\u201cUniCAR\u201d-modified off-the-shelf NK-92 cells for targeting of GD2-expressing tumour cells

Nicola Mitwasi,Dirk Jäger,Tibor Kovács,Domokos Máthé,Elke Jäger,Claudia Rössig,Claudia Arndt,Stefanie Koristka,Ralf Bergmann,Anja Feldmann,Congcong Zhang,Marc Schmitz,Nicole Berndt,Justyna Jureczek,Barbara Seliger,Winfried S Wels,Torsten Tonn,Achim Temme,Jessica C Hassel,Nikolett Hegedüs ,Pranav Oberoi,Liliana R Loureiro,Jiří Eitler ,Michael Bachmann

doi:10.1038/s41598-020-59082-4

Abstract

Antigen-specific redirection of immune effector cells with chimeric antigen receptors (CARs) demonstrated high therapeutic potential for targeting cancers of different origins. Beside CAR-T cells, natural killer (NK) cells represent promising alternative effectors that can be combined with CAR technology. Unlike T cells, primary NK cells and the NK cell line NK-92 can be applied as allogeneic off-the-shelf products with a reduced risk of toxicities. We previously established a modular universal CAR (UniCAR) platform which consists of UniCAR-expressing immune cells that cannot recognize target antigens directly but are redirected by a tumour-specific target module (TM). The TM contains an antigen-binding moiety fused to a peptide epitope which is recognized by the UniCAR molecule, thereby allowing an on/off switch of CAR activity, and facilitating flexible targeting of various tumour antigens depending on the presence and specificity of the TM. Here, we provide proof of concept that it is feasible to generate a universal off-the-shelf cellular therapeutic based on UniCAR NK-92 cells targeted to tumours expressing the disialoganglioside GD2 by GD2-specific TMs that are either based on an antibody-derived single-chain fragment variable (scFv) or an IgG4 backbone. Redirected UniCAR NK-92 cells induced specific killing of GD2-expressing cells in vitro and in vivo, associated with enhanced production of interferon-γ. Analysis of radiolabelled proteins demonstrated that the IgG4-based format increased the in vivo half-life of the TM markedly in comparison to the scFv-based molecule. In summary, UniCAR NK-92 cells represent a universal off-the-shelf platform that is highly effective and flexible, allowing the use of different TM formats for specific tumour targeting.

Highlights

Antigen-specific targeting of cancer in a safe and effective manner is challenging since many of the tumour-associated antigens (TAAs) known to date are expressed to some extent by healthy tissues
natural killer (NK)-92 cells were modified to express the universal chimeric antigen receptor (UniCAR) by transduction with a lentiviral vector encoding a chimeric antigen receptors (CARs) consisting of an E5B9-specific single-chain fragment variable (scFv), a flexible hinge region, the CD28 transmembrane domain and a composite of CD28-CD3ζ signalling domains (UniCAR 28/ζ), followed by an EGFP marker gene
We previously described the generation of the recombinant scFv-based anti-GD2 (α-GD2) target module (TM), which is based on the variable domains of the light and heavy chains (VL and VH) of GD2-specific CAR connected by a flexible peptide linker (Fig. 3A)[38,43]

Summary

Introduction

Antigen-specific targeting of cancer in a safe and effective manner is challenging since many of the tumour-associated antigens (TAAs) known to date are expressed to some extent by healthy tissues. The safety of infusion of irradiated NK-92 cells was demonstrated in early phase clinical trials, with some of the treated cancer patients experiencing long-lasting responses[27,28,29,30] This makes NK-92 cells an interesting option for CAR engineering which provides the cells with antigen-specific targeting, further enhancing their anti-tumour activity[31,32]. In the case of UniCAR-modified T cells, small antibody derivatives such as a scFv are preferred as a TM to allow rapid clearance from the system in case on-target/off-tumour toxicity occurs This may be less relevant for NK-92 cells which are typically irradiated before application, limiting in vivo persistence and preventing expansion in the host[27,28]. In addition to the relatively short-lived scFv-based TM38, we tested a novel homodimeric TM format in which the E5B9 epitope is connected to the GD2-specific antibody domain via an IgG4 Fc region to achieve an extended in vivo half-life adapted to the activity half-life of irradiated NK-92 cells (Fig. 1)

Methods

Results

Conclusion