Development and activity of a single chain CD70 dimer-of-trimer and its potential as a therapeutic immunostimulatory agent

Abstract

Clinically, cancer immunotherapy treatments which use monoclonal antibodies (mAbs) to augment adaptive immunity have largely focussed on blocking inhibitory receptors such as PD-1. To be fully activated, T cells need activating signals and recent evidence shows this to be lacking in a tumour environment. Tumour necrosis factor receptor superfamily (TNFRSF) members are key co-stimulators of T cells and require high level clustering for initiation of signalling. Agonist anti-TNFR mAbs have been used to improve the adaptive immune response but they require cross-linking by Fc gamma receptors (FcγR) to induce clustering of the target receptor. However, the availability of FcγRs in certain tissues can be limiting. CD27 is a co-stimulatory TNFRSF member expressed constitutively on T cells. We have sought to develop a multimeric CD27 ligand, CD70, to bypass the requirement for FcγRs. We developed a co-stimulatory recombinant protein consisting of three CD70 extracellular domains as a single chain, fused with the hinge, CH2 and CH3 domains of mouse IgG1 Fc to facilitate improved stability in circulation. This single chain mouse CD70-m1 (scmCD70-m1) protein was expressed stably in CHO-K1S cells. The protein was expressed as a dimer-of-trimer but additional oligomeric structures were noted. The homogeneous dimer-of-trimer form was isolated and shown to interact with recombinant mouse CD27. The protein also promoted in vitro T-cell activation and proliferation in combination with T cell receptor (TCR) stimulation, but not when given in the absence of the TCR signal. Similarly, when given in vivo with OVA257-264 peptide, scmCD70-m1 significantly promoted the expansion of adoptively transferred OVA-specific T cells and contributed to memory T-cell differentiation. Importantly, and unlike anti-CD27 mAb, this expansion was independent of FcγRIIB. Despite these promising results however, scmCD70-m1 was rapidly cleared from the circulation due to the presence of high oligomannose-type glycans on the protein. Enzymatic removal of the oligomannose residues significantly improved the in vivo half-life and the ability of scmCD70-m1 to enhance in vivo expansion of antigen-specific T cells. Furthermore, a version of scmCD70-m1 lacking FcγR engagement, scmCD70-mm1, could stimulate in vivo expansion of antigen specific T cells purely based on dimer-of-trimeric interactions after enzymatic removal of oligomannose residues. However, the activity induced by scmCD70-m1 was higher than scmCD70-mm1 as scmCD70-m1 protein could receive FcγR engagement which further enhanced its activity. The enzyme-treated versions of both scmCD70-m1 and scmCD70-mm1 proteins were highly efficacious in the B-cell lymphoma model (BCL1). These data reveal that rationally-designed natural ligands can be more active than co-stimulatory mAbs and have the advantage of not requiring additional cross-linking by FcγRs.

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Identifiers

ORCID for Aymen Al-Shamkhani: orcid.org/0000-0003-0727-4189

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