A phase I study of an adenoviral vector delivering a MUC1/CD40-ligand fusion protein in patients with advanced adenocarcinoma.

Cancer vaccines as immunotherapy for solid tumours are currently in development with promising results. We report a phase 1 study of Ad-sig-hMUC1/ecdCD40L (NCT02140996), an adenoviral-vector vaccine encoding the tumour-associated antigen MUC1 linked to CD40 ligand, in patients with advanced adenocarcinoma. The primary objective of this study is safety and tolerability. We also study the immunome in vaccinated patients as a secondary outcome. This trial, while not designed to determine clinical efficacy, reports an exploratory endpoint of overall response rate. The study meets its pre-specified primary endpoint demonstrating safety and tolerability in a cohort of 21 patients with advanced adenocarcinomas (breast, lung and ovary). The maximal dose of the vaccine is 1 ×1011 viral particles, with no dose limiting toxicities. All drug related adverse events are of low grades, most commonly injection site reactions in 15 (71%) patients. Using exploratory high-dimensional analyses, we find both quantitative and relational changes in the cancer immunome after vaccination. Our data highlights the utility of high-dimensional analyses in understanding and predicting effective immunotherapy, underscoring the importance of immune competency in cancer prognosis.

In-situ scalable manufacturing of Epstein-Barr virus-specific T-cells using bioreactor with an expandable culture area (BECA).

The ex-vivo expansion of antigen-specific T-cells for adoptive T-cell immunotherapy requires active interaction between T-cells and antigen-presenting cells therefore culture density and environment become important variables to control. Maintenance of culture density in a static environment is traditionally performed by the expansion of the culture area through splitting of culture from a single vessel into multiple vessels-a highly laborious process. This study aims to validate the use and efficacy of a novel bioreactor, bioreactor with an expandable culture area-dual chamber (BECA-D), that was designed and developed with a cell chamber with expandable culture area (12-108 cm2) and a separate media chamber to allow for in-situ scaling of culture with maintenance of optimum culture density and improved nutrient and gas exchange while minimizing disturbance to the culture. The performance of BECA-D in the culture of Epstein-Barr virus-specific T-cells (EBVSTs) was compared to the 24-well plate. BECA-D had 0.9-9.7 times the average culture yield of the 24-well plates across 5 donor sets. BECA-D was able to maintain the culture environment with relatively stable glucose and lactate levels as the culture expanded. This study concludes that BECA-D can support the culture of ex-vivo EBVSTs with lower manufacturing labour and time requirements compared to the use of the 24-well plate. BECA-D and its adaptation into a closed system with an automated platform (currently being developed) provides cell therapy manufacturers and developers with a closed scale-out solution to producing adoptive cell therapy for clinical use.

TCR repertoire characteristics predict clinical response to adoptive CTL therapy against nasopharyngeal carcinoma.

The past decade has witnessed the gradual and steady progress of adoptive T cell therapy in treating various types of cancer. In combination with gemcitabine and carboplatin chemotherapy, we previously conducted a clinical trial, NCT00690872, to treat Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) patients with autologous EBV-expanded cytotoxic T lymphocytes (CTLs). While achieving a 2-year overall survival rate of 62.9%, this trial failed to induce an anti-tumor response in a sizable fraction of patients. Thus, the identification of benchmarks capable of evaluating CTL products and predicting clinical immunotherapeutic efficacy remains an urgent need. We conducted T cell receptor (TCR) repertoire sequencing to assess EBV-expanded infusion-ready CTL products. To depict the overall repertoire landscape, we evaluated the individual repertoire diversity by Shannon entropy, and, compared the inter-patient CDR3 similarity to estimate T cells expanded by common antigens. With a recently developed bioinformatics algorithm, termed Motif Analysis, we made a machine-learning prediction of structural regions within the CDR3 of TCRß that associate with CTL therapy prognosis. We found that long term survivors, defined as patients surviving longer than two years, had a higher CTL repertoire diversity with reduced inter-patient similarity. Furthermore, TCR Motif Analysis identified 11 structural motifs distinguishing long term survivors from short term survivors. Specifically, two motifs with a high area under the curve (AUC) values were identified as potential predictive benchmarks for efficacious CTL production. Together, these results reveal that the presence of diverse TCR sequences containing a common core motif set is associated with a favorable response to CTL immunotherapy against EBV-positive NPC.

Monocytic Myeloid-Derived Suppressor Cells Underpin Resistance to Adoptive T Cell Therapy in Nasopharyngeal Carcinoma.

Advanced, late-stage Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) is incurable, and its treatment remains a clinical and therapeutic challenge. Results from a phase II clinical trial in advanced NPC patients employing a combined chemotherapy and EBV-specific T cell (EBVST) immunotherapy regimen showed a response rate of 71.4%. Longitudinal analysis of patient samples showed that an increase in EBV DNA plasma concentrations and the peripheral monocyte-to-lymphocyte ratio negatively correlated with overall survival. These parameters were combined into a multivariate analysis to stratify patients according to risk of death. Immunophenotyping at serial time points showed that low-risk individuals displayed significantly decreased amounts of monocytic myeloid-derived suppressor cells postchemotherapy, which subsequently influenced successful cytotoxic T-lymphocyte (CTL) immunotherapy. Examination of the low-risk group, 2 weeks post-EBVST infusion, showed that individuals with a greater overall survival possessed an increased frequency of CD8 central and effector memory T cells, together with higher levels of plasma interferon (IFN)-γ, and cytotoxic lymphocyte-associated transcripts. These results highlight the importance of the rational selection of chemotherapeutic agents and consideration of their impact on both systemic immune responses and downstream cellular immunotherapy outcomes.

A Perspective on Cell Therapy and Cancer Vaccine in Biliary Tract Cancers (BTCs).

Biliary tract cancer (BTC) is a rare, but aggressive, disease that comprises of gallbladder carcinoma, intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma, with heterogeneous molecular profiles. Advanced disease has limited therapeutic options beyond first-line platinum-based chemotherapy. Immunotherapy has emerged as a viable option for many cancers with a similar unmet need. Therefore, we reviewed current understanding of the tumor immune microenvironment and recent advances in cellular immunotherapy and therapeutic cancer vaccines against BTC. We illustrated the efficacy of dendritic cell vaccination in one patient with advanced, chemorefractory, melanoma-associated antigen (MAGE)-positive gallbladder carcinoma, who was given multiple injections of an allogenic MAGE antigen-positive melanoma cell lysate (MCL)-based autologous dendritic cell vaccine combined with sequential anti-angiogenic therapy. This resulted in good radiological and tumor marker response and an overall survival of 3 years from diagnosis. We postulate the potential synergism of adding anti-angiogenic therapy, such as bevacizumab, to immunotherapy in BTC, as a rational scientific principle to positively modulate the tumor microenvironment to augment antitumor immunity.

Tailoring precision immunotherapy: coming to a clinic soon?

Cell-based and antibody-based cancer immunotherapies have been widely tested across increasing numbers of cancers with an unprecedented number of successful practice-changing immunotherapy clinical trials, achieving significant survival outcomes and, characteristically, some very long-term survivors. Still, a sizeable proportion of patients, especially with solid tumours, do not benefit from immunotherapy. Here, we summarise key literature on immunotherapy biomarkers and resistance mechanisms and discuss potential strategies to overcome such resistance to improve patient outcomes. The ever-expanding understanding of the tumour-immune interaction and the tumour microenvironment allows a real opportunity to identify predictive biomarkers and tailor immune-based therapies, including designing rational combination drugs to enhance clinical outcomes, and to identify patients most likely to benefit from immunotherapy. Where there has never been a precision chemotherapy clinic in the last 70 years since its inception, even with no shortage of trying, the hope and evolution of a functional precision immunotherapy cancer clinic is a much more likely reality.

Cancer Immunotherapy - The Target is Precisely on The Cancer and Also Not.

In recent years, the impressive number of cancer immunotherapy drugs approved has been unprecedented-building on over a century of understanding on how the immune system combats cancer, and how cancer evades it. Leading the charge are the immune checkpoint inhibitor monoclonal antibodies, and adoptive cell therapy with chimeric- antigen-receptor (CAR)-T cell therapy. These breakthrough therapies have led to improved survival in patients with many advanced cancers. Some of the clinical outcomes have been striking, and may even be potentially curative in some terminal cancer patients. While immune checkpoint inhibitors work by blocking regulatory immune checkpoint signals between cancer and the immune cells to awaken an effective anticancer immunity, CAR-T cell therapy targets specific molecules on cancer cells. Tumour antigens as cancer targets take many forms and may not necessarily be proteins related to known functional cellular mechanisms. The convergence of cutting edge omics, bioinformatics, protein synthesis, immunobiology and immunotherapy have led to novel, potentially highly effective cancer targeting against neoantigens, hence reviving the quest for anticancer vaccines. Early clinical trials of neoantigen vaccines have provided proof-of-principle efficacy, especially in melanoma patients. Combinations of immunotherapies through rational design are underway aiming to further improve clinical outcomes. Moving forward, cancer immunotherapy will gain even more momentum from the discovery of more cancer targets-both on the cancer itself and in the tumour microenvironment as well as the identification of biomarkers of treatment resistance and efficacy.

Coriolus versicolor (Yunzhi) Use as Therapy in Advanced Hepatocellular Carcinoma Patients with Poor Liver Function or Who Are Unfit for Standard Therapy.

BACKGROUND: The majority of patients with hepatocellular carcinoma (HCC) are inoperable and results with conventional chemotherapy are dismal. Many end up with no treatment options and resort to alternative medicine. The authors report the use of Coriolus versicolor (CV) in advanced HCC patients with poor liver function or who were unfit to receive standard therapy. METHODS: Fifteen eligible cases were randomized 2:1 to either CV or placebo. The primary endpoint was the median time to progression (TTP) between both arms. Secondary endpoints include evaluating response rates, toxicity, quality of life (QOL), progression-free survival (PFS), and overall survival (OS). Further correlative studies were performed looking at the effect of CV on the immune system. RESULTS: The median treatment duration was 1.5 cycles and 3 cycles on the placebo and CV arm, respectively. Median TTP was 2.5 (1.4-5.3) months compared to 4.2 (0.4-4.2) months in the CV and placebo arm, respectively, hazard ratio (HR) 0.70 (0.16-3.05 p = 0.634). Median PFS was 2.5 (1.4-5.3) months in the CV and 1.1 (0.4-4.2) months in the placebo arm, HR 0.42 (0.13-1.34, p = 0.144). Median OS was 6.5 (3.3-24.1) and 2.2 (0.8-23.3) months, respectively, HR 0.35 (0.10-1.25, p = 0.105). Social and emotional functioning scores were higher in the CV group compared to placebo group on treatment. CV subjects had less appetite loss and pain symptoms compared to placebo subjects during treatment. CONCLUSIONS: There was no difference in TTP with use of CV compared to placebo. CV subjects generally had better QOL on treatment compared to placebo subjects. The utility of this supplement in patients whose primary treatment goal is palliation should be further explored.

Longitudinal metabolic imaging of hepatocellular carcinoma in transgenic mouse models identifies acylcarnitine as a potential biomarker for early detection.

The cumulative effects of hepatic injury due to hepatitis B virus (HBV) infections and aflatoxin-B1 (AFB1) exposure are the major risk factors of HCC. Understanding early metabolic changes involving these risk factors in an animal model closely resembling human hepatocellular carcinoma (HCC) is critical for biomarker discovery and disease therapeutics. We have used the hepatitis B surface antigen (HBsAg) transgenic mouse model that mimics HBV carriers with and without AFB1 treatment. We investigated early metabolic changes from preneoplastic state to HCC by non-invasive longitudinal imaging in three HCC groups of mice: HBsAg + AFB1(Gp-I), AFB1 alone (Gp-II), HBsAg alone (Gp-III) and a control group (wild-type untreated; Gp-IV). For the first time, we have identified acylcarnitine signals in vivo in the liver prior to the histological manifestation of the tumors in all three groups. Acylcarnitine concentration increased with increase in tumor growth in all HCC mouse models, indicating elevated metabolic activity and increased cell turnover. This was confirmed in a pilot study using human serum from HCC patients, which revealed a higher concentration of acylcarnitine compared with normal subjects. Translational clinical studies can be designed to detect acylcarnitine in patients with high risk factors for HCC.

Adoptive T-cell transfer and chemotherapy in the first-line treatment of metastatic and/or locally recurrent nasopharyngeal carcinoma.

The outcomes for patients with metastatic or locally recurrent Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) remain poor. Adoptive immunotherapy with EBV-specific cytotoxic T lymphocytes (EBV-CTLs) has proven clinical efficacy, but it has never been evaluated in the first-line treatment setting in combination with chemotherapy. To evaluate the safety and efficacy of a chemotherapy in combination with adoptive EBV-CTL transfer, we conducted a phase 2 clinical trial consisting of four cycles of gemcitabine and carboplatin (GC) followed by up to six doses of EBV-CTL. Thirty-eight patients were enrolled, and 35 received GC and EBV-CTL. GC-CTL therapy resulted in a response rate of 71.4% with 3 complete responses and 22 partial responses. With a median follow up of 29.9 months, the 2-year and 3-year overall survival (OS) rate was 62.9 and 37.1%, respectively. Five patients did not require further chemotherapy for more than 34 months since initiation of CTL. Infusion of CTL products containing T cells specific for LMP2 positively correlated with OS (hazard ratio: 0.35; 95% confidence interval: 0.14-0.84; P = 0.014). Our study achieved one of the best survival outcomes in patients with advanced NPC, setting the stage for a future randomized study of chemotherapy with and without EBV-CTL.

Identification of serum monocyte chemoattractant protein-1 and prolactin as potential tumor markers in hepatocellular carcinoma.

Early diagnosis of hepatocellullar carcinoma (HCC) remains a challenge. The current practice of serum alpha-fetoprotein (AFP) measurement is inadequate. Here we utilized a proteomic approach to identify novel serum biomarkers for distinguishing HCC patients from non-cancer controls. We profiled the serum proteins in a group of 58 resectable HCC patients and 11 non-HCC chronic hepatitis B (HBV) carrier samples from the Singapore General Hospital (SGH) using the RayBio® L-Series 507 Antibody Array and found 113 serum markers that were significantly modulated between HCC and control groups. Selected potential biomarkers from this list were quantified using a multiplex sandwich enzyme-linked immunosorbent assay (ELISA) array in an expanded SGH cohort (126 resectable HCC patients and 115 non-HCC chronic HBV carriers (NC group)), confirming that serum prolactin and monocyte chemoattractant protein-1 (MCP-1) were significantly upregulated in HCC patients. This finding of serum MCP-1 elevation in HCC patients was validated in a separate cohort of serum samples from the Mochtar Riady Institute for Nanotechnology, Indonesia (98 resectable HCC, 101 chronic hepatitis B patients and 100 asymptomatic HBV/HCV carriers) by sandwich ELISA. MCP-1 and prolactin levels were found to correlate with AFP, while MCP-1 also correlated with disease stage. Subsequent receiver operating characteristic (ROC) analysis of AFP, prolactin and MCP-1 in the SGH cohort and comparing their area under the ROC curve (AUC) indicated that neither prolactin nor MCP-1 on their own performed better than AFP. However, the combination of AFP+MCP-1 (AUC, 0.974) had significantly superior discriminative ability than AFP alone (AUC, 0.942; p<0.001). In conclusion, prolactin and MCP-1 are overexpressed in HCC and are conveniently quantifiable in patients' sera by ELISA. MCP-1 appears to be a promising complementary biomarker for HCC diagnosis and this MCP-1+AFP model should be further evaluated as potential biomarker on a larger scale in patients at-risk of HCC.

Clinical Benefit of Allogeneic Melanoma Cell Lysate-Pulsed Autologous Dendritic Cell Vaccine in MAGE-Positive Colorectal Cancer Patients.

PURPOSE: We evaluated the clinical benefit of an allogeneic melanoma cell lysate (MCL)-pulsed autologous dendritic cell (DC) vaccine in advanced colorectal cancer patients expressing at least one of six MAGE-A antigens overexpressed by the cell line source of the lysate. EXPERIMENTAL DESIGN: DCs were cultured from peripheral blood mononuclear cells (PBMC), pulsed with the allogeneic MCL, and matured using cytokines that achieved high CD83- and CCR7-expressing DCs. Each patient received up to 10 intradermal vaccinations (3-5 x 10(6) cells per dose) at biweekly intervals. RESULTS: Twenty patients received a total of 161 vaccinations. Treatment was well tolerated and quality of life measurements did not vary much across time. One patient experienced partial response [5%; 95% confidence interval (CI), 1-24%] and seven achieved stable disease (35%; 95% CI, 18-57%), one of whom also achieved late tumor regression, yielding a clinical benefit response rate of 40% (95% CI, 22-61%). Although overall median progression-free survival was 2.4 months (95% CI, 1.9-4.1 months), five patients (25%) experienced prolonged progression-free survival (>6 months), two of whom (10%) remain progression-free for >27 and >37 months, respectively. This result is particularly meaningful as all patients had progressive disease before treatment. Overall, DC vaccination was associated with a serial decline in regulatory T cells. Using an antibody array, we characterized plasma protein profiles in responding patients that may correlate with vaccine activity and report a prevaccination protein signature distinguishing responders from nonresponders. CONCLUSION: This phase II vaccine study using mature, MCL-pulsed DCs has shown promising results and warrants further evaluation in a prospective randomized setting. (Clin Cancer Res 2009;15(24):7726-36).