Cargando…

Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA...

Descripción completa

Detalles Bibliográficos
Autores principales: Soldierer, Maren, Bister, Arthur, Haist, Corinna, Thivakaran, Aniththa, Cengiz, Sevgi Can, Sendker, Stephanie, Bartels, Nina, Thomitzek, Antonia, Smorra, Denise, Hejazi, Maryam, Uhrberg, Markus, Scheckenbach, Kathrin, Monzel, Cornelia, Wiek, Constanze, Reinhardt, Dirk, Niktoreh, Naghmeh, Hanenberg, Helmut
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9022481/
https://www.ncbi.nlm.nih.gov/pubmed/35464442
http://dx.doi.org/10.3389/fimmu.2022.847008
_version_ 1784690095074312192
author Soldierer, Maren
Bister, Arthur
Haist, Corinna
Thivakaran, Aniththa
Cengiz, Sevgi Can
Sendker, Stephanie
Bartels, Nina
Thomitzek, Antonia
Smorra, Denise
Hejazi, Maryam
Uhrberg, Markus
Scheckenbach, Kathrin
Monzel, Cornelia
Wiek, Constanze
Reinhardt, Dirk
Niktoreh, Naghmeh
Hanenberg, Helmut
author_facet Soldierer, Maren
Bister, Arthur
Haist, Corinna
Thivakaran, Aniththa
Cengiz, Sevgi Can
Sendker, Stephanie
Bartels, Nina
Thomitzek, Antonia
Smorra, Denise
Hejazi, Maryam
Uhrberg, Markus
Scheckenbach, Kathrin
Monzel, Cornelia
Wiek, Constanze
Reinhardt, Dirk
Niktoreh, Naghmeh
Hanenberg, Helmut
author_sort Soldierer, Maren
collection PubMed
description The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy—CD19, CD33, and CD123—to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.
format Online
Article
Text
id pubmed-9022481
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-90224812022-04-22 Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies Soldierer, Maren Bister, Arthur Haist, Corinna Thivakaran, Aniththa Cengiz, Sevgi Can Sendker, Stephanie Bartels, Nina Thomitzek, Antonia Smorra, Denise Hejazi, Maryam Uhrberg, Markus Scheckenbach, Kathrin Monzel, Cornelia Wiek, Constanze Reinhardt, Dirk Niktoreh, Naghmeh Hanenberg, Helmut Front Immunol Immunology The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy—CD19, CD33, and CD123—to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further. Frontiers Media S.A. 2022-04-07 /pmc/articles/PMC9022481/ /pubmed/35464442 http://dx.doi.org/10.3389/fimmu.2022.847008 Text en Copyright © 2022 Soldierer, Bister, Haist, Thivakaran, Cengiz, Sendker, Bartels, Thomitzek, Smorra, Hejazi, Uhrberg, Scheckenbach, Monzel, Wiek, Reinhardt, Niktoreh and Hanenberg https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Soldierer, Maren
Bister, Arthur
Haist, Corinna
Thivakaran, Aniththa
Cengiz, Sevgi Can
Sendker, Stephanie
Bartels, Nina
Thomitzek, Antonia
Smorra, Denise
Hejazi, Maryam
Uhrberg, Markus
Scheckenbach, Kathrin
Monzel, Cornelia
Wiek, Constanze
Reinhardt, Dirk
Niktoreh, Naghmeh
Hanenberg, Helmut
Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies
title Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies
title_full Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies
title_fullStr Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies
title_full_unstemmed Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies
title_short Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies
title_sort genetic engineering and enrichment of human nk cells for car-enhanced immunotherapy of hematological malignancies
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9022481/
https://www.ncbi.nlm.nih.gov/pubmed/35464442
http://dx.doi.org/10.3389/fimmu.2022.847008
work_keys_str_mv AT soldierermaren geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT bisterarthur geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT haistcorinna geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT thivakarananiththa geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT cengizsevgican geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT sendkerstephanie geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT bartelsnina geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT thomitzekantonia geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT smorradenise geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT hejazimaryam geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT uhrbergmarkus geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT scheckenbachkathrin geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT monzelcornelia geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT wiekconstanze geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT reinhardtdirk geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT niktorehnaghmeh geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies
AT hanenberghelmut geneticengineeringandenrichmentofhumannkcellsforcarenhancedimmunotherapyofhematologicalmalignancies