100 results on '"Paul M. Armistead"'
Search Results
2. Evaluation of the performance of a prior tacrolimus population pharmacokinetic kidney transplant model among adult allogeneic hematopoietic stem cell transplant patients
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Jing Zhu, Olivia Campagne, Chad D. Torrice, Gabrielle Flynn, Jordan A. Miller, Tejendra Patel, Oscar Suzuki, Jonathan R. Ptachcinski, Paul M. Armistead, Tim Wiltshire, Donald E. Mager, Daniel L. Weiner, and Daniel J. Crona
- Subjects
Therapeutics. Pharmacology ,RM1-950 ,Public aspects of medicine ,RA1-1270 - Abstract
Abstract Tacrolimus is a calcineurin inhibitor used to prevent acute graft versus host disease in adult patients receiving allogeneic hematopoietic stem cell transplantation (HCT). Previous population pharmacokinetic (PK) models have been developed in solid organ transplant, yet none exists for patients receiving HCT. The primary objectives of this study were to (1) use a previously published population PK model in adult patients who underwent kidney transplant and apply it to allogeneic HCT; (2) evaluate model‐predicted tacrolimus steady‐state trough concentrations and simulations in patients receiving HCT; and (3) evaluate covariates that affect tacrolimus PK in allogeneic HCT. A total of 252 adult patients receiving allogeneic HCT were included in the study. They received oral tacrolimus twice daily (0.03 mg/kg) starting 3 days prior to transplant. Data for these analyses included baseline clinical and demographic data, genotype data for single nucleotide polymorphisms in CYP3A4/5 and ABCB1, and the first tacrolimus steady‐state trough concentration. A dosing simulation strategy based on observed trough concentrations (rather than model‐based predictions) resulted in 12% more patients successfully achieving tacrolimus trough concentrations within the institutional target range (5–10 ng/ml). Stepwise covariate analyses identified HLA match and conditioning regimen (myeloablative vs. reduced intensity) as significant covariates. Ultimately, a previously published tacrolimus population PK model in kidney transplant provided a platform to help establish a model‐based dose adjustment strategy in patients receiving allogenic HCT, and identified HCT‐specific covariates to be considered for future prospective studies. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Tacrolimus is a cornerstone immunosuppressant used in patients who undergo organ transplantations. However, because of its narrow therapeutic index and wide interpatient pharmacokinetic (PK) variability, optimizing its dose is crucial to maximize efficacy and minimize tacrolimus‐induced toxicities. Prior to this study, no tacrolimus population PK models have been developed for adult patients receiving allogeneic hematopoietic stem cell transplantation (HCT). Therefore, research effort was warranted to develop a population PK model that begins to propose more precision tacrolimus dosing and begins to address both a clinical and scientific gap in this patient population. WHAT QUESTION DID THIS STUDY ADDRESS? The study addressed whether there is value in utilizing the observed tacrolimus steady‐state trough concentrations from patients receiving allogeneic HCT within the context of a pre‐existing population PK model developed for kidney transplant. The study also addressed whether there are clinically relevant covariates specific to adult patients receiving allogeneic HCT. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? Inclusion of a single steady‐state tacrolimus trough concentration is beneficial to model predictions. The dosing simulation strategy based on observed tacrolimus concentration, rather than the model‐predicted concentration, resulted in more patients achieving the target range at first steady‐state collection. Future studies should evaluate HLA matching and myeloablative conditioning versus reduced intensity conditioning regimens as covariates. These data and model‐informed dose adjustments should be included in future prospective studies. This research could also serve as a template as to how to assess the utility of prior information for other disease settings. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? The M2 model fitting method and D2 dosing simulation method can be applied to other clinical pharmacology studies where only a single steady‐state trough concentration is available per patient in the presence of a previously published population PK model.
- Published
- 2021
- Full Text
- View/download PDF
3. Targeted therapy of human leukemia xenografts in immunodeficient zebrafish
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Ranganatha R. Somasagara, Xiaoyan Huang, Chunyu Xu, Jamil Haider, Jonathan S. Serody, Paul M. Armistead, and TinChung Leung
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Medicine ,Science - Abstract
Abstract Personalized medicine holds tremendous promise for improving safety and efficacy of drug therapies by optimizing treatment regimens. Rapidly developed patient-derived xenografts (pdx) could be a helpful tool for analyzing the effect of drugs against an individual’s tumor by growing the tumor in an immunodeficient animal. Severe combined immunodeficiency (SCID) mice enable efficient in vivo expansion of vital tumor cells and generation of personalized xenografts. However, they are not amenable to large-scale rapid screening, which is critical in identifying new compounds from large compound libraries. The development of a zebrafish model suitable for pdx could facilitate large-scale screening of drugs targeted against specific malignancies. Here, we describe a novel strategy for establishing a zebrafish model for drug testing in leukemia xenografts. We used chronic myelogenous leukemia and acute myeloid leukemia for xenotransplantation into SCID zebrafish to evaluate drug screening protocols. We showed the in vivo efficacy of the ABL inhibitor imatinib, MEK inhibitor U0126, cytarabine, azacitidine and arsenic trioxide. We performed corresponding in vitro studies, demonstrating that combination of MEK- and FLT3-inhibitors exhibit an enhanced effect in vitro. We further evaluated the feasibility of zebrafish for transplantation of primary human hematopoietic cells that can survive at 15 day-post-fertilization. Our results provide critical insights to guide development of high-throughput platforms for evaluating leukemia.
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- 2021
- Full Text
- View/download PDF
4. Computational modeling and confirmation of leukemia-associated minor histocompatibility antigens
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Jefferson L. Lansford, Udara Dharmasiri, Shengjie Chai, Sally A. Hunsucker, Dante S. Bortone, James E. Keating, Ian M. Schlup, Gary L. Glish, Edward J. Collins, Gheath Alatrash, Jeffrey J. Molldrem, Paul M. Armistead, and Benjamin G. Vincent
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Specialties of internal medicine ,RC581-951 - Abstract
Abstract: T-cell responses to minor histocompatibility antigens (mHAs) mediate both antitumor immunity (graft-versus-leukemia [GVL]) and graft-versus-host disease (GVHD) in allogeneic stem cell transplant. Identifying mHAs with high allele frequency, tight binding affinity to common HLA molecules, and narrow tissue restriction could enhance immunotherapy against leukemia. Genotyping and HLA allele data from 101 HLA-matched donor-recipient pairs (DRPs) were computationally analyzed to predict both class I and class II mHAs likely to induce either GVL or GVHD. Roughly twice as many mHAs were predicted in HLA-matched unrelated donor (MUD) stem cell transplantation (SCT) compared with HLA-matched related transplants, an expected result given greater genetic disparity in MUD SCT. Computational analysis predicted 14 of 18 previously identified mHAs, with 2 minor antigen mismatches not being contained in the patient cohort, 1 missed mHA resulting from a noncanonical translation of the peptide antigen, and 1 case of poor binding prediction. A predicted peptide epitope derived from GRK4, a protein expressed in acute myeloid leukemia and testis, was confirmed by targeted differential ion mobility spectrometry-tandem mass spectrometry. T cells specific to UNC-GRK4-V were identified by tetramer analysis both in DRPs where a minor antigen mismatch was predicted and in DRPs where the donor contained the allele encoding UNC-GRK4-V, suggesting that this antigen could be both an mHA and a cancer-testis antigen. Computational analysis of genomic and transcriptomic data can reliably predict leukemia-associated mHA and can be used to guide targeted mHA discovery.
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- 2018
- Full Text
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5. Influence of Germline Genetics on Tacrolimus Pharmacokinetics and Pharmacodynamics in Allogeneic Hematopoietic Stem Cell Transplant Patients
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Jing Zhu, Tejendra Patel, Jordan A. Miller, Chad D. Torrice, Mehak Aggarwal, Margaret R. Sketch, Maurice D. Alexander, Paul M. Armistead, James M. Coghill, Tatjana Grgic, Katarzyna J. Jamieson, Jonathan R. Ptachcinski, Marcie L. Riches, Jonathan S. Serody, John L. Schmitz, J. Ryan Shaw, Thomas C. Shea, Oscar Suzuki, Benjamin G. Vincent, William A. Wood, Kamakshi V. Rao, Tim Wiltshire, Eric T. Weimer, and Daniel J. Crona
- Subjects
tacrolimus ,pharmacogenetics ,pharmacokinetics ,pharmacodynamics ,allogeneic hematopoietic stem cell transplant ,single nucleotide polymorphism (snp) ,germline ,cyp3a4/5 ,abcb1 ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Tacrolimus exhibits high inter-patient pharmacokinetics (PK) variability, as well as a narrow therapeutic index, and therefore requires therapeutic drug monitoring. Germline mutations in cytochrome P450 isoforms 4 and 5 genes (CYP3A4/5) and the ATP-binding cassette B1 gene (ABCB1) may contribute to interindividual tacrolimus PK variability, which may impact clinical outcomes among allogeneic hematopoietic stem cell transplantation (HSCT) patients. In this study, 252 adult patients who received tacrolimus for acute graft versus host disease (aGVHD) prophylaxis after allogeneic HSCT were genotyped to evaluate if germline genetic variants associated with tacrolimus PK and pharmacodynamic (PD) variability. Significant associations were detected between germline variants in CYP3A4/5 and ABCB1 and PK endpoints (e.g., median steady-state tacrolimus concentrations and time to goal tacrolimus concentration). However, significant associations were not observed between CYP3A4/5 or ABCB1 germline variants and PD endpoints (e.g., aGVHD and treatment-emergent nephrotoxicity). Decreased age and CYP3A5*1/*1 genotype were independently associated with subtherapeutic tacrolimus trough concentrations while CYP3A5*1*3 or CYP3A5*3/*3 genotypes, myeloablative allogeneic HSCT conditioning regimen (MAC) and increased weight were independently associated with supratherapeutic tacrolimus trough concentrations. Future lines of prospective research inquiry are warranted to use both germline genetic and clinical data to develop precision dosing tools that will optimize both tacrolimus dosing and clinical outcomes among adult HSCT patients.
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- 2020
- Full Text
- View/download PDF
6. Assay and Isolation of Single Proliferating CD4+ Lymphocytes Using an Automated Microraft Array Platform.
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Cody A. LaBelle, Raymond J. Zhang, Paul M. Armistead, and Nancy L. Allbritton
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- 2020
- Full Text
- View/download PDF
7. Shared graft-versus-leukemia minor histocompatibility antigens in DISCOVeRY-BMT
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Kelly S. Olsen, Othmane Jadi, Sarah Dexheimer, Dante S. Bortone, Steven P. Vensko, Sarah Bennett, Hancong Tang, Marisa Diiorio, Tanvi Saran, David Dingfelder, Qianqian Zhu, Yiwen Wang, Christopher A. Haiman, Loreall Pooler, Xin Sheng, Amy Webb, Marcelo C. Pasquini, Philip L. McCarthy, Stephen R. Spellman, Eric Weimer, Theresa Hahn, Lara Sucheston-Campbell, Paul M. Armistead, and Benjamin G. Vincent
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Hematology - Abstract
T-cell responses to minor histocompatibility antigens (mHAs) mediate graft-versus-leukemia (GVL) effects and graft-versus-host disease (GVHD) in allogeneic hematopoietic cell transplantation. Therapies that boost T-cell responses improve allogeneic hematopoietic cell transplant (alloHCT) efficacy but are limited by concurrent increases in the incidence and severity of GVHD. mHAs with expression restricted to hematopoietic tissue (GVL mHAs) are attractive targets for driving GVL without causing GVHD. Prior work to identify mHAs has focused on a small set of mHAs or population-level single-nucleotide polymorphism–association studies. We report the discovery of a large set of novel GVL mHAs based on predicted immunogenicity, tissue expression, and degree of sharing among donor-recipient pairs (DRPs) in the DISCOVeRY-BMT data set of 3231 alloHCT DRPs. The total number of predicted mHAs varied by HLA allele, and the total number and number of each class of mHA significantly differed by recipient genomic ancestry group. From the pool of predicted mHAs, we identified the smallest sets of GVL mHAs needed to cover 100% of DRPs with a given HLA allele. We used mass spectrometry to search for high-population frequency mHAs for 3 common HLA alleles. We validated 24 predicted novel GVL mHAs that are found cumulatively within 98.8%, 60.7%, and 78.9% of DRPs within DISCOVeRY-BMT that express HLA-A∗02:01, HLA-B∗35:01, and HLA-C∗07:02, respectively. We confirmed the immunogenicity of an example novel mHA via T-cell coculture with peptide-pulsed dendritic cells. This work demonstrates that the identification of shared mHAs is a feasible and promising technique for expanding mHA-targeting immunotherapeutics.
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- 2023
8. Utilization of a population pharmacokinetic model to improve a busulfan test‐dose strategy in allogeneic hematopoietic cell transplant recipients
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Tyler C. Dunlap, Daniel L. Weiner, Ryan M. Kemper, Miramar Kardouh, Susanna C. DeVane, Allison Symonds, J. Ryan Shaw, Paul M. Armistead, Jonathan R. Ptachcinski, and Daniel J. Crona
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Pharmacology ,Pharmacology (medical) - Published
- 2023
9. Data from Peptide/MHC Tetramer–Based Sorting of CD8+ T Cells to a Leukemia Antigen Yields Clonotypes Drawn Nonspecifically from an Underlying Restricted Repertoire
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Paul M. Armistead, Gary L. Glish, Jeffrey A. Frelinger, Adam S. Buntzman, Gregory Lizée, Tania Rodriguez-Cruz, Gheath Alatrash, Jonathan S. Serody, Thomas C. Shea, Stefanie Sarantopoulos, Don A. Gabriel, William A. Wood, James M. Coghill, Patricia A. Ropp, Lisa M. Bixby, Karen P. McKinnon, Jennifer P. Waugh, Atim A. Enyenihi, Benjamin G. Vincent, Colleen S. McGary, and Sally A. Hunsucker
- Abstract
Testing of T cell–based cancer therapeutics often involves measuring cancer antigen–specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8+ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer+ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor β (TCRβ) sequencing, we identified recurrent UNC-CDK4-1 tetramer–associated TCRβ clonotypes in a patient with a UNC-CDK4-1 tetramer+ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRβ repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer–associated TCRβ clonotypes represented >17% of the entire TCRβ repertoire—far in excess of the UNC-CDK4-1 tetramer+ frequency—indicating that the recurrent TCRβ clonotypes identified from UNC-CDK-4-1 tetramer+ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1–driven clonal T-cell expansion. Mapping recurrent TCRβ clonotype sequences onto TCRβ repertoires can help confirm or refute antigen-specific T-cell expansion in vivo. Cancer Immunol Res; 3(3); 228–35. ©2015 AACR.
- Published
- 2023
10. Supplementary Tables 1 - 4, Figures 1 - 2 from Peptide/MHC Tetramer–Based Sorting of CD8+ T Cells to a Leukemia Antigen Yields Clonotypes Drawn Nonspecifically from an Underlying Restricted Repertoire
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Paul M. Armistead, Gary L. Glish, Jeffrey A. Frelinger, Adam S. Buntzman, Gregory Lizée, Tania Rodriguez-Cruz, Gheath Alatrash, Jonathan S. Serody, Thomas C. Shea, Stefanie Sarantopoulos, Don A. Gabriel, William A. Wood, James M. Coghill, Patricia A. Ropp, Lisa M. Bixby, Karen P. McKinnon, Jennifer P. Waugh, Atim A. Enyenihi, Benjamin G. Vincent, Colleen S. McGary, and Sally A. Hunsucker
- Abstract
The document contains 4 supplemental tables: 1. RT-PCR Primers for TCRbeta sequencing 2. HPLC-MS identified peptide sequences 3. The amino acid sequences of recurrent TCRbeta clonotypes 4. The distribution of the CDR3 amino acid length in the analyzed patient. There are 2 supplementary figures: 1. Tetramer flow cytometry showing the generation of an oligoclonal T-cell population with a UNC-CDK4-1 high affinity T-cell population. 2. More detailed information regarding the tetramer gating strategy employed.
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- 2023
11. Supplementary Figures 1 - 4, Tables 1 - 2 from A Novel HLA-A*0201 Restricted Peptide Derived from Cathepsin G Is an Effective Immunotherapeutic Target in Acute Myeloid Leukemia
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Gheath Alatrash, Paul M. Armistead, Gary L. Glish, Jeffrey J. Molldrem, Sijie Lu, Karen Clise-Dwyer, Gregory Lizee, Tania Rodriguez-Cruz, Haroon Jakher, Patricia A. Ropp, Zein Al-Atrache, Kathryn Ruisaard, Anna Sergeeva, Elizabeth A. Mittendorf, Sally A. Hunsucker, Lisa S. St. John, Atim A. Enyenihi, Pariya Sukhumalchandra, and Mao Zhang
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PDF file - 706k, Figure S1. CG is ubiquitinated in AML. Figure S2. CG1 is a naturally processed epitope that is eluted from leukemia cell surface. Figure S3. CG expression by each of the AML samples used in the cytotoxicity assays. Figure S4. Gating strategy used to determine CG1-CTL frequency in AML patient blood. Supplementary Table S1. Characteristics of cathepsin G-derived peptides with highest binding affinity to HLA-A*0201 Supplementary Table S2. AML samples used in determining cathepsin G expression
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- 2023
12. Data from A Novel HLA-A*0201 Restricted Peptide Derived from Cathepsin G Is an Effective Immunotherapeutic Target in Acute Myeloid Leukemia
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Gheath Alatrash, Paul M. Armistead, Gary L. Glish, Jeffrey J. Molldrem, Sijie Lu, Karen Clise-Dwyer, Gregory Lizee, Tania Rodriguez-Cruz, Haroon Jakher, Patricia A. Ropp, Zein Al-Atrache, Kathryn Ruisaard, Anna Sergeeva, Elizabeth A. Mittendorf, Sally A. Hunsucker, Lisa S. St. John, Atim A. Enyenihi, Pariya Sukhumalchandra, and Mao Zhang
- Abstract
Purpose: Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy.Experimental Design: We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients.Results: CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL–mediated cytotoxicity, further confirming HLA-A*0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation.Conclusion: CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development. Clin Cancer Res; 19(1); 247–57. ©2012 AACR.
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- 2023
13. Correlation of Engraftment and Time from Melphalan Administration to Stem Cell Infusion
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Anthony D. Sung, Brandi Anders, Maurice Alexander, Paul M. Armistead, J. Ryan Shaw, LeAnne Kennedy, Dominic T. Moore, Shannon Palmer, Kaci Shuman, Lauren Bohannon, Tatjana Grgic, and Jonathan R. Ptachcinski
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Melphalan ,Transplantation ,business.industry ,Medicine ,Molecular Medicine ,Immunology and Allergy ,Cell Biology ,Hematology ,Stem cell ,Pharmacology ,business ,Administration (government) ,medicine.drug - Abstract
Single-agent, high-dose melphalan continues to be the most commonly used conditioning regimen for transplantation-eligible patients with multiple myeloma undergoing autologous stem cell transplantation. The timing of melphalan administration with respect to stem cell infusion has not been clearly defined. Many institutions require a minimum of 24 hours between melphalan administration and stem cell infusion; however, some institutions have adopted shorter intervals based on melphalan's short half-life. Some studies have suggested that shortening the interval between melphalan administration and stem cell infusion may contribute to delays in engraftment, but this correlation has not been clearly evaluated or defined. This multicenter retrospective cohort study evaluated the times to neutrophil and platelet engraftment in patients who received stem cells at least 24 hours after melphalan (≥24 hours cohort) compared with those who received stem cells within 24 hours of melphalan (24 hours cohort. The study included a total of 723 adult patients, 502 patients in the ≥24 hours cohort and 221 in the24 hours cohort, treated at 3 transplantation centers between January 1, 2016, and September 30, 2019. Patient characteristics were summarized using descriptive statistics. The Fisher exact test was used to compare nominal categorical variables between the 2 cohorts, and the nonparametric van der Waerden test or Mood median test was used to compare ordinal or continuous variables. The median time to neutrophil engraftment was 12 days for both the ≥24 hours cohort (interquartile range [IQR], 11 to 12 days) and the24 hours cohort (IQR, 11 to 13 days) (P = .07). The median time to platelet engraftment was 19 days for both the ≥24 hours cohort (IQR, 17 to 22 days) and24 hours cohort (IQR, 17 to 20 days) (P = .25). The median time between melphalan administration and stem cell infusion in the24 hours cohort was 18 hours, with a minimum time of 12 hours. The existing literature has not clearly defined the impact of the timing between melphalan administration and stem cell infusion on engraftment in autologous transplantation. The ability to safely shorten the interval between chemotherapy and transplantation could increase logistical flexibility and/or decrease the length of hospital stay. This large multicenter retrospective study did not identify a statistical or clinical impact on engraftment when melphalan was infused24 hours or ≥24 hours before autologous stem cell infusion.
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- 2022
14. Abrogation of Immune Effector Cell Neurotoxicity Syndrome (ICANS) By Rimiducid (RIM) in Patients Treated with CD19-Specific Chimeric Antigen Receptor Modified T-Cells (CAR-T) Engineered with an Inducible Caspase 9 (iC9 CAR.19)-Clinical and Pharmacodynamic Correlates
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Matthew C. Foster, Barbara Savoldo, Winnie Lau, Clio Rubinos, Natalie S. Grover, Paul M. Armistead, James M. Coghill, Katarzyna Joanna Jamieson, Robert Hagan, J. Kaitlin Morrison, Faith Brianne Buchanan, Catherine Joyce Arago Cheng, Anastasia Ivanova, Tammy Cavallo, John West, Megan Gonzalez, Jonathan S. Serody, and Gianpietro Dotti
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
15. Targeted therapy of human leukemia xenografts in immunodeficient zebrafish
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Xiaoyan Huang, Chunyu Xu, Ranganatha R. Somasagara, Paul M. Armistead, Jonathan S. Serody, Jamil Haider, and TinChung Leung
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0301 basic medicine ,Cancer therapy ,Cell Survival ,MAP Kinase Signaling System ,medicine.medical_treatment ,Science ,Azacitidine ,Transplantation, Heterologous ,Antineoplastic Agents ,Article ,Targeted therapy ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,Nitriles ,Butadienes ,Medicine ,Animals ,Humans ,Molecular Targeted Therapy ,Cancer models ,Protein Kinase Inhibitors ,Zebrafish ,Cell Proliferation ,Mitogen-Activated Protein Kinase Kinases ,Haematological cancer ,Multidisciplinary ,ABL ,Cell Death ,business.industry ,Myeloid leukemia ,Imatinib ,Zebrafish Proteins ,medicine.disease ,Xenograft Model Antitumor Assays ,Transplantation ,Leukemia ,030104 developmental biology ,fms-Like Tyrosine Kinase 3 ,030220 oncology & carcinogenesis ,Cancer research ,business ,Chronic myelogenous leukemia ,medicine.drug - Abstract
Personalized medicine holds tremendous promise for improving safety and efficacy of drug therapies by optimizing treatment regimens. Rapidly developed patient-derived xenografts (pdx) could be a helpful tool for analyzing the effect of drugs against an individual’s tumor by growing the tumor in an immunodeficient animal. Severe combined immunodeficiency (SCID) mice enable efficient in vivo expansion of vital tumor cells and generation of personalized xenografts. However, they are not amenable to large-scale rapid screening, which is critical in identifying new compounds from large compound libraries. The development of a zebrafish model suitable for pdx could facilitate large-scale screening of drugs targeted against specific malignancies. Here, we describe a novel strategy for establishing a zebrafish model for drug testing in leukemia xenografts. We used chronic myelogenous leukemia and acute myeloid leukemia for xenotransplantation into SCID zebrafish to evaluate drug screening protocols. We showed the in vivo efficacy of the ABL inhibitor imatinib, MEK inhibitor U0126, cytarabine, azacitidine and arsenic trioxide. We performed corresponding in vitro studies, demonstrating that combination of MEK- and FLT3-inhibitors exhibit an enhanced effect in vitro. We further evaluated the feasibility of zebrafish for transplantation of primary human hematopoietic cells that can survive at 15 day-post-fertilization. Our results provide critical insights to guide development of high-throughput platforms for evaluating leukemia.
- Published
- 2021
16. Analysis of an Early Assessment of Medical and Psychosocial Issues to Improve Outcomes Following Allogeneic Hematopoietic Cell Transplantation
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Natalie Schmidt, Paul M. Armistead, Marcie L. Riches, Dominic T Moore, and Ally Wardell
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Transplantation ,Molecular Medicine ,Immunology and Allergy ,Cell Biology ,Hematology - Published
- 2023
17. Evaluation of the performance of a prior tacrolimus population pharmacokinetic kidney transplant model among adult allogeneic hematopoietic stem cell transplant patients
- Author
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Gabrielle Flynn, Jonathan R. Ptachcinski, Chad Torrice, Jing Zhu, Donald E. Mager, Olivia Campagne, Jordan A Miller, Daniel Weiner, Tim Wiltshire, Daniel J. Crona, Paul M. Armistead, Tejendra Patel, and Oscar Suzuki
- Subjects
Adult ,Male ,Oncology ,medicine.medical_specialty ,Transplantation Conditioning ,medicine.medical_treatment ,Calcineurin Inhibitors ,Population ,Administration, Oral ,Graft vs Host Disease ,Context (language use) ,RM1-950 ,Hematopoietic stem cell transplantation ,Models, Biological ,Article ,Tacrolimus ,General Biochemistry, Genetics and Molecular Biology ,law.invention ,Young Adult ,law ,Internal medicine ,Humans ,Medicine ,Computer Simulation ,Dosing ,General Pharmacology, Toxicology and Pharmaceutics ,Prospective cohort study ,education ,Aged ,education.field_of_study ,Clinical pharmacology ,Dose-Response Relationship, Drug ,business.industry ,Research ,General Neuroscience ,Hematopoietic Stem Cell Transplantation ,Articles ,General Medicine ,Middle Aged ,Kidney Transplantation ,Calcineurin ,surgical procedures, operative ,Biological Variation, Population ,Female ,Therapeutics. Pharmacology ,Public aspects of medicine ,RA1-1270 ,business - Abstract
Tacrolimus is a calcineurin inhibitor used to prevent acute graft versus host disease in adult patients receiving allogeneic hematopoietic stem cell transplantation (HCT). Previous population pharmacokinetic (PK) models have been developed in solid organ transplant, yet none exists for patients receiving HCT. The primary objectives of this study were to (1) use a previously published population PK model in adult patients who underwent kidney transplant and apply it to allogeneic HCT; (2) evaluate model‐predicted tacrolimus steady‐state trough concentrations and simulations in patients receiving HCT; and (3) evaluate covariates that affect tacrolimus PK in allogeneic HCT. A total of 252 adult patients receiving allogeneic HCT were included in the study. They received oral tacrolimus twice daily (0.03 mg/kg) starting 3 days prior to transplant. Data for these analyses included baseline clinical and demographic data, genotype data for single nucleotide polymorphisms in CYP3A4/5 and ABCB1, and the first tacrolimus steady‐state trough concentration. A dosing simulation strategy based on observed trough concentrations (rather than model‐based predictions) resulted in 12% more patients successfully achieving tacrolimus trough concentrations within the institutional target range (5–10 ng/ml). Stepwise covariate analyses identified HLA match and conditioning regimen (myeloablative vs. reduced intensity) as significant covariates. Ultimately, a previously published tacrolimus population PK model in kidney transplant provided a platform to help establish a model‐based dose adjustment strategy in patients receiving allogenic HCT, and identified HCT‐specific covariates to be considered for future prospective studies. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Tacrolimus is a cornerstone immunosuppressant used in patients who undergo organ transplantations. However, because of its narrow therapeutic index and wide interpatient pharmacokinetic (PK) variability, optimizing its dose is crucial to maximize efficacy and minimize tacrolimus‐induced toxicities. Prior to this study, no tacrolimus population PK models have been developed for adult patients receiving allogeneic hematopoietic stem cell transplantation (HCT). Therefore, research effort was warranted to develop a population PK model that begins to propose more precision tacrolimus dosing and begins to address both a clinical and scientific gap in this patient population. WHAT QUESTION DID THIS STUDY ADDRESS? The study addressed whether there is value in utilizing the observed tacrolimus steady‐state trough concentrations from patients receiving allogeneic HCT within the context of a pre‐existing population PK model developed for kidney transplant. The study also addressed whether there are clinically relevant covariates specific to adult patients receiving allogeneic HCT. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? Inclusion of a single steady‐state tacrolimus trough concentration is beneficial to model predictions. The dosing simulation strategy based on observed tacrolimus concentration, rather than the model‐predicted concentration, resulted in more patients achieving the target range at first steady‐state collection. Future studies should evaluate HLA matching and myeloablative conditioning versus reduced intensity conditioning regimens as covariates. These data and model‐informed dose adjustments should be included in future prospective studies. This research could also serve as a template as to how to assess the utility of prior information for other disease settings. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? The M2 model fitting method and D2 dosing simulation method can be applied to other clinical pharmacology studies where only a single steady‐state trough concentration is available per patient in the presence of a previously published population PK model.
- Published
- 2021
18. Microraft Arrays for Serial-Killer CD19 Chimeric Antigen Receptor T Cells and Single Cell Isolation
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Cody A. LaBelle, Raymond J. Zhang, Sally A. Hunsucker, Paul M. Armistead, and Nancy L. Allbritton
- Subjects
Histology ,Cell Biology ,Pathology and Forensic Medicine - Abstract
Chimeric antigen receptor T (CAR-T) cell immunotherapies have seen success in treating hematological malignancies in recent years; however, the results can be highly variable. Single cell heterogeneity plays a key role in the variable efficacy of CAR-T cell treatments yet is largely unexplored. A major challenge is to understand the killing behavior and phenotype of individual CAR-T cells which are able to serially kill targets. Thus, a platform capable of measuring time-dependent CAR-T cell mediated killing and then isolating single cells for downstream assays would be invaluable in characterizing CAR-T cells. An automated microraft array platform was designed to track CD19 CAR-T cell killing of CD19+ target cells and CAR-T cell motility over time followed by CAR-T cell collection based on killing behavior. The platform demonstrated automated CAR-T cell counting with up to 98% specificity and 96% sensitivity, and single cells were isolated with 89% efficiency. On average, 2.3% of single CAR-T cells were shown to participate in serial-killing of target cells, killing a maximum of 3 target cells in a 6 h period. The cytotoxicity and motility of7,000 individual CAR-T cells was tracked across 4 microraft arrays. The automated microraft array platform measured temporal cell-mediated cytotoxicity, CAR-T cell motility, CAR-T cell death, and CAR-T cell to target cell distances, followed by the capability to sort any desired CAR-T cell. The pipeline has the potential to further our understanding of T cell-based cancer immunotherapies and improve cell-therapy products for better patient outcomes. This article is protected by copyright. All rights reserved.
- Published
- 2022
19. Alkali Metal Cationization of Tumor-associated Antigen Peptides for Improved Dissociation and Measurement by Differential Ion Mobility-Mass Spectrometry
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Gary L. Glish, Benjamin G. Vincent, James E. Keating, Chris Chung, Paul M. Armistead, Jans F. Prins, Shengjie Chai, and Sally A. Hunsucker
- Subjects
chemistry.chemical_classification ,Spectrometry, Mass, Electrospray Ionization ,Electrospray ,Metals, Alkali ,Chemistry ,Ion-mobility spectrometry ,Peptide ,General Chemistry ,Alkali metal ,Photochemistry ,Tandem mass spectrometry ,Biochemistry ,Article ,Dissociation (chemistry) ,Ion ,Metal ,Tandem Mass Spectrometry ,Cations ,visual_art ,Ion Mobility Spectrometry ,visual_art.visual_art_medium ,Peptides - Abstract
Tandem mass spectrometry (MS/MS) is a highly sensitive and selective method for the detection of tumor-associated peptide antigens. These short, nontryptic sequences may lack basic residues, resulting in the formation of predominantly [peptide + H]+ ions in electrospray. These singly charged ions tend to undergo inefficient dissociation, leading to issues in sequence determination. Addition of alkali metal salts to the electrospray solvent can drive the formation of [peptide + H + metal]2+ ions that have enhanced dissociation characteristics relative to [peptide + H]+ ions. Both previously identified tumor-associated antigens and predicted neoantigen sequences were investigated. The previously reported rearrangement mechanism in MS/MS of sodium-cationized peptides is applied here to demonstrate complete C-terminal sequencing of tumor-associated peptide antigens. Differential ion mobility spectrometry (DIMS) is shown to selectively enrich [peptide + H + metal]2+ species by filtering out singly charged interferences at relatively low field strengths, offsetting the decrease in signal intensity associated with the use of alkali metal cations.
- Published
- 2020
20. NeoSplice: a bioinformatics method for prediction of splice variant neoantigens
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Shengjie Chai, Christof C Smith, Tavleen K Kochar, Sally A Hunsucker, Wolfgang Beck, Kelly S Olsen, Steven Vensko, Gary L Glish, Paul M Armistead, Jan F Prins, and Benjamin G Vincent
- Subjects
General Medicine - Abstract
Motivation Splice variant neoantigens are a potential source of tumor-specific antigen (TSA) that are shared between patients in a variety of cancers, including acute myeloid leukemia. Current tools for genomic prediction of splice variant neoantigens demonstrate promise. However, many tools have not been well validated with simulated and/or wet lab approaches, with no studies published that have presented a targeted immunopeptidome mass spectrometry approach designed specifically for identification of predicted splice variant neoantigens. Results In this study, we describe NeoSplice, a novel computational method for splice variant neoantigen prediction based on (i) prediction of tumor-specific k-mers from RNA-seq data, (ii) alignment of differentially expressed k-mers to the splice graph and (iii) inference of the variant transcript with MHC binding prediction. NeoSplice demonstrates high sensitivity and precision (>80% on average across all splice variant classes) through in silico simulated RNA-seq data. Through mass spectrometry analysis of the immunopeptidome of the K562.A2 cell line compared against a synthetic peptide reference of predicted splice variant neoantigens, we validated 4 of 37 predicted antigens corresponding to 3 of 17 unique splice junctions. Lastly, we provide a comparison of NeoSplice against other splice variant prediction tools described in the literature. NeoSplice provides a well-validated platform for prediction of TSA vaccine targets for future cancer antigen vaccine studies to evaluate the clinical efficacy of splice variant neoantigens. Availability and implementation https://github.com/Benjamin-Vincent-Lab/NeoSplice Supplementary information Supplementary data are available at Bioinformatics Advances online.
- Published
- 2022
21. Impact of Common Graft Versus Leukemia Minor Histocompatibility Antigens on the Outcomes of Allogeneic Hematopoietic Cell Transplantation (alloHCT)
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Sarah Entwistle, Tao Wang, Kelly Olsen, Hancong Tang, Steven Vensko, Stephen R. Spellman, Junke Wang, Yiwen Wang, Xin Sheng, David Van Den Berg, Dante S. Bortone, Benjamin G. Vincent, Christopher A. Haiman, Theresa Hahn, Othmane Jadi, Loreall Pooler, Lara E. Sucheston-Campbell, and Paul M. Armistead
- Subjects
Transplantation ,Hematopoietic cell ,business.industry ,Cell Biology ,Hematology ,medicine.disease ,Leukemia ,Immunology ,Minor histocompatibility antigen ,medicine ,Molecular Medicine ,Immunology and Allergy ,business - Published
- 2021
22. Insights and lessons learned from a prospective clinical pharmacology study in allogeneic hematopoietic stem cell transplant during the COVID-19 pandemic
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Daniel Weiner, Daniel J. Crona, Jonathan R. Ptachcinski, Jing Zhu, Tim Wiltshire, Paul M. Armistead, and Gauri Rao
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2019-20 coronavirus outbreak ,Clinical pharmacology ,Coronavirus disease 2019 (COVID-19) ,business.industry ,General Neuroscience ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,MEDLINE ,Hematopoietic Stem Cell Transplantation ,COVID-19 ,General Medicine ,General Biochemistry, Genetics and Molecular Biology ,law.invention ,law ,Pandemic ,Immunology ,Pharmacology, Clinical ,Commentary ,Medicine ,Humans ,Allogeneic hematopoietic stem cell transplant ,Prospective Studies ,General Pharmacology, Toxicology and Pharmaceutics ,business ,Pandemics - Published
- 2021
23. Population Distribution of GvL and GvH Minor Histocompatibility Antigens
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David Van Den Berg, Xin Sheng, Christopher A. Haiman, Kelly Olsen, Hancong Tang, Lara E. Sucheston-Campbell, Dante S. Bortone, Stephen R. Spellman, Loreall Pooler, Sarah Entwistle, Steven Vensko, Junke Wang, Benjamin G. Vincent, Paul M. Armistead, and Theresa Hahn
- Subjects
education.field_of_study ,Immunology ,Population ,Minor histocompatibility antigen ,Distribution (pharmacology) ,Cell Biology ,Hematology ,Biology ,education ,Biochemistry - Abstract
Background: Donor-derived T cells that target minor histocompatibility antigens (mHAs) in allogeneic hematopoietic cell transplant (HCT) mediate graft versus leukemia (GvL) and graft versus host (GvH) effects. Prediction of mHAs that drive GvL has garnered interest for targeted immunotherapy, but there have been few large-scale studies of population prevalence of predicted mHAs. Prioritization of mHAs that are shared among patients would allow for treatment of more individuals with mHA-targeting therapies. We report here population metrics of predicted mHAs in a dataset of over 3000 patients treated with HCT and reported to CIBMTR from 2000-2011. Our goal is to identify the most common mHAs within leukemia and Myelodysplastic Syndrome (MDS) patient populations to target with T cell immunotherapies or graft engineering techniques. Methods: Data is derived from two cohorts of donor recipient HCT pairs (DRPs) treated for Acute Myeloid Leukemia (AML), Acute Lymphocytic Leukemia (ALL), and MDS from the CIBMTR and previously analyzed in DISCOVeRY-BMT. Cohort 1 included 2609 10/10 HLA-matched DRPs treated from 2000-2008, and Cohort 2 included 572 10/10 HLA-matched DRPs treated from 2009-2011 plus 351 8/8 HLA-matched DRPs treated from 2000-2011 (Hahn et al. 2015, Biol Blood Marrow Transplant). Cohorts were combined for analyses. Approximately 20,000 missense SNPs were extracted from Illumina HumanOmni Express genotyping data. Computational mHA prediction was performed according to prior work from our lab (Lansford et al. 2018, Blood Adv.). Minor mismatches were predicted based on coding SNPs present in the recipient but not donor. mHAs were defined as mismatches that would lead to variant peptides predicted to bind at least 1 recipient HLA molecule and be expressed in leukemia cells (GvL mHA) and/or acute GvHD target organs (GvH mHA). GvL mHAs were categorized as "GvL,No_GvH" or "GvL" based on transcripts per million (TPM) corresponding to GvH organs, with "GvL" indicating between 5-50 TPM and "GvL,No_GvH" indicating Results: Patient demographics and number of total predicted mHAs from each ethnic group are shown in Table 1. Number of predicted mHAs per patient varied widely both within and between HLA types (Figure 1). Despite underrepresentation of some ethnic groups in our dataset, we identified thousands of potential mHAs in each group (Figure 2A). GvL mHA and GvH mHA proportions were similar across recipient ethnicities (Figure 2A-B). GvL mHA made up approximately half of predicted mHAs for each ethnic group (Figure 2B). Total numbers of mHAs per HCT recipient were significantly different between recipient ethnic groups within each cohort (Figure 2C). Although proportions of GvL vs GvH mHAs were stable across HLA alleles, there were substantial differences in number of predicted mHA by allele (Figure 3). Despite limited representation of some HLA types in our dataset, we were able to identify GvL mHAs for potential therapeutic targeting corresponding to 56 HLA alleles. We generated ranked lists of the most common shared mHA for each HLA allele, using an implementation of the standard greedy algorithm solution to the maximum set coverage problem. With this method, we identified the fewest number of mHA peptides needed to cover desired percentages of the recipient population with at least one mHA. For example, for HLA A*02:01, HLA*B07:02, and HLA*C07:01, engineering T cells to target the top nine to twelve peptides would allow for treatment of 80% of the patient population in our cohorts (Figure 4). These represent common HLA alleles in Caucasian, African American, Hispanic, and Asian populations, indicating that this technique can identify targets that could be therapeutically beneficial for a greater diversity of patients than standard treatments. mHA pools can also be filtered on peptide expression or HLA binding to ensure that the targeted peptides are highly expressed and presented. Conclusions: Despite differences in predicted number of mHA by ethnicity and HLA alleles, shared GvL mHA exist across common HLA. To the extent that these are targetable by adoptive cellular therapy, we can expand equal access to mHA targeted immunotherapies, improving upon traditional models where only the most prevalent HLA types are covered. Disclosures Armistead: Cell Microsystems: Patents & Royalties: Patent application U.S. 16/347,104 "Automated collection of a specified number of cells"; GeneCentric: Consultancy. Vincent:GeneCentric Therapeutics: Consultancy.
- Published
- 2020
24. Alternative tumour-specific antigens
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Shengjie Chai, Benjamin G. Vincent, Paul M. Armistead, Jonathan S. Serody, Sara R. Selitsky, and Christof C. Smith
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Multiple tumours ,viruses ,General Mathematics ,medicine.medical_treatment ,Genomics ,Computational biology ,Biology ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,INDEL Mutation ,Antigen ,Cancer immunotherapy ,Antigens, Neoplasm ,Neoplasms ,medicine ,Animals ,Humans ,Frameshift Mutation ,Applied Mathematics ,Alternative splicing ,Computational Biology ,Cancer ,medicine.disease ,Alternative Splicing ,030220 oncology & carcinogenesis ,Mutation ,Cancer vaccine ,Gene Fusion ,Tumor immunology - Abstract
The study of tumour-specific antigens (TSAs) as targets for antitumour therapies has accelerated within the past decade. The most commonly studied class of TSAs are those derived from non-synonymous single-nucleotide variants (SNVs), or SNV neoantigens. However, to increase the repertoire of available therapeutic TSA targets, ‘alternative TSAs’, defined here as high-specificity tumour antigens arising from non-SNV genomic sources, have recently been evaluated. Among these alternative TSAs are antigens derived from mutational frameshifts, splice variants, gene fusions, endogenous retroelements and other processes. Unlike the patient-specific nature of SNV neoantigens, some alternative TSAs may have the advantage of being widely shared by multiple tumours, allowing for universal, off-the-shelf therapies. In this Opinion article, we will outline the biology, available computational tools, preclinical and/or clinical studies and relevant cancers for each alternative TSA class, as well as discuss both current challenges preventing the therapeutic application of alternative TSAs and potential solutions to aid in their clinical translation. To date, very few actionable tumour-specific antigens (TSAs) have been identified that have successfully translated into therapeutic cancer vaccines. This Opinion article provides both examples of TSAs alternative to the traditional single-nucleotide variant neoantigens and details about the novel computational tools used to identify them, with the view to broaden the number of targetable antigens that can be used for cancer vaccine development.
- Published
- 2019
25. Evaluation of a Test Dose Strategy for Pharmacokinetically-Guided Busulfan Dosing for Hematopoietic Stem Cell Transplantation
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Jonathan S. Serody, Yunro Chung, Paul M. Armistead, Benjamin G. Vincent, Anastasia Ivanova, Kamakshi V. Rao, Jessica M. Davis, James M. Coghill, Jonathan R. Ptachcinski, Marcie L. Riches, Maurice Alexander, Katarzyna Jamieson, J. Ryan Shaw, Andrew Sharf, William A. Wood, and Thomas C. Shea
- Subjects
Adult ,Male ,medicine.medical_specialty ,Transplantation Conditioning ,Test dose ,medicine.medical_treatment ,Urology ,Hematopoietic stem cell transplantation ,Disease-Free Survival ,03 medical and health sciences ,0302 clinical medicine ,Pharmacokinetics ,Humans ,Medicine ,Dosing ,Busulfan ,Transplantation ,Adult patients ,business.industry ,Hematopoietic Stem Cell Transplantation ,Area under the curve ,Hematology ,Middle Aged ,Myeloablative Agonists ,030220 oncology & carcinogenesis ,Toxicity ,Female ,business ,030215 immunology ,medicine.drug - Abstract
Targeted busulfan dosing helps limit chemotherapy-related toxicity and optimize disease outcomes in hematopoietic stem cell transplantation (HCT). The objective of this study was to evaluate busulfan exposure from a pharmacokinetic (PK)-guided dosing strategy using a test dose. This retrospective evaluation included adult patients who underwent HCT at our institution with busulfan-based myeloablative (>9 mg/kg) conditioning between January 2014 and October 2015. A weight-based test dose of 0.8 mg/kg was used with PK assessments to predict area under the curve (AUCpred) achieved with weight-based dosing, with a target AUC of 4800 µM*minute (AUCtarget). PK from the test dose was then used to calculate a PK-guided first myeloablative busulfan dose. PK assessments were also done after the first dose to assess if the goal area under the curve (AUC) had been achieved (AUCfirst). A PK-guided first dose resulted in achievement of target AUC with target ranges of ±10% in 50% of patients, ±15% in 75%, and ±20% in 94%. This was an improved rate of target achievement compared with the 33%, 44%, and 63% of patients who achieved the desired AUC for these respective target ranges when using weight-based dosing (P = .12, .004, and
- Published
- 2019
26. Genetics of HLA Peptide Presentation and Impact on Outcomes in HLA-Matched Allogeneic Hematopoietic Cell Transplantation
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Shahinaz M. Gadalla, Tao Wang, Malek Kamoun, Marcie L. Riches, Stephen R. Spellman, Johannes Schetelig, Steven G.E. Marsh, Michelle Kuxhausen, Valerie I. Brown, Minoo Battiwalla, David B. Miklos, Edmund K. Waller, Lee Ann Baxter-Lowe, Loren Gragert, Joannis Mytilineos, Sophie Paczesny, Stephanie J. Lee, Vijaya Raj Bhatt, Paul M. Armistead, Sherif M. Badawy, Jefferson L Lansford, Benjamin G. Vincent, and Charlotte McIlwaine Story
- Subjects
Transplantation ,Surrogate endpoint ,business.industry ,Hematopoietic Stem Cell Transplantation ,Context (language use) ,Cell Biology ,Hematology ,Human leukocyte antigen ,medicine.disease ,Article ,Graft-versus-host disease ,Antigen ,Immunology ,Minor histocompatibility antigen ,Molecular Medicine ,Immunology and Allergy ,Medicine ,Humans ,Cumulative incidence ,Neoplasm Recurrence, Local ,business ,Peptides ,Unrelated Donors ,Retrospective Studies - Abstract
Minor histocompatibility antigens (mHAs), recipient-derived peptide epitopes presented on the cell surface, are known to mediate graft-versus-host disease (GVHD); however, there are no current methods to associate mHA features with GVHD risk. This deficiency is due in part to the lack of technological means to accurately predict, let alone confirm, the tremendous number of potential mHAs in each individual transplant. Previous studies have shown that different HLA molecules present varying fractions of candidate peptide epitopes; however, the genetic “distance” between HLA-matched donors and recipients is relatively constrained. From these 2 observations, it is possible that the HLA type for a donor-recipient pair (DRP) would provide a surrogate measurement of the number of predicted mHAs, which could be related to GVHD risk. Because different HLA molecules present variable numbers of peptide antigens, a predicted cumulative peptide-binding efficiency can be calculated for individual DRP based on the pair's HLA type. The purpose of this study was to test whether cumulative peptide-binding efficiency is associated with the risk of acute GVHD (aGVHD) or relapse. In this retrospective Center for International Blood and Marrow Transplant Research study, a total of 3242 HLA-matched DRPs were analyzed for predicted cumulative peptide-binding efficiency using their HLA types and were divided into tertiles based on their scores. Univariable and multivariable analyses was performed to test for associations between cumulative peptide-binding efficiency for DRPs, divided into the HLA-matched related donor (MRD) and HLA-matched unrelated donor (MUD) cohorts, and the primary outcomes of aGVHD and relapse. Secondary outcomes investigated included overall survival, disease-free survival, and transplantation-related mortality. Using a computationally generated peptidome as a test dataset, the tested series of HLA class I displayed peptide-binding frequencies ranging from 0.1% to 3.8% of the full peptidome, and HLA class II molecules had peptide-binding frequencies of 12% to 77% across the HLA-DRB1 allotypes. By increasing binding efficiency tertile, the cumulative incidence of aGVHD at 6 months for MUD patients was 41%, 41%, and 45% for HLA class I (P = .336) and 44%, 41%, and 42% for HLA class II (P = .452). The cumulative incidences of relapse at 3 years for MUD transplant recipients were 36%, 38%, and 38% for HLA class I (P = .533) and 37%, 37%, and 38% for HLA class II (P = .896). The findings were similar for MRD transplant recipients. Multivariable analysis did not identify any impact of peptide-binding efficiency on aGVHD or relapse in MUD or MRD transplant recipients. Whereas GVHD is mediated by minor antigen mismatches in the context of HLA-matched allo-HCT, peptide-binding efficiency, which was used as a surrogate measurement for predicted number of binding antigens, did not provide additional clinical information for GVHD risk assessment. The negative result may be due to the limitations of this surrogate marker, or it is possible that GVHD is driven by a subset of immunogenic mHAs. Further research should be directed at direct mHA epitope and immunogenicity prediction.
- Published
- 2021
27. Utility of a safety switch to abrogate CD19.CAR T-cell-associated neurotoxicity
- Author
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Faith Brianne Buchanan, Winnie W. Y. Lau, Kaitlin Morrison, Natalie S Grover, Robert S. Hagan, Barbara Savoldo, Spencer Laing, James M. Coghill, John A. West, Matthew C. Foster, Gianpietro Dotti, Paul M. Armistead, Clio Rubinos, Catherine Cheng, Aaron E. Foster, Jonathan S. Serody, and Anastasia Ivanova
- Subjects
Adult ,biology ,business.industry ,Lymphoblastic Leukemia ,Immunology ,Antigens, CD19 ,Neurotoxicity ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Immunotherapy, Adoptive ,CD19 ,Neoplasm Proteins ,Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ,medicine ,biology.protein ,Cancer research ,Humans ,Neurotoxicity Syndromes ,Car t cells ,business ,Letter to Blood ,Burkitt's lymphoma - Published
- 2021
28. Influence of Germline Genetics on Tacrolimus Pharmacokinetics and Pharmacodynamics in Allogeneic Hematopoietic Stem Cell Transplant Patients
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James M. Coghill, Tim Wiltshire, J. Ryan Shaw, Paul M. Armistead, Thomas C. Shea, Marcie L. Riches, Chad Torrice, Jordan A Miller, Mehak Aggarwal, Kamakshi V. Rao, William A. Wood, Jing Zhu, Oscar Suzuki, Tatjana Grgic, Margaret R Sketch, John L. Schmitz, Daniel J. Crona, Eric T. Weimer, Tejendra Patel, Benjamin G. Vincent, Katarzyna Jamieson, Maurice Alexander, Jonathan S. Serody, and Jonathan R. Ptachcinski
- Subjects
Male ,Oncology ,medicine.medical_treatment ,Graft vs Host Disease ,Hematopoietic stem cell transplantation ,germline ,030226 pharmacology & pharmacy ,Germline ,lcsh:Chemistry ,0302 clinical medicine ,allogeneic hematopoietic stem cell transplant ,Databases, Genetic ,Odds Ratio ,Cytochrome P-450 CYP3A ,Medicine ,tacrolimus ,lcsh:QH301-705.5 ,Spectroscopy ,pharmacogenetics ,medicine.diagnostic_test ,Hematopoietic Stem Cell Transplantation ,ABCB1 ,General Medicine ,Middle Aged ,Computer Science Applications ,surgical procedures, operative ,030220 oncology & carcinogenesis ,Female ,pharmacokinetics ,Immunosuppressive Agents ,Adult ,CYP3A4/5 ,medicine.medical_specialty ,Genotype ,chemical and pharmacologic phenomena ,Polymorphism, Single Nucleotide ,Article ,Catalysis ,Inorganic Chemistry ,Young Adult ,03 medical and health sciences ,Germline mutation ,single nucleotide polymorphism (SNP) ,Internal medicine ,pharmacodynamics ,Humans ,Transplantation, Homologous ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,Physical and Theoretical Chemistry ,CYP3A5 ,Molecular Biology ,Germ-Line Mutation ,Aged ,business.industry ,Organic Chemistry ,Tacrolimus ,Logistic Models ,lcsh:Biology (General) ,lcsh:QD1-999 ,Therapeutic drug monitoring ,Pharmacodynamics ,business ,Pharmacogenetics - Abstract
Tacrolimus exhibits high inter-patient pharmacokinetics (PK) variability, as well as a narrow therapeutic index, and therefore requires therapeutic drug monitoring. Germline mutations in cytochrome P450 isoforms 4 and 5 genes (CYP3A4/5) and the ATP-binding cassette B1 gene (ABCB1) may contribute to interindividual tacrolimus PK variability, which may impact clinical outcomes among allogeneic hematopoietic stem cell transplantation (HSCT) patients. In this study, 252 adult patients who received tacrolimus for acute graft versus host disease (aGVHD) prophylaxis after allogeneic HSCT were genotyped to evaluate if germline genetic variants associated with tacrolimus PK and pharmacodynamic (PD) variability. Significant associations were detected between germline variants in CYP3A4/5 and ABCB1 and PK endpoints (e.g., median steady-state tacrolimus concentrations and time to goal tacrolimus concentration). However, significant associations were not observed between CYP3A4/5 or ABCB1 germline variants and PD endpoints (e.g., aGVHD and treatment-emergent nephrotoxicity). Decreased age and CYP3A5*1/*1 genotype were independently associated with subtherapeutic tacrolimus trough concentrations while CYP3A5*1*3 or CYP3A5*3/*3 genotypes, myeloablative allogeneic HSCT conditioning regimen (MAC) and increased weight were independently associated with supratherapeutic tacrolimus trough concentrations. Future lines of prospective research inquiry are warranted to use both germline genetic and clinical data to develop precision dosing tools that will optimize both tacrolimus dosing and clinical outcomes among adult HSCT patients.
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- 2020
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29. Computational modeling and confirmation of leukemia-associated minor histocompatibility antigens
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Jeffrey J. Molldrem, Jefferson L. Lansford, Edward J. Collins, Shengjie Chai, Udara Dharmasiri, Gheath Alatrash, Paul M. Armistead, Benjamin G. Vincent, Ian M. Schlup, James E. Keating, Gary L. Glish, Dante S. Bortone, and Sally A. Hunsucker
- Subjects
Male ,0301 basic medicine ,Graft vs Host Disease ,Graft vs Leukemia Effect ,Human leukocyte antigen ,Biology ,Epitope ,Minor Histocompatibility Antigens ,03 medical and health sciences ,Antigen ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,Minor histocompatibility antigen ,medicine ,Humans ,Computer Simulation ,Allele frequency ,Transplantation ,Hematopoietic Stem Cell Transplantation ,Models, Immunological ,Myeloid leukemia ,Hematology ,Allografts ,medicine.disease ,Leukemia, Myeloid, Acute ,Leukemia ,030104 developmental biology ,Myelodysplastic Syndromes ,Immunology ,Female ,Unrelated Donors - Abstract
T-cell responses to minor histocompatibility antigens (mHAs) mediate both antitumor immunity (graft-versus-leukemia [GVL]) and graft-versus-host disease (GVHD) in allogeneic stem cell transplant. Identifying mHAs with high allele frequency, tight binding affinity to common HLA molecules, and narrow tissue restriction could enhance immunotherapy against leukemia. Genotyping and HLA allele data from 101 HLA-matched donor-recipient pairs (DRPs) were computationally analyzed to predict both class I and class II mHAs likely to induce either GVL or GVHD. Roughly twice as many mHAs were predicted in HLA-matched unrelated donor (MUD) stem cell transplantation (SCT) compared with HLA-matched related transplants, an expected result given greater genetic disparity in MUD SCT. Computational analysis predicted 14 of 18 previously identified mHAs, with 2 minor antigen mismatches not being contained in the patient cohort, 1 missed mHA resulting from a noncanonical translation of the peptide antigen, and 1 case of poor binding prediction. A predicted peptide epitope derived from GRK4, a protein expressed in acute myeloid leukemia and testis, was confirmed by targeted differential ion mobility spectrometry-tandem mass spectrometry. T cells specific to UNC-GRK4-V were identified by tetramer analysis both in DRPs where a minor antigen mismatch was predicted and in DRPs where the donor contained the allele encoding UNC-GRK4-V, suggesting that this antigen could be both an mHA and a cancer-testis antigen. Computational analysis of genomic and transcriptomic data can reliably predict leukemia-associated mHA and can be used to guide targeted mHA discovery.
- Published
- 2018
30. Assay and Isolation of Single Proliferating CD4+ Lymphocytes Using an Automated Microraft Array Platform
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Raymond Zhang, Paul M. Armistead, Cody A. LaBelle, and Nancy L. Allbritton
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CD4-Positive T-Lymphocytes ,Lymphoblast ,T cell ,medicine.medical_treatment ,0206 medical engineering ,T-cell receptor ,Biomedical Engineering ,02 engineering and technology ,Cell Separation ,Biology ,Cell sorting ,Lymphocyte Activation ,020601 biomedical engineering ,Molecular biology ,Article ,Immune system ,medicine.anatomical_structure ,Single-cell analysis ,Cancer immunotherapy ,Antigen ,medicine ,Humans ,Cells, Cultured - Abstract
Objective: While T lymphocytes have been employed as a cancer immunotherapy, the development of effective and specific T-cell-based therapeutics remains challenging. A key obstacle is the difficulty in identifying T cells reactive to cancer-associated antigens. The objective of this research was to develop a versatile platform for single cell analysis and isolation that can be applied in immunology research and clinical therapy development. Methods: An automated microscopy and cell sorting system was developed to track the proliferative behavior of single-cell human primary CD4+ lymphocytes in response to stimulation using allogeneic lymphoblastoid feeder cells. Results: The system identified single human T lymphocytes with a sensitivity of 98% and specificity of 99% and possessed a cell collection efficiency of 86%. Time-lapse imaging simultaneously tracked 4,534 alloreactive T cells on a single array; 19% of the arrayed cells formed colonies of ≥2 cells. From the array, 130 clonal colonies were isolated and 7 grew to colony sizes of >10,000 cells, consistent with the known proliferative capacity of T cells in vitro and their tendency to become exhausted with prolonged stimulation. The isolated colonies underwent ELISA assay to detect interferon-γ secretion and Sanger sequencing to determine T cell receptor β sequences with a 100% success rate. Conclusion: The platform is capable of both identification and isolation of proliferative T cells in an automated manner. Significance: This novel technology enables the identification of TCR sequences based on T cell proliferation which is expected to speed the development of future cancer immunotherapies.
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- 2019
31. Age-Associated Changes in the Respiratory Epithelial Response to Influenza Infection
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Subhashini A. Sellers, William A. Fischer, Paul M. Armistead, Louisa E Brighton, Sally A. Hunsucker, Ilona Jaspers, Kelly D. Chason, and Joel S. Parker
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Adult ,Male ,0301 basic medicine ,Aging ,Immunosenescence ,medicine.medical_treatment ,Blotting, Western ,Antigen presentation ,Enzyme-Linked Immunosorbent Assay ,Major histocompatibility complex ,Risk Assessment ,Statistics, Nonparametric ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Influenza, Human ,medicine ,Humans ,Cytotoxic T cell ,RNA, Messenger ,Cells, Cultured ,Aged ,biology ,Antigen processing ,business.industry ,Epithelial Cells ,Orthomyxoviridae ,Acquired immune system ,Immunity, Innate ,Nasal Mucosa ,030104 developmental biology ,Cytokine ,Immunology ,biology.protein ,The Journal of Gerontology: Translational Section ,Female ,Geriatrics and Gerontology ,business ,030217 neurology & neurosurgery - Abstract
Older adults suffer a disproportionate burden of influenza-related morbidity and mortality typically attributed to defects in the aging immune system collectively known as immunosenescence. While the age-related decline in the adaptive immune system has been well characterized, little is known about how aging affects the principal site of influenza infection—the nasal epithelium. In human nasal epithelial cell cultures (hNECs) from older adults, we found similar or increased levels of cytokines during influenza infection compared with hNECs from younger individuals. However, hNECs from older individuals demonstrated decreased mRNA expression for several key proteins that affect clearance of infected cells, including MHC-I and transporter associated with antigen presentation (TAP). These findings were confirmed at the level of protein expression. In vivo studies corroborated the in vitro differences in MHC-I and TAP gene expression and also revealed important decreases in the expression of key influenza-specific antiviral mediators MX1 and IFITM1. Furthermore, epithelial cell-cytotoxic T lymphocyte co-cultures demonstrate that CTL cytotoxic activity is dose-dependent on MHC-I antigen presentation. Taken together, these results indicate that aging is associated with important changes in the nasal epithelium, including antigen presentation and antiviral pathways, which may contribute to increased severity of disease in older adults through impaired clearance of infected cells.
- Published
- 2018
32. Safety and Antitumor Effects of CD19-Specific Autologous Chimeric Antigen Receptor-Modified T (CAR-T) Cells Expressing the Inducible Caspase 9 Safety Switch (iC9-CAR19 T Cells) in Adult Acute Lymphoblastic Leukemia (ALL)
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Philip A. Roehrs, Katarzyna Jamieson, J. Kaitlin Morrison, Gianpietro Dotti, Barbara Savoldo, Natalie S Grover, George E Hucks, Paul M. Armistead, Spencer Laing, Matthew C. Foster, Faith Brianne Buchanan, Jonathan S. Serody, and Catherine Cheng
- Subjects
Caspase-9 ,biology ,Immunology ,biology.protein ,Cancer research ,Adult Acute Lymphoblastic Leukemia ,Cell Biology ,Hematology ,Car t cells ,Biochemistry ,CD19 ,Chimeric antigen receptor - Abstract
Introduction: CAR-T cells targeting the CD19 antigen are approved to treat children and young adults with relapsed and refractory B-cell ALL, in whom response rates are >80%. Acute toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) complicate CAR-T therapy in most patients. Though most patients with CRS or ICANS experience toxicities that can be managed with supportive care including corticosteroids and tocilizumab, 46% experience grade 3-4 CRS and 13% experience grade 3 ICANS (Maude SL et al N Engl J Med 2018; 37:439). Thus, novel approaches to address the management of high grade toxicities are needed. Donor T lymphocytes engineered to express human caspase 9 fused to a modified human FK-binding protein that induces caspase-dependent apoptosis when exposed to the dimerizing drug rimiducid (Di Stasi A et al. N Engl J Med 2011; 365:1673). We hypothesized that the inducible caspase 9 safety switch (iC9) coupled with CAR19 could mitigate severe CRS or ICANS in patients treated with CAR-T cells. We initiated a phase I trial to test the safety and efficacy of autologous T lymphocytes, genetically modified to express both iC9 and CAR19 administered to patients with relapsed and refractory B-ALL. Methods: Subjects with B-cell ALL in 2nd or greater bone marrow (BM) relapse, relapse >100 days after allogeneic stem cell transplant, disease refractory to ≥2 induction therapies, or with measurable residual disease (MRD) persistence/recurrence were enrolled in a phase I dose escalation trial. Autologous T-lymphocytes were collected, and CAR-T cell products generated by gene modification with a γ-retroviral vector encoding for iC9, ΔNGFR (for selection and tracking purposes) and CAR.CD19 (encoding 4-1BB) genes (Diaconu I et al Mol Ther 2017; 25:580). Subjects underwent lymphodepletion with fludarabine and cyclophosphamide and CAR-T cells were subsequently infused at one of two dose levels (DL1: 5 x 105 CAR-T cells/kg; DL2: 1 x 106 CAR-T cells/kg). Toxicities were graded by CTCAE v5 or ASBMT consensus grading for CRS and ICANS. Dose limiting toxicities (DLT) were grade 3-4 CRS or ICANS lasting >7 days despite standard of care intervention or grade 3 or higher autoimmune or non-CRS/ICANS organ toxicity. CAR-T cell expansion in peripheral blood (PB) was determined by flow cytometry (FC) and Q-PCR. Leukemia response was determined by NCCN criteria at 4 and 8 weeks after CAR-T infusion. Results: Nine products from 9 consecutive patients have been successfully manufactured in a median of 14 days (range 13-22), with transduction efficiency of 83 ± 6% after specific ΔNGFR selection. Six subjects have been enrolled to date, three in each cohort. Median age of subjects was 32 years (range 21-41). Median number of prior therapies was 3 (range 2-5). One subject had Philadelphia chromosome positive ALL. Maximum grade CRS was 2 in two subjects and 1 in three subjects. Median duration of any grade CRS was 4 days (range 2-7). One subject in DL1 experienced grade 1 ICANS for 2 days. All CRS/ICANS resolved with standard of care supportive measures, and no subject received rimiducid. No subject experienced DLT. CAR-T cells were found to be increased by PB Q-PCR, peaking at week 2 post infusion (7.9 x 104 ± 3.4 x 104 copies/μg of DNA). This trend paralleled the detection of CAR-T cells by FC. At 4 weeks post infusion, CAR-T cell signals were also detectable in BM samples, and BM B cells comprised Conclusions: iC9-CAR19 cells can be safely administered to adult patients with relapsed and refractory B-ALL. The use of iC9 as a safety switch has not been required due to the absence of patients with high-grade CRS/ICANS . Preliminary antileukemic activity was observed. The recommended phase 2 cell dose, 1 x 106 transduced cells/kg is being tested in an expansion cohort. A dose finding study to determine the effects of rimiducid on CRS/ICANS grade, CAR-T cell persistence and cytokine levels is being conducted among expansion cohort patients who experience CRS/ICANS not responding to standard supportive care. Introduction: CAR-T cells targeting the CD19 antigen are approved to treat children and young adults with relapsed and refractory B-cell ALL, in whom response rates are >80%. Acute toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) complicate CAR-T therapy in most patients. Though most patients with CRS or ICANS experience toxicities that can be managed with supportive care including corticosteroids and tocilizumab, 46% experience grade 3-4 CRS and 13% experience grade 3 ICANS (Maude SL et al N Engl J Med 2018; 37:439). Thus, novel approaches to address the management of high grade toxicities are needed. Donor T lymphocytes engineered to express human caspase 9 fused to a modified human FK-binding protein that induces caspase-dependent apoptosis when exposed to the dimerizing drug rimiducid (Di Stasi A et al. N Engl J Med 2011; 365:1673). We hypothesized that the inducible caspase 9 safety switch (iC9) coupled with CAR19 could mitigate severe CRS or ICANS in patients treated with CAR-T cells. We initiated a phase I trial to test the safety and efficacy of autologous T lymphocytes, genetically modified to express both iC9 and CAR19 administered to patients with relapsed and refractory B-ALL. Methods: Subjects with B-cell ALL in 2nd or greater bone marrow (BM) relapse, relapse >100 days after allogeneic stem cell transplant, disease refractory to ≥2 induction therapies, or with measurable residual disease (MRD) persistence/recurrence were enrolled in a phase I dose escalation trial. Autologous T-lymphocytes were collected, and CAR-T cell products generated by gene modification with a γ-retroviral vector encoding for iC9, ΔNGFR (for selection and tracking purposes) and CAR.CD19 (encoding 4-1BB) genes (Diaconu I et al Mol Ther 2017; 25:580). Subjects underwent lymphodepletion with fludarabine and cyclophosphamide and CAR-T cells were subsequently infused at one of two dose levels (DL1: 5 x 105 CAR-T cells/kg; DL2: 1 x 106 CAR-T cells/kg). Toxicities were graded by CTCAE v5 or ASBMT consensus grading for CRS and ICANS. Dose limiting toxicities (DLT) were grade 3-4 CRS or ICANS lasting >7 days despite standard of care intervention or grade 3 or higher autoimmune or non-CRS/ICANS organ toxicity. CAR-T cell expansion in peripheral blood (PB) was determined by flow cytometry (FC) and Q-PCR. Leukemia response was determined by NCCN criteria at 4 and 8 weeks after CAR-T infusion. Results: Nine products from 9 consecutive patients have been successfully manufactured in a median of 14 days (range 13-22), with transduction efficiency of 83 ± 6% after specific ΔNGFR selection. Six subjects have been enrolled to date, three in each cohort. Median age of subjects was 32 years (range 21-41). Median number of prior therapies was 3 (range 2-5). One subject had Philadelphia chromosome positive ALL. Maximum grade CRS was 2 in two subjects and 1 in three subjects. Median duration of any grade CRS was 4 days (range 2-7). One subject in DL1 experienced grade 1 ICANS for 2 days. All CRS/ICANS resolved with standard of care supportive measures, and no subject received rimiducid. No subject experienced DLT. CAR-T cells were found to be increased by PB Q-PCR, peaking at week 2 post infusion (7.9 x 104 ± 3.4 x 104 copies/μg of DNA). This trend paralleled the detection of CAR-T cells by FC. At 4 weeks post infusion, CAR-T cell signals were also detectable in BM samples, and BM B cells comprised Conclusions: iC9-CAR19 cells can be safely administered to adult patients with relapsed and refractory B-ALL. The use of iC9 as a safety switch has not been required due to the absence of patients with high-grade CRS/ICANS . Preliminary antileukemic activity was observed. The recommended phase 2 cell dose, 1 x 106 transduced cells/kg is being tested in an expansion cohort. A dose finding study to determine the effects of rimiducid on CRS/ICANS grade, CAR-T cell persistence and cytokine levels is being conducted among expansion cohort patients who experience CRS/ICANS not responding to standard supportive care. Disclosures Foster: Bellicum Pharmaceuticals: Research Funding; Daiichi Sankyo: Consultancy; Macrogenics: Consultancy, Research Funding. Roehrs:Spark Therapeutics: Consultancy; Gamifant: Speakers Bureau. Grover:Tessa: Consultancy; Genentech: Research Funding. Armistead:GeneCentric: Consultancy; Cell Microsystems: Patents & Royalties: Patent application U.S. 16/347,104 "Automated collection of a specified number of cells". Morrison:Vesselon: Consultancy. Dotti:Tessa Therapeutics: Consultancy, Research Funding; Bellicum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Savoldo:Bellicum Inc: Research Funding; Bluebirdbio: Research Funding; Tessa theraputics: Consultancy, Patents & Royalties, Research Funding; Cell Medica: Ended employment in the past 24 months, Research Funding. OffLabel Disclosure: Rimiducid will be mentioned in management of toxicities of cellular therapies.
- Published
- 2020
33. Cellular therapy against public neoantigens
- Author
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Paul M. Armistead
- Subjects
0301 basic medicine ,NPM1 ,Myeloid ,T cell ,medicine.medical_treatment ,Cell therapy ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,hemic and lymphatic diseases ,medicine ,Humans ,Antigens ,Nucleophosmin ,integumentary system ,business.industry ,Nuclear Proteins ,General Medicine ,Immunotherapy ,medicine.disease ,Leukemia ,Leukemia, Myeloid, Acute ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Cancer research ,business ,Research Article - Abstract
The most frequent subtype of acute myeloid leukemia (AML) is defined by mutations in the nucleophosmin 1 (NPM1) gene. Mutated NPM1 (ΔNPM1) is an attractive target for immunotherapy, since it is an essential driver gene and 4 bp frameshift insertions occur in the same hotspot in 30%–35% of AMLs, resulting in a C-terminal alternative reading frame of 11 aa. By searching the HLA class I ligandome of primary AMLs, we identified multiple ΔNPM1-derived peptides. For one of these peptides, HLA-A*02:01–binding CLAVEEVSL, we searched for specific T cells in healthy individuals using peptide-HLA tetramers. Tetramer-positive CD8(+) T cells were isolated and analyzed for reactivity against primary AMLs. From one clone with superior antitumor reactivity, we isolated the T cell receptor (TCR) and demonstrated specific recognition and lysis of HLA-A*02:01–positive ΔNPM1 AML after retroviral transfer to CD8(+) and CD4(+) T cells. Antitumor efficacy of TCR-transduced T cells was confirmed in immunodeficient mice engrafted with a human AML cell line expressing ΔNPM1. In conclusion, the data show that ΔNPM1-derived peptides are presented on AML and that CLAVEEVSL is a neoantigen that can be efficiently targeted on AML by ΔNPM1 TCR gene transfer. Immunotherapy targeting ΔNPM1 may therefore contribute to treatment of AML.
- Published
- 2019
34. Application of a Tacrolimus Population Pharmacokinetic Model to Adult Allogeneic Hematopoietic Stem Cell Transplant Patients
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Oscar Suzuki, Gabrielle Flynn, Daniel Weiner, Tejendra Patel, Tim Wiltshire, Jordan A Miller, Paul M. Armistead, Olivia Campagne, Chad Torrice, Daniel J. Crona, Jing Zhu, Donald E. Mager, and Jonathan R. Ptachcinski
- Subjects
Transplantation ,education.field_of_study ,business.industry ,Population ,Cell Biology ,Hematology ,Tacrolimus ,Pharmacokinetics ,Immunology ,Molecular Medicine ,Immunology and Allergy ,Medicine ,Allogeneic hematopoietic stem cell transplant ,education ,business - Published
- 2021
35. Associations of Clinical Outcomes after Allogeneic Hematopoietic Cell Transplantation with Number of Predicted Class II Restricted mHA
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Marcelo C. Pasquini, Hancong Tang, Junke Wang, Othmane Jadi, Theresa Hahn, Stephen R. Spellman, David Van Den Berg, Steven Vensko, Xin Sheng, Christopher A. Haiman, Benjamin G. Vincent, Lara E. Sucheston-Campbell, Loreall Pooler, Dante S. Bortone, and Paul M. Armistead
- Subjects
Oncology ,medicine.medical_specialty ,Proportional hazards model ,business.industry ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Human leukocyte antigen ,medicine.disease ,Biochemistry ,Transplantation ,Leukemia ,surgical procedures, operative ,medicine.anatomical_structure ,immune system diseases ,Acute lymphocytic leukemia ,Internal medicine ,Minor histocompatibility antigen ,Medicine ,Bone marrow ,business - Abstract
Background: Leukemia relapse after allogeneic hematopoietic cell transplantation (alloHCT) has been associated with loss of heterozygosity of class II HLA, suggesting that T cells targeting leukemia via class II interactions may be critical for graft-vs-leukemia (GvL) effects and protection from relapse (Zeiser & Vago, 2019). Therefore, we aimed to determine whether the number of predicted class-II restricted minor histocompatibility antigens (mHAs) in alloHCT recipients associates with clinical outcome. Specifically, we investigated: (1) if the number of mHAs expressed on leukemia and bone marrow cells (GvL mHAs) is correlated with incidence of leukemia relapse, and/or (2) if mHAs expressed on acute graft versus host disease (GvHD) target organs (GvH mHAs) correlated with incidence of GvHD. We expected increased GvL mHAs to be associated with increased survival, and increased GvH mHAs to be associated with increased incidence of GvHD. Methods: The study population was derived from donor-recipient alloHCT pairs (DRPs) treated for Acute Myeloid Leukemia (AML), Acute Lymphocytic Leukemia (ALL), and Myelodysplastic Syndrome (MDS) from the CIBMTR and previously analyzed in DISCOVeRY-BMT. To predict HLA class II restricted mHAs in alloHCT recipients, we translated nonsynonymous single nucleotide variants present in each recipient, but absent in their respective donor, into all possible peptides 15-24 amino acids long. We used netMHCIIpan to score these peptides and select those most likely to be presented (Reynisson et al., 2020). Predicted mHAs highly expressed in skin, hepatobiliary, and bowel tissues were classified as GvH mHAs, and those highly expressed highly in, testis, and bone marrow were classified as GvL mHAs. The impact of mHA on disease related mortality (DRM), leukemia free survival (LFS), disease relapse and death due to GVHD were assessed with competing risk models. We next determined whether knowledge of the number of GvL and GvH mHAs improves the ability of models to predict clinical outcome. We fit optimal logistic regression models (elastic net regularization with Monte Carlo cross validation) to predict GvHD mortality and relapse mortality at 1 year after alloHCT using: (1) clinical data only, and (2) both clinical data and number of GvH/GvL mHAs. Lastly, using the same regularization and cross-validation procedures as above, we determined which variables contributed most to the Cox regression models of overall survival. Results: The number of GvL-restricted and GvH-restricted class-II mHAs were significantly correlated (Figure 1). Patients who died of disease before 1 year after alloHCT had significantly fewer GvL mHAs when controlling for covariates (HR=.89, 95% CI .89, .99, P= 0.04) (Figure 2). There was no association between GvL mHAs and LFS or relapse. Surprisingly, death due to GvHD by 1 year after alloHCT was lower for recipients with more GvL mHAs (HR=.88, 95% CI= .76, 1.01 , P=.08) (Figure 3), however, because GvH and GvL mHAs are highly correlated, we believe this reflects better overall survival associated with increased GvL mHAs. Neither the predictive models that included the number of GvH/GvL mHAs nor the Cox regression models fitted with GvL mHA numbers performed better than models trained on clinical data alone (Figure 4). Conclusions: GvL mHAs were inversely associated with DRM at 1-year post-alloHCT as expected. Contrary to our hypothesis, the number of GvH mHAs was inversely associated with GvHD mortality at 1 year. This may reflect better overall survival associated with increased GvL mHAs, as GvH and GvL mHAs are highly correlated. The potential association between class-II mHAs and DRM warrant further investigation and validation. If validated, this supports the development of class-II mHA targeted immunotherapies. Disclosures Pasquini: Bristol Myers Squibb: Consultancy; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Other; Novartis: Research Funding; Kite: Research Funding. Armistead:GeneCentric: Consultancy; Cell Microsystems: Patents & Royalties: Patent application U.S. 16/347,104 "Automated collection of a specified number of cells". Vincent:GeneCentric Therapeutics: Consultancy.
- Published
- 2020
36. Cathepsin G is broadly expressed in acute myeloid leukemia and is an effective immunotherapeutic target
- Author
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L.S. St. John, Greg Lizee, Gheath Alatrash, Haroon Jakher, Jason Roszik, Pariya Sukhumalchandra, Paul M. Armistead, Dean A. Lee, Hong He, Anna Sergeeva, Suk-Young Yoo, Steve Kornblau, Qing Ma, David H. Hawke, Jeffrey J. Molldrem, Minying Zhang, Alexander A. Perakis, Haven R. Garber, Amjad H. Talukder, Kevin R. Coombes, Elizabeth A. Mittendorf, Yihua Qiu, Lisa M. Becker, Karen C. Dwyer, and Vladimir Senyukov
- Subjects
0301 basic medicine ,Cancer Research ,medicine.medical_specialty ,Cathepsin G ,Myeloid ,medicine.medical_treatment ,Gene Expression ,Article ,Fusion gene ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Animals ,Humans ,Hematology ,business.industry ,Myeloid leukemia ,Immunotherapy ,medicine.disease ,Leukemia, Myeloid, Acute ,Haematopoiesis ,Leukemia ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,chemistry ,Immunology ,Heterografts ,business - Abstract
Cathepsin G is broadly expressed in acute myeloid leukemia and is an effective immunotherapeutic target
- Published
- 2016
37. Array-Based Platform To Select, Release, and Capture Epstein–Barr Virus-Infected Cells Based on Intercellular Adhesion
- Author
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Christopher E. Sims, Paul M. Armistead, Nancy L. Allbritton, Peter J. Attayek, Yuli Wang, and Sally A. Hunsucker
- Subjects
food.ingredient ,Chemistry ,Cell ,medicine.disease_cause ,Gelatin ,Epstein–Barr virus ,Molecular biology ,Analytical Chemistry ,medicine.anatomical_structure ,food ,Single-cell analysis ,medicine ,Microrafts ,Cell adhesion ,Intracellular ,K562 cells - Abstract
Microraft arrays were developed to select and separate cells based on a complex phenotype, weak intercellular adhesion, without knowledge of cell-surface markers or intracellular proteins. Since the cells were also not competent to bind to a culture surface, a method to encapsulate nonadherent cells within a gelatin plug on the concave microraft surface was developed, enabling release and collection of the cells without the need for cell attachment to the microraft surface. After microraft collection, the gelatin was liquified to release the cell(s) for culture or analysis. A semiautomated release and collection device for the microrafts demonstrated 100 ± 0% collection efficiency of the microraft while increasing throughput 5-fold relative to that of manual release and collection. Using the microraft array platform along with the gelatin encapsulation method, single cells that were not surface-attached were isolated with a 100 ± 0% efficiency and a 96 ± 4% postsort single-cell cloning efficiency. As a demonstration, Epstein-Barr virus-infected lymphoblastoid cell lines (EBV-LCL) were isolated based on their intercellular adhesive properties. The identified cell colonies were collected with a 100 ± 0% sorting efficiency and a postsort viability of 87 ± 3%. When gene expression analysis of the EBV latency-associated gene, EBNA-2, was performed, there was no difference in expression between blasting or weakly adhesive cells and nonblasting or nonadhesive cells. Microraft arrays are a versatile method enabling separation of cells based on complicated and as yet poorly understood cell phenotypes.
- Published
- 2015
38. Detection of Measurable Residual Disease (MRD) in Peripheral Blood: First Report of a Novel Microfluidic Platform in Patients with Acute Myeloid Leukemia (AML)
- Author
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Rolf Müller, Rachel Toughiri, Yuri Fedoriw, Paul M. Armistead, Craig Carson, Emily Mirkin, Matthew C. Foster, Catherine C. Coombs, Joshua F. Zeidner, Maryam Zomorrodi, Will Pulley, and Alena Bartakova
- Subjects
Oncology ,medicine.medical_specialty ,Myeloid ,biology ,business.industry ,CD117 ,Immunology ,CD33 ,CD34 ,Induction chemotherapy ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Leukemia ,Immunophenotyping ,medicine.anatomical_structure ,Internal medicine ,biology.protein ,Medicine ,Bone marrow ,business - Abstract
Background: Despite therapeutic advances, AML remains a disease in which the majority of patients relapse after attaining remission. The presence of bone marrow MRD is associated with impending AML relapse. Prediction and prevention of relapse could improve outcomes, but most current MRD tests either require bone marrow aspirate or detect individualized molecular genetic features present in a minority of AML. Furthermore, bone marrow MRD assessments are impractical if performed frequently enough to detect most early relapses. We developed and tested a novel microfluidic chip device (MCD) that can quantitate cell numbers using automated methods and explored its ability to detect low levels of peripheral blood leukemic cells with aberrant immunophenotypes. Methods: The MCD contains sinusoidal capture channels that were coated with antibodies with specificity towards one of the commonly expressed markers found on immature myeloid cells--CD33, CD34 and CD117 (capture antigens). Initial spiking experiments used fluorescently labeled leukemia cell lines HL60 (CD33+), KG1 (CD34+), Kasumi1 (CD117+) spiked into a 5000 cell/mL suspension. Cell suspensions were passed through MCDs coated with a capture antigen known to be expressed on the tested cell line. These experiments established an efficient capture using a flow rate of 1mL/sec. Then, AML patients with an aberrant immunophenotype were enrolled in a pilot study either at the start of induction chemotherapy or prior to allogeneic stem cell transplant (SCT). For patients receiving induction chemotherapy, whole blood samples were obtained monthly starting at the time of remission assessment, or monthly starting prior to SCT. Buffered whole blood was passed through MCDs coated with a capture antigen known to be expressed on patient myeloblasts. The captured cells were then released, eluted, centrifuged and plated on a glass slide. Plated cell pellets were then labeled with fluorescent antibodies targeting surface proteins known to be aberrantly (either by lineage infidelity or asynchronous expression) expressed on the patients' AML blasts. Automated fluorescence microscopy was used to identify and quantify captured cells with the known aberrant immunophenotype of the AML blasts. Descriptive statistics described serial cell counts in patients maintaining remission and relapsing patients. Results: Of 31 patients who have been enrolled in the study to date, 13 had at least 3 post-remission MCD analyses. Of these patients, 6 had either morphologic relapse or persistent/rising marrow MRD. In these patients, there was a trend towards higher initial aberrant immunophenotype cell counts, with mean initial count = 59 (95% CI 1, 108), compared to other patients with mean initial count = 15 (95% CI 5, 25). Of 5 patients who relapsed with MCD data within 1 month prior to relapse, the mean absolute rise prior to relapse above minimum MCD cell count was 54 (95% CI 2, 105), in comparison to non-relapsing patients with mean rise of 9 (95% CI 3, 15). From the initial 16 patients, 10 underwent induction therapy (the other 6 were enrolled prior to SCT). In these 10 patients there was a non-significant association between peripheral blood aberrant immunophenotype cells and remission status following induction. A total of 8 patients underwent allogeneic SCT. Two of these patients had known bone marrow MRD at the time of SCT and had a statistically significant greater number of aberrant immunophenotypic cells pre-SCT (48 and 60) compared to the 6 MRD negative patients (median = 12, range 9, 42). Conclusions: A novel MCD assay can reliably capture and detect low numbers of AML blasts from peripheral blood using immunofluorescent imaging and automated cell counts to quantify leukemia cells with aberrant immunophenotypes. Because this method uses peripheral blood, frequent sampling is feasible and of minimal risk to patients. An ongoing clinical trial will further explore the associations between MCD-based cell enumeration and clinical endpoints in AML patients that were suggested in the pilot phase of this study. Because the MCD releases trace populations of viable cells, additional experiments, such as primary cell culturing and single cell sequencing, are possible. Figure Disclosures Foster: Bellicum Pharmaceuticals, Inc: Research Funding; Daiichi Sankyo: Consultancy; MacroGenics: Research Funding; Celgene: Research Funding. Fedoriw:Alexion Pharmaceuticals: Consultancy, Speakers Bureau. Zeidner:Celgene: Consultancy, Honoraria, Research Funding; AsystBio Laboratories: Consultancy; Merck: Research Funding; Covance: Consultancy; Pfizer: Honoraria; Agios: Honoraria; Daiichi Sankyo: Honoraria; Tolero: Honoraria, Research Funding. Coombs:Covance: Consultancy; Octopharma: Honoraria; Medscape: Honoraria; Cowen & Co.: Consultancy; Loxo: Honoraria; H3 Biomedicine: Honoraria; Dedham Group: Consultancy; Pharmacyclics: Honoraria; Abbvie: Consultancy. Mirkin:BioFluidica: Employment. Zomorrodi:BioFluidica: Employment. Toughiri:BioFluidica: Employment. Bartakova:BioFluidica: Employment. Carson:BioFluidica: Employment. Muller:BioFluidica: Employment.
- Published
- 2019
39. Drug-Drug Interaction Between Isavuconazole and Tacrolimus: Is Empiric Dose Adjustment Necessary?
- Author
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Lindsay M. Daniels, J. Ryan Shaw, Paul M. Armistead, Maurice Alexander, Wesley D Kufel, and Jonathan R. Ptachcinski
- Subjects
medicine.medical_specialty ,Posaconazole ,Antifungal Agents ,Pyridines ,chemical and pharmacologic phenomena ,030226 pharmacology & pharmacy ,Tacrolimus ,03 medical and health sciences ,0302 clinical medicine ,Pharmacokinetics ,Internal medicine ,Nitriles ,Medicine ,Humans ,Pharmacology (medical) ,Trough Concentration ,Drug Interactions ,030212 general & internal medicine ,Dosing ,Dose Reduced ,medicine.diagnostic_test ,Drug Tapering ,business.industry ,Middle Aged ,Triazoles ,Allografts ,surgical procedures, operative ,Therapeutic drug monitoring ,Pharmacodynamics ,Female ,Drug Monitoring ,business ,Immunosuppressive Agents ,medicine.drug - Abstract
A paucity of data currently exists regarding drug–drug interaction (DDI) with tacrolimus and isavuconazole coadministration. Current literature provides conflicting recommendations on whether an empiric tacrolimus dose reduction is necessary when coadministered with isavuconazole. A 47-year-old African American female with acute lymphoblastic leukemia underwent an allogenic stem cell transplant (alloSCT) and was subsequently placed on routine posttransplant therapy including tacrolimus for immunosuppression and posaconazole for antifungal prophylaxis. Tacrolimus was empirically dose reduced due to the expected DDI with posaconazole based on current recommendations. Due to a persistently prolonged QTc interval and need for mold coverage, antifungal prophylaxis was ultimately changed to isavuconazole at standard recommended dosing. Tacrolimus was empirically dose reduced by 40% based on limited available literature at the time; however, tacrolimus trough concentrations subsequently declined, requiring an increase in tacrolimus dose to maintain therapeutic trough concentrations. Adequate isavuconazole absorption was documented through pharmacokinetic and pharmacodynamic data by measuring an isavuconazole trough concentration and directly observing isavuconazole’s shortening effect on the QTc interval, respectively. Our experience in an alloSCT patient suggests that an empiric tacrolimus dose reduction is not required when isavuconazole is initiated, but close tacrolimus therapeutic drug monitoring should rather be performed to guide tacrolimus dosing.
- Published
- 2018
40. Myeloid Leukemia Vaccines
- Author
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Jonathan S. Serody and Paul M. Armistead
- Subjects
business.industry ,Cancer research ,Myeloid leukemia ,Medicine ,business ,medicine.disease ,Chronic myelogenous leukemia - Published
- 2018
41. Enzymatic cleavage of uracil-containing single-stranded DNA linkers for the efficient release of affinity-selected circulating tumor cells
- Author
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Paul M. Armistead, Steven A. Soper, Victoria L. Bae-Jump, Peter M. Voorhees, Joshua M. Jackson, Mateusz L. Hupert, Małgorzata A. Witek, Sally A. Hunsucker, Travis Sapp, Weiya Z. Wysham, Caroline E. Perry, Paola A. Gehrig, Maria A. M. Lindell, and Soumya V. Nair
- Subjects
DNA, Single-Stranded ,Antigens, CD34 ,Cleavage (embryo) ,Article ,Catalysis ,chemistry.chemical_compound ,Circulating tumor cell ,Antigens, Neoplasm ,Cell Line, Tumor ,Endopeptidases ,Materials Chemistry ,Humans ,Viability assay ,Uracil ,Uracil-DNA Glycosidase ,chemistry.chemical_classification ,Endodeoxyribonucleases ,Cell adhesion molecule ,Serine Endopeptidases ,Metals and Alloys ,Antibodies, Monoclonal ,Membrane Proteins ,General Chemistry ,Epithelial Cell Adhesion Molecule ,Neoplastic Cells, Circulating ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Enzyme ,chemistry ,Biochemistry ,Gelatinases ,Uracil-DNA glycosylase ,Ceramics and Composites ,Cell Adhesion Molecules ,DNA - Abstract
We report a novel strategy to enzymatically release affinity-selected cells, such as circulating tumor cells (CTCs), from surfaces with high efficiency (~90%) while maintaining cell viability (>85%). The strategy utilizes single-stranded DNAs that link a capture antibody to the surfaces of a CTC selection device. The DNA linkers contain a uracil residue that can be cleaved.
- Published
- 2015
42. Type 2 innate lymphoid cells treat and prevent acute gastrointestinal graft-versus-host disease
- Author
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John T. Woosley, Warren D. Shlomchik, Trisha A. Dant, Shannon Reisdorf, Andrew Neil James Mckenzie, Karen P. McKinnon, Jenny P.-Y. Ting, David A. Serody, Danny W. Bruce, Heather E. Stefanski, Dietmar M. W. Zaiss, Bruce R. Blazar, James M. Coghill, Justin E. Wilson, Jonathan S. Serody, Paul M. Armistead, Hemamalini Bommiasamy, and Benjamin G. Vincent
- Subjects
0301 basic medicine ,Gastrointestinal Diseases ,Graft vs Host Disease ,Proinflammatory cytokine ,03 medical and health sciences ,Mice ,Immune system ,Medicine ,Animals ,Myeloid Cells ,Lymphocytes ,Bone Marrow Transplantation ,Mice, Knockout ,Gastrointestinal tract ,business.industry ,Innate lymphoid cell ,General Medicine ,medicine.disease ,Allografts ,Transplantation ,030104 developmental biology ,Graft-versus-host disease ,surgical procedures, operative ,Immunology ,Acute Disease ,Stem cell ,business ,Homeostasis ,Research Article - Abstract
Acute graft-versus-host disease (aGVHD) is the most common complication for patients undergoing allogeneic stem cell transplantation. Despite extremely aggressive therapy targeting donor T cells, patients with grade III or greater aGVHD of the lower GI tract, who do not respond to therapy with corticosteroids, have a dismal prognosis. Thus, efforts to improve understanding of the function of local immune and non-immune cells in regulating the inflammatory process in the GI tract during aGVHD are needed. Here, we demonstrate, using murine models of allogeneic BMT, that type 2 innate lymphoid cells (ILC2s) in the lower GI tract are sensitive to conditioning therapy and show very limited ability to repopulate from donor bone marrow. Infusion of donor ILC2s was effective in reducing the lethality of aGVHD and in treating lower GI tract disease. ILC2 infusion was associated with reduced donor proinflammatory Th1 and Th17 cells, accumulation of donor myeloid-derived suppressor cells (MDSCs) mediated by ILC2 production of IL-13, improved GI tract barrier function, and a preserved graft-versus-leukemia (GVL) response. Collectively, these findings suggest that infusion of donor ILC2s to restore gastrointestinal tract homeostasis may improve treatment of severe lower GI tract aGVHD.
- Published
- 2017
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43. Effectiveness of an Algorithm-Based Approach to the Utilization of Plerixafor in Patients Undergoing Chemotherapy-Based Stem Cell Mobilization
- Author
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Yara A. Park, Jay S. Raval, Eric Chow, Deborah L. Covington, James M. Coghill, Katarzyna Jamieson, Jonathan S. Serody, Paul M. Armistead, Kamakshi V. Rao, William A. Wood, Thomas C. Shea, and Don A. Gabriel
- Subjects
Adult ,Male ,Benzylamines ,medicine.medical_specialty ,medicine.medical_treatment ,CD34 ,Autologous stem cell transplantation ,Cyclams ,Transplantation, Autologous ,Young Adult ,Autologous stem-cell transplantation ,Heterocyclic Compounds ,medicine ,Humans ,Etoposide ,Multiple myeloma ,Aged ,Chemotherapy ,Transplantation ,business.industry ,Plerixafor ,Hematology ,Middle Aged ,medicine.disease ,Hematopoietic Stem Cell Mobilization ,Surgery ,Stem cell mobilization ,Blood Component Removal ,Female ,Stem cell ,business ,Algorithm ,Algorithms ,medicine.drug - Abstract
Autologous stem cell transplantation remains a mainstay of therapy for diseases such as multiple myeloma and relapsed lymphoma. The use of plerixafor has been shown to augment the ability to collect adequate stem cells, but the optimal use of this agent when used with chemotherapy is not yet clear. We utilized an algorithm-based approach with the addition of plerixafor to 54 patients undergoing chemomobilization with reduced-dose etoposide who had a less than optimal preapheresis CD34+ cell count. We used a CD34+ precount of 20 cells/μL as a threshold to initiate stem cell apheresis. Ninety-four percent of patients were successfully collected and proceeded to transplantation. Fourteen of 51 (28%) patients who successfully collected required plerixafor to augment stem cell yield. Of the patients who successfully collected, 94% (89% of the entire population) were able to collect in 2 or fewer days. Compared with previous data from our institution, the rate of patients collecting > 4 × 106 CD34+ cells/kg in a single collection was increased from 39% to 69%. The safety profile of this approach was acceptable. The use of this algorithm-based method to determine when and whether to add plerixafor to chemomobilization was shown to be a successful and cost-effective approach to stem cell collection.
- Published
- 2014
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44. Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device
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Katrina N. Battle, Paul M. Armistead, Steven A. Soper, Małgorzata A. Witek, Mateusz L. Hupert, Sally A. Hunsucker, and Joshua M. Jackson
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Ultraviolet Rays ,Biochemistry ,Article ,Analytical Chemistry ,chemistry.chemical_compound ,Biotin ,Cell Line, Tumor ,Electrochemistry ,Humans ,Polymethyl Methacrylate ,Environmental Chemistry ,Biotinylation ,Solid phase extraction ,Spectroscopy ,Downstream processing ,Chromatography ,biology ,Chemistry ,Solid Phase Extraction ,Extraction (chemistry) ,Membrane Proteins ,NeutrAvidin ,Equipment Design ,Microfluidic Analytical Techniques ,Solubility ,Membrane protein ,biology.protein ,Linker - Abstract
We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3,600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (~20 fmol) of membrane proteins could be isolated and recovered with ~89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.
- Published
- 2014
45. Automated microraft platform to identify and collect non-adherent cells successfully gene-edited with CRISPR-Cas9
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Philip J. Grayeski, Christopher E. Sims, Peter J. Attayek, Jennifer P. Waugh, Sally A. Hunsucker, Paul M. Armistead, and Nancy L. Allbritton
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0301 basic medicine ,Green Fluorescent Proteins ,Biomedical Engineering ,Biophysics ,Computational biology ,Biosensing Techniques ,Gene mutation ,Biology ,Transfection ,Article ,03 medical and health sciences ,0302 clinical medicine ,Software ,Genome editing ,Genes, Reporter ,Electrochemistry ,Image Processing, Computer-Assisted ,CRISPR ,Humans ,Leukemia ,business.industry ,General Medicine ,Equipment Design ,Cell sorting ,Splicing Factor U2AF ,Molecular biology ,030104 developmental biology ,Tissue Array Analysis ,030220 oncology & carcinogenesis ,Mutation ,Microrafts ,CRISPR-Cas Systems ,business ,K562 Cells ,Cytometry ,Biotechnology - Abstract
Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware released microrafts with 98% efficiency and collected released microrafts with 100% efficiency. The automated system was used to examine the temporal variation in EGFP expression in cells transfected with CRISPR-Cas9 components for gene editing. Of 11,499 microrafts possessing a single cell, 220 microrafts were identified as possessing temporally varying EGFP-expression. Candidate cells (n=172) were released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies were successfully gene edited demonstrating the desired mutation.
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- 2016
46. Identification and isolation of antigen-specific cytotoxic T lymphocytes with an automated microraft sorting system
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Nancy L. Allbritton, Christopher E. Sims, Peter J. Attayek, Sally A. Hunsucker, and Paul M. Armistead
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0301 basic medicine ,CD4-Positive T-Lymphocytes ,T cell ,Biophysics ,Streptamer ,Cell Separation ,Biology ,CD8-Positive T-Lymphocytes ,Biochemistry ,Article ,03 medical and health sciences ,Antigen ,Lab-On-A-Chip Devices ,medicine ,Cytotoxic T cell ,Humans ,Antigen-presenting cell ,Antigens, Viral ,Cells, Cultured ,Immunoassay ,Equipment Design ,Cell biology ,Equipment Failure Analysis ,Killer Cells, Natural ,030104 developmental biology ,Cell killing ,medicine.anatomical_structure ,Tissue Array Analysis ,T cell mediated cytotoxicity ,CD8 ,Epitope Mapping ,T-Lymphocytes, Cytotoxic - Abstract
The simultaneous measurement of T cell function with recovery of individual T cells would greatly facilitate characterizing antigen-specific responses both in vivo and in model systems. We have developed a microraft array methodology that automatically measures the ability of individual T cells to kill a population of target cells and viably sorts specific cells into a 96-well plate for expansion. A human T cell culture was generated against the influenza M1p antigen. Individual microrafts on a 70 × 70 array were loaded with on average 1 CD8+ cell from the culture and a population of M1p presenting target cells. Target cell killing, measured by fluorescence microscopy, was quantified in each microraft. The rates of target cell death among the individual CD8+ T cells varied greatly; however, individual T cells maintained their rates of cytotoxicity throughout the time course of the experiment enabling rapid identification of highly cytotoxic CD8+ T cells. Microrafts with highly active CD8+ T cells were individually transferred to wells of a 96-well plate, using a needle-release device coupled to the microscope. Three sorted T cells clonally expanded. All of these expressed high-avidity T cell receptors for M1p/HLA*02:01 tetramers, and 2 of the 3 receptors were sequenced. While this study investigated single T cell cytotoxicity rates against simple targets with subsequent cell sorting, future studies will involve measuring T cell mediated cytotoxicity in more complex cellular environments, enlarging the arrays to identify very rare antigen specific T cells, and measuring single cell CD4+ and CD8+ T cell proliferation.
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- 2016
47. Microfluidic Chemical Cytometry of Peptide Degradation in Single Drug-Treated Acute Myeloid Leukemia Cells
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Paul M. Armistead, Nancy L. Allbritton, Pavak K. Shah, and Michelle L. Kovarik
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Drug ,Proteolysis ,media_common.quotation_subject ,Antineoplastic Agents ,Peptide ,Aminopeptidase ,Article ,Analytical Chemistry ,Cell Line, Tumor ,medicine ,Humans ,Amino Acid Sequence ,Peptide sequence ,media_common ,chemistry.chemical_classification ,medicine.diagnostic_test ,Myeloid leukemia ,Microfluidic Analytical Techniques ,Molecular biology ,Kinetics ,Leukemia, Myeloid, Acute ,chemistry ,Cell culture ,Peptides ,Cytometry - Abstract
Microfluidic systems show great promise for single-cell analysis; however, as these technologies mature, their utility must be validated by studies of biologically relevant processes. An important biomedical application of these systems is characterization of tumor cell heterogeneity. In this work, we used a robust microfluidic platform to explore the heterogeneity of enzyme activity in single cells treated with a chemotherapeutic drug. Using chemical cytometry, we measured peptide degradation in the U937 acute myeloid leukemia (AML) cell line in the presence and absence of the aminopeptidase inhibitor Tosedostat (CHR-2797). The analysis of 99 untreated cells revealed rapid and consistent degradation of the peptide reporter within 20 min of loading. Results from drug-treated cells showed inhibited, but ongoing degradation of the reporter. Because the device operates at an average sustained throughput of 37 ± 7 cells/h, we were able to sample cells over the course of this time-dependent degradation. In data from 498 individual drug-treated cells, we found a linear dependence of degradation rate on amount of substrate loaded superimposed upon substantial heterogeneity in peptide processing in response to inhibitor treatment. Importantly, these data demonstrated the potential of microfluidic systems to sample biologically relevant analytes and time-dependent processes in large numbers of single cells.
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- 2013
48. Automated Capillary Electrophoresis System for Fast Single-Cell Analysis
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Alexandra J. Dickinson, Nancy L. Allbritton, and Paul M. Armistead
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Lysis ,Sphingosine ,Kinase ,Cell ,Sphingosine kinase ,Electrophoresis, Capillary ,PC12 Cells ,Molecular biology ,Article ,Rats ,Analytical Chemistry ,Automation ,chemistry.chemical_compound ,Capillary electrophoresis ,medicine.anatomical_structure ,chemistry ,Single-cell analysis ,Tumor Cells, Cultured ,medicine ,Animals ,Single-Cell Analysis ,Fluorescein - Abstract
Capillary electrophoresis (CE) is a promising technique for single-cell analysis, but its use in biological studies has been limited by low throughput. This paper presents an automated platform employing microfabricated cell traps and a three-channel system for rapid buffer exchange for fast single-cell CE. Cells loaded with fluorescein and Oregon green were analyzed at a throughput of 3.5 cells/min with a resolution of 2.3 ± 0.6 for the fluorescein and Oregon green. Cellular protein kinase B (PKB) activity, as measured by immunofluorescence staining of phospho-PKB, was not altered, suggesting that this stress-activated kinase was not upregulated during the CE experiments and that basal cell physiology was not perturbed prior to cell lysis. The activity of sphingosine kinase (SK), which is often upregulated in cancer, was measured in leukemic cells by loading a sphingosine-fluorescein substrate into cells. Sphingosine fluorescein (SF), sphingosine-1-phosphate fluorescein (S1PF), and a third fluorescent species were identified in single cells. A single-cell throughput of 2.1 cells/min was achieved for 219 total cells. Eighty-eight percent of cells possessed upregulated SK activity, although subpopulations of cells with markedly different SK activity relative to that of the population average were readily identified. This system was capable of stable and reproducible separations of biological compounds in hundreds of adherent and nonadherent cells, enabling measurements of previously uncharacterized biological phenomena.
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- 2013
49. Effectiveness of etoposide chemomobilization in lymphoma patients undergoing auto-SCT
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Thomas C. Shea, Amber N Essenmacher, Paul Brown, Andrew Sharf, William A. Wood, Kamakshi V. Rao, Jonathan S. Serody, James M. Coghill, Robert Irons, Paul M. Armistead, Julia Whitley, Stefanie Sarantopoulos, Ravi K. Goyal, and D. A. Gabriel
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Oncology ,Transplantation ,medicine.medical_specialty ,business.industry ,Plerixafor ,medicine.medical_treatment ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Surgery ,Granulocyte colony-stimulating factor ,Lymphoma ,Regimen ,Internal medicine ,medicine ,Autologous transplantation ,business ,Etoposide ,Hematopoietic Stem Cell Mobilization ,medicine.drug - Abstract
The effectiveness of stem cell mobilization with G-CSF in lymphoma patients is suboptimal. We reviewed our institutional experience using chemomobilization with etoposide (VP-16; 375 mg/m2 on days +1 and +2) and G-CSF (5 μg/kg twice daily from day +3 through the final day of collection) in 159 patients with lymphoma. This approach resulted in successful mobilization (>2 × 106 CD34+ cells collected) in 94% of patients (83% within 4 apheresis sessions). Fifty-seven percent of patients yielded at least 5 × 106 cells in ⩽2 days and were defined as good mobilizers. The regimen was safe with a low rate of rehospitalization. Average costs were $14 923 for good mobilizers and $27 044 for poor mobilizers (P
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- 2012
50. The Role of Antigen Cross-presentation From Leukemia Blasts on Immunity to the Leukemia-associated Antigen PR1
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Eric D. Wieder, Tania G. Rodriguez-Cruz, Gheath Alatrash, Shoudan Liang, Lisa S. St. John, Hong He, Na Qiao, Gregory Lizée, Jeffrey J. Molldrem, Paul M. Armistead, Karen Clise-Dwyer, Sijie Lu, Anna Sergeeva, Elizabeth A. Mittendorf, Tian Hui Yang, Mao Zhang, Yoko Ono, Kathryn Ruisaard, and Pariya Sukhumalchandra
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Proteasome Endopeptidase Complex ,Cancer Research ,Myeloid ,medicine.drug_class ,Myeloblastin ,Immunology ,Antigen-Presenting Cells ,Biology ,Monoclonal antibody ,Article ,Cross-Priming ,Immune system ,Antigen ,Antigens, Neoplasm ,Immunity ,Cell Line, Tumor ,medicine ,Humans ,Immunology and Allergy ,Pharmacology ,B-Lymphocytes ,Leukemia ,Histocompatibility Antigens Class I ,Ubiquitination ,Cross-presentation ,Myeloid leukemia ,Dendritic Cells ,medicine.disease ,Protein Transport ,medicine.anatomical_structure ,Leukocyte Elastase ,Lysosomes ,Signal Transduction - Abstract
Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Because neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination, and proteasome processing for cross-presentation. Using anti-PR1/human leukocyte antigen-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, whereas DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classic major histocompatibility class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia.
- Published
- 2012
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