Replication competent retrovirus testing (RCR) in the National Gene Vector Biorepository: No evidence of RCR in 1,595 post-treatment peripheral blood samples obtained from 60 clinical trials.

The clinical impact of any therapy requires the product be safe and effective. Gammaretroviral vectors pose several unique risks, including inadvertent exposure to replication competent retrovirus (RCR) that can arise during vector manufacture. The US FDA has required patient monitoring for RCR, and the National Gene Vector Biorepository is an NIH resource that has assisted eligible investigators in meeting this requirement. To date, we have found no evidence of RCR in 338 pre-treatment and 1,595 post-treatment blood samples from 737 patients associated with 60 clinical trials. Most samples (75%) were obtained within 1 year of treatment, and samples as far out as 9 years after treatment were analyzed. The majority of trials (93%) were cancer immunotherapy, and 90% of the trials used vector products produced with the PG13 packaging cell line. The data presented here provide further evidence that current manufacturing methods generate RCR-free products and support the overall safety profile of retroviral gene therapy.

Adoptive transfer of personalized neoantigen-reactive TCR-transduced T cells in metastatic colorectal cancer: phase 2 trial interim results.

Adoptive cell transfer (ACT) with neoantigen-reactive T lymphocytes can mediate cancer regression. Here we isolated unique, personalized, neoantigen-reactive T cell receptors (TCRs) from tumor-infiltrating lymphocytes of patients with metastatic gastrointestinal cancers and incorporated the TCR α and ß chains into gamma retroviral vectors. We transduced autologous peripheral blood lymphocytes and adoptively transferred these cells into patients after lymphodepleting chemotherapy. In a phase 2 single-arm study, we treated seven patients with metastatic, mismatch repair-proficient colorectal cancers who had progressive disease following multiple previous therapies. The primary end point of the study was the objective response rate as measured using RECIST 1.1, and the secondary end points were safety and tolerability. There was no prespecified interim analysis defined in this study. Three patients had objective clinical responses by RECIST criteria including regressions of metastases to the liver, lungs and lymph nodes lasting 4 to 7 months. All patients received T cell populations containing ≥50% TCR-transduced cells, and all T cell populations were polyfunctional in that they secreted IFNγ, GM-CSF, IL-2 and granzyme B specifically in response to mutant peptides compared with wild-type counterparts. TCR-transduced cells were detected in the peripheral blood of five patients, including the three responders, at levels ≥10% of CD3+ cells 1 month post-ACT. In one patient who responded to therapy, ~20% of CD3+ peripheral blood lymphocytes expressed transduced TCRs more than 2 years after treatment. This study provides early results suggesting that ACT with T cells genetically modified to express personalized neoantigen-reactive TCRs can be tolerated and can mediate tumor regression in patients with metastatic colorectal cancers. ClinicalTrials.gov registration: NCT03412877 .

Epstein-Barr virus (EBV) antibody changes over time in a general population cohort in rural Uganda, 1992-2008.

BACKGROUND: Epstein-Barr virus (EBV) infection is ubiquitous and in sub-Saharan Africa, occurs early in life. In a population-based rural African cohort, we leveraged historical samples from the General Population Cohort (GPC) in Uganda to examine the epidemiology of infection with EBV over time, in the era of HIV. METHODS: We used 9024 serum samples collected from the GPC in 1992, 2000, 2008, from 7576 participants across the age range (0-99 years of age) and tested for anti-EBV immunoglobulin G (IgG) antibodies to EAd, VCA, and EBNA-1 using a multiplex bead-based assay. The related gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV) seropositivity was also determined by detection of anti-KSHV IgG antibodies to K8.1 or ORF73 measured by recombinant protein enzyme-linked immunosorbent assay. Data on sex, age, and HIV serostatus were also collected. EBV seropositivity was modeled with age (excluding those under one year, who may have had maternal antibodies), sex, HIV serostatus, and KSHV serostatus using generalized linear mixed effects models to produce beta estimates. RESULTS: More than 93% of children were EBV seropositive by one year of age. EBV seropositivity was significantly associated with KSHV seropositivity. Anti-EBNA-1 antibody levels decreased with increasing age and were lower on average in people living with HIV. In general, anti-EAd antibody levels increased with age, were higher in males and KSHV seropositive persons, but decreased over calendar time. Anti-VCA antibody levels increased with age and with calendar time and were higher in KSHV seropositive persons but lower in males. CONCLUSIONS: This is the first study to identify factors associated with EBV antibodies across the entire life-course in rural sub-Saharan Africa. Consistent with other studies, EBV was near ubiquitous in the population by age one year. Patterns of antibodies show changes by age, sex and calendar time, but no association with HIV was evident, suggesting no relationship between EBV sero-epidemiology and the spread of HIV in the population over time in Uganda.

Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers.

The accurate identification of antitumor T cell receptors (TCRs) represents a major challenge for the engineering of cell-based cancer immunotherapies. By mapping 55 neoantigen-specific TCR clonotypes (NeoTCRs) from 10 metastatic human tumors to their single-cell transcriptomes, we identified signatures of CD8+ and CD4+ neoantigen-reactive tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TILs exhibited tumor-specific expansion with dysfunctional phenotypes, distinct from blood-emigrant bystanders and regulatory TILs. Prospective prediction and testing of 73 NeoTCR signature-derived clonotypes demonstrated that half of the tested TCRs recognized tumor antigens or autologous tumors. NeoTCR signatures identified TCRs that target driver neoantigens and nonmutated viral or tumor-associated antigens, suggesting a common metastatic TIL exhaustion program. NeoTCR signatures delineate the landscape of TILs across metastatic tumors, enabling successful TCR prediction based purely on TIL transcriptomic states for use in cancer immunotherapy.

Targeting eIF4A-Dependent Translation of KRAS Signaling Molecules.

Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5' untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC. SIGNIFICANCE: These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.

Estimating the Threshold Value for Change for the Six Dimensions of the Impairment Inventory of the Chedoke-McMaster Stroke Assessment.

Purpose: Our purpose was to estimate a threshold value for change for the six dimensions of the Impairment Inventory of the Chedoke-McMaster Stroke Assessment and the confidence in labelling a person as having improved or not. Method: Secondary analysis of two data sets, previously reported by two research teams, consisted of two statistical analyses. The first analysis used a multiple of the standard error of measurement to calculate the threshold value for change for the six dimensions. The second analysis used the diagnostic test method to calculate a threshold improvement value and the confidence a clinician had in labelling a person as having improved or not on the leg, foot, and postural control dimensions. Results: The threshold value for change was determined to be 1 impairment point (i.e., stage) for the arm, hand, leg, foot, and postural control dimensions and 2 impairment points for the shoulder pain dimension. The positive predictive values associated with the leg, foot, and postural control dimensions were 74%, 59%, and 65%, respectively. Conclusions: Clinicians can use a change of 1 impairment point for the arm, hand, leg, foot, and postural control dimensions and a change of 2 impairment points for the shoulder pain dimension to identify true change in a patient's motor recovery.

Objectif : parvenir à une valeur seuil de changement aux six dimensions de l'inventaire des déficiences de l'évaluation Chedoke-McMaster de l'AVC ainsi que de la confiance à déclarer que l'état d'une personne s'est amélioré ou non. Méthodologie : l'analyse secondaire de deux ensembles de données, dont deux équipes de recherche avaient déjà rendu compte, s'est déclinée en deux analyses statistiques. La première faisait appel à un multiple de l'écart-type de mesure pour calculer la valeur seuil de changement aux six dimensions. La seconde puisait dans la méthode de test diagnostique pour calculer une valeur d'amélioration du seuil et la confiance du clinicien à déclarer que l'état d'une personne s'est amélioré ou non dans les dimensions du contrôle de la jambe, du pied et de la posture. Résultats : les chercheurs ont établi que la valeur seuil de changement correspondait à 1 point de déficience (phase) pour les dimensions du contrôle du bras, de la main, de la jambe, du pied et de la posture et de 2 points de déficience pour la dimension de la douleur de l'épaule. Les valeurs prédictives positives associées aux dimensions de contrôle de la jambe, du pied et de la posture s'élevaient à 74 %, 59 % et 65 %, respectivement. Conclusions : les cliniciens peuvent utiliser un changement d'1 point de déficience des dimensions de contrôle du bras, de la main, de la jambe, du pied et de la posture, et un changement de 2 points de déficience pour la dimension de la douleur de l'épaule pour déclarer un véritable changement dans le rétablissement moteur du patient.