Drug development of an affinity enhanced, broadly neutralizing heavy chain-only antibody that restricts SARS-CoV-2 in rodents

Abstract

We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and −2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and −2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.

SciScore for 10.1101/2021.03.08.433449: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	IACUC: All experiments were in compliance with the German animal protection law and approved by the animal welfare committee of the Regierungspräsidium Freiburg (permit G-20/91). IRB: Housing conditions and experimental procedures were approved by the ethics committee of animal experimentation of KU Leuven (license P065-2020).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	In brief, female Syrian hamsters (Mesocricetus auratus) of 6-8 weeks’ old were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 µL containing 2×106 TCID50 SARS-CoV-2 BetaCov/Belgium/GHB-03021/2020.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Open reading frames corresponding to the light and heavy chains of the hIgG1 anti-SARS-CoV-2 antibody S309 were ordered synthetically at IDT.	anti-SARS-CoV-2 suggested: None
Binding was detected by incubating the plates sequentially with either mouse anti-Histidine Tag antibody (MCA1396, Abd Serotec) followed by horseradish peroxidase (HRP)-linked anti-mouse IgG (1/2000, NXA931, GE Healthcare) or Streptavidin-HRP (554066, BD Biosciences) or by HRP-linked rabbit anti-human IgG (A8792, Sigma).	anti-Histidine Tag suggested: None MCA1396 suggested: None anti-mouse IgG ( 1/2000 , NXA931 , GE Healthcare ) suggested: None Streptavidin-HRP suggested: (Cell Signaling Technology Cat# 3999, RRID:AB_10830897)
Binding of human monoclonal antibodies palivizumab, CB6, and S309 and VHH72-Fc or variants thereof was detected with an AF633 conjugated goat anti-human IgG antibody (Invitrogen).	CB6 suggested: None VHH72-Fc suggested: None
Binding of monomeric VHHs to SARS-CoV-1 or SARS-CoV-2 S was detected with a mouse anti-HisTag antibody (AbD Serotec) and an AF647 conjugated donkey anti-mouse IgG antibody (Invitrogen).	SARS-CoV-1 suggested: None anti-HisTag suggested: (RevMAb Biosciences Cat# 54-1161-00, RRID:AB_2716428)
Subsequently, the cells were washed 3 times with PBS containing 0.5% BSA and stained with an AF647 conjugated donkey anti-mouse IgG antibody (Invitrogen) for 1 h.	anti-mouse IgG suggested: None
First, a pre-screen was undertaken to determine the levels of background binding of the test antibody to untransfected HEK293 cells, and to slides spotted with gelatin +/- SARS-CoV-2 spike protein.	SARS-CoV-2 spike protein . suggested: None
Anti-His monoclonal antibody was immobilized on S sensor chip CM3 by covalent coupling and used to capture the His-tagged Fcγ receptors.	Anti-His suggested: None
After washing in PBS, the cells were fixed in 1% PFA, washed twice with PBS, blocked with 1% BSA and stained with dilution series of anti-RBD antibodies or palivizumab.	anti-RBD suggested: None
Binding of the antibodies was detected using Alexa fluor 633 conjugated anti-human IgG antibodies (Invitrogen).	anti-human IgG suggested: None
Expression of the surface-displayed myc-tagged RBDs was detected using a FITC conjugated chicken anti-myc antibody (Immunology Consultants Laboratory, Inc.).	anti-myc suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Where data are labeled as ‘XVR011’, protein material manufactured from a stable CHO cell line has been used in the experiments, of identical sequence as humVHH_S56A/LALA-Fc/Gen2 (which is the naming we use for the protein produced in ExpiCHO transient transfection).	CHO suggested: None
Two days after transfecting HEK293T cells or HEK293S cells with spike expression plasmids each combined with a GFP expression plasmid, the cells were collected, washed once with PBS and fixed with 1% PFA for 30 minutes.	HEK293S suggested: RRID:CVCL_A784)
SARS-CoV pseudovirus neutralization assay: To generate replication-deficient VSV pseudotyped viruses, HEK293T cells, transfected with SARS-CoV-1 S or SARS-CoV-2 S were inoculated with a replication deficient VSV vector containing eGFP and firefly luciferase expression cassettes77, 78.	HEK293T suggested: None
The incubated pseudoviruses were subsequently added to subconfluent monolayers of VeroE6 cells.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
Human HEK293 cells were used for reverse transfection/expression.	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
Experimental Models: Organisms/Strains
Sentences	Resources
Mouse challenge experiments: Transgenic (K18-hACE2)2Prlmn mice were originally purchased from The Jackson Laboratory.	K18-hACE2)2Prlmn suggested: None
Software and Algorithms
Sentences	Resources
Simulations of the VHH72(mutant)-RBD complex were with Gromacs version 2020.141 using the Amber ff99SB-ILDN force field70 and were run for 5 nanoseconds.	Gromacs suggested: (GROMACS, RRID:SCR_014565)
Spike coding sequences were retrieved by aligning the genomes to the reference spike sequence annotated in NC_045512.2 (Wuhan-Hu-1 isolate, NCBI RefSeq).	RefSeq suggested: (RefSeq, RRID:SCR_003496)
For this purpose, pairwise alignments were performed using the R package Biostrings version 2.54.0 , a fixed substitution matrix in the “overlap” mode with the following parameters according to Biostrings documentation: 1 and -3 for match and mismatch substitution scores; 5 as gap opening and 2 as gap extension penalties.	Biostrings suggested: (Biostrings, RRID:SCR_016949)
Data collected for spike protein RBD (positions 333 – 516) was visualized using ggplot2 version 3.3.0.	ggplot2 suggested: (ggplot2, RRID:SCR_014601)
Samples were prepared by mixing the following: 30 µl sample (from a 1 mg/ml stock in HPLC grade water), 35 µL of 1% methylcellulose solution (ProteinSimple, 101876), 4 µl pH 3-10 pharmalytes (ProteinSimple, 042-848), 0.5 µl of 4.65, 0.5 µl 9.77 synthetic pI markers (ProteinSimple, 102223 and 102219), and 12.5 µl of 8 M urea solution (Sigma Aldrich®)	Sigma Aldrich® suggested: (Sigma-Aldrich, RRID:SCR_008988)
The resulting MS/MS spectra were analyzed with the BioPharma FinderTM 3.0 software (Thermo Fischer Scientific) and mapped onto the appropriate protein sequence.	BioPharma suggested: (TransCelerate BioPharma, RRID:SCR_003728)
Data analysis was carried out using the ASTRA 7.3.2 software.	ASTRA suggested: (ASTRA, RRID:SCR_016255)
In brief, 2019-nCoV S protein RBD that was biotinylated through an Avi-tag (AcroBiosystems, Cat nr.	AcroBiosystems suggested: (ACRObiosystems, RRID:SCR_012550)
This is achieved by visual inspection using the images gridded on the ImageQuant software.	ImageQuant suggested: (ImageQuant, RRID:SCR_014246)
Statistical evaluation (mean, SD) was conducted using Excel (Microsoft).	Excel suggested: None
The binding curves were fitted using nonlinear regression (Graphpad 8.0).	Graphpad suggested: (GraphPad, RRID:SCR_000306)
Concentrations were back-calculated by 4PL interpolation using Graphpad Prism (9.0).	Graphpad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

While the field of SARS-CoV-2 antibody development currently sees arguments in both directions with regard to the desired extent of Fc effector functionality in diverse settings (prophylactic, therapeutic, route and frequency of administration), our LALA-Fc choice attempts to strike an optimal balance between safety and efficacy in diverse application modalities, within the limitations of relevant current clinical data, while ascertaining an established regulatory track record and freedom to operate. It was recently reported that in a therapeutic setting, some human neutralizing antibodies require intact Fc effector functions to control SARS-CoV-2 replication in the K18-hACE2 transgenic mouse and Syrian hamster challenge models68, using the LALAPG mutations to remove effector function of those antibodies. In contrast to that report, we found that Fc effector silent humVHH_S56A/LALA(PG)-Fc/Gen2 administered 4, 16 or 24 hours after viral challenge resulted in a very strong reduction of lung viral RNA and infectious virus levels. This suggests that the non-RBM binding mode of neutralization, likely including spike destabilization, perhaps combined with a faster biodistribution compared with a conventional antibody, allows for efficacious viral control. The requirement of effector functionality for therapeutic efficacy appears to be very antibody-dependent even with human antibodies, as a very recent study also demonstrated therapeutic efficacy in the same hamster model of other ...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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No protocol registration statement was detected.

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Drug development of an affinity enhanced, broadly neutralizing heavy chain-only antibody that restricts SARS-CoV-2 in rodents

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