key: cord-1040267-ji2eaa13 authors: Ferrari, Mathieu; Mekkaoui, Leila; Ilca, F. Tudor; Akbar, Zulaikha; Bughda, Reyisa; Lamb, Katarina; Ward, Katarzyna; Parekh, Farhaan; Karattil, Rajeev; Allen, Christopher; Wu, Philip; Baldan, Vania; Mattiuzzo, Giada; Bentley, Emma M.; Takeuchi, Yasuhiro; Sillibourne, James; Datta, Preeta; Kinna, Alexander; Pule, Martin; Onuoha, Shimobi C. title: Characterisation of a novel ACE2-based therapeutic with enhanced rather than reduced activity against SARS-CoV2 variants date: 2021-03-17 journal: bioRxiv DOI: 10.1101/2021.03.17.435802 sha: c79bc8fb851709e331ba677205af18898201b71a doc_id: 1040267 cord_uid: ji2eaa13 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. The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 1 has caused a major coronavirus disease world-wide pandemic outbreak, totalling > 100 million confirmed cases and > 2 million associated deaths as of January 2021 (www.covid19.who.int). The rapid replication of SARS-CoV-2 has been shown in some patients to trigger an aggressive inflammatory response in the lung and acute respiratory disease syndrome (ARDS), leading to a cytokine release syndrome (CRS) due to the elevated expression of pro-inflammatory cytokines [2] [3] [4] . Similar to SARS-CoV-1 5 , this enveloped virus belongs to the β -coronavirus genus with a positive-strand RNA genome and utilises angiotensin-converting enzyme 2 (ACE2) as the receptor for host cell entry by binding to its glycoprotein Spike (S) 1, 6 . The S protein is arranged as a trimeric complex of heterodimers composed of S1, containing the receptor binding domain (RBD) and S2, responsible for viral fusion and cell entry, which are generated from the proteolytical cleavage of the S precursor via furin in the host cell 6, 7 . Currently, more than 1100 monoclonal antibodies (mAb) against SARS-CoV-2 have been reported in the literature, with over 20 currently in clinical evaluation 8, 9 . The antibodies LY-CoV555 and LY-CoV016 suggesting convergent evolution in SARS-CoV-2 due to similar selective pressures 16 . These variants have already been shown to impact on mAbs neutralisation potency 17,18 . 4 We first explored the binding kinetic between SARS-CoV-2 S1 and ACE2. Inhouse purified recombinant S1 domains from WT, D614G, B.1.1.7 and B.1.351 variants demonstrated similar properties to commercially sourced S1 WT protein (Supplementary Figure 1A and 1B) . Interestingly, the WT and D614G variants displayed a similar thermal unfolding profile with the first transition event (Tm) at 42.9 and 42.2°C, respectively, while the B.1.1.7 and B.1.351 resulted in a 6.9 and 11.5°C increase compared to S1 WT, respectively (Supplementary Figure 1C) . The binding affinity of the spike variants for the ACE2 receptor was assessed by surface plasmon resonance (SPR) using the recombinant S1 domains to allow for a monovalent binding interaction. The SARS-CoV-2 S1 WT, D614G and B.1.351 displayed overall similar kinetic affinities, although the latter showed a 1.5-fold slower off-rate (k d ) compared to WT S1, which was compensated by a slightly slower on-rate (k a ). The B.1.1.7 S1 variant however, showed a > 3-fold increase in affinity compared to WT, mainly driven by a 3.8-fold slower k d ( Figure 1A and Table 1 ). To assess the infectivity conferred by the SARS-CoV-2 spike variants, we engineered replication The extracellular domain of human ACE2 (aa 18-740, Uniprot Q9BYF1) was fused to the human IgG1 Fc via the human IgG1 hinge region to allow for homodimer stabilisation (Figure 2A) This mutation is predicted to remove interaction with zinc ions (Zn +2 ) mediated by the two original Histidine (His) residues, with a spatially conservative mutation ( Figure 2B) . We first set out to confirm inactivation of the ACE2 component. In vitro testing using a fluorogenic substrate for ACE2, Mca-APK(Dnp), showed complete abrogation of enzymatic activity for the ACE2-Fc construct carrying the HH:NN mutation, while the wild-type (WT) active ACE2-Fc molecule was able to efficiently process the peptide ( Figure 2C) Figure 2D ). We next explored whether the ACE2 mutations impacted SP binding. Both WT and mutated ACE2 showed comparable binding capacity for recombinant SARS-CoV-2 full S trimer and S1 domain by ELISA ( Figure 2E ). SPR measurements of kinetic interaction for the S1 domain of SARS-CoV-2 showed comparable kinetic profiles between active WT and HH:NN ACE2 (Table 1) , further suggesting the preservation of an unaltered Spike binding domain. To overcome the risk of activating the host immune system, thus exacerbating the hyperinflammatory response often associated with severe COVID-19 development 27 , the Fc domain was engineered to remove FcγR interactions. The well-established L234A/L235A (LALA) 28 mutations of the CH2 domain and the LALA combination with P329G (LALA-PG) 29 were introduced in the human IgG1 Fc portion of the ACE2-Fc fusion protein. We first investigated the expression yields of the ACE2(HH:NN) with WT Fc, LALA Fc and LALA-PG Fc and ACE2 domain activity. All constructs showed comparable expression and purification efficiencies using protein A affinity chromatography (Supplementary Figure 2) . Mutations on the Fc domain did not affect the binding capacity of ACE2 for SARS-CoV-2 S-protein and all three versions showed highly comparable dose/response curves to recombinant SARS-CoV-2 full S trimer or S1 domain by ELISA ( Figure 3A) . Similarly, all three variants were able to bind SupT1 cell lines expressing SARS-CoV-2 full S trimer as a transmembrane protein (Figure 3B ), further confirming binding capacity for the glycoprotein in a more physiological environment. Figure 3E ). Binding specificity and cross-reactivity of the ACE2(HH:NN)-Fc LALA-PG construct was assessed using a cell-based protein microarray assay, screening 5477 full length plasma membrane and cell surfacetethered human secreted proteins, 371 human heterodimers, and the SARS-CoV-2 S-protein (Supplementary Table 1 ). The test construct showed strong specific binding to the target protein SARS-CoV-2 S, while no other interaction was detected across the comprehensive panel of human protein ( Figure 4A ). An Fc LALA-PG only construct with the ACE2 domain omitted did not display any interaction with SARS-CoV-2 S-protein or any other target tested. The control fusion protein CTLA4-hFc instead, showed strong interaction for its predicted target CD86, and the FcγRIa, due to the presence of a WT IgG1 Fc domain. A secondary anti-human Fc antibody interaction with human IgG3 was detected across all conditions tested ( Figure 4A ). As this receptor decoy has the potential to bind S glycoproteins of viruses that utilise ACE2 as host-cell receptor, binding kinetics were generated for the S1 spike domain of SARS-CoV showed specificity only for the SARS-CoV-2 related S-proteins ( Figure 4B ). The ACE2(HH:NN)-Fc and ACE2(HH:NN)-Fc LALA-PG showed comparable affinities for the tested S1 domains, confirming no effect of the Fc mutations on ACE2 binding ( Figure 4B and Table 1 ). Similar to the active ACE2-Fc, the inactive ACE2-Fc constructs also displayed a 3-fold enhanced affinity for the SARS-CoV-2 S1 B.1.1.7. While the monoclonal antibody REGN10987 maintained a similar affinity for the SARS-CoV-2 S1 variants tested, the LY-CoV555 and REGN10933 were dramatically affected by the B.1.351 variant with 12-and 23-fold reduction in affinity compared to S1 WT, respectively ( Figure 4B and Table 1 ). We first assessed the neutralisation capacity of our decoy receptor against live replicating SARS-CoV-2. Figure 5B ). It has been previously reported that Syrian hamsters (Mesocricetus auratus) are a relevant small animal also showed an overall reduction in lung damage for the ACE2(HH:NN)-Fc LALA-PG treated groups, characterised by fewer lesions and blood clotting ( Figure 6C ). Finally, i.p. administered ACE2(HH:NN)-Fc LALA-PG was still detectable in the hamster sera at day 7, with levels almost 20-fold higher for the high dose compared to low dose treatment ( Figure 6D ). To define a suitable formulation considering manufacturing scale-up for clinical application, the well- Figure 7A ). When tested in 20 mM His buffer, the first unfolding event occurred at a Tm between 42.3 and 51.6°C, with the lowest Tm associated with pH 3.5 and the most stable Tm obtained at pH 6.5 ( Figure 7A ). A crucial phase during manufacturing of monoclonal antibodies lies in the viral inactivation step, often carried out at low pH 34 , which can affect the stability and aggregation state of the proteins in solution. To investigate this, the ACE2(HH:NN)-Fc was exposed to pH 3.5 for 90 minutes before dialysis in 20 mM His pH 6.5. Thermal stability comparison of ACE2(HH:NN)-Fc at pH 3.5, 6.5 and 3.5 dialysed to 6.5 showed how the initial instability due to pH 3.5 could efficiently be restored to that of the ACE2(HH:NN)-Fc following dialysis at pH 6.5 ( Figure 7B ). The distribution of particles within the solution showed a predominantly monodispersed profile for the ACE2(HH:NN)-Fc in PBS and 20 mM His pH 6.5, with an average diameter of 13.5 and 13.3 nm, respectively, in agreement with a molecule of predicted MW of 219 kDa. The suspension in a low pH buffer of 3.5 did not significantly enhance 9 aggregation of ACE2(HH:NN)-Fc ( Figure 7C) . Furthermore, the change of buffer from PBS pH 7.4 to 20 mM His pH 6.5 and, crucially, the viral inactivation step in at pH 3.5 with subsequent dialysis to pH 6.5, did not affect the capacity of the ACE2(HH:NN)-Fc to bind the SARS-CoV-2 S1 protein, further validating the proposed process ( Figure 7D ). The ACE2(HH:NN)-Fc LALA-PG also showed an increased thermal stability when in 20 mM His pH 6.5 buffer, with Tm moving from 48.1°C to 52.0°C, and CH2 CH3 unfolding happening at 64.3°C and 81.8°C, respectively ( Figure 7E ). The ACE2(HH:NN)-Fc LALA-PG was also characterised by a monodispersed particle profile with an average diameter size of 13.6 nm in 20 mM His pH 6.5 ( Figure 7F ). Finally, the formulation in 20 mM His pH 6.5 of ACE2(HH:NN)-Fc LALA-PG did not alter the SARS-CoV-2 neutralisation capacity of the construct (Supplementary figure 4) . 1 0 We have described the generation of a catalytically inactive ACE2 receptor decoy fused to an engineered human Fc domain with abrogated FcγR engagement, showing optimal biophysical properties and manufacturability. The construct showed strong neutralisation potency against several SARS-CoV-2 variants of concern in vitro and evidence of efficacy as a therapeutic administration in a live viral challenge model in vivo. Monoclonal antibodies developed for the treatment of COVID-19 have shown efficacy in the treatment of early phases of the infection, potentially useful in prophylaxis or as an alternative for people who cannot be vaccinated 35 . However, cumulative spike-protein mutants may render therapeutic mAbs ineffective. For instance, the variants of concern B.1.351 and P.1 have been shown to affect the neutralisation capacity of the approved antibody therapeutics. The LY-CoV555 antibody reported an almost complete abrogation of neutralisation, while the antibody cocktail REGN-COV2 showed a severe impairment for one of its components, suggesting preservation of limited therapeutic efficacy 17, 18 . Although not yet present in naturally occurring variants, the single amino acid mutation E406W has recently been shown to be able to escape both antibodies in the REGN-COV2 cocktail 36 . "Receptor traps" are an established therapeutic approach, e.g. the anti-TNF Etanercept 19 As a fusion construct, unexpected off-target binding events could manifest, representing a liability over its safety profile. To this end, a cross-reactivity study against a comprehensive panel of close to 6000 human soluble and membrane-bound proteins has highlighted the exquisite specificity of this construct for the target protein, providing confidence over its safety profile. Finally, we achieved high expression yields for our ACE2 receptor decoy, using a non-optimised transient transfection system and single-step protein-A affinity purification. The stability of the construct at pH 3.5, while showing no change in its aggregation profile and binding capacity, lends to its suitability for antibody-like purification processes such as low pH viral inactivation 45 . We have also determined an optimal pH to enhance stability of the protein using a commonly adopted His buffer for clinical-stage monoclonal antibodies 46 , raising the Tm to 52°C. These characteristics would allow for standard manufacturing scale-up required for clinical grade material. In conclusion, we describe detailed in vitro and in vivo characterisation of a soluble catalytically inactive ACE2-Fc receptor decoy molecule resistant to spike protein mutation. We also demonstrate that our decoy molecule has the potential for rapid upscale manufacturability. In theory, our decoy should be active against any new ACE2-tropic virus which might emerge in the future. In this phase of the SARS-CoV-2 pandemic where viral variants are exerting pressure over the efficacy of vaccines and monoclonal antibodies, the development of biotherapeutics which are inherently resistant to SARS-CoV-2 mutations may be prudent. Human ACE2 amino acid 18-740 (Uniprot Q9BYF1) was fused to the human IgG1 hinge and Fc (Uniprot P01857). Inactive ACE2 was generated by introducing H374N and H378N Thermal stability was determined by differential scanning fluorimetry nano(DSF) on a Prometheus NT.48 instrument (Nanotemper) using first derivative of 350/330nm ratio to determine the melting temperature (Tm) value. Samples were loaded on a glass capillary and temperature scanned from 20 to 95°C at 1°C/min. Aggregation propensity and average particle size of the test proteins was determined using a Zetasizer Ultra device and ZS Xplorer software (Malvern Panalytical) by MADLS. Samples were loaded into a low volume quartz cuvette (Malvern Panalytical -ZEN2112) at a concentration of 1 mg/ml or 20 mg/ml. Triplicate measurements were taken for each sample. Particle size of Aldolase (158 kDa) was used as reference. Enzymatic activity of active ACE2-Fc (ACRO biosystems -AC2-H5257) and ACE2(HH:NN)-Fc was For SARS-CoV-1 S1 (ACRO biosystems -S1N-S52H5), HCoV-NL63 S1 (SIN-V52H3), SARS-CoV-2 S1 WT (ACRO biosystems -S1N-C52H3) and in-house expressed SARS-CoV-2 S1 WT, D614G, All experiments were performed at 25°C with a flow rate of 30 µl/ml. Flow cell 1 was unmodified and used for reference subtraction. A '0 concentration' sensogram of buffer alone was used as a double reference subtraction to factor for drift. Data were fit to a 1:1 Langmuir binding model using Biacore insight evaluation software (GE Healthcare). SARS-CoV-1 S1 sensograms were also fit to a two-state 1 8 kinetics. Since a capture system was used, a local Rmax parameter was used for the data fitting in each case. Manufacturer's protocol was followed, and samples were assayed in triplicates. Briefly, after incubation with kit's ViraBind TM reagents and virus inactivation, samples were incubated in microwell plates precoated with anti-p24 antibodies followed by a subsequent incubation with secondary FITC-conjugated anti-HIV p24 monoclonal antibody (1:1000). Subsequently, well were exposed to HRP-conjugated anti- Functional infectious viral titres were determined by flow cytometry analysis (BD LSRFORTESSA X-20 cell analyser) of transgene expression in transduced HEK-293T cells that were previously engineered to express human ACE2 and TMPRSS2. Experiments were performed in 24-well plates (50,000 cells/well). Serially diluted viral supernatants were added onto seeded cells in the presence of 8 μ g/mL polybrene. Transduction efficiencies were determined 72 h later using BD LSRFORTESSA X-20 cell analyser and eGFP expression between 0.5% -20% were used in the following equation to determine viral titer: Proteins was serially diluted in PBS to 7 decreasing concentrations ranging from 100 mg/mL to 6. Cell microarray test 5477 expression vectors, encoding both ZsGreen1 and a full-length human plasma membrane protein or a cell surface-tethered human secreted protein, and 371 human heterodimers were co-arrayed across a microarray slide in duplicate (Supplementary Table 1 All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software). Specific analysis is detailed in figure legends. A p value < 0.05 was considered significant. 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(Mean ± SD) LALA Fc (green) or LALA-PG Fc (orange), detected with biotinylated SARS-CoV-2 S1 and streptavidin conjugated secondary agent. No binding was detected with ACE2-Fc constructs carrying the LALA or LALA-PG mutations (Mean ± SD). D) Representative flow cytometry of Fc-mediated binding of ACE2(HH:NN) WT Fc (blue) and LALA-PG Fc (orange) on human monocyte-derived M1 No binding detected with Fc carrying the LALA-PG mutation (n=4). E) SPR binding kinetic of ACE2(HH:NN) WT Fc, LALA Fc or LALA-PG Fc on human FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb. LALA-PG mutations mediated a complete abrogation of FcγR interaction ACE2(HH:NN)-Fc LALA-PG shows strong specific interaction with SARS-CoV-2 spike protein only. B) SPR binding kinetics of ACE2(HH:NN) WT Fc, LALA-PG Fc, LY-CoV555, REGN10933 and REGN10987 against SARS-CoV-1, SARS-CoV-2 variants All sensograms were fitted with Langmuir 1:1 binding model, except for SARS-CoV-1 S1 kinetics which were fitted with two-state kinetics. 2-fold serial dilutions starting from 250 nM HCoV-NL63 2.97E+03 3.55E-03 ACE2(HH:NN) Fc SARS-CoV-1 ACE2(HH:NN)