Jeremy L. Davis

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.

T Cells Targeting Carcinoembryonic Antigen Can Mediate Regression of Metastatic Colorectal Cancer but Induce Severe Transient Colitis

Autologous T lymphocytes genetically engineered to express a murine T cell receptor (TCR) against human carcinoembryonic antigen (CEA) were administered to three patients with metastatic colorectal cancer refractory to standard treatments. All patients experienced profound decreases in serum CEA levels (74-99%), and one patient had an objective regression of cancer metastatic to the lung and liver. However, a severe transient inflammatory colitis that represented a dose limiting toxicity was induced in all three patients. This report represents the first example of objective regression of metastatic colorectal cancer mediated by adoptive T cell transfer and illustrates the successful use of a TCR, raised in human leukocyte antigen (HLA) transgenic mice, against a human tumor associated antigen. It also emphasizes the destructive power of small numbers of highly avid T cells and the limitations of using CEA as a target for cancer immunotherapy.Autologous T lymphocytes genetically engineered to express a murine T cell receptor (TCR) against human carcinoembryonic antigen (CEA) were administered to three patients with metastatic colorectal cancer refractory to standard treatments. All patients experienced profound decreases in serum CEA levels (74-99%), and one patient had an objective regression of cancer metastatic to the lung and liver. However, a severe transient inflammatory colitis that represented a dose limiting toxicity was induced in all three patients. This report represents the first example of objective regression of metastatic colorectal cancer mediated by adoptive T cell transfer and illustrates the successful use of a TCR, raised in human leukocyte antigen (HLA) transgenic mice, against a human tumor associated antigen. It also emphasizes the destructive power of small numbers of highly avid T cells and the limitations of using CEA as a target for cancer immunotherapy.

Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.

No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.

Tremelimumab in combination with ablation in patients with advanced hepatocellular carcinoma

BACKGROUND & AIMS Tremelimumab is a fully human monoclonal antibody that binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on the surface of activated T lymphocytes. Ablative therapies induce a peripheral immune response which may enhance the effect of anti-CTLA4 treatment in patients with advanced hepatocellular carcinoma (HCC). This study aimed to demonstrate whether tremelimumab could be combined safely and feasibly with ablation. METHODS Thirty-two patients with HCC were enrolled: male:female: 28:4; median age: 62 (range 36-76). Patients were given tremelimumab at two dose levels (3.5 and 10mg/kg i.v.) every 4weeks for 6 doses, followed by 3-monthly infusions until off-treatment criteria were met. On day 36, patients underwent subtotal radiofrequency ablation or chemoablation. Staging was performed by contrast-enhanced CT or MRI scan every 8weeks. RESULTS No dose-limiting toxicities were encountered. The most common toxicity was pruritus. Of the 19 evaluable patients, five (26.3%; 95% CI: 9.1-51.2%) achieved a confirmed partial response. Twelve of 14 patients with quantifiable HCV experienced a marked reduction in viral load. Six-week tumor biopsies showed a clear increase in CD8+ T cells in patients showing a clinical benefit only. Six and 12-month probabilities of tumor progression free survival for this refractory HCC population were 57.1% and 33.1% respectively, with median time to tumor progression of 7.4months (95% CI 4.7 to 19.4months). Median overall survival was 12.3months (95% CI 9.3 to 15.4months). CONCLUSIONS Tremelimumab in combination with tumor ablation is a potential new treatment for patients with advanced HCC, and leads to the accumulation of intratumoral CD8+ T cells. Positive clinical activity was seen, with a possible surrogate reduction in HCV viral load. LAY SUMMARY Studies have shown that the killing of tumors by direct methods (known as ablation) can result in the immune system being activated or switched on. The immune system could potentially also recognize and kill the cancer that is left behind. There are new drugs available known as immune checkpoint inhibitors which could enhance this effect. Here, we test one of these drugs (tremelimumab) together with ablation. CLINICAL TRIAL NUMBER ClinicalTrials.gov: NCT01853618.

Expression profiling of TCR-engineered T cells demonstrates overexpression of multiple inhibitory receptors in persisting lymphocytes.

Despite significant progress in the development of adoptive cell-transfer therapies (ACTs) using gene-engineered T cells, little is known about the fate of cells following infusion. To address that, we performed a comparative analysis of gene expression between T-cell receptor-engineered lymphocytes persisting in the circulation 1 month after administration and the product that was infused. We observed that 156 genes related to immune function were differentially expressed, including underexpression of stimulators of lymphocyte function and overexpression of inhibitory genes in postinfusion cells. Of genes overexpressed postinfusion, the product of programmed cell death 1 (PDCD1), coinhibitory receptor PD-1, was expressed at a higher percentage in postinfusion lymphocytes than in the infusion product. This was associated with a higher sensitivity to inhibition of cytokine production by interaction with its ligand PD-L1. Coinhibitory receptor CD160 was also overexpressed in persisting cells, and its expression was associated with decreased reactivity, which surprisingly was found to be ligand-independent. These results contribute to a deeper understanding of the properties of transgenic lymphocytes used to treat human malignancies and may provide a rationale for the development of combination therapies as a method to improve ACT.

Prospective randomized trial evaluating mandatory second look surgery with HIPEC and CRS vs. standard of care in patients at high risk of developing colorectal peritoneal metastases

BackgroundThe standard of care for colorectal peritoneal carcinomatosis is evolving from chemotherapy to cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) for patients with disease limited to the peritoneum. Peritoneal carcinomatosis from colorectal cancer treated with chemotherapy alone results in median survival of 5 to 13 months, whereas CRS with HIPEC for early peritoneal carcinomatosis from colorectal cancer resulted in median survival of 48-63 months and 5 year survival of 51%.Completeness of cytoreduction and limited disease are associated with longer survival, yet early peritoneal carcinomatosis is undetectable by conventional imaging. Exploratory laparotomy can successfully identify early disease, but this approach can only be justified in patients with high risk of peritoneal carcinomatosis. Historical data indicates that patients presenting with synchronous peritoneal carcinomatosis, ovarian metastases, perforated primary tumor, and emergency presentation with bleeding or obstructing lesions are at high risk of peritoneal carcinomatosis. Approximately 55% of these patient populations will develop peritoneal carcinomatosis. We hypothesize that performing a mandatory second look laparotomy with CRS and HIPEC for patients who are at high risk for developing peritoneal carcinomatosis from colorectal cancer will lead to improved survival as compared to patients who receive standard of care with routine surveillance.Methods/DesignThis study is a prospective randomized trial designed to answer the question whether mandatory second look surgery with CRS and HIPEC will prolong overall survival compared to the standard of care in patients who are at high risk for developing peritoneal carcinomatosis from colorectal cancer (CRC). Patients with CRC at high risk for developing peritoneal carcinomatosis who underwent curative surgery and subsequently received standard of care adjuvant chemotherapy will be evaluated. The patients who remain without evidence of disease by imaging, physical examination, and tumor markers for 12 months after the primary operation will be randomized to mandatory second look surgery or standard-of-care surveillance. At laparotomy, CRS and HIPEC will be performed with intraperitoneal oxaliplatin with concurrent systemic 5-fluorouracil and leucovorin. Up to 100 patients will be enrolled to allow for 35 evaluable patients in each arm; accrual is expected to last 5 years.Trial RegistrationClinicalTrials.gov ID: NCT01095523

Identification and characterization of a tumor infiltrating CD56+/CD16- NK cell subset with specificity for pancreatic and prostate cancer cell lines

In a recent clinical trial, a patient exhibited regression of several pancreatic cancer metastases following the administration of the immune modulator Ipilimumab (anti-CTLA-4 antibody). We sought to characterize the immune cells responsible for this regression. Tumor infiltrating lymphocytes (TIL-2742) and an autologous tumor line (TC-2742) were expanded from a regressing metastatic lesion excised from this patient. Natural killer (NK) cells predominated in the TIL (92% CD56+) with few T cells (12% CD3+). A majority (88%) of the NK cells were CD56brightCD16−. TIL-2742 secreted IFN-γ and GM-CSF following co-culture with TC-2742 and major histocompatibility complex mismatched pancreatic tumor lines. After sorting TIL-2742, the purified CD56+CD16−CD3− subset showed reactivity similar to TIL-2742 while the CD56−CD16−CD3+ cells exhibited no tumor recognition. In co-culture assays, TIL-2742 and the NK subset expressed high reactivity to several pancreatic and prostate cancer cell lines and could lyse the autologous tumor as well as pancreas and prostate cancer lines. Reactivity was partially abrogated by blockade of TRAIL. We thus identified a unique subset of NK cells (CD56brightCD16dim) isolated from a regressing metastatic pancreatic cancer in a patient responding to Ipilimumab. This represents the first report of CD56+CD16− NK cells with apparent specificity for pancreatic and prostate cancer cell lines and associated with tumor regression following the treatment with an immune modulating agent.

Liver Directed Therapy for Renal Cell Carcinoma

Background: Metastatic renal cell carcinoma (RCC) to the liver portrays a poor prognosis and liver directed therapy remains controversial. We aimed to determine potential selection criteria for patients who might benefit from this strategy. Materials and Methods: We evaluated 247 consecutive patients with RCC metastatic to the liver from a prospectively maintained database. Results: Eighteen patients received liver directed therapy (18/247, 7%). Ten patients underwent liver resection (10/247, 4%) and eight patients underwent radiofrequency ablation (RFA, 8/247, 3%). All were rendered free of disease in the liver. Five had synchronous liver disease and underwent synchronous resections with their primary. Mortality was 0%. Fourteen had single (surgery 7, RFA 7) and four (surgery 3, RFA 1) had multiple liver lesions, respectively. Median size of lesions was 5cm (0.5 - 10cm) and 2.5cm (1 - 6cm) in the surgery and RFA groups, respectively. Median DFI was 10 months, and no difference was observed in those with a longer vs. shorter than median DFI (p = 0.95); liver specific progression free survival for the surgery and RFA groups were 4 and 6 months, respectively (p= 0.93). 1, 3 and 5-year actuarial survivals for the whole group were 89%, 40%, 27%. Median survival for the surgery group was 24 (3 to 254+) months, and for the RFA group 15.6 (7-56+) months (p = 0.56). Metachronous liver disease was associated with prolonged survival (p = 0.02). Conclusions: Liver directed therapy for RCC is safe. For highly selected patients with metachronous liver RCC metastases, liver directed therapy should be considered in a multidisciplinary manner.

Liver Resection for Metastatic Melanoma with Postoperative Tumor-Infiltrating Lymphocyte Therapy

BackgroundPatients with metastatic melanoma to the liver (MML) have a median survival of 4 to 6 months. This study evaluated patients who underwent liver resection with intent to receive postoperative tumor-infiltrating lymphocyte (TIL) therapy.MethodsRetrospective analysis of a prospective database identified patients with MML who underwent liver resection from 1980 to 2008.ResultsA total of 539 patients had MML, and 39% (204 of 539) had tumor collected for TIL. A total of 17% (35 of 204) underwent liver resection for TIL. The 3-year overall survival was 53%. Lack of extrahepatic disease (P = .026), negative margin (P = .056), and single hepatic metastasis (P = .04) predicted survival after univariate analysis. Only lack of extrahepatic disease remained a significant predictor of survival after multivariate analysis (P = .043). A total of 31% (11 of 35) underwent complete resection without TIL, and 69% (24 of 35) underwent resection with synchronous intrahepatic and extrahepatic disease with intent to receive TIL. For 9 of 11 patients (2 of 11 excluded for gene therapy), 3-year survival was 80%. A total of 4 (44%) of 9 experienced recurrence, with a median disease-free survival of 1.2 years. For 24 patients (69%) with residual disease, 3-year survival was 51% (2 of 24 excluded for gene therapy). A total of 63% (15 of 24) received postoperative TIL (3-year survival 65%), and 29% (7 of 24) did not. A total of 40% (6 of 15) had disease that partially responded to TIL; the disease of 67% (4 of 6) had not progressed at median follow-up of 55 months (range, 42–197+ months). The seven patients who did not receive TIL had a median survival of 4.6 months.ConclusionsResection of MML with TIL should be considered because it can result in prolonged survival in a highly selected group of patients.

Regional Chemotherapy in Locally Advanced Pancreatic Cancer: RECLAP Trial

BackgroundPancreatic cancer is the fourth leading cause of cancer death in the United States. Surgery offers the only chance for cure. However, less than twenty percent of patients are considered operative candidates at the time of diagnosis. A common reason for being classified as unresectable is advanced loco-regional disease.A review of the literature indicates that almost nine hundred patients with pancreatic cancer have received regional chemotherapy in the last 15 years. Phase I studies have shown regional administration of chemotherapy to be safe. The average reported response rate was approximately 26%. The average 1-year survival was 39%, with an average median survival of 9 months. Of the patients that experienced a radiographic response to therapy, 78 (78/277, 28%) patients underwent exploratory surgery following regional chemotherapy administration; thirty-two (41%) of those patients were amenable to pancreatectomy. None of the studies performed analyses to identify factors predicting response to regional chemotherapy.Progressive surgical techniques combined with current neoadjuvant chemoradiotherapy strategies have already yielded emerging support for a multimodality approach to treatment of advanced pancreatic cancer.Intravenous gemcitabine is the current standard treatment of pancreatic cancer. However, >90% of the drug is secreted unchanged affecting toxicity but not the cancer per se. Gemcitabine is converted inside the cell into its active drug form in a rate limiting reaction. We hypothesize that neoadjuvant regional chemotherapy with continuous infusion of gemcitabine will be well tolerated and may improve resectability rates in cases of locally advanced pancreatic cancer.DesignThis is a phase I study designed to evaluate the feasibility and toxicity of super-selective intra-arterial administration of gemcitabine in patients with locally advanced, unresectable pancreatic adenocarcinoma. Patients considered unresectable due to locally advanced pancreatic cancer will receive super-selective arterial infusion of gemcitabine over 24 hours via subcutaneous indwelling port. Three to six patients will be enrolled per dose cohort, with seven cohorts, plus an additional six patients at the maximum tolerated dose; accrual is expected to last 36 months. Secondary objectives will include the determination of progression free and overall survival, as well as the conversion rate from unresectable to potentially resectable pancreatic cancer.Trial RegistrationClinicalTrials.gov ID: NCT01294358