Authors:	Wernersbach, Jan
Title:	Evaluation of T-cell receptor reprogrammed natural killer cells as an "off the shelf" adoptive cellular immunotherapy against acute myeloid leukemia Evaluation T-Zell-Rezeptor reprogrammierter Natürlicher Killerzellen für eine adoptive zelluläre ’off the shelf’ Immuntherapie bei akuter myeloischer Leukämie
Online publication date:	13-Nov-2023
Year of first publication:	2023
Language:	english
Abstract:	Background: Adoptive cellular therapy (ACT) with redirected T cells expressing a chimeric antigen receptor (CAR) or transgenic T-cell-receptor (tTCR) has revolutionized cellular immunotherapy to hematological neoplasia, in particular to acute lymphoid leukemia, and also shows great promise as therapy for solid tumors. While CARs can only detect fully cell surface expressed target structures, TCR-mediated recognition is not limited to surface antigens, but covers processed tumor neoantigens derived from the whole proteome. However, mispairing of transgenic and endogenous TCRs and restriction to patient-derived, autologous T lymphocytes with variable “fitness” and T cell subsets due to individual health conditions and age of the patient exemplify the current limitations encountered in TCR-redirected ACT. The natural killer (NK) cell line NK92 elicits lytic activity comparable to T cells, has been approved by the FDA for ACT and shown not to cause graft-vs-host disease. Since NK cell redirection is currently limited to CARs, NK92 cells engineered to express a CD3/TCR or CD3/CD8/TCR complex might evolve as an attractive, standardized cellular source for off-the-shelf TCR- based ACT. Aims: Thus, the goal of this study was to explore NK92-CD3+ and NK92-CD3+CD8+ variants redirected to acute myeloid leukemia (AML) and mdm-2 expressing tumor targets by expression of different reactive TCRs for antitumoral immunity in vitro. Methods: NK92CD3+cells (provided by Dr. C. Wölfel, III. Dept. of Med.) were further engineered to express human CD8. Upon viral gene transfer of optimized TCRs recognizing primary AML-blasts or EBV-BLCL from patient MZ580 (TCRs from CTL 5H11, 25F2 and 5B2) and the mdm-2 peptide (anti-mdm2 TCR) NK92 CD3+ and NK92CD3+CD8+ were expanded and enriched for >90% TCR expression (referred to as NK92TCR+). Additional expression profiling of checkpoint molecules (e.g. CD80/86, PD1/PDL1, NKG2A, TIM3, TIGIT) and NCRs was performed by FACS. IFN-γ release and cytolytic activity was tested by ELISpot and bioluminescence-based assays, respectively. Results: Upon coculture with MZ580 BLCL or mdm-2 expressing IM9 (myeloma) and mdm-2 peptide loaded HLA-A*02:01+ K562 targets NK92TCR+ cells elicited strong TCR-dependent IFN–γ release and cytotoxicity. This reactivity was greatly enhanced upon CD8 coexpression for TCR 5H11 and indispensable for the mdm-2 TCR clearly demonstrating CD8 coreceptor dependency for both TCRs. Cytolytic activity of 5H11 T cells and 5H11TCR+NK92 was comparable. Intriguingly, NK92TCR+ cells shifted to a more T cell-like phenotype and showed reduced NKp30-mediated killing of K562. First adoptive transfer studies of NK92TCR+ cells into a NSG-AML PDX model are in progress. Summary/Conclusion: These studies demonstrate that the established and FDA approved NK92 cell line can be redirected to elicit TCR-mediated antitumoral immunity to AML. NK92TCR+ cells might thus represent a promising universal tool for an ‘off-the-shelf’ ACT product. TCR-redirected NK92CD3+CD8+ cells elicit antitumoral immunity comparable to original cytotoxic T cell clones. Co-expression of CD8 broadens the spectrum of therapeutic TCRs applicable to adoptive NK92TCR+ cell therapy. Thus, TCR-redirected NK92 cells might represent a promising new tool for an ‘off-the-shelf’ ACT with limited off-target effects.
DDC:	500 Naturwissenschaften 500 Natural sciences and mathematics 570 Biowissenschaften 570 Life sciences 610 Medizin 610 Medical sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 04 Medizin
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-9642
URN:	urn:nbn:de:hebis:77-openscience-561a62c0-e150-419a-9df6-da77b27653b61
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	http://rightsstatements.org/vocab/InC/1.0/
Extent:	124 Seiten ; Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen