levels ( P = .001) and remissions of lymphoma ( P < .001). Conclusion CAR-19 T cells preceded by low-dose chemotherapy induced remission of advanced-stage lymphoma, and high serum IL-15 levels were associated with the effectiveness of this treatment regimen. CAR-19 T cells will likely become an important treatment for patients with relapsed lymphoma.

Simplified method of the growth of human tumor infiltrating lymphocytes in gas-permeable flasks to numbers needed for patient treatment.

Adoptive cell therapy of metastatic melanoma with autologous tumor infiltrating lymphocytes (TIL) is clinically effective, but TIL production can be challenging. Here we describe a simplified method for initial TIL culture and rapid expansion in gas-permeable flasks. TIL were initially cultured from tumor digests and fragments in 40 mL capacity flasks with a 10 cm2 gas-permeable silicone bottom, G-Rex10. A TIL rapid expansion protocol (REP) was developed using 500 mL capacity flasks with a 100 cm2 gas-permeable silicone bottom, G-Rex100. TIL growth was successfully initiated in G-Rex10 flasks from tumor digests from 13 of 14 patients and from tumor fragments in all 11 tumor samples tested. TIL could then be expanded to 8–10×109 cells in a 2-step REP that began by seeding 5×106 TIL into a G-Rex100 flask, followed by expansion at day 7 into 3 G-Rex100 flasks. To obtain the 30–60×109 cells used for patient treatment, we seeded 6 G-Rex100 flasks with 5×106 cells and expanded into 18 G-Rex100 flasks. Large-scale TIL REP in gas-permeable flasks requires approximately 9–10 L of media, about 3–4 times less than other methods. In conclusion, TIL initiation and REP in gas-permeable G-Rex flasks require fewer total vessels, less media, less incubator space, and less labor than initiation and REP in 24-well plates, tissue culture flasks, and bags. TIL culture in G-Rex flasks will facilitate the production of TIL at the numbers required for patient treatment at most cell processing laboratories.

Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the WAVE® bioreactor.

BackgroundTo simplify clinical scale lymphocyte expansions, we investigated the use of the WAVE®, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes.MethodsWe have developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant numbers of cells for our adoptive cell transfer clinical protocols.ResultsTIL and genetically modified PBL were rapidly expanded to clinically relevant scales in both static bags and the WAVE bioreactor. Both bioreactors produced comparable numbers of cells; however the cultures generated in the WAVE bioreactor had a higher percentage of CD4+ cells and had a less activated phenotype.ConclusionsThe WAVE bioreactor simplifies the process of rapidly expanding tumor reactive lymphocytes under GMP conditions, and provides an alternate approach to cell generation for ACT protocols.

Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two-stage, single-arm, phase 2 study

BACKGROUND Uveal melanoma is a rare tumour with no established treatments once metastases develop. Although a variety of immune-based therapies have shown efficacy in metastatic cutaneous melanoma, their use in ocular variants has been disappointing. Recently, adoptive T-cell therapy has shown salvage responses in multiple refractory solid tumours. Thus, we sought to determine if adoptive transfer of autologous tumour-infiltrating lymphocytes (TILs) could mediate regression of metastatic uveal melanoma. METHODS In this ongoing single-centre, two-stage, phase 2, single-arm trial, patients (aged ≥16 years) with histologically confirmed metastatic ocular melanoma were enrolled. Key eligibility criteria were an Eastern Cooperative Oncology Group performance status of 0 or 1, progressive metastatic disease, and adequate haematological, renal, and hepatic function. Metastasectomies were done to procure tumour tissue to generate autologous TIL cultures, which then underwent large scale ex-vivo expansion. Patients were treated with lymphodepleting conditioning chemotherapy (intravenous cyclophosphamide [60 mg/kg] daily for 2 days followed by fludarabine [25 mg/m2] daily for 5 days, followed by a single intravenous infusion of autologous TILs and high-dose interleukin-2 [720 000 IU/kg] every 8 h). The primary endpoint was objective tumour response in evaluable patients per protocol using Response to Evaluation Criteria in Solid Tumors, version 1.0. An interim analysis of this trial is reported here. The trial is registered at ClinicalTrials.gov, number NCT01814046. FINDINGS From the completed first stage and ongoing expansion stage of this trial, a total of 21 consecutive patients with metastatic uveal melanoma were enrolled between June 7, 2013, and Sept 9, 2016, and received TIL therapy. Seven (35%, 95% CI 16-59) of 20 evaluable patients had objective tumour regression. Among the responders, six patients achieved a partial response, two of which are ongoing and have not reached maximum response. One patient achieved complete response of numerous hepatic metastases, currently ongoing at 21 months post therapy. Three of the responders were refractory to previous immune checkpoint blockade. Common grade 3 or worse toxic effects were related to the lymphodepleting chemotherapy regimen and included lymphopenia, neutropenia, and thrombocytopenia (21 [100%] patients for each toxicity); anaemia (14 [67%] patients); and infection (six [29%] patients). There was one treatment-related death secondary to sepsis-induced multiorgan failure. INTERPRETATION To our knowledge, this is the first report describing adoptive transfer of autologous TILs to mediate objective tumour regression in patients with metastatic uveal melanoma. These initial results challenge the belief that metastatic uveal melanoma is immunotherapy resistant and support the further investigation of immune-based therapies for this cancer. Refinement of this T-cell therapy is crucial to improve the frequency of clinical responses and the general applicability of this treatment modality. FUNDING Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.