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mAbs Volume 13, 2021 - Issue 1
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Bispecific VH/Fab antibodies targeting neutralizing and non-neutralizing Spike epitopes demonstrate enhanced potency against SARS-CoV-2

Shion A. Lima Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USAView further author information
,
Josef A. Gramespachera Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USAView further author information
,
Katarina Pancea Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USAView further author information
,
Nicholas J. Rettkoa Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USAView further author information
,
Paige Solomona Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USAView further author information
,
Jing Jinb Vitalant Research Institute and Department of Laboratory Medicine, University of California San Francisco, University of California San Francisco, California, USAView further author information
,
Irene Luia Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USAView further author information
,
Susanna K. Elledgea Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USAView further author information
,
Jia Liua Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USAView further author information
,
Colton J. Brackena Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USAView further author information
,
Graham Simmonsb Vitalant Research Institute and Department of Laboratory Medicine, University of California San Francisco, University of California San Francisco, California, USAView further author information
,
Xin X. Zhoua Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USAView further author information
,
Kevin K. Leunga Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USAView further author information
&
James A. Wellsa Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USA;c Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California, USA;d Chan Zuckerberg Biohub, San Francisco, California, USACorrespondence[email protected]
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Article: 1893426 | Received 28 Oct 2020, Accepted 17 Feb 2021, Published online: 05 Mar 2021

Figures & data

Table 1. In vitro binding affinities of antibodies against SARS-CoV-2 Spike

Figure 1. Fabs identified by phage display bind Spike RBD and Secto with high affinity outside of the ACE2 binding site

(a) Schematic of phage display used to isolate binders to Spike RBD-Fc from an in-house Fab-phage library. (b) Phage ELISA used to characterize binders shows that a majority of binders isolated did not bind similarly to Spike RBD in complex with ACE2 as to RBD alone. Multipoint BLI measurements of (c) Fab C01 and (d) Fab D01 on Spike RBD-Fc demonstrate high affinity binding. (e) Sequential epitope binning BLI demonstrates when Spike RBD-Fc is pre-saturated with ACE2-Fc, both Fabs C01 and D01 can still bind, indicating a non-overlapping epitope with ACE2-Fc. Multipoint BLI measurements of (f) IgG C01 and (g) IgG D01 show that conversion of Fab to IgG increases affinity to both Spike RBD-Fc (top) and trimeric Secto (bottom).
Figure 1. Fabs identified by phage display bind Spike RBD and Secto with high affinity outside of the ACE2 binding site

Figure 2. Bispecific VH/Fab IgGs bind with high affinity to trimeric Secto. (a) Cartoon schematic of the bispecific VH/Fab IgG antibody scaffold utilized in this study. (b-e) Multipoint BLI measurements (10 nM, 5 nM, and 2.5 nM) of the indicated bispecific antibody on Secto. (b) Bis1 (VH A01/FabC01) (c) Bis2 (VH B01/FabC01) (d) Bis3 (VH A01/FabD01) (e) Bis4 (VH B01/FabD01)

Figure 2. Bispecific VH/Fab IgGs bind with high affinity to trimeric Secto. (a) Cartoon schematic of the bispecific VH/Fab IgG antibody scaffold utilized in this study. (b-e) Multipoint BLI measurements (10 nM, 5 nM, and 2.5 nM) of the indicated bispecific antibody on Secto. (b) Bis1 (VH A01/FabC01) (c) Bis2 (VH B01/FabC01) (d) Bis3 (VH A01/FabD01) (e) Bis4 (VH B01/FabD01)

Table 2. SARS-CoV-2 Pseudovirus Neutralization IC50

Figure 3. Bispecific VH/Fab IgGs are more potent in neutralizing SARS-CoV-2 pseudovirus than the mono-specific counterparts

Pseudovirus neutralization curves for (a) Bis1 (VH A01/Fab C01) compared to VH-Fc A01 and IgG C01, (b) Bis2 (VH B01/Fab C01) compared to VH-Fc B01 and IgG C01, (c) Bis3 (VH A01/Fab D01) compared to VH-Fc A01 and IgG D01, (d) Bis4 (VH B01/Fab D01) compared to VH-Fc A01 and IgG D01. Data represent the average and standard deviation of three independent experiments and were fit to a non-linear regression using Prism 8 software to obtain IC50 values
Figure 3. Bispecific VH/Fab IgGs are more potent in neutralizing SARS-CoV-2 pseudovirus than the mono-specific counterparts

Table 3. Authentic SARS-CoV-2 Virus Neutralization IC50

Figure 4. Bispecific VH/Fab IgGs neutralize authentic SARS-CoV-2 virus more potently than the mono-specific VH-Fcs

Authentic SARS-CoV-2 virus neutralization curves of VH-Fc A01, VH-Fc B01, Bis1 (VH A01/Fab C01), Bis2 (VH B01/Fab C01), Bis3 (VH A01/Fab D01), Bis4 (VH B01/Fab D01). Vero E6 cells were incubated with the virus and threefold dilution of binder and assessed for cytopathic effect (CPE) 7 d after infection. Data represent the average and standard deviation of two independent experiments. Data were fit to a non-linear regression using Prism 8 software to obtain IC50 values.
Figure 4. Bispecific VH/Fab IgGs neutralize authentic SARS-CoV-2 virus more potently than the mono-specific VH-Fcs

Table

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Data availability statement:

All data supporting the findings of this study are available from the corresponding author upon request.

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