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Graduate students entering entry-level occupational therapy programs are confronted by new ways of learning and interacting for which they may be ill-prepared. Confronted with the need to change their approach to learning, students may become frustrated and lose motivation, resulting in resistance. This article describes a pilot first term group advisement seminar, informed by Tolman and Kremling. Integrated Model of Student Resistance (IMSR), and designed to prepare students for these necessary changes. The article describes topics addressed, strategies implemented, and insights and reflections on the process and outcomes of participation in the seminar.

U.S. military Veterans aging with HIV represent a unique special population. The aim of this qualitative study was to investigate risk and protective factors associated with Veterans’ ability to age well with HIV. Participants included 25 Veterans (≥ age 50) participating in the Veterans Aging Cohort Study who were recruited from the Atlanta Veterans Affairs (VA) Medical Center. This study conducted semi-structured interviews, social network mapping, and sociodemographic and health surveys. Participants ranged in age from 50 to 72, with a mean age of 59. Most (80%) were male, and more than half (60%) were African American. Findings showed that many participants experienced adversity in childhood or early adulthood, including military sexual violence, childhood abuse, and non-military domestic violence, as well as harassment based on sexual identity. Many also had histories of substance abuse. Although military life provided stability for some, most experienced some form of instability after leaving the service, including financial difficulties and loss of valued military ties. Timing of diagnosis, whether in the military or after the military, impacted resiliency. Receiving an HIV diagnosis was an important turning point in participants’ lives characterized by either maladaptive (e.g., suicide ideation) or therapeutic (e.g., health promoting behaviors) coping strategies. Positive social support, including close relationships many developed with providers at the VA HIV clinic, was an important protective factor. The sample experienced cumulative life events that shaped their ability to age with HIV. Findings have important implications for interventions to promote Veterans’ ability to age well with HIV.

Social isolation and loneliness are common at end of life and important contributors to psychological distress, particularly among older adults. Despite evidence that isolation and loneliness are generally prevalent in assisted living (AL), little research has examined these states within the context of end of life (EOL). This study uses secondary data from an EOL study in assisted living to qualitatively characterize the experience of isolation and loneliness in African American AL residents with varying levels of psychological distress. We performed a thematic analysis using ethnographic and interview data collected from 25 residents (64% female) in a large (90+ bed) all-African American AL community in metropolitan Atlanta. We assessed psychological distress using the PHQ-4 and used this measure in qualitative analysis to explore varying patterns of social isolation and loneliness across residents. Findings showed high variability in psychological distress based on PHQ-4 scores (range 0–12, mean = 3.5, SD = 3.75). Across themes of social isolation and loneliness, we compared experiences of residents with low and high psychological distress. The maintenance of agency in the face of isolation and loneliness was the predominant pattern among the low-distress group, while passive withdrawal was consistently identified among the high-distress group. We classified these themes in terms of individual interpersonal relationships characterized by self-isolation and negotiation of family connections, as well as AL community-level relationships characterized by the liminality of the lived AL experience. Results have important implications for multilevel interventions designed to reduce social isolation, loneliness, and psychological distress among this population.

The past two decades have brought significant medical advances in antiretroviral therapies (ART) for people living with HIV/AIDS (PLWHA). This has enabled PLWHA to live longer and healthier lives than ever before. In fact, nearly half of all PLWHA in the United States are age 50+ and HIV is treated as a chronic disease, instead of a terminal illness. Despite these advancements older adults living with HIV/AIDS (OALWHA) are still a highly stigmatized population who faces challenges with their health and overall well-being. This symposium will highlight recent research on factors that play a role in successful aging among OALWHA. Our first presentation leverages a quantitative dataset of OALWHA and examines the relationship between childhood sexual abuse and ART adherence, with a particular focus on substance abuse as a potential mediator. Our second presentation features a qualitative study with OALWHA that explores their conceptions of successful aging. Our third presentation includes a sample of medical case managers who serve OALWHA and examines the feasibility of using a cognitive screening tool with this population. Cognitive screening with OALWHA can help provide early indicators of cognitive decline and initiate early intervention. Finally, our fourth presentation includes a sample of older women living with HIV/AIDS and examines their support needs and resources, particularly to help understand their complex caregiving needs. Findings from these four studies advance our understanding of OALWHA and their needs to ensure continued successful aging. This symposium informs further research and direct practice with this population.

The role of dysregulated intracellular creatine (Cr) metabolism in disuse atrophy is unknown. In this study, skeletal muscle biopsy samples were obtained after 7 days of unilateral leg immobilization (IMMOB) and from the nonimmobilized control limb (CTRL) of 15 healthy men (23.1 ± 3.5 yr). Samples were analyzed for fiber type cross-sectional area (CSA) and creatine transporter (CreaT) at the cell membrane periphery (MEM) or intracellular (INT) areas, via immunofluorescence microscopy. Creatine kinase (CK) and AMP-activated protein kinase (AMPK) were determined via immunoblot. Phosphocreatine (PCr), Cr, and ATP were measured via enzymatic analysis. Body composition and maximal isometric knee extensor strength were assessed before and after disuse. Leg strength and fat-free mass were reduced in IMMOB (~32% and 4%, respectively; P < 0.01 for both). Type II fiber CSA was smaller (~12%; P = 0.028) and intramuscular PCr lower (~13%; P = 0.015) in IMMOB vs. CTRL. CreaT protein was greater in type I fibers in both limbs ( P < 0.01). CreaT was greater in IMMOB vs. CTRL ( P < 0.01) and inversely associated with PCr concentration in both limbs ( P < 0.05). MEM CreaT was greater than INT CreaT in type I and II fibers of both limbs (~14% for both; P < 0.01 for both). Type I fiber CreaT tended to be greater in IMMOB vs. CTRL ( P = 0.074). CK was greater and phospho-to-total AMPKThr172tended to be greater,in IMMOB vs. CTRL ( P = 0.013 and 0.051, respectively). These findings suggest that modulation of intracellular Cr metabolism is an adaptive response to immobilization in young healthy skeletal muscle.

Design Thinking provides an empathy-based creative problem-solving approach to intervention and/or patient-centered care design that seeks to articulate the right problem space and propose appropriate solutions to challenges encountered by individuals in specific contexts, such as healthcare. This abstract illustrates the application of the Design Thinking approach in the context of dementia caregiving. Emory’s Roybal Center for Caregiving Mastery conducted a three-part design studio workshop with African American dementia family caregivers to understand and identify prioritized solutions to their caregiving challenges. The workshops followed the Double-Diamond design process model that is divided into four phases (Discover, Define, Develop, and Deliver) and that emphasizes divergent and convergent modes of thinking. The process first encourages divergent thinking to explore a problem space and then employs convergent thinking to identify the salient problem and develop the right solution. The workshop’s Discover stage provided a broad and deep contextual understanding of the range of unmet needs African American caregivers experience in their interactions with healthcare personnel and systems. The Define phase enabled the convergent review and prioritization of the list of needs to be addressed. The Develop phase generated 17 intervention ideas. Finally, the Deliver phase focused on converging and validating the proposed interventions. The workshops concluded that having access to caregiver-focused resources supporting mental/physical self-care, such as support groups and peer programs, can be beneficial interventions to potentially improve knowledge, awareness, and caregiving mastery among African Americans American caregivers.

Background The transgender population is composed of subgroups that are diverse in gender identity (e.g., transgender women[TW], transgender men[TM], nonbinary[NB] individuals). Compared to cisgender adults, transgender adults are more likely to report subjective cognitive decline (SCD). It remains unclear if SCD prevalence and related limitations vary by transgender subgroups. Methods 2015-2020 Behavioral Risk Factor Surveillance System data, representing 38 U.S. states that assessed SCD (confusion/memory loss happening more often/getting work over previous 12months) and gender identity were used to examine differences in SCD prevalence and SCD-related limitations by transgender subgroups, TW(n=442), TM(n=298), and NB(n=183). Age-adjusted odds ratios (OR) along with 95% confidence intervals (CI) were calculated to investigate group differences in SCD prevalence. Separate analyses compared SCD-related limitations, demographics, and health across groups among participants reporting SCD. Results SCD prevalence was highest among NB(21.3%), followed by TW(16.3%) and TM(14.1%). After accounting for age, subgroup differences remained; odds of SCD were 1.6x higher among TW compared to TM (CI:1.1–2.4, p=0.012). Among those with SCD, TW were less likely to receive help they needed with day-to-day activities when compared to TM (OR=7.9; CI:0.1–0.2, p< 0.001) and NB (OR=5.0; CI:0.1–0.4, p=0.001); and TW were more likely to be deaf (OR=4.2; CI:1.7–10.1, p=0.002) and have asthma (OR=2.8; CI:1.4–5.7, p=0.005) when compared to NB adults. No other differences were found. Conclusion Health and social inequities are not uniformly experienced across transgender subgroups, and it is important to understand how these factors impact the brain health of TW, TM, and NB adults.

Measurement of social relationships is challenging because constructs have dimensions that can be assessed at multiple levels. Consequently, researchers often operationalize social relationships differently, making it difficult to compare results with the same outcome across studies. The National Institutes of Health (NIH) commissioned the Toolbox Assessment of Neurological and Behavioral Function to create a set of harmonized measures to use across studies. The NIH Toolbox-Emotions Battery (NIHTB-EB) was normed in English and Spanish-speaking populations, which found support for a three-factor structure (negative affect, social satisfaction, and psychological well-being). Here we investigated the factor structure of the NIHTB-EB among a sample of 862 people aged 17 to 82 with and without HIV and methamphetamine use in comparison to the factor structure found in the NIH Toolbox norming project. Initial confirmatory factor analysis did not support the three-factor structure identified previously for the full sample or subsamples. Subsequent exploratory factor analyses showed a good model fit for a four-factor solution for the full sample, with slight variations in factor loading across subgroups. In addition to the three factors described in the norming project, our solution contains an additional factor, “rejection and hostility,” which includes scores from social satisfaction and negative affect in the normative population. The four-factor solution will be utilized for subsequent analyses using the NIHTB-EB in these study samples. Our analyses highlight the importance of assessing measures in distinct sub-populations to understand possible challenges to measurement invariance and ultimately allow the use of NIHTB-EB across groups.

“Design Thinking,” an innovative, human-centric approach to problem-solving, seeks to ensure that design efforts “solve the right problem.” This presentation describes the Design Thinking process and illustrates its use in the context of three design studio sessions with of family caregivers of patients at the Integrated Memory Care Clinic (IMCC), a comprehensive medical home for persons living with dementia. The Design Thinking process entails five steps – Empathize, Define, Ideate, Prototype, Test – that engage consumers/end-users to identify, as precisely as possible, the issues or concerns that are most important to them and to further identify the possible solutions that seem to most fully address these concerns. The process can be described as one of divergent and convergent thinking. In the first session, the Empathize phase, IMCC caregivers were asked to think as broadly as possible about needs not being met by IMCC. These topics were reviewed more convergently in the second session, the Define phase; here the participants agreed on a shorter, prioritized list of needs to be addressed. In the third session (that combined the Ideate and pre-Prototype stages), participants identified 14 topics (interventions) they felt should be included in this program. Finally, in the Test phase, they assessed the topics and agreed that the most important need IMCC could address would be to provide a comprehensive orientation program for new caregivers. IMCC clinicians concurred with the salience of the problem to be solved and saw addressing it as contributing substantially to the improvement of IMCC clinical care.

Anti-CD19 CAR T cell therapies have improved outcomes for non-Hodgkin lymphoma (NHL) patients. However, only 30-40% of patients treated with commercially available CART cell therapies obtain long term remission, highlighting the need for more efficacious and durable therapies. Emerging clinical data suggest several failure modes for CD19 CAR T cell therapies: including loss or downregulation of CD19 antigen, loss of co-stimulation pathways on tumor cells, exhaustion of CAR-T cells, and immunosuppressive microenvironments. To overcome these hurdles, we devised the next-generation autologous CAR-T cell therapy bbT369. bbT369 is dual targeted (CD79a/CD20) CAR T cell therapy that uses an OR gate design to limit antigen escape, has split 41BB and CD28 co-stimulatory domain architecture to augment T cell activation, and contains a knock-out of the CBLB gene to enhance potency and reduce T cell exhaustion. Here we report the first results with bbT369, demonstrating anti-lymphoma activity in in vitro assays and in vivo using xenograft mouse models. We demonstrate that CD79a and CD20 expression is B cell lineage restricted in normal human tissue and confirm that these proteins are co-expressed in diffuse large B cell samples. To target these antigens, we show a split dual-targeting CAR configuration is optimal for bbT369-directed tumor cell killing. Using an engineered megaTAL, we demonstrate high on-target activity of greater than 75% insertions and deletions (Indels) at the CBLB target site using clinical-scale manufacturing processes and low off-target activity (all off-targets less than 0.2%). In in vitro tumor co-culture assays, we show that inclusion of the CBLB gene edit in bbT369 increases Interleukin (IL)-2 production relative to an unedited anti-CD79a/CD20 CAR T cell control. Using various xenograft mouse models, we showed that bbT369 has similar or improved efficacy compared to anti-CD19 CAR drug product, including in low tumor-antigen models. In the Toledo subcutaneous xenograft model, bbT369 showed a 3-fold increase in T cell expansion compared with an unedited anti-CD79a/CD20 dual-targeting CAR T cell control. Furthermore, while a fraction of mice (3/5) receiving the unedited anti-CD79a/CD20 dual-targeting CAR T cells experienced late relapses (between 60-80 days following initial tumor clearance), all mice (n=5) receiving bbT369 were fully protected from late relapses (up to day 104 of follow-up). Collectively, the data support a first-in-human trial for bbT369 to evaluate initial safety and efficacy in NHL patients. Citation Format: Michael Certo, Christopher Baldeviano, Sharlene Adams, Martin Asimis, Alexander Astrakhan, Andy Chavkin, Maria L. Cabral, Jimmy Chu, Marie Debrue, Devina Desai, John Evans, Pinky Htun, Amanda Iniguez, Jordan Jarjour, Carl Johnson, Harini Kantamneni, Sema Kurtulus, Michael Magee, Unja Martin, Seamus McKenney, Sara Miller, Prashant Nambiar, Vinh Khang Nguyen, Mauris Nnamani, Jen Obrigewitch, Lisa Pechilis, Molly Perkins, Christopher Petersen, Jason Pinger, Cindy Rogers, Nick Rouillard, Kendal Sanson, Emily Thompson, Collin Walter, Roslyn Yi, Sarah Voytek, Philip Gregory. bbT369, a dual-targeted and CBLB gene-edited autologous CART product, demonstrates anti-lymphoma activity in preclinical mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 581.

Developing a Research Participation Enhancement and Advocacy Training Program for Diverse Seniors (DREAMS) is a two-part health education (part 1 [DP1]) and research advocacy training (part 2 [DP2]) program grounded in CBPR principles designed to address underrepresentation of socioeconomically disadvantaged and/or minority older adults in research.We examine the effects of DREAMS on health literacy, psychosocial function, beliefs/attitudes toward research, and self-efficacy in diverse older adults age 65 and older who completed only part 1 of DREAMS (n = 51) versus those who completed both parts 1 and 2 (DREAMS Team [DT]; n = 24). We hypothesized that benefits would be observed in both groups, with greater benefits seen among DT completers versus those who completed only part 1.Participants completed pre- and post-course questionnaires, including a satisfaction survey. DREAMS versus DT data were analyzed and compared using t tests, χ2 tests, analyses of variance/repeated measures analyses of variance, and Mann-Whitney U tests as appropriate. Themes were identified from open-ended survey questions and provide context for quantitative data.Findings showed significant group × time interaction in depression, participation/autonomy, and physical composite scores as well as trends toward improved attitudes toward research participation (P = .07), decreased depression (Plt; .001), and increased participation (P = .03) of the DT group. Results from the satisfaction questionnaire indicated that both programs were well-received.Findings provide strong evidence of DREAMS and particularly the DT's success in achieving our key objectives.

BACKGROUND Unavoidable periods of disuse lead to muscle atrophy and functional decline. Preventing such declines can reduce the risk of re-injury and improve recovery of normal physiological functioning. OBJECTIVES We aimed to determine the effectiveness of high-dose leucine supplementation on muscle morphology and strength during 7 d of unilateral lower-limb immobilization, and the role of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis in disuse atrophy. METHODS Sixteen healthy males (mean ± SEM age: 23 ± 1 y) underwent 7 d of unilateral lower-limb immobilization, with thrice-daily leucine (LEU; n = 8) or placebo (PLA; n = 8) supplementation (15 g/d). Before and after immobilization, muscle strength and compartmental tissue composition were assessed. A primed continuous infusion of l-[ring-13C6]-phenylalanine with serial muscle biopsies was used to determine postabsorptive and postprandial (20 g milk protein) MyoPS and MitoPS, fiber morphology, markers of protein turnover, and mitochondrial function between the control leg (CTL) and the immobilized leg (IMB). RESULTS Leg fat-free mass was reduced in IMB (mean ± SEM: -3.6% ± 0.5%; P = 0.030) but not CTL with no difference between supplementation groups. Isometric knee extensor strength declined to a greater extent in IMB (-27.9% ± 4.4%) than in CTL (-14.3% ± 4.4%; P = 0.043) with no difference between groups. In response to 20 g milk protein, postprandial MyoPS rates were significantly lower in IMB than in CTL (-22% ± 4%; P

Maintaining adequate daily protein intake is important to maintain muscle mass throughout the lifespan. In this regard, the overnight period has been identified as a window of opportunity to increase protein intake in the elderly. However, it is unknown whether pre-sleep protein intake affects next-morning appetite and, consequently, protein intake. Therefore, the purpose of the current study was to investigate the effects of a pre-sleep protein drink on next-morning appetite, energy intake and metabolism. Twelve older individuals (eight males, four females, age: 71.3 &plusmn, 4.2 years) took part in a single-blind randomised cross-over study. After a standardised dinner, participants consumed either a 40-g protein drink, isocaloric maltodextrin drink, or placebo water control before bedtime. Next-morning appetite, energy intake, resting metabolic rate (RMR), respiratory exchange rate (RER), and plasma acylated ghrelin, leptin, glucose, and insulin concentrations were assessed. No between-group differences were observed for appetite and energy intake at breakfast. Furthermore, RMR, RER, and assessed blood markers were not significantly different between any of the treatment groups. Pre-sleep protein intake does not affect next-morning appetite and energy intake and is therefore a viable strategy to increase daily protein intake in an older population.

The impact of Adverse Childhood Experiences (ACEs) on the physical and mental well–being of older adults with Opioid Use Disorder (OUD) is unclear, and we know even less about gender differences. This study explores this association and investigates additional factors (e.g., pain, depression) that may affect physical and mental well–being in this population with a focus on gender. The sample for the present analysis includes 90 adults aged 50 and older from a larger study focused on aging with OUD across eight opioid treatment programs in Georgia. We performed multivariable linear regression analyses by gender. There was a small, but significant, association between ACEs and mental well–being for men only. Other significant predictors of physical and mental well-being (e.g., insurance status, pain, satisfaction with social role, stigma) varied by gender. We discuss the importance of these gender differences in identifying appropriate areas for intervention to improve physical and mental well–being.

Regressive evolution is a widespread phenomenon that affects every living organism, yet the mechanisms underlying trait loss remain largely unknown. Cave animals enable the study of degenerative disorders, owing to the frequent loss of eyes and pigmentation among lineages evolving in the subterranean habitat. Here, we utilize the blind Mexican cavefish, Astyanax mexicanus, to investigate regressive loss of pigmentation because “ancestral” surface-dwelling morphs allow direct comparisons with cave-dwelling forms. Two genes (Oca2-albinism and Mc1r-brown) have been linked to specific pigmentation alterations in several cavefish populations. Pigment cell (melanophore) number is a complex trait governed by multiple genes, and variation in this trait may contribute to pigmentation diversity in Astyanax. To uncover genes associated with this trait, we assembled a high-resolution linkage map and used automated phenotypic scoring to quantify melanophore number variation across seven body regions in a surface × Pachon cave F2 pedigree. QTL mapping yielded several markers strongly associated with melanophore number variation in the dorsal mid-lateral stripe area and superior head region, which anchor to regions of the Astyanax genome and the zebrafish genome. Within these syntenic regions, we identified two candidate genes, Tyrp1b and Pmela, with known roles in pigmentation based on gene ontology annotation. Mutant forms of these candidate genes in other organisms cause global and regional pigmentation variation, respectively. In Astyanax, these genes harbor coding sequence mutations and demonstrate differential expression in Pachon cavefish compared to surface morphs. In sum, this work identifies genes involved with complex aspects of Astyanax pigmentation and provides insight into genetic mechanisms governing regressive phenotypic change.

Numerous animals have invaded subterranean caverns and evolved remarkably similar features. These features include loss of vision and pigmentation, and gains in nonvisual sensation. This broad convergence echoes smaller-scale convergence, in which members of the same species repeatedly evolve the same cave-associated phenotypes. The blind Mexican tetra of the Sierra de El Abra region of northeastern Mexico has a complex origin, having recurrently colonized subterranean environments through numerous invasions of surface-dwelling fish. These colonizations likely occurred ∼1-5 MYa. Despite evidence of historical and contemporary gene flow between cave and surface forms, the cave-associated phenotype appears to remain quite stable in nature. This model system has provided insight to the mechanisms of phenotypic regression, the genetic basis for constructive trait evolution, and the origin of behavioral novelties. Here, we document the rise of this model system from its discovery by a Mexican surveyor in 1936, to a powerful system for cave biology and contemporary genetic research. The recently sequenced genome provides exciting opportunities for future research, and will help resolve several long-standing biological problems. Developmental Dynamics 244:1031-1038, 2015. © 2015 Wiley Periodicals, Inc.

Chimeric antigen receptor (CAR) T cell therapy has proven to be highly effective in treating hematologic malignancies, and major efforts are being made to achieve similar efficacy in solid tumors. These efforts face multiple challenges, including off-tumor target expression and checkpoint inhibition of CAR T cell activity in the tumor microenvironment. CAR T cells are much more potent compared to antibody therapeutics, therefore there is a need for more stringent CAR T target safety assessment to avoid adverse events resulting from “on-target/off-tumor” activity. Furthermore, it is critical to track and monitor CAR+ T cells within the context of intact tissue and tumor to understand the mechanisms underlying off-tumor toxicity and efficacy in tumor killing. In this study we employed the RNAscope in situ hybridization (ISH) technology to assess target expression specificity and to track CAR T cell distribution and activation in xenograft and host tissues using the RPMI-8226 xenograft mouse model, which expresses both BCMA and ROR1. The anti-ROR1 CAR T cells used in this study recognized both the mouse and human proteins whereas the anti-BCMA CAR T cells recognized only the human protein. RNA ISH revealed that BCMA was only expressed in the xenograft tumor and in no mouse organs, while ROR1 was found to be expressed in the xenograft tumor as well as at low levels in mouse lung and liver. Duplex RNA ISH assay with probes targeting the CAR 3’ UTR and either IFNG or GZMB mRNA allowed highly sensitive and specific detection of CAR T cells and their activation state in both tumor and normal tissues from vehicle, anti-ROR1 CAR T cell, or anti-BCMA CAR T cell treated mice. Activated anti-BCMA CAR T cells expressing GZMB and IFNG were found only in the xenograft tumor, where BCMA was expressed. In contrast, activated anti-ROR1 CAR T cells were found almost exclusively in mouse lung and liver with very few anti-ROR1 CAR T cells being found in the xenograft tumor, consistent with the previously observed pulmonary and hepatic toxicity of anti-ROR1 CAR T cells that was not predicted by IHC analysis of ROR1 protein presumably due to lack of antibody sensitivity. Lastly, we employed a multiplex ISH-IHC approach to confirm the presence of activated anti-BCMA CAR T cells in the xenograft tumor through simultaneous detection of the BCMA CAR 3’ UTR, IFNG, GZMB, and CD3, validating the anti-tumor activity of anti-BCMA CAR T cells. These data thus demonstrate how the RNAscope ISH assay can be utilized for CAR T efficacy and safety/toxicity assessment in preclinical models by detecting very low levels of target antigen expression in off-tumor tissues and monitoring CAR T cell pharmacodynamics and activation in tumor models. This technology has equal utility in understanding both CAR T and TCR-T cell activity in patient tumors. Citation Format: Helly Pimentel, Helen Jarnagin, Hailing Zong, Courtney Todorov, Kenneth Ganley, Fay Eng, Kevin Friedman, Molly Perkins, Shannon Grande, Courtney M. Anderson, Bingqing Zhang, Christopher Bunker, James B. Rottman, Xiao-Jun Ma. Preclinical safety, biodistribution, and tumor infiltration analysis of CAR T cell targets using in situ hybridization technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2324.

Numerous animals have invaded subterranean caverns and evolved remarkably similar features. These features include loss of vision and pigmentation, and gains in nonvisual sensation. This broad convergence echoes smaller-scale convergence, in which members of the same species repeatedly evolve the same cave-associated phenotypes. The blind Mexican tetra of the Sierra de El Abra region of northeastern Mexico has a complex origin, having recurrently colonized subterranean environments through numerous invasions of surface-dwelling fish. These colonizations likely occurred ∼1-5 MYa. Despite evidence of historical and contemporary gene flow between cave and surface forms, the cave-associated phenotype appears to remain quite stable in nature. This model system has provided insight to the mechanisms of phenotypic regression, the genetic basis for constructive trait evolution, and the origin of behavioral novelties. Here, we document the rise of this model system from its discovery by a Mexican surveyor in 1936, to a powerful system for cave biology and contemporary genetic research. The recently sequenced genome provides exciting opportunities for future research, and will help resolve several long-standing biological problems. Developmental Dynamics 244:1031-1038, 2015. © 2015 Wiley Periodicals, Inc.

Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells for treatment requires ex vivo culture to facilitate CAR gene transfer and to achieve a therapeutic dose of the modified cells. Recent data suggests that specific T cell subtypes can provide enhanced anti-tumor efficacy, spurring efforts to optimize the production of therapeutic T cells via the cumbersome physical isolation of central memory T cells or culture in cytokines such as IL-7 and IL-15. Here we explored the potential for a simple culture modification to improve the therapeutic potential of CAR T cells without adding manufacturing complexity. To this end, we produced CAR T cells specific to B cell maturation antigen (BCMA) using standard IL-2 culture conditions supplemented with a PI3K inhibitor, or with IL-7 and IL-15 in place of IL-2. The in vivo activity of CAR T cells was studied in mouse models of human Burkitt's lymphoma (Daudi) and multiple myeloma (RPMI-8226), both of which express BCMA. In the Daudi model, NSG mice were injected intravenously with 2 × 106 tumor cells and allowed to accumulate a large tumor burden to model late stage disease observed in relapsed and refractory lymphoma. In this advanced disease model, anti-BCMA CAR T cells (4 × 106/mouse) cultured either in IL-2 or IL-7 and IL-15 had little or no effect on tumor growth (p = 0.22 and 0.23, respectively) and all mice succumbed to tumors within two weeks of treatment. In contrast, all animals treated with the same number of anti-BCMA CAR T cells cultured with PI3K inhibition survived and had complete long-term tumor regression (p = 0.003). The same anti-BCMA CAR T cells were studied in a model of multiple myeloma. NSG mice were injected subcutaneously with 107 RPMI-8226 cells and 22 days later received a single administration of anti-BCMA CAR T cells (4 × 105/mouse) cultured under various conditions. In this model, tumor regression occurred regardless of in vitro culture conditions. To model tumor relapse and evaluate CAR T cell durability, surviving animals were re-challenged with RPMI-8226 cells on the opposite flank two weeks after initial tumor clearance. In contrast to other conditions, all animals treated with anti-BCMA CAR T cells cultured with PI3K inhibition were protected against subsequent tumor challenge (p = 0.005). This improved therapeutic activity of anti-BCMA CAR T cells cultured with PI3K inhibition was associated with an increased frequency of CD62L+ CD8+ T cells in the drug product (p < 0.001) suggesting enrichment of this distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion with IL-2 may generate an improved anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Citation Format: Shannon Grande, Molly R. Perkins, Amanda Hamel, Holly M. Horton, Fay Eng, Claire J. Rhodes, Tracy E. Garrett, Sara M. Miller, John W. Evans, Howard J. Latimer, Christopher Horvath, Michael Kuczewski, Kevin Friedman, Richard A. Morgan. Inhibition of the PI3K/Akt pathway during CAR T cell production results in enhanced efficacy across multiple in vivo tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2296.

B cell maturation antigen (BCMA) is expressed on most multiple myeloma (MM) and some lymphoma cells, yet normal tissue expression is limited to plasma and some B cells. Here we describe the development of a CAR T cell that targets BCMA. We tested 19 BCMA-specific single chain variable fragments (scFv) linked to CD137 (4-1BB) and CD3zeta T cell signaling domains. The activity of each CAR T cell candidate differed despite targeting the same antigen. One anti-BCMA CAR T cell candidate (bb2121) was selected based on strong surface CAR expression, superior biological activity to multiple BCMA+ cell lines, and low antigen-independent activity. In vitro analysis demonstrated that antigen-independent activity was associated with T cell differentiation characterized by significantly lower CD62L expression (p=0.003). To determine the impact on tumor control, CAR T cells with antigen-independent activity, or bb2121 CAR T cells, were used to treat an NSG mouse model of MM (RPMI-8226). Immunohistochemical analysis found MM-infiltrated bb2121 T cells within 5 days and mice showed complete regressions by 12 days post treatment. In contrast, mice that received CAR T cells with antigen-independent activity had delayed regressions that occurred 20 days post treatment. Robust recognition of as few as 220 BCMA molecules/cell (compared to >10,000 molecules on MM cell lines) permitted reactivity to primary chronic lymphocytic leukemia (CLL) and other CD19+ B cell tumor lines. Indeed comparable tumor regressions were observed after administration of bb2121 or anti-CD19 CAR T cells into an NSG mouse model of disseminated Burkitt's lymphoma (Daudi: 1,170 BCMA and 267,000 CD19 molecules). Prior investigators have shown improved therapeutic efficacy by enriching for memory CAR T cells, yet current antibody-based selection methods are expensive and difficult to scale. Unexpectedly, we found bb2121 manufacture in the presence of a PI3-kinase inhibitor enriched for memory-like CAR T cells without a complicated cell sorting procedure. bb2121 CAR T cells cultured with a PI3K inhibitor expressed markers associated with T cell memory including CD62L, CD127, CD197, and CD38. In a first “stress test,” of advanced disseminated lymphoma (Daudi), NSG mice were administered an amount of bb2121 CAR T cells that failed to control tumor outgrowth. Mice administered the same number of bb2121 CAR T cells cultured with a PI3K inhibitor resulted in complete tumor regressions. A defining property of memory T cells is durability despite multiple antigen encounters. In a second “stress test,” bb2121 CAR T cell durability was evaluated after initial MM (RPMI-8226) clearance. Two weeks post-tumor elimination, mice were re-challenged with tumor on the opposite flank without an additional dose of CAR T cells. None of the mice treated with bb2121 CAR T cells prevented tumor outgrowth in this model. In contrast, all mice treated with bb2121 CAR T cells cultured with a PI3K inhibitor were able to control the tumor re-challenge. These data demonstrate that a potent, antigen-dependent, memory-like BCMA CAR T cell produced with an industrially scalable manufacturing process has promise for robust tumor regressions in clinical applications.

Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells is challenged by the necessity to produce a unique drug product for each patient. Each treatment requires ex vivo culture of patient T cells to facilitate CAR gene transfer and to achieve therapeutic amounts of T cells. Paradoxically, ex vivo culture with IL-2 also decreases CAR T cell activity. Some investigators have proposed isolating central memory T cells (thought to be enriched for therapeutic T cells), yet isolation techniques are cumbersome and costly to scale commercially. Culture of T cells in IL-7 and IL-15 has also been shown by several investigators to improve therapeutic activity. Here we explored the potential for culture modifications to improve the therapeutic potential of CAR T cells without adding complexity to manufacturing. We tested this hypothesis using CAR T cells specific to B cell maturation antigen (BCMA) manufactured using standard IL-2 culture with an inhibitor of PI3K added to the media, or with IL-7 and IL-15 in place of IL-2. The in vivo activity was studied in NSG mouse models of human Burkitt's lymphoma (Daudi), and multiple myeloma (RPMI-8226), both of which express BCMA. In the lymphoma model, NSG mice were injected intravenously (IV) with 2 x 106 Daudi cells and allowed to accumulate a large tumor burden before being treated with 4 x 106 CAR+ T cells on day 18 post-tumor injection. At this late time point post implantation, mice had highly disseminated Daudi tumor (our goal was to model late stage disease observed in relapsed and refractory lymphoma). In this model of advanced disease, IL-2 cultured anti-BCMA CAR T cells had no effect on tumor growth (p = 0.22) and all mice succumbed to the tumors within two weeks after treatment. Anti-BCMA CAR T cells grown in IL-7 and IL-15 also failed to control tumor growth (p = 0.23). In sharp contrast, all animals treated with anti-BCMA CAR T cells cultured with the PI3K inhibitor survived and experienced complete long-term tumor regression (p=0.003). The same anti-BCMA CAR T cells were used in a model of multiple myeloma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells, and at 22 days post-implantation mice received a single IV administration of anti-BCMA CAR T cells (4 x 105 CAR+ T cells/mouse) cultured under various conditions. In this model, all treatment groups demonstrated tumor regression, regardless of the in vitro culture conditions. To evaluate CAR T cell durability, two weeks after initial tumor clearance, surviving animals were then re-challenged with RPMI-8226 cells on the opposite flank to model tumor relapse. We found that only animals that had been treated with anti-BCMA CAR T cells cultured with PI3K inhibition were immune to subsequent tumor challenge (p=0.005). Given the superior in vivo efficacy of anti-BCMA CAR T cells cultured with PI3K inhibition, we sought to identify phenotypic characteristics associated with the improved therapeutic activity. Anti-BCMA CAR T cells cultured with PI3K inhibition contained an increased frequency of CD62L+ CD8 T cells in the final product (p < 0.001) suggesting improved expansion of a distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion with IL-2 may generate a superior anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Disclosures Perkins: bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.