Genetically modulating T-cell function to target cancer.

The adoptive transfer of tumor-specific T-lymphocytes holds promise for the treatment of metastatic cancer. Genetic modulation of T-lymphocytes using TCR transfer with tumor-specific TCR genes is an attractive strategy to generate anti-tumor response, especially against large solid tumors. Recently, several clinical trials have demonstrated the therapeutic potential of this approach which lead to impressive tumor regression in cancer patients. Still, several factors may hinder the clinical benefit of this approach, such as the type of cells to modulate, the vector configuration or the safety of the procedure. In the present review we will aim at giving an overview of the recent developments related to the immune modulation of the anti-tumor adaptive response using genetically engineered lymphocytes and will also elaborate the development of other genetic modifications to enhance their anti-tumor immune response.

Level of neo-epitope predecessor and mutation type determine T cell activation of MHC binding peptides.

BACKGROUND: Targeting epitopes derived from neo-antigens (or "neo-epitopes") represents a promising immunotherapy approach with limited off-target effects. However, most peptides predicted using MHC binding prediction algorithms do not induce a CD8 + T cell response, and there is a crucial need to refine the predictions to readily identify the best antigens that could mediate T-cell responses. Such a response requires a high enough number of epitopes bound to the target MHC. This number is correlated with both the peptide-MHC binding affinity and the number of peptides reaching the ER. Beyond this, the response may be affected by the properties of the neo-epitope mutated residues. METHODS: Herein, we analyzed several experimental datasets from cancer patients to elaborate better predictive algorithms for T-cell reactivity to neo-epitopes. RESULTS: Indeed, potent classifiers for epitopes derived from neo-antigens in melanoma and other tumors can be developed based on biochemical properties of the mutated residue, the antigen expression level and the peptide processing stage. Among MHC binding peptides, the present classifiers can remove half of the peptides falsely predicted to activate T cells while maintaining the absolute majority of reactive peptides. CONCLUSIONS: The classifier properties further highlight the contribution of the quantity of peptides reaching the ER and the mutation type to CD8 + T cell responses. These classifiers were then validated on neo-antigens obtained from other datasets, confirming the validity of our prediction. Algorithm Availability: http://peptibase.cs.biu.ac.il/Tcell_predictor/ or by request from the authors as a standalone code.

Adoptive Cell Therapy for Metastatic Melanoma.

Adoptive cell therapy (ACT) of tumor-infiltrating lymphocytes (TILs) is a powerful form of immunotherapy by inducing durable complete responses that significantly extend the survival of melanoma patients. Mutation-derived neoantigens were recently identified as key factors for tumor recognition and rejection by TILs. The isolation of T-cell receptor (TCR) genes directed against neoantigens and their retransduction into peripheral T cells may provide a new form of ACT.Genetic modifications of T cells with chimeric antigen receptors (CARs) have demonstrated remarkable clinical results in hematologic malignancies, but are so far less effective in solid tumors. Only very limited reports exist in melanoma. Progress in CAR T-cell engineering, including neutralization of inhibitory signals or additional safety switches, may open opportunities also in melanoma.We review clinical results and latest developments of adoptive therapies with TILs, T-cell receptor, and CAR-modified T cells and discuss future directions for the treatment of melanoma.

Selection of Shared and Neoantigen-Reactive T Cells for Adoptive Cell Therapy Based on CD137 Separation.

Adoptive cell therapy (ACT) of autologous tumor infiltrating lymphocytes (TIL) is an effective immunotherapy for patients with solid tumors, yielding objective response rates of around 40% in refractory patients with metastatic melanoma. Most clinical centers utilize bulk, randomly isolated TIL from the tumor tissue for ex vivo expansion and infusion. Only a minor fraction of the administered T cells recognizes tumor antigens, such as shared and mutation-derived neoantigens, and consequently eliminates the tumor. Thus, there are many ongoing effects to identify and select tumor-specific TIL for therapy; however, those approaches are very costly and require months, which is unreasonable for most metastatic patients. CD137 (4-1BB) has been identified as a co-stimulatory marker, which is induced upon the specific interaction of T cells with their target cell. Therefore, CD137 can be a useful biomarker and an important tool for the selection of tumor-reactive T cells. Here, we developed and validated a simple and time efficient method for the selection of CD137-expressing T cells for therapy based on magnetic bead separation. CD137 selection was performed with clinical grade compliant reagents, and TIL were expanded in a large-scale manner to meet cell numbers required for the patient setting in a GMP facility. For the first time, the methodology was designed to comply with both clinical needs and limitations, and its feasibility was assessed. CD137-selected TIL demonstrated significantly increased antitumor reactivity and were enriched for T cells recognizing neoantigens as well as shared tumor antigens. CD137-based selection enabled the enrichment of tumor-reactive T cells without the necessity of knowing the epitope specificity or the antigen type. The direct implementation of the CD137 separation method to the cell production of TIL may provide a simple way to improve the clinical efficiency of TIL ACT.

Use of HLA peptidomics and whole exome sequencing to identify human immunogenic neo-antigens.

The antigenicity of cells is demarcated by the peptides bound by their Human Leucocyte Antigen (HLA) molecules. Through this antigen presentation, T cell specificity response is controlled. As a fraction of the expressed mutated peptides is presented on the HLA, these neo-epitopes could be immunogenic. Such neo-antigens have recently been identified through screening for predicted mutated peptides, using synthetic peptides or ones expressed from minigenes, combined with screening of patient tumor-infiltrating lymphocytes (TILs). Here we present a time and cost-effective method that combines whole-exome sequencing analysis with HLA peptidome mass spectrometry, to identify neo-antigens in a melanoma patient. Of the 1,019 amino acid changes identified through exome sequencing, two were confirmed by mass spectrometry to be presented by the cells. We then synthesized peptides and evaluated the two mutated neo-antigens for reactivity with autologous bulk TILs, and found that one yielded mutant-specific T-cell response. Our results demonstrate that this method can be used for immune response prediction and promise to provide an alternative approach for identifying immunogenic neo-epitopes in cancer.

The role of RASSF1A in uveal melanoma.

PURPOSE: RASSF1A inactivation in uveal melanoma (UM) is common and methylation-induced. We investigated the effect of RASSF1A re-expression on the UM phenotype in vivo and in vitro. METHODS: The phenotypic effect of methylation-induced inactivation of RASSF1A in UM was explored using a stable RASSF1A-expressing UM-15 clone. RASSF1A expression was assessed using QRT-PCR. Proliferation was evaluated in vitro using MTT assays. Additionally, athymic NOD/SCID mice were injected subcutaneously or intraocularly with RASSF1A-expressing and -non-expressing UM-15 clones, and euthanized when tumors reached a volume of 1500 mm(3), or at 56 or 46 days, respectively. Tumor tissues, eyes, and livers were analyzed histologically. RESULTS: In vitro analysis confirmed the lack of RASSF1A expression and full methylation of the RASSF1A promoter region in the UM-15 cell line, which was reversible following treatment with 5-Aza-2-deoxycytidine. Cells expressing exogenous RASSF1A showed slower proliferation than controls and regained sensitivity to cisplatin. Compared to mice injected with control cells, mice treated with UM-15 cells expressing exogenous RASSF1A did not acquire intraocular tumors, and their subcutaneous tumors were relatively delayed and small. Neither group had liver metastases. CONCLUSIONS: UM cells reduced tumorigenicity in the presence of activated RASSF1A. RASSF1A apparently has an important role in the development of UM, and its reactivation might be applied in the development of new treatments.

PI3K/Akt pathway mutations in retinoblastoma.

PURPOSE: Many malignancies are known to be associated with abnormal activation of the PI3K-AKT pathway. Recently, a somatic mutation in the AKT1 gene (E17K) was identified in a small proportion of human tumors. This mutation activated AKT1 by means of abnormal membrane recruitment and stimulated downstream signaling. This study was designed to analyze AKT1 mutations in retinoblastoma and gain insights into the role PI3K-AKT pathway plays in the development of this tumor. METHODS: Twenty-four samples of retinoblastoma from children were analyzed for mutations in the AKT1, PTEN and K-RAS genes, using a chip-based matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometer. Mutations in the PIK3CA gene were analyzed in 16 retinoblastoma samples using direct sequencing. RESULTS: These results show that the mutation E17K/AKT1 was not detected in the 24 samples of retinoblastoma analyzed. K-RAS mutations were identified in two samples. There were no mutations in any of the other genes analyzed by a mass array system. On direct sequencing of 16 samples for the PIK3CA gene, one sample showed gain of function mutation in exon 9. In another sample, a genetic polymorphism of unknown significance (rs17849079) was detected in exon 20. CONCLUSIONS: Although the PI3K-AKT pathway is known to be dysregulated in retinoblastoma, the low frequency of oncogenic mutations in the AKT1, PIK3CA, and PTEN genes, suggests a different activating mechanism.

Hypermethylation of CpG island loci of multiple tumor suppressor genes in retinoblastoma.

Epigenetic silencing of tumor suppressor genes by methylation of discrete regions of the CpG islands is a major mechanism underlying tumorigenesis. Methylation of at least three of five specific genes may represent a distinct trait, termed the CpG island methylator phenotype (CIMP). Positive CIMP is associated with BRAF mutations. The present study sought to investigate the presence of BRAF mutations in human retinoblastoma and the role of epigenetic silencing of multiple tumor suppression genes in a search for methylation phenotype. Twenty-five archival retinoblastoma samples were analyzed for BRAF mutations with polymerase chain reaction, Mutector assay, and direct sequencing. Nineteen samples were also analyzed for the promoter methylation status of eight candidate cancer-related genes using real-time quantitative methylation-specific polymerase chain reaction after sodium bisulfite modification. The CIMP status was determined. No BRAF mutations were found. The frequencies of cancer-related gene methylation were as follows: 89% for RASSF1A, 52% for NEUROG1, 5% for DAP-kinase, RUNX3 and CACNA1G, and 0 for RAR-beta2, SOCS-1 and IGF-2. The lack of BRAF mutations in retinoblastoma is in accord with the negative CIMP status and the high hypermethylation rate for RASSF1A. The high methylation status for NEUROG1 may point to an alternative pathway in the development and progression of retinoblastoma, but further studies are needed.