Hu8F4-CAR T cells with mutated Fc spacer segment improve target-specificity and mediate anti-leukemia activity in vivo.

Hu8F4 is a T cell receptor (TCR)-like antibody with high affinity for leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is comprised of the Hu8F4 scFv, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain, and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from AML patients in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor (FcgR)-expressing cells. We generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with FcgR-expressing cells in vivo. The primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2+ PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice. Herein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor-binding-induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo.

The Global ALPL gene variant classification project: Dedicated to deciphering variants.

BACKGROUND: Hypophosphatasia (HPP) is an inherited multisystem disorder predominantly affecting the mineralization of bones and teeth. HPP is caused by pathogenic variants in ALPL, which encodes tissue non-specific alkaline phosphatase (TNSALP). Variants of uncertain significance (VUS) cause diagnostic delay and uncertainty amongst patients and health care providers. RESULTS: The ALPL gene variant database (https://alplmutationdatabase.jku.at/) is an open-access archive for interpretation of the clinical significance of variants reported in ALPL. The database contains coding and non-coding variants, including single nucleotide variants, insertions/deletions and structural variants affecting coding or non-coding sequences of ALPL. Each variant in the database is displayed with details explaining the corresponding pathogenicity, and all reported genotypes and phenotypes, including references. In 2021, the ALPL gene variant classification project was established to reclassify VUS and continuously assess and update genetic, phenotypic, and functional variant information in the database. For this purpose, the database provides a unique submission system for clinicians, geneticists, genetic counselors, and researchers to submit VUS within ALPL for classification. An international, multidisciplinary consortium of HPP experts has been established to reclassify the submitted VUS using a multi-step process adhering to the stringent ACMG/AMP variant classification guidelines. These steps include a clinical phenotype assessment, deep literature research including artificial intelligence technology, molecular genetic assessment, and in-vitro functional testing of variants in a co-transfection model to measure ALP residual activity. CONCLUSION: This classification project and the ALPL gene variant database will serve the global medical community, widen the genotypic and phenotypic HPP spectrum by reporting and characterizing new ALPL variants based on ACMG/AMP criteria and thus facilitate improved genetic counseling and medical decision-making for affected patients and families. The project may also serve as a gold standard framework for multidisciplinary collaboration for variant interpretation in other rare diseases.

Multi-faceted role of LRP1 in the immune system.

Graft versus host disease (GVHD) represents the major complication after allogeneic hematopoietic stem cell transplantation (Allo-SCT). GVHD-prone patients rely on GVHD prophylaxis (e.g. methotrexate) and generalized anti-GVHD medical regimen (glucocorticoids). New anti-GVHD therapy strategies are being constantly explored, however there is an urgent need to improve current treatment, since GVHD-related mortality reaches 22% within 5 years in patients with chronic GVHD. This review is an attempt to describe a very well-known receptor in lipoprotein studies - the low-density lipoprotein receptor related protein 1 (LRP1) - in a new light, as a potential therapeutic target for GVHD prevention and treatment. Our preliminary studies demonstrated that LRP1 deletion in donor murine T cells results in significantly lower GVHD-related mortality in recipient mice with MHC (major histocompatibility complex) -mismatched HSCT. Given the importance of T cells in the development of GVHD, there is a significant gap in scientific literature regarding LRP1's role in T cell biology. Furthermore, there is limited research interest and publications on this classical receptor molecule in other immune cell types. Herein, we endeavor to summarize existing knowledge about LRP1's role in various immune cells to demonstrate the possibility of this receptor to serve as a novel target for anti-GVHD treatment.

Procoagulant Activity of Blood and Microvesicles Is Disturbed by Pneumococcal Pneumolysin, Which Interacts with Coagulation Factors.

The coagulation and contact systems are parts of the innate immune system as they prevent bleeding and dissemination of pathogens and also contribute to microbial killing by inflammatory reactions and the release of antimicrobial peptides. Here, we investigated the influence of Streptococcus pneumoniae on the coagulation and contact system. S. pneumoniae (pneumococci), but no other investigated streptococcal species, impairs coagulation of blood by autolysis and release of pneumolysin. Defective blood coagulation results from the lysis of tissue factor-producing mononuclear cells and their procoagulant microvesicles, which are the main trigger for blood coagulation during sepsis. In addition, pneumolysin binds coagulation and contact system factors, but this does not result in activation. Thus, pneumococci modulate activation of the coagulation system by releasing pneumolysin, which could potentiate lung injury during pneumonia.

In training emergency physicians the carotid artery Doppler with passive leg raise, does previous sonographic experience influence scan time and competency?

BACKGROUND: Determination of fluid responsiveness (FR) associated with intravascular fluid resuscitation in hypotensive patients poses a challenge, with current best evidence methods fraught with poor retest reliability and difficulty in image acquisition (Osman, Crit Care Med 2007; 35: 64; Marik, Crit Care Med 2009; 37: 2642). Doppler carotid blood flow with passive leg raise (PLR) is a recent modality for determining FR (Marik, Chest 2013; 143: 364). PURPOSE: This study aimed to determine whether emergency physicians with limited ultrasound experience can reliably acquire this skill. METHOD: This prospective study recruited 60 emergency physicians with varying experience, who underwent a 3-step learning programme. Participants performed carotid velocity time integral (VTi) Doppler on healthy subjects, followed by repeat measurements in the PLR position. A 16-point checklist and time recorded were assessed for each sonographer, with each participant completing a post-study questionnaire to evaluate perceived competence and ease of skill acquisition. RESULTS: Of the 60 emergency physicians recruited, 37 (61.6%) were inexperienced and 23 (38.4%) were experienced. Against the 16-point assessment, 61% completed assessment without any errors. Fifty-six out of 60 (94.3%) completed the assessment to acceptable standard with errors recognised and corrected, and four participants (6.7%) made critical errors without correction (Figure 1). Average (±SEM) total scan time was 4:52 ± 0:19, with no significant difference found between inexperienced and experienced groups. CONCLUSIONS: This study demonstrated feasibility to train emergency physicians, demonstrating that average FR assessment was obtained within 5 min, with no difference between prior experience in scan quality/time taken. 94% completed the scan to acceptable standards, demonstrating ease of carotid Doppler flow with PLR to provide critical information in management of the hypotensive patient.

Perfluoroalkyl substances exposure and immunity, allergic response, infection, and asthma in children: review of epidemiologic studies.

BACKGROUND: Increased exposure to perfluoroalkyl substances (PFAS) potentially affects infant and childhood health through immunosuppression. Given rapidly evolving research on PFAS, it is important to comprehensively examine the impact of PFAS exposure among the pediatric population as new research becomes available due to potential fragility of the developing immune system. OBJECTIVES: This review assessed the effects of PFAS fetal, infant and childhood exposures upon the development of immune function during early life stages. METHODS: Researchers completed a literature review, searching PubMed for human studies published since 2010 for PFAS and health outcomes among infants and children. Included articles incorporated key search terms in the title or abstract; non-research reports and non-English papers were excluded. The search identified 518 studies for possible inclusion. Following hands-on review, 34 were determined relevant. Subsequent analyses found 8 additional relevant articles, totaling 42 studies. RESULTS: Major immune-related sequelae from PFAS exposures on infant and child health outcomes documented in recent literature include:• Strong indication of immunosuppression, with diminished childhood antibody response to vaccination, particularly with PFOA, PFOS and PFHxS exposures.• Some indication of increased risks of childhood infectious diseases/infections, particularly from PFOS exposures.• Limited indication of an effect of PFAS exposure on allergic reactions/allergen specific IgE antibodies.• Limited indication of an effect of PFAS exposure on atopic dermatitis (AD).• Limited indication of an effect of PFAS exposure on asthma and lung function. CONCLUSION: This review summarizes recent findings of PFAS effects on infant and childhood immune health. Evidence of immunosuppression, diminished vaccine efficacy, and increased risk of infections, allergies, asthma and AD were described following in utero, infant, and early childhood PFAS exposures. Further investigation is warranted to characterize PFAS exposure pathways and potential modes of action in relation to PFAS effects on the developing immune system. Incontrovertible proof of PFAS immunotoxic effects could optimally be obtained by a large prospective study cohort of mothers and children from infancy through school-age. Regular assessments of circulating antibodies and response to infant and childhood vaccines during growth years could prove invaluable.

Novel myeloperoxidase-derived HLA-A2-restricted peptides as therapeutic targets against myeloid leukemia.

BACKGROUND AIMS: Human myeloperoxidase has been shown to be overexpressed in many types of leukemia, such as chronic myeloid leukemia, acute myeloid leukemia and myelodysplastic syndrome. The authors identified two myeloperoxidase-derived HLA-A2-restricted peptides, MY4 and MY8, as novel leukemia-associated antigens. METHODS: Ex vivo-elicited MY4- and MY8-specific cytotoxic T lymphocytes were generated, and tested for leukemia cell lysis in vitro and in NOD/SCID AML xenograft model. RESULTS: These MY4- and MY8-specific cytotoxic T lymphocytes killed leukemic blasts while sparing healthy donor bone marrow cells. In addition, co-injection of MY4- and MY8-specific cytotoxic T lymphocytes into nonobese diabetic/severe combined immunodeficiency mice with acute myeloid leukemia drastically reduced tumor burden in vivo. The authors also found that MY4- and MY8-specific T cells could be detected in the peripheral blood mononuclear cells of allogeneic stem cell transplant recipients. CONCLUSIONS: These antigen-specific T cells were significantly increased in blood samples from patients compared with healthy donors, suggesting that both MY4 and MY8 are immunogenic and that MY4- and MY8-specific cytotoxic T lymphocytes may play a role in reducing leukemia in vivo. Thus, the discovery of MY4 and MY8 as novel leukemia-associated antigens paves the way for targeting these antigens in immunotherapy against myeloid leukemia.

What Motivates Smokers to Switch to ENDS? A Qualitative Study of Perceptions and Use.

Switching completely from cigarettes to electronic nicotine delivery systems (ENDS) may reduce health risks for addicted smokers. This paper provides information about perceptions and other factors that may influence smokers' ENDS use and substitution for cigarettes. We conducted 12 online focus groups (N = 61) among smokers who had never tried using ENDS (Never Users, N = 11), currently used both cigarettes and ENDS (Dual Users, N = 21), used but discontinued ENDS (Rejectors, N = 14), and switched completely to ENDS use (Switchers, N = 15). Thematic analysis was used to interpret the transcripts. Participants described initial interest in trying ENDS in hopes of quitting smoking and because of convenience (i.e., due to rules, regulations, or social norms). Risk perceptions and higher prices relative to cigarettes were reported as disadvantages of ENDS that discouraged initiation. Dual Users and Rejectors reported product problems (e.g., products breaking) and dissatisfaction (i.e., inability to satisfy cravings for cigarettes) as factors that lowered their substitutability for cigarettes or led to discontinuing ENDS use. Switchers indicated that satisfaction, lack of product problems, and perceived safety facilitated successfully switching from cigarette smoking to exclusive ENDS use. However, Switchers reported trying many products before they found ones that satisfied their needs. We recommend that policymakers consider the potential impact of tobacco control policies on smokers' motivation and ability to switch completely from cigarettes to ENDS.

Two unique HLA-A*0201 restricted peptides derived from cyclin E as immunotherapeutic targets in leukemia.

Immunotherapy targeting leukemia-associated antigens has shown promising results. Because of the heterogeneity of leukemia, vaccines with a single peptide have elicited only a limited immune response. Targeting several peptides together elicited peptide-specific cytotoxic T lymphocytes (CTLs) in leukemia patients, and this was associated with clinical responses. Thus, the discovery of novel antigens is essential. In the current study, we investigated cyclin E as a novel target for immunotherapy. Cyclin E1 and cyclin E2 were found to be highly expressed in hematologic malignancies, according to reverse transcription polymerase chain reaction and western blot analysis. We identified two HLA-A*0201 binding nonameric peptides, CCNE1M from cyclin E1 and CCNE2L from cyclin E2, which both elicited the peptide-specific CTLs. The peptide-specific CTLs specifically kill leukemia cells. Furthermore, CCNE1M and CCNE2L CTLs were increased in leukemia patients who underwent allogeneic hematopoietic stem cell transplantation, and this was associated with desired clinical outcomes. Our findings suggest that cyclin E1 and cyclin E2 are potential targets for immunotherapy in leukemia.

Sexually Transmitted Disease Partner Services Costs, Other Resources, and Strategies Across Jurisdictions to Address Unique Epidemic Characteristics and Increased Incidence.

BACKGROUND: Sexually transmitted disease (STD) partner services (PS) are a core component of STD programs. Data on costs are needed to support PS programming. METHODS: In Washington State STD PS programs, disease intervention specialists (DIS) conduct telephone-based interviews and occasional field visits, offer expedited partner therapy to heterosexuals with gonorrhea or chlamydia, and promote human immunodeficiency virus (HIV) testing, preexposure prophylaxis, and HIV care. We conducted activity-based microcosting of PS, including: observational and self-reported time studies and interviews. We analyzed cost, surveillance, and service delivery data to determine costs per program outcomes. RESULTS: In King, Pierce, and Spokane counties, respectively, DIS allocated 6.5, 6.4, and 28.8 hours per syphilis case and 1.5, 1.6, and 2.9 hours per gonorrhea/chlamydia case, on average. In 2016, each full-time DIS investigated 270, 268, and 61 syphilis and 1177, 1105, and 769 gonorrhea/chlamydia cases. Greater than 80% of syphilis cases in King and Pierce were among men who have sex with men versus 38% in Spokane. Disease intervention specialists spent 12% to 39% of their time actively interviewing cases and notifying partners (clients), and the remaining time locating clients, coordinating and verifying care, and managing case reports. Time spent on expedited partner therapy, HIV testing, and referrals to HIV treatment or preexposure prophylaxis, was minimal (<5 minutes per interview) at locations with resources outside PS staff. Program cost-per-interview ranged from US $527 to US $2210 for syphilis, US $219 to US $484 for gonorrhea, and US $164 to US $547 for chlamydia. DISCUSSION: The STD PS resource needs depended on epidemic characteristics and program models. Integrating HIV prevention objectives minimally impacted PS-specific program costs. Results can inform program planning, future budget impact, and cost-effectiveness analyses.

Role of Structural Distortion in Stabilizing Electrosynthesized Blue-Emitting Phosphorene Quantum Dots.

Luminescent phosphorene quantum dots (PQDs) have emerged as fascinating nanomaterials for potential applications in optoelectronics, catalysis, and sensing. Herein, we investigate the structural distortion of black phosphorus (BP) under an applied electric field to yield blue luminescent PQDs [average diameter 8 ± 1.5 nm ( N = 60)]. The electrosynthesized PQDs exhibit photoluminescence emission independent of excitation wavelength with 84% quantum efficiency. Structural distortion that occurred during the transformation of BP to PQDs is confirmed by results obtained during transmission electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. Further, using first-principles-based density functional theory, calculations on oxygenated and nonoxygenated PQDs augment the experimental observations that an optimum oxygen content maintains the structural integrity of PQDs, above which the structural robustness of PQDs is drastically diminished.

Fucosylation Enhances the Efficacy of Adoptively Transferred Antigen-Specific Cytotoxic T Lymphocytes.

PURPOSE: Inefficient homing of adoptively transferred cytotoxic T lymphocytes (CTLs) to tumors is a major limitation to the efficacy of adoptive cellular therapy (ACT) for cancer. However, through fucosylation, a process whereby fucosyltransferases (FT) add fucose groups to cell surface glycoproteins, this challenge may be overcome. Endogenously fucosylated CTLs and ex vivo fucosylated cord blood stem cells and regulatory T cells were shown to preferentially home to inflamed tissues and marrow. Here, we show a novel approach to enhance CTL homing to leukemic marrow and tumor tissue. EXPERIMENTAL DESIGN: Using the enzyme FT-VII, we fucosylated CTLs that target the HLA-A2-restricted leukemia antigens CG1 and PR1, the HER2-derived breast cancer antigen E75, and the melanoma antigen gp-100. We performed in vitro homing assays to study the effects of fucosylation on CTL homing and target killing. We used in vivo mouse models to demonstrate the effects of ex vivo fucosylation on CTL antitumor activities against leukemia, breast cancer, and melanoma. RESULTS: Our data show that fucosylation increases in vitro homing and cytotoxicity of antigen-specific CTLs. Furthermore, fucosylation enhances in vivo CTL homing to leukemic bone marrow, breast cancer, and melanoma tissue in NOD/SCID gamma (NSG) and immunocompetent mice, ultimately boosting the antitumor activity of the antigen-specific CTLs. Importantly, our work demonstrates that fucosylation does not interfere with CTL specificity. CONCLUSIONS: Together, our data establish ex vivo CTL fucosylation as a novel approach to improving the efficacy of ACT, which may be of great value for the future of ACT for cancer.

Comparative Validation Study to Demonstrate the Equivalence of an Alternate Next-Day Enrichment Protocol for VIP® Gold for Salmonella Method to Culture Methods for the Detection of Salmonella in Selected Foods and Environmental Surfaces.

Background: VIP® Gold for Salmonella is a lateral flow immunodetection device that was validated by AOAC in 1999 as Official Method of Analysis 999.09. It was improved upon in 2009 by introducing gold colloid as the detection method. Objective: A simple next-day enrichment protocol using modified enterohemorrhagic Escherichia coli media was developed for the VIP Gold for Salmonella to improve the time-to-results and laboratory work flow. Methods: We tested 128 Salmonella strains, representing all serotypes from A to Z and 51 to 66 as well as 50 non-Salmonella strains for inclusivity/exclusivity. Performance of the VIP using the new enrichment protocol was compared with the U.S. Department of Agriculture (USDA) Microbiology Laboratory Guidebook reference culture procedure for the detection of Salmonella in ready-to-eat poultry, roast beef, and chicken carcass rinsate. VIP performance was also compared with the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) for the detection of Salmonella from raw spinach, raw almonds, raw pasta, and environmental surfaces (stainless steel, rubber, and plastic). Results: The VIP detected all 128 of Salmonella strains and none of the 50 non-Salmonella strains. There was no statistically significant difference in the numbers of positive results with VIP Gold for Salmonella protocol compared with appropriate USDA-Food Safety and Inspection Service or FDA-BAM reference methods for any of these matrixes. Conclusions: This new enrichment protocol has met all the requirements to be approved as a Performance Tested MethodSM. Highlights: The new enrichment protocol will improve the time-to-results and allow quicker decisions about the contamination of food products.

Evaluation of the TRANSIA® PLATE Staphylococcal Enterotoxins Kit for the Detection of Staphylococcal Enterotoxins in Selected Foods.

The TRANSIA® PLATE Staphylococcal Enterotoxins enzyme immunoassay (EIA) was validated according to AOAC INTERNATIONAL guidelines for validating qualitative binary chemistry and microbiological methods. Five food matrixes were analyzed to determine the probability of detection (POD) for staphylococcal enterotoxins (SE), including SEA, SEB, SEC1, SEC2, SEC3, SED, and SEE, by the TRANSIA ^{PLATE Staphylococcal Enterotoxins EIA. The food matrixes tested were food types implicated in staphylococcal enterotoxin outbreaks and included raw milk cheese, liquid infant formula, eclairs, ready-to-eat ham, and canned mushrooms. Cheese and infant formula were tested with and without dialysis/concentration. The infant formula was also tested by an independent laboratory. Each food matrix was inoculated with specific toxins at low levels to yield fractional recoveries (0.015-0.20 ng/g of food) for POD analysis. One hundred percent recovery was achieved at concentrations ranging from <0.10 ng/g to 0.25 ng/g of toxin in the various food matrixes. At the same time, 50 Staphylococcus aureus strains known to produce toxins and 30 non-toxin producing bacteria (including 22 Staphylococcus strains) were grown and tested. All the SE toxin-producing strains yielded positive results and all of the exclusivity strains were negative. Robustness studies showed that changes in sample volume, sample pH, and EIA assay temperature had no significant effect on performance. Stability studies showed that kits stored for 12+ months performed as well as newly made kits. This assay has been approved as a Performance Tested MethodSM.}

Comparative Validation Study to Demonstrate the Equivalence of an Alternate Next-Day Enrichment Protocol for the TRANSIA® PLATE Salmonella Gold Method to Culture Methods for the Detection of Salmonella in Selected Foods and Environmental Surfaces.

TRANSIA® PLATE Salmonella Gold is an ELISA that was validated by Association Française de Normalisation (AFNOR) in 2001 and as a Performance Tested MethodSM (PTM) by AOAC in 2006 (PTM No. 010602) as a two-step enrichment protocol requiring 48 h. A simple next-day enrichment protocol using modified Enterohemorrhagic Escherichia coli media was developed for the TRANSIA PLATE Salmonella Gold to improve the time-to-results and laboratory work flow. We tested 128 Salmonella strains, representing all serotypes from A though Z and 51-66. TRANSIA PLATE Salmonella Gold detected all 128 of these strains. None of the 50 non-Salmonella strains were detected by TRANSIA PLATE Salmonella Gold. Performance of TRANSIA PLATE Salmonella Gold using the new enrichment protocol was compared with U.S. Department of Agriculture Microbiology Laboratory Guidebook reference culture procedure for the detection of Salmonella in ready-to-eat poultry, ready-to-eat beef, and chicken carcass rinsate. In addition, TRANSIA PLATE Salmonella Gold performance was compared with U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) for the detection of Salmonella from raw spinach, raw almonds, raw pasta, and environmental surfaces (stainless steel, rubber, and plastic). There was no statistically significant difference in the numbers of positive results TRANSIA PLATE Salmonella Gold protocol compared with the appropriate U.S. Department of Agriculture Food Safety and Inspection Service or FDA-BAM reference methods for any of these matrixes. Robustness testing demonstrated that the introduction of small changes in the normal assay parameters had no impact on the method performance. This new enrichment protocol has been approved as a Third Level modification to Performance Tested Method 010602.

Membrane-Associated Proteinase 3 on Granulocytes and Acute Myeloid Leukemia Inhibits T Cell Proliferation.

Proteinase 3 (P3), a serine protease expressed by myeloid cells, localized within azurophil granules, and also expressed on the cellular membrane of polymorphonuclear neutrophils (PMN), is the target of autoimmunity in granulomatosis with polyangiitis. PR1, an HLA-A2 restricted nonameric peptide derived from P3, has been targeted effectively in myeloid leukemia. We previously showed (Molldrem et al. 2003. JClinInvest 111: 639-647) that overexpression of P3 in chronic myeloid leukemia induces apoptosis of high-affinity PR1-specific T cells, leading to deletional tolerance and leukemia outgrowth. In this study, we investigated the effect of membrane P3 (mP3)-expressing PMN and acute myeloid leukemia (AML) blasts on the proliferation of CD4 and CD8 T cells in vitro. We demonstrate that mP3-expressing PMN significantly inhibits autologous healthy donor T cell proliferation but does not affect cytokine production in activated T cells and that this effect requires cell proximity and was abrogated by P3 blockade. This inhibition required P3 enzyme activity. However, suppression was not reversed by either the addition of catalase or the inhibition of arginase I. In addition to P3 blockade, anti-low density lipoprotein receptor-related protein 1 (LRP1) Ab also restored T cells' capacity to proliferate. Last, we show dose-dependent inhibition of T cell proliferation by mP3-expressing AML blasts. Together, our findings demonstrate a novel mechanism whereby PMN- and AML-associated mP3 inhibits T cell proliferation via direct LRP1 and mP3 interaction, and we identify P3 as a novel target to modulate immunity in myeloid leukemia and autoimmune disease.

Targeting the Leukemia Antigen PR1 with Immunotherapy for the Treatment of Multiple Myeloma.

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies.Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples.Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivoConclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386-96. ©2018 AACR.

A Novel T-Cell Engaging Bi-specific Antibody Targeting the Leukemia Antigen PR1/HLA-A2.

Despite substantial advances in the treatment of acute myeloid leukemia (AML), only 30% of patients survive more than 5 years. Therefore, new therapeutics are much needed. Here, we present a novel therapeutic strategy targeting PR1, an HLA-A2 restricted myeloid leukemia antigen. Previously, we have developed and characterized a novel T-cell receptor-like monoclonal antibody (8F4) that targets PR1/HLA-A2 and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC). To improve the potency of 8F4, we adopted a strategy to link T-cell cytotoxicity with a bi-specific T-cell-engaging antibody that binds PR1/HLA-A2 on leukemia and CD3 on neighboring T-cells. The 8F4 bi-specific antibody maintained high affinity and specific binding to PR1/HLA-A2 comparable to parent 8F4 antibody, shown by flow cytometry and Bio-Layer Interferometry. In addition, 8F4 bi-specific antibody activated donor T-cells in the presence of HLA-A2+ primary AML blasts and cell lines in a dose dependent manner. Importantly, activated T-cells lysed HLA-A2+ primary AML blasts and cell lines after addition of 8F4 bi-specific antibody. In conclusion, our studies demonstrate the therapeutic potential of a novel bi-specific antibody targeting the PR1/HLA-A2 leukemia-associated antigen, justifying further clinical development of this strategy.

Community Policies and Programs to Prevent Obesity and Child Adiposity.

INTRODUCTION: Evidence regarding impact of community policies and programs (CPPs) to prevent child obesity is limited, and which combinations of strategies and components are most important is not understood. The Healthy Communities Study was an observational study to assess relationships of characteristics and intensity of CPPs with adiposity, diet, and physical activity in children, taking advantage of variation across the U.S. in community actions to prevent child obesity. The study examined the association of CPPs to prevent child obesity with measured BMI and waist circumference, hypothesizing that communities with more-comprehensive CPPs would have children with lower adiposity. METHODS: The study included 130 communities selected by probability-based sampling or because of known CPPs targeting child obesity. Data were collected at home visits on 5,138 children during 2013-2015. CPPs were scored for multiple attributes to create a CPP intensity score. A CPP target behavior score reflected the number of distinct target behaviors addressed. Scores were standardized with the smallest observed score across communities being 0 and the largest 1. Multilevel regression analysis in 2016 adjusted for community, household, and individual characteristics. RESULTS: Higher CPP target behavior score was significantly associated with lower BMI and waist circumference in a dose-response relationship, with magnitude for the past 3 years of CPPs of 0.843 (p=0.013) for BMI and 1.783 cm (p=0.020) for waist circumference. CONCLUSIONS: This study provides plausible evidence that comprehensive CPPs targeting a greater number of distinct physical activity and nutrition behaviors were associated with lower child adiposity.

Neuropilin-1 mediates neutrophil elastase uptake and cross-presentation in breast cancer cells.

Neutrophil elastase (NE) can be rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer, which results in cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapy target in hematological and solid tumor malignancies. The mechanism of NE uptake, however, remains unknown. Using the mass spectrometry-based approach, we identify neuropilin-1 (NRP1) as a NE receptor that mediates uptake and PR1 cross-presentation in breast cancer cells. We demonstrated that soluble NE is a specific, high-affinity ligand for NRP1 with a calculated Kd of 38.7 nm Furthermore, we showed that NRP1 binds to the RRXR motif in NE. Notably, NRP1 knockdown with interfering RNA or CRISPR-cas9 system and blocking using anti-NRP1 antibody decreased NE uptake and, subsequently, susceptibility to lysis by PR1-specific cytotoxic T cells. Expression of NRP1 in NRP1-deficient cells was sufficient to induce NE uptake. Altogether, because NRP1 is broadly expressed in tumors, our findings suggest a role for this receptor in immunotherapy strategies that target cross-presented antigens.