Decreased neutralization of SARS-CoV-2 global variants by therapeutic anti-spike protein monoclonal antibodies

TLDR: In this paper, the authors tested monoclonal antibodies REGN10933 and REGN10987 that are used in combination, for their ability to neutralize SARS-CoV-2 variants B.1.7, mink cluster 5 and COH.

Abstract: Monoclonal antibodies against the SARS-CoV-2 spike protein, notably, those developed by Regeneron Pharmaceuticals and Eli Lilly and Company have proven to provide protection against severe COVID-19. The emergence of SARS-CoV-2 variants with heavily mutated spike proteins raises the concern that the therapy could become less effective if any of the mutations disrupt epitopes engaged by the antibodies. In this study, we tested monoclonal antibodies REGN10933 and REGN10987 that are used in combination, for their ability to neutralize SARS-CoV-2 variants B.1.1.7, B.1.351, mink cluster 5 and COH.20G/677H. We report that REGN10987 maintains most of its neutralization activity against viruses with B.1.1.7, B.1.351 and mink cluster 5 spike proteins but that REGN10933 has lost activity against B.1.351 and mink cluster 5. The failure of REGN10933 to neutralize B.1.351 is caused by the K417N and E484K mutations in the receptor binding domain; the failure to neutralize the mink cluster 5 spike protein is caused by the Y453F mutation. The REGN10933 and REGN10987 combination was 9.1-fold less potent on B.1.351 and 16.2-fold less potent on mink cluster 5, raising concerns of reduced efficacy in the treatment of patients infected with variant viruses. The results suggest that there is a need to develop additional monoclonal antibodies that are not affected by the current spike protein mutations.

Figures

Figure. 2

Figure. 3

Full text

1
Decreased neutralization of SARS-CoV-2 global variants by
therapeutic anti-spike protein monoclonal antibodies
Takuya Tada
1
, Belinda M. Dcosta
1
, Hao Zhou
1
, Ada Vaill
2
, Wes Kazmierski
2
and
Nathaniel R. Landau
1,3
1
Department of Microbiology, NYU Grossman School of Medicine, New York, NY
10016, USA
2
Biohaven Pharmaceuticals, Inc., 215 Church Street, New Haven, CT 06510, USA
3
Lead contact
*Correspondence: nathaniel.landau@med.nyu.edu
Nathaniel R. Landau, Ph.D.
NYU Langone Medical Center
430 East 29th Street, Alexandria West Building, Rm 509, New York, NY 10016
Phone: (212) 263-9197
Email: nathaniel.landau@med.nyu.edu
Short Title: S. Africa variant is not neutralized by REGN10933 anti-spike protein
monoclonal antibody
Key words: SARS-CoV-2, spike protein variants, B.1.1.7, B.1.351, Mink cluster 5
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.18.431897doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.18.431897doi: bioRxiv preprint

2
Abstract
Monoclonal antibodies against the SARS-CoV-2 spike protein, notably, those developed
by Regeneron Pharmaceuticals and Eli Lilly and Company have proven to provide
protection against severe COVID-19. The emergence of SARS-CoV-2 variants with
heavily mutated spike proteins raises the concern that the therapy could become less
effective if any of the mutations disrupt epitopes engaged by the antibodies. In this study,
we tested monoclonal antibodies REGN10933 and REGN10987 that are used in
combination, for their ability to neutralize SARS-CoV-2 variants B.1.1.7, B.1.351, mink
cluster 5 and COH.20G/677H. We report that REGN10987 maintains most of its
neutralization activity against viruses with B.1.1.7, B.1.351 and mink cluster 5 spike
proteins but that REGN10933 has lost activity against B.1.351 and mink cluster 5. The
failure of REGN10933 to neutralize B.1.351 is caused by the K417N and E484K
mutations in the receptor binding domain; the failure to neutralize the mink cluster 5 spike
protein is caused by the Y453F mutation. The REGN10933 and REGN10987 combination
was 9.1-fold less potent on B.1.351 and 16.2-fold less potent on mink cluster 5, raising
concerns of reduced efficacy in the treatment of patients infected with variant viruses. The
results suggest that there is a need to develop additional monoclonal antibodies that are
not affected by the current spike protein mutations.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.18.431897doi: bioRxiv preprint

3
Introduction
Monoclonal antibody therapies for the treatment of COVID-19 have been found to reduce
virus loads and alleviate symptoms when given shortly after diagnosis
1,2
. The REGN-
COV2 therapy developed by Regeneron Pharmaceuticals is a two recombinant
monoclonal antibody cocktail consisting of REGN10933 and REGN10987
3,4
while the Eli
Lilly therapy is based on a single antibody LY-CoV016
5
. The antibodies bind epitopes
within the receptor binding domain (RBD) of the Wuhan-Hu-1 spike protein. The rapid
evolution of SARS-CoV-2 variants with mutations in the viral S gene that encodes the
spike protein raises concerns that monoclonal antibody therapies could lose effectiveness
against viruses for which the spike protein has mutations that alter the amino acid
sequences of the epitopes bound by the antibodies.
Following the isolation of Wuhan-Hu1 SARS-CoV-2 in December 2019, the virus
has continued to further evolve as it adapts to the human host. A variant with a D614G
mutation
6
the spike protein which was identified in January, 2020 and by May became
the predominant strain world-wide with a prevalence of >97%. The amino acid residue,
which is located near the S1:S2 processing site, reduces S1 subunit shedding from
virions, has increased infectivity and results in higher virus loads
7-9
. Additional variants
containing the D614G mutation with increased transmissibility were subsequently
identified. The B.1.1.7 lineage (VOC-202012/01) variant identified in patients in the United
Kingdom
10-12
encodes a spike protein with 8 mutations in addition to D614G (Δ69-70,
Y144Del, N501Y, A570D, P681H, T716I, S982A and D1118H). N501Y is one of six ACE2
contact residues and has been shown to increase affinity for ACE2
13
by hydrogen bonding
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.18.431897doi: bioRxiv preprint

4
with ACE2 Y41
14
; the Δ69-70 deletion in the N-terminal domain is found in multiple
independent lineages
15
; and P681H lies adjacent to the furin cleavage site suggesting a
role in spike protein processing. The B.1.351 lineage variant identified in patients in South
Africa rapidly became the predominant circulating genotype
16
. The virus encodes a spike
protein that is more heavily mutated than B.1.1.7 with 9 mutations (L18F, D80A, D215G,
L242-244del, R246I, K417N, E484K, N501Y and A701V) three of which (K417N, E484K
and N501Y) are in the RBD. E484K, like N501Y, lies in the receptor binding motif (RBM)
that directly contacts specific ACE2 residues. K417N, while not contributing to ACE2
binding, is an epitope for neutralizing antibodies, as is E484K, and thus may have been
selected for evasion of the humoral response
17-21
. Based on phylogenetic tree branch-
length, it has been suggested that the variant arose through the prolonged virus
replication in an immunocompromised individual
15
. Additional variants found to be
circulating in the human population include the European isolate 20A.EU2
22
, Columbus,
Ohio variant COH.20G/677H and the mink cluster 5 variant found in domesticated minks
in Denmark with the potential for transfer into humans
23
.
Recent findings have demonstrated partial escape of the B.1.351 variant, and to a
lesser extent, B.1.1.7, from neutralization by the serum antibodies of convalescent
patients and by antibodies elicited by the Pfizer-BioNtech BNT162b2 and Moderna
mRNA-1273 mRNA vaccines that encode trimerized spike proteins
24
25
. The decreased
neutralizing titers against B.1.351 were largely the result of the E484K mutation, an amino
acid residue that serves as a contact point for ACE2
26-28
.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.18.431897doi: bioRxiv preprint

5
In this study, we analyzed neutralizing titers of REGN10933 and REGN10987 for
viruses with the SARS-CoV-2 variant spike proteins. The results showed that
REGN10933 maintains neutralizing activity against B.1.1.7 but has lost neutralizing
activity against virus with the B.1.351 and mink cluster 5 spike proteins. Analysis of
viruses with the individual B.1.351 mutations mapped the escape to E484K and K417N,
residues that lie within the RBD. REGN10987 maintains most of its neutralizing activity
against virus with the B.1.1.7, B.1.351 and mink cluster 5 variants, although a small but
significant decrease in neutralizing titer was noted against B.1.351 and mink cluster 5
spike proteins. As a result of the decreased activity of both antibodies, the combination
of REGN10933 and REGN10987 was decreased in neutralizing titer by 9.1-fold against
B.1.351 and 16.2-fold against mink cluster 5.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted February 19, 2021. ; https://doi.org/10.1101/2021.02.18.431897doi: bioRxiv preprint

Frequently asked questions

Q1. What are the contributions in "Decreased neutralization of sars-cov-2 global variants by therapeutic anti-spike protein monoclonal antibodies" ?

In this paper, a two recombinant monoclonal antibody cocktail consisting of REGN10933 and REGN109873,4 was used for the treatment of SARS-CoV-2. 

Q2. What is the effect of the recombinant monoclonal antibody on COVI?

Monoclonal antibody therapies for the treatment of COVID-19 have been found to reduce virus loads and alleviate symptoms when given shortly after diagnosis1,2. 

Q3. What is the reason for the rapid evolution of SARS-CoV-2 variants?

The rapid evolution of SARS-CoV-2 variants with mutations in the viral S gene that encodes the spike protein raises concerns that monoclonal antibody therapies could lose effectiveness against viruses for which the spike protein has mutations that alter the amino acid sequences of the epitopes bound by the antibodies. 

Q4. What is the amino acid residue that reduces S1 subunit shedding?

The amino acid residue, which is located near the S1:S2 processing site, reduces S1 subunit shedding from virions, has increased infectivity and results in higher virus loads7-9. 

Q5. What is the predominant strain of SARS-CoV-2?

A variant with a D614G mutation6 the spike protein which was identified in January, 2020 and by May became the predominant strain world-wide with a prevalence of >97%. 

Q6. What is the author/funder of this preprint?

The relative sensitivity of REGN10933 to mutations in spike protein variants may result from selective pressure to optimize ACE2 interacting amino(which was not certified by peer review) is the author/funder. 

Q7. What is the effect of the variant spike proteins on the neutralizing activity of REGN10933?

Neutralizing activity was measured with lentiviral virions pseudotyped with the variant spike proteins, an approach that allows for accurate measurement of neutralizing titers without the need for BSL-3 containment and provides a means to rapidly generate viruses with novel spike proteins variants. 

Q8. What is the author/funder of the research?

While REGN10987 has so far been unaffected by spike protein mutations, it would be advantageous to develop additional monoclonal antibodies that(which was not certified by peer review) is the author/funder. 

Q9. What is the effect of the two antibodies on the ACE2 binding site?

As a result of the decreased potency of both antibodies, the combined REGN10933 and REGN10987 cocktail had a decrease in neutralizing titer of 9.1-fold against B.1.351 and 16.2-fold against mink cluster 5.REGN10933 and REGN10987 bind to non-overlapping sites on the RBD3. 

Q10. What is the effect of the regn10933 on the virus?

REGN10987 maintains most of its neutralizing activity against virus with the B.1.1.7, B.1.351 and mink cluster 5 variants, although a small but significant decrease in neutralizing titer was noted against B.1.351 and mink cluster 5 spike proteins. 

Q11. What was the titer of the B.1.351 that was neutralized?

Analysis of the single mutations of B.1.351 showed that escape from REGN10933 was due to the K417N and E484K, each of which on its own was sufficient. 

Q12. What is the significance of the results presented here?

The results presented here highlight the importance of continued surveillance for SARS-CoV-2 variants and for testing the sensitivity of variants to antispike protein neutralizing antibodies in clinical use as well as their ability to be neutralized by vaccine elicited antibodies. 

Q13. Who is the author/funder of this preprint?

3. Location of amino acid residue causing escape from REGN10933 in the SARS-CoV-2 spike protein.(which was not certified by peer review) is the author/funder. 

Q14. What is the effect of a two antibody cocktail on the B.1.351 variant?

The findings highlight the benefit of a two antibody cocktail as therapy with the single REGN10933 would have lost effectiveness for use in patients infected with the B.1.351 variant and would be problematic in populations in which the variant was prevalent. 

Q15. What was the titer of the resenn10933?

Neutralizing titers for the mixture against B.1.351 and mink cluster 5 were reduced 9.14- and 16.2-fold compared to D614G, respectively, a result that reflects the large decrease in neutralizing titer for REGN10933 on both variants combined with a minor decrease in neutralizing titer by REGN10987 on both variants (Figure. 2 and Table. 1). 

Q16. What is the phylogenetic tree branch length of the B.1.351 variant?

Based on phylogenetic tree branchlength, it has been suggested that the variant arose through the prolonged virus replication in an immunocompromised individual15. 

Q17. What is the effect of the two antibody cocktail on the B.1.351 variant?

It is not clear whether the reduced neutralizing titer of the REGN10933 andREGN10987 cocktail will translate into a loss of effectiveness of REGN-COV2 therapy for individuals infected with the B.1.351 variant. 

Q18. What was the luciferase activity measured after 2 days?

Luciferase activity was measured after 2 days using Nano-Glo luciferase substrate (Promega) and plates were read in an Envision 2103 microplate luminometer (PerkinElmer).