HLA Class I Knockout Converts Allogeneic Primary NK Cells Into Suitable Effectors for "Off-the-Shelf" Immunotherapy.

Keven Hoerster,Helmut Hanenberg,Constanze Wiek,Stefan Heinrichs,Peter A Horn,Markus Uhrberg

doi:10.3389/fimmu.2020.586168

Abstract

Cellular immunotherapy using chimeric antigen receptors (CARs) so far has almost exclusively used autologous peripheral blood-derived T cells as immune effector cells. However, harvesting sufficient numbers of T cells is often challenging in heavily pre-treated patients with malignancies and perturbed hematopoiesis and perturbed hematopoiesis. Also, such a CAR product will always be specific for the individual patient. In contrast, NK cell infusions can be performed in non-HLA-matched settings due to the absence of alloreactivity of these innate immune cells. Still, the infused NK cells are subject to recognition and rejection by the patient’s immune system, thereby limiting their life-span in vivo and undermining the possibility for multiple infusions. Here, we designed genome editing and advanced lentiviral transduction protocols to render primary human NK cells unsusceptible/resistant to an allogeneic response by the recipient’s CD8+ T cells. After knocking-out surface expression of HLA class I molecules by targeting the B2M gene via CRISPR/Cas9, we also co-expressed a single-chain HLA-E molecule, thereby preventing NK cell fratricide of B2M-knockout (KO) cells via “missing self”-induced lysis. Importantly, these genetically engineered NK cells were functionally indistinguishable from their unmodified counterparts with regard to their phenotype and their natural cytotoxicity towards different AML cell lines. In co-culture assays, B2M-KO NK cells neither induced immune responses of allogeneic T cells nor re-activated allogeneic T cells which had been expanded/primed using irradiated PBMNCs of the respective NK cell donor. Our study demonstrates the feasibility of genome editing in primary allogeneic NK cells to diminish their recognition and killing by mismatched T cells and is an important prerequisite for using non-HLA-matched primary human NK cells as readily available, “off-the-shelf” immune effectors for a variety of immunotherapy indications in human cancer.

Highlights

Adoptive cell transfer (ACT) of autologous genetically modified immune cells has emerged as an attractive treatment option for various malignancies of hematologic origin
Despite the high gene transfer efficiencies that can be achieved in natural killer (NK) cells with baboon envelope-pseudotyped lentiviral vectors [68], we observed only approximately 16% HLA class I-negative cells (Figure 1C) four days after transduction of primary NK cells with the CRISPR/Cas9 HLA class I targeting vector
As the forced expression of HLA-E on neighboring cells might lead to tonic engagement of CD94/ NKG2A, which can signal via downstream targets [82, 83], we investigated if the genetic modifications in HLA expression impacted the NK cell phenotype and functions

Summary

Introduction

Adoptive cell transfer (ACT) of autologous genetically modified immune cells has emerged as an attractive treatment option for various malignancies of hematologic origin. For a significant number of patients, an autologous final product cannot be generated in time for treatment [reviewed in [1]] To alleviate these problems, research in the field is moving towards “off-the-shelf” products, making use of immune effector cells from healthy donors. A severe side effect of allogeneic cellular therapy is Graft-versus-Host-Disease (GvHD), a life-threatening complication caused by the transplanted alloreactive T cells and known since the early days of hematopoietic stem cell transplantation (HSCT) [2,3,4,5] To circumvent this complication, several approaches have been developed. Virus-specific cytotoxic T (VST) cells, for example, have successfully been used to control latent infections post HSCT without causing GvHD [6, 7] They have been proposed as a potential T cell population to create “off-theshelf” therapeutic products [8, 9]. An interesting approach to abrogate unwanted or alloreactive signaling from the endogenous T cell receptors (TCRs) in chimeric antigen receptor (CAR) T cells uses genome editing on common TCR domains [12, 13], this genomic editing will require additional gene transfer systems and will add several layers of complexities to CAR T cell clinical trials

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Conclusion