Sartorius_May21_NeutAntiMakingTheir - 35

NEUTRALIZING ANTIBODIES MAKING THEIR MARK IN NEXT WAVE OF BIOLOGICS

(mAbs) into bins based upon the antigen region

spike promoters when the RBD is in the down

or epitope bound by each antibody (Figure 1).

conformation but were more accessible in the

Furthermore, an Octet® competition assay revealed

up confirmation. Octet® assays observed CR3022

that 240CD or CR3022 antibody binding to SARS

binding to non-stabilized S-glycoprotein (S1)

CoV-2 RBD did not perturb ACE2 recognition.

conformations and much weaker binding to S2

Crystallography data revealed that CR3022

P trimers. To assess whether minimal proteolytic

engaged the RBD at a site that is conserved

action or receptor binding could increase the

in both SARS CoV and SARS CoV-2 explaining

availability of the obscure CR3022 epitope, S2

the cross-reactivity. However, a comparison of

trimers were treated with trypsin or incubated

SARS-CoV and MERS-CoV epitope regions from

with ACE2. Incubation of S2 P trimer with

previous structural data to SARS CoV-2 identified

human ACE2 did not dramatically affect CR3022

in this work revealed that CR3022 was binding to

binding. However, trypsin treatment of the S2

a novel epitope within SARS CoV-2 S.

P conformation resulted in increased binding

A specific RBD knockout mutant introduced

levels similar to non-stabilized S glycoprotein

within this epitope region (glycan sequon at

binding, and the level of binding was titratable

position 384) eliminated binding to both CR3022

with increasing proteolytic action, inferring to the

and 240CD antibodies according to Octet®

cryptic nature of the CR3022 binding epitope.

data confirming shared epitopes between both

Similar observations were reported by Yuan et al.,

antibodies.

using the crystal structure of neutralizing antibody

To understand the SARS CoV-2 Spike
conformations with respect to receptor binding

CR3022 bound to SARS-CoV-2 RBD14.
Octet® binding data showed that despite high

and cell entry, structures of CR3022 binding

sequence conservation between SARS-CoV and

to trimeric units of SARS-CoV-2, SARS-CoV and

SARS CoV 2 RBDs, CR3022 FABs binds SARS-CoV

MERS-CoV were modeled5. Data indicated that

with ~100-fold higher affinity than to SARS

CR3022 epitopes were obstructed by adjacent

CoV-2 RBD possibly driven by the non-conserved

Figure 1: Assay formats of epitope binning assays and recommendation guide to select the most suitable epitope binning assay format.
Octet® systems enable multiple assay formats for cross-competition assessment and provide dedicated high-throughput software analysis
tools for epitope binning. (A) In Tandem Assay: Antigen is immobilized on the biosensor, followed by the binding of the saturating mAb
(Ab1) and competing mAb (Ab2), respectively. (B) Classical Sandwich Assay: One mAb is immobilized on the biosensor (Ab1), antigen is
captured using this mAb and then a second sandwiching mAb is tested (Ab2). (C) Premix Assay: One mAb is immobilized on the biosensor
(Ab1), the biosensor is then exposed to a premix solution containing the antigen and a large molar excess of the second mAb (Ab2). (D)
Guidance for selecting the most suitable epitope binning assay format. Please read the Octet® assay guide to learn more about developing
and analyzing cross-competition screens.

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| 35


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Sartorius_May21_NeutAntiMakingTheir

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