Pan-cancer proteogenomics expands the landscape of therapeutic targets.

Fewer than 200 proteins are targeted by cancer drugs approved by the Food and Drug Administration (FDA). We integrate Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomics data from 1,043 patients across 10 cancer types with additional public datasets to identify potential therapeutic targets. Pan-cancer analysis of 2,863 druggable proteins reveals a wide abundance range and identifies biological factors that affect mRNA-protein correlation. Integration of proteomic data from tumors and genetic screen data from cell lines identifies protein overexpression- or hyperactivation-driven druggable dependencies, enabling accurate predictions of effective drug targets. Proteogenomic identification of synthetic lethality provides a strategy to target tumor suppressor gene loss. Combining proteogenomic analysis and MHC binding prediction prioritizes mutant KRAS peptides as promising public neoantigens. Computational identification of shared tumor-associated antigens followed by experimental confirmation nominates peptides as immunotherapy targets. These analyses, summarized at https://targets.linkedomics.org, form a comprehensive landscape of protein and peptide targets for companion diagnostics, drug repurposing, and therapy development.

Targeting IDH2R140Q and other neoantigens in acute myeloid leukemia.

ABSTRACT: For patients with high-risk or relapsed/refractory acute myeloid leukemia (AML), allogeneic stem cell transplantation (allo-HSCT) and the graft-versus-leukemia effect mediated by donor T cells, offer the best chance of long-term remission. However, the concurrent transfer of alloreactive T cells can lead to graft-versus-host disease that is associated with transplant-related morbidity and mortality. Furthermore, ∼60% of patients will ultimately relapse after allo-HSCT, thus, underscoring the need for novel therapeutic strategies that are safe and effective. In this study, we explored the feasibility of immunotherapeutically targeting neoantigens, which arise from recurrent nonsynonymous mutations in AML and thus represent attractive targets because they are exclusively present on the tumor. Focusing on 14 recurrent driver mutations across 8 genes found in AML, we investigated their immunogenicity in 23 individuals with diverse HLA profiles. We demonstrate the immunogenicity of AML neoantigens, with 17 of 23 (74%) reactive donors screened mounting a response. The most immunodominant neoantigens were IDH2R140Q (n = 11 of 17 responders), IDH1R132H (n = 7 of 17), and FLT3D835Y (n = 6 of 17). In-depth studies of IDH2R140Q-specific T cells revealed the presence of reactive CD4+ and CD8+ T cells capable of recognizing distinct mutant-specific epitopes restricted to different HLA alleles. These neo-T cells could selectively recognize and kill HLA-matched AML targets endogenously expressing IDH2R140Q both in vitro and in vivo. Overall, our findings support the clinical translation of neoantigen-specific T cells to treat relapsed/refractory AML.

Incongruity between T cell receptor recognition of breast cancer hotspot mutations ESR1 Y537S and D538G following exogenous peptide loading versus endogenous antigen processing.

T cell receptor engineered T cell (TCR T) therapies have shown recent efficacy against certain types of solid metastatic cancers. However, to extend TCR T therapies to treat more patients across additional cancer types, new TCRs recognizing cancer-specific antigen targets are needed. Driver mutations in AKT1, ESR1, PIK3CA, and TP53 are common in patients with metastatic breast cancer (MBC) and if immunogenic could serve as ideal tumor-specific targets for TCR T therapy to treat this disease. Through IFN-γ ELISpot screening of in vitro expanded neopeptide-stimulated T cell lines from healthy donors and MBC patients, we identified reactivity towards 11 of 13 of the mutations. To identify neopeptide-specific TCRs, we then performed single-cell RNA sequencing of one of the T cell lines following neopeptide stimulation. Here, we identified an ESR1 Y537S specific T cell clone, clonotype 16, and an ESR1 Y537S/D538G dual-specific T cell clone, clonotype 21, which were HLA-B*40:02 and HLA-C*01:02 restricted, respectively. TCR Ts expressing these TCRs recognized and killed target cells pulsed with ESR1 neopeptides with minimal activity against ESR1 WT peptide. However, these TCRs failed to recognize target cells expressing endogenous mutant ESR1. To investigate the basis of this lack of recognition we performed immunopeptidomics analysis of a mutant-overexpressing lymphoblastoid cell line and found that the ESR1 Y537S neopeptide was not endogenously processed, despite binding to HLA-B*40:02 when exogenously pulsed onto the target cell. These results indicate that stimulation of T cells that likely derive from the naïve repertoire with pulsed minimal peptides may lead to the expansion of clones that recognize non-processed peptides, and highlights the importance of using methods that selectively expand T cells with specificity for antigens that are efficiently processed and presented.

Mesenchymal stromal cell delivery of oncolytic immunotherapy improves CAR-T cell antitumor activity.

The immunosuppressive tumor microenvironment (TME) is a formidable barrier to the success of adoptive cell therapies for solid tumors. Oncolytic immunotherapy with engineered adenoviruses (OAd) may disrupt the TME by infecting tumor cells, as well as surrounding stroma, to improve the functionality of tumor-directed chimeric antigen receptor (CAR)-T cells, yet efficient delivery of OAds to solid tumors has been challenging. Here we describe how mesenchymal stromal cells (MSCs) can be used to systemically deliver a binary vector containing an OAd together with a helper-dependent Ad (HDAd; combinatorial Ad vector [CAd]) that expresses interleukin-12 (IL-12) and checkpoint PD-L1 (programmed death-ligand 1) blocker. CAd-infected MSCs deliver and produce functional virus to infect and lyse lung tumor cells while stimulating CAR-T cell anti-tumor activity by release of IL-12 and PD-L1 blocker. The combination of this approach with administration of HER.2-specific CAR-T cells eliminates 3D tumor spheroids in vitro and suppresses tumor growth in two orthotopic lung cancer models in vivo. Treatment with CAd MSCs increases the overall numbers of human T cells in vivo compared to CAR-T cell only treatment and enhances their polyfunctional cytokine secretion. These studies combine the predictable targeting of CAR-T cells with the advantages of cancer cell lysis and TME disruption by systemic MSC delivery of oncolytic virotherapy: incorporation of immunostimulation by cytokine and checkpoint inhibitor production through the HDAd further enhances anti-tumor activity.

The immunotherapy era of myeloma: monoclonal antibodies, vaccines, and adoptive T-cell therapies.

The treatment of multiple myeloma has evolved significantly over the last decades from primarily alkylator-based chemotherapeutic agents with minimal efficacy to the introduction of more effective agents including immune modulators and proteasome inhibitors, which have changed the landscape of therapy for this disease. We are now entering a new era that will increasingly integrate immunotherapy into standard treatment. This review discusses the current immune-based strategies currently approved, as well as various immune approaches being actively investigated including monoclonal antibodies, checkpoint inhibitors, vaccines, and adoptive T-cell therapies.

An Inducible Caspase-9 Suicide Gene to Improve the Safety of Therapy Using Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSC) hold promise for regenerative therapies, though there are several safety concerns including the risk of oncogenic transformation or unwanted adverse effects associated with hiPSC or their differentiated progeny. Introduction of the inducible caspase-9 (iC9) suicide gene, which is activated by a specific chemical inducer of dimerization (CID), is one of the most appealing safety strategies for cell therapies and is currently being tested in multicenter clinical trials. Here, we show that the iC9 suicide gene with a human EF1α promoter can be introduced into hiPSC by lentiviral transduction. The transduced hiPSC maintain their pluripotency, including their capacity for unlimited self-renewal and the potential to differentiate into three germ layer tissues. Transduced hiPSC are eliminated within 24 hours of exposure to pharmacological levels of CID in vitro, with induction of apoptosis in 94-99% of the cells. Importantly, the iC9 suicide gene can eradicate tumors derived from hiPSC in vivo. In conclusion, we have developed a direct and efficient hiPSC killing system that provides a necessary safety mechanism for therapies using hiPSC. We believe that our iC9 suicide gene will be of value in clinical applications of hiPSC-based therapy.

Mesenchymal Stromal Cells for Linked Delivery of Oncolytic and Apoptotic Adenoviruses to Non-small-cell Lung Cancers.

Oncolytic adenoviruses (OAdV) represent a promising strategy for cancer therapy. Despite their activity in preclinical models, to date the clinical efficacy remains confined to minor responses after intratumor injection. To overcome these limitations, we developed an alternative approach using the combination of the OAdv ICOVIR15 with a replication incompetent adenoviral vector carrying the suicide gene of inducible Caspase 9 (Ad.iC9), both of which are delivered by mesenchymal stromal cells (MSCs). We hypothesized that coinfection with ICOVIR15 and Ad.iC9 would allow MSCs to replicate both vectors and deliver two distinct types of antitumor therapy to the tumor, amplifying the cytotoxic effects of the two viruses, in a non-small-cell lung cancer (NSCLC) model. We showed that MSCs can replicate and release both vectors, enabling significant transduction of the iC9 gene in tumor cells. In the in vivo model using human NSCLC xenografts, MSCs homed to lung tumors where they released both viruses. The activation of iC9 by the chemical inducer of dimerization (CID) significantly enhanced the antitumor activity of the ICOVIR15, increasing the tumor control and translating into improved overall survival of tumor-bearing mice. These data support the use of this innovative approach for the treatment of NSCLC.

Health warning labels on cannabis products. What is the best design?

BACKGROUND: Health warning label on cannabis products has been recently studied, and with the latest trends of regulation around the world, there is a need to determine the most effective ways to apply this strategy. The current study aimed to examine the effects of different health warning label designs (pictorial vs text-only, background color, warning themes) on cannabis products. METHODS: An online experiment study (N=533) was carried out in Colombia with a between-subject design. Participants were randomly assigned to five package conditions: without warning, text-only white warning, text-only yellow warning, pictorial white warning, and pictorial yellow warning. Participants performed an attention task and rated each of the stimuli based on product appeal, perceived addictiveness, harm perception, and interest in trying cannabis products. RESULTS: Pictorial health warnings were generally the most effective. Especially, pictorial health warnings with a yellow background were found to decrease product appeal and interest in trying cannabis products, as well as increase harm perception compared to other designs. The most effective warning themes were mental health, smoke toxicity, aesthetic implications, and traffic accidents. CONCLUSION: The current study provides empirical evidence on the effectiveness of different designs of cannabis health warnings. Our results suggest that graphic yellow warnings are the most effective in communicating the risks of cannabis use.

High-avidity cytotoxic T lymphocytes specific for a new PRAME-derived peptide can target leukemic and leukemic-precursor cells.

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies, but is absent on normal tissues, including hematopoietic progenitor cells, and may therefore be an appropriate candidate for T cell-mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity, PRAME-specific cytotoxic T lymphocytes (CTLs), we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal, PRAME-specific CTL lines and elicited high-avidity CTLs, with a high proportion of cells recognizing a previously uninvestigated HLA-A*02-restricted epitope, P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME(+) hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts, but also against leukemic progenitor cells as assessed by colony-forming-inhibition assays, which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors, indicating that this approach may be of value for immunotherapy of PRAME(+) hematologic malignancies.

In vitro and in vivo model of a novel immunotherapy approach for chronic lymphocytic leukemia by anti-CD23 chimeric antigen receptor.

Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19(+)CD5(+)CD20(dim) B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23.CAR). CD23.CAR(+) T cells showed specific cytotoxic activity against CD23(+) tumor cell lines (average lysis 42%) and primary CD23(+) CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR(+) T cells released inflammatory cytokines (1445-fold more TNF-ß, 20-fold more TNF-α, and 4-fold more IFN-γ). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR(+) T cells. Redirected T cells were also effective in vivo in a CLL Rag2(-/-)γ(c)(-/-) xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23.CAR(+) T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23.CAR(+) T cells represent a selective immunotherapy for the elimination of CD23(+) leukemic cells in patients with CLL.

Engineered Adoptive T-Cell Therapies for Breast Cancer: Current Progress, Challenges, and Potential.

Breast cancer remains a significant health challenge, and novel treatment approaches are critically needed. This review presents an in-depth analysis of engineered adoptive T-cell therapies (E-ACTs), an innovative frontier in cancer immunotherapy, focusing on their application in breast cancer. We explore the evolving landscape of chimeric antigen receptor (CAR) and T-cell receptor (TCR) T-cell therapies, highlighting their potential and challenges in targeting breast cancer. The review addresses key obstacles such as target antigen selection, the complex breast cancer tumor microenvironment, and the persistence of engineered T-cells. We discuss the advances in overcoming these barriers, including strategies to enhance T-cell efficacy. Finally, our comprehensive analysis of the current clinical trials in this area provides insights into the future possibilities and directions of E-ACTs in breast cancer treatment.

Genetic modification of human T lymphocytes for the treatment of hematologic malignancies.

Modern chemotherapy regimens and supportive care have produced remarkable improvements in the overall survival of patients with hematologic malignancies. However, the development of targeted small molecules, monoclonal antibodies, and biological therapies that demonstrate greater efficacy and lower toxicity remains highly desirable in hematology, and oncology in general. In the context of biological therapies, T-lymphocyte based treatments have enormous potential. Donor lymphocyte infusion in patients relapsed after allogeneic hematopoietic stem cell transplant pioneered the concept that T lymphocytes can effectively control tumor growth, and this was then followed by the development of cell culture strategies to generate T lymphocytes with selective activity against tumor cells. Over the past decade, it has become clear that the adoptive transfer of ex vivo expanded antigen-specific cytotoxic T lymphocytes promotes sustained antitumor effects in patients with virus-associated lymphomas, such as Epstein-Barr virus related post-transplant lymphomas and Hodgkin's lymphomas. Because of this compelling clinical evidence and the concomitant development of methodologies for robust gene transfer to human T lymphocytes, the field has rapidly evolved, offering new opportunities to extend T-cell based therapies. This review summarizes the most recent biological and clinical developments using genetically manipulated T cells for the treatment of hematologic malignancies.

Cancer Therapy With TCR-Engineered T Cells: Current Strategies, Challenges, and Prospects.

To redirect T cells against tumor cells, T cells can be engineered ex vivo to express cancer-antigen specific T cell receptors (TCRs), generating products known as TCR-engineered T cells (TCR T). Unlike chimeric antigen receptors (CARs), TCRs recognize HLA-presented peptides derived from proteins of all cellular compartments. The use of TCR T cells for adoptive cellular therapies (ACT) has gained increased attention, especially as efforts to treat solid cancers with ACTs have intensified. In this review, we describe the differing mechanisms of T cell antigen recognition and signal transduction mediated through CARs and TCRs. We describe the classes of cancer antigens recognized by current TCR T therapies and discuss both classical and emerging pre-clinical strategies for antigen-specific TCR discovery, enhancement, and validation. Finally, we review the current landscape of clinical trials for TCR T therapy and discuss what these current results indicate for the development of future engineered TCR approaches.

Multi-antigen-targeted T-cell therapy to treat patients with relapsed/refractory breast cancer.

Purpose: Adoptively transferred, ex vivo expanded multi-antigen-targeted T cells (multiTAA-T) represent a new, potentially effective, and nontoxic therapeutic approach for patients with breast cancer (BC). In this first-in-human trial, we investigated the safety and clinical effects of administering multiTAA T cells targeting the tumor-expressed antigens, Survivin, NY-ESO-1, MAGE-A4, SSX2, and PRAME, to patients with relapsed/refractory/metastatic BC. Materials and methods: MultiTAA T-cell products were generated from the peripheral blood of heavily pre-treated patients with metastatic or locally recurrent unresectable BC of all subtypes and infused at a fixed dose level of 2 × 107/m2. Patients received two infusions of cells 4 weeks apart and safety and clinical activity were determined. Cells were administered in an outpatient setting and without prior lymphodepleting chemotherapy. Results: All patients had estrogen receptor/progesterone receptor positive BC, with one patient also having human epidermal growth factor receptor 2-positive. There were no treatment-related toxicities and the infusions were well tolerated. Of the 10 heavily pre-treated patients enrolled and infused with multiTAA T cells, nine had disease progression while one patient with 10 lines of prior therapies experienced prolonged (5 months) disease stabilization that was associated with the in vivo expansion and persistence of T cells directed against the targeted antigens. Furthermore, antigen spreading and the endogenous activation of T cells directed against a spectrum of non-targeted tumor antigens were observed in 7/10 patients post-multiTAA infusion. Conclusion: MultiTAA T cells were well tolerated and induced disease stabilization in a patient with refractory BC. This was associated with in vivo T-cell expansion, persistence, and antigen spreading. Future directions of this approach may include additional strategies to enhance the therapeutic benefit of multiTAA T cells in patients with BC.

Predictive Biomarkers of Immune Checkpoint Inhibitor Response in Breast Cancer: Looking beyond Tumoral PD-L1.

Immune checkpoint inhibitors utilize the immune system to kill cancer cells and are now widely applied across numerous malignancies. Pembrolizumab has two breast-specific indications in triple-negative disease. Currently, programmed death ligand-1 (PD-L1) expression on tumor and surrounding immune cells is the only validated predictive biomarker for immune checkpoint inhibitors (ICIs) in breast cancer; however, it can be imprecise. Additional biomarkers are needed to identify the patient population who will derive the most benefit from these therapies. The tumor immune microenvironment contains many biomarker candidates. In tumor cells, tumor mutational burden has emerged as a robust biomarker across malignancies in general, with higher burden cancers demonstrating improved response, but will need further refinement for less mutated cancers. Preliminary studies suggest that mutations in breast cancer gene 2 (BRCA-2) are associated with increased immune infiltration and response to ICI therapy. Other genomic alterations are also being investigated as potential predictive biomarkers. In immune cells, increased quantity of tumor-infiltrating lymphocytes and CD8+ cytotoxic T cells have correlated with response to immunotherapy treatment. The role of other immune cell phenotypes is being investigated. Peripherally, many liquid-based biomarker strategies such as PD-L1 expression on circulating tumor cells and peripheral immune cell quantification are being studied; however, these strategies require further standardization and refinement prior to large-scale testing. Ultimately, multiple biomarkers utilized together may be needed to best identify the appropriate patients for these treatments.

Lumpectomy followed by radiation improves survival in HER2 positive and triple-negative breast cancer with high tumor-infiltrating lymphocytes compared to mastectomy alone.

OBJECTIVE: The goal was to compare the 5-year DFS and 5-year OS in patients with early-stage human epidermal growth factor receptor 2 breast cancer (HER2+ BC) and triple-negative breast cancer (TNBC) in relation to the amount of stromal tumor-infiltrating lymphocytes (TILs) after locoregional management by either mastectomy without radiation or lumpectomy and whole-breast radiotherapy (RT). METHODS: This was a retrospective review of HER2+ BC and TNBC patients' charts and histopathology slides with clinical stage of T1-T2 N0 who presented at our facility between January 2009 and December 2019. Locoregional treatment included either mastectomy without RT (M) or lumpectomy with RT (L+R). TILs were assessed by three pathologists using the guidelines of the 2014 TILs working group. A competing risk model and Kaplan-Meier analysis were used to analyze correlations between TILs levels and clinical outcome. RESULTS: We reviewed 211 patients' charts. Of them, 190 proceeded to the final analysis. Patients were split into groups of "low TILs" and "high TILs" based on a 50% TILs cut-off. Of them 26% had high TILs, 48% received RT, 97% received chemotherapy, all HER2+ BC patients received HER2-directed therapy and all HER2+ BC that were also hormone receptor positive (HR+) received endocrine therapy (ET). In patient with low TILs, L+R did not improve outcomes compared to M. Moreover, patients with high TILs had a significant improvement of their DFS and OS with L+R when compared to M. CONCLUSION: The results of our study reflect that a selected group of HER2+ BC and TNBC with elevated TILs, L+R is associated with improvement of 5-year DFS and 5-year OS.

Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer.

BACKGROUND: Successful targeting of solid tumors such as breast cancer (BC) using chimeric antigen receptor (CAR) T cells has proven challenging, largely attributed to the immunosuppressive tumor microenvironment (TME). Myeloid-derived suppressor cells (MDSCs) inhibit CAR T cell function and persistence within the breast TME. To overcome this challenge, we have developed CAR T cells targeting tumor-associated mucin 1 (MUC1) with a novel chimeric costimulatory receptor that targets tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TR2) expressed on MDSCs. METHODS: The function of the TR2.41BB costimulatory receptor was assessed by exposing non-transduced (NT) and TR2.41BB transduced T cells to recombinant TR2, after which nuclear translocation of NFκB was measured by ELISA and western blot. The cytolytic activity of CAR.MUC1/TR2.41BB T cells was measured in a 5-hour cytotoxicity assay using MUC1+ tumor cells as targets in the presence or absence of MDSCs. In vivo antitumor activity was assessed using MDSC-enriched tumor-bearing mice treated with CAR T cells with or without TR2.41BB. RESULTS: Nuclear translocation of NFκB in response to recombinant TR2 was detected only in TR2.41BB T cells. The presence of MDSCs diminished the cytotoxic potential of CAR.MUC1 T cells against MUC1+ BC cell lines by 25%. However, TR2.41BB expression on CAR.MUC1 T cells induced MDSC apoptosis, thereby restoring the cytotoxic activity of CAR.MUC1 T cells against MUC1+ BC lines. The presence of MDSCs resulted in an approximately twofold increase in tumor growth due to enhanced angiogenesis and fibroblast accumulation compared with mice with tumor alone. Treatment of these MDSC-enriched tumors with CAR.MUC1.TR2.41BB T cells led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared with CAR.MUC1 (469.79±81.46 mm3) or TR2.41BB (434.86±64.25 mm3) T cells alone. CAR.MUC1.TR2.41BB T cells also demonstrated improved T cell proliferation and persistence at the tumor site, thereby preventing metastases. We observed similar results using CAR.HER2.TR2.41BB T cells in a HER2+ BC model. CONCLUSIONS: Our findings demonstrate that CAR T cells that coexpress the TR2.4-1BB receptor exhibit superior antitumor potential against breast tumors containing immunosuppressive and tumor promoting MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.

Oncolytic adenovirus and gene therapy with EphA2-BiTE for the treatment of pediatric high-grade gliomas.

BACKGROUND: Pediatric high-grade gliomas (pHGGs) are among the most common and incurable malignant neoplasms of childhood. Despite aggressive, multimodal treatment, the outcome of children with high-grade gliomas has not significantly improved over the past decades, prompting the development of innovative approaches. METHODS: To develop an effective treatment, we aimed at improving the suboptimal antitumor efficacy of oncolytic adenoviruses (OAs) by testing the combination with a gene-therapy approach using a bispecific T-cell engager (BiTE) directed towards the erythropoietin-producing human hepatocellular carcinoma A2 receptor (EphA2), conveyed by a replication-incompetent adenoviral vector (EphA2 adenovirus (EAd)). The combinatorial approach was tested in vitro, in vivo and thoroughly characterized at a molecular level. RESULTS: After confirming the relevance of EphA2 as target in pHGGs, documenting a significant correlation with worse clinical outcome of the patients, we showed that the proposed strategy provides significant EphA2-BiTE amplification and enhanced tumor cell apoptosis, on coculture with T cells. Moreover, T-cell activation through an agonistic anti-CD28 antibody further increased the activation/proliferation profiles and functional response against infected tumor cells, inducing eradication of highly resistant, primary pHGG cells. The gene-expression analysis of tumor cells and T cells, after coculture, revealed the importance of both EphA2-BiTE and costimulation in the proposed system. These in vitro observations translated into significant tumor control in vivo, in both subcutaneous and a more challenging orthotopic model. CONCLUSIONS: The combination of OA and EphA2-BiTE gene therapy strongly enhances the antitumor activity of OA, inducing the eradication of highly resistant tumor cells, thus supporting the clinical translation of the approach.

Cytotoxic T lymphocytes directed to the preferentially expressed antigen of melanoma (PRAME) target chronic myeloid leukemia.

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed in many hematologic malignancies, including chronic myeloid leukemia (CML). The sensitivity of CML to donor lymphocyte infusion after allogeneic stem cell transplantation suggests this tumor can be highly susceptible to cellular immunotherapy targeted to tumor associated antigens. We therefore tested whether functional PRAME-specific cytotoxic T lymphocytes (PRAME CTLs) could be generated and expanded from healthy donors and CML patients, or whether the limited immunogenicity of this CTA coupled with tumor-associated anergy would preclude this approach. Using optimized culture conditions and HLA-A*02-restricted PRAME-peptides, we have consistently generated PRAME CTLs from 8/9 healthy donors and 5/6 CML patients. These CTLs released IFNgamma in response to PRAME peptides (between 113 +/- 8 and 795 +/- 23 spot forming cells/10(5) T cells) and lysed PRAME peptide-loaded cells (45 +/- 19% at an effector:target [E:T] ratio of 20:1) in a MHC-restricted fashion. Importantly, these CTLs recognized and had cytotoxic activity against HLA-A*02(+)/PRAME(+) tumor cell lines, and could recognize and respond to primary CML cells. PRAME CTLs were generated almost exclusively from the naive T-cell compartment, and clonal analysis showed these cells could have high alphabetaTCR-peptide avidity. PRAME CTLs or vaccines may thus be of value for patients with CML.