The human angiotensin-converting enzyme 2 acts as the host cell receptor for SARS-CoV-2 and the other members of the Coronaviridae family SARS-CoV-1 and HCoV-NL63. Here we report the biophysical properties of the SARS-CoV-2 spike variants D614G, B.1.1.7, B.1.351 and P.1 with affinities to the ACE2 receptor and infectivity capacity, revealing weaknesses in the developed neutralising antibody approaches. Furthermore, we report a pre-clinical characterisation package for a soluble receptor decoy engineered to be catalytically inactive and immunologically inert, with broad neutralisation capacity, that represents an attractive therapeutic alternative in light of the mutational landscape of COVID-19. This construct efficiently neutralised four SARS-CoV-2 variants of concern. The decoy also displays antibody-like biophysical properties and manufacturability, strengthening its suitability as a first-line treatment option in prophylaxis or therapeutic regimens for COVID-19 and related viral infections. IMPORTANCE Mutational drift of SARS-CoV-2 risks rendering both therapeutics and vaccines less effective. Receptor decoy strategies utilising soluble human ACE2 may overcome the risk of viral mutational escape since mutations disrupting viral interaction with the ACE2 decoy will by necessity decrease virulence thereby preventing meaningful escape. The solution described here of a soluble ACE2 receptor decoy is significant for the following reasons: While previous ACE2-based therapeutics have been described, ours has novel features including (1) mutations within ACE2 to remove catalytical activity and systemic interference with the renin/angiotensin system; (2) abrogated FcγR engagement, reduced risk of antibody-dependent enhancement of infection and reduced risk of hyperinflammation, and (3) streamlined antibody-like purification process and scale-up manufacturability indicating that this receptor decoy could be produced quickly and easily at scale. Finally, we demonstrate that ACE2-based therapeutics confer a broad-spectrum neutralisation potency for ACE2-tropic viruses, including SARS-CoV-2 variants of concern in contrast to therapeutic mAb.

Lentiviral vectors (LVs) have recently witnessed an increasing demand in research and clinical applications. Their current purification processes represent the main bottleneck in their widespread use, as the methods used are cumbersome and yield low recoveries. We aimed to develop a one-step method to specifically purify LVs, with high yields and reduced levels of impurities, using the biotin-streptavidin system. Herein, packaging HEK293T cells were genetically engineered with a cyclical biotin-mimicking peptide displayed on a CD8α stalk, termed cTag8. LVs were modified with cTag8 by its passive incorporation onto viral surfaces during budding, without viral protein engineering or hindrance on infectivity. Expression of cTag8 on LVs allowed complete capture of infectious particles by streptavidin magnetic beads. As cTag8 binds streptavidin in the nanomolar range, the addition of micromolar concentrations of biotin resulted in the release of captured LVs by competitive elution, with overall yields of ≥60%. Analysis of eluted LVs revealed high purity with a >3-log and 2-log reduction in DNA contamination and host cell proteins, respectively. This one-step purification was also tested for scalable vector processing using monolith affinity chromatography, with an encouraging preliminary overall yield of 20%. This method will be of valuable use for both research and clinical applications of LVs.

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The human angiotensin-converting enzyme 2 acts as the host cell receptor for SARS-CoV-2 and the other members of the Coronaviridae family SARS-CoV-1 and HCoV-NL63. Here we report the biophysical properties of the SARS-CoV-2 spike variants D614G, B.1.1.7 and B.1.351 with affinities to the ACE2 receptor and infectivity capacity, revealing weaknesses in the developed neutralising antibody approaches. Furthermore, we report a pre-clinical characterisation package for a soluble receptor decoy engineered to be catalytically inactive and immunologically inert, with broad neutralisation capacity, that represents an attractive therapeutic alternative in light of the mutational landscape of COVID-19. This construct efficiently neutralised four SARS-CoV-2 variants of concern. The decoy also displays antibody-like biophysical properties and manufacturability, strengthening its suitability as a first-line treatment option in prophylaxis or therapeutic regimens for COVID-19 and related viral infections.

Antibody phage display is a powerful platform for discovery of clinically applicable high affinity monoclonal antibodies against a broad range of targets. Libraries generated from immunized animals offer the advantage of in vivo affinity-maturation of V regions prior to library generation. Despite advantages, few studies have described isolation of antibodies from rats using immune phage display. In our study, we describe a novel primer set, covering the full rat heavy chain variable and kappa light chain variable regions repertoire for the generation of an unbiased immune libraries. Since the immune repertoire of rats is poorly understood, we first performed a deep sequencing analysis of the V(D)J regions of VH and VLK genes, demonstrating the high abundance of IGVH2 and IGVH5 families for VH and IGVLK12 and IGVLK22 for VLK. The comparison of gene’s family usage in naïve rats have been used to validate the frequency’s distribution of the primer set, confirming the absence of PCR-based biases. The primers were used to generate and assemble a phage display library from human CD160-vaccinated rats. CD160 represents a valid therapeutic target as it has been shown to be expressed on chronic lymphocytic leukaemia cells and on the surface of newly formed vessels. We utilised a novel phage display panning strategy to isolate a high affinity pool (KD range: 0.399–233 nM) of CD160 targeting monoclonal antibodies. Subsequently, identified binders were tested for function as third generation Chimeric Antigen Receptors (CAR) T cells demonstrating specific cytolytic activity. Our novel primer set coupled with a streamlined strategy for phage display panning enable the rapid isolation and identification of high affinity antibodies from immunised rats. The therapeutic utility of these antibodies was demonstrated in CAR format.

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The hostile tumour microenvironment limits the efficacy of adoptive cell therapies. Activation of the Fas death receptor initiates apoptosis and disrupting these receptors could be key to increase CAR T-cell efficacy. We screened a library of Fas-TNFR proteins identifying several novel chimeras that not only prevented Fas ligand-mediated kill, but also enhanced CAR T-cell efficacy by signalling synergistically with the CAR. Upon binding Fas ligand, Fas-CD40 activated the NF-kappaB pathway, inducing greatest proliferation and IFNgamma release out of all Fas-TNFRs tested. Fas-CD40 induced profound transcriptional modifications, particularly genes relating to the cell cycle, metabolism, and chemokine signalling. Co-expression of Fas-CD40 with either 4-1BB- or CD28-containing CARs increased in vitro efficacy by eliciting maximal CAR T-cell proliferation and cancer target cytotoxicity, and enhanced tumour killing and overall mouse survival in vivo. Functional activity of the Fas-TNFRs were dependent on the co-stimulatory domain within the CAR, highlighting crosstalk between signalling pathways. Furthermore, we show that a major source for Fas-TNFR activation derives from CAR T cells themselves via activation-induced Fas ligand upregulation, highlighting a universal role of Fas-TNFRs in augmenting CAR T-cell responses. We have identified Fas-CD40 as the optimal chimera for overcoming Fas ligand-mediated kill and enhancing CAR T-cell efficacy.

The development of multicistronic vectors enabling differential transgene expression is a goal of gene therapy and poses a significant engineering challenge. Current approaches rely on the insertion of long regulatory sequences that occupy valuable space in vectors, which have a finite and limited packaging capacity. Here we describe a simple method of achieving differential transgene expression by inserting stop codons and translational readthrough motifs (TRMs) to suppress stop codon termination. TRMs reduced downstream transgene expression ∼sixfold to ∼140-fold, depending on the combination of stop codon and TRM used. We show that a TRM can facilitate the controlled secretion of the highly potent cytokine IL-12 at therapeutically beneficial levels in an aggressive immunocompetent mouse melanoma model to prevent tumor growth. Given their compact size (6 bp) and ease of introduction, we envisage that TRMs will be widely adopted in recombinant DNA engineering to facilitate differential transgene expression.