January 2016, Vol. 3, No. 1
Certain Types of T-Cells Show Clinical Benefit in Patients with Glioblastoma And other news from the 2015 Society for Immunotherapy of CancerSociety for Immunotherapy of Cancer
Although the current standards of care cannot cure glioblastoma (GBM), an adoptive T-cell transfer may help improve outcomes in patients with GBM.
At the 2015 Society for Immunotherapy of Cancer 30th Anniversary Annual Meeting, researchers reported their findings from a phase 1 clinical trial, NCT01109095. They observed patients with progressive GBM receiving autologous cytomegalovirus (CMV).pp65 T-cells that were grafted with a second-generation HER2 chimeric antigen receptor (CAR) with a CD28.zeta signaling domain.
The trial comprised 17 CMV-seropositive patients with radiologically progressive HER2-positive GBM. The median age was 49 years (range, 11-71 years), and included 6 children and 11 adults. The children who were enrolled had significantly larger tumor volumes at the time of infusion. With a peripheral blood draw (90 mL maximum), a cell product was successfully generated for all patients.
The researchers found that a median of 67% (range, 46%-82%) of T-cells expressed the HER2 CAR, and demonstrated a median of 985.5 (range, 390-1292) CMV.pp65 reactivity in an interferon-γ Elispot assay (spot-forming cells/105 T cells). Infusions of 1×106/m2 to 1×108/m2 were well tolerated, and there were no severe adverse events or cytokine release syndrome. HER2 CMV T-cells were identified in the peripheral blood for ≤2 weeks postinfusion, according to the real-time polymerase chain reaction of a CAR-specific amplicon.
Of the 16 evaluable patients, 8 patients showed progressive disease and 8 patients had objective responses. One patient experienced a partial response with an approximately 62% reduction in tumor volume lasting 8 months. A total of 7 patients experienced stable disease for >6 weeks (of these, 5 were durable for >10 weeks), and 3 patients currently have a follow-up of 24 to >30 months following T-cell infusion. The median survival was 11.6 months from the time of infusion, and 24.8 months from the time of diagnosis. The median survival for the adults in the trial was 30 months from the time of diagnosis.
The researchers concluded that systemically administered HER2 CAR CMV bispecific T-cells are safe, and that approximately 38% of patients experienced a durable clinical benefit.
Epacadostat plus Pembrolizumab Shows Promise in Patients with Advanced Cancers
Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolizing enzyme that induces immune tolerance by suppressing T-cell responses, and is found in many cancer types. Epacadostat is a potent, selective oral inhibitor of IDO1. A dose-escalation study of patients with advanced melanoma receiving epacadostat plus ipilimumab showed a favorable overall response rate (ORR), disease control rate (DCR), and progression-free survival in participants who were immunotherapy-naïve (Gibney G, et al. Abstract presented at European Cancer Congress; September 25-29, 2015; Vienna, Austria).
Researchers presented their own ongoing dose-escalation and dose-expansion study at the 2015 Society for Immunotherapy of Cancer 30th Anniversary Annual Meeting. This study analyzes epacadostat plus pembrolizumab in patients with stage IIIB, stage IV, or recurrent non–small-cell lung cancer (NSCLC), melanoma, transitional cell carcinoma (TCC), renal-cell carcinoma (RCC), endometrial adenocarcinoma (EA), or squamous-cell carcinoma of the head and neck (SCCHN). Patients were excluded from the study if they were previously treated with anti–PD-1 or anti–CTLA-4 therapies. Enrollment is complete in the epacadostat 25-mg twice-daily, 50-mg twice-daily, and 100-mg twice-daily groups with pembrolizumab 2 mg/kg intravenously every 3 weeks. Expansion groups are currently being enrolled for epacadostat 50 mg twice daily, 100 mg twice daily, and 300 mg twice daily with pembrolizumab 200 mg.
A total of 54 patients were enrolled in the study as of August 21, 2015. The researchers included safety data on 28 patients (melanoma, n = 11; RCC, n = 5; NSCLC, n = 5; TCC, n = 3; EA, n = 2; and SCCHN, n = 2); 19 patients are evaluable for efficacy as of July 13, 2015. A dose-limiting, grade 3 rash was observed in 1 of 8 patients receiving epacadostat 50 mg twice daily and pembrolizumab 2 mg/kg. No dose-limiting toxicities were observed in patients receiving epacadostat 100 mg and pembrolizumab 2 mg/kg. The most common (≥20%) adverse events were fatigue, diarrhea, rash, arthralgia, and nausea; the majority were grade 1 or 2. Immune-related adverse events grade ≥3 were mucosal inflammation and rash.
Tumor burden reductions were observed in 15 of the 19 evaluable patients. The responses are ongoing, and were observed in all tumor types. In 7 evaluable patients with melanoma, the ORR was 57%, and the DCR was 86%, including 2 complete responses. In 5 evaluable patients with RCC, the ORR was 40% and the DCR was 80%. Based on a pharmacokinetic–pharmacodynamic model for epacadostat, nearly all patients’ Cavg exceeded the IC50, which is the range of active drug exposure seen in preclinical models.
The researchers concluded that epacadostat plus pembrolizumab was well-tolerated, and that the efficacy data suggest encouraging clinical activity. They are evaluating the correlations between biomarker expression and response, and will present the updated data.
Making Headway with Autologous T-Cells in Metastatic Cancer
In patients with metastatic melanoma who have received an adoptive cell transfer of patient-derived tumor-infiltrating lymphocytes (TILs), durable complete response rates of 20% have been observed.
To further explore these findings, researchers conducted an analysis of TIL that was administered to a patient with metastatic melanoma who had a complete response for >3 years. Results of the analysis, which were presented at the 2015 Society for Immunotherapy of Cancer 30th Anniversary Annual Meeting, showed that the autologous TIL recognized 10 specific somatically mutated gene products.
The investigators identified >4000 nonsynonymous somatic mutation variants by employing whole exome and ribonucleic acid sequencing, with a bioinformatic analysis of the patient’s matched tumor and normal gDNA. A total of 745 somatically mutated genes were screened with tandem minigene constructs that expressed transcripts expressed in autologous tumor cells with expression levels >0.1% of the β-actin levels. Following this, the tandem minigenes were transfected into autologous B-cells, and analyzed for their capacity to stimulate the T-cells that were administered.
The results of this analysis showed that the autologous TIL recognized 10 somatically mutated gene products, and each of these gene products was recognized in the context of 3 separate human leukocyte antigen class I restriction elements that were expressed by the patient’s tumor. A detailed T-cell clonal analysis showed that 9 of 20 major clones that were present in the infused TIL encompassed >25% of the total infused cells. These clones also recognized mutated antigens.
The results of this analysis helped further support the researchers’ efforts toward identifying mutation-reactive T cells for treating patients with metastatic cancer.
Significance of Mitochondrial Activity in CD8T–NK-Cell Interaction
Patients with an increasingly diverse collection of major histocompatibility complex (MHC) class I molecules are likely to have more responsive natural killer (NK)-cells. In addition, NK-cells with a larger number of inhibitory receptors that recognize surrounding MHC class I molecules respond to stimuli better than NK-cells that have less recognition of the surrounding MHC.
At the 2015 Society for Immunotherapy of Cancer 30th Anniversary Annual Meeting, investigators shared their observations of the mechanisms of local tumor antigen-specific T-cell-NK-cell collaboration. This collaboration appeared to be essential for eliminating tumor cells, including antigen-deficient tumor escape variants that appear before metastasis occurs.
The investigators observed a mouse model of mastocytoma expressing a self-tumor–antigen P1A. The effector CD8T-cells assisted dormant NK-cells by triggering their antitumor effector function. After an adoptive transfer of P1A-specific T-cells in RAG-/- and RAG-/-γc-/- in mice, bioluminescence imaging of mastocytoma tumors showed that NK-cell antitumor activity requires cytolytic T-cells, where the T-cells can function without NK-cells.
Phorbol myristate acetate and ionomycin-stimulated CD8T have cells formed multiple links with naïve NK lymphocytes in 2D and 3D coculture systems. Data have shown that NK-cells that interact with activated CD8T cells display an upregulation of CD25 and CD69 expression that is mediated by intercellular contacts, as well as the activation of NKG2D receptors and Stat2, Stat6, Jak1, Jak3, Tyk2, and PTEN signaling molecules, with a decrease in the phosphorylation of Stat1, PKB/Akt, SAPK/JNK, and p38.
However, the investigators noted that interacting NK-cells downregulate the CD25 molecule expression on CD8T-cells, and encourage the distinction of central-memory CD44+CD62L+T-cells. CD8T-cells demonstrate an elevation in the phosphorylation of Stat1, and a downregulation of Stat5 with stimulated PKB/Akt, Lck, mTOR, and p42/p44. Changes in the phosphorylation status of multiple signaling proteins during CD8T–NK-cell interaction imply a cellular remodeling, where NK-cells polarize activated CD8T-cells toward a central-memory phenotype, and activated CD8T lymphocytes prompt naïve NK-cells toward an effector or regulatory phenotype.
In addition, significant cytosolic and mitochondrial Ca2+ changes, mitochondrial reactive oxygen species production, mitochondrial membrane potential, mitochondrial permeability transition pore, and synthesis of nitric oxide and nonprotein thiols—mostly reduced glutathione—were observed in a mutual CD8T–NK-cell interaction.
The investigators’ findings underscore the significance of mitochondrial activity in the renovation of activation signaling and memory distinction of CD8T–NK-cell interaction. This has the potential to enhance strategies for cancer immunotherapy.
Anti-CD123 Plus Anti-CD19 Chimeric Antigen Receptor T-Cells Effective
Anti-Cluster of Differentiation 19(CD19) chimeric antigen receptor T-cells (CART19) and bispecific anti-CD19/CD3 antibodies (blinatumomab) are now generating complete responses in patients with relapsing/refractory B-cell acute lymphoblastic leukemia (ALL). However, some of these patients still relapse as a result of a loss of detectable CD19 (Maude SL, et al. N Engl J Med. 2014;371:1507-1517). The interleukin-3 receptor alpha (CD123) was shown to be expressed in several hematologic neoplasms, including acute myeloid leukemia, and, recently, B-cell acute ALL.
Marco Ruella, MD, Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, and colleagues evaluated the impact of targeting CD19 and CD123 with chimeric antigen receptor T-cells on treatment and prevention of CD19-negative relapses in patients who have previously received CD19-directed therapies.
The authors analyzed CD123 expression with flow cytometry in 36 relapsing/refractory B-cell ALL samples. They found that CD123 was highly expressed (81.75%, range: 5.10-99.60); therefore, CD123 was shown to be a promising option for targeted therapy in patients with B-cell acute ALL. The authors also found that CD123 was expressed in putative leukemia stem cells, identified as CD34-positive and CD38-negative. In addition, an expression of CD123 was detected in all (N = 6) CD19-negative B-cell acute ALL blasts that were analyzed after patients relapsed with CART19 treatment. Thus, Dr Ruella and colleagues produced CART123, which was costimulated with 4-1-BB using a lentiviral vector. They then evaluated the antileukemia efficacy of CART123 in vitro and in vivo against primary B-cell acute ALL blasts, and the NALM-6 cell line.
CART123 showed intense antileukemia activity, defined by a specific CD107a degranulation, cytokine production, cytotoxicity, and proliferation. However, these results were not statistically different from that of CART19. To test the role of CART123 in targeting CD19-negative relapses, the authors developed a new in vivo model, engrafting immunodeficient NOD/SCID/gamma mice with blasts from a patient with CD19-negative disease who relapsed after treatment with CART19. At day 14, the mice were randomized to receive control T cells, CART19, or CART123 in combination with CART19. CART19 and control T-cell–treated mice showed no antitumor activity; however, CART123 plus CART19 resulted in long-term survival, and complete elimination of the disease.
“Here we demonstrate that CD123 is expressed in CD19-negative [B-cell acute ALL] relapses occurring after CD19-directed therapies,” the study authors explained. “Combining CART123 cells with CART19 cells is an effective therapy for the treatment and prevention of antigen-loss relapses in [B-cell acute ALL] murine xenografts.”
Targeted Treatment Possible in Patients with TNBC Who Overexpress the B7-H4 Receptor
B7-H4, a coregulatory receptor that is thought to negatively regulate T-cell function, has been associated with poor prognosis in patients with renal-cell and ovarian cancers. Donald R. Shaffer, PhD, Jounce Therapeutics, Cambridge, MA, and colleagues analyzed gene expression data from the Cancer Genome Atlas, and found that of all the tumors that were analyzed, patients with triple-negative breast cancer (TNBC) had the greatest absolute B7-H4 messenger ribonucleic acid level.
Recent clinical trials of anti–programmed cell death-1 (PD-1) or programmed death-ligand 1 (PD-L1) therapies have shown promising activity in patients with TNBC. As a result, the study authors investigated archived samples from a cohort of 96 patients with TNBC, which were collected at Yale University, New Haven, CT. Based on these data, checkpoint molecule expression (PD-1, PD-L1, and B7-H4) and immune-cell infiltration expression (CD8, Fox P3) were explored in this patient sample.
“We developed a specific and sensitive immunohistochemistry (IHC) assay for evaluating B7-H4 protein, and used an immunofluorescence-based multiplex IHC for assessing combinations of checkpoint molecules in the TNBC samples,” Shaffer and colleagues described. They also noted that they were able to detect expression of B7-H4 in the majority of tumors, but expression of PD-L1 was limited to a subset of patients with TNBC; approximately 20% of patients had >5% of PD-L1–positive cells.
An analysis of multiplex IHC and flow cytometry showed that the majority of B7-H4 expression was restricted to the tumor epithelial cells, whereas the CD45+ immune cells were shown to be negative for B7-H4 expression. Of note, a majority of the tumors that highly expressed B7-H4 were negative, or showed little PD-L1 staining. In addition, the cells that were B7-H4–positive were negative for PD-L1 staining, which suggests that B7-H4 and PD-L1 checkpoint proteins may act in a mutually exclusive fashion. B7-H4 expression was not associated with overall survival, disease stage, nodal status, or other clinical characteristics; however, PD-L1, PD-1, and CD8 expression all presented a significant survival advantage for patients with TNBC, underscoring the importance of the immune response in this patient population.
When the authors further investigated these data, they were unable to find a definitive immunosuppressive role of B7-H4, which is contrary to the results of previously published literature. However, overexpression of B7-H4 in a CT-26 syngeneic in vivo model accelerated tumor growth. The unique expression pattern of B7-H4 in patients with TNBC suggests that there is an opportunity to incorporate targeted treatment approaches with possible immunomodulatory activity. More research is needed on this topic to further explain the immunologic mechanisms of B7-H4. However, the study authors suggest that the unique expression pattern of B7-H4 means that it could be an appealing option for the targeted treatment of patients with TNBC.
CD8+PD-1+ Is Enhanced in Tumor-Reactive, Mutation-Specific Cancer Cells
T-cells that target unique somatic mutations seem to play a significant role in antitumor responses that occur after T-cell transfer. Furthermore, the isolation of mutation-specific lymphocytes and T-cell receptors has become a hindrance in the development of more effective immunotherapies. Identifying tumor-reactive and mutation-specific cells in patients with cancer has been mainly limited to tumor-infiltrating lymphocytes, but mutation-specific cells are thought to be considerably less prevalent in peripheral blood, which is a more accessible and ample source of T-cells.
Alena Gros, PhD, National Cancer Institute, National Institutes of Health, Bethesda, MD, and colleagues previously found that anti–programmed cell death-1 (PD-1) expression identifies the patient-specific catalog of tumor-reactive cells that infiltrate melanoma tumors. Based on these findings, the authors analyzed the utility of PD-1 expression with peripheral blood lymphocytes to detect tumor- and neoantigen-specific lymphocytes, and presented their analysis.
The authors separated peripheral blood CD8+ lymphocytes based on PD-1 expression into CD8+PD-1–, CD8+PD-1+, and CD8+PD-1hi cells, and expanded them in vitro for 15 days. They subsequently screened circulating T-cell subsets to acknowledge mutated antigens identified by whole exome sequencing. A high throughput and personalized approach that allows the expression of all potential tumor neoantigens in the autologous antigen-presenting cells was used. The recognition of shared melanoma differentiation antigens, and cancer germline antigens was also evaluated.
“PD-1+ lymphocytes represented a small percentage of all the circulating CD8+ cells in patients with metastatic melanoma,” Gros and colleagues explained. “We found that selection of CD8+PD-1+ lymphocytes circulating in peripheral blood, but not the CD8+ or CD8+PD-1- cells, led to direct enrichment of tumor-reactive cells from peripheral blood in all 4 patients studied.”
In 3 of 4 patients with melanoma, peripheral blood CD8+PD-1+ and PD-1hi cells included mutation-specific lymphocytes that targeted 3, 3, and 1 unique patient-specific neoantigens, respectively. Circulating CD8+PD-1+ and PD-1hi lymphocytes in the 4 patients with melanoma were also enriched in T-cells that targeted ≥1 cancer germline antigens, including NY-ESO-I, MAGE-A3, and SSX2. Mutation- and cancer germline-specific lymphocytes were not detected in the peripheral blood CD8+, or in the CD8+PD-1– group.
These results show that peripheral blood CD8+PD-1+ in patients with cancer is enhanced in naturally occurring, tumor-reactive and mutation-specific cells, and provides a new approach for developing personalized T-cell–based therapies to treat patients with cancer.
Local Immunotherapy Injection Targets Unresponsive Tumors, Increases Survival
With the growing anticipation for new approaches to immunotherapy, viral cancer therapy has shifted from mainly providing oncolysis, to immunotherapy. “Adenoviruses have a unique ability to prime and boost immune responses,” Sari Pesonen, Oncos Therapeutics, Helsinki, Finland, and colleagues noted. “Granulocyte-macrophage colony-stimulating factor coding adenovirus ONCOS-102 causes immunogenic cancer cell death whereupon tumor antigens are presented into the immunogenic environment.”
Previously, ONCOS-102 has been shown to initiate CD8+ T-cell responses against tumor-derived antigens in patients with chemotherapy-refractory cancer. To further explore the impact of local immunotherapy with ONCOS-102, the study authors conducted a phase 1 study, and presented their findings at the 2015 Society for Immunotherapy of Cancer 30th Anniversary Annual Meeting.
The study included 12 patients with cancer who received recurring ONCOS-102 intratumoral injections. Researchers collected biopsies at baseline, 1 month, and 2 months after the initial treatment. Using immunohistochemistry (IHC), the biopsy results were analyzed for the presence of immune cells. The expression levels for innate immune (CD68, CD163, CD11c), T- (CD3, CD4, CD8), and B- (CD19) cells were analyzed in tumorous regions with an image analysis algorithm based on color deconvolution and division of the IHC-stained cells. When the authors conducted an exploratory analysis, they found a correlation between the total expression levels of immune-cell markers in the tumors.
The correlation between overall survival (OS) and absolute expression levels of different immune-cell markers in tumors was assessed with Spearman’s rank correlation analysis. The absolute expression level of macrophage marker CD68 negatively correlated with OS (P = .04) at baseline. This suggests that tumor-associated macrophages (TAMs) were tumorigenic in untreated tumors. No correlation was observed between other immune-cell markers and OS at baseline.
Of the 12 total patients included, 11 showed an increase in tumor-infiltrating innate and adaptive immune cells posttreatment. The most evident increase was observed in CD8+ cells. Posttreatment samples showed a positive correlation between CD68+ cell expression levels and OS (P = .01) compared with baseline. This suggested that the CD68+ macrophages that were drawn to tumors after ONCOS-102 injection displayed a different utility than pretreatment TAMs. In addition, the total expression levels of T-cell markers CD3 (P = .006), CD4 (P = .004), and CD8 (P =.007) all positively correlated with OS in posttreatment biopsies.
Ultimately, the ONCOS-102 injection generated infiltration of CD68+ macrophages and T-cells. This was associated with increased OS, whereas pretreatment with CD68+ TAMs was associated with decreased OS. These results suggest that local immunotherapy with ONCOS-102 can activate immunologically unresponsive tumors, and reduce local immune suppression in patients with metastatic tumors.
In their review article, Carin I. M. Dahlberg, PhD, from the Cell Therapies Institute, Nova Southeastern University, Fort Lauderdale, FL, and the Cell and Gene Therapy Group, Center for Hematology and Regenerative Medicine, Karolinska University Hospital Huddinge, NOVUM, Stockholm, Sweden, and colleagues evaluated natural killer (NK)-cell–based therapies targeting cancer. In [ Read More ]
In 3 phase 1 and 2 studies, improved overall response rates (ORRs) were shown in patients with metastatic melanoma taking pembrolizumab (Keytruda) in combination with 3 other immunotherapies, including epacadostat, talimogene laherparepvec (Imlygic), and ipilimumab (Yervoy). All 3 studies demonstrated significant antitumor activity. In addition, long-term follow-up data in a [ Read More ]