Kristen M. Cunanan

Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection

Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumour rejection. The most successful CARs used to date are those targeting CD19 (ref. 2), which offer the prospect of complete remission in patients with chemorefractory or relapsed B-cell malignancies. CARs are typically transduced into the T cells of a patient using γ-retroviral vectors or other randomly integrating vectors, which may result in clonal expansion, oncogenic transformation, variegated transgene expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific interventions in human cells, including targeted gene delivery to the CCR5 and AAVS1 loci. Here we show that directing a CD19-specific CAR to the T-cell receptor α constant (TRAC) locus not only results in uniform CAR expression in human peripheral blood T cells, but also enhances T-cell potency, with edited cells vastly outperforming conventionally generated CAR T cells in a mouse model of acute lymphoblastic leukaemia. We further demonstrate that targeting the CAR to the TRAC locus averts tonic CAR signalling and establishes effective internalization and re-expression of the CAR following single or repeated exposure to antigen, delaying effector T-cell differentiation and exhaustion. These findings uncover facets of CAR immunobiology and underscore the potential of CRISPR/Cas9 genome editing to advance immunotherapies.

Prevalence of Incontinence by Race and Ethnicity of Older People Admitted to Nursing Homes

OBJECTIVE While admissions of minorities to nursing homes (NHs) are increasing and prevalence of incontinence in NHs remains high, little is known about incontinence among racial-ethnic groups of NH admissions other than blacks. The purpose of this study was to describe the prevalence of incontinence among older adults admitted to NHs by race/ethnicity at three levels of measurement: individual resident, NH, and Census division. DESIGN Cross-sectional and descriptive. PARTICIPANTS AND SETTING Admissions of persons age 65 or older to 1 of 457 NHs of a national, for-profit chain over 3 years 2000-2002 (n = 111,640 residents). METHODS Data sources were the Minimum Data Set v. 2.0 and 2000 US Census. Prevalence of the following definitions of incontinence was analyzed: Only Urinary Incontinence (UI), Only Fecal Incontinence (FI), Dual Incontinence (DI; UI and FI), Any UI (UI with or without FI), Any FI (FI with or without UI), and Any Incontinence (UI and/or FI and/or DI). RESULTS Asian patients, black patients, and Hispanic patients had a higher prevalence of Any Incontinence (67%, 66%, and 58%, respectively) compared to white patients (48%) and American Indian patients (46%). At the NH level, all prevalence measures of incontinence (except Only UI) appear to trend in the opposite direction from the percentage of NH admissions who were white. Among Asian and white patients, there was a higher prevalence of all types of incontinence in men compared with women except for Only UI. Among Census divisions, the prevalence of all types of incontinence, except Only UI, was lowest in the 2 divisions with the highest percentage of white admissions to their NHs. CONCLUSIONS NHs admitting more racial/ethnic minorities may be faced with managing more incontinence and needing additional staffing resources. The association of the prevalence of most types of incontinence with the race/ethnicity of NH admissions at all levels of measurement lend support to the growing evidence that contextual factors beyond individual resident characteristics may contribute to NH differences.

Basket Trials in Oncology: A Trade-Off Between Complexity and Efficiency

The current oncology drug development landscape is dominated by efforts to create therapies that are mechanistically designed to improve outcomes for patients with cancers that harbor specific molecular aberrations, which often occur across a variety of tumor types. In the evaluation of targeted therapies, basket trials have emerged as an approach to test the hypothesis that targeted therapies may be effective independent of tumor histology, as long as the molecular target is present. However, the term basket has been applied broadly, and there is little uniformity in the design or goals of these trials. Furthermore, the scientific goals frequently are not specified with the precision conventionally used for clinical trials, leading to some difficulties in design and interpretation. For instance, many investigative teams use the popular Simon two-stage design, independently in each basket, thus effectively treating the trial overall as a series of independent phase II clinical trials. However, the actual goals are typically more complex than those of simple phase II clinical trials of new agents. In this commentary, we present an overview of the various trials described as basket trials, clarify the distinctive goals that basket trials seek to address, discuss the inherent hidden complexities, and offer general recommendations regarding their design. Several approaches to evaluating targeted therapies in multiple tumor types have been described as basket trials (Fig 1). The first prototype in Figure 1 is the basket trial of vemurafenib. Vemurafenib is an oral inhibitor of BRAF that has greater selectivity for the BRAF mutant form of the kinase than for wildtype BRAF, which had been previously approved for patients with BRAF mutation–positive metastatic melanoma. Vemurafenib was targeted at a single variant in a variety of cancers with different primary disease sites and histologies, thereby defining diseasespecific baskets. The second prototype in Figure 1 is the CREATE trial, which evaluated the use of the anaplastic lymphoma kinase and/or mesenchymal-epithelial transition factor inhibitor crizotinib. Here, although again there was a single agent under investigation, the drug inhibits multiple oncokinases including c-Met and anaplastic lymphoma kinase. Thus the baskets reflect a combination of diseases and targets. The last prototype in Figure 1 is the CUSTOM trial. In this trial, investigators planned to enroll patients with one of three diseases and allocate them to one of five targeted therapies, resulting in 15 disease-drug-mutation–specific baskets. Thus, this study tests the efficacy of a variety of drugs in a variety of targets and disease sites. We note that more complex trials than those presented in Figure 1, such as the NCI-MATCH, Genentech MyPathway, Novartis Signature, and American Society of Clinical Oncology’s Targeted Agent and Profiling Utilization Registry Study (TAPUR) basket trials, fall into this general framework. These ongoing studies define drug-mutation–specific baskets because their aim is to determine the efficacy of drugs that target certain pathways, typically with postmarketed drugs in nonindicated solid tumor types. The NCI-MATCH trial is even more complex in that genomic screening is incorporated into the therapeutic study itself and treatment assignment is determined by a matching algorithm that uses predefined levels of evidence of the gene variants. The trial aims to assess the activity of multiple drugs used in mutation-specific baskets (mutations, amplifications, or translocations), regardless of tumor origin, using a single stage design for each biomarker-defined subgroup (ie, mutation-specific basket). As the clinical setting becomes more complex for a study, the terms basket trial and umbrella trial begin to overlap. For example, the NCI-MATCH trial has been referred to as both an umbrella trial as a result of the multiple drugs under evaluation and as a basket trial because of the multiple disease populations for screening. Moreover, many basket studies evaluate multiple genomic variants in a given gene, which further complicates the clinical setting, and these variants may individually influence the likelihood of response to therapy. All the trials in Figure 1 were constructed as a series of independent phase II trials, using a conventional two-stage design, such as the Simon design, within each basket, individually controlling type I error at a nominal level. However, the design aspects and performance characteristics of these trials are not well understood, and the nature of the scientific goals is more complex than those of traditional disease-specific studies. Most clinical trials are constructed to address a single primary objective. Although secondary objectives may be articulated in the protocol, the chosen study design may not be ideal for addressing all of them, although in conventional clinical trials, the overall design is frequently suitable for addressing typical secondary goals. In the context of a basket trial, the ideal design options are not necessarily well aligned for the numerous questions being asked. Thus, careful consideration is needed to identify which question is paramount and to design the study accordingly. To focus our discussion, we limit attention to the seemingly most straightforward setting in which there is one target mutation and one drug targeting that mutation, evaluated in several disease types. In this setting, the questions the investigators seek to address may be one or more of

Establishment of the In Vivo Efficacy of Pretargeted Radioimmunotherapy Utilizing Inverse Electron Demand Diels-Alder Click Chemistry

The pretargeting system based on the inverse electron demand Diels-Alder reaction (IEDDA) between trans-cyclooctene (TCO) and tetrazine (Tz) combines the favorable pharmacokinetic properties of radiolabeled small molecules with the affinity and specificity of antibodies. This strategy has proven to be an efficient method for the molecularly targeted delivery of pharmaceuticals, including isotopes for radiological imaging. Despite encouraging results from in vivo PET imaging studies, this promising system has yet to be thoroughly evaluated for pretargeted radioimmunotherapy (PRIT). Toward that end, we synthesized two novel 177Lu-labeled tetrazine-bearing radioligands. Next, we compared the usefulness of our ligands for PRIT when paired with TCO-modified 5B1—a human, anti-CA19.9 mAb—in preclinical murine models of pancreatic cancer. The exemplary ligand, 177Lu-DOTA-PEG7-Tz, showed rapid (4.6 ± 0.8% ID/g at 4 hours) and persistent (16.8 ± 3.9% ID/g at 120 hours) uptake in tumors while concurrently clearing from blood and nontarget tissues. Single-dose therapy studies using 5B1-TCO and varying amounts of 177Lu-DOTA-PEG7-Tz (400, 800, and 1,200 μCi) showed that our system elicits a dose-dependent therapeutic response in mice bearing human xenografts. Furthermore, dosimetry calculations suggest that our approach is amenable to clinical applications with its excellent dosimetric profile in organs of clearance (i.e., liver and kidneys) as well as in dose-limiting tissues, such as red marrow. This study established that a pretargeted methodology utilizing the IEDDA reaction can rapidly and specifically deliver a radiotherapeutic payload to tumor tissue, thus illustrating its excellent potential for clinical translation. Mol Cancer Ther; 16(1); 124–33. ©2016 AACR.

Evaluating the performance of copula models in phase I-II clinical trials under model misspecification

BackgroundTraditionally, phase I oncology trials are designed to determine the maximum tolerated dose (MTD), defined as the highest dose with an acceptable probability of dose limiting toxicities(DLT), of a new treatment via a dose escalation study. An alternate approach is to jointly model toxicity and efficacy and allow dose escalation to depend on a pre-specified efficacy/toxicity tradeoff in a phase I-II design. Several phase I-II trial designs have been discussed in the literature; while these model-based designs are attractive in their performance, they are potentially vulnerable to model misspecification.MethodsPhase I-II designs often rely on copula models to specify the joint distribution of toxicity and efficacy, which include an additional correlation parameter that can be difficult to estimate. We compare and contrast three models for the joint probability of toxicity and efficacy, including two copula models that have been proposed for use in phase I-II clinical trials and a simple model that assumes the two outcomes are independent. We evaluate the performance of the various models through simulation both when the models are correct and under model misspecification.ResultsBoth models exhibited similar performance, as measured by the probability of correctly identifying the optimal dose and the number of subjects treated at the optimal dose, regardless of whether the data were generated from the correct or incorrect copula, even when there is substantial correlation between the two outcomes. Similar results were observed for a simple model that assumes independence, even in the presence of strong correlation. Further simulation results indicate that estimating the correlation parameter in copula models is difficult with the sample sizes used in Phase I-II clinical trials.ConclusionsOur simulation results indicate that the operating characteristics of phase I-II clinical trials are robust to misspecification of the copula model but that a simple model that assumes independence performs just as well due to difficulty in estimating the copula model correlation parameters from binary data.

Prevalence of Pressure Ulcers by Race and Ethnicity for Older Adults Admitted to Nursing Homes

Little is known about the prevalence of pressure ulcers (PUs) among racial and ethnic groups of older individuals admitted to nursing homes (NHs). NHs admitting higher percentages of minority individuals may face resource challenges for groups with more PUs or ones of greater severity. This study examined the prevalence of PUs (Stages 2 to 4) among older adults admitted to NHs by race and ethnicity at the individual, NH, and regional levels. Results show that the prevalence of PUs in Black older adults admitted to NHs was greater than that in Hispanic older adults, which were both greater than in White older adults. The PU rate among admissions of Black individuals was 1.7 times higher than White individuals. A higher prevalence of PUs was observed among NHs with a lower percentage of admissions of White individuals. [Journal of Gerontological Nursing, 40(3), 20-26.].

Non-invasive Interrogation of DLL3 Expression in Metastatic Small Cell Lung Cancer

The Notch ligand DLL3 has emerged as a novel therapeutic target expressed in small cell lung cancer (SCLC) and high-grade neuroendocrine carcinomas. Rovalpituzumab teserine (Rova-T; SC16LD6.5) is a first-in-class DLL3-targeted antibody-drug conjugate with encouraging initial safety and efficacy profiles in SCLC in the clinic. Here we demonstrate that tumor expression of DLL3, although orders of magnitude lower in surface protein expression than typical oncology targets of immunoPET, can serve as an imaging biomarker for SCLC. We developed 89Zr-labeled SC16 antibody as a companion diagnostic agent to facilitate selection of patients for treatment with Rova-T based on a noninvasive interrogation of the in vivo status of DLL3 expression using PET imaging. Despite low cell-surface abundance of DLL3, immunoPET imaging with 89Zr-labeled SC16 antibody enabled delineation of subcutaneous and orthotopic SCLC tumor xenografts as well as distant organ metastases with high sensitivity. Uptake of the radiotracer in tumors was concordant with levels of DLL3 expression and, most notably, DLL3 immunoPET yielded rank-order correlation for response to SC16LD6.5 therapy in SCLC patient-derived xenograft models. Cancer Res; 77(14); 3931-41. ©2017 AACR.

Exploring Structural Parameters for Pretargeting Radioligand Optimization

Pretargeting offers a way to enhance target specificity while reducing off-target radiation dose to healthy tissue during payload delivery. We recently reported the development of an 18F-based pretargeting strategy predicated on the inverse electron demand Diels-Alder reaction as well as the use of this approach to visualize pancreatic tumor tissue in vivo as early as 1 h postinjection. Herein, we report a comprehensive structure: pharmacokinetic relationship study of a library of 25 novel radioligands that aims to identify radiotracers with optimal pharmacokinetic and dosimetric properties. This investigation revealed key relationships between molecular structure and in vivo behavior and produced two lead candidates exhibiting rapid tumor targeting with high target-to-background activity concentration ratios at early time points. We believe this knowledge to be of high value for the design and clinical translation of next-generation pretargeting agents for the diagnosis and treatment of disease.

Statistical Approaches to Assessing Health and Healthcare Disparities

Determining whether racial and ethnic disparities exist for a health-related outcome requires first specifying how outcomes will be measured and disparities calculated. We explain and contrast two common approaches for quantifying racial/ethnic disparities in health, with an applied example from nursing research. Data from a national for-profit chain of nursing homes in the US were analyzed to estimate racial/ethnic disparities in incidence of pressure ulcer within 90 days of nursing home admission. Two approaches were used and then compared: logistic regression and Peters-Belson. Advantages and disadvantages of each approach are given. Logistic regression can be used to quantify disparities as the odds of the outcome for one group relative to another. Peters-Belson can be used to quantify an overall disparity between groups as a risk difference and also provides the proportion of that disparity that is explained by available risk factors. Extensions to continuous outcomes, to survival outcomes, and to clustered data are outlined. Both logistic regression and Peters-Belson are easily implementable and interpretable and provide information on the predictors associated with the outcome. These disparity estimation methods have different interpretations, assumptions, strengths, and weaknesses, of which the researcher should be aware when planning an analytic approach.

A Bayesian adaptive phase I–II trial design for optimizing the schedule of therapeutic cancer vaccines

Phase I-II clinical trials refer to the class of designs that evaluate both the safety and efficacy of a novel therapeutic agent in a single trial. Typically, Phase I-II oncology trials take the form of dose-escalation studies, where initial subjects are treated at the lowest dose level and subsequent subjects are treated at progressively higher doses until the optimal dose is identified. While dose-escalation designs are well-motivated in the case of traditional chemotherapeutic agents, an alternate approach may be considered for therapeutic cancer vaccines, where an investigators main objective is to evaluate the safety and efficacy of a set of dosing schedules or adjuvant combinations rather than to compare the safety and efficacy of progressively higher dose levels. We present a two-stage, Bayesian adaptive Phase I-II trial design to evaluate the safety and efficacy of therapeutic cancer vaccines. In the first stage, we determine whether a vaccination schedule achieves a minimum level of performance by comparing the toxicity and immune response rates to historical benchmarks. Vaccination schedules that achieve a minimum level of performance are compared using their magnitudes of immune response. If the superiority of a single schedule cannot be established after the first stage, Bayesian posterior predictive probabilities are used to determine the additional sample size required to identify the optimal vaccination schedule in a second stage. Copyright