Delivering co-stimulatory tumor necrosis factor receptor agonism for cancer immunotherapy: past, current and future perspectives

The tumor necrosis factor superfamily (TNFSF) and their receptors (TNFRSF) are important regulators of the immune system, mediating proliferation, survival, differentiation, and function of immune cells. As a result, their targeting for immunotherapy is attractive, although to date, under-exploited. In this review we discuss the importance of co-stimulatory members of the TNFRSF in optimal immune response generation, the rationale behind targeting these receptors for immunotherapy, the success of targeting them in pre-clinical studies and the challenges in translating this success into the clinic. The efficacy and limitations of the currently available agents are discussed alongside the development of next generation immunostimulatory agents designed to overcome current issues, and capitalize on this receptor class to deliver potent, durable and safe drugs for patients

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

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

Delivering co-stimulatory tumor necrosis factor receptor agonism for cancer immunotherapy: past, current and future perspectives

The tumor necrosis factor superfamily (TNFSF) and their receptors (TNFRSF) are important regulators of the immune system, mediating proliferation, survival, differentiation, and function of immune cells. As a result, their targeting for immunotherapy is attractive, although to date, under-exploited. In this review we discuss the importance of co-stimulatory members of the TNFRSF in optimal immune response generation, the rationale behind targeting these receptors for immunotherapy, the success of targeting them in pre-clinical studies and the challenges in translating this success into the clinic. The efficacy and limitations of the currently available agents are discussed alongside the development of next generation immunostimulatory agents designed to overcome current issues, and capitalize on this receptor class to deliver potent, durable and safe drugs for patients

Fcγ receptor binding is required for maximal immunostimulation by CD70-Fc

Introduction: T cell expressed CD27 provides costimulation upon binding to inducible membrane expressed trimeric CD70 and is required for protective CD8 T cell responses. CD27 agonists could therefore be used to bolster cellular vaccines and anti-tumour immune responses. To date, clinical development of CD27 agonists has focussed on anti-CD27 antibodies with little attention given to alternative approaches.Methods: here, we describe the generation and activity of soluble variants of CD70 that form either trimeric (t) or dimer-of-trimer proteins and conduct side-by-side comparisons with an agonist anti-CD27 antibody. To generate a dimer-of-trimer protein (dt), we fused three extracellular domains of CD70 to the Fc domain of mouse IgG1 in a ‘string of beads’ configuration (dtCD70-Fc).Results: whereas tCD70 failed to costimulate CD8 T cells, both dtCD70-Fc and an agonist anti-CD27 antibody were capable of enhancing T cell proliferation in vitro. Initial studies demonstrated that dtCD70-Fc was less efficacious than anti-CD27 in boosting a CD8 T cell vaccine response in vivo, concomitant with rapid clearance of dtCD70-Fc from the circulation. The accelerated plasma clearance of dtCD70-Fc was not due to the lack of neonatal Fc receptor binding but was dependent on the large population of oligomannose type glycosylation. Enzymatic treatment to reduce the oligomannose-type glycans in dtCD70-Fc improved its half-life and significantly enhanced its T cell stimulatory activity in vivo surpassing that of anti-CD27 antibody. We also show that whereas the ability of the anti-CD27 to boost a vaccine response was abolished in Fc gamma receptor (FcγR)-deficient mice, dtCD70-Fc remained active. By comparing the activity of dtCD70-Fc with a variant (dtCD70-Fc(D265A)) that lacks binding to FcγRs, we unexpectedly found that FcγR binding to dtCD70-Fc was required for maximal boosting of a CD8 T cell response in vivo. Interestingly, both dtCD70-Fc and dtCD70-Fc(D265A) were effective in prolonging the survival of mice harbouring BCL1 B cell lymphoma, demonstrating that a substantial part of the stimulatory activity of dtCD70-Fc in this setting is retained in the absence of FcγR interaction.Discussion: these data reveal that TNFRSF ligands can be generated with a tunable activity profile and suggest that this class of immune agonists could have broad applications in immunotherapy

Agonistic CD27 antibody potency is determined by epitope-dependent receptor clustering augmented through Fc-engineering

Agonistic CD27 monoclonal antibodies (mAb) have demonstrated impressive anti-tumour efficacy in multiple preclinical models but modest clinical responses. This might reflect current reagents delivering suboptimal CD27 agonism. Here, using a novel panel of CD27 mAb including a clinical candidate, we investigate the determinants of CD27 mAb agonism. Epitope mapping and in silico docking analysis show that mAb binding to membrane-distal and external-facing residues are stronger agonists. However, poor epitope-dependent agonism could partially be overcome by Fc-engineering, using mAb isotypes that promote receptor clustering, such as human immunoglobulin G1 (hIgG1, h1) with enhanced affinity to Fc gamma receptor (FcγR) IIb, or hIgG2 (h2). This study provides the critical knowledge required for the development of agonistic CD27 mAb that are potentially more clinically efficacious

Complex interplay between epitope specificity and isotype dictates the biological activity of anti-human CD40 antibodies

Anti-CD40 monoclonal antibodies (mAbs) that promote or inhibit receptor function hold promise as therapeutics for cancer and autoimmunity. Rules governing their diverse range of functions, however, are lacking. Here we determined characteristics of nine hCD40 mAbs engaging epitopes throughout the CD40 extracellular region expressed as varying isotypes. All mAb formats were strong agonists when hyper-crosslinked; however, only those binding the membrane-distal cysteine-rich domain 1 (CRD1) retained agonistic activity with physiological Fc gamma receptor crosslinking or as human immunoglobulin G2 isotype; agonistic activity decreased as epitopes drew closer to the membrane. In addition, all CRD2-4 binding mAbs blocked CD40 ligand interaction and were potent antagonists. Thus, the membrane distal CRD1 provides a region of choice for selecting CD40 agonists while CRD2-4 provides antagonistic epitopes. CD40 agonist mAbs are being investigated for cancer treatment, whereas antagonistic mAbs are under investigation for the treatment of autoimmune and inflammatory conditions. Yu et al. show that the activity of a CD40 mAb is determined by an interplay between the location of its epitope within CD40 and its isotype.</p