Title: CAR T-cells that target acute B-lineage leukemia irrespective of CD19 expression
Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(−) disease. We report that CD19(−) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(−) escape BL-ALL while preserving their upfront efficacy. We created a CD19/20/22-targeting CAR T-cell by coexpressing individual CAR molecules on a single T-cell using one tricistronic transgene. CD19/20/22CAR T-cells killed CD19(−) blasts from patients who relapsed after CD19CAR T-cell therapy and CRISPR/Cas9 CD19 knockout primary BL-ALL both in vitro and in an animal model, while CD19CAR T-cells were ineffective. At the subcellular level, CD19/20/22CAR T-cells formed dense immune synapses with target cells that mediated effective cytolytic complex formation, were efficient serial killers in single-cell tracking studies, and were as efficacious as CD19CAR T-cells against primary CD19(+) disease. In conclusion, independent of CD19 expression, CD19/20/22CAR T-cells could be used as salvage or front-line CAR therapy for patients with recalcitrant disease. more »« less
Bandey, Irfan N; Adolacion, Jay R; Romain, Gabrielle; Paniagua, Melisa Martinez; An, Xingyue; Saeedi, Arash; Liadi, Ivan; You, Zheng; Rajanayake, Rasindu B; Hwu, Patrick; et al(
, Journal for ImmunoTherapy of Cancer)
null
(Ed.)
Background Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive. Methods To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling. Results With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107a pos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice. Conclusions Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.
2938 Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy
63rd ASH Annual Meeting and Exposition, December 11-14, 2021, Georgia World Congress Center, Atlanta, GA
Program: Oral and Poster Abstracts
Session: 801. Gene Therapies: Poster II
Hematology Disease Topics & Pathways:
Bleeding and Clotting, Biological, Translational Research, Hemophilia, Genetic Disorders, Clinically Relevant, Diseases, Gene Therapy, Therapies
Sunday, December 12, 2021, 6:00 PM-8:00 PM
Ritu M Ramamurthy1*, Wen Ting Zheng2*, Sunil George, PhD1*, Meimei Wan1*, Yu Zhou, PhD1*, Baisong Lu, PhD1*, Colin E Bishop, PhD1*, Anthony Atala, M.D.1*, Christopher D Porada, PhD1* and M. Graca Almeida-Porada, MD3
1Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC
2Massachusetts Institute of Technology, Cambridge, MA
3Fetal Research and Therapy Program, Wake Forest Institute For Regenerative Medicine, Winston-Salem, NC
Clinical trials employing AAV vectors for hemophilia A have been hindered by unanticipated immunological and/or inflammatory responses in some of the patients. Also, these trials have often yielded lower levels of transgene expression than were expected based upon preclinical studies, highlighting the poor correlation between the transduction efficiency observed in traditional 2D cultures of primary cells in vitro, and that observed in those same cell types in vivo. It has been also recognized that there are marked species-specific differences in AAV-vector tropism, raising the critical question of the accuracy with which various animal models will likely predict tropism/vector transduction efficiency, and eventual treatment success in humans. Human liver tissue equivalents (hLTEs) are comprised of major cell types in the liver in physiologically relevant frequencies and possess the ability to recapitulate the biology and function of native human liver. Here, we hypothesize that hLTEs can be used as a better model to predict the efficacy and safety of AAV gene therapy in humans. We fabricated hLTEs using 75% hepatocytes, 10% stellate cells, 10% Kupffer cells, and 5% liver sinusoid-derived endothelial cells in 96-well Elplasia plates with 79 microwells per well. hLTEs were transduced at an MOI of 105vg/cell, on the day of fabrication, with the clinically relevant serotypes AAV5 (hLTE-5) or AAV3b (hLTE-3b), both encoding a GFP reporter. After 4 days of self-aggregation, live/dead assay was performed to confirm viability. Non-transduced hLTEs served as negative controls (hLTE(-)), and hLTEs exposed to 20 mM acetaminophen were used as positive controls for liver inflammation/damage. Incucyte® Live-Cell Imaging system was used to track the aggregation and GFP expression of hLTEs. Over the course of the next 5 days, media was collected to determine hepatic functionality, RNA was isolated to assess dysregulation of genes involved in inflammation and fibrosis, DNA was isolated to determine whether AAV vectors integrate into the genome of human hepatocytes and, if so, to define the frequency at which this occurs and the genomic loci of integration, and hLTEs were fixed and processed at appropriate times for histological analyses and transmission electron microscopy (TEM). TEM analysis revealed that all groups exhibited microvilli and bile-canaliculus-like structures, demonstrating the formation of a rudimentary biliary system and, more importantly, proving that hLTEs resemble native liver structure. Incucyte® imaging showed that AAV5 and AAV3b transduction impaired formation of hLTEs (57.57 ± 2.42 and 24.57 ± 4.01 spheroids/well, respectively) in comparison with hLTE(-) (74.86 ± 3.8 spheroids/well). Quantification of GFP expression demonstrated that AAV5 yielded the most efficient transduction of hLTEs (fold change in GFP expression compared to control: 2.73 ± 0.09 and 1.19 ± 0.03 for hLTE-5 and hLTE-3b, respectively). Chromogenic assays showed decreased urea production in cell culture supernatants of AAV transduced groups compared to the non-transduced hLTEs on days 6 and 10 of culture, demonstrating decreased hepatocyte functionality. However, ALT and AST levels were similar in all groups. On day 10, hLTEs were either used for RNA isolation or fixed in 4% PFA and processed for histology. Masson’s Trichrome and Alcian Blue/Sirius Red staining was performed to detect fibrosis, which was then quantified using ImageJ. These analyses showed no significant increase in fibrosis in either hLTE-5 or hLTE-3b compared to hLTE(-). Nevertheless, RT2 PCR Array for Human Fibrosis detected dysregulation of several genes involved in fibrosis/inflammation in both hLTE-5 and hLTE-3b (16/84 and 26/84, respectively). In conclusion, data collected thus far show successful recapitulation of native liver biology and demonstrate that AAV5 transduces hLTEs more efficiently than AAV3b. However, impaired self-aggregation and decreased hepatocyte functionality was observed in both AAV-transduced groups. Studies to address the incidence and location(s) of AAV integration are ongoing. We have thus shown that the hLTE system can provide critical new knowledge regarding the efficacy and safety of AAV gene therapy in the human liver.
Disclosures: No relevant conflicts of interest to declare.
Liu, Lunan; Ma, Chao; Zhang, Zhuoyu; Witkowski, Matthew T; Aifantis, Iannis; Ghassemi, Saba; Chen, Weiqiang(
, Journal for ImmunoTherapy of Cancer)
Background Adaptive CD19-targeted chimeric antigen receptor (CAR) T-cell transfer has become a promising treatment for leukemia. Although patient responses vary across different clinical trials, reliable methods to dissect and predict patient responses to novel therapies are currently lacking. Recently, the depiction of patient responses has been achieved using in silico computational models, with prediction application being limited. Methods We established a computational model of CAR T-cell therapy to recapitulate key cellular mechanisms and dynamics during treatment with responses of continuous remission (CR), non-response (NR), and CD19-positive (CD19 + ) and CD19-negative (CD19 − ) relapse. Real-time CAR T-cell and tumor burden data of 209 patients were collected from clinical studies and standardized with unified units in bone marrow. Parameter estimation was conducted using the stochastic approximation expectation maximization algorithm for nonlinear mixed-effect modeling. Results We revealed critical determinants related to patient responses at remission, resistance, and relapse. For CR, NR, and CD19 + relapse, the overall functionality of CAR T-cell led to various outcomes, whereas loss of the CD19 + antigen and the bystander killing effect of CAR T-cells may partly explain the progression of CD19 − relapse. Furthermore, we predicted patient responses by combining the peak and accumulated values of CAR T-cells or by inputting early-stage CAR T-cell dynamics. A clinical trial simulation using virtual patient cohorts generated based on real clinical patient datasets was conducted to further validate the prediction. Conclusions Our model dissected the mechanism behind distinct responses of leukemia to CAR T-cell therapy. This patient-based computational immuno-oncology model can predict late responses and may be informative in clinical treatment and management.
Harris, Emily; Elmer, Jacob J.(
, Biotechnology Progress)
Abstract
CAR‐T therapy is a particularly effective treatment for some types of cancer that uses retroviruses to deliver the gene for a chimeric antigen receptor (CAR) to a patient's T cells ex vivo. The CAR enables the T cells to bind and eradicate cells with a specific surface marker (e.g., CD19+B cells) after they are transfused back into the patient. This treatment was proven to be particularly effective in treating non‐Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL), but the current CAR‐T cell manufacturing process has a few significant drawbacks. For example, while lentiviral and gammaretroviral transduction are both relatively effective, the process of producing viral vectors is time‐consuming and costly. Additionally, patients must undergo follow up appointments for several years to monitor them for any unanticipated side effects associated with the virus. Therefore, several studies have endeavored to find alternative non‐viral gene delivery methods that are less expensive, more precise, simple, and safe. This review focuses on the current state of the most promising non‐viral gene delivery techniques, including electroporation and transfection with cationic polymers or lipids.
Lin, Chih-Wei; Wang, Yu-Jen; Lai, Ting-Yen; Hsu, Tsui-Ling; Han, Shin-Ying; Wu, Han-Chung; Shen, Chia-Ning; Dang, Van; Chen, Ming-Wei; Chen, Lan-Bo; et al(
, Proceedings of the National Academy of Sciences)
Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease.
Fousek, Kristen, Watanabe, Junji, Joseph, Sujith K., George, Ann, An, Xingyue, Byrd, Tiara T., Morris, Jessica S., Luong, Annie, Martínez-Paniagua, Melisa A., Sanber, Khaled, Navai, Shoba A., Gad, Ahmed Z., Salsman, Vita S., Mathew, Pretty R., Kim, Hye Na, Wagner, Dimitrios L., Brunetti, Lorenzo, Jang, Albert, Baker, Matthew L., Varadarajan, Navin, Hegde, Meenakshi, Kim, Yong-Mi, Heisterkamp, Nora, Abdel-Azim, Hisham, and Ahmed, Nabil. CAR T-cells that target acute B-lineage leukemia irrespective of CD19 expression. Retrieved from https://par.nsf.gov/biblio/10195220. Leukemia . Web. doi:10.1038/s41375-020-0792-2.
Fousek, Kristen, Watanabe, Junji, Joseph, Sujith K., George, Ann, An, Xingyue, Byrd, Tiara T., Morris, Jessica S., Luong, Annie, Martínez-Paniagua, Melisa A., Sanber, Khaled, Navai, Shoba A., Gad, Ahmed Z., Salsman, Vita S., Mathew, Pretty R., Kim, Hye Na, Wagner, Dimitrios L., Brunetti, Lorenzo, Jang, Albert, Baker, Matthew L., Varadarajan, Navin, Hegde, Meenakshi, Kim, Yong-Mi, Heisterkamp, Nora, Abdel-Azim, Hisham, and Ahmed, Nabil.
"CAR T-cells that target acute B-lineage leukemia irrespective of CD19 expression". Leukemia (). Country unknown/Code not available. https://doi.org/10.1038/s41375-020-0792-2.https://par.nsf.gov/biblio/10195220.
@article{osti_10195220,
place = {Country unknown/Code not available},
title = {CAR T-cells that target acute B-lineage leukemia irrespective of CD19 expression},
url = {https://par.nsf.gov/biblio/10195220},
DOI = {10.1038/s41375-020-0792-2},
abstractNote = {Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(−) disease. We report that CD19(−) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(−) escape BL-ALL while preserving their upfront efficacy. We created a CD19/20/22-targeting CAR T-cell by coexpressing individual CAR molecules on a single T-cell using one tricistronic transgene. CD19/20/22CAR T-cells killed CD19(−) blasts from patients who relapsed after CD19CAR T-cell therapy and CRISPR/Cas9 CD19 knockout primary BL-ALL both in vitro and in an animal model, while CD19CAR T-cells were ineffective. At the subcellular level, CD19/20/22CAR T-cells formed dense immune synapses with target cells that mediated effective cytolytic complex formation, were efficient serial killers in single-cell tracking studies, and were as efficacious as CD19CAR T-cells against primary CD19(+) disease. In conclusion, independent of CD19 expression, CD19/20/22CAR T-cells could be used as salvage or front-line CAR therapy for patients with recalcitrant disease.},
journal = {Leukemia},
author = {Fousek, Kristen and Watanabe, Junji and Joseph, Sujith K. and George, Ann and An, Xingyue and Byrd, Tiara T. and Morris, Jessica S. and Luong, Annie and Martínez-Paniagua, Melisa A. and Sanber, Khaled and Navai, Shoba A. and Gad, Ahmed Z. and Salsman, Vita S. and Mathew, Pretty R. and Kim, Hye Na and Wagner, Dimitrios L. and Brunetti, Lorenzo and Jang, Albert and Baker, Matthew L. and Varadarajan, Navin and Hegde, Meenakshi and Kim, Yong-Mi and Heisterkamp, Nora and Abdel-Azim, Hisham and Ahmed, Nabil},
}
Warning: Leaving National Science Foundation Website
You are now leaving the National Science Foundation website to go to a non-government website.
Website:
NSF takes no responsibility for and exercises no control over the views expressed or the accuracy of
the information contained on this site. Also be aware that NSF's privacy policy does not apply to this site.
