Note: Descriptions are shown in the official language in which they were submitted.
WO 2022/067269
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ANTIBODIES AGAINST SARS-COV-2
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text
format
in lieu of a paper copy, and is hereby incorporated by reference into the
specification.
The name of the text file containing the Sequence Listing is
930585 418W0 SEQUENCE LISTING.txt. The text file is 330 KB, was created on
September 26, 2021, and is being submitted electronically via EFS-Web.
BACKGROUND
A novel betacoronavirus emerged in Wuhan, China, in late 2019. As of
September 22, 2021, approximately 230 million cases of infection by this virus
(termed,
among other names, SARS-CoV-2 and Wuhan coronavirus), were confirmed
worldwide, and had resulted in over 4.7 million deaths. Therapies for
preventing,
treating, or diagnosing SARS-CoV-2 infection are needed.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-1C show binding of certain antibodies of the present disclosure to
SARS-CoV-2 Domain A. Human monoclonal antibodies isolated from donors were
expressed recombinantly and were tested for binding by EL1SA. The boxes on the
right
side of each figure indicate the calculated EC50 value (ng/mL) for the
indicated
antibody.
Figure 2A shows frequency of antibodies specific for SARS CoV-2 RBD, Spike
protein (non-RBD), or Domain A from sera of three donors. Figure 2B shows
percent
identity to IGHV germline sequence of certain antibodies. Figure 2C shows
percent
identity to IGLV germline sequence of certain antibodies.
Figures 3A-3E show additional functional characterization of certain
antibodies. (3A) Neutralization on Domain A by ELISA. (3B, 3C) Neutralization
of
SARS CoV-2 pseudoparticles. (3D) Maximum neutralization plateau. (3E)
Neutralization EC50.
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Figures 4A-4B show neutralization by antibodies 418_i (4A) and 418 5 (4B)
against authentic SARS-CoV-2 virus. Other antibodies were used as comparators.
Figures 5A-5C show results from epitope binning studies using biolayer
interferometry (BLI). In (5B), RBD-specific antibodies (top) were used as
comparators.
Figures 6A and 6B relate to binding of certain antibodies of the present
disclosure to transiently transfected ExpiCHO cells expressing various
sarbecoviruses
(Clade 2, Clade 1, or Clade 3), embecoviruses, merbecovirus, or mock. Antibody
S2X259 was included as a comparator in Figure 6A (flow cytometry study).
Figures 7A-7D show neutralization of infection by certain antibodies expressed
as recombinant Fab or full IgG. Figure 7E shows results from binding on
binding
assays using ACE2 (left) or spike (bottom, right). In the bottom panel, data
from
comparator antibodies S309, S2E12, and S2M11 is also shown
Figures 8A and 8B show effector functions of certain antibodies of the present
disclosure, along with a comparator antibody. Figure 8C shows antibody-
mediated
shedding of CoV-2 Si protein from infected cells. In Figure 8C, antibodies
S309,
S2E12, and S2M11v1 were used as comparators
Figures 9A-9C show data from neutralization experiments testing antibody
combinations with antibody 418 4 and another antibody (S309, S2E12, or S2M11)
against MLV pscudotypc with SARS-CoV2.
Figures 10A and 10B show binding of certain antibodies of the present
disclosure to SARS-CoV-2 Domain A, as measured by ELISA.
Figure 11 shows data from neutralization experiments using certain antibodies
of the present disclosure and SARS-CoV-2 pseudoparticles
Figure 12 shows binding of certain antibodies of the present disclosure to to
SARS-CoV-2 Domain A, as measured by ELISA.
Figure 13 shows neutralization of SARS CoV-2 pseudoparticles by certain
antibodies of the present disclosure.
Figures 14A-14C show binding of certain antibodies of the present disclosure
to SARS-CoV-2 Domain A, as measured by ELISA.
Figures 15A and 15B shows data from neutralization experiments using certain
antibodies of the present disclosure and SARS CoV-2 pseudoparticles.
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Figures 16A-16C show binding of certain antibodies of the present disclosure
to SARS-CoV-2 spike protein and to SARS-CoV-2 Domain A, as measured by ELISA.
Figures 17A-17C show kinetics of binding by three antibodies of the present
disclosure to SARS-CoV-2 spike protein. Calculated Kon, Koff, and KD values
are
shown in the boxes on the right side of each figure.
Figure 18 shows data from neutralization experiments using certain antibodies
of the present disclosure and SARS-CoV-2 virus pseudoparticles.
Figures 19A-19F show kinetics of binding by certain antibodies of the present
disclosure to SARS-CoV-2 Domain A, as measured by BLI.
Figure 20 shows the frequency of antibodies recognizing the SARS-CoV-2 N-
terminal domain (NTD, also referred to herein as Domain A), RBD, or other S
regions
for monoclonal antibodies cloned from IgG+ memory B cells of three donors.
Figure 21 shows binding and neutralization data for certain NTD-specific
antibodies. The left panel shows binding of 41 anti-NTD antibodies to
immobilized
SARS-CoV-2 S protein, NTD, or RBD, as determined by ELISA. The center panel
shows neutralization of infection by 1V1LV pseudotyped with SARS-CoV-2 S
protein for
each of 15 NTD-specific antibodies. The right panel shows maximal
neutralization
plateau for the same 15 NTD-specific antibodies.
Figure 22 shows neutralization of authentic SARS-CoV-2-Nluc infection for
certain antibodies assessed after 6 hours, using an MOT of 0.1. Error bars
indicate
standard deviation of triplicates.
Figure 23 shows neutralization of authentic SARS-CoV-2-Nluc infection for
certain antibodies assessed after 24 hours, using an MOT of 0.01. Error bars
indicate
standard deviation of triplicates.
Figures 24A-24D show binding kinetic analysis of SARS-CoV-2 NTD to
immobilized antibodies, as measured using BLI.
Figure 25 shows V gene usage for heavy chain (left panel) and light chain
(right
panel) of certain NTD-specific antibodies
Figure 26 shows further characterization of certain NTD-specific antibodies.
The left panel shows nucleotide sequence identity of the antibodies relative
to their
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respective V germline genes. The right panel shows the HCDR3 amino acid length
for
the antibodies.
Figure 27 shows results from a cell-to-cell fusion inhibition assay using Vero
E6 cells expressing SARS-CoV-2 S protein incubated with varying concentrations
of
each of four NTD-specific antibodies or RBD-specific antibody S2M11.
Figures 28A-28F show binding of each of 41 NTD-specific antibodies to
immobilized SARS-CoV-2 S protein ("Spike"), NTD ("Dom A"), or RBD as measured
by ELISA.
Figures 29A-29F show neutralization of infection by MLV pseudotyped with
SARS-CoV-2 S protein for each of 41 NTD-specific antibodies.
Figure 30 shows six antigenic sites (i) ¨ (vi) identified by epitope binning
of 41
NTD-specific antibodies based on competition binding assays using biolayer
interferometry (BLI).
Figures 31A-31I show the results of competition binding assays for 41 NTD-
specific antibodies using BLI. Results for antibodies identified as binding
Site i are
shown in Figures 31A-31C. Results for antibodies identified as binding Site ii
are
shown in Figure 31D. Results for antibodies identified as binding Site iii are
shown in
Figures 31E-31H. Results for antibodies identified as binding Site iv, Site v,
and Site vi
arc shown in Figure 3H.
Figure 32 shows competition by each of four NTD-specific antibodies and
RBD-specific antibody S2E12 with ACE2 for binding to SARS-CoV-2 S protein as
measured by BLI. ACE2 was immobilized at the surface of the biosensors before
incubation with S protein alone or in complex with antibody. The vertical
dashed line
in each graph indicates the start of the association of S/antibody complex or
free S with
solid-phase ACE2.
Figure 33 shows neutralization of authentic SARS-CoV-2-Nluc infection by
IgG or Fab of each of four NTD-specific antibodies and of comparator
antibodies S309
and S2M11 Symbols are means SD of triplicates Dotted lines in each graph
indicate IC50 and IC90 values.
Figure 34 shows results of SPR analysis of antibodies binding to SARS-CoV-2
S protein ectodomain trimer. The gray dashed line in each graph indicates a
fit to a 1:1
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binding model. The equilibrium dissociation constant (KD) or apparent
equilibrium
dissociation constant (KD, app) are indicated on each graph. White and gray
stripes on
each graph indicate association and dissociation phases, respectively.
Figure 35 shows activation of FciRlla H131 (left panel) and FcyRIIIa V158
(right panel) induced by the NTD-specific antibodies indicated and by RBD-
specific
antibody S309.
Figure 36 shows a matrix assessing synergistic activity of S2X333 and S309
antibody cocktails for in vitro neutralization of authentic SARS-CoV-2-Nluc
infection.
Data are from one representative example performed in triplicate.
Figures 37A-37D show data from Syrian hamsters injected with the indicated
amount of S2X333 antibody 48 hours before intra-nasal challenge with SARS-CoV-
2.
Figure 37A shows quantification of viral RNA in the lungs four days post-
infection
Figure 37B shows quantification of replicating virus in lung homogenates
harvested
four days post infection using a TCID50 assay. Figures 37C and 37D show viral
RNA
load (Figure 37C) and replicating virus titers (Figure 37D) in the lung four
days post
infection plotted as a function of serum antibody concentration before
infection (day 0)
Figure 38 shows infection of HEK293T cells transfected to over-express ACE2
or one of a panel of selected lectins and receptor candidates by VSV-SARS-CoV-
2
pscudovirus.
Figure 39 shows micrographs of stable TIEK293T cell lines overexpressing DC-
SIGN, L-SIGN, SIGLEC1, or ACE2 infected with authentic SARS-CoV-2 (MOI of
0.1), then fixed and immunostained for 24 hours for SARS-CoV-2 nucleoprotein
(red)
Figure 40 shows quantification of luciferase levels in stable HEK293T cell
lines
overexpressing DC-SIGN, L-SIGN, SIGLEC1, or ACE2, as measured 24 hours after
infection with SARS-CoV-2-Nluc.
Figure 41 shows quantification of luciferase levels in stable HEK293T cell
lines
overexpressing DC-SIGN, L-SIGN, SIGLEC1, or ACE2 after incubation with
different
concentrations of anti-SIGLEC1 monoclonal antibody (clone 7-239) and infection
with
SARS-CoV-2-Nluc.
Figure 42 shows infection of cells transiently transduced to overexpress DC-
SIGN, L-SIGN, SIGLEC1, or ACE2 by VSV-SARS-CoV-2 pseudovirus. Results for
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HEK293T cells (left panel), HeLa cells (center panel), and MRCS cells (right
panel) are
shown.
Figure 43 shows infection of stable HEK293T cell lines overexpressing DC-
SIGN, L-SIGN, SIGLEC1, or ACE2 after treatment with ACE2 siRNA followed by
infection with VSV-SARS-CoV-2 pseudovirus.
Figure 44 shows infection of stable HEK293T cell lines overexpressing DC-
SIGN, L-SIGN, SIGLEC1, or ACE2 after treatment with different concentrations
of
anti-ACE2 antibody (polyclonal serum) followed by infection with VSV-SARS-CoV-
2
pseudovirus.
Figure 45 shows the distribution and expression of ACE2, DC-SIGN (CD209),
L-SIGN (CLEC4M), and SIGLEC1 in the human lung cell atlas.
Figure 46 shows analysis of major cell types with detectable SARS-CoV-2
genome in bronchoalveolar lavage fluid or sputum of severe COVID-19 patients.
The
single cell gene expression profiles are shown as a t-SNE (t-distributed
stochastic
neighbor embedding) plot, sized by viral load.
Figure 47 shows analysis of major cell types with detectable SARS-CoV-2
genome in bronchoalveolar lavage fluid or sputum of severe COVID-19 patients.
The
cumulative fraction of cells (y-axis) with detected viral RNA per cell up to
the
corresponding logCPM (log(counts per million); x-axis) is shown for each of
the
indicated cell types.
Figure 48 shows a heatmap matrix of counts for cells with detected transcripts
for the receptor genes shown on the x-axis and SARS-CoV-2+ cell types on the y-
axis.
Total n=3,085 cells from eight subjects. See Ren, X. et al. COVID-19 immune
features
revealed by a large-scale single cell transcriptome atlas. Cell,
doi:10.1016/j.ce11.2021.01.053 (2021).
Figure 49 shows the correlation of receptor transcript counts (y-axis of each
plot) with SARS-CoV-2 RNA counts (x-axis of each plot) in macrophages and in
secretory cells Correlation is based on counts before log transformation from
Ren et al
Figure 50 shows the results of trans-infection with VSV-SARS-CoV-2. A
schematic of the trans-infection process is shown in the left panel. HeLa
cells
transduced with DC-SIGN, L-SIGN, or SIGLEC1 were incubated with VSV-SARS-
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CoV-2, extensively washed, and co-cultured with Vero-E6-TMPRSS2 susceptible
target cells. Results in the presence or absence of target cells are shown in
the right
panel.
Figure 51 shows the results of trans-infection, where VSV-SARS-CoV-2 viral
adsorption was performed in the presence or absence of an anti-SIGLEC1
blocking
antibody.
Figure 52 shows neutralization of SARS-CoV-2 infection of Vero-E6 cells by
antibodies S309, S2E12, and S2X33.
Figure 53 shows neutralization of SARS-CoV-2 infection of Vero-E6-
T1VIPRSS2 cells by antibodies S309, S2E12, and S2X33.
Figure 54 shows quantification of binding of purified, fluorescently-labeled
SARS-CoV-2 spike protein or RED to the indicated cell lines, as measured by
flow
cytometry. "A" indicates cell line overexpressing ACE2; "T" indicates cell
line
overexpressing TMPRSS2.
Figure 55 shows quantification of cellular ACE2 and TIVfPRSS2 transcripts in
the indicated cell lines, as measured by RT-qPCR "A" indicates cell line
overexpressing ACE2, "T" indicates cell line overexpressing TMPRSS2.
Figure 56 shows neutralization of SARS-CoV-2-Nluc infection by antibodies
S309, S2E12, or S2X333. Each of the seven cell lines indicated was tested.
Luciferase
signal was quantified 24 hours post infection.
Figure 57 shows neutralization of VSV-SARS-CoV-2 pseudovirus infection by
antibodies S309, S2E12, or S2X333. Each of the seven cell lines indicated was
tested.
Luciferase signal was quantified 24 hours post infection.
Figure 58 shows S2E12-induced uni-directional fusion (also referred to as
trans-fusion) of S-positive CHO-S cells with fluorescently labelled S-negative
CHO
cells in the absence of ACE2. Nuclei were stained with Hoechst dye; cytoplasm
was
stained with CellTracker Green.
Figure 59 shows neutralization of infection of a stable HEK293T cell line
overexpressing ACE2 by authentic SARS-CoV-2 pre-incubated with the indicated
monoclonal antibodies. Infection was measured by immunostaining at 24 hours
for the
SARS-CoV-2 nucleoprotein.
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Figure 60 shows neutralization of infection of a stable HEK293T cell line
overexpressing SIGLEC1 by authentic SARS-CoV-2 pre-incubated with the
indicated
monoclonal antibodies. Infection was measured by immunostaining at 24 hours
for the
SARS-CoV-2 nucleoprotein.
Figure 61 shows neutralization of infection of a stable HEK293T cell line
overexpressing DC-SIGN by authentic SARS-CoV-2 pre-incubated with the
indicated
monoclonal antibodies. Infection was measured by immunostaining at 24 hours
for the
SARS-CoV-2 nucleoprotein.
Figure 62 shows neutralization of infection of a stable HEK293T cell line
overexpressing L-SIGN by authentic SARS-CoV-2 pre-incubated with the indicated
monoclonal antibodies. Infection was measured by immunostaining at 24 hours
for the
SARS-CoV-2 nucleoprotein.
Figure 63 shows analysis of binding of antibodies targeting DC/L-SIGN, DC-
SIGN, SIGLEC1, or ACE2 on HEK293T cells stably over-expressing the respective
attachment receptor, as measured by flow cytometry.
Figure 64 shows analysis of binding of antibodies targeting DC/L-SIGN, DC-
SIGN, SIGLEC1, or ACE2 on HEK293T cells stably over-expressing the respective
attachment receptor, as measured by immunofluorescence.
Figure 65 shows infection of HEK293T cells stably over-expressing the
indicated attachment receptor by VSV-SARS-CoV-2 pseudotyped with wild type
spike
protein (grey bars), or VSV-SARS-CoV-2 pseudotyped with spike protein bearing
the
mutations of the B1.1.7 lineage (red bars). Luminescence was analyzed one day
post
infection.
Figure 66 shows neutralization of SARS-CoV-2 infection of Vero-E6 or Vero-
E6-TMPRS S2 cells by 10 mg/m1 of S309, S2E12, and S2X333. Cells were infected
with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the
indicated antibodies. Cells were fixed 24h post infection and viral
nucleocapsid protein
was immunostained
Figure 67 shows quantification of binding of purified, fluorescently labelled
SARS-CoV-2 spike protein (left panels) or RBD (right panels) to the indicated
cell
lines, as measured by flow cytometry.
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Figure 68 shows quantification of binding of purified, fluorescently labelled
SARS-CoV-2 spike protein (left panels) or RBD (right panels) to the indicated
cell
lines, as measured by flow cytometry.
Figure 69 shows an analysis of the correlation between ACE2 transcript levels
(x-axis) and maximum antibody-related neutralization of infection (y-axis) in
SARS-
CoV-2-susceptible cell lines for antibody S309 (left panel) and antibody
S2X333 (right
panel).
Figure 70 shows binding of immunocomplexes to hamster splenocytes. Alexa-
488 fluorescent immunocomplexes (IC) were titrated (0-200 nM range) and
incubated
with total naive hamster splenocytes. Binding was revealed with a cytometer
upon
exclusion of dead/apoptotic cells and physical gating on bona fide monocyte
population. Left panel shows the fluorescent intensity associated to hamster
cells of IC
made with either hamster or human Fe antibodies. A single replicate of two is
shown.
Right panel shows the relative Alexa-488 mean fluorescent intensity of the
replicates
measured on the entire monocyte population.
Figure 71 shows analysis of the role of host effector function in SARS-CoV-2
challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of
hamster
IgG2a S309, either wt or Fe silenced (S309-N297A). Top panel shows
quantification
of viral RNA in the lung 4 days post infection. Center panel shows
quantification of
replicating virus in the lung 4 days post infection. Bottom panel shows
histopathological score in the lung 4 days post infection. Control animals
(white
symbols) were injected with 4 mg/kg unrelated control isotype antibody. * p<
0.05, **
p< 0.01, *** p< 0.001, **** p< 0.0001 vs control animals, using Mann-Whitney
test.
Figure 72 shows neutralization of SARS-CoV-2 infection of HEK293T cells
stably expressing ACE2 (top panel) or DC-SIGN (bottom panel) in the presence
of the
indicated antibodies. Cells were infected at MOI of 0.02. Cells were fixed 24h
post
infection, viral nucleocapsid protein was immunostained and positive cells
were
quantified
Figure 73 shows neutralization of SARS-CoV-2 infection of HEK293T cells
stably expressing SIGLEC I (top panel) or L-SIGN (bottom panel) in the
presence of
the indicated antibodies. Cells were infected at MOI of 0.02. Cells were fixed
24h post
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infection, viral nucleocapsid protein was immunostained and positive cells
were
quantified.
DETAILED DESCRIPTION
Provided herein are antibodies and antigen-binding fragments that bind to
SARS-CoV-2 coronavirus (e.g., a SARS-CoV-2 Domain A, in a SARS-CoV-2 virion
and/or expressed on the surface of a cell infected by the SARS-CoV-2
coronavirus). In
certain embodiments, presently disclosed antibodies and antigen-binding
fragments can
neutralize a SARS-CoV-2 infection in an in vitro model of infection and/or in
a human
subject. Also provided are polynucleotides that encode the antibodies and
antigen-
binding fragments, vectors, host cells, and related compositions, as well as
methods of
using the antibodies, nucleic acids, vectors, host cells, and related
compositions to treat
(e.g., reduce, delay, eliminate, or prevent) a SARS-CoV-2 infection in a
subject and/or
in the manufacture of a medicament for treating a SARS-CoV-2 infection in a
subject.
Prior to setting forth this disclosure in more detail, it may be helpful to an
understanding thereof to provide definitions of certain terms to be used
herein.
Additional definitions are set forth throughout this disclosure.
As used herein, "SARS-CoV-2", also referred to herein as "Wuhan seafood
market phenomia virus", or "Wuhan coronavirus" or "Wuhan CoV", or "novel CoV",
or
"nCoV", or "2019 nCoV", or "Wuhan nCoV" is a betacoronavirus believed to be of
lineage B (sarbecovirus). SARS-CoV-2 was first identified in Wuhan, Hubei
province,
China, in late 2019 and spread within China and to other parts of the world by
early
2020. Symptoms of SARS-CoV-2 infection include fever, dry cough, and dyspnea.
The genomic sequence of SARS-CoV-2 isolate Wuhan-Hu-1 is provided in SEQ
ID NO.:1 (see also GenBank MN908947.3, January 23, 2020), and the amino acid
translation of the genome is provided in SEQ ID NO. :2 (see also GenBank
QHD43416.1, January 23, 2020). Like other coronavinises (e.g., SARS- CoV-1),
SARS-CoV-2 comprises a "spike" or surface ("S") type I transmembrane
glycoprotein
containing a receptor binding domain (RBD). RBD is believed to mediate entry
of the
lineage B SARS coronavirus to respiratory epithelial cells by binding to the
cell surface
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receptor angiotensin-converting enzyme 2 (ACE2). In particular, a receptor
binding
motif (RBM) in the virus RBD is believed to interact with ACE2. SARS CoV-2 S
protein also includes, N-terminal to the RBD and C-terminal to the S protein
signal
peptide, domain A (also referred-to as the N-terminal Domain or "NTD").
Antibodies
of the present disclosure are specific for domain A.
The amino acid sequence of the Wuhan-Hu-1 surface glycoprotein is provided
in SEQ ID NO.:3. The amino acid sequence of SARS-CoV-2 RBD is provided in SEQ
ID NO.:4. SARS-CoV-2 S protein has approximately 73% amino acid sequence
identity with SARS-CoV-1. The amino acid sequence of SARS-CoV-2 RBM is
provided in SEQ ID NO.:5. SARS-CoV-2 RBD has approximately 75% to 77% amino
acid sequence similarity to SARS¨CoV-1 RBD, and SARS-CoV-2 RBM has
approximately 50% amino acid sequence similarity to SARS-CoV-1 RBM.
Unless otherwise indicated herein, SARS-CoV-2 Wuhan-Hu-1 refers to a virus
comprising the amino acid sequence set forth in any one or more of SEQ ID
NOs.:2, or
3, optionally with the genomic sequence set forth in SEQ ID NO.:1.
There have been a number of emerging SARS-CoV-2 variants. Some SARS-
CoV-2 variants contain an N439K mutation, which has enhanced binding affinity
to the
human ACE2 receptor (Thomson, E.C., et al., The circulating SARS-CoV-2 spike
variant N439K maintains fitness while evading antibody-mediated immunity.
bioRxiv,
2020). Some SARS-CoV-2 variants contain an N501Y mutation, which is associated
with increased transmissibility, including the lineages B.1.1.7 (also known as
20I/501Y.V1 and VOC 202012/01) and B.1.351 (also known as 20H/501Y.V2), which
were discovered in the United Kingdom and South Africa, respectively (Tegally,
H., et
al., Emergence and rapid spread of a new severe acute respiratory syndrome-
related
coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South
Africa.
medRxiv, 2020: p. 2020.12.21.20248640; Leung, K., et al., Early empirical
assessment
of the N50 IY mutant strains of SAPS-CoV-2 in the United Kingdom, October to
November 2020. medRxiv, 2020: p. 2020.12 20.20248581). B.1.351 also include
two
other mutations in the RBD domain of SARS-CoV2 spike protein, K417N and E484K
(Tegally, H., et al., Emergence and rapid spread of a new severe acute
respiratory
syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike
mutations in
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South Africa. medRxiv, 2020: p. 2020.12.21.20248640). Other SARS-CoV-2
variants
include the Lineage B.1.1.28, which was first reported in Brazil; the Variant
P.1,
lineage B.1.1.28 (also known as 20J/501Y.V3), which was first reported in
Japan;
Variant L452R, which was first reported in California in the United States
(Pan
American Health Organization, Epidemiological update: Occurrence of variants
of
SARS-CoV-2 in the Americas, January 20, 2021, available at
https://reliefweb.int/sites/reliefweb.int/files/resources/2021-jan-20-phe-epi-
update-
SARS-CoV-2.pdf). Other SARS-CoV-2 variants include a SARS CoV-2 of clade 19A;
SARS CoV-2 of clade 19B; a SARS CoV-2 of clade 20A; a SARS CoV-2 of clade 20B;
a SARS CoV-2 of clade 20C; a SARS CoV-2 of clade 20D; a SARS CoV-2 of clade
20E (EU1); a SARS CoV-2 of clade 20F; a SARS CoV-2 of clade 20G; and SARS
CoV-2 B1.1.207; and other SARS CoV-2 lineages described in Rambaut, A., et
al., A
dynamic nomenclature proposal .for SARS-CoV-2 lineages to assist genomic
epidemiology. Nat Microbiol 5, 1403-1407 (2020). The Alpha (B.1.1.7), Beta
(B.1.351, B.1.351.2, B.1.351.3), Delta (B.1.617.2, AY.1, AY.2, AY.3), and
Gamma
(P.1, P.1.1, P.1.2) variants of SARS-CoV-2 circulating in the United States
are
classified as variants of concern by the U.S. Centers for Disease Control and
Prevention
(see https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html).
Treating a
SARS CoV-2 infection in accordance with the present disclosure includes
treating
infection by any one or more of the aforementioned SARS-CoV-2 viruses. In
certain
embodiments, treating a SARS-CoV-2 infection comprises treating any one or
more of:
SARS CoV-2 Wuhan-Hu-1; a SARS-CoV-2 variant comprising a N439K mutation; a
SARS-CoV-2 variant comprising a N501 Y mutation; a SARS-CoV-2 variant
comprising a K417N mutation and/or a E484K mutation; a SARS-CoV-2 comprising a
L452R mutation; B.1.1.28; B.1.1.7 (also referred-to as the "alpha" variant);
B.1.351
(also referred-to as the "beta" variant); P.1 (also referred-to as the "gamma"
variant);
B.1.617.1 (also referred-to as the "kappa" variant); B.1.429 (also referred-to
as the
"epsilon" variant); B.1.525 (also referred-to as the "eta" variant); B.1.526
(also referred-
to as the "iota" variant); B.1.258; a variant of Wuhan-Hu-1 comprising a N440K
mutation; B.1.243.1; B.1.258 with a K417N mutation; A.27.1; R.1; P.2; R.2;
B.1.1.519;
A.23.1; B.1.318; B.1.619; A.VOI.V2; B.1.618; a variant of Wuhan-Hu-1
comprising
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N440K and E484K mutations; B.1.617.2 (also referred-to as the "delta"
variant);
B.1.1.298; B.1.617.2-AY.1; B.1.617.2-AY.2; C.37 (also referred-to as the
"lambda"
variant); a SARS CoV-2 of clade 19A; SARS CoV-2 of clade 19B; a SARS CoV-2 of
clade 20A; a SARS CoV-2 of clade 20B; a SARS CoV-2 of clade 20C; a SARS CoV-2
of clade 20D; a SARS CoV-2 of clade 20E (EU1); a SARS CoV-2 of clade 20F; and
a
SARS CoV-2 of clade 20G. Other coronaviruses are believed to enter cells by
binding
to other receptors (e.g., 9-0-Ac-Sia receptor analog; DPP4; APN).
In the present description, any concentration range, percentage range, ratio
range, or integer range is to be understood to include the value of any
integer within the
recited range and, when appropriate, fractions thereof (such as one tenth and
one
hundredth of an integer), unless otherwise indicated. Also, any number range
recited
herein relating to any physical feature, such as polymer subunits, size or
thickness, are
to be understood to include any integer within the recited range, unless
otherwise
indicated. As used herein, the term "about" means 20% of the indicated
range, value,
or structure, unless otherwise indicated. In particular embodiments, "about"
comprises
5%, 10%, or 15%.
It should be understood that the terms "a" and "an" as used herein refer to
"one
or more" of the enumerated components. The use of the alternative (e.g., "or")
should
be understood to mean either one, both, or any combination thereof of the
alternatives.
As used herein, the terms "include," "have," and "comprise" are used
synonymously,
which terms and variants thereof are intended to be construed as non-limiting.
"Optional" or "optionally" means that the subsequently described element,
component, event, or circumstance may or may not occur, and that the
description
includes instances in which the element, component, event, or circumstance
occurs and
instances in which they do not.
In addition, it should be understood that the individual constructs, or groups
of
constructs, derived from the various combinations of the structures and
subunits
described herein, are disclosed by the present application to the same extent
as if each
construct or group of constructs was set forth individually. Thus, selection
of particular
structures or particular subunits is within the scope of the present
disclosure.
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The term "consisting essentially of' is not equivalent to "comprising" and
refers
to the specified materials or steps of a claim, or to those that do not
materially affect the
basic characteristics of a claimed subject matter. For example, a protein
domain,
region, or module (e.g., a binding domain) or a protein "consists essentially
of' a
particular amino acid sequence when the amino acid sequence of a domain,
region,
module, or protein includes extensions, deletions, mutations, or a combination
thereof
(e.g., amino acids at the amino- or carboxy-terminus or between domains) that,
in
combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%,
4%, 3%,
2% or 1%) of the length of a domain, region, module, or protein and do not
substantially affect (i.e., do not reduce the activity by more than 50%, such
as no more
than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s),
region(s), module(s), or protein (e.g., the target binding affinity of a
binding protein).
As used herein, "amino acid" refers to naturally occurring and synthetic amino
acids, as well as amino acid analogs and amino acid mimetics that function in
a manner
similar to the naturally occurring amino acids. Naturally occurring amino
acids are
those encoded by the genetic code, as well as those amino acids that are later
modified,
e.g., hydroxyproline, y-carboxyglutamate, and 0-phosphoserine. Amino acid
analogs
refer to compounds that have the same basic chemical structure as a naturally
occurring
amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group,
an amino
group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide,
methionine
methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or
modified
peptide backbones, but retain the same basic chemical structure as a naturally
occurring
amino acid. Amino acid mimetics refer to chemical compounds that have a
structure
that is different from the general chemical structure of an amino acid, but
that functions
in a manner similar to a naturally occurring amino acid.
As used herein, "mutation" refers to a change in the sequence of a nucleic
acid
molecule or polypeptide molecule as compared to a reference or wild-type
nucleic acid
molecule or polypepti de molecule, respectively A mutation can result in
several
different types of change in sequence, including substitution, insertion or
deletion of
nucleotide(s) or amino acid(s).
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A "conservative substitution" refers to amino acid substitutions that do not
significantly affect or alter binding characteristics of a particular protein.
Generally,
conservative substitutions are ones in which a substituted amino acid residue
is replaced
with an amino acid residue having a similar side chain. Conservative
substitutions
include a substitution found in one of the following groups: Group 1: Alanine
(Ala or
A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2:
Aspartic acid
(Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N),
Glutamine (Gln
or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H);
Group 5:
Isoleucine (Ile or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val
or V); and
Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W).
Additionally or alternatively, amino acids can be grouped into conservative
substitution
groups by similar function, chemical structure, or composition (e.g., acidic,
basic,
aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping
may
include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile. Other
conservative
substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C);
acidic:
Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues:
Ala, Ser,
Thr, Pro, and Gly, polar, negatively charged residues and their amides: Asp,
Asn, Glu,
and Gln; polar, positively charged residues: His, Arg, and Lys; large
aliphatic, nonpolar
residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr,
and Trp.
Additional information can be found in Creighton (1984) Proteins, W.H Freeman
and
Company.
As used herein, "protein" or "polypeptide" refers to a polymer of amino acid
residues. Proteins apply to naturally occurring amino acid polymers, as well
as to
amino acid polymers in which one or more amino acid residue is an artificial
chemical
mimetic of a corresponding naturally occurring amino acid, and non-naturally
occurring
amino acid polymers. Variants of proteins, peptides, and polypeptides of this
disclosure
are also contemplated. In certain embodiments, variant proteins, peptides, and
polypeptides comprise or consist of an amino acid sequence that is at least
70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%
identical to an amino acid sequence of a defined or reference amino acid
sequence as
described herein.
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"Nucleic acid molecule" or "polynucleotide" or "polynucleic acid" refers to a
polymeric compound including covalently linked nucleotides, which can be made
up of
natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits
(e.g.,
morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and
xanthine,
and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid
molecules
include polyribonucleic acid (RNA), which includes mRNA, microRNA, siRNA,
viral
genomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA), which
includes cDNA, genomic DNA, and synthetic DNA, either of which may be single
or
double stranded. If single-stranded, the nucleic acid molecule may be the
coding strand
or non-coding (anti-sense) strand. A nucleic acid molecule encoding an amino
acid
sequence includes all nucleotide sequences that encode the same amino acid
sequence.
Some versions of the nucleotide sequences may also include intron(s) to the
extent that
the intron(s) would be removed through co- or post-transcriptional mechanisms.
In
other words, different nucleotide sequences may encode the same amino acid
sequence
as the result of the redundancy or degeneracy of the genetic code, or by
splicing.
Variants of nucleic acid molecules of this disclosure are also contemplated.
Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are
preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule
of a
defined or reference polynucleotide as described herein, or that hybridize to
a
polynucleotide under stringent hybridization conditions of 0.015M sodium
chloride,
0.0015M sodium citrate at about 65-68 C or 0.015M sodium chloride, 0.0015M
sodium
citrate, and 50% formamide at about 42 C. Nucleic acid molecule variants
retain the
capacity to encode a binding domain thereof having a functionality described
herein,
such as binding a target molecule.
"Percent sequence identity" refers to a relationship between two or more
sequences, as determined by comparing the sequences. Preferred methods to
determine
sequence identity are designed to give the best match between the sequences
being
compared For example, the sequences are aligned for optimal comparison
purposes
(e.g., gaps can be introduced in one or both of a first and a second amino
acid or nucleic
acid sequence for optimal alignment). Further, non-homologous sequences may be
disregarded for comparison purposes. The percent sequence identity referenced
herein
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is calculated over the length of the reference sequence, unless indicated
otherwise.
Methods to determine sequence identity and similarity can be found in publicly
available computer programs. Sequence alignments and percent identity
calculations
may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or
BLASTX). The mathematical algorithm used in the BLAST programs can be found in
Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Within the context of
this
disclosure, it will be understood that where sequence analysis software is
used for
analysis, the results of the analysis are based on the "default values" of the
program
referenced. "Default values" mean any set of values or parameters which
originally
load with the software when first initialized.
The term "isolated" means that the material is removed from its original
environment (e.g., the natural environment if it is naturally occurring). For
example, a
naturally occurring nucleic acid or polypeptide present in a living animal is
not isolated,
but the same nucleic acid or polypeptide, separated from some or all of the co-
existing
materials in the natural system, is isolated. Such nucleic acid could be part
of a vector
and/or such nucleic acid or polypeptide could be part of a composition (e.g.,
a cell
lysate), and still be isolated in that such vector or composition is not part
of the natural
environment for the nucleic acid or polypeptide. "Isolated" can, in some
embodiments,
also describe an antibody, antigen-binding fragment, polynucleotide, vector,
host cell,
or composition that is outside of a human body.
The term "gene" means the segment of DNA or RNA involved in producing a
polypeptide chain; in certain contexts, it includes regions preceding and
following the
coding region (e.g., 5' untranslated region (UTR) and 3' UTR) as well as
intervening
sequences (introns) between individual coding segments (exons).
A "functional variant" refers to a polypeptide or polynucleotide that is
structurally similar or substantially structurally similar to a parent or
reference
compound of this disclosure, but differs slightly in composition (e.g., one
base, atom or
functional group is different, added, or removed), such that the polypeptide
or encoded
polypeptide is capable of performing at least one function of the parent
polypeptide
with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%,
85%, 90%,
95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent
polypeptide.
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In other words, a functional variant of a polypeptide or encoded polypeptide
of this
disclosure has "similar binding," "similar affinity" or "similar activity"
when the
functional variant displays no more than a 50% reduction in performance in a
selected
assay as compared to the parent or reference polypeptide, such as an assay for
measuring binding affinity (e.g., Biacore or tetramer staining measuring an
association (Ka) or a dissociation (Ku) constant).
As used herein, a "functional portion" or "functional fragment" refers to a
polypeptide or polynucleotide that comprises only a domain, portion or
fragment of a
parent or reference compound, and the polypeptide or encoded polypeptide
retains at
least 50% activity associated with the domain, portion or fragment of the
parent or
reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%,
95%,
96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent
polypeptide, or
provides a biological benefit (e.g., effector function). A "functional
portion" or
"functional fragment" of a polypeptide or encoded polypeptide of this
disclosure has
"similar binding" or "similar activity" when the functional portion or
fragment displays
no more than a 50% reduction in performance in a selected assay as compared to
the
parent or reference polypeptide (preferably no more than 20% or 10%, or no
more than
a log difference as compared to the parent or reference with regard to
affinity).
As used herein, the term "engineered," "recombinant," or "non-natural" refers
to
an organism, microorganism, cell, nucleic acid molecule, or vector that
includes at least
one genetic alteration or has been modified by introduction of an exogenous or
heterologous nucleic acid molecule, wherein such alterations or modifications
are
introduced by genetic engineering (i.e., human intervention). Genetic
alterations
include, for example, modifications introducing expressible nucleic acid
molecules
encoding functional RNA, proteins, fusion proteins or enzymes, or other
nucleic acid
molecule additions, deletions, substitutions, or other functional disruption
of a cell's
genetic material. Additional modifications include, for example, non-coding
regulatory
regions in which the modifications alter expression of a polynucleotide, gene,
or
operon.
As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to
any gene, protein, compound, nucleic acid molecule, or activity that is not
native to a
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host cell or a subject, or any gene, protein, compound, nucleic acid molecule,
or activity
native to a host cell or a subject that has been altered. Heterologous, non-
endogenous,
or exogenous includes genes, proteins, compounds, or nucleic acid molecules
that have
been mutated or otherwise altered such that the structure, activity, or both
is different as
between the native and altered genes, proteins, compounds, or nucleic acid
molecules.
In certain embodiments, heterologous, non-endogenous, or exogenous genes,
proteins,
or nucleic acid molecules (e.g., receptors, ligands, etc.) may not be
endogenous to a
host cell or a subject, but instead nucleic acids encoding such genes,
proteins, or nucleic
acid molecules may have been added to a host cell by conjugation,
transformation,
transfection, electroporation, or the like, wherein the added nucleic acid
molecule may
integrate into a host cell genome or can exist as extra-chromosomal genetic
material
(e.g., as a plasmid or other self-replicating vector) The term "homologous" or
"homolog" refers to a gene, protein, compound, nucleic acid molecule, or
activity found
in or derived from a host cell, species, or strain. For example, a
heterologous or
exogenous polynucleotide or gene encoding a polypeptide may be homologous to a
native polynucleotide or gene and encode a homologous polypeptide or activity,
but the
polynucleotide or polypeptide may have an altered structure, sequence,
expression
level, or any combination thereof. A non-endogenous polynucleotide or gene, as
well
as the encoded polypeptide or activity, may be from the same species, a
different
species, or a combination thereof.
In certain embodiments, a nucleic acid molecule or portion thereof native to a
host cell will be considered heterologous to the host cell if it has been
altered or
mutated, or a nucleic acid molecule native to a host cell may be considered
heterologous if it has been altered with a heterologous expression control
sequence or
has been altered with an endogenous expression control sequence not normally
associated with the nucleic acid molecule native to a host cell. In addition,
the term
"heterologous" can refer to a biological activity that is different, altered,
or not
endogenous to a host cell As described herein, more than one heterologous
nucleic
acid molecule can be introduced into a host cell as separate nucleic acid
molecules, as a
plurality of individually controlled genes, as a polycistronic nucleic acid
molecule, as a
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single nucleic acid molecule encoding a fusion protein, or any combination
thereof.
When
As used herein, the term "endogenous" or "native" refers to a polynucleotide,
gene, protein, compound, molecule, or activity that is normally present in a
host cell or
a subject.
The term "expression", as used herein, refers to the process by which a
polypeptide is produced based on the encoding sequence of a nucleic acid
molecule,
such as a gene. The process may include transcription, post-transcriptional
control,
post-transcriptional modification, translation, post-translational control,
post-
translational modification, or any combination thereof An expressed nucleic
acid
molecule is typically operably linked to an expression control sequence (e.g.,
a
promoter).
The term "operably linked" refers to the association of two or more nucleic
acid
molecules on a single nucleic acid fragment so that the function of one is
affected by
the other. For example, a promoter is operably linked with a coding sequence
when it is
capable of affecting the expression of that coding sequence (i.e., the coding
sequence is
under the transcriptional control of the promoter). "Unlinked" means that the
associated
genetic elements are not closely associated with one another and the function
of one
does not affect the other.
As described herein, more than one heterologous nucleic acid molecule can be
introduced into a host cell as separate nucleic acid molecules, as a plurality
of
individually controlled genes, as a polycistronic nucleic acid molecule, as a
single
nucleic acid molecule encoding a protein (e.g., a heavy chain of an antibody),
or any
combination thereof. When two or more heterologous nucleic acid molecules are
introduced into a host cell, it is understood that the two or more
heterologous nucleic
acid molecules can be introduced as a single nucleic acid molecule (e.g., on a
single
vector), on separate vectors, integrated into the host chromosome at a single
site or
multiple sites, or any combination thereof The number of referenced
heterologous
nucleic acid molecules or protein activities refers to the number of encoding
nucleic
acid molecules or the number of protein activities, not the number of separate
nucleic
acid molecules introduced into a host cell.
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The term "construct" refers to any polynucleotide that contains a recombinant
nucleic acid molecule (or, when the context clearly indicates, a fusion
protein of the
present disclosure). A (polynucleotide) construct may be present in a vector
(e.g., a
bacterial vector, a viral vector) or may be integrated into a genome. A
"vector" is a
nucleic acid molecule that is capable of transporting another nucleic acid
molecule.
Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a
linear or
circular DNA or RNA molecule that may include chromosomal, non-chromosomal,
semi-synthetic or synthetic nucleic acid molecules. Vectors of the present
disclosure
also include transposon systems (e.g., Sleeping Beauty, see, e.g., Geurts et
al., Mol.
Ther. 8:108, 2003: Mates et al, Nat. Genet. 41:753, 2009). Exemplary vectors
are
those capable of autonomous replication (episomal vector), capable of
delivering a
polynucleotide to a cell genome (e.g., viral vector), or capable of expressing
nucleic
acid molecules to which they are linked (expression vectors).
As used herein, "expression vector" or "vector" refers to a DNA construct
containing a nucleic acid molecule that is operably linked to a suitable
control sequence
capable of effecting the expression of the nucleic acid molecule in a suitable
host. Such
control sequences include a promoter to effect transcription, an optional
operator
sequence to control such transcription, a sequence encoding suitable mRNA
ribosome
binding sites, and sequences which control termination of transcription and
translation.
The vector may be a plasmid, a phage particle, a virus, or simply a potential
genomic
insert. Once transformed into a suitable host, the vector may replicate and
function
independently of the host genome, or may, in some instances, integrate into
the genome
itself or deliver the polynucleotide contained in the vector into the genome
without the
vector sequence. In the present specification, "plasmid," "expression
plasmid," "virus,"
and "vector" are often used interchangeably.
The term "introduced" in the context of inserting a nucleic acid molecule into
a
cell, means "transfection", "transformation," or "transduction" and includes
reference to
the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic
cell
wherein the nucleic acid molecule may be incorporated into the genome of a
cell (e.g.,
chromosome, plasmid, plastid, or mitochondrial DNA), converted into an
autonomous
replicon, or transiently expressed (e.g., transfected mRNA).
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In certain embodiments, polynucleotides of the present disclosure may be
operatively linked to certain elements of a vector. For example,
polynucleotide
sequences that are needed to effect the expression and processing of coding
sequences
to which they are ligated may be operatively linked. Expression control
sequences may
include appropriate transcription initiation, termination, promoter, and
enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation
signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance
translation
efficiency (i.e., Kozak consensus sequences); sequences that enhance protein
stability;
and possibly sequences that enhance protein secretion. Expression control
sequences
may be operatively linked if they are contiguous with the gene of interest and
expression control sequences that act in trans or at a distance to control the
gene of
interest.
In certain embodiments, the vector comprises a plasmid vector or a viral
vector
(e.g., a lentiviral vector or a y-retroviral vector). Viral vectors include
retrovirus,
1.5 adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus,
negative strand
RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus
(e.g., rabies
and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai),
positive
strand RNA viruses such as picornavirus and alphavirus, and double-stranded
DNA
viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1
and 2,
Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccini a, fowlpox,
and
canarypox). Other viruses include, for example, Norwalk virus, togavirus,
flavivirus,
reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of
retroviruses
include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type
viruses,
HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The
viruses and
their replication, In Fundamental Virology, Third Edition, B. N. Fields et
al., Eds.,
Lippincott-Raven Publishers, Philadelphia, 1996).
"Retroviruses" are viruses having an RNA genome, which is reverse-transcribed
into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is
then
incorporated into the host cell genome. "Gammaretrovirus" refers to a genus of
the
retroviridae family. Examples of gammaretroviruses include mouse stem cell
virus,
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murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian
reticuloendotheliosis viruses.
"Lentiviral vectors" include HIV-based lentiviral vectors for gene delivery,
which can be integrative or non-integrative, have relatively large packaging
capacity,
and can transduce a range of different cell types. Lentiviral vectors are
usually
generated following transient transfection of three (packaging, envelope, and
transfer)
or more plasmids into producer cells. Like HIV, lentiviral vectors enter the
target cell
through the interaction of viral surface glycoproteins with receptors on the
cell surface.
On entry, the viral RNA undergoes reverse transcription, which is mediated by
the viral
reverse transcriptase complex. The product of reverse transcription is a
double-stranded
linear viral DNA, which is the substrate for viral integration into the DNA of
infected
cells.
In certain embodiments, the viral vector can be a gammaretrovirus, e.g.,
Moloney murine leukemia virus (MLV)-derived vectors. In other embodiments, the
viral vector can be a more complex retrovirus-derived vector, e.g., a
lentivirus-derived
vector. HIV-1-derived vectors belong to this category. Other examples include
lentivirus vectors derived from HIV-2, Fly, equine infectious anemia virus,
Sly, and
Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and
lentiviral viral
vectors and packaging cells for transducing mammalian host cells with viral
particles
containing transgenes are known in the art and have been previous described,
for
example, in: U.S. Patent 8,119,772; Walchli et at., PLoS One 6:327930, 2011;
Zhao et
at., J. Immunol. /74:4415, 2005; Engels et at., Hum. Gene Ther. 14:1155, 2003;
Frecha
et at., Mot. Ther. 18:1748, 2010; and Verhoeyen et at., Methods Mot. Biol.
506:97,
2009. Retroviral and lentiviral vector constructs and expression systems are
also
commercially available. Other viral vectors also can be used for
polynucleotide delivery
including DNA viral vectors, including, for example adenovirus-based vectors
and
adeno-associated virus (AAV)-based vectors; vectors derived from herpes
simplex
viruses (HSVs), including amplicon vectors, replication-defective HSV and
attenuated
HSV (Krisky et at., Gene Ther. 5:1517 , 1998).
Other vectors that can be used with the compositions and methods of this
disclosure include those derived from baculoviruses and a-viruses. (Jolly, D
J. 1999.
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Emerging Viral Vectors. pp 209-40 in Friedmann T. ed. The Development of Human
Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as
sleeping beauty or other transposon vectors).
When a viral vector genome comprises a plurality of polynueleotides to be
expressed in a host cell as separate transcripts, the viral vector may also
comprise
additional sequences between the two (or more) transcripts allowing for
bicistronic or
multicistronic expression. Examples of such sequences used in viral vectors
include
internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide,
or any
combination thereof.
Plasmid vectors, including DNA-based antibody or antigen-binding fragment-
encoding plasmid vectors for direct administration to a subject, are described
further
herein.
As used herein, the term "host" refers to a cell or microorganism targeted for
genetic modification with a heterologous nucleic acid molecule to produce a
polypeptide of interest (e.g., an antibody of the present disclosure).
A host cell may include any individual cell or cell culture which may receive
a
vector or the incorporation of nucleic acids or express proteins. The term
also
encompasses progeny of the host cell, whether genetically or phenotypically
the same
or different. Suitable host cells may depend on the vector and may include
mammalian
cells, animal cells, human cells, simian cells, insect cells, yeast cells, and
bacterial cells.
These cells may be induced to incorporate the vector or other material by use
of a viral
vector, transformation via calcium phosphate precipitation, DEAE-dextran,
el ectroporati on, mi croinj ecti on, or other methods. See, for example,
Sambrook etal.,
Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory,
1989).
In the context of a SARS-CoV-2 infection, a "host" refers to a cell or a
subject
infected with the SARS-CoV-2 coronavirus.
"Antigen" or "Ag", as used herein, refers to an immunogenic molecule that
provokes an immune response. This immune response may involve antibody
production, activation of specific immunologically-competent cells, activation
of
complement, antibody dependent cytotoxicicity, or any combination thereof An
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antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide,
polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the
like. It is
readily apparent that an antigen can be synthesized, produced recombinantly,
or derived
from a biological sample. Exemplary biological samples that can contain one or
more
antigens include tissue samples, stool samples, cells, biological fluids, or
combinations
thereof. Antigens can be produced by cells that have been modified or
genetically
engineered to express an antigen. Antigens can also be present in a SARS-CoV-2
coronavirus (e.g., a surface glycoprotein or portion thereof), such as present
in a virion,
or expressed or presented on the surface of a cell infected by SARS-CoV-2.
The term "epitope" or "antigenic epitope" includes any molecule, structure,
amino acid sequence, or protein determinant that is recognized and
specifically bound
by a cognate binding molecule, such as an immunoglobulin, or other binding
molecule,
domain, or protein. Epitopic determinants generally contain chemically active
surface
groupings of molecules, such as amino acids or sugar side chains, and can have
specific
three-dimensional structural characteristics, as well as specific charge
characteristics.
Where an antigen is or comprises a peptide or protein, the epitope can be
comprised of
consecutive amino acids (e.g., a linear epitope), or can be comprised of amino
acids
from different parts or regions of the protein that are brought into proximity
by protein
folding (e.g., a discontinuous or conformational epitope), or non-contiguous
amino
acids that are in close proximity irrespective of protein folding.
Antibodies, Antigen-Binding Fragments, and Compositions
In one aspect, the present disclosure provides an isolated antibody, or an
antigen-binding fragment thereof, that comprises a heavy chain variable domain
(VH)
comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain
(VL) comprising a CDRL1, a CDRL2, and a CDRL3, and is capable of binding to a
surface glycoprotein of SARS-CoV-2, in particular in an epitope that is at
least partially
comprised in or defined by Domain A. In certain embodiments, the antibody or
antigen-binding fragment is capable of binding to a surface glycoprotein of
SARS-
CoV-2 expressed on a cell surface of a host cell and/or on a SARS-CoV-2 virion
In certain embodiments, an antibody or antigen-binding fragment of the present
disclosure associates with or unites with a SARS-CoV-2 surface glycoprotein
Domain
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A epitope or antigen comprising the epitope, while not significantly
associating or
uniting with any other molecules or components in a sample.
In certain embodiments, an antibody or antigen binding fragment of the present
disclosure is cross-reactive for SARS-CoV-2 and one or more additional
sarbecovirus
of clade 2, but not of clade 1 or clade 3. In certain embodiments, an antibody
or antigen
binding fragment of the present disclosure is not cross-reactive against an
embecovirus,
a merbecovirus, or both.
In certain embodiments, an antibody or antigen-binding fragment of the present
disclosure specifically binds to a SARS-CoV-2 surface glycoprotein. As used
herein,
"specifically binds" refers to an association or union of an antibody or
antigen-binding
fragment to an antigen with an affinity or Ka (i.e., an equilibrium
association constant of
a particular binding interaction with units of 1/M) equal to or greater than
1051\44
(which equals the ratio of the on-rate [Km] to the off rate [Karr] for this
association
reaction), while not significantly associating or uniting with any other
molecules or
components in a sample. Alternatively, affinity may be defined as an
equilibrium
dissociation constant (Ka) of a particular binding interaction with units of M
(e.g., 10-5
M to 10-13 M). Antibodies may be classified as "high-affinity" antibodies or
as "low-
affinity" antibodies. "High-affinity" antibodies refer to those antibodies
having a Ka of
at least 107M4, at least 1081\41, at least 109 M-1, at least 1010 M-1, at
least 1011 M4, at
least 1012M4, or at least 10'3 M4 "Low-affinity" antibodies refer to those
antibodies
having a Ka of up to 107M4, up to 106 M-", up to 105 M1. Alternatively,
affinity may
be defined as an equilibrium dissociation constant (Ka) of a particular
binding
interaction with units of M (e.g., le M to I 013 M)
In some contexts, antibody and antigen-binding fragments may be described
with reference to affinity and/or to avidity for antigen. Unless otherwise
indicated,
avidity refers to the total binding strength of an antibody or antigen-binding
fragment
thereof to antigen, and reflects binding affinity, valency of the antibody or
antigen-
binding fragment (e.g., whether the antibody or antigen-binding fragment
comprises
one, two, three, four, five, six, seven, eight, nine, ten, or more binding
sites), and, for
example, whether another agent is present that can affect the binding (e.g., a
non-
competitive inhibitor of the antibody or antigen-binding fragment).
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A variety of assays are known for identifying antibodies of the present
disclosure that bind a particular target, as well as determining binding
domain or
binding protein affinities, such as Western blot, ELISA (e.g., direct,
indirect, or
sandwich), analytical ultracentrifugation, spectroscopy, and surface plasmon
resonance
(Biacoreg) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660,
1949;
Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and
U.S.
Patent Nos. 5,283,173, 5,468,614, or the equivalent). Assays for assessing
affinity or
apparent affinity or relative affinity are also known.
In certain examples, binding can be determined by recombinantly expressing a
SARS-CoV-2 antigen in a host cell (e.g., by transfection) and immunostaining
the (e.g.,
fixed, or fixed and permeabilized) host cell with antibody and analyzing
binding by
flow cytometry (e.g., using a ZE5 Cell Analyzer (BioRadg) and FlowJo software
(TreeStar). In some embodiments, positive binding can be defined by
differential
staining by antibody of SARS-CoV-2 -expressing cells versus control (e.g.,
mock) cells.
In some embodiments an antibody or antigen-binding fragment of the present
disclosure binds to SARS-CoV-2 S protein, as measured using biolayer
interferometry.
In certain embodiments, an antibody or antigen-binding fragment of the present
disclosure binds to SARS-CoV-2 S protein with a KD of less than about 4.5x109
M, less
than about 5x109 M, less than about 1x104 M, less than about 5x104 M, less
than
about 1x10-11 M, less than about 5x10-11 M, less than about 1x10'2 M, or less
than
about 5x1012 M.
Certain characteristics of presently disclosed antibodies or antigen-binding
fragments may be described using IC50 or EC50 values. In certain embodiments,
the
IC50 is the concentration of a composition (e.g., antibody) that results in
half-maximal
inhibition of the indicated biological or biochemical function, activity, or
response. In
certain embodiments, the EC50 is the concentration of a composition that
provides the
half-maximal response in the assay. In some embodiments, e.g., for describing
the
ability of a presently disclosed antibody or antigen-binding fragment to
neutralize
infection by SARS-CoV-2, IC50 and EC50 are used interchangeably.
In certain embodiments, an antibody of the present disclosure is capable of
neutralizing infection by SARS-CoV-2. As used herein, a "neutralizing
antibody" is
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one that can neutralize, i.e., prevent, inhibit, reduce, impede, or interfere
with, the
ability of a pathogen to initiate and/or perpetuate an infection in a host.
Neutralization
may be quantified by, for example, assessing SARS-CoV-2 RNA levels in a(n e.g.
lung) sample, assessing SARS-CoV-2 viral load in a(n e.g. lung) sample,
assessing
histopathology of a(n e.g. lung) sample, or the like. The terms "neutralizing
antibody"
and "an antibody that neutralizes" or "antibodies that neutralize" are used
interchangeably herein. In any of the presently disclosed embodiments, the
antibody or
antigen-binding fragment is capable of preventing and/or neutralizing a SARS-
CoV-2
infection in an in vitro model of infection and/or in an in vivo animal model
of infection
(e.g., using a Syrian hamster model with intranasal delivery of SARS-CoV-2)
and/or in
a human.
In certain embodiments, the antibody or antigen-binding fragment (i)
recognizes
an epitope in the Domain A of SARS-CoV-2; (ii) is capable of neutralizing a
SARS
CoV-2 infection; (iii) is capable of eliciting at least one immune effector
function
against SARS CoV-2; (iv) is capable of preventing shedding, from a cell
infected with
SARS CoV-2, of Si protein; or (v) any combination of (i)-(iv).
Terms understood by those in the art of antibody technology are each given the
meaning acquired in the art, unless expressly defined differently herein. For
example,
the term "antibody" refers to an intact antibody comprising at least two heavy
(H)
chains and two light (L) chains inter-connected by disulfide bonds, as well as
any
antigen-binding portion or fragment of an intact antibody that has or retains
the ability
to bind to the antigen target molecule recognized by the intact antibody, such
as an
scFv, Fab, or Fab'2 fragment. Thus, the term "antibody" herein is used in the
broadest
sense and includes polyclonal and monoclonal antibodies, including intact
antibodies
and functional (antigen-binding) antibody fragments thereof, including
fragment
antigen binding (Fab) fragments, F(abl)2 fragments, Fab fragments, Fv
fragments,
recombinant IgG (rIgG) fragments, single chain antibody fragments, including
single
chain variable fragments (scFv), and single domain antibodies (e.g., sdAb,
sdFv,
nanobody) fragments. The term encompasses genetically engineered and/or
otherwise
modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric
antibodies, fully human antibodies, humanized antibodies, and heteroconjugate
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antibodies, multispecific, e.g., bispecific antibodies, diabodies, triabodies,
tetrabodies,
tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term
"antibody"
should be understood to encompass functional antibody fragments thereof. The
term
also encompasses intact or full-length antibodies, including antibodies of any
class or
sub-class, including IgG and sub-classes thereof (IgGl, IgG2, IgG3, IgG4),
IgM, IgE,
IgA, and IgD.
The terms "VL" or "VL" and "VH" or "VH" refer to the variable binding region
from an antibody light chain and an antibody heavy chain, respectively. In
certain
embodiments, a VL is a kappa (lc) class (also "VK" herein). In certain
embodiments, a
VL is a lambda (X) class. The variable binding regions comprise discrete, well-
defined
sub-regions known as "complementarity determining regions" (CDRs) and
"framework
regions" (FRs). The terms "complementarity determining region," and "CDR," are
synonymous with "hypervariable region" or "HVR," and refer to sequences of
amino
acids within antibody variable regions, which, in general, together confer the
antigen
specificity and/or binding affinity of the antibody, wherein consecutive CDRs
(i.e.,
CDR1 and CDR2, CDR2 and CDR3) are separated from one another in primary
structure by a framework region. There are three CDRs in each variable region
(HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also referred to as CDRHs and
CDRLs, respectively). In certain embodiments, an antibody VH comprises four
FRs
and three CDRs as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and an
antibody VL comprises four FRs and three CDRs as follows: FR1-LCDR1-FR2-
LCDR2-FR3-LCDR3-FR4. In general, the VH and the VL together form the antigen-
binding site through their respective CDRs.
As used herein, a "variant" of a CDR refers to a functional variant of a CDR
sequence having up to 1-3 amino acid substitutions (e.g., conservative or non-
conservative substitutions), deletions, or combinations thereof.
Numbering of CDR and framework regions may be according to any known
method or scheme, such as the Kabat, Chothia, EU, IMGT, and AHo numbering
schemes (see, e.g., Kabat et al., " Sequences of Proteins of Immunological
Interest, US
Dept. Health and Human Services, Public Health Service National Institutes of
Health,
1991, 5th ed.; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); Lefranc et
al., Dev.
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Comp. Immunol. 27:55, 2003; Honegger and Pltickthun, J. Mol. Bio. 309:657-670
(2001)). Equivalent residue positions can be annotated and for different
molecules to
be compared using Antigen receptor Numbering And Receptor Classification
(ANARCI) software tool (2016, Bioinformatics 15:298-300). Accordingly,
identification of CDRs of an exemplary variable domain (VH or VL) sequence as
provided herein according to one numbering scheme is not exclusive of an
antibody
comprising CDRs of the same variable domain as determined using a different
numbering scheme. In certain embodiments, an antibody or antigen-binding
fragment
is provided that comprises CDRs in a VH sequence according to any one of SEQ
ID
NOs.: 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142,152, 162, 172,
182 192,
202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342,
352, 362,
372, 382, 392, 402, 412, 422, and 432, and in a VL sequence according to any
one of
SEQ ID NOs.: 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156,
166, 176,
186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326,
336, 346,
356, 366, 376, 386, 396, 406, 416, 426, and 436, as determined using any known
CDR
numbering method, including the Kabat, Chothia, EU, IMGT, Martin (Enhanced
Chothia), Contact, and AHo numbering methods. In certain embodiments, CDRs are
according to the IMGT numbering method. In certain embodiments, CDRs are
according to the antibody numbering method developed by the Chemical Computing
Group (CCG); e.g., using Molecular Operating Environment (MOE) software
(www.chemcomp.com).
In certain embodiments, an antibody or an antigen-binding fragment is provided
that comprises a heavy chain variable domain (VH) comprising a CDRH I , a
CDRH2,
and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a
CDRL2,
and a CDRL3, wherein: (i) the CDRH1 comprises or consists of the amino acid
sequence according to any one of SEQ ID NOs.: 23, 33, 43, 53, 63, 73, 83, 93,
103,
113, 123, 133, 143, 153, 163, 173, 183, 193, 203, 213, 223, 233, 243, 253,
263, 273,
283, 293, 303, 313, 323, 333, 343, 353, 363, 373, 383, 393, 403, 413, 423, or
433, or a
sequence variant thereof comprising one, two, or three acid substitutions, one
or more
of which substitutions is optionally a conservative substitution and/or is a
substitution to
a germline-encoded amino acid; (ii) the CDRH2 comprises or consists of the
amino
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acid sequence according to any one of SEQ ID NOs.: 24, 34, 44, 54, 64, 74, 84,
94, 104,
114, 124, 134, 144, 154, 164, 174, 184, 194, 204, 214, 224, 234, 244, 254,
264, 274,
284, 294, 304, 314, 324, 334, 344, 354, 364, 374, 384, 394, 404, 414, 424, or
434, or a
sequence variant thereof comprising one, two, or three amino acid
substitutions, one or
more of which substitutions is optionally a conservative substitution and/or
is a
substitution to a germline-encoded amino acid; (iii) the CDRH3 comprises or
consists
of the amino acid sequence according to any one of SEQ ID NOs.. 25, 35, 45,
55, 65,
75, 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225,
235, 245,
255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395,
405, 415,
425, or 435, or a sequence variant thereof comprising one, two, or three amino
acid
substitutions, one or more of which substitutions is optionally a conservative
substitution and/or is a substitution to a germline-encoded amino acid, (iv)
the CDRL1
comprises or consists of the amino acid sequence according to any one of SEQ
ID
NOs.: 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177,
187, 197,
207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347,
357, 367,
377, 387, 397, 407, 417, 427, or 437, or a sequence variant thereof comprising
one, two,
or three amino acid substitutions, one or more of which substitutions is
optionally a
conservative substitution and/or is a substitution to a germline-encoded amino
acid; (v)
the CDRL2 comprises or consists of the amino acid sequence according to any
one of
SEQ ID NOs 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 148, 158, 168,
178,
188, 198, 208, 218, 228, 238, 248, 258, 268, 278, 288, 298, 308, 318, 328,
338, 348,
358, 368, 378, 388, 398, 408, 418, 428, or 438, or a sequence variant thereof
comprising one, two, or three amino acid substitutions, one or more of which
substitutions is optionally a conservative substitution and/or is a
substitution to a
germline-encoded amino acid, and/or (vi) the CDRL3 comprises or consists of
the
amino acid sequence according to any one of SEQ ID NOs.: 29, 39, 49, 59, 69,
79, 89,
99, 109, 119, 129, 139, 149, 159, 169, 179, 189, 199, 209, 219, 229, 239, 249,
259, 269,
279, 289, 299, 309, 319, 329, 339, 349, 359, 369, 379, 389, 399, 409, 419,
429, or 439,
or a sequence variant thereof comprising having one, two, or three amino acid
substitutions, one or more of which substitutions is optionally a conservative
substitution and/or is a substitution to a germline-encoded amino acid,
wherein the
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antibody or antigen binding fragment is capable of binding to a surface
glycoprotein of
SARS-CoV-2. In some embodiments, the SARS-CoV-2 surface glycoprotein is
expressed on a cell surface of a host cell and/or is present in a virion. In
certain
embodiments, the CDRs are according to the IMGT numbering method.
In any of the presently disclosed embodiments, the antibody or antigen-binding
fragment is capable of preventing and/or neutralizing a SARS-CoV-2 infection
in an in
vitro model of infection and/or in an in vivo animal model of infection and/or
in a
human.
In any of the presently disclosed embodiments, the antibody or antigen-binding
fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino
acid sequences according to SEQ ID NOs.: (i) 23-25 and 27-29, respectively;
(ii) 33-35
and 37-39, respectively; (iii) 43-45 and 47-49, respectively; (iv) 53-55 and
57-59,
respectively; (v) 63-65 and 67-69, respectively; (vi) 73-75 and 77-79,
respectively; (vii)
83-85 and 87-89, respectively; (viii) 93-95 and 97-99, respectively; (ix) 103-
105 and
107-109, respectively; (x) 113-115 and 117-119, respectively; (xi) 123-125 and
127-
129, respectively; (xii) 133-135 and 137-139, respectively, (xiii) 143-145 and
147-149,
respectively, (xiv) 153-155 and 157-159, respectively, (xv) 163-165 and 167-
169,
respectively; (xvi) 173-175 and 177-179, respectively; (xvii) 183-185 and 187-
189,
respectively; (xviii) 193-195 and 197-199, respectively; (xix) 203-205 and 207-
209,
respectively; (xx) 213-215 and 217-219, respectively; (xxi) 223-225 and 227-
229,
respectively; (xxii) 233-235 and 237-239, respectively; (xxiii) 243-245 and
247-249,
respectively; (xxiv) 253-255 and 257-259, respectively; (xxv) 263-265 and 267-
269,
respectively; (xxvi) 273-275 and 277-279, respectively; (xxvii) 283-285 and
287-289,
respectively; (xxviii) 293-295 and 297-299, respectively; (xxix) 303-305 and
307-309,
respectively; (xxx) 313-315 and 317-319, respectively; (xxxi) 323-325 and 327-
329,
respectively; (xxxii) 333-335 and 337-339, respectively; (xxxiii) 343-345 and
347-349,
respectively; (xxxiv) 353-355 and 357-359, respectively; (xxxv) 363-365 and
367-369,
respectively; (xxxvi) 373-375 and 377-379, respectively; (xxxvii) 383-385 and
387-
389, respectively; (xxxviii) 393-395 and 397-399, respectively; (xxxix) 403-
405 and
407-409, respectively; (xxxx) 413-415 and 417-419, respectively; (xxxxi) 423-
425 and
427-429, respectively; or (xxxxii) 433-435 and 437-439, respectively.
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In some embodiments, an antibody or antigen-binding fragment is provided that
comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid
sequences as set forth in SEQ ID NOs.:163-165 and 167-169, respectively. In
certain
embodiments, the antibody or antigen-binding fragment comprises VH and VL
amino
acid sequences as set forth in SEQ ID NOs.:162 and 166, respectively.
In some embodiments, an antibody or antigen-binding fragment is provided that
comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid
sequences as set forth in SEQ ID NOs.:103-105 and 107-109, respectively. In
certain
embodiments, the antibody or antigen-binding fragment comprises VH and VL
amino
acid sequences as set forth in SEQ ID NOs.:102 and 106, respectively.
In some embodiments, an antibody or antigen-binding fragment is provided that
comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid
sequences as set forth in SEQ ID NOs.:73-75 and 77-79, respectively. In
certain
embodiments, the antibody or antigen-binding fragment comprises VH and VL
amino
acid sequences as set forth in SEQ ID NOs.:72 and 76, respectively.
In some embodiments, an antibody or antigen-binding fragment is provided that
comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid
sequences as set forth in SEQ ID NOs.:63-65 and 67-69, respectively. In
certain
embodiments, the antibody or antigen-binding fragment comprises VH and VL
amino
acid sequences as set forth in SEQ ID NOs.:62 and 66, respectively.
In some embodiments, an antibody or antigen-binding fragment is provided that
comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid
sequences as set forth in SEQ ID NOs.:23-25 and 27-29, respectively. In
certain
embodiments, the antibody or antigen-binding fragment comprises VH and VL
amino
acid sequences as set forth in SEQ ID NOs.:22 and 26, respectively.
In some embodiments, an antibody or antigen-binding fragment is provided that
comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid
sequences as set forth in SEQ ID NOs .33-35 and 37-39, respectively In certain
embodiments, the antibody or antigen-binding fragment comprises VH and VL
amino
acid sequences as set forth in SEQ ID NOs.:32 and 36, respectively.
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In some embodiments, an antibody or antigen-binding fragment is provided that
comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid
sequences as set forth in SEQ ID NOs.:53-55 and 57-59, respectively. In
certain
embodiments, the antibody or antigen-binding fragment comprises VH and VL
amino
acid sequences as set forth in SEQ ID NOs.:52 and 56, respectively.
In some embodiments, an antibody or antigen-binding fragment is provided that
comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid
sequences as set forth in SEQ ID NOs.:363-365 and 367-369, respectively. In
certain
embodiments, the antibody or antigen-binding fragment comprises VH and VL
amino
acid sequences as set forth in SEQ ID NOs.:362 and 366, respectively.
In certain embodiments, an antibody or an antigen-binding fragment of the
present disclosure comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and
a CDRL3, wherein each CDR is independently selected from a corresponding CDR
of
Antibody 418_i, Antibody 4182, Antibody 4183, Antibody 4184, Antibody 4185,
Antibody 4186, Antibody 4187, Antibody 4188, Antibody 418_9, Antibody
41810, Antibody 418 11, Antibody 41812, Antibody 41813, Antibody 41814,
Antibody 41815, Antibody 41816, Antibody 41817, Antibody 41818, Antibody
41819, Antibody 41820, Antibody 41821, Antibody 41822, Antibody 41823,
Antibody 41824, Antibody 41825, Antibody 41826, Antibody 41827, Antibody
41828, Antibody 418_29, Antibody 418_30, Antibody 41831, Antibody 418_33,
Antibody 41834, Antibody 41835, Antibody 41837, Antibody 41838, Antibody
41839, Antibody 41840, Antibody 41841, Antibody 41842, Antibody 41843, or
Antibody 4 18 44, as provided in Table I . That is, all combinations of CDRs
from
SARS-CoV-2 mAbs and the variant sequences thereof provided in Table 1 are
contemplated.
Antibody 418 1 is also referred to herein as S2X28. Antibody 418 2 is also
referred to herein as S2X303. Antibody 418 3 is also referred to herein as
S2X320.
Antibody 418_4 is also referred to herein as S2X333 Antibody 418_5 is also
referred
to herein as S2M28. Antibody 418 6 is also referred to herein as S2M24 or
S2M24v2.
Antibody 418 7 is also referred to herein as S2L7. Antibody 4 [8 8 is also
referred to
herein as S2L24. Antibody 418 9 is also referred to herein as S2L28. Antibody
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418 10 is also referred to herein as S2X310. Antibody 418 11 is also referred
to herein
as S2X94. Antibody 418_12 is also referred to herein as S2X169. Antibody 418
13 is
also referred to herein as S2L11. Antibody 418_14 is also referred to herein
as S2L12.
Antibody 418 15 is also referred to herein as S2X186. Antibody 418 16 is also
referred to herein as S2X175. Antibody 418 17 is also referred to herein as
S2X170.
Antibody 418 18 is also referred to herein as S2X125. Antibody 418_19 is also
referred to herein as S2X107. Antibody 418 20 is also referred to herein as
S2X105.
Antibody 418 21 is also referred to herein as S2X102. Antibody 418_22 is also
referred to herein as S2X15. Antibody 418 23 is also referred to herein as
S2X49.
Antibody 418 24 is also referred to herein as S2X51. Antibody 418 25 is also
referred
to herein as S2X72. Antibody 418 26 is also referred to herein as S2X91.
Antibody
418 27 is also referred to herein as S2X98. Antibody 418 28 is also referred
to herein
as S2X124. Antibody 418 29 is also referred to herein as S2X158. Antibody 418
30
is also referred to herein as S2X161. Antibody 418_31 is also referred to
herein as
S2X165. Antibody 418_33 is also referred to herein as S2X173. Antibody 418_34
is
also referred to herein as S2X176. Antibody 418 35 is also referred to herein
as
S2X316. Antibody 418 37 is also referred to herein as S2X90. Antibody 418 38
is
also referred to herein as S2X93. Antibody 418_39 is also referred to herein
as S2L14.
Antibody 418 40 is also referred to herein as S2L20 or S2L20v1. Antibody
418_41 is
also referred to herein as S2L26. Antibody 418_42 is also referred to herein
as S2L35.
Antibody 418 43 is also referred to herein as S2L38. Antibody 418 44 is also
referred
to herein as S2L50.
The term "CL" refers to an "immunoglobulin light chain constant region" or a
"light chain constant region," i.e., a constant region from an antibody light
chain. The
term "CH" refers to an "immunoglobulin heavy chain constant region" or a
"heavy
chain constant region," which is further divisible, depending on the antibody
isotype
into CHL CH2, and CH3 (IgA, IgD, IgG), or CHL CH2, CH3, and CH4 domains (IgE,
IgM) The Fc region of an antibody heavy chain is described further herein In
any of
the presently disclosed embodiments, an antibody or antigen-binding fragment
of the
present disclosure comprises any one or more of CL, a CHI, a CH2, and a CH3.
In
certain embodiments, a CL comprises an amino acid sequence having 90%, 91%,
92%,
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93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence
of
SEQ ID NO. :8 or SEQ ID NO.: 9. In certain embodiments, a CH1-CH2-CH3
comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO. :6 or
SEQ
ID NO.:7.
It will be understood that, for example, production in a mammalian cell line
can
remove one or more C-terminal lysine of an antibody heavy chain (see, e.g.,
Liu et al.
mAbs 6(5):1145-1154 (2014)). Accordingly, an antibody or antigen-binding
fragment
of the present disclosure can comprise a heavy chain, a CH1-CH3, a CH3, or an
Fc
polypeptide wherein a C-terminal lysine residue is present or is absent; in
other words,
encompassed are embodiments where the C-terminal residue of a heavy chain, a
CH1-
CH3, or an Fc polypeptide is not a lysine (e.g., is a glycine), and
embodiments where a
lysine is the C-terminal residue. In certain embodiments, a composition
comprises a
plurality of an antibody and/or an antigen-binding fragment of the present
disclosure,
wherein one or more antibody or antigen-binding fragment does not comprise a
lysine
residue at the C-terminal end of the heavy chain, CH1-CH3, or Fc polypeptide,
and
wherein one or more antibody or antigen-binding fragment comprises a lysine
residue at
the C-terminal end of the heavy chain, CH1-CH3, or Fc polypeptide.
A "Fab" (fragment antigen binding) is the part of an antibody that binds to
antigens and includes the variable region and CH1 of the heavy chain linked to
the light
chain via an inter-chain disulfide bond. Each Fab fragment is monovalent with
respect
to antigen binding, i.e., it has a single antigen-binding site. Pepsin
treatment of an
antibody yields a single large F(ab')2 fragment that roughly corresponds to
two
disulfide linked Fab fragments having divalent antigen-binding activity and is
still
capable of cross-linking antigen. Both the Fab and F(ab')2 are examples of
"antigen-
binding fragments." Fab' fragments differ from Fab fragments by having
additional few
residues at the carboxy terminus of the CH1 domain including one or more
cysteines
from the antibody hinge region Fab'-SH is the designation herein for Fab' in
which the
cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2
antibody
fragments originally were produced as pairs of Fab' fragments that have hinge
cysteines
between them. Other chemical couplings of antibody fragments are also known.
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Fab fragments may be joined, e.g., by a peptide linker, to form a single chain
Fab, also referred to herein as "scFab." In these embodiments, an inter-chain
disulfide
bond that is present in a native Fab may not be present, and the linker serves
in full or in
part to link or connect the Fab fragments in a single polypeptide chain. A
heavy chain-
derived Fab fragment (e.g., comprising, consisting of, or consisting
essentially of VH +
CH1, or "Fd") and a light chain-derived Fab fragment (e.g., comprising,
consisting of,
or consisting essentially of VL + CL) may be linked in any arrangement to form
a
scFab. For example, a scFab may be arranged, in N-terminal to C-terminal
direction,
according to (heavy chain Fab fragment ¨ linker ¨ light chain Fab fragment) or
(light
chain Fab fragment ¨ linker ¨ heavy chain Fab fragment). Peptide linkers and
exemplary linker sequences for use in scFabs are discussed in further detail
herein.
"Fv" is a small antibody fragment that contains a complete antigen-recognition
and antigen-binding site. This fragment generally consists of a dimer of one
heavy- and
one light-chain variable region domain in tight, non-covalent association.
However,
even a single variable domain (or half of an Fv comprising only three CDRs
specific for
an antigen) can have the ability to recognize and bind antigen, although
typically at a
lower affinity than the entire binding site.
"Single-chain Fv" also abbreviated as "sFv" or "scFv", are antibody fragments
that comprise the VH and VL antibody domains connected into a single
polypeptide
chain. In some embodiments, the scFv polypeptide comprises a polypeptide
linker
disposed between and linking the Vu and VL domains that enables the scFv to
retain or
form the desired structure for antigen binding. Such a peptide linker can be
incorporated into a fusion polypeptide using standard techniques well known in
the art.
For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal
Antibodies,
vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994);
Borrebaeck 1995, infra. In certain embodiments, the antibody or antigen-
binding
fragment comprises a scFv comprising a VH domain, a VL domain, and a peptide
linker
linking the VH domain to the VL domain In particular embodiments, a scFv
comprises
a VH domain linked to a VL domain by a peptide linker, which can be in a VH-
linker-
VL orientation or in a VL-linker-VH orientation. Any scFv of the present
disclosure
may be engineered so that the C-terminal end of the VL domain is linked by a
short
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peptide sequence to the N-terminal end of the VH domain, or vice versa (i.e.,
(N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C). Alternatively, in some
embodiments, a linker may be linked to an N-terminal portion or end of the VH
domain, the VL domain, or both.
Peptide linker sequences may be chosen, for example, based on: (1) their
ability
to adopt a flexible extended conformation; (2) their inability or lack of
ability to adopt a
secondary structure that could interact with functional epitopes on the first
and second
polypeptides and/or on a target molecule; and/or (3) the lack or relative lack
of
hydrophobic or charged residues that might react with the polypeptides and/or
target
molecule. Other considerations regarding linker design (e.g., length) can
include the
conformation or range of conformations in which the VH and VL can form a
functional
antigen-binding site. In certain embodiments, peptide linker sequences
contain, for
example, Gly, Asn and Ser residues. Other near neutral amino acids, such as
Thr and
Ala, may also be included in a linker sequence. Other amino acid sequences
which may
be usefully employed as linker include those disclosed in Maratea et al., Gene
40:39 46
(1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S.
Pat. No.
4,935,233, and U.S. Pat. No. 4,751,180. Other illustrative and non-limiting
examples of
linkers may include, for example, Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-
Lys-
Val-Asp (SEQ ID NO: 19) (Chaudhary et al., Proc. Natl. Acad. Sci. USA 87:1066-
1070 (1990)) and Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-
Ser-Leu-Asp (SEQ ID NO: 20) (Bird et al., Science 242:423-426 (1988)) and the
pentamer Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 21) when present in a single
iteration or
repeated Ito 5 or more times, or more; see, e.g., SEQ ID NO: 17. Any suitable
linker
may be used, and in general can be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16,
17, 18, 19, 20, 21, 22, 15 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80,
90, 100
amino acids in length, or less than about 200 amino acids in length, and will
preferably
comprise a flexible structure (can provide flexibility and room for
conformational
movement between two regions, domains, motifs, fragments, or modules connected
by
the linker), and will preferably be biologically inert and/or have a low risk
of
immunogenicity in a human. Exemplary linkers include those comprising or
consisting
of the amino acid sequence set forth in any one or more of SEQ ID NOs: 10-21.
In
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certain embodiments, the linker comprises or consists of an amino acid
sequence
having at least 75% (i.e., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%,
95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set
forth in
any one of SEQ ID NOs: 10-21.
scFvs can be constructed using any combination of the VH and VL sequences or
any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3
sequences disclosed herein.
In some embodiments, linker sequences are not required; for example, when the
first and second polypeptides have non-essential N-terminal amino acid regions
that can
be used to separate the functional domains and prevent steric interference.
During antibody development, DNA in the germline variable (V), joining (J),
and diversity (D) gene loci may be rearranged and insertions and/or deletions
of
nucleotides in the coding sequence may occur. Somatic mutations may be encoded
by
the resultant sequence, and can be identified by reference to a corresponding
known
germline sequence. In some contexts, somatic mutations that are not critical
to a
desired property of the antibody (e.g., binding to a SARS-CoV-2 antigen), or
that
confer an undesirable property upon the antibody (e.g., an increased risk of
immunogenicity in a subject administered the antibody), or both, may be
replaced by
the corresponding germline-encoded amino acid, or by a different amino acid,
so that a
desirable property of the antibody is improved or maintained and the
undesirable
property of the antibody is reduced or abrogated. Thus, in some embodiments,
the
antibody or antigen-binding fragment of the present disclosure comprises at
least one
more germline-encoded amino acid in a variable region as compared to a parent
antibody or antigen-binding fragment, provided that the parent antibody or
antigen
binding fragment comprises one or more somatic mutations. Variable region and
CDR
amino acid sequences of exemplary anti-SARS-CoV-2 antibodies of the present
disclosure are provided in Table 1 herein.
In certain embodiments, an antibody or antigen-binding fragment comprises an
amino acid modification (e.g., a substitution mutation) to remove an undesired
risk of
oxidation, deamidation, and/or isomerization.
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Also provided herein are variant antibodies that comprise one or more amino
acid alterations in a variable region (e.g., VH, VL, framework or CDR) as
compared to
a presently disclosed ("parent") antibody, wherein the variant antibody is
capable of
binding to a SARS-CoV-2 antigen.
In certain embodiments, the VH comprises or consists of an amino acid
sequence having at least 85% (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97,
98, 99, or 100%) identity to the amino acid sequence according to any one of
SEQ ID
NOs.: 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172,
182 192,
202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342,
352, 362,
372, 382, 392, 402, 412, 422, or 432, wherein the variation is optionally
limited to one
or more framework regions and/or the variation comprises one or more
substitution to a
germline-encoded amino acid, and/or (ii) the VL comprises or consists of an
amino acid
sequence having at least 85% (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97,
98, 99, or 100%) identity to the amino acid sequence according to any one of
SEQ ID
NOs.: 26, --------------------------------------------------------------------
36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196,
206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346,
356, 366,
376, 386, 396, 406, 416, 426, or 436, wherein the variation is optionally
limited to one
or more framework regions and/or the variation comprises one or more
substitution to a
germline-encoded amino acid.
In certain embodiments, the VH comprises or consists of any VH amino acid
sequence set forth in Table 1, and the VL comprises or consists of any VL
amino acid
sequence set forth in Table 1. In particular embodiments, the VH and the VL
comprise
amino acid sequences having at least have at least 85% (i.e., 85%, 86, 87, 88,
89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to, or comprise or
consist of, the
amino acid sequences according to SEQ ID NOs.. (i) 22 and 26, respectively,
(ii) 32
and 36, respectively; (iii) 42 and 46, respectively; (iv) 52 and 56,
respectively; (v) 62
and 66, respectively; (vi) 72 and 76, respectively; (vii) 82 and 86,
respectively; (viii) 92
and 96, respectively; (ix) 102 and 106, respectively; (x) 112 and 116,
respectively; (xi)
122 and 126, respectively, (xii) 132 and 136, respectively, (xiii) 142 and
146,
respectively; (xiv) 152 and 156, respectively; (xv) 162 and 166, respectively;
(xvi) 172
and 176, respectively; (xvii) 182 and 186, respectively; (xviii) 192 and 196,
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respectively; (xix) 202 and 206, respectively; (xx) 212 and 216, respectively;
(xxi) 222
and 226, respectively, (xxii) 232 and 236, respectively, (xxiii) 242 and 246,
respectively; (xxiv) 252 and 256, respectively; (xxv) 262 and 266,
respectively; (xxvi)
272 and 276, respectively, (xxvii) 282 and 286, respectively, (xxviii) 292 and
296,
respectively; (xxix) 302 and 306, respectively; (xxx) 312 and 316,
respectively; (xxxi)
322 and 326, respectively; (xxxii) 332 and 336, respectively; (xxxiii) 342 and
346,
respectively, (xxxiv) 352 and 356, respectively, (xxxv) 362 and 366,
respectively,
(xxxvi) 372 and 376, respectively; (xxxvii) 382 and 386, respectively;
(xxxviii) 392 and
396, respectively; (xxxix) 402 and 406, respectively; (xxxx) 412 and 416,
respectively;
(xxxxi) 422 and 426, respectively; or (xxxxii) 432 and 436, respectively.
In certain embodiments, an antibody or antigen-binding fragment of the present
disclosure is monospecific (e.g., binds to a single epitope) or is
multispecific (e.g.,
binds to multiple epitopes and/or target molecules). Antibodies and antigen
binding
fragments may be constructed in various formats. Exemplary antibody formats
are
disclosed in Spiess et al., Mol. Immunol. 67(2):95 (2015), and in Brinkmann
and
Kontermann, mAbs 9(2).182-212 (2017), which formats and methods of making the
same are incorporated herein by reference and include, for example, Bispecific
T cell
Engagers (BiTEs), DARTs, Knobs-Into-Holes (KIH) assemblies, scFv-CH3-KIH
assemblies, KIH Common Light-Chain antibodies, TandAbs, Triple Bodies, TriBi
Minibodies, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs,
Intrabodies, CrossMabs, Dual Action Fabs (DAFs) (two-in-one or four-in-one),
DutaMabs, DT-IgG, Charge Pairs, Fab-arm Exchange, SEEDbodies, Triomabs, LUZ-Y
assemblies, Fcabs, la-bodies, orthogonal Fabs, DVD-Igs (e.g., US Patent No.
8,258,268, which formats are incorporated herein by reference in their
entirety),
IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V,
V(H)-
IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig,
Zybody,
and DVI-IgG (four-in-one), as well as so-called FIT-Ig (e.g., PCT Publication
No. WO
2015/103072, which formats are incorporated herein by reference in their
entirety), so-
called WuxiBody formats (e.g., PCT Publication No. WO 2019/057122, which
formats
are incorporated herein by reference in their entirety), and so-called In-
Elbow-Insert Ig
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formats (IEI-Ig; e.g., PCT Publication Nos. WO 2019/024979 and WO 2019/025391,
which formats are incorporated herein by reference in their entirety).
In certain embodiments, the antibody or antigen-binding fragment comprises
two or more of VH domains, two or more VL domains, or both (i.e., two or more
VH
domains and two or more VL domains). In particular embodiments, an antigen-
binding
fragment comprises the format (N-terminal to C-terminal direction) VH-linker-
VL-
linker-VH-linker-VL, wherein the two VH sequences can be the same or different
and
the two VL sequences can be the same or different. Such linked scFvs can
include any
combination of VH and VL domains arranged to bind to a given target, and in
formats
comprising two or more VH and/or two or more VL, one, two, or more different
eptiopes or antigens may be bound. It will be appreciated that formats
incorporating
multiple antigen-binding domains may include VH and/or VL sequences in any
combination or orientation. For example, the antigen-binding fragment can
comprise
the format VL-linker-VH-linker-VL-linker-VH, VH-linker-VL-linker-VL-linker-VH,
or
VL-linker-VH-linker-VH-linker-VL.
Monospecific or multispecific antibodies or antigen-binding fragments of the
present disclosure constructed comprise any combination of the VH and VL
sequences
and/or any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and
CDRL3 sequences disclosed herein. A bispecific or multispecific antibody or
antigen-
binding fragment may, in some embodiments, comprise one, two, or more antigen-
binding domains (e.g., a VH and a VL) of the instant disclosure. Two or more
binding
domains may be present that bind to the same or a different SARS-CoV-2
epitope, and
a bispecific or multi specific antibody or antigen-binding fragment as
provided herein
can, in some embodiments, comprise a further SARS-CoV-2 binding domain, and/or
can comprise a binding domain that binds to a different antigen or pathogen
altogether.
In any of the presently disclosed embodiments, the antibody or antigen-binding
fragment can be multispecific; e.g., bispecific, trispecific, or the like.
In certain embodiments, the antibody or antigen-binding fragment comprises.
(i)
a first VH and a first VL; and (ii) a second VH and a second VL, wherein the
first VH
and the second VH are different and each independently comprise an amino acid
sequence having at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%,
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94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence set
forth in any one of SEQ ID NOs.: 22, 32, 42, 52, 62, 72, 82, 92, 102, 112,
122, 132,
142, 152, 162, 172, 182 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292,
302,
312, 322, 332, 342, 352, 362, 372, 382, 392, 402, 412, 422, or 432, and
wherein the first
VL and the second VL are different and each independently comprise an amino
acid
sequence having at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence set
forth in any one of SEQ ID NOs.: 26, 36, 46, 56, 66, 76, 86, 96, 106, 116,
126, 136,
146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286,
296, 306,
316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, or 436, and
wherein the first
VH and the first VL together form a first antigen-binding site, and wherein
the second
VH and the second VL together form a second antigen-binding site.
In certain embodiments, the antibody or antigen-binding fragment comprises:
(i)
a first VH and a first VL; and (ii) a second VH and a second VL, wherein the
first VH
comprises an amino acid sequence having at least 85% (i.e., 85%, 86%, 87%,
88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to
the
amino acid sequence set forth in SEQ ID NO: 52 and the first VL comprises an
amino
acid sequence haying at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid
sequence
set forth in SEQ ID NO: 56; and a) the second VH comprises an amino acid
sequence
having at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence set
forth in
SEQ ID NO: 442 and the second VL comprises an amino acid sequence having at
least
85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO:
446;
b) the second VH comprises an amino acid sequence having at least 85% (i.e.,
85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100%) identity to the amino acid sequence set forth in SEQ ID NO: 450 and the
second
VL comprises an amino acid sequence having at least 85% (i.e., 85%, 86%, 87%,
88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to
the
amino acid sequence set forth in SEQ ID NO: 454; or c) the second VH comprises
an
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amino acid sequence having at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid
sequence set forth in SEQ ID NO: 458 and the second VL comprises an amino acid
sequence having at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence set
forth in SEQ ID NO: 462; and wherein the first VH and the first VL together
form a
first antigen-binding site, and wherein the second VH and the second VL
together form
a second antigen-binding site.
In certain embodiments, the antibody or antigen-binding fragment comprises a
Fc polypeptide, or a fragment thereof. The "Fc" fragment or Fc polypeptide
comprises
the carboxy-terminal portions (i.e., the CH2 and CH3 domains of IgG) of both
antibody
H chains held together by disulfides. Antibody "effector functions" refer to
those
biological activities attributable to the Fc region (a native sequence Fc
region or amino
acid sequence variant Fc region) of an antibody, and vary with the antibody
isotype.
Examples of antibody effector functions include: Clq binding and complement
dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors
(e.g., B
cell receptor); and B cell activation. As discussed herein, modifications
(e.g., amino
acid substitutions) may be made to an Fc domain in order to modify (e.g.,
improve,
reduce, or ablate) one or more functionality of an Fc-containing polypeptide
(e.g., an
antibody of the present disclosure). Such functions include, for example, Fc
receptor
(FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC
function, protein A binding, protein G binding, and complement binding. Amino
acid
modifications that modify (e.g., improve, reduce, or ablate) Fc
functionalities include,
for example, the T250Q/M428L, M252Y/S254T/T256E, H433K/N434F,
M428L/N434S, E233P/L234V/L235A/G236 + A327G/A330S/P331S, E333A,
S239D/A330L/I332E, P2571/Q311, K326W/E333S, S239D/I332E/G236A, N297Q,
K322A, S228P, L235E + E318A/K320A/K322A, L234A/L235A (also referred to
herein as "LALA"), and L234A/L235A/P329G mutations, which mutations are
summarized and annotated in "Engineered Fc Regions", published by InvivoGen
(2011)
and available online at invivogen.com/PDF/review/review-Engineered-Fc-Regions-
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invivogen.pdf?utm source=review&utm medium=pdf&utm
campaign=review&utm content=Engineered-Fc-Regions, and are incorporated herein
by reference. Unless the context indicates otherwise, Fc amino acid residues
are
numbered herein according to the EU numbering system.
For example, to activate the complement cascade, the Clq protein complex can
bind to at least two molecules of IgG1 or one molecule of IgM when the
immunoglobulin molecule(s) is attached to the antigenic target (Ward, E. S.,
and
Ghetie, V., Ther. Immunol 2 (1995) 77-94). Burton, D. R., described (Mol.
Immtmol.
22 (1985) 161-206) that the heavy chain region comprising amino acid residues
318 to
337 is involved in complement fixation. Duncan, A. R., and Winter, G. (Nature
332
(1988) 738-740), using site directed mutagenesis, reported that Glu318, Lys320
and
Lys322 form the binding site to Clq. The role of Glu318, Lys320 and Lys 322
residues
in the binding of Clq was confirmed by the ability of a short synthetic
peptide
containing these residues to inhibit complement mediated lysis.
For example, FcR binding can be mediated by the interaction of the Fc moiety
(of an antibody) with Fc receptors (FcRs), which are specialized cell surface
receptors
on cells including hematopoietic cells. Fe receptors belong to the
immunoglobulin
superfamily, and shown to mediate both the removal of antibody-coated
pathogens by
phagocytosis of immune complexes, and the lysis of erythrocytes and various
other
cellular targets (e.g. tumor cells) coated with the corresponding antibody,
via antibody
dependent cell mediated cytotoxicity (ADCC; Van de Winkel, J. G., and
Anderson, C.
L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity
for
immunoglobulin classes; Fc receptors for IgG antibodies are referred to as
FcyR, for
IgE as Fcall, for IgA as FcaR and so on and neonatal Fc receptors are referred
to as
FcRn. Fc receptor binding is described for example in Ravetch, J. V., and
Kinet, J. P.,
Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., et al., Immunomethods 4
(1994)
25-34; de Haas, M., et al., J Lab. Cl/n. Med. 126 (1995) 330-341; and Gessner,
J. E., et
al., Ann. Hematol. 76 (1998) 231-248
Cross-linking of receptors by the Fc domain of native IgG antibodies (FcyR)
triggers a wide variety of effector functions including phagocytosis, antibody-
dependent
cellular cytotoxicity, and release of inflammatory mediators, as well as
immune
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complex clearance and regulation of antibody production. Fc moieties providing
cross-
linking of receptors (e.g., FcyR) are contemplated herein. In humans, three
classes of
FcyR have been characterized to-date, which are: (i) FcyRI (CD64), which binds
monomeric IgG with high affinity and is expressed on macrophages, monocytes,
neutrophils and eosinophils; (ii) FcyRII (CD32), which binds complexed IgG
with
medium to low affinity, is widely expressed, in particular on leukocytes, is
believed to
be a central player in antibody-mediated immunity, and which can be divided
into
FcyRITA, FcyRII,B and FeyRITC, which perform different functions in the immune
system, but bind with similar low affinity to the IgG-Fe, and the ectodomains
of these
receptors are highly homologuous; and (iii) FcyRIII (CD16), which binds IgG
with
medium to low affinity and has been found in two forms: FeyRITIA, which has
been
found on NK cells, macrophages, eosinophils, and some monocytes and T cells,
and is
believed to mediate ADCC; and FcyRIII,B, which is highly expressed on
neutrophils.
FcyRITA is found on many cells involved in killing (e.g. macrophages,
monocytes, neutrophils) and seems able to activate the killing process. FcyRIM
seems
to play a role in inhibitory processes and is found on B-cells, macrophages
and on mast
cells and eosinophils. Importantly, it has been shown that 75% of all FcyRIIB
is found
in the liver (Ganesan, L. P. et al., 2012: "FcyRIIb on liver sinusoidal
endothelium clears
small immune complexes," Journal of Immunology 189: 4981-4988). FeyMIB is
abundantly expressed on Liver Sinusoidal Endothelium, called LSEC, and in
Kupffer
cells in the liver and LSEC are the major site of small immune complexes
clearance
(Ganesan, L. P. et al., 2012: FcyRIIb on liver sinusoidal endothelium clears
small
immune complexes. Journal of Immunology 189: 4981-4988).
In some embodiments, the antibodies disclosed herein and the antigen-binding
fragments thereof comprise an Fc polypeptide or fragment thereof for binding
to
FeyRIIb, in particular an Fc region, such as, for example IgG-type antibodies.
Moreover, it is possible to engineer the Fc moiety to enhance FcyRIM binding
by
introducing the mutations S267E and L328F as described by Chu, S. Y. et al.,
2008:
Inhibition of B cell receptor-mediated activation of primary human B cells by
coengagement of CD 19 and FcgammaRII13 with Fc-engineered antibodies.
Molecular
Immunology 45,3926-3933. Thereby, the clearance of immune complexes can be
46
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enhanced (Chu, S., et al., 2014: Accelerated Clearance of IgE In Chimpanzees
Is
Mediated By Xmab7195, An Fc-Engineered Antibody With Enhanced Affinity For
Inhibitory Receptor FcyRIIb. Am J Respir Crit, American Thoracic Society
International Conference Abstracts). In some embodiments, the antibodies of
the
present disclosure, or the antigen binding fragments thereof, comprise an
engineered Fc
moiety with the mutations S267E and L328F, in particular as described by Chu,
S. Y. et
al., 2008: Inhibition of B cell receptor-mediated activation of primary human
B cells by
coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular
Immunology 45, 3926-3933.
On B cells, FcyRIIB may function to suppress further immunoglobulin
production and isotype switching to, for example, the IgE class. On
macrophages,
FcyRIIB is thought to inhibit phagocytosis as mediated through FcyRIIA. On
eosinophils and mast cells, the B form may help to suppress activation of
these cells
through IgE binding to its separate receptor.
Regarding FcyRI binding, modification in native IgG of at least one of E233-
G236, P238, D265, N297, A327 and P329 reduces binding to FcyRI. IgG2 residues
at
positions 233-236, substituted into corresponding positions IgG1 and IgG4,
reduces
binding of IgG1 and IgG4 to FcyRI by 103-fold and eliminated the human
monocyte
response to antibody-sensitized red blood cells (Armour, K. L., et al. Eur. J.
1111117111101.
29(1999) 2613-2624)
Regarding FcyRII binding, reduced binding for FcyRIIA is found, e.g., for IgG
mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270,
Q295,
A327, R292 and K4I4.
Two allelic forms of human FcyRIIA are the "H131" variant, which binds to
IgG1 Fc with high affinity, and the "R131" variant, which binds to IgG1 Fc
with low
affinity. See, e.g., Bruhns et al., Blood/13:3716-3725 (2009).
Regarding FcyRIII binding, reduced binding to FcyRIIIA is found, e.g., for
mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270,
Q295,
A327, S239, E269, E293, Y296, V303, A327, K338 and D376. Mapping of the
binding
sites on human IgG 1 for Fc receptors, the above-mentioned mutation sites, and
methods
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for measuring binding to FcyRI and FeyRIIA, are described in Shields, R. L.,
et al., J.
Biol. Chem. 276 (2001) 6591-6604.
Two allelic forms of human FcyRIIIA are the "F158" variant, which binds to
IgG1 Fc with low affinity, and the "V158" variant, which binds to IgG1 Fc with
high
affinity. See, e.g., Bruhns et al., Blood/13:3716-3725 (2009).
Regarding binding to FcyRII, two regions of native IgG Fc appear to be
involved in interactions between FcyRIIs and IgGs, namely (i) the lower hinge
site of
IgG Fc, in particular amino acid residues L, L, G, G (234 ¨ 237, EU
numbering), and
(ii) the adjacent region of the CH2 domain of IgG Fc, in particular a loop and
strands in
the upper CH2 domain adjacent to the lower hinge region, e.g. in a region of
P331
(Wines, B.D., et al., J. Immunol. 2000; 164: 5313 ¨5318). Moreover, FcyR1
appears to
bind to the same site on IgG Fc, whereas FcRn and Protein A bind to a
different site on
IgG Fc, which appears to be at the CH2-CH3 interface (Wines, B.D., et al., J.
Immunol.
2000; 164: 5313 ¨ 5318).
Also contemplated are mutations that increase binding affinity of an Fc
polypeptide or fragment thereof of the present disclosure to a (i.e., one or
more) Fcy
receptor (e.g., as compared to a reference Fe polypeptide or fragment thereof
or
containing the same that does not comprise the mutation(s)). See, e.g.,
Delillo and
Raveteh, Cell 161(5):1035-1045 (2015) and Ahmed et al., J. Struc. Biol.
194(1):78
(2016), the Fc mutations and techniques of which are incorporated herein by
reference.
In any of the herein disclosed embodiments, an antibody or antigen-binding
fragment can comprise a Fc polypeptide or fragment thereof comprising a
mutation
selected from G236A; S239D; A330L; and 1332E; or a combination comprising any
two or more of the same; e.g., S239D/I332E; S239D/A330L/I332E;
G236A/S239D/I332E; G236A/A330L/I332E (also referred to herein as "GAALIE"), or
G236A/S239D/A330L/I332E. In some embodiments, the Fc polypeptide or fragment
thereof does not comprise S239D. In some embodiments, the Fc polypeptide or
fragment thereof comprises S at position 239 (EU numbering)
In certain embodiments, the Fc polypeptide or fragment thereof may comprise
or consist of at least a portion of an Fc polypeptide or fragment thereof that
is involved
in binding to FcRn binding. In certain embodiments, the Fc polypeptide or
fragment
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thereof comprises one or more amino acid modifications that improve binding
affinity
for (e.g., enhance binding to) FcRn (e.g., at a pH of about 6.0) and, in some
embodiments, thereby extend in vivo half-life of a molecule comprising the Fc
polypeptide or fragment thereof (e.g., as compared to a reference Fc
polypeptide or
fragment thereof or antibody that is otherwise the same but does not comprise
the
modification(s)). In certain embodiments, the Fc polypeptide or fragment
thereof
comprises or is derived from a IgG Fc and a half-life-extending mutation
comprises any
one or more of: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E;
T250Q; P257I Q31 11; D376V; T307A; E380A (EU numbering). In certain
embodiments, a half-life-extending mutation comprises M428L/N434S (also
referred to
herein as "MLNS"). In certain embodiments, a half-life-extending mutation
comprises
M252Y/S254T/T256E. In certain embodiments, a half-life-extending mutation
comprises T250Q/M428L. In certain embodiments, a half-life-extending mutation
comprises P257I/Q3111. In certain embodiments, a half-life-extending mutation
comprises P257I/N434H. In certain embodiments, a half-life-extending mutation
comprises D376V/N434H. In certain embodiments, a half-life-extending mutation
comprises T307A/E380A/N434A.
In some embodiments, an antibody or antigen-binding fragment includes a Fc
moiety that comprises the substitution mtuations M428L/N434S. In some
embodiments, an antibody or antigen-binding fragment includes a Fc polypeptide
or
fragment thereof that comprises the substitution mtuations G236A/A330L/I332E.
In
certain embodiments, an antibody or antigen-binding fragment includes a (e.g.,
IgG) Fc
moiety that comprises a G236A mutation, an A330L mutation, and a 1332E
mutation
(GAALIE), and does not comprise a S239D mutation (e.g., comprises a native S
at
position 239). In particular embodiments, an antibody or antigen-binding
fragment
includes an Fc polypeptide or fragment thereof that comprises the substitution
mutations: M428L/N434S and G236A/A330L/1332E, and optionally does not comprise
S239D (e.g., comprises S at 239) In certain embodiments, an antibody or
antigen-
binding fragment includes a Fc polypeptide or fragment thereof that comprises
the
substitution mutations: M428L/N434S and G236A/S239D/A330L/1332E.
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In certain embodiments, the antibody or antigen-binding fragment comprises a
mutation that alters glycosylation, wherein the mutation that alters
glycosylation
comprises N297A, N297Q, or N297G, and/or the antibody or antigen-binding
fragment
is partially or fully aglycosylated and/or is partially or fully afucosylated.
Host cell
lines and methods of making partially or fully aglycosylated or partially or
fully
afucosylated antibodies and antigen-binding fragments are known (see, e.g.,
PCT
Publication No. WO 2016/181357; Suzuki et al. Clin. Cancer Res. 13(6):1875-82
(2007); Huang et al. MAbs 6:1-12 (2018)).
In certain embodiments, the antibody or antigen-binding fragment is capable of
eliciting continued protection in vivo in a subject even once no detectable
levels of the
antibody or antigen-binding fragment can be found in the subject (i.e., when
the
antibody or antigen-binding fragment has been cleared from the subject
following
administration). Such protection is referred to herein as a vaccinal effect.
Without
wishing to be bound by theory, it is believed that dendritic cells can
internalize
complexes of antibody and antigen and thereafter induce or contribute to an
endogenous
immune response against antigen. In certain embodiments, an antibody or
antigen-
binding fragment comprises one or more modifications, such as, for example,
mutations
in the Fc comprising G236A, A330L, and 1332E, that are capable of activating
dendritic
cells that may induce, e.g., T cell immunity to the antigen.
In any of the presently disclosed embodiments, the antibody or antigen-binding
fragment comprises a Fc polypeptide or a fragment thereof, including a CH2 (or
a
fragment thereof, a CH3 (or a fragment thereof), or a CH2 and a CH3, wherein
the
CH2, the CH3, or both can be of any isotype and may contain amino acid
substitutions
or other modifications as compared to a corresponding wild-type CH2 or CH3,
respectively. In certain embodiments, a Fc polypeptide of the present
disclosure
comprises two CH2-CH3 polypeptides that associate to form a dimer.
In any of the presently disclosed embodiments, the antibody or antigen-binding
fragment can be monoclonal The term "monoclonal antibody" (mAb) as used herein
refers to an antibody obtained from a population of substantially homogeneous
antibodies, i.e., individual antibodies comprising the population are
identical except for
possible naturally occurring mutations that may be present, in some cases in
minor
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amounts. Monoclonal antibodies are highly specific, being directed against a
single
antigenic site. Furthermore, in contrast to polyclonal antibody preparations
that include
different antibodies directed against different epitopes, each monoclonal
antibody is
directed against a single epitope of the antigen. In addition to their
specificity, the
monoclonal antibodies are advantageous in that they may be synthesized
uncontaminated by other antibodies. The term "monoclonal" is not to be
construed as
requiring production of the antibody by any particular method. For example,
monoclonal antibodies useful in the present invention may be prepared by the
hybridoma methodology first described by Kohler et al., Nature 256:495 (1975),
or
may be made using recombinant DNA methods in bacterial, eukaryotic animal, or
plant
cells (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be
isolated
from phage antibody libraries using the techniques described in Clackson et
al., Nature,
352:624-628 (1991) and Marks et
Mol Biol., 222:581-597 (1991), for example.
Monoclonal antibodies may also be obtained using methods disclosed in PCT
Publication No. WO 2004/076677A2.
Antibodies and antigen-binding fragments of the present disclosure include
"chimeric antibodies" in which a portion of the heavy and/or light chain is
identical
with or homologous to corresponding sequences in antibodies derived from a
particular
species or belonging to a particular antibody class or subclass, while the
remainder of
the chain(s) is identical with or homologous to corresponding sequences in
antibodies
derived from another species or belonging to another antibody class or
subclass, as well
as fragments of such antibodies, so long as they exhibit the desired
biological activity
(see, U.S. Pat. Nos. 4,816,567; 5,530,101 and 7,498,415; and Morrison et al.,
Proc.
Natl. Acad. Sci. USA, 81:6851-6855 (1984)). For example, chimeric antibodies
may
comprise human and non-human residues. Furthermore, chimeric antibodies may
comprise residues that are not found in the recipient antibody or in the donor
antibody.
These modifications are made to further refine antibody performance. For
further
details, see Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature
332:323-
329 (1988); and Presta, C 117T . Op. StrucL Biol. 2:593-596 (1992). Chimeric
antibodies
also include primatized and humanized antibodies.
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A "humanized antibody" is generally considered to be a human antibody that
has one or more amino acid residues introduced into it from a source that is
non-human.
These non-human amino acid residues are typically taken from a variable
domain.
Humanization may be performed following the method of Winter and co-workers
(Jones et at., Nature, 321:522-525 (1986); Reichmann et at., Nature, 332:323-
327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting non-
human
variable sequences for the corresponding sequences of a human antibody.
Accordingly,
such "humanized" antibodies are chimeric antibodies (U.S. Pat. Nos. 4,816,567;
5,530,101 and 7,498,415) wherein substantially less than an intact human
variable
domain has been substituted by the corresponding sequence from a non-human
species.
In some instances, a -humanized" antibody is one which is produced by a non-
human
cell or animal and comprises human sequences, e.g., Hc domains.
A "human antibody" is an antibody containing only sequences that are present
in
an antibody that is produced by a human (i.e., sequences that are encoded by
human
antibody-encoding genes). However, as used herein, human antibodies may
comprise
residues or modifications not found in a naturally occurring human antibody
(e.g., an
antibody that is isolated from a human), including those modifications and
variant
sequences described herein. These are typically made to further refine or
enhance
antibody performance. In some instances, human antibodies are produced by
transgenic
animals. For example, see U.S. Pat. Nos. 5,770,429; 6,596,541 and 7,049,426.
In certain embodiments, an antibody or antigen-binding fragment of the present
disclosure is chimeric, humanized, or human.
Polynucleotides, Vectors, and Host cells
In another aspect, the present disclosure provides isolated polynucleotides
that
encode any of the presently disclosed antibodies or an antigen-binding
fragment
thereof, or a portion thereof (e.g., a CDR, a VH, a VL, a heavy chain, or a
light chain).
In certain embodiments, the polynucleotide is codon-optimized for expression
in a host
cell. Once a coding sequence is known or identified, codon optimization can be
performed using known techniques and tools, e.g., using the GenScript
OptimiumGeneTM tool; see also Scholten et at., Cl/n. 1111171111101 119:135,
2006).
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Codon-optimized sequences include sequences that are partially codon-optimized
(i.e.,
one or more codon is optimized for expression in the host cell) and those that
are fully
codon-optimized.
It will also be appreciated that polynucleotides encoding antibodies and
antigen-
binding fragments of the present disclosure may possess different nucleotide
sequences
while still encoding a same antibody or antigen-binding fragment due to, for
example,
the degeneracy of the genetic code, splicing, and the like.
In certain embodiments, the polynucleotide comprises a polynucleotide having
at least 50% (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the polynucleotide sequence
according to any one or more of SEQ ID NOs.:30, 31, 40, 41, 50, 51, 60, 61,
70, 71, 80,
81, 90, 91, 100, 101, 110, 111, 120, 121, 130, 131, 140, 141, 150, 151, 160,
161, 170,
171, 180, 181, 190, 191, 200, 201, 210, 211, 220, 221, 230, 231, 240, 241,
250, 251,
260, 261, 270, 271, 280, 281, 290, 291, 300, 301, 310, 311, 320, 321, 330,
331, 340,
341, 350, 351, 360, 361, 370, 371, 380, 381, 390, 391, 400, 401, 410, 411,
420, 421,
430, 431, 440, and 441, or any combination thereof (e.g., a polynucleotide
comprises a
polynucleotide having at least 50% identity to to SEQ ID NO. :30 and a
polynucleotide
having at least 50% identity to SEQ ID NO. :31).
It will be appreciated that in certain embodiments, a polynucleotide encoding
an
antibody or antigen-binding fragment is comprised in a polynucleotide that
includes
other sequences and/or features for, e.g-., expression of the antibody or
antigen-binding
fragment in a host cell. Exemplary features include a promoter sequence, a
polyadenylation sequence, a sequence that encodes a signal peptide (e.g.,
located at the
N-terminus of a expressed antibody heavy chain or light chain), or the like.
In any of the presently disclosed embodiments, the polynucleotide can comprise
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments,
the
RNA comprises messenger RNA (mRNA).
Vectors are also provided, wherein the vectors comprise or contain a
polynucleotide as disclosed herein (e.g., a polynucleotide that encodes an
antibody or
antigen-binding fragment that binds to SARS-CoV-2). A vector can comprise any
one
or more of the vectors disclosed herein. In particular embodiments, a vector
is provided
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that comprises a DNA plasmid construct encoding the antibody or antigen-
binding
fragment, or a portion thereof (e.g., so-called "DMAb"; see, e.g., Muthumani
et al., J
infect Dis. 214(3):369-378 (2016); Muthumani et al., Hum Vaccin Immunother
9:2253-
2262 (2013)); Flingai et al., Sci Rep. 5:12616 (2015); and Elliott et al., NPJ
Vaccines
18 (2017), which antibody-coding DNA constructs and related methods of use,
including administration of the same, are incorporated herein by reference).
In certain
embodiments, a DNA plasmid construct comprises a single open reading frame
encoding a heavy chain and a light chain (or a VH and a VL) of the antibody or
antigen-
binding fragment, wherein the sequence encoding the heavy chain and the
sequence
encoding the light chain are optionally separated by polynucleotide encoding a
protease
cleavage site and/or by a polynucleotide encoding a self-cleaving peptide. In
some
embodiments, the substituent components of the antibody or antigen-binding
fragment
are encoded by a polynucleotide comprised in a single plasmid. In other
embodiments,
the substituent components of the antibody or antigen-binding fragment are
encoded by
a polynucleotide comprised in two or more plasmids (e.g., a first plasmid
comprises a
polynucleotide encoding a heavy chain, VH, or VH+CH, and a second plasmid
comprises a polynucleotide encoding the cognate light chain, VL, or VL+CL). In
certain embodiments, a single plasmid comprises a polynucleotide encoding a
heavy
chain and/or a light chain from two or more antibodies or antigen-binding
fragments of
the present disclosure. An exemplary expression vector is pVaxl, available
from
Invitrogen . A DNA plasmid of the present disclosure can be delivered to a
subject by,
for example, electroporation (e.g., intramuscular electroporation), or with an
appropriate formulation (e.g., hyaluronidase).
In a further aspect, the present disclosure also provides a host cell
expressing an
antibody or antigen-binding fragment according to the present disclosure; or
comprising
or containing a vector or polynucleotide according the present disclosure.
Examples of such cells include but are not limited to, eukaryotic cells, e.g.,
yeast
cells, animal cells, insect cells, plant cells; and prokaryotic cells,
including E. coli In
some embodiments, the cells are mammalian cells. In certain such embodiments,
the
cells are a mammalian cell line such as CHO cells (e.g., DHFR- CHO cells
(Urlaub et
al., PNAS 77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells),
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PER.C6 cells, YO cells, Sp2/0 cells. NSO cells, human liver cells, e.g. Hepa
RG cells,
myeloma cells or hybridoma cells. Other examples of mammalian host cell lines
include mouse sertoli cells (e.g., TNI4 cells); monkey kidney CV1 line
transformed by
SV40 (COS-7); baby hamster kidney cells (BHK); African green monkey kidney
cells
(VERO-76); monkey kidney cells (CV1); human cervical carcinoma cells (BELA);
human lung cells (W138); human liver cells (Hep G2); canine kidney cells
(MDCK;
buffalo rat liver cells (BRL 3A); mouse mammary tumor (MMT 060562); TRI
cells; MRC 5 cells; and FS4 cells. Mammalian host cell lines suitable for
antibody
production also include those described in, for example, Yazaki and Wu,
Methods in
Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.),
pp. 255-
268 (2003).
In certain embodiments, a host cell is a prokaryotic cell, such as an E. coll.
The
expression of peptides in prokaryotic cells such as E. coil is well
established (see, e.g.,
Pluckthun, A. Bio/Technology 9:545-551 (1991). For example, antibodies may be
produced in bacteria, in particular when glycosylation and Fc effector
function are not
needed. For expression of antibody fragments and polypeptides in bacteria,
see, e.g.,
U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523.
In particular embodiments, the cell may be transfected with a vector according
to the present description with an expression vector. The term "transfection"
refers to
the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA)
molecules, into cells, such as into eukaryotic cells. In the context of the
present
description, the term "transfection" encompasses any method known to the
skilled
person for introducing nucleic acid molecules into cells, such as into
eukaryotic cells,
including into mammalian cells. Such methods encompass, for example,
electroporation, lipofection, e.g., based on cationic lipids and/or liposomes,
calcium
phosphate precipitation, nanoparticle based transfection, virus based
transfection, or
transfection based on cationic polymers, such as DEAE-dextran or
polyethylenimine,
etc In certain embodiments, the introduction is non-viral
Moreover, host cells of the present disclosure may be transfected stably or
transiently with a vector according to the present disclosure, e.g. for
expressing an
antibody, or an antigen-binding fragment thereof, according to the present
disclosure.
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In such embodiments, the cells may be stably transfected with the vector as
described
herein. Alternatively, cells may be transiently transfected with a vector
according to the
present disclosure encoding an antibody or antigen-binding fragment as
disclosed
herein. In any of the presently disclosed embodiments, a polynucleotide may be
heterologous to the host cell.
Accordingly, the present disclosure also provides recombinant host cells that
heterologously express an antibody or antigen-binding fragment of the present
disclosure. For example, the cell may be of a species that is different to the
species
from which the antibody was fully or partially obtained (e.g., CHO cells
expressing a
human antibody or an engineered human antibody). In some embodiments, the cell
type of the host cell does not express the antibody or antigen-binding
fragment in
nature Moreover, the host cell may impart a post-translational modification
(PTM,
e.g., glysocylation or fucosylation) on the antibody or antigen-binding
fragment that is
not present in a native state of the antibody or antigen-binding fragment (or
in a native
state of a parent antibody from which the antibody or antigen binding fragment
was
engineered or derived) Such a PTM may result in a functional difference (e.g.,
reduced
immunogenicity). Accordingly, an antibody or antigen-binding fragment of the
present
disclosure that is produced by a host cell as disclosed herein may include one
or more
post-translational modification that is distinct from the antibody (or parent
antibody) in
its native state (e.g., a human antibody produced by a CHO cell can comprise a
more
post-translational modification that is distinct from the antibody when
isolated from the
human and/or produced by the native human B cell or plasma cell).
Insect cells useful expressing a binding protein of the present disclosure are
known in the art and include, for example, ,S'podoptera frugipera Sf9 cells,
Trichoplusia
ni BTI-TN5B1-4 cells, and Spodoptera fruppera SfSWTO1 "Mimi CTM" cells. See,
e.g.,
Palmberger et at., J. Biotechnot /53(3-4):160-166 (2011). Numerous baculoviral
strains have been identified which may be used in conjunction with insect
cells,
particularly for transfecti on of S'podopterafrugiperda cells
Eukaryotic microbes such as filamentous fungi or yeast are also suitable hosts
for cloning or expressing protein-encoding vectors, and include fungi and
yeast strains
with "humanized" glycosylation pathways, resulting in the production of an
antibody
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with a partially or fully human glycosylation pattern. See Gerngross, Nat.
Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006).
Plant cells can also be utilized as hosts for expressing a binding protein of
the
present disclosure. For example, PLANTIBODIESTm technology (described in, for
example, U.S. Pat. Nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; and
6,417,429)
employs transgenic plants to produce antibodies.
In certain embodiments, the host cell comprises a mammalian cell. In
particular
embodiments, the host cell is a CHO cell, a HEK293 cell, a PER.C6 cell, a YO
cell, a
Sp2/0 cell, a NSO cell, a human liver cell, a myeloma cell, or a hybridoma
cell.
In a related aspect, the present disclosure provides methods for producing an
antibody, or antigen-binding fragment, wherein the methods comprise culturing
a host
cell of the present disclosure under conditions and for a time sufficient to
produce the
antibody, or the antigen-binding fragment. Methods useful for isolating and
purifying
recombinantly produced antibodies, by way of example, may include obtaining
supernatants from suitable host cell/vector systems that secrete the
recombinant
antibody into culture media and then concentrating the media using a
commercially
available filter. Following concentration, the concentrate may be applied to a
single
suitable purification matrix or to a series of suitable matrices, such as an
affinity matrix
or an ion exchange resin. One or more reverse phase HPLC steps may be employed
to
further purify a recombinant polypeptide. These purification methods may also
be
employed when isolating an immunogen from its natural environment. Methods for
large scale production of one or more of the isolated/recombinant antibody
described
herein include batch cell culture, which is monitored and controlled to
maintain
appropriate culture conditions. Purification of soluble antibodies may be
performed
according to methods described herein and known in the art and that comport
with laws
and guidelines of domestic and foreign regulatory agencies.
Compositions
Also provided herein are compositions that comprise any one or more of the
presently disclosed antibodies, antigen-binding fragments, polynucleotides,
vectors, or
host cells, singly or in any combination, and can further comprise a
pharmaceutically
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acceptable carrier, excipient, or diluent. Carriers, excipients, and diluents
are discussed
in further detail herein.
In certain embodiments, a composition comprises a plurality of an antibody
and/or an antigen-binding fragment of the present disclosure, wherein one or
more
antibody or antigen-binding fragment does not comprise a lysine residue at the
C-
terminal end of the heavy chain, CH1-CH3, or Fc polypeptide, and wherein one
or more
antibody or antigen-binding fragment comprises a lysine residue at the C-
terminal end
of the heavy chain, CH1-CH3, or Fc polypeptide.
In certain embodiments, a composition comprises two or more different
antibodies or antigen-binding fragments according to the present disclosure.
In certain
embodiments, antibodies or antigen-binding fragments to be used in a
combination
each independently have one or more of the following characteristics
neutralize
naturally occurring SARS-CoV-2 variants; do not compete with one another for
Spike
protein binding; bind distinct Spike protein epitopes; have a reduced
formation of
resistance to SARS-CoV-2; when in a combination, have a reduced formation of
resistance to SARS-CoV-2; potently neutralize live SARS-CoV-2 virus, exhibit
additive or synergistic effects on neutralization of live SARS-CoV-2 virus
when used in
combination; exhibit effector functions; are protective in relevant animal
model(s) of
infection; are capable of being produced in sufficient quantities for large-
scale
production
In certain embodiments, a composition comprises (a) antibody S2X333 (or an
antigen-binding fragment thereof) or an antibody or antigen-binding fragment
that
competes with antibody S2X333 for SARS-CoV-2 S protein binding and (b)
antibody
S309 (or an antigen-binding fragment thereof) or an antibody or antigen-
binding
fragment that competes with antibody S309 for SARS-CoV-2 S protein binding.
In certain embodiments, a composition comprises (a) antibody S2X333 (or an
antigen-binding fragment thereof) or an antibody or antiben-binding fragment
that
competes with antibody S2X333 for SARS-CoV-2 S protein binding and (11)
antibody
S2E12 (or an antigen-binding fragment thereof) or an antibody or antigen-
binding
fragment that competes with antibody S2E12 for SARS-CoV-2 S protein binding.
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In certain embodiments, a composition comprises (a) antibody S2X333 (or an
antigen-binding fragment thereof) or an antibody or antigen-binding fragment
that
competes with antibody S2X333 for SARS-CoV-2 S protein binding and (b)
antibody
S2M11 (or an antigen-binding fragment thereof) or an antibody or antigen-
binding
fragment that competes with antibody S2M11 for SARS-CoV-2 S protein binding.
Antibody S2X333 comprises the VH amino acid sequence of SEQ ID NO. :52
and the VL amino acid sequence of SEQ ID NO. :56.
Antibody S2E12 comprises the VH amino acid sequence of SEQ ID NO.450
and the VL amino acid sequence of SEQ ID NO.454.
Antibody S309 comprises the VH amino acid sequence of SEQ ID NO. :442 and
the VL amino acid sequence of SEQ ID NO. :446. A variant VH of antibody S309
comprises the amino acid sequence of SEQ ID NO. 466.
Antibody S2M11 comprises the VH amino acid sequence of SEQ ID NO.458
and the VL amino acid sequence of SEQ ID NO. :462.
In certain embodiments, a composition comprises two or more different
antibodies or antigen-binding fragments according to the present disclosure.
In certain embodiments, a composition comprises a first vector comprising a
first plasmid, and a second vector comprising a second plasmid, wherein the
first
plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH, and a
second plasmid comprises a polynucleotide encoding the cognate light chain,
VL, or
VL+CL of the antibody or antigen-binding fragment thereof. In certain
embodiments, a
composition comprises a polynucleotide (e.g., mRNA) coupled to a suitable
delivery
vehicle or carrier. Exemplary vehicles or carriers for administration to a
human subject
include a lipid or lipid-derived delivery vehicle, such as a liposome, solid
lipid
nanoparticle, oily suspension, submicron lipid emulsion, lipid microbubble,
inverse
lipid micelle, cochlear liposome, lipid microtubule, lipid microcylinder, or
lipid
nanoparticle (LNP) or a nanoscale platform (see, e.g., Li et at. Wilery
Interdi.5cip Rev.
Nanomed Nanohiotechnol. 1/(2).e1530 (2019)). Principles, reagents, and
techniques
for designing appropriate mRNA and and formulating mRNA-LNP and delivering the
same are described in, for example, Pardi et at. (I Control Release 2/7345-351
(2015));
Thess et al. (Mol Ther 23: 1456-1464 (2015)); Thran et al. (EMBO Mol Med
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9(10):1434-1448 (2017); Kose et al. (Sc. Immunol. 4 eaaw6647 (2019); and
Sabnis et
at. (Mol. Ther. 26:1509-1519 (2018)), which techniques, include capping, codon
optimization, nucleoside modification, purification of mRNA, incorporation of
the
mRNA into stable lipid nanoparticles (e.g., ionizable cationic
lipid/phosphatidylcholine/cholesterol/PEG-lipid; ionizable lipid:distearoyl
PC:cholesterol:polyethylene glycol lipid), and subcutaneous, intramuscular,
intradermal, intravenous, intraperitoneal, and intratracheal administration of
the same,
are incorporated herein by reference.
Methods and Uses
Also provided herein are methods for use of an antibody or antigen-binding
fragment, nucleic acid, vector, cell, or composition of the present disclosure
in the
detection or diagnosis of SARS-CoV-2 infection (e.g., in a human subject, or
in a
sample obtained from a human subject).
Methods of diagnosis (e.g., in vitro, ex vivo) may include contacting an
antibody
or antibody fragment (e.g., antigen binding fragment) with a sample. Such a
sample
may be isolated from a subject, for example an isolated (e.g., fluid, tissue,
or secretion)
sample from a nasal passage, a sinus cavity, a salivary gland, a lung, a
liver, a trachea, a
bronchiole, a pancreas, a kidney, an ear, an eye, a placenta, an alimentary
tract, a heart,
an ovary, a pituitary gland, an adrenal, a thyroid gland, a brain, sera,
plasma, skin, or
blood. In some embodiments, the sample may comprise a nasal secretion, sputum,
bronchial lavage, urine, stool, saliva, sweat, or any combination thereof.
Methods of
diagnosis may also include the detection of an antigen/antibody complex, in
particular
following the contacting of an antibody or antibody fragment with a sample.
Such a
detection step can be performed at the bench, i.e. without any contact to the
human or
animal body. Examples of detection methods are well-known to the person
skilled in
the art and include, e.g., ELISA (enzyme-linked immunosorbent assay),
including
direct, indirect, and sandwich ELISA.
Also provided herein are methods of treating a subject using an antibody or
antigen-binding fragment of the present disclosure, or a composition
comprising the
same, wherein the subject has, is believed to have, or is at risk for having
an infection
by SARS-CoV-2. "Treat," "treatment," or "ameliorate" refers to medical
management
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of a disease, disorder, or condition of a subject (e.g., a human or non-human
mammal,
such as a primate, horse, cat, dog, goat, mouse, or rat). In general, an
appropriate dose
or treatment regimen comprising an antibody or composition of the present
disclosure is
administered in an amount sufficient to elicit a therapeutic or prophylactic
benefit.
Therapeutic or prophylactic/preventive benefit includes improved clinical
outcome;
lessening or alleviation of symptoms associated with a disease; decreased
occurrence of
symptoms, improved quality of life, longer disease-free status, diminishment
of extent
of disease, stabilization of disease state; delay or prevention of disease
progression;
remission; survival; prolonged survival; or any combination thereof. In
certain
embodiments, therapeutic or prophylactic/preventive benefit includes reduction
or
prevention of hospitalization for treatment of a SARS-CoV-2 infection (i.e.,
in a
statistically significant manner) In certain embodiments, therapeutic or
prophylactic/preventive benefit includes a reduced duration of hospitalization
for
treatment of a SARS-CoV-2 infection (i.e., in a statistically significant
manner). In
certain embodiments, therapeutic or prophylactic/preventive benefit includes a
reduced
or abrogated need for respiratory intervention, such as intubation and/or the
use of a
respirator device. In certain embodiments, therapeutic or
prophylactic/preventive
benefit includes reversing a late-stage disease pathology and/or reducing
mortality.
A "therapeutically effective amount" or "effective amount" of an antibody,
antigen-binding fragment, polynucleotide, vector, host cell, or composition of
this
disclosure refers to an amount of the composition or molecule sufficient to
result in a
therapeutic effect, including improved clinical outcome, lessening or
alleviation of
symptoms associated with a disease; decreased occurrence of symptoms; improved
quality of life; longer disease-free status; diminishment of extent of
disease,
stabilization of disease state, delay of disease progression, remission,
survival, or
prolonged survival in a statistically significant manner. When referring to an
individual
active ingredient, administered alone, a therapeutically effective amount
refers to the
effects of that ingredient or cell expressing that ingredient alone When
referring to a
combination, a therapeutically effective amount refers to the combined amounts
of
active ingredients or combined adjunctive active ingredient with a cell
expressing an
active ingredient that results in a therapeutic effect, whether administered
serially,
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sequentially, or simultaneously. A combination may comprise, for example, two
different antibodies that specifically bind a SARS-CoV-2 antigen, which in
certain
embodiments, may be the same or different SARS-CoV-2 antigen, and/or can
comprise
the same or different epitopes.
Accordingly, in certain embodiments, methods are provided for treating a
SARS-CoV-2 infection in a subject, wherein the methods comprise administering
to the
subject an effective amount of an antibody, antigen-binding fragment,
polynucleotide,
vector, host cell, or composition as disclosed herein.
Subjects that can be treated by the present disclosure are, in general, human
and
other primate subjects, such as monkeys and apes for veterinary medicine
purposes.
Other model organisms, such as mice and rats, may also be treated according to
the
present disclosure. In any of the aforementioned embodiments, the subject may
be a
human subject. The subjects can be male or female and can be any suitable age,
including infant, juvenile, adolescent, adult, and geriatric subjects.
A number of criteria are believed to contribute to high risk for severe
symptoms
or death associated with a SARS CoV-2 infection These include, but are not
limited to,
age, occupation, general health, pre-existing health conditions, and lifestyle
habits. In
some embodiments, a subject treated according to the present disclosure
comprises one
or more risk factors.
In certain embodiments, a human subject treated according to the present
disclosure is an infant, a child, a young adult, an adult of middle age, or an
elderly
person. In certain embodiments, a human subject treated according to the
present
disclosure is less than 1 year old, or is 1 to 5 years old, or is between 5
and 125 years
old (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100,
105, 110, 115, or 125 years old, including any and all ages therein or
therebetween). In
certain embodiments, a human subject treated according to the present
disclosure is 0-
19 years old, 20-44 years old, 45-54 years old, 55-64 years old, 65-74 years
old, 75-84
years old, or 85 years old, or older Persons of middle, and especially of
elderly age are
believed to be at particular risk. In particular embodiments, the human
subject is 45-54
years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old,
or older.
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In some embodiments, the human subject is biologically male. In some
embodiments,
the human subject is biologically female.
In certain embodiments, a human subject treated according to the present
disclosure is a resident of a nursing home or a long-term care facility, is a
hospice care
worker, is a healthcare provider or healthcare worker, is a first responder,
is a family
member or other close contact of a subject diagnosed with or suspected of
having a
SARS-CoV-2 infection, is overweight or clinically obese, is or has been a
smoker, has
or had chronic obstructive pulmonary disease (COPD), is asthmatic (e.g.,
having
moderate to severe asthma), has an autoimmune disease or condition (e.g.,
diabetes),
and/or has a compromised or depleted immune system (e.g., due to AIDS/HIV
infection, a cancer such as a blood cancer, a lymphodepleting therapy such as
a
chemotherapy, a bone marrow or organ transplantation, or a genetic immune
condition),
has chronic liver disease, has cardiovascular disease, has a pulmonary or
heart defect,
works or otherwise spends time in close proximity with others, such as in a
factory,
shipping center, hospital setting, or the like.
In certain embodiments, a subject treated according to the present disclosure
has
received a vaccine for SARS-CoV-2 and the vaccine is determined to be
ineffective,
e.g., by post-vaccine infection or symptoms in the subject, by clinical
diagnosis or
scientific or regulatory consensus.
In certain embodiments, treatment is administered as pen -exposure prophylaxis
In certain embodiments, treatment is administered to a subject with mild-to-
moderate
disease, which may be in an outpatient setting. In certain embodiments,
treatment is
administered to a subject with moderate-to-severe disease, such as requiring
hospitalization.
Typical routes of administering the presently disclosed compositions thus
include, without limitation, oral, topical, transdermal, inhalation,
parenteral, sublingual,
buccal, rectal, vaginal, and intranasal. The term "parenteral", as used
herein, includes
subcutaneous injections, intravenous, intramuscular, intrasternal injection or
infusion
techniques. In certain embodiments, administering comprises administering by a
route
that is selected from oral, intravenous, parenteral, intragastric,
intrapleural,
intrapulmonary, intrarectal, intradermal, intraperitoneal, intratumoral,
subcutaneous,
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topical, transdermal, intracisternal, intrathecal, intranasal, and
intramuscular. In
particular embodiments, a method comprises orally administering the antibody,
antigen-
binding fragment, polynucleotide, vector, host cell, or composition to the
subject.
Pharmaceutical compositions according to certain embodiments of the present
invention are formulated so as to allow the active ingredients contained
therein to be
bioavailable upon administration of the composition to a patient. Compositions
that
will be administered to a subject or patient may take the form of one or more
dosage
units, where for example, a tablet may be a single dosage unit, and a
container of a
herein described an antibody or antigen-binding in aerosol form may hold a
plurality of
dosage units. Actual methods of preparing such dosage forms are known, or will
be
apparent, to those skilled in this art; for example, see Remington: The
Science and
Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and
Science,
2000). The composition to be administered will, in any event, contain an
effective
amount of an antibody or antigen-binding fragment, polynucleotide, vector,
host cell_
or composition of the present disclosure, for treatment of a disease or
condition of
interest in accordance with teachings herein.
A composition may be in the form of a solid or liquid. In some embodiments,
the carrier(s) are particulate, so that the compositions are, for example, in
tablet or
powder form. The carrier(s) may be liquid, with the compositions being, for
example,
an oral oil, injectable liquid or an aerosol, which is useful in, for example,
inhalatory
administration. When intended for oral administration, the pharmaceutical
composition
is preferably in either solid or liquid form, where semi solid, semi liquid,
suspension
and gel forms are included within the forms considered herein as either solid
or liquid
As a solid composition for oral administration, the pharmaceutical composition
may be formulated into a powder, granule, compressed tablet, pill, capsule,
chewing
gum, wafer or the like. Such a solid composition will typically contain one or
more
inert diluents or edible carriers. In addition, one or more of the following
may be
present. binders such as carboxymethylcellulose, ethyl cellulose,
microcrystalline
cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or
dextrins,
disintegrating agents such as alginic acid, sodium alginate, Primogel, corn
starch and
the like; lubricants such as magnesium stearate or Sterotex; glidants such as
colloidal
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silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring
agent such
as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
When the
composition is in the form of a capsule, for example, a gelatin capsule, it
may contain,
in addition to materials of the above type, a liquid carrier such as
polyethylene glycol or
Oil.
The composition may be in the form of a liquid, for example, an elixir, syrup,
solution, emulsion or suspension. The liquid may be for oral administration or
for
delivery by injection, as two examples. When intended for oral administration,
preferred compositions contain, in addition to the present compounds, one or
more of a
sweetening agent, preservatives, dye/colorant and flavor enhancer. In a
composition
intended to be administered by injection, one or more of a surfactant,
preservative,
wetting agent, dispersing agent, suspending agent, buffer, stabilizer and
isotonic agent
may be included.
Liquid pharmaceutical compositions, whether they be solutions, suspensions or
other like form, may include one or more of the following adjuvants: sterile
diluents
such as water for injection, saline solution, preferably physiological saline,
Ringer's
solution, isotonic sodium chloride, fixed oils such as synthetic mono or
diglycerides
which may serve as the solvent or suspending medium, polyethylene glycols,
glycerin,
propylene glycol or other solvents; antibacterial agents such as benzyl
alcohol or methyl
paraben; antioxidants such as ascorbic acid or sodium bi sulfite; chelating
agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates or
phosphates and
agents for the adjustment of tonicity such as sodium chloride or dextrose. The
parenteral preparation can be enclosed in ampoules, disposable syringes or
multiple
dose vials made of glass or plastic. Physiological saline is a preferred
adjuvant. An
injectable pharmaceutical composition is preferably sterile.
A liquid composition intended for either parenteral or oral administration
should
contain an amount of an antibody or antigen-binding fragment as herein
disclosed such
that a suitable dosage will be obtained Typically, this amount is at least
001% of the
antibody or antigen-binding fragment in the composition. When intended for
oral
administration, this amount may be varied to be between 0.1 and about 70% of
the
weight of the composition. Certain oral pharmaceutical compositions contain
between
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about 4% and about 75% of the antibody or antigen-binding fragment. In certain
embodiments, pharmaceutical compositions and preparations according to the
present
invention are prepared so that a parenteral dosage unit contains between 0.01
to 10% by
weight of antibody or antigen-binding fragment prior to dilution.
The composition may be intended for topical administration, in which case the
carrier may suitably comprise a solution, emulsion, ointment or gel base. The
base, for
example, may comprise one or more of the following: petrolatum, lanolin,
polyethylene glycols, bee wax, mineral oil, diluents such as water and
alcohol, and
emulsifiers and stabilizers. Thickening agents may be present in a composition
for
topical administration. If intended for transdermal administration, the
composition may
include a transdermal patch or iontophoresis device. The pharmaceutical
composition
may be intended for rectal administration, in the form, for example, of a
suppository,
which will melt in the rectum and release the drug. The composition for rectal
administration may contain an oleaginous base as a suitable nonirritating
excipient.
Such bases include, without limitation, lanolin, cocoa butter and polyethylene
glycol.
A composition may include various materials which modify the physical form
of a solid or liquid dosage unit. For example, the composition may include
materials
that form a coating shell around the active ingredients. The materials that
form the
coating shell arc typically inert, and may be selected from, for example,
sugar, shellac,
and other enteric coating agents. Alternatively, the active ingredients may be
encased
in a gelatin capsule. The composition in solid or liquid form may include an
agent that
binds to the antibody or antigen-binding fragment of the disclosure and
thereby assists
in the delivery of the compound. Suitable agents that may act in this capacity
include
monoclonal or polyclonal antibodies, one or more proteins or a liposome. The
composition may consist essentially of dosage units that can be administered
as an
aerosol. The term aerosol is used to denote a variety of systems ranging from
those of
colloidal nature to systems consisting of pressurized packages. Delivery may
be by a
liquefied or compressed gas or by a suitable pump system that dispenses the
active
ingredients. Aerosols may be delivered in single phase, bi phasic, or tri
phasic systems
in order to deliver the active ingredient(s). Delivery of the aerosol includes
the
necessary container, activators, valves, subcontainers, and the like, which
together may
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form a kit. One of ordinary skill in the art, without undue experimentation,
may
determine preferred aerosols.
It will be understood that compositions of the present disclosure also
encompass
carrier molecules for polynucleotides, as described herein (e.g., lipid
nanoparticles,
nanoscale delivery platforms, and the like).
The pharmaceutical compositions may be prepared by methodology well known
in the pharmaceutical art. For example, a composition intended to be
administered by
injection can be prepared by combining a composition that comprises an
antibody,
antigen-binding fragment thereof, or antibody conjugate as described herein
and
optionally, one or more of salts, buffers and/or stabilizers, with sterile,
distilled water so
as to form a solution. A surfactant may be added to facilitate the formation
of a
homogeneous solution or suspension. Surfactants are compounds that non-
covalently
interact with the peptide composition so as to facilitate dissolution or
homogeneous
suspension of the antibody or antigen-binding fragment thereof in the aqueous
delivery
system.
In general, an appropriate dose and treatment regimen provide the
composition(s) in an amount sufficient to provide therapeutic and/or
prophylactic
benefit (such as described herein, including an improved clinical outcome
(e.g., a
decrease in frequency, duration, or severity of diarrhea or associated
dehydration, or
inflammation, or longer disease-free and/or overall survival, or a lessening
of symptom
severity). For prophylactic use, a dose should be sufficient to prevent, delay
the onset
of, or diminish the severity of a disease associated with disease or disorder.
Prophylactic benefit of the compositions administered according to the methods
described herein can be determined by performing pre-clinical (including in
vitro and in
vivo animal studies) and clinical studies and analyzing data obtained
therefrom by
appropriate statistical, biological, and clinical methods and techniques, all
of which can
readily be practiced by a person skilled in the art.
Compositions are administered in an effective amount (e.g., to treat a Wuhan
coronavirus infection), which will vary depending upon a variety of factors
including
the activity of the specific compound employed; the metabolic stability and
length of
action of the compound; the age, body weight, general health, sex, and diet of
the
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subject; the mode and time of administration; the rate of excretion; the drug
combination; the severity of the particular disorder or condition; and the
subject
undergoing therapy. In certain embodiments, tollowing administration of
therapies
according to the formulations and methods of this disclosure, test subjects
will exhibit
about a 10% up to about a 99% reduction in one or more symptoms associated
with the
disease or disorder being treated as compared to placebo-treated or other
suitable
control subjects.
Generally, a therapeutically effective daily dose of an antibody or antigen
binding fragment is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07
mg) to
about 100 mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is
(for a 70 kg
mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg (i.e., 3.5 g);
more
preferably a therapeutically effective dose is (for a 70 kg mammal) from about
1 mg/kg
(i.e., 70 mg) to about 25 mg/kg (i.e., 1.75 g). For polynucleotides, vectors,
host cells,
and related compositions of the present disclosure, a therapeutically
effective dose may
be different than for an antibody or antigen-binding fragment.
In certain embodiments, a method comprises administering the antibody,
antigen-binding fragment, polynucleotide, vector, host cell, or composition to
the
subject at 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more.
In certain embodiments, a method comprises administering the antibody,
antigen-binding fragment, or composition to the subject a plurality of times,
wherein a
second or successive administration is performed at about 6, about 7, about 8,
about 9,
about 10, about 11, about 12, about 24, about 48, about 74, about 96 hours, or
more,
following a first or prior administration, respectively.
In certain embodiments, a method comprises administering the antibody,
antigen-binding fragment, polynucleotide, vector, host cell, or composition at
least one
time prior to the subject being infected by SARS-CoV-2.
Compositions comprising an antibody, antigen-binding fragment,
polynucleotide, vector, host cell, or composition of the present disclosure
may also be
administered simultaneously with, prior to, or after administration of one or
more other
therapeutic agents. Such combination therapy may include administration of a
single
pharmaceutical dosage formulation which contains a compound of the invention
and
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one or more additional active agents, as well as administration of
compositions
comprising an antibody or antigen-binding fragment of the disclosure and each
active
agent in its own separate dosage formulation. For example, an antibody or
antigen-
binding fragment thereof as described herein and the other active agent can be
administered to the patient together in a single oral dosage composition such
as a tablet
or capsule, or each agent administered in separate oral dosage formulations.
Similarly,
an antibody or antigen-binding fragment as described herein and the other
active agent
can be administered to the subject together in a single parenteral dosage
composition
such as in a saline solution or other physiologically acceptable solution, or
each agent
administered in separate parenteral dosage formulations. Where separate dosage
formulations are used, the compositions comprising an antibody or antigen-
binding
fragment and one or more additional active agents can be administered at
essentially the
same time, i.e., concurrently, or at separately staggered times, i.e.,
sequentially and in
any order; combination therapy is understood to include all these regimens.
In certain embodiments, a combination therapy is provided that comprises one
or more anti-SARS-CoV-2 antibody (or one or more nucleic acid, host cell,
vector, or
composition) of the present disclosure and one or more anti-inflammatory agent
and/or
one or more anti-viral agent. In particular embodiments, the one or more anti-
inflammatory agent comprises a corticosteroid such as, for example,
dexamethasone,
predni sone, or the like. In some embodiments, the one or more anti-
inflammatory
agents comprise a cytokine antagonist such as, for example, an antibody that
binds to
IL6 (such as siltuximab), or to IL-6R (such as tocilizumab), or to IL-13, IL-
7, IL-8, IL-
9, IL- I 0, FGF, G-CSF, GM-CSF, IFN-y, IP- I 0, MCP-I, MIP- I A, MIP I -B,
PDGR,
TNF-a, or VEGF. In some embodiments, anti-inflammatory agents such as
leronlimab,
ruxolitinib and/or anakinra are used. In some embodiments, the one or more
anti-viral
agents comprise nucleotide analogs or nucelotide analog prodrugs such as, for
example,
remdesivir, sofosbuvir, acyclovir, and zidovudine. In particular embodiments,
an anti-
viral agent comprises lopinavir, ritonavir, favipiravir, or any combination
thereof
Other anti-inflammatory agents for use in a combination therapy of the present
disclosure include non-steroidal anti-inflammatory drugs (NSAIDS). It will be
appreciated that in such a combination therapy, the one or more antibody (or
one or
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more nucleic acid, host cell, vector, or composition) and the one or more anti-
inflammatory agent and/or one or the more antiviral agent can be administered
in any
order and any sequence, or together.
In some embodiments, an antibody (or one or more nucleic acid, host cell,
vector, or composition) is administered to a subject who has previously
received one or
more anti-inflammatory agent and/or one or more antiviral agent. In some
embodiments, one or more anti-inflammatory agent and/or one or more antiviral
agent
is administered to a subject who has previously received an antibody (or one
or more
nucleic acid, host cell, vector, or composition).
In certain embodiments, a combination therapy is provided that comprises two
or more anti-SARS-CoV-2 antibodies of the present disclosure. A method can
comprise administering a first antibody to a subject who has received a second
antibody, or can comprise administering two or more antibodies together. For
example,
in particular embodiments, a method is provided that comprises administering
to the
subject (a) a first antibody or antigen-binding fragment, when the subject has
received a
second antibody or antigen-binding fragment; (b) the second antibody or
antigen-
binding fragment, when the subject has received the first antibody or antigen-
binding
fragment; or (c) the first antibody or antigen-binding fragment, and the
second antibody
or antigen-binding fragment.
In a related aspect, uses of the presently disclosed antibodies, antigen-
binding
fragments, vectors, host cells, and compositions are provided.
In certain embodiments, an antibody, antigen-binding fragment, polynucleotide,
vector, host cell, or composition is provided for use in a method of treating
a SARS-
CoV-2 infection in a subject.
In certain embodiments, an antibody, antigen-binding fragment, or composition
is provided for use in a method of manufacturing or preparing a medicament for
treating
a SARS-CoV-2 infection in a subject.
In certain embodiments, an antibody or antigen-binding fragment is provided
for
use in a method of detecting SARS-CoV-2 in a sample. In some embodiments, the
method comprises contacting the sample with the antibody or antigen-binding
fragment
and detecting binding of the antibody or antigen-binding fragment to a SARS-
CoV-2
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protein or polypeptide in the sample. In some embodiments, binding to SARS-CoV-
2
protein or polypeptide is detected by immunohistochemistry, ELISA,
agglutination,
immuno-dot, immuno-chromatography, and/or immuno-filtration.
In certain embodiments, an antibody or antigen-binding fragment is provided
for
use in a method of diagnosing a SARS-CoV-2 infection in a subject. In some
embodiments, the method comprises testing a biological sample from the subject
for the
presence of a SARS-CoV-2 protein or polypeptide. In some embodiments, the
testing
comprises contacting the sample with the antibody or antigen-binding fragment
and
detecting binding of the antibody or antigen-binding fragment to the SARS-CoV-
2
protein or polypeptide. In some embodiments, binding to SARS-CoV-2 protein or
polypeptide is detected by immunohistochemistry, ELISA, agglutination, immuno-
dot,
immuno-chromatography, and/or immuno-filtration.
In some embodiments, a detection and/or diagnostic method as provided herein
(such as using a disclosed antibody, antigen-binding fragment, composition,
and/or kit)
can provide a result within 1, 5, 10, 20, 30, 45, 60, 75, 90, or 120 minutes,
or within one
day, of beginning the method.
In another aspect, the present disclosure provides kits comprising materials
useful for carrying out detection or diagnostic methods. In certain aspects, a
kit
comprising an antibody or antigen-binding fragment as described herein is
provided. In
some embodiments, the kit is used for detecting the presence of SARS-CoV-2 in
a
biological sample. In some embodiments, the kit is used for detecting the
presence of a
SARS-CoV-2 protein or polypeptide, for example, SARS-CoV-2 spike protein, in a
biological sample. In some embodiments, the presence of a SARS-CoV-2 protein
is
detected by immunohistochemistry, immunoblot, ELISA, agglutination, immuno-
dot,
immuno-chromatography, and/or immuno-filtration. In some embodiments, the kit
includes a secondary antibody detectably labeled with, for example,
horseradish
peroxidase (HRP), and/or instructions and/or other reagents for performing a
detection
method as provided herein
In further aspects, a kit comprising a composition is provided, wherein the
composition comprises an antibody or antigen-binding fragment as described
herein and
a carrier or excipient. In some embodiments, the kit is used for detecting the
presence
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of SARS-CoV-2 in a biological sample. In some embodiments, the kit is used for
detecting the presence of a SARS-CoV-2 protein or polypeptide, for example,
SARS-
CoV-2 spike protein, in a biological sample. In some embodiments, the presence
of a
SARS-CoV-2 protein is detected by immunohistochemistry, immunoblot, ELISA,
agglutination, immuno-dot, immuno-chromatography, and/or immuno-filtration. In
some embodiments, the kit includes a secondary antibody detectably labeled
with, for
example, horseradish peroxidase (I-1RP) and/or instructions and/or other
reagents for
performing a detection method as provided herein.
The methods for detecting the presence of a SARS-CoV-2 protein or
polypeptide described herein may be performed by a diagnostic laboratory, an
experimental laboratory, or a clinician, or they may be performed in-home by a
caregiver or by a subject providing the sample. Provided herein are kits that
can be
used in one or more of these settings. Materials and reagents for
characterizing
biological samples and diagnosis a SARS-CoV-2 infection in a subject according
to the
methods herein by be assembled together as a kit. In some embodiments, a kit
comprises an antibody or antigen-binding fragment according to the present
disclosure
and instructions for using the kit.
Kits comprising an antibody or antigen-binding fragment as described herein
may futher comprise one or more substrates to anchor the antigen binding
molecules,
including membranes, beads, plastic tubes, or other surfaces, secondary
antibodies,
sample buffer, labeling buffer or reagents, wash buffers or reagents,
immunodetection
buffer or reagents, and detection means. In some embodiments, the kit
comprises a
substrate to which antibodies or antigen-binding fragments are anchored.
Protocols for
using these buffers and reagents for performing different steps of the
procedure may be
included in the kit. The reagents may be supplied in a solid (e.g.,
lyophilized) or liquid
form. Kits of the present disclosure may optionally comprise different
containers (e.g.,
vial, ampoule, test tube, flask or bottle) for each individual buffer or
reagent. Each
component will generally be suitable as aliquoted in its respective container
or provided
in a concentrated form. Other containers suitable for conducting certain steps
of the
disclosed methods may also be provided. The individual containers of the kit a
preferably maintained in close confinement for commercial sale.
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In some embodiments, kits of the present disclosure further include control
samples, reference samples, or any combination thereof. Instructions for using
the kit,
according to one or more methods of this disclosure, may comprise instructions
for
processing the biological sample obtained from a subject, performing the test,
interpreting the results, or any combination thereof. Kits of the present
disclosure may
further include a notice in the form prescribed by a governmental agency
(e.g., FDA)
regulating the manufacture, use, or sale of pharmaceuticals or biological
products.
In any of the presently disclosed embodiments, an antibody or antigen-binding
fragment for use in a detection and/or diagnostic method can comprise a
detectable
agent. Exemplary detectable agents include enzymes (e.g., a chromogenic
reporter
enzyme, such as horseradish peroxidase (HRP) or an alkaline phosphatase (AP)),
dyes,
(e.g., cyanin dye, coumarin, rhodamine, xanthene, fluorescein or a sulfonated
derivative
thereof, and fluorescent proteins, including those described by Shaner et al.,
Nature
Methods (2005)), fluorescent labels or moieties (e.g., PE, Pacific blue, Alexa
fluor,
APC, and FITC) DNA barcodes (e.g., ranging from five up to 75 nucleotides
long), and
peptide tags (e.g., Strep tag, Myc tag, His tag, Flag tag, Xpress tag, Avi
tag, Calmodulin
tag, Polyglutamate tag, HA tag, Nus tag, S tag, X tag, SBP tag, Softag, V5
tag, CBP,
GST, MBP, GFP, Thioredoxin tag).
The present disclosure also provides the following non-limiting Embodiments.
Embodiment 1. An antibody, or antigen-binding fragment
thereof,
comprising a heavy chain variable domain (VH) comprising a CDRH I , a CDRH2,
and
a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2,
and
a CDRL3, wherein:
(i) the CDRH1 comprises or consists of the amino acid
sequence according
to any one of SEQ ID NOs.: 53, 23, 33, 43, 63, 73, 83, 93, 103, 113, 123, 133,
143, 153,
163, 173, 183, 193, 203, 213, 223, 233, 243, 253, 263, 273, 283, 293, 303,
313, 323,
333, 343, 353, 363, 373, 383, 393, 403, 413, 423, or 433, or a sequence
variant thereof
comprising one, two, or three acid substitutions, one or more of which
substitutions is
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optionally a conservative substitution and/or is a substitution to a germline-
encoded
amino acid,
(ii) the CDRH2 comprises or consists of the amino acid
sequence according
to any one of SEQ ID NOs.. 54, 24, 34, 44, 64, 74, 84, 94, 104, 114, 124, 134,
144, 154,
164, 174, 184, 194, 204, 214, 224, 234, 244, 254, 264, 274, 284, 294, 304,
314, 324,
334, 344, 354, 364, 374, 384, 394, 404, 414, 424, or 434, or a sequence
variant thereof
comprising one, two, or three amino acid substitutions, one or more of which
substitutions is optionally a conservative substitution and/or is a
substitution to a
germline-encoded amino acid;
(iii) the CDRH3 comprises or consists of the amino acid sequence according
to any one of SEQ ID NOs.: 55, 25, 35, 45, 65, 75, 85, 95, 105, 115, 125, 135,
145, 155,
165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305,
315, 325,
335, 345, 355, 365, 375, 385, 395, 405, 415, 425, or 435, or a sequence
variant thereof
comprising one, two, or three amino acid substitutions, one or more of which
substitutions is optionally a conservative substitution and/or is a
substitution to a
germline-encoded amino acid;
(iv) the CDRL1 comprises or consists of the amino acid
sequence according
to any one of SEQ ID NOs.: 57, 27, 37, 47, 67, 77, 87, 97, 107, 117, 127, 137,
147, 157,
167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307,
317, 327,
337, 347, 357, 367, 377, 387, 397, 407, 417, 427, or 437, or a sequence
variant thereof
comprising one, two, or three amino acid substitutions, one or more of which
substitutions is optionally a conservative substitution and/or is a
substitution to a
germline-encoded amino acid;
(v) the CDRL2 comprises or consists of the amino acid
sequence according
to any one of SEQ ID NOs.. 58, 28, 38, 48, 68, 78, 88, 98, 108, 118, 128, 138,
148, 158,
168, 178, 188, 198, 208, 218, 228, 238, 248, 258, 268, 278, 288, 298, 308,
318, 328,
338, 348, 358, 368, 378, 388, 398, 408, 418, 428, or 438, or a sequence
variant thereof
comprising one, two, or three amino acid substitutions, one or more of which
substitutions is optionally a conservative substitution and/or is a
substitution to a
germline-encoded amino acid; and/or
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(vi) the CDRL3 comprises or consists of the amino acid
sequence according
to any one of SEQ ID NOs.: 59, 29, 39, 49, 69, 79, 89, 99, 109, 119, 129, 139,
149, 159,
169, 179, 189, 199, 209, 219, 229, 239, 249, 259, 269, 279, 289, 299, 309,
319, 329,
339, 349, 359, 369, 379, 389, 399, 409, 419, 429, or 439, or a sequence
variant thereof
comprising having one, two, or three amino acid substitutions, one or more of
which
substitutions is optionally a conservative substitution and/or is a
substitution to a
germline-encoded amino acid,
wherein the antibody or antigen binding fragment is capable of binding to a
surface glycoprotein of a SARS-CoV-2, optionally when the surface glycoprotein
is
expressed on a cell surface of a host cell and/or on a virion.
Embodiment 2. The antibody or antigen-binding fragment
of Embodiment
1, which is capable of neutralizing a SARS-CoV-2 infection in an in vitro
model of
infection and/or in an in vivo animal model of infection and/or in a human.
Embodiment 3. The antibody or antigen-binding fragment
of any one of
Embodiments 1-2, comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and
CDRL3 amino acid sequences according to SEQ ID NOs.:
(i) 53-55 and 57-59, respectively,
(ii) 33-35 and 37-39, respectively;
(iii) 43-45 and 47-49, respectively;
(iv) 23-25 and 27-29, respectively;
(v) 63-65 and 67-69, respectively;
(vi) 73-75 and 77-79, respectively;
(vii) 83-85 and 87-89, respectively;
(viii) 93-95 and 97-99, respectively;
(ix) 103-105 and 107-109, respectively
(x) 113-115 and 117-119, respectively;
(xi) 123-125 and 127-129, respectively;
(xii) 133-135 and 137-139, respectively;
(xiii) 143-145 and 147-149, respectively,
(xiv) 153-155 and 157-159, respectively;
(xv) 163-165 and 167-169, respectively;
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(xvi) 173-175 and 177-179, respectively;
(xvii) 183-185 and 187-189, respectively,
(xviii) 193-195 and 197-199, respectively;
(xix) 203-205 and 207-209, respectively;
(xx) 213-215 and 217-219, respectively;
(xxi) 223-225 and 227-229, respectively;
(xxii) 233-235 and 237-239, respectively,
(xxiii) 243-245 and 247-249, respectively;
(xxiv) 253-255 and 257-259, respectively;
(xxv) 263-265 and 267-269, respectively;
(xxvi) 273-275 and 277-279, respectively;
(xxvii) 283-285 and 287-289, respectively,
(xxviii) 293-295 and 297-299, respectively;
(xxix) 303-305 and 307-309, respectively;
(xxx) 313-315 and 317-319, respectively;
(xxxi) 323-325 and 327-329, respectively;
(xxxii) 333-335 and 337-339, respectively,
(xxxiii) 343-345 and 347-349, respectively;
(xxxiv) 353-355 and 357-359, respectively;
(xxxv) 363-365 and 367-369, respectively;
(xxxvi) 373-375 and 377-379, respectively;
(xxxvii) 383-385 and 387-389, respectively,
(xxxviii) 393-395 and 397-399, respectively;
(xxxix) 403-405 and 407-409, respectively;
(xxxx) 413-415 and 417-419, respectively,
(xxxxi) 423-425 and 427-429, respectively; or
(xxxxii) 433-435 and 437-439, respectively.
Embodiment 4
The antibody or antigen-binding fragment of any one of
Embodiments 1-3, wherein.
(i) the VH comprises or consists of an amino acid sequence having at least
85% identity to the amino acid sequence according to any one of SEQ ID NOs.:
52, 22,
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32, 42, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182 192, 202,
212, 222,
232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372,
382, 392,
402, 412, 422, and 432, wherein the variation is optionally limited to one or
more
framework regions and/or the variation comprises one or more substitution to a
germline-encoded amino acid; and/or
(ii) the VL comprises or consists of an amino acid
sequence having at least
85% identity to the amino acid sequence according to any one of SEQ ID NOs..
56, 26,
36, 46, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206,
216, 226,
236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376,
386, 396,
406, 416, 426, and 436, wherein the variation is optionally limited to one or
more
framework regions and/or the variation comprises one or more substitution to a
germline-encoded amino acid
Embodiment 5.
The antibody or antigen-binding fragment of any one of
Embodiments 1-4, wherein the VH and the VL comprise or consist of the amino
acid
sequences according to SEQ ID NOs.:
(i) 52 and 56, respectively;
(ii) 32 and 36, respectively,
(iii) 42 and 46, respectively;
(iv) 22 and 26, respectively;
(v) 62 and 66, respectively;
(vi) 72 and 76, respectively;
(vii) 82 and 86, respectively,
(viii) 92 and 96, respectively;
(ix) 102 and 106, respectively;
(x) 112 and 116, respectively,
(xi) 122 and 126, respectively;
(xii) 132 and 136, respectively;
(xiii) 142 and 146, respectively;
(xiv) 152 and 156, respectively,
(xv) 162 and 166, respectively;
(xvi) 172 and 176, respectively;
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(xvii) 182 and 186, respectively;
(xviii) 192 and 196, respectively;
(xix) 202 and 206, respectively;
(xx) 212 and 216, respectively;
(xxi) 222 and 226, respectively;
(xxii) 232 and 236, respectively;
(xxiii) 242 and 246, respectively;
(xxiv) 252 and 256, respectively;
(xxv) 262 and 266, respectively;
(xxvi) 272 and 276, respectively;
(xxvii) 282 and 286, respectively;
(xxviii) 292 and 296, respectively;
(xxix) 302 and 306, respectively;
(xxx) 312 and 316, respectively;
(xxxi) 322 and 326, respectively;
(xxxii) 332 and 336, respectively;
(xxxiii) 342 and 346, respectively,
(xxxiv) 352 and 356, respectively;
(xxxv) 362 and 366, respectively;
(xxxvi) 372 and 376, respectively;
(xxxvii) 382 and 386, respectively;
(xxxviii) 392 and 396, respectively;
(xxxix) 402 and 406, respectively;
(xxxx) 412 and 416, respectively;
(xxxxi) 422 and 426, respectively; or
(xxxxii) 432 and 436, respectively.
Embodiment 6.
The antibody or antigen-binding fragment of any one of
Embodiments 1-5, which. (i) recognizes an epitope in a Domain A of SARS-CoV-2;
(ii)
is capable of neutralizing a SARS CoV-2 infection; (iii) is capable of
eliciting at least
one immune effector function against SARS CoV-2; (iv) is capable of preventing
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shedding, from a cell infected with SARS CoV-2, of Si protein; or (v) any
combination
of (i)-(iv).
Embodiment 7. The antibody or antigen-binding fragment
of any one of
Embodiments 1-6, which is a IgG, IgA, IgM, IgE, or IgD isotype.
Embodiment 8. The antibody or antigen-binding fragment of any one of
Embodiments 1-7, which is an IgG isotype selected from IgGl, IgG2, IgG3, and
IgG4.
Embodiment 9. The antibody or antigen-binding fragment
of any one of
Embodiments 1-8, which is human, humanized, or chimeric.
Embodiment 10. The antibody or antigen-binding fragment
of any one of
Embodiments 1-9, wherein the antibody, or the antigen-binding fragment,
comprises a
human antibody, a monoclonal antibody, a purified antibody, a single chain
antibody, a
Fab, a Fab', a F(ab')2, a Fv, a scFv, or a scFab.
Embodiment 11. The antibody or antigen-binding fragment
of Embodiment
10, wherein the scFv comprises more than one VH domain and more than one VL
domain.
Embodiment 12. The antibody or antigen-binding fragment
of any one of
Embodiments 1-11, wherein the antibody or antigen-binding fragment is a
multi-specific antibody or antigen binding fragment.
Embodiment 13. The antibody or antigen-binding fragment
of Embodiment
12, wherein the antibody or antigen binding fragment is a bi specific antibody
or
antigen-binding fragment.
Embodiment 14. The antibody or antigen-binding fragment
of Embodiment
12 or 13, comprising:
(i) a first VH and a first VL; and
(ii) a second VH and a second VL,
wherein the first VH and the second VH are different and each independently
comprise an amino acid sequence having at least 85% identity to the amino acid
sequence set forth in any one of SEQ ID NOs 52, 22, 32, 42, 62, 72, 82, 92,
102, 112,
122, 132, 142, 152, 162, 172, 182 192, 202, 212, 222, 232, 242, 252, 262, 272,
282,
292, 302, 312, 322, 332, 342, 352, 362, 372, 382, 392, 402, 412, 422, and 432,
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wherein the first VL and the second VL are different and each independently
comprise an amino acid sequence having at least 85% identity to the amino acid
sequence set forth in any one of SEQ ID NOs.: 56, 26, 36, 46, 66, 76, 86, 96,
106, 116,
126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266,
276, 286,
296, 306, 316, 326, 336, 346, 356, 366, 376, 386, 396, 406, 416, 426, and 436,
and wherein the first VH and the first VL together form a first antigen-
binding
site, and wherein the second VH and the second VL together form a second
antigen-
binding site.
Embodiment 15. The antibody or antigen-binding fragment
of any one of
Embodiments 1-14, wherein the antibody or antigen-binding fragment further
comprises a Fc polypeptide or a fragment thereof
Embodiment 16. The antibody or antigen-binding fragment
of Embodiment
15, wherein the Fc polypeptide or fragment thereof comprises:
(i) a mutation that enhances binding to a FcRn as compared to a reference
Fc polypeptide that does not comprise the mutation; and/or
(ii) a mutation that enhances binding to a FcyR as compared to a reference
Fe polypeptide that does not comprise the mutation.
Embodiment 17. The antibody or antigen-binding fragment
of Embodiment
16, wherein the mutation that enhances binding to a FcRn comprises: M428L;
N434S;
N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P2571; Q311 I; D376V;
T307A; or E380A; or any combination thereof.
Embodiment 18. The antibody or antigen-binding fragment
of Embodiment
16 or 17, wherein the mutation that enhances binding to FcRn comprises:
(i) M428L/N434S;
(ii) M252Y/S254T/T256E;
(iii) T250Q/M428L;
(iv) P257I/Q3111;
(v) P2571/N434H;
(vi) D376V/N434H;
(vii) T307A/E380A/N434A; or
(viii) any combination of (i)-(vii).
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Embodiment 19. The antibody or antigen-binding fragment
of any one of
Embodiments 16-18, wherein the mutation that enhances binding to FcRn
comprises
M428L/N434S.
Embodiment 20. The antibody or antigen-binding fragment
of any one of
Embodiments 16-19, wherein the mutation that enhances binding to a FcyR
comprises
S239D; 1332E; A330L; G236A; or any combination thereof
Embodiment 21. The antibody or antigen-binding fragment
of any one of
Embodiments 16-20, wherein the mutation that enhances binding to a FcyR
comprises:
(i) S239D/I332E;
(ii) S239D/A330L/1332E;
(iii) G236A/S239D/I332E; or
(iv) G236A/A330L/1332E.
Embodiment 22. The antibody or antigen-binding fragment
of any one of
Embodiments 16-21, wherein the Fc polypeptide comprises a L234A mutation and a
L235A mutation.
Embodiment 23. The antibody or antigen-binding fragment
of any one of
Embodiments 1-22, which comprises a mutation that alters glycosylation,
wherein the
mutation that alters glycosylation comprises N297A, N297Q, or N297G, and/or
which
is aglycosylated and/or afucosylated.
Embodiment 24. An isolated polynucleotide encoding the antibody or
antigen-binding fragment of any one of Embodiments 1-23, or encoding a VH, a
heavy
chain, a VL, and/or a light chain of the antibody or the antigen-binding
fragment.
Embodiment 25. The polynucleotide of Embodiment 24,
wherein the
polynucleotide comprises deoxyribonucleic acid (DNA) or ribonucleic acid
(RNA),
wherein the RNA optionally comprises messenger RNA (mRNA).
Embodiment 26. The polynucleotide of Embodiment 24 or
25, which is
codon-optimized for expression in a host cell.
Embodiment 27 The polynucleotide of any one of
Embodiments 24-26,
comprising a polynucleotide having at least 50% identity to the polynucleotide
sequence according to any one or more of SEQ ID NOs.: 60, 61, 30, 31, 40, 41,
50, 51,
70, 71, 80, 81, 90, 91, 100, 101, 110, 111, 120, 121, 130, 131, 140, 141, 150,
151, 160,
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161, 170, 171, 180, 181, 190, 191, 200, 201, 210, 211, 220, 221, 230, 231,
240, 241,
250, 251, 260, 261, 270, 271, 280, 281, 290, 291, 300, 301, 310, 311, 320,
321, 330,
331, 340, 341, 350, 351, 360, 361, 370, 371, 380, 381, 390, 391, 400, 401,
410, 411,
420, 421, 430, 431, 440, and 441, or any combination thereof.
Embodiment 28. A recombinant vector comprising the polynucleotide of
any one of Embodiments 24-27.
Embodiment 29. A host cell comprising the
polynucleotide of any one of
Embodiments 24-27 and/or the vector of Embodiment 28, wherein the
polynucleotide is
heterologous to the host cell.
Embodiment 30. A human B cell comprising the polynucleotide of any one
of Embodiments 24-28, wherein polynucleotide is heterologous to the human B
cell
and/or wherein the human B cell is immortalized.
Embodiment 31. A composition or combination comprising:
(i) the antibody or antigen-binding fragment of any one of Embodiments 1-
23;
(ii) the polynucleotide of any one of Embodiments 24-27;
(iii) the recombinant vector of Embodiment 28,
(iv) the host cell of Embodiment 29; and/or
(v) the human B cell of Embodiment 30,
and an optional pharmaceutically acceptable excipient, carrier, or diluent.
Embodiment 32. The composition or combination of
Embodiment 31,
comprising two or more antibodies or antigen-binding fragments of any one of
Embodiments 1-23, and/or comprising one or more antibody according to any one
of
Embodiments 1-23 and an antibody or antigen-binding fragment that binds to a
SARS
CoV-2 surface glycoprotein RBD.
Embodiment 33. A composition comprising the
polynucleotide of any one
of Embodiments 24-27 encapsulated in a carrier molecule, wherein the carrier
molecule
optionally comprises a lipid, a lipid-derived delivery vehicle, such as a
liposome, a solid
lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid
microbubble,
an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid
microcylinder,
lipid nanoparticle (LNP), or a nanoscale platform.
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Embodiment 34. A method of treating a SARS-CoV-2
infection in a
subject, the method comprising administering to the subject an effective
amount of
(i) the antibody or antigen-binding fragment of any one
of Embodiments 1-
23;
(ii) the polynucleotide of any one of Embodiments 24-27;
(iii) the recombinant vector of Embodiment 28;
(iv) the host cell of Embodiment 29;
(v) the human B cell of Embodiment 30; and/or
(vi) the composition or combination of any one of Embodiments 31-33.
Embodiment 35. The antibody or antigen-binding fragment of any one of
Embodiments 1-23, the polynucleotide of any one of Embodiments 24-27, the
recombinant vector of Embodiment 28, the host cell of Embodiment 29, the human
B
cell of Embodiment 30, and/or the composition or combination of any one of
Embodiments 31-33 for use in a method of treating a SARS-CoV-2 infection in a
subject.
Embodiment 36. The antibody or antigen-binding fragment
of any one of
Embodiments 1-23, the polynucleotide of any one of Embodiments 24-27, the
recombinant vector of Embodiment 28, the host cell of Embodiment 29, the human
B
cell of Embodiment 30, and/or the composition or combination of any one of
Embodiments 31-33 for use in the preparation of a medicament for the treatment
of a
SARS-CoV-2 infection in a subject.
Embodiment 37. A method for in vitro or ex vivo
diagnosis of a SARS-
CoV-2 infection, the method comprising:
(i) contacting a sample from a subject with an antibody or antigen-binding
fragment of any one of Embodiments 1-23; and
(ii) detecting a complex comprising an antigen and the antibody, or
comprising an antigen and the antigen binding fragment.
Embodiment 38 The method of Embodiment 37, wherein the
sample
comprises blood isolated from the subject.
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Embodiment 39. An antibody, or an antigen-binding
fragment thereof, that
competes for binding to a SARS-CoV-2 surface glycoprotein with the antibody or
antigen-binding fragment of any one of Embodiments 1-23.
Embodiment 40. A method of preventing or treating or
neutralizing a
coronavirus infection in a subject, the method comprising administering to a
subject an
effective amount of (i) an antibody or antigen-binding fragment of any one of
Embodiments 1-23 or 39 and (ii) an antibody or antigen-binding fragment that
is
capable of specifically binding to a SARS CoV-2 S protein RBD.
Embodiment 41. A method of detecting a SARS-CoV-2
protein or
polypeptide in a sample, comprising contacting the sample with the antibody or
antigen-binding fragment of any one of Embodiments 1-23 or 39 and detecting
binding
of the antibody or antigen-binding fragment to the SARS-CoV-2 protein or
polypeptide
Embodiment 42. The method of Embodiment 41, wherein
detecting
binding of the antibody or antigen-binding fragment to the SARS-CoV-2 protein
or
polypeptide comprises immunohistochemistry, ELISA, agglutination, immuno-dot,
immuno-chromatography, and/or immuno-filtration
Embodiment 43. The antibody or antigen-binding fragment
thereof of any
one of Embodiments 1-23 for use in a method of detecting a SARS-CoV-2 protein
or
polypeptide in a sample, the method comprising contacting the sample with the
antibody or antigen-binding fragment and detecting binding of the antibody or
antigen-
binding fragment to the SARS-CoV-2 protein or polypeptide, wherein,
optionally,
detecting binding of the antibody or antigen-binding fragment to the SARS-CoV-
2
protein or polypepti de comprises immunohistochemistry, ELISA, agglutination,
immuno-dot, immuno-chromatography, and/or immuno-filtration.
Embodiment 44. A method of diagnosing a SARS-CoV-2 infection in a
subject, comprising testing a biological sample from the subject for the
presence of a
SARS-CoV-2 protein or polypeptide, wherein the testing comprises contacting
the
sample with the antibody or antigen-binding fragment of any one of Embodiments
1-23
and detecting binding of the antibody or antigen-binding fragment to the SARS-
CoV-2
protein or polypeptide, wherein, optionally, detecting binding of the antibody
or
antigen-binding fragment to the SARS-CoV-2 protein or polypeptide comprises
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immunohistochemistry, ELISA, agglutination, immuno-dot, immuno-chromatography,
and/or immuno-filtration.
Embodiment 45. The method of Embodiment 44, wherein the
SARS-CoV-
2 protein or polypeptide is detected by immunohistochemistry.
Embodiment 46. The method of any one of Embodiments 41-45, wherein
the sample comprises a nasal secretion, sputum, a bronchial lavage, urine,
stool, saliva,
sweat, or any combination thereof.
Embodiment 47. An antibody or antigen-binding fragment
thereof for use
in a method of diagnosing a SARS-CoV-2 infection in a subject, the method
comprising
testing a biological sample from the subject for the presence of a SARS-CoV-2
protein
or polypeptide, wherein the testing comprises contacting the sample with the
antibody
or antigen-binding fragment and detecting binding of the antibody or antigen-
binding
fragment to the SARS-CoV-2 protein or polypeptide, wherein, optionally,
detecting
binding of the antibody or antigen-binding fragment to the SARS-CoV-2 protein
or
polypeptide comprises immunohistochemistry, ELISA, agglutination, immuno-dot,
immuno-chromatography, and/or immuno-filtration, wherein, optionally, the
antibody
or antigen-binding fragment is the antibody or antigen-binding fragment
thereof of any
one of Embodiments 1-23.
Embodiment 48. The antibody or antigen-binding fragment
of any one of
Embodiments 1-23 or the antibody or antigen-binding fragment for use of
Embodiment
43 or 47, or the method of any one of Embodiments 41, 42, or 44-46, wherein
the
antibody or antigen-binding fragment comprises a detectable agent.
Embodiment 49. A kit comprising the antibody or antigen-
binding
fragment thereof of any one of Embodiments 1-23, and optional instructions for
using
the antibody or antigen-binding fragment to detect the presence of a SARS-CoV-
2
protein or polypeptide in a biological sample.
Embodiment 50. The kit according to Embodiment 49 for
use in a method
of detecting the presence of a SARS-CoV-2 protein or polypeptide in a
biological
sample.
Embodiment 5L The kit of for use of Embodiment 50, wherein the method
comprises detecting the presence of a SARS-CoV-2 protein or polypeptide by
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immunohistochemistry, ELISA, agglutination, immuno-dot, immuno-chromatography,
and/or immuno-filtration.
Embodiment 52. The kit of Embodiment 49 or the kit for
use of any one of
Embodiments 50 or 51, further comprising a detectably labeled secondary
antibody.
Embodiment 53. The kit of Embodiment 49 or the kit for use of any one of
Embodiments 50-52, further comprising one or more of a sample buffer, a wash
buffer,
an immunodetection buffer, a substrate, detection means, a control sample, a
reference
sample, and instructions for use.
Embodiment 54. The kit of Embodiment 49 or the kit for
use of any one of
Embodiments 50-53, wherein the sample comprises a nasal secretion, sputum,
bronchial
lavage, urine, stool, saliva, and/or sweat.
Embodiment 55. The composition or combination of
Embodiment 32,
comprising (a) antibody S2X333 (or an antigen-binding fragment thereof) or an
antibody or antigen-binding fragment thereof that competes with antibody
S2X333 for
SARS-CoV-2 S protein binding and (b) antibody S309 (or an antigen-binding
fragment
thereof) or an antibody or antigen-binding fragment thereof that competes with
antibody S309 for SARS-CoV-2 S protein binding.
Embodiment 56. The composition of Embodiment 32,
comprising a)
antibody S2X333 (or an antigen-binding fragment thereof) or an antibody or an
antigen-
binding fragment thereof that competes with antibody S2X333 for SARS-CoV-2 S
protein binding and b) antibody S2E12 (or an antigen-binding fragment thereof)
or an
antibody or an antigen-binding fragment thereof that competes with antibody
S2E12 for
SARS-CoV-2 S protein binding.
Embodiment 57. The composition of Embodiment 32,
comprising (a)
antibody S2X333 (or an antigen-binding fragment thereof) or an antibody or an
antigen-
binding fragment thereof that competes with antibody S2X333 for SARS-CoV-2 S
protein binding and (b) antibody S2M11 (or an antigen-binding fragment
thereof) or an
antibody or an antigen-binding fragment thereof that competes with antibody
S2M11
for SARS-CoV-2 S protein binding.
Embodiment 58. The antibody or antigen-binding fragment of Embodiment
12 or 13, comprising (i) a first VH and a first VL; and (ii) a second VH and a
second
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VL, wherein the first VH comprises an amino acid sequence having at least 85%
(i.e.,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 52 and
the first
VL comprises an amino acid sequence having at least 85% (i.e., 85%, 86%, 87%,
88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to
the
amino acid sequence set forth in SEQ ID NO: 56; and
a) the second VH comprises an amino acid sequence having at least 85% (i.e.,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 442 and
the
second VL comprises an amino acid sequence having at least 85% (i.e., 85%,
86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)
identity to the amino acid sequence set forth in SEQ ID NO: 446;
b) the second VH comprises an amino acid sequence having at least 85% (i.e.,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 450 and
the
second VL comprises an amino acid sequence having at least 85% (i.e., 85%,
86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)
identity to the amino acid sequence set forth in SEQ ID NO: 454; or
c) the second VH comprises an amino acid sequence having at least 85% (i.e.,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 458 and
the
second VL comprises an amino acid sequence having at least 85% (i.e., 85%,
86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)
identity to the amino acid sequence set forth in SEQ ID NO: 462; and
wherein the first VH and the first VL together form a first antigen-binding
site,
and wherein the second VH and the second VL together form a second antigen-
binding
site.
Embodiment 59. A method of treating or preventing SARS-
CoV-2
infection comprising administering a composition or combination of any one of
Embodiments 55-57 or the antibody or antigen-binding fragment of Embodiment
58.
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Embodiment 60. The composition or combination of any
one of
Embodiments 55-57, wherein, optionally the antibody or antigen-binding
fragment of a)
and/or b) comprises (i) a Fc polypeptide comprising a mutation that enhances
binding to
a FcRn as compared to a reference Fc polypeptide that does not comprise the
mutation;
and/or (ii) a Fc polypeptide comprising a mutation that enhances binding to a
FcyR as
compared to a reference Fc polypeptide that does not comprise the mutation.
Embodiment 61. The antibody or antigen-binding fragment
of Embodiment
58, or the method of Embodiment 59, wherein, optionally, the antibody or
antigen-
binding fragment comprises (i) a Fc polypeptide comprising a mutation that
enhances
binding to a FcRn as compared to a reference Fc polypeptide that does not
comprise the
mutation; and/or (ii) a Fc polypeptide comprising a mutation that enhances
binding to a
FcyR as compared to a reference Fc polypeptide that does not comprise the
mutation.
Table 1. Sequences
SEQ
Sequence
ID Sequence
Description
NO.
1 attaaaggtt tataccttcc caggtaacaa accaaccaac tttcgatctc
ttgtagatct 61 gttctctaaa cgaactttaa aatctgtgtg gctgtcactc
ggctgcatgc ttagtgcact 121 cacgcagtat aattaataac
taattactgt cgttgacagg acacgagtaa ctcgtctatc 181
ttctgcaggc tgcttacggt ttcgtccgtg ttgcagccga tcatcagcac
atctaggttt 241 cgtccgggtg tgaccgaaag gtaagatgga
SARS-CoV-2 gagccttgtc cctgghtca acgagaaaac 301
acacgtccaa
Wuhan seafood ctcagthgc ctghttaca ggttcgcgac
gtgctcgtac gtggctttgg
market pneumonia 361 agactccgtg gaggaggtct tatcagaggc
cgtcaacat
virus isolate cttaaagatg gcacttgtgg 421 cttagtagaa
gttgaaaaag
Wuhan-Hu-1 1 gcgttttgcc tcaacttgaa cagccctatg
tgttcatcaa 481
genomic sequence acgttcggat gctcgaactg cacctcatgg
tcatgttatg gttgagctgg
(GenBank: tagcagaact 541 cgaaggcatt cagtacggtc
gtagtggtga
MN908947.3;Janu gacacttggt gtccttgtcc ctcatgtggg 601
cgaaatacca
ary 23, 2020) gtggcttacc gcaaggttct tcttcgtaag
aacggtaata aaggagctgg
661 tggccatagt tacggcgccg atctaaagtc atttgactta
ggcgacgagc ttggcactga 721 tccttatgaa gattlicaag
aaaactggaa cactaaacat agcagtggtg ttacccgtga 781
actcatgcgt gag cttaacg gaggggcata cactcgctat gtcgataaca
acttctgtgg 841 ccctgatggc taccctcttg agtgcattaa
agaccttcta gcacgtgctg gtaaagcttc 901 atgcactttg
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SEQ
Sequence
ID Sequence
Description
NO.
tccgaacaac tggactttat tgacactaag aggggtgtat actgctgccg
961 tgaacatgag catgqnattg cttggtacac ggaacgttct
gaaaagagct atgaattgca 1021 gacacc Lilt gaaattaaat
tggcaaagaa atttgacacc ttcaatgggg aatgtccaaa 1081
ttttgtattt cccttaaatt ccataatcaa gactattcaa ccaagggttg
aaaagaaaaa 1141 gcttgatggc tttatgggta gaattcgatc
tgtctatcca gttgcgtcac caaatgaatg 1201 caaccaaatg
tgcctttcaa ctctcatgaa gtgtgatcat tgtggtgaaa cttcatggca
1261 gacgggcgat tttgttaaag ccacttgcga attilgtggc
actgagaatt tgactaaaga 1321 aggtgccact acttgtggtt
acttacccca aaatgctgtt gttaaaattt attgtccagc 1381
atgtcacaat tcagaagtag gacctgagca tagtcttgcc gaataccata
atgaatctgg 1441 cttgaaaacc attcttcgta agggtggtcg
cactattgcc tttggaggct gtgtgttctc
1501 ttatgttggt tgccataaca agtgtgccta ttgggttcca
cgtgctagcg ctaacatagg 1561 ttgtaaccat acaggtgttg
ttggagaagg ttccgaaggt cttaatgaca accttcttga 1621
aatactccaa aaagagaaag tcaacatcaa tattgttggt gactttaaac
ttaatgaaga 1681 gatcgccatt attliggcat clittictgc ttccacaagt
gcttttgtgg aaactgtgaa 1741 aggtttggat tataaagcat
tcaaacaaat tgttgaatcc tgtggtaatt ttaaagttac
1801 aaaaggaaaa gctaannaag gtgcctggaa tattggtgaa
cagaaatcaa tactgagtcc 1861 tctttatgca tttgcatcag
aggctgctcg tgttgtacga tcaattttct cccgcactct 1921
tgaaactgct caaaattctg tgcgtgtttt acagaaggcc gctataacaa
tactagatgg 1981 aatttcacag tattcactga gactcattga
tgctatgatg ttcacatctg atttggctac 2041 taacaatcta
gttgtaatgg cctacattac aggtggtgtt gttcagttga cttcgcagtg
2101 gctaactaac atctttggca ctgtttatga annactcaaa
cccgtccttg attggcttga 2161 agagaagttt aaggaaggtg
tagagtact tagagacggt tgggaaattg ttaaatttat 2221
ctcaacctgt gcttgtgaaa ttgtcggtgg acaaattgtc acctgtgcaa
aggaaattaa 2281 ggagagtgtt cagacattct ttaagcttgt
aaataaattt ttggctttgt gtgctgactc 2341 tatcattatt ggtggagcta
aacttaaagc cttgaattta ggtgaaacat ttgtcacgca
2401 ctcaaaggga ttgtacagaa agtgtgttaa atccagagaa
gaaactggcc tactcatgcc 2461 tctaaaagcc ccaaaagaaa
ttatcttctt agagggagaa acacttccca cagaagtgtt 2521
aacagaggaa gttgtcttga aaactggtga tttacaacca ttagaacaac
ctactagtga 2581 agctgttgaa gctccattgg ttggtacacc
agtttgtatt aacgggctta tgttgctcga 2641 aatcaaagac
acagaaaagt actgtgccct tgcacctaat atgatggtaa caaacaatac
2701 cttcacactc aaaggcggtg caccaacaaa ggttactitt
ggtgatgaca ctgtgataga 2761 agtgcaaggt tacaagagtg
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SEQ
Sequence
ID Sequence
Description
NO.
tgaatatcac ttttgaactt gatgaaagga ttgataaagt 2821
acttaatgag aagtgctctg cctatacagt tgaactcggt acagaagtaa
atgagttcgc 2881 ctgtgttgtg gcagatgctg tcataaaaac
tttgcaacca gtatctgaat tacttacacc
2941 actgggcatt gatttagatg agtggagtat ggctacatac
tacttatttg atgagtctgg 3001 tgagtttaaa ttggcttcac atatgtattg
ttctttctac cctccagatg aggatgaaga 3061 agaaggtgat
tgtgaagaag aagagifiga gccatcaact caatatgagt atggtactga
3121 agatgattac caaggtaaac ctttggaatt tggtgccact
tctgctgctc ttcaacctga 3181 agaagagcaa gaagaagatt
ggttagatga tgatagtcaa caaactgttg gtcaacaaga 3241
cggcagtgag gacaatcaga caactactat tcaaacaatt gttgaggttc
aacctcaatt 3301 agagatggaa cttacaccag ttgttcagac
tattgaagtg aatagtitta gtggttatrt 3361 aaaacttact gacaatgtat
acattaaaaa tgcagacatt gtggaagaag ctaaaaaggt 3421
aaaaccaaca gtggttgtta atgcagccaa tgtttacctt aaacatggag
gaggtgttgc
3481 aggagcctta aataaggcta ctaacaatgc catgcaagtt
gaatctgatg attacatagc 3541 tactaatgga ccacttaaag
tgggtggtag ttgtgtttta agcggacaca atcttgctaa 3601
acactgtctt catgttgtcg gcccaaatgt taacaaaggt gaagacattc
aacttcttaa 3661 gagtgcttat ganaatttta atcagcacga
agttctactt gcaccattat tatcagctgg 3721 tatilliggt gctgacccta
tacattcttt aagagtttgt gtagatactg ttcgcacaaa
3781 tgtctactta gctgtattg ataaaaatct ctatgacaaa cttgtttcaa
gcitiligga 3841 aatgaagagt gaaaagcaag ttgaacaaaa
gatcgctgag attcctaaag aggaagttaa
3901 gccatttata actgaaagta aaccttcagt tgaacagaga
aaacaagatg ataagaaaat 3961 caaagcttgt gttgaagaag
ttacaacaac tctggaagaa actaagttcc tcacagaaaa 4021
cttgttactt tatattgaca ttaatggcaa tcttcatcca gattctgcca
ctcttgttag 4081 tgacattgac atcactttct taaagaaaga
tgctccatat atagtgggtg atgttgttca 4141 agagggtgtt
ttaactgctg tggttatacc tactaaaaag gctggtggca ctactgaaat
4201 gctagcgaaa gctttgagaa aagtgccaac agacaattat
ataaccactt acccgggtca 4261 gggtttaaat ggttacactg
tagaggaggc aaagacagtg cttaaaaagt gtaaaagtgc
4321 caltacatt ctaccatcta ttatctctaa tgagaagcaa gaaattcttg
gaactgtttc 4381 ttggaatttg cgagaaatgc ttgcacatgc
agaagaaaca cgcaaattaa tgcctgtctg
4441 tgtggaaact aaagccatag tttcaactat acagcgtaaa
tataagggta ttaaaataca 4501 agagggtgtg gttgattatg
gtgetagatt ttaattlac accagtaaaa caactgtagc 4561
gtcacttatc aacacactta acgatctaaa tgaaactctt gttacaatgc
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SEQ
Sequence
ID Sequence
Description
NO.
cacttggcta 4621 tgtaacacat ggcttaaatt tggaagaagc
tgctcggtat atgagatctc tcaaagtgcc 4681 agctacagtt
tctgtttctt cacctgatgc tgttacagcg tataatggtt atcttacttc
4741 acttctaaa acacctgaag aacattttat tgaaaccatc
tcacttgctg gttcctataa 4801 agattggtcc tattctggac
aatctacaca actaggtata gaatttctta agagaggtga
4861 taanagtgta tattacacta gtaatcctac cacattccac
ctagatggtg aagttatcac 4921 ctttgacaat cttaagacac ttctttcttt
gagagaagtg aggactatta aggtgtttac 4981 aacagtagac
aacattaacc tccacacgca agttgtggac atgtcaatga catatggaca
5041 acagt-ttggt ccaacttatt tggatggagc tgatgttact
aaaataaaac ctcataattc 5101 acatgaaggt aaaacatill
atglittacc taatgatgac actctacgtg ttgaggcttt 5161
tgagtactac cacacaactg atcctagttt tctgggtagg tacatgtcag
cattaaatca 5221 cactaaaaag tggaaatacc cacaagt-taa
tggtttaact tctattinnt gggcagataa
5281 caactgttat cttgccactg cattgttaac actccaacaa
atagagttga agtttaatcc 5341 acctgctcta caagatgctt
attacagagc aagggctggt gaagctgcta actIllgtgc
5401 acttatctta gcctactgta ataagacagt aggtgagtta
ggtgatgtta gaganacaat 5461 gagttacttg tttcaacatg
ccaatttaga ttcttgcaaa agagtcttga acg-tggtgtg 5521
taaaacttgt ggacaacagc agacaaccct taagggtgta gaagctgtta
tgtacatggg 5581 cacacifict tatgaacaat ttaagaaagg
tgttcagata ccttgtacgt gtggtaaaca 5641 agctacaaaa
tatctagtac aacaggagtc accttttgtt atgatgtcag caccacctgc
5701 tcagtatgaa cttaagcatg gtacatttac ttgtgctagt
gagtacactg gtaattacca 5761 gtgtggtcac tataaacata
taacttctaa agaaactttg tattgcatag acggtgcttt
5821 acttacaaag tcctcagaat acaaaggtcc tattacggat
g ttlictaca aagaaaacag 5881 ttacacaaca accataaaac
cagttactta taaattggat ggtgttgttt gtacagaaat 5941
tgaccctaag ttggacaatt attataagaa agacaattct tatttcacag
agcaaccaat 6001 tgatcttgta ccaaaccaac catatccaaa
cgcaagcttc gataatitta agtttgtatg 6061 tgataatatc aaatttgctg
atgatttaaa ccagttaact ggttataaga aacctgcttc
6121 aagagagctt aaagttacat ttttccctga cttaaatggt
gatgtggtgg ctattgatta 6181 taaacactac acaccctctt
ttaagaaagg agctaaattg ttacataaac ctattgtttg 6241
gcatgttaac aatgcaacta ataaagccac gtataaacca aatacctggt
gtatacgttg 6301 tctttggagc acaaaaccag ttganacatc
aaattcgttt gatgtactga agtcagagga 6361 cgcgcaggga
atggataatc ttgcctgcga agatctaaaa ccagtctctg aagaagtagt
6421 gganaatcct accatacaga aagacglict tgagtgtaat
91
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/ITS2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
gtgaaaacta ccgaagttgt 6481 aggagacatt atacttaaac
cagcaaataa tagtttaaaa attacagaag aggttggcca 6541
cacagatcta atggctgctt atgtagacaa ttctagtctt actattaaga
aacctaatga 6601 attatctaga gtattaggtt tgaaaaccct
tgctactcat ggtttagctg ctgttaatag
6661 tgtcccttgg gatactatag ctaattatgc taagcctttt
cttaacaaag ttgttagtac 6721 aactactaac atagttacac
ggtgtttaaa ccgtgtttgt actaattata tgccttattt 6781 ctttacttta
ttgctacaat tgtgtacttt tactagaagt acaaattcta gaattaaagc
6841 atctatgccg actactatag caaagaatac tgttaagagt
gtcggtaaat ttigtctaga 6901 ggcttcattt aattatttga agtcacctaa
lltttctaaa ctgataaata ttataatttg 6961 gttlttacta ttaagtgttt
gcctaggttc tttaatctac tcaaccgctg ctttaggtgt 7021 tttaatgtct
aatttaggca tgccttctta ctgtactggt tacagagaag gctatttgaa
7081 ctctactaat gtcactattg caacctactg tactggttct ataccttgta
gtgtttgtct
7141 tagtggttta gattctttag acacctatcc ttctttagaa actatacaaa
ttaccatttc 7201 atcttttaaa tgggatttaa ctgcttttgg cttagttgca
gagtggtttt tggcatatat
7261 tcttttcact aggittlict atgtacttgg attggctgca atcatgcaat
tglitticag 7321 ctattligca gtacatttta ttagtaattc ttggcttatg
tggttaataa ttaatcttgt 7381 acaaatggcc ccgatttcag
ctatggttag aatgtacatc ttctttg cat cattttatta 7441 tgtatggaaa
agttatgtgc atgttgtaga cggttgtaat tcatcaactt gtatgatgtg
7501 ttacaaacgt aatagagcaa caagagtcga atgtacaact
attgttaatg gtgttagaag
7561 gtccttttat gtctatgcta atggaggtaa aggcttttgc
aaactacaca attggaattg 7621 tgttaattgt gatacattct
gtgctggtag tacatttatt agtgatgaag ttgcgagaga 7681
cttgtcacta cagtttaaaa gaccaataaa tcctactgac cagtcttctt
acatcgttga 7741 tagtgttaca gtgaagaatg gttccatcca
tctttacttt gataaagctg gtcaaaagac 7801 ttatgaaaga
cattctctct ctcattttgt taacttagac aacctgagag ctaataacac
7861 taaagglica ttgcctatta atgttatagt ttitgatggt aaatcaaaat
gtgaagaatc 7921 atctgcaaaa tcagcgtctg tttactacag
tcagcttatg tgtcaaccta tactgttact 7981 agatcaggca
ttagtgtctg atgttggtga tagtgcggaa gttgcagtta aaatgtttga
8041 tgcttacgtt aatacg LIII catcaacttt taacgtacca
atggaa,aaac tcaaaacact 8101 agttgcaact gcagaagctg
aacttgcaaa gaatgtgtcc ttagacaatg tcttatctac
8161 ttttatttca gcagctcggc aagggtttgt tgattcagat
gtagaaacta aagatgttgt 8221 tgaatgtctt aaattgtcac
atcaatctga catagaagtt actggcgata gttgtaataa 8281
ctatatgctc acctataaca aagttgaaaa catgacaccc cgtgaccttg
92
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
gtgcttgtat 8341 tgactgtagt gcgcgtcata ttaatgcgca
ggtagcqnaa agtcacaaca ttgctttgat 8401 atggaacgtt
aaagatttca tgtcattgtc tgaacaacta cgaaaacaaa tacgtagtgc
8461 tgctaaaaag aataacttac cttttaagtt gacatgtgca
actactagac aagttgttaa 8521 tgttgtaaca acaaagatag
cacttaaggg tggtaaaatt gttaataatt ggttgaagca 8581
gttaattaaa gttacacttg tgttcctttt tgttgctgct attttctatt
taataacacc 8641 tgttcatgtc atgtctaaac atactgactt
ttcaagtgaa atcataggat acaaggctat 8701 tgatggtggt
gtcactcgtg acatagcatc tacagatact tgttttgcta acaaacatgc
8761 tgaLLILgac acatggttta gccagcgtgg tggtagttat
actaatgaca aagcttgccc 8821 attgattgct gcagtcataa
caagagaagt gggittlgtc gtgcctggtt tgcctggcac 8881
gatattacgc acaactaatg gtgacttlit gcatttctta cctagagttt
ttagtgcagt 8941 tggtaacatc tgttacacac catcaaaact
tatagagtac actgactttg caacatcagc 9001 ttgtgttttg
gctgctgaat gtacaatill taaagatgct tctggtaagc cagtaccata
9061 ttgttatgat accaatgtac tagaaggttc tgttgcttat gaaagtttac
gccctgacac 9121 acgttatgtg ctcatggatg gctctattat
tcaatttcct aacacctacc ttgaaggttc 9181 tgttagagtg
gtaacaactt ttgattctga gtactgtagg cacggcactt gtgaaagatc
9241 agaagctggt gtttgtgtat ctactagtgg tag atgggta
cttaacaatg attattacag 9301 atctttacca ggaglitict
gtggtgtaga tgctgtaaat ttacttacta atatgtttac
9361 accactaatt caacctattg gtgclligga catatcagca
tctatagtag ctggtggtat 9421 tgtagctatc gtagtaacat
gccttgccta ctattttatg aggtttagaa gagclitAgg 9481
tgaatacagt catgtagttg cctttaatac tttactattc cttatgtcat
tcactgtact 9541 ctgtttaaca ccagtttact cattcttacc tggtgtttat
tctgttattt acttgtactt 9601 gacattttat cttactaatg atgtttcttt
tttagcacat attcagtgga tggttatgtt 9661 cacaccttta gtacctttct
ggataacaat tgcttatatc atttgtattt ccacaaagca 9721 tttctattgg
ttctttagta attacctaaa gagacgtgta gtctttaatg gtgtttcctt 9781
tagtactill gaagaagctg cgctgtgcac cittligtta aataaagaaa
tgtatctaaa
9841 gttgcgtagt gatgtgctat tacctcttac gcaatataat
agatacttag ctctttataa 9901 taagtacaag tattttagtg
gagcaatgga tacaactagc tacagagaag ctgcttgttg 9961
tcatctcgca aaggctctca atgacttcag taactcaggt tctgatgttc
tttaccaacc 10021 accacaaacc tctatcacct cagctgtttt
gcagagtggt tttagaaaaa tggcattccc 10081 atctggtana
gttgagggtt gtatggtaca agtaacttgt ggtacaacta cacttaacgg
10141 tctttggctt gatgacgtag tttactgtcc aagacatgtg
atctgcacct ctgaagacat 10201 gcttaaccct aattatgaag
93
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/ITS2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
atttactcat tcgtaagtct aatcataatt tcttggtaca 10261
ggctggtaat gttcaactca gggttattgg acattctatg caaaattgtg
tacttaagct 10321 taaggttgat acagccaatc ctaagacacc
taagtataag tttgttcgca ttcaaccagg 10381 acagactttt
tcagtgttag cttgttacaa tggttcacca tctggtgttt accaatgtgc
10441 tatgaggccc aatttcacta ttaagggttc attccttaat
ggttcatgtg gtagtgttgg 10501 ttttaacata gattatgact
gtgtctcttt ttgttacatg caccatatgg aattaccaac 10561
tggagttcat gctggcacag acttagaagg taacttttat ggaccillig
ttgacaggca 10621 aacagcacaa gcagctggta cggacacaac
tattacagtt aatglittag cttggttgta 10681 cgctgctgtt
ataaatggag acaggtggtt tctcaatcga tttaccacaa ctcttaatga
10741 ctttaacctt gtggctatga agtacaatta tgaacctcta
acacaagacc atgttgacat 10801 actaggacct ctttctgctc
aaactggaat tgccglltla gatatgtgtg cttcattaaa 10861
agaattactg caaaatggta tgaatggacg taccatattg ggtagtgctt
tattagaaga 10921 tgaatttaca ccttttgatg ttgttagaca
atgctcaggt gttactttcc aaagtgcagt 10981 gagaagaaca
atcaagggta cacaccactg gttgttactc acaalittga cttcactitt
11041 aglillagtc cagagtactc aatggtcttt gttc111111 ttgtatgaaa
atgccILLIL 11101 acctillgct atgggtatta ttgctatgtc tgcttttgca
atgatgtttg tcaaacataa 11161 gcatgcattt ctctgtttgt ttttgttacc
ttctcttgcc actgtagctt attttaatat 11221 ggtctatatg cctgctagtt
gggtgatgcg tattatgaca tggttggata tggttgatac 11281
tagtttgtct ggttttaagc taaaagactg tgttatgtat gcatcagctg
tagtgttact
11341 aatccttatg acagcaagaa ctgtgtatga tgatggtgct
aggagagtgt ggacacttat 11401 gaatgtcttg acactcgttt
ataaagttta ttatggtaat gctttagatc aagccatttc 11461
catgtgggct cttataatct ctgttacttc taactactca ggtgtagtta
caactgtcat 11521 gtttliggcc agaggtattg ittilatgtg
tgttgagtat tgccctattt tcttcataac 11581 tggtaataca
cttcagtgta taatgctagt ttattgtttc ttaggctatt tttgtacttg
11641 ttactttggc ctcttttgtt tactcaaccg ctactttaga ctgactcttg
gtgtttatga 11701 ttacttagtt tctacacagg agtttagata
tatgaattca cagggactac tcccacccaa 11761 gaatagcata
gatgccttca aactcaacat taaattgttg ggtgttggtg gcanaccttg
11821 tatcaaagta gccactgtac agtctaaaat gtcagatgta
aagtgcacat cagtagtctt 11881 actctcagtt ttgcaacaac
tcagagtaga atcatcatct aaattgtggg ctcaatgtgt
11941 ccagttacac aatgacattc tcttagctaa agatactact
gaagcctttg aaaaaatggt 12001 ttcactactt tctgtLLLgc
tttccatgca gggtgctgta gacataaaca agctttgtga 12061
agaaatgctg gacaacaggg caaccttaca agctatagcc tcagagttta
94
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
gttcccttcc 12121 atcatatgca gcttttgcta ctgctcaaga
agcttatgag caggctgttg ctaatggtga 12181 ttctgaagtt
gttcttaaaa agttgaagaa gtctttgaat gtggctaaat ctgaatttga
12241 ccgtgatgca gccatgcaac gtaagttgga aaagatggct
gatcaagcta tgacccaaat 12301 gtataaacag gctagatctg
aggacaagag ggcaaaagtt actagtgcta tgcagacaat 12361
gcttttcact atgcttagaa agttggataa tgatgcactc aacaacatta
tcaacaatgc 12421 aagagatggt tgtgttccct tgaacataat
acctcttaca acagcagcca aactaatggt
12481 tgtcatacca gactataaca catataaaaa tacgtgtgat
ggtacaacat ttacttatgc 12541 atcagcattg tgggaaatcc
aacaggttgt agatgcagat agtaaaattg ttcaacttag 12601
tgaaattagt atggacaatt cacctaattt agcatggcct cttattgtaa
cagctttaag 12661 ggccaattct gctgtcaaat tacagaataa
tgagcttagt cctgttgcac tacgacagat 12721 gtcttgtgct
gccggtacta cacaaactgc ttgcactgat gacaatgcgt tagcttacta
12781 caacacaaca aagggaggta ggtttgtact tgcactgtta
tccgatttac aggatttgaa 12841 atgggctaga ttccctaaga
gtgatggaac tggtactatc tatacagaac tggaaccacc 12901
ttgtaggttt gttacagaca cacctaaagg tcctaaagtg aagtatttat
actttattaa 12961 aggattaaac aacctaaata gaggtatggt
acttggtagt ttagctgcca cagtacgtct 13021 acaagctggt
aatgcaacag aagtgcctgc caattcaact gtattatctt tctg(gcttt
13081 tgctgtagat gctgctaaag cttacaaaga ttatctagct
agtgggggac naccaatcac 13141 taattgtgtt aagatgttgt
gtacacacac tggtactggt caggcaataa cagttacacc 13201
ggaagccaat atggatcaag aatcctttgg tggtgcatcg tgttgtctgt
actgccgttg 13261 ccacatagat catccaaatc ctaaaggatt
ttgtgactta aaaggtaagt atgtacnnat 13321 acctacaact
tgtgctaatg accctgtggg tillacactt aaaaacacag tctgtaccgt
13381 ctgcggtatg tggaaaggtt atggctgtag ttgtgatcaa
ctccgcgaac ccatgcttca 13441 gtcagctgat gcacaatcgt
ttttnnacgg gtttgcggtg taagtgcagc ccgtcttaca 13501
ccgtgcggca caggcactag tactgatgtc gtatacaggg cttttgacat
ctacaatgat 13561 aaagtagctg glittgctaa attcctaaaa
actaattgtt gtcgcttcca agnnaaggac
13621 gaagatgaca atttaattga ttcttacttt gtagttaaga
gacacacttt ctctaactac 13681 caacatgaag aaacaattta
taatttactt aaggattgtc cagctgttgc taaacatgac 13741
ttctttaagt ttagaataga cggtgacatg gtaccacata tatcacgtca
acgtcttact 13801 aaatacacaa tggcagacct cgtctatgct
ttaaggcatt ttgatgaagg taattgtgac 13861 acattaaaag
nnatacttgt cacatacaat tgttgtgatg atgattattt caatannaag
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
13921 gactggtatg attligtaga aaacccagat atattacgcg
tatacgccaa cttaggtgaa 13981 cgtgtacgcc aagctttgtt
aaaaacagta caattctgtg atgccatgcg aaatgctggt 14041
attgttggtg tactgacatt agataatcaa gatctcaatg gtaactggta
tgatttcggt 14101 gatttcatac aaaccacgcc aggtagtgga
gttcctgttg tagattctta ttattcattg 14161 ttaatgccta tattaacctt
gaccagggct ttaactgcag agtcacatgt tgacactgac
14221 ttaacaaagc cttacattaa gtgggatttg ttaaaatatg
acttcacgga agagaggtta 14281 aaactattg accgttattt
taaatattgg gatcagacat accacccaaa ttgtgttaac 14341
tgtt-tggatg acagatgcat tctgcattgt gcaaactt-ta atgttl-tatt
ctctacagtg 14401 ttcccaccta caagttttgg accactagtg
agaaaaatat ttgttgatgg tgttccattt 14461 gtagtttcaa
ctggatacca cttcagagag ctaggtgttg tacataatca ggatgtaaac
14521 ttacatagct ctagacttag altaaggaa ttacttgtgt
atgctgctga ccctgctatg 14581 cacgctgctt ctggtaatct
attactagat aaacgcacta cgtgcttttc agtagctgca 14641
cttactaaca atgttgctl-t tcaaactgtc aaacccggta att-ttaacaa
agacttctat 14701 gactttgctg tgtctaaggg tttctttaag
gaaggaagtt ctgttgaatt aaaacacttc 14761 ttctttgctc
aggatggtaa tgctgctatc agcgattatg actactatcg ttataatcta
14821 ccaacaatgt gtgatatcag acaactacta tttgtagttg
aagttgttga taagtacttt 14881 gattgttacg atggtggctg
tattaatgct aaccaagtca tcgtcaacaa cctagacaaa 14941
tcagctggtt ttccatt-taa tagatggggt aaggctagac tttattatga
ttcaatgagt 15001 tatgaggatc aagatgcact tttcgcatat
acaanacgta atgtcatccc tactataact 15061 caaatgaatc
ttaagtatgc cattagtgca aagaatagag ctcgcaccgt agctggtgtc
15121 tctatctgta gtactatgac caatagacag tttcatcaaa
aattattgaa atcaatagcc 15181 gccactagag gagctactgt
agtaattgga acaagcaaat tctatggtgg ttggcacaac 15241
atgttaaaaa ctgtttatag tgatgtagaa aaccctcacc ttatgggttg
ggattatcct 15301 aaatgtgata gagccatgcc taacatgctt
agaattatgg cctcacttgt tcttgctcgc
15361 aaacatacaa cgtgttgtag cttgtcacac cgtttctata
gattagctaa tgagtgtgct 15421 caagtattga gtgaaatggt
catgtgtggc ggttcactat atgttaaacc aggtggaacc 15481
tcatcaggag atgccacaac tgcttatgct aatagtgt[t ttaacatttg
tcaagctgtc 15541 acggccaatg ttaatgcact tttatctact
gatggtaaca aaattgccga taagtatgtc 15601 cgcaatttac
aacacagact ttatgagtgt ctctatagaa atagagatgt tgacacagac
15661 tttgtgaatg agattacgc atatrtgcgt aaacatact
caatgatgat actctctgac 15721 gatgctgttg tgtgtttcaa
tagcacttat gcatctcaag gtctagtggc tagcataaag 15781
96
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
aactttaagt cagttcttta ttatcaaaac aatgattla tgtctgaagc
qaaatgttgg 15841 actgagactg accttactaa aggacctcat
gaatttlgct ctcaacatac aatgctagtt 15901 aaacagggtg
atgattatgt gtaccttcct tacccagatc catcaagaat cctaggggcc
15961 ggctgittig tagatgatat cgtaaaaaca gatggtacac
ttatgattga acggttcgtg 16021 tctttagcta tagatgctta
cccacttact aaacatccta atcaggagta tgctgatgtc 16081
tttcatttgt acttacaata cataagaaag ctacatgatg agttaacagg
acacatgtta 16141 gacatgtatt ctgttatgct tactaatgat
aacacttcaa ggtattggga acctgagttt 16201 tatgaggcta
tgtacacacc gcatacagtc ttacaggctg ttggggcttg tgttctttgc
16261 aattcacaga cttcattaag atgtggtgct tgcatacgta
gaccattctt atgttgtaaa 16321 tgctgttacg accatgtcat
atcaacatca cataaattag tcttgtctgt taatccgtat 16381
gtttgcaatg ctccaggttg tgatgtcaca gatgtgactc aactttactt
aggaggtatg 16441 agctattatt gtaaatcaca taaaccaccc
attaglittc cattgtgtgc taatggacaa 16501 glitaggtt
tatataqaaa tacatgtgtt ggtagcgata atgttactga ctttaatgca
16561 attgcaacat gtgactggac aaatgctggt gattacattt
tag ctaacac ctgtactgaa 16621 agactcaagc Ettagcagc
agaaacgctc aaagctactg aggagacatt taaactgtct 16681
tatggtattg ctactgtacg tgaagtgctg tctgacagag aattacatct
ttcatgggaa 16741 gttggtaaac ctagaccacc acttaaccga
aattatgtct ttactggtta tcgtgtaact 16801 aaaaacagta
aagtacaqat aggagagtac acctttgaaa aaggtgacta tggtgatgct
16861 gttgtttacc gaggtacaac aacttacaaa ttaaatgttg
gtgattattt tgtgctgaca 16921 tcacatacag taatgccatt
aagtgcacct acactagtgc cacaagagca ctatgttaga
16981 attactggct tatacccaac actcaatatc tcagatgagt
tttctagcaa tgttgcaaat 17041 tatcqaaagg ttggtatgca
aaagtattct acactccagg gaccacctgg tactggtaag
17101 agtcatittg ctattggcct agctctctac tacccttctg
ctcgcatagt gtatacagct 17161 tgctctcatg ccgctgttga
tgcactatgt gagaaggcat taaaatattt gcctatagat 17221
aaatgtagta gaattatacc tgcacgtgct cgtgtagagt gttttgataa
attcaaagtg 17281 aattcaacat tagaacagta tgtctittgt
actgtaaatg cattgcctga gacgacagca 17341 gatatagttg
tctttgatga aatttcaatg gccacaaatt atgatttgag tgttgtcaat
17401 gccagattac gtgctaagca ctatgtgtac attggcgacc
ctgctcaatt acctgcacca 17461 cgcacattgc taactaaggg
cacactagaa ccagaatatt tcaattcagt gtgtagactt 17521
atgaaaacta taggtccaga catgttcctc ggaacttgtc ggcgttgtcc
tgctgaaatt 17581 gttgacactg tgagtgcttt ggtttatgat
97
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
aataagctta aagcacataa agacaaatca 17641 gctcaatgct
ttaaaatgtt ttataagggt gttatcacgc atgatgtttc atctgcaatt
17701 aacaggccac anataggcgt ggtaagagaa ttccttacac
gtaaccctgc ttggagaaaa 17761 gctgtcttta tttcacctta
taattcacag aatgctgtag cctcaaagat tttgggacta
17821 ccaactcaaa ctgttgattc atcacagggc tcagaatatg
actatgtcat attcactcaa 17881 accactgaaa cagctcactc
ttgtaatgta aacagattta atgttgctat taccagagca 17941
aaagtaggca tactttgcat aatgtctgat agagaccttt atgacapEtt
gcaatttaca 18001 agtcttgaaa ttccacgtag gaatgtggca
actttacaag ctgaaaatgt aacaggactc 18061 tttaaagatt
gtagtaaggt aatcactggg ttacatccta cacaggcacc tacacacctc
18121 agtgttgaca ctaaattcaa aactgaaggt Uatgtgttg
acatacctgg catacctaag 18181 gacatgacct atagaagact
catctctatg atgggtttta aaatgaatta tcaagttaat
18241 ggttacccta acatgtttat cacccgcgaa gaagctataa
gacatgtacg tgcatggatt 18301 ggcttcgatg tcgaggggtg
tcatgctact agagaagctg ttggtaccaa tttaccttta 18361
cagctaggtt tttctacagg tgttaaccta gttgctgtac ctacaggtta
tgttgataca 18421 cctaataata cagattlitc cagagttagt
gctaaaccac cgcctggaga tcaatttaaa 18481 cacctcatac
cacttatgta caaaggactt ccttggaatg tagtgcgtat aaagattgta
18541 caaatgttaa gtgacacact taaaaatctc tctgacagag
tcgtatttgt cttatgggca 18601 catggctttg agttgacatc
tatgaagtat tftgtgaaaa taggacctga gcgcacctgt
18661 tgtctatgtg atagacgtgc cacatgcttt tccactgctt
cagacactta tgcctgttgg 18721 catcattcta ttggatttga
ttacgtctat aatccgttta tgattgatgt tcaacaatgg 18781
ggttttacag gtaacctaca aagcaac cat gatctgtatt gtcaagtcca
tggtaatgca 18841 catgtagcta gttgtgatgc aatcatgact
aggtgtctag ctgtccacga gtgctttgtt 18901 aagcgtgttg
actggactat tgaatatcct ataattggtg atgaactgaa gattaatgcg
18961 gcttgtagaa aggttcaaca catggttgtt aaagctgcat
tattagcaga caaattccca 19021 gttcttcacg acattggtaa
ccctaaagct attaagtgtg tacctcaagc tgatgtagaa
19081 tggaagttct atgatgcaca gccttgtagt gacaaagctt
ataaaataga agaattattc 19141 tattcttatg ccacacattc
tgacaaattc acagatggtg tatgcctatt ttggaattgc 19201
aatgtcgata gatatcctgc taattccatt gtttgtagat ttgacactag
agtgctatct 19261 aaccttaact tgcctggttg tgatggtggc
agtttgtatg taaataaaca tgcattccac 19321 acaccagctt
ttgataaang tgcttttgtt aatttaaaac aattaccatt tttctattac
19381 tctgacagtc catgtgagtc tcatggaaaa caagtagtgt
cagatataga ttatgtacca 19441 ctaaagtctg ctacgtgtat
98
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/ITS2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
aacacgttgc aatttaggtg gtgctgtctg tagacatcat 19501
gctaatgagt acagattgta tctcgatgct tataacatga tgatctcagc
tggctttagc 19561 ttgtgggttt acaaacaatt tgatacttat
aacctctgga acacttttac aagacttcag 19621 agtttagaaa
atgtggcttt taatgttgta aataagggac actttgatgg acaacagggt
19681 gaagtaccag tttctatcat taataacact gtttacacaa
aagttgatgg tgttgatgta 19741 gaattgifig aaaataaaac
aacattacct gttaatgtag catttgagct ttgggctaag 19801
cgcaacatta aaccagtacc agaggtgaaa atactcaata atttgggtgt
ggacattgct 19861 gctaatactg tgatctggga ctacaaaaga
gatgctccag cacatatatc tactattggt 19921 gtagttcta
tgactgacat agccaagaaa ccaactgaaa cgatttgtgc accactcact
19981 gtctlattg atggtagagt tgatggtcaa gtagacttat
ttagaaatgc ccgtaatggt 20041 gttcttatta cagaaggtag
tgttonaggt ttacaaccat ctgtaggtcc caaacaagct 20101
agtcttaatg gagtcacatt aattggagaa gccgtaaaaa cacagttcaa
ttattataag 20161 aaagttgatg gtgttgtcca acaattacct
gaaacttact ttactcagag tagaaattta
20221 caagaattta aacccaggag tcanatggaa attgatttct
tagaattagc tatggatgaa 20281 ttcattgaac ggtataaatt
agaaggctat gccttcgaac atatcgttta tggagatttt 20341
agtcatagtc agttaggtgg tttacatcta ctgattggac tagctaaacg
ilitaaggaa 20401 tcaccittig aattagaaga ititattcct
atggacagta cagttaaaaa ctatttcata 20461 acagatgcgc
aaacaggttc atctaagtgt gtgtgttctg ttattgattt attacttgat
20521 galitigttg anataatana atcccaagat ttatctgtag
tttctaaggt tgtcaaagtg 20581 actattgact atacagaaat
ttcatttatg attggtgta aagatggcca tgtagaaaca 20641
ttttacccaa aattacaatc tagtcaagcg tggcaaccgg gtgttgctat
gcctaatctt 20701 tacaaaatgc aaagaatgct attagaaaag
tgtgaccttc aaaattatgg tgatagtgca 20761 acattaccta
aaggcataat gatgaatgtc gcaaaatata ctcaactgtg tcaatattta
20821 aacacattaa cattagctgt accctataat atgagagtta
tacattligg tgctggttct 20881 gataaaggag ttgcaccagg
tacagctgtt ttaagacagt ggttgcctac gggtacgctg 20941
cttgtcgatt cagatcttaa tgactttgtc tctgatgcag attcaacttt
gattggtgat 21001 tgtgcaactg tacatacagc taataaatgg
gatctcatta ttagtgatat gtacgaccct 21061 aagactaa a
atgttacaaa agaaaatgac tctaaagagg gttttttcac ttacatttgt
21121 gggtttatac aacaaaagct agctcttgga ggttccgtgg
ctataaagat aacagaacat 21181 tcttggaatg ctgatcttta
taagctcatg ggacacttcg catggtggac agcctrtgtt
21241 actaatgtga atgcgtcatc atctgaagca tlittaattg
gatgtaatta tcttggcaaa 21301 ccacgcgaac aaatagatgg
99
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
ttatgtcatg catgcaaatt acatattttg gaggaataca 21361
aatccaattc agttgtcttc ctattcttta tttgacatga gtaaatttcc
ccttaaatta 21421 aggggtactg ctgttatgtc tttaaaagaa
ggtcaaatca atgatatgat tttatctctt 21481 cttagtaaag
gtagacttat aattagagaa aacaacagag ttgttatttc tagtgatgtt
21541 cttgttaaca actaaacgaa caatgtttgt ttttcttgtt
ttattgccac tagtctctag
21601 tcagtgtgtt aatcttacaa ccagaactca attaccccct
gcatacacta attctttcac 21661 acgtggtgtt tattaccctg
acaaag tilt cagatcctca galtacatt caactcagga 21721
cttgactta cctLicalL ccaatgttac ttggttccat gctatacatg
tctctgggac 21781 caatggtact aagaggtttg ataaccctgt
cctaccattt aatgatggtg Matittgc 21841 ttccactgag
aagtctaaca taataagagg ctggattat ggtactactt tagattcgaa
21901 gacccagtcc ctacttattg ttaataacgc tactaatgtt
gttattaaag tctgtgaatt 21961 tcaattttgt aatgatccat ttttgggtgt
ttattaccac aaanacaaca aaagttggat 22021 gganagtgag
ttcagagttt attctagtgc gaataattgc acttttgaat atgtctctca
22081 gcctIttctt atggaccttg aaggaaaaca gggtaatttc
aaaaatctta gggaatttgt 22141 gtttaagaat attgatggtt
aLittaaaat atattctaag cacacgccta ttaatttagt
22201 gcgtgatctc cctcagggtt tttcggcttt agaaccattg
gtagatttgc caataggtat 22261 taacatcact aggtttcaaa
ctttacttgc tttacataga agttatttga ctcctggtga 22321 ttcttcttca
ggttggacag ctggtgctgc agcttattat gtgggttatc ttcaacctag
22381 gactlacta ttaaaatata atgaaaatgg aaccattaca
gatgctgtag actgtgcact 22441 tgaccctctc tcagaaacaa
agtgtacgtt gaaatccttc actgtagaaa aaggaatcta
22501 tcaaacttct aactttagag tccaaccaac agaatctatt
gttagatttc ctaatattac 22561 aaacttgtgc cctaggtg aagtLittaa
cgccaccaga tttgcatctg tttatgcttg 22621 gaacaggaag
agaatcagca actgtgttgc tgattattct gtcctatata attccgcatc
22681 attttccact tttaagtgtt atggagtgtc tcctactaaa
ttaaatgatc tctgctttac 22741 taatgtctat gcagattcat ttgtaattag
aggtgatgaa gtcagacaaa tcgctccagg 22801 gcaaactgga
aagattgctg attataatta taaattacca gatgatttta caggctgcgt
22861 tatagcttgg aattctaaca atcttgattc taaggttggt
ggtaattata attacctgta
22921 tagattgttt aggaagtcta atctcaaacc Ltttgagaga
gatatttcaa ctgaaatcta 22981 tcaggccggt agcacacctt
gtaatggtgt tgaagglitt aattgttact ttcctttaca 23041 atcatatggt
ttccaaccca ctaatggtgt tggttaccaa ccatacagag tagtagtact
23101 ttcttttgaa cttctacatg caccagcaac tgtttgtgga
100
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
cctaaaaagt ctactaattt 23161 ggttaaaaac aaatgtgtca
atttcaactt caatggttta acaggcacag gtgttcttac
23221 tgagtctaac aaanagtttc tgcctttcca acaatttggc
agagacattg ctgacactac 23281 tgatgctgtc cgtgatccac
agacacttga gattcttgac attacaccat gttcttttgg 23341
tggtgtcagt gttataacac caggaacaaa tacttctaac caggttgctg
ttctttatca 23401 ggatgttaac tgcacagaag tccctgttgc
tattcatgca gatcaactta ctcctacttg 23461 gcgtgtttat
tctacaggtt ctaatgtttt tcaaacacgt gcaggctgtt taataggggc
23521 tgaacatgtc aacaactcat atgagtgtga catacccatt
ggtgcaggta tatgcgctag 23581 ttatcagact cagactaatt
ctcctcggcg ggcacgtagt gtagctagtc aatccatcat 23641
tgcctacact atgtcacttg gtgcagaaaa ttcagttgct tactctaata
actctattgc 23701 catacccaca aattttacta ttagtgttac
cacagaaatt ctaccagtgt ctatgaccaa 23761 gacatcagta
gattgtacaa tgtacatttg tggtgattca actgaatgca gcaatctttt
23821 gttgcaatat ggcaglittt gtacacaatt anaccgtgct
ttaactggaa tagctgttga 23881 acaagacaaa aacacccaag
aagitlitgc acaagtcaaa caaatttaca aaacaccacc 23941
aattaaagat tttggtggtt ttaattlitc acaaatatta ccagatccat
caaaaccaag 24001 caagaggtca tttattgaag atctac Litt
caacaaagtg acacttgcag atgctggctt 24061 catcaaacaa
tatggtgatt gccttggtga tattgctgct agagacctca tttgtgcaca
24121 anagtttaac ggccttactg ttttgccacc tttgctcaca
gatgaaatga ttgctcaata 24181 cacttctgca ctgttagcgg
gtacaatcac ttctggttgg acctttggtg caggtgctgc 24241
attacaaata ccatttgcta tgcaaatggc ttataggttt aatggtattg
gagttacaca 24301 gaatgttctc tatgagaacc aaaaattgat
tgccaaccaa tttaatagtg ctattggcaa 24361 aattcaagac
tcactttctt ccacagcaag tgcacttgga aaacttcaag atgtggtcaa
24421 ccarmatgca caagctttaa acacgcttgt tanacaactt
agctccaatt ttggtgcaat 24481 ttcaagtgtt ttaaatgata
tcctttcacg tcttgacaaa gttgaggctg aagtgcaaat 24541
tgataggttg atcacaggca gacttcaaag tttgcagaca tatgtgactc
aacaattaat 24601 tagagctgca gaaatcagag cttctgctaa
tcttgctgct actaaaatgt cagagtgtgt 24661 acttggacaa
tcaaanagag ttgailittg tggaaagggc tatcatctta tgtccttccc
24721 tcagtcagca cctcatggtg tagtcttctt gcatgtgact
tatgtccctg cacaagaaaa 24781 gaacttcaca actgctcctg
ccatttgtca tgatggaaaa gcacactttc ctcgtgaagg
24841 tgtctttgtt tcaaatggca cacactggtt tgtaacacaa
aggaaltitt atgaaccaca 24901 aatcattact acagacaaca
catttgtgtc tggtaactgt gatgttgtaa taggaattgt 24961
caacaacaca gtttatgatc ctttgca,9cc tgaattagac tcattcaagg
101
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
aggagttaga 25021 taaatattlt aagaatcata catcaccaga
tgttgattta ggtgacatct ctggcattaa 25081 tgcttcagtt
gtaaacattc aaaaagaaat tgaccgcctc aatgaggttg ccaagaattt
25141 aaatgaatct ctcatcgatc tccaagaact tggaaagtat
gagcagtata taaaatggcc 25201 atggtacatt tggctaggtt
ttatagctgg cttgattgcc atagtaatgg tgacaattat 25261
gctttgctgt atgaccagtt gctgtagttg tctcaagggc tgttgttctt
gtggatcctg 25321 ctgcaaattt gatgaagacg actctgagcc
agtgctcaaa ggagtcaaat tacattacac 25381 ataaacgaac
ttatggattt gtttatgaga atcttcacaa ttggaactgt aactttgaag
25441 caaggtgaaa tcaaggatgc tactccttca gattligttc
gcgctactgc aacgataccg 25501 atacaagcct cactccatt
cggatggctt attgttggcg ttgcacttct tgctg UM 25561
cagagcgctt ccaaaatcat aaccctcaaa aagagatggc aactagcact
ctccaagggt 25621 gttcact-ttg tttgcaactt gctgttgttg
tttgtaacag tttactcaca ccttttgctc 25681 gttgctgctg
gccttgaagc ccctlitctc tatctttatg ctttagtcta cttcttgcag 25741
agtataaqct ttgtaagaat aataatgagg ct-ttggcttt gctggapatg
ccgttccaaa 25801 aacccattac tttatgatgc caactatitt
ctttgctggc atactaattg ttacgactat
25861 tgtatacctt acaatagtgt aacttcttca attgtcatta
cttcaggtga tggcacaaca 25921 agtcctattt ctgaacatga
ctaccagatt ggtggttata ctgaaaaatg ggaatctgga 25981
gtaaaagact gtgttgtatt acacagttac ttcacttcag actattacca
gctgtactca 26041 actcaattga gtacagacac tggtgttgaa
catgttacct tcttcatcta caataaaatt 26101 gttgatgagc
ctgaagaaca tgtccaaatt cacacaatcg acggttcatc cggagttgtt
26161 aatccagtaa tggaaccaat ttatgatgaa ccgacgacga
ctactagcgt gcctttgtaa 26221 gcacaagctg atgagtacga
acttatgtac tcattcgttt cggaagagac aggtacgtta 26281
atagttaata gcgtacttct ttlicttgct ttcgtggtat tcttgctagt
tacactagcc 26341 atccttactg cgcttcgatt gtgtgcgtac
tgctgcaata ttgttaacgt gagtcttgta 26401 aaaccttctt tttacgttta
ctctcgtgtt aaaaatctga attcttctag agttcctgat
26461 cttctggtct aaacgaacta aatattatat tag ittitct gtttggaact
ttaatittag 26521 ccatggcaga ttccaacggt actattaccg
ttgaagagct taaanagctc cttgaacaat 26581 ggaacctagt
aataggtttc ctattcctta catggatttg tcttctacaa tttgcctatg 26641
ccaacaggaa taggittlig tatataatta agttaatttt ectctggctg
ttatggccag 26701 taactttagc ttglitigtg cttgctgctg
tttacagaat anattggatc accggtggaa
26761 ttgctatcgc aatggcttgt cttgtaggct tgatgtggct
cagctacttc attgcttctt 26821 tcagactgtt tgcgcgtacg
cgttccatgt ggtcattcaa tccagaaact aacattcttc 26881
102
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
tcaacgtgcc actccatggc actattctga ccagaccgct tctagaaagt
gaactcgtaa 26941 tcggagctgt gatccttcgt ggacatcttc
gtattgctgg acaccatcta ggacgctgtg 27001 acatcaagga
cctgcctaaa gaaatcactg ttgctacatc acgaacgctt tcttattaca
27061 aattgggagc ttcgcagcgt gtagcaggtg actcaggttt
tgctgcatac agtcgctaca 27121 ggattggcaa ctataaatta
aacacagacc attccagtag cagtgacaat attgctttgc 27181
ttgtacagta agtgacaaca gatgtttcat ctcgttgact ttcaggttac
tatagcagag 27241 atattactaa ttattatgag gactittaaa
gtttccattt ggaatcttga ttacatcata 27301 aacctcataa
ttaaaaatit atctaagtca ctaactgaga ataaatattc tcaattagat
27361 gaagagcaac caatggagat tgattaaacg aacatgaaaa
ttattctitt cttggcactg 27421 ataacactcg ctacttgtga
gctttatcac taccaagagt gtgttagagg tacaacagta 27481
clataaaag aaccttgctc ttctggaaca tacgagggca attcaccatt
tcatcctcta 27541 gctgataaca aatttgcact gacttgcttt
agcactcaat ttgalligc ttgtcctgac 27601 ggcgtaaaac
acgtctatca gttacgtgcc agatcagttt cacctaact gttcatcaga
27661 caagaggaag ttcaagaact ttactctcca alltlictta
ttgttgcggc aatagtgttt 27721 ataacacttt gcttcacact
caaaagaaag acagaatgat tgaactttca ttaattgact 27781
tctatttgtg ctttttagcc tttctgctat tecttgitti aattatgctt attatctttt
27841 ggttctcact tgaactgcaa gatcataatg aaacttgtca
cgcctaaacg aacatgaaat 27901 ttcttglitt cttaggaatc
atcacaactg tagctgcatt tcaccaagaa tgtagtttac
27961 agtcatgtac tcaacatcaa ccatatgtag ttgatgaccc
gtgtcctatt cacttctatt 28021 ctaaatggta tattagagta
ggagctagaa aatcagcacc tttaattgaa ttgtgcgtgg 28081
atgaggctgg ttctaaatca cccattcagt acatcgatat cggtaattat
acagtttcct 28141 gtttaccttt tacaattaat tgccaggaac
ctaaattggg tagtcttgta gtgcgttgtt 28201 cgttctatga
agactatta gagtatcatg acgttcgtgt tgttttagat ttcatctaaa
28261 cgaacaaact aaaatgtctg ataatggacc ccaaaatcag
cgaaatgcac cccgcattac 28321 gtttggtgga ccctcagatt
caactggcag taaccagaat ggagaacgca g-tggggcgcg 28381
atcaaaacaa cgtcggcccc aaggtttacc caataatact gcgtcttggt
tcaccgctct 28441 cactcaacat ggcaaggaag accttanatt
ccctcgagga caaggcgttc caattaacac 28501 caatagcagt
ccagatgacc aaattggcta ctaccgaaga gctaccagac gaattcgtgg
28561 tggtgacggt aaaatgaaag atctcagtcc aagatggtat
ttctactacc taggaactgg 28621 gccagaagct ggacttccct
atggtgctaa caaagacggc atcatatggg ttgcaactga 28681
gggagccttg aatacaccaa aagatcacat tggcacccgc aatcctgcta
acaatgctgc 28741 aatcgtgcta caacttcctc aaggaacaac
103
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
attgccaaaa ggcttctacg cagaagggag 28801 cagaggcggc
agtcaagcct cttctcgttc ctcatcacgt agtcgcaaca gttcaagaaa
28861 ttcaactcca ggcagcagta ggggaacttc tcctgctaga
atggctggca atggcggtga 28921 tgctgctctt gctttgctgc
tgcttgacag attgaaccag cttgagagca aaatgtctgg 28981
taaaggccaa caacaacaag gccaaactgt cactaagaaa tctgctgctg
aggcttctaa 29041 gaagcctcgg caaaaacgta ctgccactaa
agcatacaat gtaacacaag ctttcggcag 29101 acgtggtcca
gaacaaaccc aaggaaattt tggggaccag gaactaatca
gacaaggaac 29161 tgattacaaa cattggccgc aaattgcaca
atttgccccc agcgcttcag cgttcttcgg 29221 aatgtcgcgc
attggcatgg aagtcacacc ttcgggaacg tggttgacct acacaggtgc
29281 catcaaattg gatgacaaag atccaaattt caaagatcaa
gtcaltilgc tgaataagca
29341 tattgacgca tacaaaacat tcccaccaac agagcctaaa
aaggacaaaa agaagaaggc 29401 tgatgaaact caagccttac
cgcagagaca gaagaaacag caaactgtga ctcttcttcc 29461
tgctgcagat ttggatgatt tctccaaaca attgcaacaa tccatgagca
gtgctgactc 29521 aactcaggcc taaactcatg cagaccacac
aaggcagatg ggctatataa acgattcgc 29581 ttttccgttt
acgatatata gtctactctt gtgcagaatg aattctcgta actacatagc
29641 acaagtagat gtagttaact ttaatctcac atagcaatct
ttaatcagtg tgtaacatta 29701 gggaggactt gaaagagcca
ccacattttc accgaggcca cgcggagtac gatcgagtgt
29761 acagtgaaca atgctaggga gagctgccta tatggaagag
ccctaatgtg taaaattaat 29821 tttagtagtg ctatccccat
gtgattltaa tagcttctta ggagaatgac aaaaaaaaaa 29881
aaaaaaaaaa aaaaaaaaaa aaa
ME S I ,VPGFNEKTHVQI , ST ;PVT ,QVR DVI ,VR GEGDS
VEEVLSEARQHLKDGTCGLVEVEKGVLPQLEQPY
VFIKRSDARTAPHGHVMVELVAELEGIQYGRSGE
SARS-00V-2
TLGVLVPHVGEIPVAYRKVLLRKNGNKGAGGHS
Wuhan seafood
YGADLKSFDLGDELGTDPYEDFQEN
market pneumonia
WNTKHSSGVTRELMRELNGGAYTRYVDNNFCGP
virus isolate
DGYPLECIKDLLARAGKASCTLSEQLDFIDTKRGV
Wuhan-Hu-1
YCCREHEHEIAWYTERSEKSYELQTPFEIKLAKKF
genomic sequence 2
DTENGECPNFVFPLNSIIKTIQPRVEKKKLDGFMG
(GenBank:
RIRSVYPVASPNECNQMCLSTLMKCDHCGETSWQ
MN908947.3janu
TGDFVKATCEFCGTENLTKEGATTCGYLPQNAVV
ary 23, 2020) ¨
KIY CPACHN S E V GPEHSLAEYHN ESGLKTILRKGG
amino acid
RTIAFGGCVESYVGCHNKCAYWVPRASANIGCNH
translation
TGVVGEGSEGLNDNL
LEILQKEKVNINIVGDFKLNEEIAIILASFSASTSAF
VETVKGLDYKAFKQIVESCGNEKVTKGKAKKGA
104
CA 03194162 2023- 3- 28
WO 2022/067269
PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
WNIGEQKSILSPLYAFASEAARVVRSIFSRTLETAQ
N SVRVLQKAAITILD GIS QYSLRLIDAMMFTSDLA
TNNLVVMAYITGGVVQLTSQWLTNIFGTVYEKLK
PVLDWLEEKFKEGVEFLRDGWEIVKFISTCACEIV
GGQIVTCAKEIKE S V QTFFKLVNKFLALCAD SIIIG
GAKLKALNLGETFVTHSKGLYRKCVKSREETGLL
MPLKAPKEIIFLEGETLPTEVLTEEVVLKTGDLQPL
EQPTSEAVEAPLVGTPVCINGLMLLEIKDTEKYCA
LAPNMMVTNNTFTLKGGAPTKVTFGDDTVIEVQ
GYKSVNITFELDERIDKVLNEKC SAYTVELGTEVN
EFACVVADAVIKTL QPV S ELLTPLGIDLDEW S MA
TYYLFDESGEFKLASHMY C SFYPPDEDEEEGDCE
EEEFEPSTQYEYGTEDDYQGKPLEFGATSAALQPE
EEQEEDWLDDDS QQTVGQQDGSEDNQTTTIQTIV
EV QPQLEMELTP V V QTIEVN SFSGYLKLTDN VY IK
NADIVEEAKKVKP'TVVVNAANVYLKHGGGVAG
A LNK A TNNAMQVESDDYIATNGPLKVGGSCVLS
GHNLAKHCLHVVGPN VNKGEDIQLLKSAYENFN
QHEVLLAPLLSAGIFGADPIHSLRVCVDTVRTNVY
LAVFDKNLYDKLVSSFLEMKSEKQVEQKIAEIPKE
EVKPFITESKPSVEQRKQDDKKIKACVEEVTTTLE
ETKFLTENLLLYIDINGNLHPD S A TLV SDIDITFLK
KDAPYIVGDVVQEGVLTAVVIPTKKAGGTTEMLA
KALRKVPTDNYITTYPGQGLNGYTVEEAKTVLKK
CK S A FYILP SIISNEKQEILG'TVSWNLREMLAHAEE
TRKLMPVCVETKAIVSTIQRKYKGIKIQEGVVDYG
ARFYFYTSKTTVASLINTLNDLNETLVTMPLGYVT
HGLNLEEAARYMRSLKVPATVSVS SPDAVTAYN
GYLTSS SKTPEEHFIETISLAGSYKDWSYSGQ STQL
GIEFLKRGDKS VYYTSN PTTFHLDGEVITFDNLKT
LLSLREVRTIKVFT'TVDN1NLHTQVVDMSMTYGQ
QFGPTYLDGADVTKIKPHNSHEGKTFYVLPNDDT
LRVEAFEYYHTTDPSFLGRYMSALNHTKKWKYP
QVNGLTSIKWADNNCYLATALLTLQQIELKFNPP
AL QDAYYRARAGEAANFCALILAYCNKTVGELG
DVRETMSYLFQHANLDSCKRVLNVVCKTCGQQQ
TTLKGVEAVMYMGTLSYEQFKKGVQIPCTCGKQ
A TKYLVQ QE S PFVMM S A PP A QYELKHGTFTC A SE
YTGNYQ CGHYKHITSKETLYCIDGALLTKSSEYK
GPITDVFYKENSYTTTIKPVTYKLDGVVCTEIDPK
LDNYYKKDNSYFTEQPIDLVPNQPYPNASFDNFK
FVCDNIKFADDLNQLTGYKKPASRELKVTFFPDL
NGDVVAIDYKHYTPSFKKGAKLLHKPI
ATNKATYKPNTWCIRCLWSTKPVETSNSFDVLKS
EDAQGMDNLACEDLKPVSEEVVENPTIQKDVLEC
105
CA 03194162 2023- 3- 28
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SEQ
Sequence
ID Sequence
Description
NO.
NVKTTEVVGDIILKPANNSLKITEEVGHTDLMAA
YVDNSSLTIKKPNELSRVLGLKTLATHGLAAVNS
VPWDTIANYAKPFLNKVV STTTNIVTRCLNRV CT
NYMPYFFTLLLQLCTFTRSTNSRIKASMPTTIAKN
TVKSVGKFCLEASFNYLKSPNFSKLINIIIWFLLLS
V CLGSL1Y STAALGVLMSNLGMP SY CTGYREGYL
NSTNVTIATYCTGSIPCSVCLSGLDSLDTYPSLETI
QITIS SFKWDLTAFGLVAEWFLAYILFTRFFYVLG
LAAIMQLFFSYFAVHFISNSWLMWLIINLVQMAPI
SAMVRMYIFFASFYYVWKSYVHVVDGCNSSTCM
MCYKRNRATRVECTTIVNGVRRSFYVYANGGKG
FCKLHNWN CVN CDTFCAGSTF1SDEVARDLSLQF
KRPINPTDQSSYIVDSVTVKNGSIHLYFDKAGQKT
YERHSLSHFVNLDNLRANNTKGSLPINVIVFDGKS
KCEES SAKSASVYY SQLMCQPILLLDQALVSDVG
DSAEVAVKMFDAYVNTF SSTFNVPMEKLKTLVA
TAEAELAKNVSLDNVLSTFISA A RQGFVDSDVET
KDVVECLKLSHQ SD1EVTGD S CNN Y MLTYN KVE
NMTPRDLGACIDC SARHINAQVAKSHNIALIWNV
KDFMSLSEQLRKQIRSAAKKNNLPFKLTCATTRQ
VVNVVTTKIALKGGKIVNNWLKQLIKVTLVFLFV
A A IFYLITPVHVMSKHTDF S SEIIGYK A IDGGVTRD
IA STDTCFANKHADFDTWF S QRGGSYTNDKACPL
IAAVITREVGFVVPGLPGTILRTTNGDFLHFLPRVF
SAVGNICY'TPSKLIEYTDFATSACVLAAECTIFKD
A SGKPVPYCYDTNVLEGSVAYE SLRPDTRYVLM
DGSIIQFPNTYLEGSVRVVTTFDSEYCRHGTCERS
EAGVCVSTSGRWVLNNDYYRSLPGVFCGVDAVN
LLTNMFTPLIQPIGALDISA SIVAGGIVAIVVTC LA
YYFMRFRRAFGEY SHVVAFNTLLFLMSFTVLCLT
PVYSFLPGVYSVIYLYLTFYLTNDVSFLAHIQWM
VMFTPLVPFWITIAYIICISTKHFYWFFSNYLKRRV
VFNGV SF STFEEAALCTFLLNKEMYLKLRSDVLLP
LTQYNRYLALYNKYKYF SGAMDTTSYREAAC CH
LAKALNDFSNSGSDVLYQPPQTSITSAVLQSGFRK
MAFPSGKVEGCMVQVTCGTTTLNGLWLDDVVY
CP RHVICT SEDMLNPNYEDLLIRKSNHNFLVQAG
NVQLRVIGHSMQNCVLKLKVDTANPKTPKYKFV
RIQ PGQTF SVLACYNGSP SGVYQ CA MRPNFTIKGS
FLNGS CGSVGFNIDYDCV SF CYMITHMELPTGVHA
GTDLEGNFYGPFVDRQTAQAAGTDTTITVNVLA
WLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNY
EPLTQDHVDILGPLSAQTGIAVLDMCASLKELLQN
GMNGRTILGSALLEDEFTPFDVVRQC SGVTFQ SA
VKRTIKGTHHWLLLTILTSLLVLVQSTQWSLFFFL
106
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SEQ
Sequence
ID Sequence
Description
NO.
YENAFLPFAMGIIAMSAFAMMFVKHKHAFLCLFL
LP S LATVAYFNMVYMPA SWVMRIMTWLDMVDT
SL S GFKLKD CVMYA SAVVLLILMTARTVYDD GA
RRVWTLMNVLTLVYKVYYGNALDQAISMWALII
SVTSNYSGVVTTVMFLARGIVFMCVEYCPIFFITG
NTLQCIMLVYCFLGYFCTCYFGLFCLLNRYFRLTL
GVYDYLVSTQEFRYMNSQGLLPPKNSIDAFKLNI
KLLGVGGKPCIKVATVQSKMSDVKCTSVVLL SVL
QQLRVES SSKLWAQCVQLHNDILLAKDTTEAFEK
MVSLLSVLLSMQGAVDINKLCEEMLDNRATLQAI
A SEF S S LP SYAAFATAQEAYEQAVANGD SEVVLK
KLKKSLN VAKSEFDRDAAMQRKLEKMADQAMT
QMYKQARSEDKRAKVTSAMQTMLFTMLRKLDN
DALNNIINNARDGCVPLNIIPLTTAAKLMVVIPDY
NTYKN TCDGTTFTYASALWEIQQVVDADSKIVQL
SEISMDNSPNLAWPLIVTALRANSAVKLQNNEL SP
VALRQMS CA AGTTQTACTDDNALAYYNTTKGGR
FVLALLSDLQDLKWARFPKSDGTGTIYTELEPPCR
FVTDTPKGPKVKYLYFIKGLNNLNRGMVLGSLAA
TVRLQAGNATEVPAN S TVL SF CAFAVDAAKAYK
DYLASGGQPITNCVKMLCTHTGTGQAITVTPEAN
MD QESFGGA SC CLYCRCHIDHPNPKGFCDLKGKY
VQIPTTCANDPVGFTLKN'TVCTVCGMWKGYGC S
CD QLREPMLQ SADA Q SFLNRVCGVSAARLTPCGT
GTSTDVVYRAFDIYNDKVA GFAKFLK'TNCCRFQE
KDEDDNLID SYFVVKRHTFSNYQHEETIYNLLKD
CPAVAKHDFFKFRIDGDMVPHISRQRLTKYTMAD
LVYALRHFDEGNCDTLKEILVTYNCCDDDYFNKK
DWYDFVENPDILRVYANLGERVRQALLKTVQFC
DAMRNAGIVGVLILDN QDLNGN W YDFGDFIQ TT
PG SGVPVV
D SYY S LLMPILTLTR A LTA E SHVDTDLTKPYIKWD
LLKYDFTEERLKLFDRYFKYWDQTYHPNCVNCL
DDRCILHCANFNVLFSTVFPPTSFGPLVRKIFVDG
VPFVVSTGYHFRELGVVHNQDVNLHSSRLSFKEL
LVYAADPAMHAASGNLLLDKRTTCFSVAALTNN
VAFQTVKPGNFNKDFYDFAVSKGFFKEGS SVELK
HFFFAQDGNA A IS DYDYYRYNLPTMC DIRQ LLFV
VEVVDKYFDCYDGGCINANQVIVNNLDKSAGFPF
NKWGKARLYYDSMSYEDQDALFAYTKRNVIPTIT
QMNLKYAISAKNRARTVAG V SIC STMTNRQFHQ
KLLKSIAATRGATVVIGTSKFYGGWHNMLKTVYS
DVENPHLMGWDYPKCDRAMPNMLRIMASLVLA
RKHTTCCSL SHRFYRLANECAQVLSEMVMCGGS
LYVKPGGTSSGDATTAYANSVFNICQAVTANVNA
107
CA 03194162 2023- 3- 28
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SEQ
Sequence
ID Sequence
Description
NO.
LLSTDGNKIADKYVRNLQHRLYECLYRNRDVDT
DFVNEFYAYLRKHFSMMILSDDAVVCFNSTYASQ
GLVASIKNFKSVLYYQNNVFMSEAKCWTETDLT
KGPHEFCSQHTMLVKQGDDYVYLPYPDPSRILGA
GCFVDDIVKTDGTLMIERFVSLAIDAYPLTKHPNQ
EYADVFHLYLQYIRKLHDELTGHMLDMY S V MLT
NDNTSRYWEPEFYEAMYTPHTVLQAVGACVLCN
SQTSLRCGACIRRPFLCCKCCYDHVISTSHKLVLS
VNPYVCNAPGCDVTDVTQLYLGGMSYYCKSHKP
PISFPLCANGQVFGLYKNTCVGSDNVTDFNAIATC
DWTNAGDYILANTCTERLKLFAAETLKATEETFK
L SY GIATVREVL SDRELHL SW EV GKPRPPLN RN Y
VFTGYRVTKNSKVQIGEYTFEKGDYGDAVVYRG
TTTYKLNVGDYFVLTSHTVMPLSAPTLVPQEHYV
RITGLYPTLNISDEFSSN VAN Y QKVGMQKY STLQ
GPPGTGKSHFAIGLALYYPSARIVYTACSHAAVD
ALCEKALKYLPIDKC SRIIPARARVECFDKFKVNS
TLEQYVFCTVNALPETTADIVVFDEISMATNYDLS
VVNARLRAKHYVYIGDPAQLPAPRTLLTKGTLEP
EYFNSVCRLMKTIGPDMFLGTCRRCPAEIVDTV SA
LVYDNKLKAHKDKSAQCFKMFYKGVITHDVS SAT
NRPQIGVVREFLTRNPAWRK AVFISPYNSQNAVA
SKILGLPTQTVDSSQGSEYDYVIFTQTTETAHSCN
VNRFNVAI I RAKVGILCIMSDRDLYDKLQFTSLEI
PRRNVA TLQ A ENVTGLFKDC SKVITGLHPTQ A PT
HLSVDTKFKTEGLCVDIPGIPKDMTYRRLISMMGF
KMNYQVNGYPNMFITREEAIRHVRAWIGFDVEG
CHATREAVGTNLPLQLGFSTGVNLVAVPTGYVDT
PNNTDFSRVSAKPPPGDQFKHLIPLMYKGLPWNV
VRIKIVQMLSDILKNLSDRVVFVLWAHGFELTSM
KYFVKIGPERTCCLCDRRATCF STA SDTYACWHEI
SIGFDYVYNPFMIDVQQWGFTGNLQSNHDLYCQ
VHGNAHVASCDAIMTRCLAVHECFVKRVDWTIE
YPIIGDELKINAACRKVQHMVVKAALLADKFPVL
HDIGNPKAIKCVPQADVEWKFYDAQPCSDKAYKI
EELFYSYATHSDKFTDGVCLFWNCNVDRYPAN ST
VCRFDTRVLSNLNLPGCDGGSLYVNKHAFHTPAF
DK SAFVNLKQLPFFYYSDSPCESHGKQVVSDIDY
VPLKSATCITRCNLGGAVCRHHANEYRLYLDAYN
MMISAGFSLWVYKQFDTYNLWNTFTRLQSLENV
AFNVVNKGHFDGQQGEVPVSIINNTVYTKVDGV
DVELFENKTTLPVNVAFELWAKRNIKPVPEVKILN
NLGVDIAANTVIWDYKRDAPAHISTIGVCSMTDIA
KKPTETICAPLTVFFDGRVDGQVDLFRNARNGVLI
TEGSVKGLQPSVGPKQASLNGVTLIGEAVKTQFN
108
CA 03194162 2023- 3- 28
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PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
YYKKVDGVVQQLPETYFTQSRNLQEFKPRSQMEI
DFLELAMDEFIERYKLEGYAFEHIVYGDFSHSQLG
GLHLLIGLAKRFKESPFELEDFIPMDSTVKNYFITD
AQTGSSKCVCSVIDLLLDDFVEIIKSQDLSVVSKV
VKVTIDYTEISFMLWCKDGHVETFYPKLQSSQAW
QPGVAMPNLYKMQRMLLEKCDLQNYGDSATLP
KGIMMNVAKYTQLCQYLNTLTLAVPYNMRVIHF
GAGSDKGVAPGTAVLRQWLPTGTLLVDSDLNDF
VSDADSTLIGDCATVHTANKWDLIISDMYDPKTK
NVTKENDSKEGFFTYICGFIQQKLALGGSVAIKITE
HSWNADLYKLMGHFAWWTAFVTNVNASSSEAF
LIGCNYLGKPREQIDGYVMHANYIFWRNTNPIQLS
SYSLFDMSKFPLKLRGTAVMSLKEGQINDMILSLL
SKGRLIIRENNRVVISSDVLVNN
mfvflvllpl vssqcvnitt rtqlppaytn sftrgvyypd kvfrssvlhs
tqdlflpffs 61 nvtwfhaihv sgtngtkrfd npvlpfndgv
yfasteksni irgwifgttl dsktqslliv 121 nnatnvvikv
cefqfcndpf lgvyyhknnk swmesefrvy ssannctfey
vsqpflmdle181 gkqgnfknlr efvflmidgy fkiyskhtpi
nlvrdlpqgf saleplvdlp iginitrfqt 241 llallirsylt
pgdsssgwta gaaayyvgyl qprtfllkyn engtitdavd caldplsetk
301 ctlksftvck giyqtsnfry qptcsivrfp nitnlcpfgc
vfilatrfasv yawnrkrisn 361 cvadysvlyn sasfstfkcy
gvsptklndl cftnvyadsf virgdevrqi apgqtgkiad 421
ynyklpddft gcviawnsnn ldskvggnyn ylvrlfrksn lkpferdist
surface eiyqagstpc 481 ngvegfitcyf plqsygfqpt
ngvgyqpyry
glycoprotein vvlsfellha patvcgpkks tnlvknkcvn 541
frifngligtg
1SARS-CoV-2 vltesnkkfl pfqqfgrdia dttdavrdpq
tleilditpc sfggvsvitp
Wuhan seafood 601 gtntsnqvav lyqdvnctev pvaihadqlt ptwrvystgs
'3
market pneumonia - nvfqtragcl igaehvnnsy 661 eedipigagi
casyqtqlns
virus]; GenBank: prrarsvasq siiaytmslg aensvaysnn
siaiptnfti 721
QHD43416 .1 ; svt-teilpvs mtktsvdctm yicgdstecs
nlllqygsfc tqlnraltgi
January 23, 2020 aveqdkntqe 781 vfaqvkqiyk tppikdfggf
nfsqilpdps
kpskrsfied llfnkvtlad agfikqygdc 841 lgdiaardli
caqkfngltv 1pplltdemi aqytsallag titsgw-tfga gaalqipfam
901 qmayrfngig vtqnvlyenq klianqfnsa igkiqdslss
tasalgklqd vvnqnaqaln 961 tivkqlssnf gaissvindi
lsrldkveae vqidrlitgr lqslqtyvtq qliraaeira
1021 sanlaatkms ecvlgqskry dfcgkgyhlm sfpqsaphgv
vflhvtyvpa qeknfttapa 1081 ichdgkahfp regvfvsngt
hwfvtqmfy epqiittdnt fvsgncdvvi givnntvydp 1141
lqpeldsfice eldkyficnht spdvdlgdis ginasvvniq keidfineva
knlneslidl 1201 qelgkyeqyi kwpwyiwlgf iagliaivmv
timlccmtsc csclkgccsc gscckfdedd 1261 sepvlkgvkl hyt
109
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SEQ
Sequence
ID Sequence
Description
NO.
surface
glycoprotein RBD
nitni cpfgevfn atrfasvyawn rkri sncvadysvlynsasfstfkcygvs
[SARS-CoV-2
Wuhan seafood
ptklndleftnvyadsfvirgdevrqiapgqtgkiadynyklpddftgcvia
4
wnsnnldskvggnynylyrlfrksnlkpferdisteiyqagstpcngvegfn
market pneumonia
cyfpl qsygfqptngvgyqpyrvvvl sfellh apatvcgpkk stnlvknkc
virus]; GenBank:
vnfnfngltgtg
QHD43416.1;
January 23, 2020
Receptor Binding
Motif (RBM) in
surface
glycoprotein RBD
[SARS-CoV-2
Nsnnldskvggnynylyrlfrksnlkpferdisteiyqag stpcngvegfnc
Wuhan seafood yfplqsygfqgtngvgyqpy
market pneumonia
virus]; GenBank:
QHD43416.1;
January 23, 2020
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT
SARS-CoV-2 PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
CH1-CH3 LS KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
Glm17 IgHG1*01 6VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
(aa) TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VLHEALHSHYTQKSLSLSPGK
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
DKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRT
SARS-CoV-2 PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
CH1-CH3 LS, 7 KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
ALE Glm17 VSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDE
IgHG1*01 (aa) LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVLHEALHSHYTQKSLSLSPGK
110
CA 03194162 2023- 3- 28
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SEQ
Sequence
ID Sequence
Description
NO.
GQPKAAPSVTLFPPS SEELQANKATLVCLISDFYP
GAVTVAWKADSSPVKAGVETTTPSKQSNNKYAA
SARS-CoV-2 CL SSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP
IgLC*01 (aa) 8 TECS
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR
EAKVQWKVDNALQSGNSQESVTEQDSKD STYSL
SARS-CoV-2 CL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF
(CK) klm3 9 NRGEC
IgKC*01 (aa)
Linker (aa) 10 GSTSGSGKPGSGEGSTKG
Linker (aa) 11 GSGKPGSGEG
Linker (aa) 12 GKPGSGEG
Linker (aa) 13 SGKPGSGE
Linker (aa) 14 BPXXXZ, wherein each X is
independently a glycine
(G) or serine (S). B is a positively charged amino acid
and Z is glycine (G) or a negatively charged amino acid
Linker (aa) 15 (GxS)y, wherein xis 1-10 and y is 1-
10
Linker (aa) 16 GGGGSGGGGSGGGGS
Linker (aa) 17 GGGGSGGGGSGGGGSGGGGSGGGGS
GGGGSGGGGSGGGGSGGGGSGGGGS
Linker (aa) 18 GSTSGGGSGGGSGGGGSS
Linker (aa) 19 EGKS SG SG SESKVD
Linker (aa) 20 KESGSVSSEQLAQFRSLD
Linker (aa) 21 GGGGS
EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAM
Antibody 418 1 HWVRQAPGKGLEWVAVILSDGSNKYYADSVKG
22
VH (aa) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDR
SPLVGFGDNYGMDVWGQGTTVTVSS
Antibody 418 1 23 GFTFSTYA
CDRH1 (aa)
Antibody 418_i 24 ILSDGSNK
CDRH2 (aa)
111
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SEQ
Sequence
ID Sequence
Description
NO.
Antibody 418 1 25 ARDRSPLVGFGDNYGMDV
CDRH3 (aa)
SYELTQPPSVSVSPGQTARITCSGDALPKKYAYW
Antibody 418 1 YQQKSGQAPVLVIYEDSKRPSGIPERFSGSSSGTM
VL (aa) 26 ATLTISGAQVEDEADYYCSSTDSSGNQGVFGGGT
KLTVL
Antibody 418_i 27 ALPKKY
CDRL1 (aa)
Antibody 418 1
28 EDS
CDRL2 (aa)
Antibody 418 1 29 SSTDSSGNQGV
CDRL3 (aa)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGT
GGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTG
TGCAGCCTCTGGATTCACCTTCAGTACCTATG
CTATGCACTGGGTCCGCCAGGCTCCAGGCAAG
GGGCTAGAGTGGGTGGCAGTTATATTATCTGA
Antibody 418 1 TGGAAGTAATAAATATTACGCAGACTCTGTGA
30 H (nt AGGGC CGATTCAC CATC TC CAGAGACAATTC CA
)
V
AGAACACGCTGTATCTGCAAATGAACAGCCTGA
GAGCTGAGGACACGGCTGTGTATTACTGTGCG
AGAGATCGAAGTCCCCTCGTGCCATTCGCGC
ACAACTATGGTATGGACGTCTGGGGCCAAGG
GACCACGGTCACCGTCTCCTCA
TCCTATGAGCTGACACAGCCACCCTCGGTGTCA
GTGTCCCCAGGACAAACGGCCAGGATCACCTGC
TCTGGAGATGCATTGCCAAAAAAATATGCTTA
TTGGTACCAGCAGAAGTCAGGCCAGGCCCCTGT
Antibody 418 1 GCTGGTCATCTATGAGGACAGCAAACGACCCT
VL (nt) 31 CCGGGATC CC TGAGAGATTC TCTGGC TCCAGC T
CAGGGACAATGGCCACCTTGACTATCAGTGGGG
CCCAGGTGGAGGATGAAGCTGACTACTACTGTT
CCTCAACAGACACCACTGGTAATCAAGGGGT
ATTCGGCGGAGGGACCAAGCTGACCGTCCTAG
QITLKESGPTLVKPTQTLTLTCKLSGFSVNTGGVG
Antibody 418 2 VGWIRQPPGKALEWLALIYWNDDKLYSP SLKSRL
VH (aa) 32 TVTKDTSKNQVVLTMTNMDPVDTATYYCAHVL
VWFGEVLPDAFDVWGQGTMVTVSS
Antibody 418_2 33 GFSVNTGGVG
CDRH1 (aa)
112
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SEQ
Sequence
ID Sequence
Description
NO.
Antibody 418 2 34 IYWNDDK
CDRH2 (aa)
Antibody 418 2
35 AHVLVWFGEVLPDAFDV
CDRH3 (aa)
SYELTQPPSVSVSPGQTASITCSGDKLGETYASW
Antibody 418 2 YQQKPGQSPILVIYQDNKRPSGIPERFSGSNSENTA
VL (aa) 36TLTISGTQTMDEADYYCQAWDKTIAGFGGGTKL
TVL
Antibody 418_2 37 KLGETY
CDRL1 (aa)
Antibody 418 2 38 QDN
CDRL2 (aa)
Antibody 418_2 39 QAWDKTIAG
CDRL2 (aa)
CAGATCACCTTGAAGGAGTCTGGTCCTACGCTG
GTGAAACCCACACAGACCCTCACGCTGACCTGC
AAATTATCTGGGTTTTCAGTCAACACTCGTGC
AGTGGGTGTGGGCTGGATCCGTCAGCCCCCAG
GAAAGGCCCTGGAGTGGCTTGCACTCATTTATT
Antibody 418 2 GGAATGATGATAAGTTGTACAGCCCATCTCTG
VH (nt) 40AAGAGCAGGCTCACCGTCACCAAGGACACATC
CAAAAACCAGGTGGTCCTTACAATGACCAACAT
GGACCCTGTGGACACAGCCACATATTACTGTGC
ACACCTATTACTTTCGTTCGCCGAGGTATTA
CCCGATGCTTTTGATGTGTGGGGCCAAGGGAC
AATGGTCACCGTCTCTTCAG
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCC
GTGTCCCCAGGACAGACAGCCAGCATCACCTGC
TCTGGAGATAAATTGGGGGAGACATATGCTAG
TTGGTATCAGCAGAAGCCAGGCCAGTCCCCTAT
Antibody 418 2 TCTAGTCATCTATCAAGATAACAAGCGGCCCTC
VL (nt) 41 AGGGATCCCTGAGCGATTCTCTGGCTCCAACTC
TGAGAACACAGCCACTCTGACCATCAGCGGGA
CCCAGACTATGGATGAGGCTGACTATTACTGTC
AGGCGTCCGACAAGACCATCGCCGCATTCGG
CGGAGGGACCAAGCTGACCGTCCTAG
EVQLVESGGGVVQPGRSLRLSCAASGFIFSTYGM
Antibody 418 3 HWVRQAPGKGLEWVAlIWYDGTKKYYADSVKG
VH (aa) 42RFTISRDNSKNTLYLQMNILRAEDTAVYYCASNR
YHYASSGYYQLDYWGQGTLVTVSS
113
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SEQ
Sequence
ID Sequence
Description
NO.
Antibody 418 3
43 GFIFSTYG
CDRHI (aa)
Antibody 418 3
44 1WYDGTKK
CDRH2 (aa)
Antibody 418 3
45 ASNRYHYASSGYYQLDY
CDRH3 (aa)
DIQMTQSPSSLSASVGDRVTITCQASQDISNSLNW
Antibody 418 3 YQQKPGKAPNLLIYDASNLETGVPSRFSGSGSGTD
VL (aa) 46 FTFTISSLQPEDVATYYCQHYDHLPLTFGGGTKV
EIK
Antibody 418 3 47 QDISNS
CDRLI (aa)
Antibody 418 3
48 DAS
CDRL2 (aa)
Antibody 418_3
49 QHYDHLPL T
CDRL3 (aa)
GAGGTGCAGCTGGTGGAGTCGGGGGGAGGCGT
GGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTG
TGCAGCGTCTGGATTCATCTTCAGTACCTATG
GCATGCACTGGGTCCGCCAGGCTCCAGGCAAG
GGGCTGGAGTGGGTGGCTATTATATGGTATGA
Antibody 418 3 TGGAACTAAAAAATACTATGCAGACTCCGTGA
VH (nt) 5 AGGGCCGATTCACCATCTCCAGAGACAATTCCA
AGAACACGCTGTATCTACAAATGAACATCCTGA
GAGCCGAGGACACGGCTGTGTATTACTGTGCG
AGTAACCGGTA ICACTATGCTAGTAGTGUI
ATTATCAACTTGACTACTGGGGCCAGGGAACC
CTGGTCACCGTCTCCTCAG
GACATCCAGATGACCCAGTCTCCATCCTCCCTG
TCTGCATCTGTTGGAGACAGAGTCACCATCACT
TGCCAGGCGAGTCAGGACATTAGCAACTCTTT
AAATTGGTATCAGCAGAAACCAGGGAAAGCCC
Antibody 418 3 CTAACCTCCTGATCTACGATGCATCCAATTTGG
VL (nt) 51 AAACAGGGGTCCCATCAAGGTTCAGTGGAAGT
GGATCTGGGACAGATTTTACTTTCACCATCAGC
AGCCTGCAGCCTGAAGATGTTGCAACATATTAC
TGTCAACATTATGATCATCTCCCTCTCACTTT
CGGCGGAGGGACCAAGGTGGAGATCAAAC
EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYG
Antibody 418_4 52
MI-IWVRQAPGKGLEWVAVIWYDGSNKFYADSV
114
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SEQ
Sequence
ID Sequence
Description
NO.
VH (aa) KGRFTISRDNSKNSLYLQMNSLRAEDTAVYFCAR
AFPDSSSWSGFTIDYWGQGTLVTVSS
Antibody 418_4 53 GFTFSNYG
CDRH1 (aa)
Antibody 418 4 54 IWYDGSNK
CDRH2 (aa)
Antibody 418 4 55 ARAFPDSSSWSGF TIDY
CDRH3 (aa)
SYELTQPPSVSVAPGQTARITCGGNNIERKSVHW
Antibody 418_4 CQQKPGQAPALVVYDDSDRPSGIPERFSGSNSGNT
VL (aa) 56 ATLTISRVEAGDEADYYCQVWDSGSDQVIFGGG
TKLTVL
Antibody 418 4 57 NIERKS
CDRL 1 (aa)
Antibody 418_4
58 DDS
CDRL2 (aa)
Antibody 418_4 59 QVWDSGSDQVI
CDRL3 (aa)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGT
GGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTG
TGCAGCGTCTGGATTCACCTTCAGTAATTATG
GCATGCACTGGGTCCGCCAGGCTCCAGGCAAG
GGACTGGAGTGGGTGGCAGTTATATGGTATGA
Antibody 418 4 TGGAAGTAATAAATTCTATGCAGACTCCGTGA
VH (nt) ¨ 60AGGGCCGATTCACCATCTCCAGAGACAATTCCA
AGAACAGTCTCTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACGGCTGTTTATTTCTGTGCGA
GGGCCTTTCCCGATAGCAGCAGCTGGTCCGG
CTTCACTATTGACTACTGGGGCCAGGGAACCC
TGGTCACCGTCTCCTCAG
TCCTATGAGCTGACTCAGCCACCCTCGGTGTCA
GTGGCCCCAGGACAGACGGCCAGGATTACCTGT
GGGGGAAACAACATTGAGAGGAAAAGTGTGC
ACTGGTGCCAGCAGAAGCCAGGCCAGGCCCCT
GCGCTGGTCGTCTATGATGATAGCGACCGGCC
Antibody 418 4
61 CTCAGGGATCCCTGAGCGATTCTCTGGCTCCAA
VL (nt) CTCTGGGAACACGGCCACCCTGACCATCAGCAG
GGTCGAAGCCGGGGATGAGGCCGACTATTACT
GTCAGGTGTGGGATAGTGGTAGTGATCAGGT
GATATTCGGCGGAGGGACCAAGCTGACCGTCC
TAG
115
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SEQ
Sequence
ID Sequence
Description
NO.
EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGM
HWVRQAPGKGLEWVTVIWYDGSNRYYADSVKG
VU Antibody
62 RFTISRDNSKNTLYLQMDSLRAEDTAVYYCARAV
Anti4a)18-5
AGEWYFDYWGQGTLVTVSS
Antibody 418_5 63 GFTFSSYC
CDRH1 (aa)
Antibody 418_5 64 IWYDGSNR
CDRH2 (aa)
Antibody 418 5 65 ARAVAGEWYFDY
CDRH3 (aa)
SYELTQPPSVSVSPGQTARITCSGDALAKHYAYW
YRQKPGQAPVLVIYKDSERPSGIPERFSGSSSGTTV
Antibody 418 5
TLTISGVQAEDEADYYCQSADSIGSSWVFGGGTK
VL (aa) 66LTVL
Antibody 418 5 67 ALAKHY
CDRL1 (aa)
Antibody 418 5 68 KDS
CDRL2 (aa)
Antibody 418_S 69 QSADSIGSSWV
CDRL3 (aa)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGT
GGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTG
TGCAGCGTCTGGATTCACCTTCAGTAGCTATG
GCATGCACTGGGTCCGCCAGGCTCCAGGCAAG
GGGCTGGAGTGGGTGACAGTTATTTGGTATGA
TGGAAGTAATCGATACTATGCAGACTCCGTGA
Antibody 418-5
VH (nt 70 AGGGCCGATTCACCATCTCCAGAGACAATTCCA
) AGAACACGCTGTATCTGCAAATGGACAGCCTGA
GAGCCGAGGACACGGCTGTTTATTACTGTGCGA
GAGCAGTGGCCGGGGAATGGTACTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC
AG
TCCTATGAGCTGACACAGCCACCCTCGGTGTCA
GTGTCCCCAGGACAGACGGCCAGGATCACCTGC
TCCGGAGATGCATTGGCAAAACACTATGCTTA
Antibody 418 5
71 TTGGTACCGGCAGAAGCCAGGCCAGGCCCCTGT
VL (nt)
GCTGGTGATATATAAAGACAGTGAGAGGCCCT
CAGGGATCCCTGAGCGATTCTCTGGCTCCAGCT
CAGGGACAACAGTCACGTTGACCATCAGTGGA
116
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SEQ
Sequence
ID Sequence
Description
NO.
GTCCAGGCAGAAGACGAGGCTGACTATTACTGT
CAATCAGCAGACAGCATTGGTAGTTCTTGGG
TGTTCGGCGGAGGGACCAAGCTGACCGTCCTA
QVQLQESGPGLVKPSETLSLTCTVSGGSVNSGSY
Antibody 418 6 YWSWIRQPPGKGLEWIGYIFYS GS TYYNP SLKSR
¨ 72 VTISIDTSKNQF SLKLS SVTAADTAVYYCAREVAP
VH (aa)
VAGTAHQTTYYFDYWGQGTLVTVSS
Antibody 418_6 73 GGS VN SGS Y Y
CDRH1 (aa)
Antibody 418_6 74 IFYSGST
CDRH2 (aa)
Antibody 418 6 75 AREVAPVAGTAHQTTYYFDY
CDRH3 (aa)
DIVMTQSPSSLSVSVGDRVSITCRASQSISTYLNW
Antibody 418 6 YQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTD
VL ( ¨ 76 FTLTISSLQPEDFATYYCQQSRPLEEGICRYTFGQ
aa)
GTKLEIK
Antibody 418 6
77 QSISTY
CDRL1 (aa)
Antibody 418 6 78 AAS
CDRL2 (aa)
Antibody 418 6 QQSRPLEEGICRYT
79
CDRL3 (aa)
CAGGTGCAGCTACAGGAGTCGGGCCCAGGATT
GGTGAAGCCTTCGGAGACCCTGTCCCTCACCTG
CACTGTCTCTGGTGGCTCCGTCAACAGTGGCA
GTTACTACTGGAGCTGGATCCGGCAGCCCCCA
GGGAAGGGACTGGAGTGGATTGGGTATATCTT
TTACAGTGGGAGCACCTACTACAACCCCTCCC
Antibody 418-6 80 TCAAGAGTCGAGTCACCATATCAATAGACACGT
VH (nt)
CCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTG
TGACCGCTGCGGACACAGCCGTGTATTACTGTG
CGAGAGAGGTTGCGCCAGTGGCTGGTACTG
CCCACCAAACAACGTACTACTTTGACTACTG
GGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
GACATCGTGATGACCCAGTCTCCATCCTCCCTG
Antibody 418 6
81 TCTGTGTCTGTAGGAGACAGAGTCAGCATCACT
VL (nt) TGCCGGGCAAGTCAGAGCATTAGCACCTATTT
117
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SEQ
Sequence
ID Sequence
Description
NO.
AAATTGGTATCAGCAGAAACCAGGGAAAGCCC
CTAAGCTCCTGATCTATGCTGCATCCAGTTTGC
ACAGTGGGGTCCCATCAAGGTTCAGTGGCAGTG
GATCTGGGACAGATTTCACTCTCACCATCAGCA
GTCTGCAACCTGAAGATTTTGCAACTTACTACT
GTCAACAGAGTCGGCCGCTCGAAGAAGGCAA
AAGGTACACTTTTGGCCAGGGGACCAAGCTGG
AGATCAAAC
EVQLVQ SGAEVNKPGS SVKVSCKASGGTFSSYAI
Antibody 418 7 SWVRQAPGQGLEWMGGIIPIFHTANYAQKFHGR
VH ( ¨ 82 VTITADESTSTAYMELN SLRSEDTAVY Y CAGD SG
aa)
SSTWLGPFDIWGQGTMVTVS S
Antibody 418 7 83 GGTFSSYA
CDRHI (aa)
Antibody 418 7 84 IIPIFHTA
CDRH2 (aa)
Antibody 418_7 85 AGDSGSSTWLGPFDI
CDRH3 (aa)
VIWMTQ S P S TL SA SVGDRVTITC RA S QGISSYLAW
Antibody 418 7 YQQKPGKAPKLLIYDASTLQ SGVP SRFSGSGSGTE
¨ VL ( 86 FTLTI S SLQPGDF A TYYC QQYNSYP YTFGQGTKLE
aa)
IK
Antibody 418 7
87 QGISSY
CDRL 1 (aa)
Antibody 418 7 88 DAS
CDRL2 (aa)
Antibody 418 7 89 QQYNSYPYT
CDRL3 (aa)
GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGT
GAACAAGCCTGGGTCCTCGGTGAAGGTCTCCTG
CAAGGCTTCTGGAGGCACCTTCAGCAGCTAT
GCCATCAGCTGGGTGCGACAGGCCCCTGGACA
Antibody 418 7 90 AGGGCTTGAGTGGATGGGAGGGAT CATCCC TA
VH (nt) TCTTTCATACAGCAAACTACGCACAGAAGTTC
CACGGCAGAGTCACGATTACCGCGGACGAATC
CACGAGCACAGCCTACATGGAGCTGAACAGCC
TGAGATCTGAGGACACGGCCGTGTATTACTGTG
CGGGGGATAGTGGGAGCTCAACCTGGCTCG
118
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SEQ
Sequence
ID Sequence
Description
NO.
GACCTTTTGATATCTGGGGCCAAGGGACAATG
GTCACCGTCTCTTCAG
GTCATCTGGATGACCCAGTCTCCTTCCACCCTGT
CTGCATCTGTAGGAGACAGAGTCACCATCACTT
GCCGGGCCAGTCAGGGCATTAGCAGTTATTTA
GCCTGGTATCAGCAAAAACCAGGGAAAGCCCC
Antibody 418 7 TAAGCTCCTGATCTATGATGCATCCACTTTGCA
VL (nt 91 AAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG
)
GATCTGGGACAGAATTCACTCTCACCATCAGCA
GCCTGCAGCCTGGTGATTTTGCAACTTATTACT
GCCAACAGTATAATAGTTACCCGTACACTTTT
GGCCAGGGGACCAAGCTGGAGATCAAAC
QVQLVQSGAEVKKPGA SVKVSCKVSGYTLIEIS
Antibody 418 8 MHWVRQAPGKGLEWNIGGFDPEDAETIYAQKFQ
9")
VH (aa) GRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT
QYAILTHSYFDYWGQGTLYTVSS
Antibody 418 8
93 GYTLIELS
CDRH1 (aa)
Antibody 418_8
94 FDPEDAET
CDRH2 (aa)
Antibody 418 8
95 ATQYAILTBSYFDY
CDRH3 (aa)
DIQLTQSPSSLSASVGDRVITIVRASQGISNYLAW
Antibody 418 8 YQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTD
VL (aa) 96 FTLTISSIAREDVATYYCQKYNSAPQTFGQGTKV
EIK
Antibody 418_8
97 QGISNY
CDRL1 (aa)
Antibody 418 8
98 AAS
CDRL2 (aa)
Antibody 418_8
99 QKYNSAPQT
CDRL3 (aa)
CAGGTGCAGCTGGTACAGTCTGGGGCTGAGGICT
A A G.A A GC CTGGGGCCTCA. MU A A GCiTCTCCTGC
AAGGTTTCCG GA TACACC CTCATTGAA TTA TC
CATGCA CTGGGTGCGAC AGG CICCIGGAAAA G
Antibody 418_8
100 GGCTTGAGTGGATGGGA GGTTUT GATCCTGA A
VH (nt)
GATGCTGAAACAATCTACG CAC AGAACTIC CA
GGGCACiAGICACCATGACCGAGGACACATCTA
CAGACACAGCCTACATGGAGCTGAGCAG CCM
AG A TCTGAGGACACGG CCGTGTATTA CTGTG CA
119
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SEQ
Sequence
ID Sequence
Description
NO.
ACACAA TA CG CAA TCCTTACTCATTCCTACTT
Tc ACTACTGGGGCCAGGGAACCCTGGTCACCG
TCH:CTCAG
GACATCCAGTTGACCCAGTCTCCATCCTCCCTG
TCTGCATCTGTAGGAGACAGAGTcACCATCACT
TGCCGGGCGAGTCAGGGCATTAGCAATTATIFT
AGCCTGG'FATCAGCAGAAACCAGGGAAAGTTC
Antibody 418 8
CTAAGCTCCTGATCTATCCTGCATCCACTTTGC
VL (nt) 101AATCAGGGGTCCCATCTCGGTFCAGCGGCAGTG
GATCTGGGACAGATITCACTCTCACCATCAGCA
GCCTGCAGCCTGAAGATGTTG CAA CTTATTA CT
GTCAAAAGTATAACAGTGCCCCTCAGACGTT
CGGCCAAGGGACCAAGGTGGAAATCAAAC
EVQLVESGGGILVNPGGSLRLSCAASGFTFSDYTI
Antibody 418 9
I-11VVRQAPGKGLEAVVSSISSSSNYIYYADSVKGRF
VH (aa)
102 ,VISRDNAKNSLSWAINSLRAEDTAVYYCARDGN
AYKWLLAENVIUDYINGQGILVIVSS
Antibody 418 9
103 GFTFSDYT
CDRH1 (aa)
Antibody 418 9
104 ISSSSNY1
CDRH2 (aa)
Antibody 418 9
105 ARDGNAYKANLLAENVRFDY
CDRH3 (aa)
QTVVTQPASVSGSPGQSITISCTGTSSDVGGVNYV
Antibody 418 9
SWYQQI-IPGKAPKLM1YDVSDRPSGVSNRFSGSKS
VL (aa)
106GNTASETISGLQAEDEADY YCSSYTSSSTPNWV17
GGGTKILTV1,
Antibody 418 9
107 SSDVGGYNY
CDRL1 (aa)
Antibody 418_9
108 DVS
CDRL2 (aa)
Antibody 418 9
109 SSYTSSSTPNWV
CDRL3 (aa)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCT
GGICAACCCTGGGGGGIFCCCTGAGACTCTCCTG
TGCAGCCTCTGGATTCACCTTCAGTGACTATA
CC.:ATFCACTGGG-FCCGCCACiGcrccAGGGAAG
Antibody 418 9
110 GGGCTGGACi.MGGICICATCCATIAGTAGIAG
VH (nt) TAGTAATTATATATACTACGCGGACTCAGTGA
AGGGCCGATTCACCATCTCC.AGAGACAA.COCCA
AGAA.CTCACIGTCTCMCAAATGAACA.GCCTGA
GAGCCGA GG A C A COGCT(IFCiTA "'TA Cf(iTGCG
120
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SEQ
Sequence
ID Sequence
Description
NO.
AGA GATGGTAATG CCTACAAGTGG TTA TTGG
GAGA G AACGTTCG TTIFTGACTACTGGGGCCA
GGGAACCCTGGTCACCGTCTCCTCAG
C AGA CTGTG GTGA CTCAGCCTGCCTC CGTGTCT
GGGTCTCCTGGACAGTCCiATC AC CATCTCCTGC
ACTGGAAC CAGCA GT GACGT T GGTGGTTATA
AC TAT GTCTC CIGGTAC CAACAACAC C CAGGCA
AAGCCCCCAAA CTCATGA 111 ATCATCTCACT
Antibody 418 9
111 GATCGGCCCTCAGGGGITTCTAATCGCTTCTCT
VL (nt)
GGCTCCAAGTCTGGCAACACGGCCTCCCTGACC
ATCTCTGGGCTC CAGGCTGAGGAC GAGGCTGAT
TATTACTG CA GCTC ATATACAA GCAGCAGCA C
CCCCAATTGGG TGTTCGGCGGAGGGACCAAGC
TGACCGTCCTAG
QVQLQQRGAGLLKPSETLSLTCDVYGGSLSGYY
Antibody 418 10 WSWIRQAPGKGLEWIGEINHRGSTNYNPSLKSRV
VH (aa) 112 TISIDTSKKQFSLKLSSVTAADTAVYYCARYVVVI
VHALPMPVNWFDPWGQGTLVTVSS
Antibody 418 10
113 GGSLSGYY-
CDRH1 (aa)
Antibody 418 10
CDRH2 (aa) 114 INIIRGST
Antibody 418 10
115 ARY VVVIVHALPMPVNWFDP
CDRH3 (aa)
QSVLTQPASVSGSPGQSITISCTGTSSDVGSYNLVS
Antibody 418 10 WYQQHPAKAPKLIWEGSKRPSGVSNRFSGSKSG
vL'(aa) ¨ 116 NTASLTISGLQAEDECDYYCCSYAGSSPLIVFGTG
TKVTVL
Antibody 418 10
117 SSDVGSYNL
CDRL1 (aa)
Antibody 418 10
118 EGS
CDRL2 (aa)
Antibody 418 10
119 CSYAGSSPLIV
CDRL3 (aa)
CAGGTGCAGCTACAGCAGCGGGGCGCAGGACT
GTTGAAGCCTTCGGAGACCCTGTCCCTCACCTG
CGATGTCTATGGTGGGTCCCTCAGTGGTTACT
Antibody 418 10
120 ACTGGAGCTGGATCCGCCAGGCCCCAGGGAAG
VH (nt)
GGGCTGGAGTGGATTGGGGAAATCAATCATCG
TGGAAGCACCAACTACAACCCGTCCCTCAAGA
GTCGGGTCACCATATCAATAGACACGTCCAAGA
121
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SEQ
Sequence
ID Sequence
Description
NO.
AGCAGTTCTCCCTGAAGCTGAGCTCTGTGACCG
CCGCGGACACGGCTGTGTATTACTGTGCGAGA
TACGTTGTGGTGATCGTACATGCCCTTCCAA
TGCCAGTTAACTGGTTCGACCCCTGGGGCCA
GGGAACCCTGGTCACCGTCTCCTCAG
CAGTCTGTGCTGACTCAGCCTGCCTCCGTGTCT
GGGTCTCCTGGACAGTCGATCACCATCTCCTGC
ACTGGAACCAGCAGTGATGTTGGGAGTTATA
ACCTTGTCTCCTGGTACCAACAACACCCAGCCA
AAGCCCCCAAACTCATCATTTATGAGGGCAGT
Antibody 418 10 121 AAGCGGCCCTCAGGAGTTICTAATCGCTICTCT
VL (nt) GGCTCCAAGTCTGGCAACACGGCCTCCCTGACA
ATCTCTGGACTCCAGGCTGAGGACGAATGTGAT
TATTACTGCTGCTCATATGCAGGTAGTAGCCC
CTTGATAGTCTTCGGAACTGGGACCAAGGTCA
CCGTCCTAG
EVQLLESGGGLIQPGGSLRLSCAASGFSVSSNYM
Antibody 418 11 NWVRQAPGKGLEWVSVIYSGGSAYYADSVKGRF
VH (aa) 122 TISRDISKNTLYLQMNSLRAEDTAVYYCARAPGS
WAYWYFDLWGRGTLVTVSS
Antibody 418 11 GFSVSSNY
123
CDRH1 (aa)
Antibody 418 11 124 IYSGGSA
CDRH2 (aa)
Antibody 418 11 125 ARAPGSWAYWYFDL
CDRH3 (aa)
EIVMMQSPATLSVSPGERATLSCRASQSVRSNLA
A WYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGT
126 EFTLTISSMQSEDFAVYYCQQYNIWPTFGQGTKV
Antibody 418
11
EIK
Antibody 418 11 127 QSVRSN
CDRL1 (aa)
Antibody 418 11 GAS
128
CDRL2 (aa)
Antibody 418 11 1,9 QQYNIWPT
CDRL3 (aa)
GAGGTGCAGCTGTTGGAGTCTGGAGGAGGCTTG
Antibody 418 11 ATCCAGCCGGGGGGGTCCCTGAGACTCTCCTGT
VH (nt) 130GCAGCCTCTGGGTTCAGCGTCAGTAGCAACT
ACATGAACTGGGTCCGCCAGGCTCCAGGGAAG
122
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SEQ
Sequence
ID Sequence
Description
NO.
GGGCTGGAGTGGGTCTCAGTTATTTATAGCGG
TGGTAGTGCATACTACGCAGACTCCGTGAAGG
GCCGATTCACCATCTCCAGAGACATTTCCAAGA
ACACGCTGTATCTTCAAATGAACAGCCTGAGAG
CCGAGGACACGGCCGTGTATTACTGTGCGAGA
GCCCCCGGCAGTTGGGCCTACTGGTACTTCG
ATCTCTGGGGCCGTGGAACCCTGGTCACTGTCT
CCTCAG
GAAATAGTGATGATGCAGTCTCCAGCCACCCTG
TCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCC
TGCAGGGCCAGTCAGAGTGTTAGAAGCAACTT
AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
Antibody 418 11
CAGGCTCCTCATCTATGGTGCATCCACCAGGG
VL(VK) (nt) , ,
CCACTGGTATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGAGITCACTCTCACCATCAGCA
GCATGCAGTCTGAAGATTTTGCAGTTTATTACT
GTCAGCAGTATAATATCTGGCCGACGTTCGGC
CAAGGGACCAAGGTGGAAATCAAAC
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAM
Antibody 418 12
SWVRQAPGKGLEWVSVISGSGGDTYYADSVKGR
¨ 132 FTTSRDNSKNTLYLQ1VINSLRAEDTAVYYCAKGE
VH (aa)
RIKMIVVVTMIDYWGQGTLVTVSS
Antibody 418_12 133 GFTFSSHA
CDRH1 (aa)
Antibody 418_12 134 ISGSGGDT
CDRH2 (aa)
Antibody 418 12 135 AKGERIKMIVVVTMIDY
CDRH3 (aa)
QSVVTQPPSVSAAPGQKVTISCSGSSSNIGSNYVS
418 12 Antibody
WYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGT
vL'(aa) ¨ 136 SATLGITGLQTGDEADYYCGTWDNSLSAGVFGG
GTKLTVL
Antibody 418_12 137 SSNIGSNY
CDRL1 (aa)
Antibody 418 12 138 DNN
CDRL2 (aa)
Antibody 418 12 139 GTWDNSLSAGV
CDRL3 (aa)
123
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SEQ
Sequence
ID Sequence
Description
NO.
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTG
GTACAGCCTGGGGGGTCCCTGAGACTCTCCTGT
GCAGCCTCTGGATTCACCTTTAGCAGTCATGC
CATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG
GGCTGGAGTGGGTCTCAGTTATTAGTGGTAGT
Antibody 418 12 GGTGGTGACACATACTACGCAGACTCCGTGAA
vH (nt) ¨ 140 GGGCCGGTTCACCATCTCCAGAGACAATTCCAA
GAACACGCTGTATTTGCAAATGAACAGCCTGAG
AGC CGAGGACACGGC CGTATATTACTGTGC GA
AAGGCGAACGTATTAAAATGATAGTAGTCGT
TACTATGATTGACTACTGGGGCCAGGGAACCC
TGGTCACCGTCTCCTCAG
CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCT
GCGGCCCCAGGACAGAAGGTCACCATCTCCTGC
TCTGGAAGCAGCTCCAACATTGGGAGTAATTA
TGTATCCTGGTACCAGCAGCTCCCAGGAACAGC
CC CCAAACTCCTCATTTATGACAATAATAAGCG
Antibody 418 12 141 ACCCTCAGGGATTCCTGACCGATTCTCTGGCTC
VL (nt) CAAGTCTGGCACGTCAGC CAC CCTGGGCATCA C
CGGACTCCAGACTGGGGACGAGGCCGATTATTA
CTGCGGAACATGGGATAACAGCCTGAGTGCT
GGGGTATTCGGCGGAGGGACCAAGCTGACCGT
CCTAG
QVTLRESGPALVKPTQTLTLTCTFSGFSLSTRGM
Antibody 418_13 CVNWIRQPPGKALEWLAFIDWDDDICYYSTSLKT
VI-I 142 RLTISKDTSKNQVVLTMTNMDPVDTATYYCARIR
(aa)
GV1PAAGTVPYYHYMDVWGKGTTVTVSS
Antibody 418 13 143 GFSLSTRGMC
CDRH1 (aa)
Antibody 418_13 144 IDWDDDK
CDRH2 (aa)
Antibody 418_13 145 ARIRGVIPAAGTVPYYHYMDV
CDRH3 (aa)
DIVMTQSPLSLPVTPGEPASISCRS SQSLL HSNGY
Antibody 418 13 NYLDWYLQKPGQ SPQLLIYLGSNRASGVPDRF SG
¨ VL(VK) (aa) 146 SGSGTDFTLKISRVEAEDVGVYYCMQAL QTLSIT
FGQGTRLEIK
Antibody 418 13 147 QSLLHSNGYNY
CDRL 1 (aa)
124
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SEQ
Sequence
ID Sequence
Description
NO.
Antibody 418 13 LGS
148
CDRL2 (aa)
Antibody 418 13 149 MQALQTLSIT
CDRL3 (aa)
CAGGTCACCTTGAGGGAGTCTGGTCCTGCGTTG
GTGAAACCCACACAGACCCTCACACTGACCTGC
ACCTTCTCTGGGTTCTCACTCAGCACTCGTGG
AATGTGTGTGAACTGGATCCGTCAGCCCCCAG
GGAAGGCCCTGGAGTGGCTTGCATTCATTGATT
Antibody 418 13 GGGATGATGATAAATACTACAGCACATCTCTG
VH (nt) ¨ 150 AAGACCAGGCTCACCATCTCCAAGGACACCTCC
AAAAACCAGGTGGTCCTTACAATGACCAACATG
GACCCTGTGGACACAGCCACGTATTACTGTGCA
CGGATACGGGGGGTTATACCAGCAGCTGGTA
CAGTTCCCTACTACCACTACATGGACGTCTG
GGGC A A AGGGACCACGGTCACCGTCTCCTCA
GATATTGTGATGACTCAGTCTCCACTCTCCCTGC
CCGTCACCCCTGGAGAGCCGGCCTCCATCTCCT
GCAGGTCTAGTCAGAGCCTCCTGCATAGTAAT
CCATACAACTATTTGGATTGGTACCTGCAGAA
GCCAGGGCAGTCTCCACAGCTCCTGATCTATTT
Antibody 418 13 151 GGGTTCTAATCGGGCCTCCGGGGTCCCTGACA
VL(VK) (nt) GGTTCAGTGGCAGTGGATCAGGCACAGATTTTA
CACTGAAAATCAGCAGAGTGGAGGCTGAGGAT
GTTGGGGTTTATTACTGCATGCAAGCTCTACA
AACTCTTTCCATCACCITCGGCCAAGGGACAC
GACTGGAGATTAAAC
EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYSM
Antibody 418 14 NWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRF
( ¨ 152 TISRDNAKNSLFLQMNSLRAEDTAVYYCARWGY
aa VH )
SYDSRGYYPRELDYWGQGTLVTVSS
Antibody 418_14 153 GFTFSTYS
CDRH1 (aa)
Antibody 418 14 154 ISSSSSYI
CDRH2 (aa)
Antibody 418 14 155 ARWGYSYDSRGYYPRELDY
CDRH3 (aa)
Antibody 418 14 DIVMTQSPATLSVSPGERATLSCTASQSVSNNLA
VL(VK) (aa) 156WYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGT
125
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SEQ
Sequence
ID Sequence
Description
NO.
EFTLTISSLQSEDFAVYYCQHYYNWPPWTFGQGT
NVEIK
Antibody 418_14 157 QSVSNN
CDRL1 (aa)
Antibody 418_14 GAS
158
CDRL2 (aa)
Antibody 418 14 159 QHYYNWPPWT
CDRL3 (aa)
GAGGTGCAACTGGTGGAGTCTGGGGGAGGCCT
GGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTG
TGCAGCCTCCGGATTCACGTTCAGTACCTATA
GCATGAACTGGGTCCGCCAGGCTCCAGGGAAG
GGGCTGGAGTGGGTCTCATCCATTAGTAGTAG
Antibody 418 14 TAGTAGTTACATATACTACGCAGACTCAGTGA
vH-(nt) ¨ 160 AGGGCCGATTCACCATCTCCAGAGACAACGCCA
AGAACTCACTGTTTCTGCAAATGAACAGCCTGA
GAGCCGAGGACACGGCTGTTTATTACTGTGCGA
GGTGGGGTTATTCCTATGACAGTCGTGGCTA
TTACCCCCGGGAACTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCCTCAG
GATATTGTGATGACTCAGTCTCCAGCCACCCTG
TCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCC
TGCACGGCCAGTCAGAGTGTTAGCAACAACTT
AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
Antibody 418 14 CAGGCTCCTCATCTATGGTGCATCCACCAGGG
¨ VL(VK) (nt) 161 CCACTGGTATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGAGITCACTCTCACCATCAGCA
GCCTGCAGTCTGAAGATTTTGCAGTTTATTACT
GTCAGCACTATTATAACTGGCCTCCGTGGAC
CTTCGGCCAAGGGACCAACGTGGAAATCAAAC
EVQLVESGGGVVQPGGSLRLSCAASGFTFNSYG
Antibody 418 40 MHWVRQAPGKGLEWVAFIRYDGGNKYYADSV
VH ( ¨ 162 KGRFTISRDNSKNTLYLQMKSLRAEDTAVYYCAN
aa)
LKDSRYSGSYYDYWGQGTLVTVSS
Antibody 418 40 163 GFTFNSYG
CDRH1 (aa)
Antibody 418 40 164 IRYDGGNK
CDRH2 (aa)
Antibody 418 40 165 ANLKDSRYSGSYYDY
126
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SEQ
Sequence
ID Sequence
Description
NO.
CDRH3 (aa)
VIWMTQSPSSLSASVGDRVTITCQASQDIRFYLN
Antibody 418 40 WYQQKPGKAPKLLISDASNMETGVPSRFSGSGSG
166 TDFTFTISSLQPEDIATYYCQQYDNLPFTFGPGTK
VL(VK) (aa)
VDFK
Antibody 418_40 167 QDIRFY
CDRL1
Antibody 418 40
168 DAS
CDRL2
Antibody 418_40
169 QQYDNLPFT
CDRL3
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGT
GGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTG
TGCAGCGTCTGGATTCACCTTCAATAGTTATG
GCATGCACTGGGTCCGCCAGGCTCCAGGCAAG
GGGCTGGAGTGGGTGGCATTTATACGGTATGA
Antibody 418 40 TGGAGGTAATAAGTACTATGCAGACTCCGTGA
170 AGGGCCGATTCACCATCTCCAGAGACAATTCCA
VH(VK) (nt)
AGAACACGCTGTATCTGCAAATGAAGAGCCTG
AGAGCTGAGGACACGGCTGTGTATTACTGTGC
GAACCTGAAAGATAGCAGATATAGTGGGAGC
TATTATGACTACTGGGGCCAGGGAACCCTGGT
CACCGTCTCCTCAG
GTCATCTGGATGACCCAGTCTCCATCCTCCCTGT
CTGCATCTGTAGGAGACAGAGTCACCATCACTT
GCCAGGCGAGTCAGGACATTAGGTTCTATTTA
AATTGGTATCAGCAGAAACCAGGGAAAGCCCC
Antibody 418 40 TAAGCTCCTGATCTCCGATGCATCCAATATGGA
171 AACAGGGGTCCCATCAAGGTTCAGTGGAAGTG
VL (VK) (lit)
GATCTGGGACCGATTTTACTTTCACCATCAGCA
GCCTTCAGCCTGAAGATATTGCAACATATTACT
GTCAACAGTATGATAATCTCCCTTTCACTTTC
GGCCCTGGGACCAAGGTGGATTTCAAAC
EVQLVQSGAEVKKPGASVKVSCKASGYTFT
GYHMI-IWVRQAPGQGLEWIVIGWINPNSGGT
Antibody 418 15 172 NYVQKF QGRVTMTRDT SI S TAYMEL SRLR SD
VH (aa) DTAVYYCAKVVAVAGPFDHWGQGTLVTVS
Antibody 418 15 173 GYTFTGYH
127
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SEQ
Sequence
ID Sequence
Description
NO.
CDRH1 (aa)
Antibody 418_15 174 INPNSGGT
CDRH2 (aa)
Antibody 418 15 175 AKVVAVAGPFDH
CDRH3 (aa)
QSVLIQPASVSGSPGQSITISCIGTSSDVGSYNLVS
WYQQHPGKAPKLMIYEGSKRPSGVSNRFSGSKSG
Antibody 418-15 176 NTASLTISGLQAEDEADYYCFSYAGSSDWVEGGG
VL (aa)
TKLTVL
Antibody 418_15 177 SSDVGSYNL
CDRL1
Antibody 418 15 178 EGS
CDRL2
Antibody 418_15 179 FSYAGSSDWV
CDRL3
GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGT
GAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTG
CAAGGCTTCTGGATACACCTTCACCGGCTACC
ATATGCACTGGGTGCGACAGGCCCCTGGACAA
GGGCTTGAGTGGATGGGATGGATCAACCCTAA
CAGTGGTGGCACAAACTATGTACAGAAGTTTC
Antibody 418-15
VH ( 180 AGGGCAGGGTCACCATGACCAGGGACACGTCC
nt)
ATCAGCACAGCCTACATGGAGCTGAGCAGGCT
GAGATCTGACGACACGGCCGTGTATTACTGTGC
GAAGGTTGTAGCAGTGGCTGGCCCCTTTGAC
CACTGGGGCCAGGGAACCCTGGTCACCGTCTCC
TCAG
CAGTCTGTGTTGACTCAGCCTGCCTCCGTGTCTG
GGTCTCCTGGACAGTCGATCACCATCTCCTGCA
CTGGAACCAGCAGTGATGTTGGGAGTTATAA
CCTTGTCTCCTGGTACCAACAGCACCCAGGCAA
AGCCCCCAAACTCATGATTTATGAGGGCAGTA
Antibody 418 15 181 AGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTG
VL (nt) GCTCCAAGTCTGGCAACACGGCCTCCCTGACAA
TCTCTGGGCTCCAGGCTGAGGACGAGGCTGATT
ATTACTGCTTCTCATATGCAGGTAGTAGTGAT
TGGGTGTTCGGCGGAGGGACCAAGCTGACCGT
CCTAG
EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYSM
Antibody 418 16 182
NWVRQAPGKGLEWVSSVSISSSYIYYADSVKGRF
128
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SEQ
Sequence
ID Sequence
Description
NO.
VH (aa) TISRDNAKNSLYLQMNNVRAEDTAVYYCARVRP
HNYDSSGYYPDAFDIWGQGTMVTVS S
Antibody 418_16 183 GFTFSTYS
CDRH1 (aa)
Antibody 418_16 184 VSISSSYI
CDRH2 (aa)
Antibody 418 16 185 ARVRPHNYDSSGYYPDAFDI
CDRH3 (aa)
VIWMTQ SPA TLSVSPGFRA TLSCRA SQSVSSNLA
Antibody 418 16 WYQ QKPGQAPRLLIYGASTRAT SVPARF S GS
GSG
¨ 186 TEFTLTIS SLQSEDFAVYYCQHYYNWPPWTFGQG
VL(VK) (aa)
TKVEVK
Antibody 418 16 187 QSVSSN
CDRL 1
Antibody 418 16 GAS
188
CDRL2
Antibody 418 16 189 QHYYNWPPWT
CDRL3
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGC CT
GGTCAAGCCTGGGGGGTCCCTGAGACTTTCCTG
TGCAGCCTCTGGATTCACCTTCAGTACCTATA
GCATGAACTGGGTCCGCCAGGCTCCAGGGAAG
GGGCTGGAGTGGGTCTCATCCGTTAGTATTAG
Antibody 418 16 TAGTAGTTACATATATTACGCAGACTCAGTGA
¨ 190 AGGGCCGATTCAC CATCTCCAGAGACAA CGC CA
VH (nt)
AGAACTCACTGTATCTGCAAATGAACAACGTGA
GAGCCGAGGACACGGCCGTGTATTACTGTGCG
AGAGTTCGCCCCCATAACTATGATAGTAGTG
GTTATTATCCGGATGCTTTTGATATCTGGGGC
CAAGGGACAATGGTCA CC GTCTCTTCAG
GTCATCTGGATGAC CCAGTCTCCAGC CAC CCTG
TCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCC
TGCAGGGC CAGTCAGAGT GT TA GCA GCAA CTT
Antibody 418 16 191 AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
VL (VK) (nt) CAGGCTCCTCATCTATGGTGCATCCACCAGGG
CCACCAGTGTCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGAGTTCACTCTCACCATCAGCA
GCCTGCAGTCTGAAGATTTTGCAGTTTATTACT
129
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SEQ
Sequence
ID Sequence
Description
NO.
GTCAGCATTATTATAACTGGCCTCCGTGGAC
GTTCGGCCAAGGGACCAAGGTGGAAGTCAAAC
EVQLVESGGGLVKPGGSLRL S CAA S GFTFSSYTM
Antibody 418 17 NWVRQAPGKGLEWVS SISSSGSYIYYADSVKGRF
¨ 192 TISRDSAKTSLYLQMNSLRAEDTAVYFCARDLMS
VH (aa)
RSIFSGYYPDAFDIWGQGTMVTVSS
Antibody 418 17 193 GFTFSSYT
CDRH1 (aa)
Antibody 418 17 194 IS SSGSYI
CDRH2 (aa)
Antibody 418 17 195 ARDLMSRSIFSGYYPDAFDI
CDRH3 (aa)
EIVMMQSPVTLSVSPGERATLSCRASQSVSSNLA
Antibody 418 17 WYQ QKPGQAPRLLIY GASTRATGIPARF S GSGS
GT
¨ 196 EFTLSISSMQSEDFAVYYCQHYYNWPPWTFGQG
VL(VK) (aa)
TTVEIK
Antibody 418_17
1,7 QSVSSN
CDRL1
Antibody 418_17 198 GAS
CDRL2
Anti body 418_17 199
wyy-Nwppwi,
CDRL3
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGC CT
GGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTG
TGCAGCCTCTGGATTCACCTTCAGTTCCTATA
CCATGAACTGGGTCCGC CAGGCTCCAGGGAAG
GGGCTGGAGTGGGTCTCATCCATTAGTAGTAG
Antibody 418 17 TGGTAGTTACATATATTACGCAGACTCAGTGA
¨ 200 AGGGCCGATTCAC CATCTCCAGAGACAGCGC CA
VH (nt)
AGACCTCACTGTATCTACAAATGAACAGCCTGA
GAGC CGAGGACACGGCTGTGTATTTCTGTGC GA
GAGATCTTATGAGTAGGAGCATCTTCTCTGG
TTATTATCCTGATGCTTTTGATATCTGGGGCC
AAGGGACAATGGTCACCGTCTCTTCAG
GAAATAGTGATGATGCAGTCTCCAGTCACCCTG
TCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCC
Antibody 418 17
201 TGCAGGGCCAGTCAGAGTGTTAGCAGCAACTT
VL (VK) (nt) AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCACCAGGG
130
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SEQ
Sequence
ID Sequence
Description
NO.
CCACTGGTATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGAGTTCACTCTCAGCATCAGCA
GCATGCAGTCTGAAGATTTTGCAGTTTATTACT
GTCAGCACTATTATAACTGGCCTCCGTGGAC
GTTCGGCCAAGGGACCACGGTGGAAATCAAAC
QVQLVESGGGLVKPGGSLRLSCAASGFTFRSYSI
Antibody 418 18 HWVRQAPGKGLEWVS SISRSSNYIYYADSVKGRF
VH ( ¨ 202 TV SRDNAKD S LYLQMN GLRAEDTAVYYCARDL
aa)
QSSSGWYWDAFDIWGQGTMVTVSS
Antibody 418 18 203 GFTFRSYS
CDRH1 (aa)
Antibody 418_18 204 ISRSSNYI
CDRH2 (aa)
Antibody 418 18 205 ARDLQS SSGWYWDAFDI
CDRH3 (aa)
QSVLTQPPSVSGAPGQRVTIS CTGSSSNIGAGYDV
Antibody 418 18 HWYQHLPGTAPKLLIYGNNNRP SGVPDRFSGSKS
¨ VL ( 206 GTSA SLAITGLQAEDEADYYCQSFDNTHVVFGGG
aa )
TKLTVL
Antibody 418_18 207 SSNIGAGYD
CDRL I
Antibody 418 18
208 GNN
CDRL2
Antibody 418_18 209 Q SFDN THV V
CDRL3
CAGGTACAGCTGGTGGAGTCTGGGGGAGGCCT
GGTCAAGCCTGGGGGGTCCCTGAGACTCTCATG
TGCAGCCTCTGGATTCACCTTCCGTAGTTATA
GCATACACTGGGTCCGCCAGGCTCCAGGGAAG
GGGCTGGAGTGGGTCTCTTCCATTAGTCGTAG
Antibody 418 18 TAGTAATTACATATACTACGCAGACTCAGTGA
¨ 210 AGGGCCGATTCAC CGTCTCCAGAGACAA CGC CA
VH (nt)
AGGACTCACTGTATCTGCAAATGAACGGCCTGA
GAGCCGAGGACACGGCTGTGTATTACTGTGCG
AGAGATCTACAATCCAGCAGTGGCTGGTACT
GGGATGCTTTTGATATCTGGGGCCAAGGGACA
ATGGTCACCGTCTCTTCAG
CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCT
Antibody 418 18 211
GGGGCCCCAGGGCAGAGGGTCACCATCTCCTGC
131
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SEQ
Sequence
ID Sequence
Description
NO.
VL (nt)
ACTGGGAGCAGCTCCAACATCGGGGCAGGTT
ATGATGTACACTGGTACCAGCACCTTCCAGGAA
CAGCCCCCAAACTCCTCATCTATGGTAACAACA
ATCGGCCCTCAGGGGTCCCTGACCGATTCTCTG
GCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCA
TCACTGGGCTCCAGGCTGAGGATGAGGCTGATT
ATTACTGCCAGTCGTTTGACAACACCCATGTG
GTATTCGGCGGAGGGACCAAGCTGACCGTCCT
AG
QVQLQESGPGLVRPSETLSLTCAVSGYSISSGYY
Antibody 418 19
WGWIRQPPGKGLEWIGS1YHSGSAYYNPSLKSRL
VH ( ¨
212 TISADTSKNQFSLKLSSVTAADTAVYYCAREAVA
aa)
GTVRLNWFDPWGQGTLVTVSS
Antibody 418 19 13 GYSISSGYY
2
CDRH1 (aa)
Antibody 418 19 14 IYHSGSA
2
CDRH2 (aa)
Antibody 418 19 5 AREAVAGTVRLNWFDP
21
CDRH3 (aa)
AIRMTQSPSSLSASVGDRVTITCRPSQTISSYLNW
Antibody 418 19
YQQKPGKAPKLITYGASSLQSGVPSRFSGSESGID
¨ VL(VK) (aa) 216 FTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDI
Antibody 418 19 17 QTISSY
2
CDRL1
Antibody 418 19 GAS
218
CDRL2
Antibody 418 19 219 QQSYSTPFT
CDRL3
CAGGTCCAGCTACAGGAGTCGGGCCCAGGACT
GGTGAGGCCTTCGGAGACCCTGTCCCTCACCTG
CGCTGTCTCTGGTTACTCCATCAGCAGTGGCT
ATTACTGGGGCTGGATCCGGCAGCCCCCAGGG
Antibody 418 19
AAGGGGCTGGAGTGGATTGGGAGTATCTATCA
VH (nt)
220 TAGTGGGAGCGCCTACTACAACCCGTCCCTCA
AGAGTCGACTCACCATATCAGCAGACACGTCCA
AGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGA
CCGCCGCAGACACGGCCGTGTATTACTGTGCGA
GAGAGGCAGTGGCTGGTACCGTGCGGCTGA
132
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SEQ
Sequence
ID Sequence
Description
NO.
ACTGGTTCGACCCCTGGGGCCAGGGGACCCTG
GTCACCGTTTCCTCAG
GCCATCCGGATGACCCAGTCTCCATCCTCCCTG
TCTGCATCTGTAGGAGACAGAGTCACCATCACT
TGCCGGCCAAGTCAGACCATTAGCAGCTATTT
AAATTGGTATCAGCAGAAACCAGGGAAAGCCC
Antibody 418 19 CTAAGCTCCTCATCTATGGTGCATCCAGTTTGC
¨ 221 AAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTG
VL (VK) (nt)
AATCTGGGATAGATTTCACTCTCACCATCAGCA
GTCTGCAACCTGAAGATTTTGCAACTTACTACT
GTCAACAGAGTTACAGTACTCCATTCACTTTC
GGCCCTGGGACCAAAGTGGATATCAAAC
QVQLQESGPGLVKPSETLSLTCTVSGGSISNY
Antibody 418-20 222 YWSWIRQPPGKGLEWIGYIYHSVSTNYNPSL
VH (aa)
K SRVTI S VD T SKNQF SLKL S SVTAADTAVYYC
ARDHRFGEFGR1VISWFDPWGQGTLVTVS S
Antibody 418_20 223 GGSISNYY
CDRH1 (aa)
Antibody 418 20 224 IYHSVST
CDRH2 (aa)
Antibody 418_20 225 ARDHRFGEFGRMSWFDP
CDRH3 (aa)
EIVMMQSPATLSVSPGERATLSCRASQSVSSNLA
Antibody 418 20 WYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGT
¨ VL(VK)( )
226 EFTLTISSLQSEDFAVYYCHQYNNWPRTFGQGTK
aa
VEIK
Antibody 418 20 QSVSSN
227
CDRL1
Antibody 418 20 GAS
228
CDRL2
Antibody 418 20 HQYNNWPRT
229
CDRL3
CAGGTACAGCTGCAGGAGTCGGGCCCAGGACT
GGTGAAGCCTTCGGAGACCCTGTCCCTCACCTG
Antibody 418 20 0 CACTGTCTCTGGTGGCTCCATCAGTAATTACT
23
VU (nt) ACTGGAGCTGGATCCGGCAGCCCCCAGGGAAG
GGACTGGAATGGATTGGGTATATCTATCACAG
TGTGAGCACCAACTACAACCCCTCCCTCAAGA
133
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SEQ
Sequence
ID Sequence
Description
NO.
GTCGAGTCACCATATCAGTAGACACGTCCAAGA
ACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCG
CTGCGGACACGGC CGTGTATTACTGTGC GAGA
GATCATAGGTTCGGGGAGTTTGGGAGAATGA
GCTGGTTCGACCCCTGGGGCCAGGGAACCCTG
GTCACCGTCTCCTCAG
GAAATAGTGATGATGCAGTCTCCAGCCACCCTG
TCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCC
TGCAGGGCCAGTCAGAGTGTTAGCAGCAACTT
AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCACCAGGG
Antibody 418-20 231 CCACTGGTATCCCAGCCAGGTTCAGTGGCAGTG
VL (VK) (nt)
GGTCTGGGACAGAGTTCACTCTCACCATCAGCA
GCCTGCAGTCTGAAGATTTTGCAGTTTATTACT
GTCACCAGTATAATAACTGGCCTCGGACGTT
CGGCCAAGGGACCAAGGTGGAAATCAAAC
QVQLQESGPGLVKPSETLSLSCTVSGGSISNYYWS
Antibody 418 21 WIRQPPG KG LEWIGYIYYTGSTYYNP SLKSRVTIS
232 VDTSKNQFSLKLS SVTAADTAVYYCAREVHNWN
VH (aa)
TENWFDPWGQGTLVTVSS
Antibody 418_21
233 GGS1SN Y Y
CDRH1 (aa)
Antibody 418_21 234 1YYTGST
CDRH2 (aa)
Antibody 418 21 235 AREV1-INWNTENWFDP
CDRH3 (aa)
QPVLTQSPGTLSLSPGERATL SCRA SQSVSSTYLA
Antibody 418 21 WYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT
¨ 236 DFTLTISRLEPEDFAVYYCQQYGG SPPLITFGQGT
VL(VK) (aa)
RLEIK
Antibody 418_21
237 QSVSSTY
CDRL 1
Antibody 418 21 GAS
238
CDRL2
Antibody 418_21 239 QQYGGSPPLIT
CDRL3
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACT
Antibody 418 21
240 GGTGAAGCCTTCGGAGACCCTGTCCCTCAGCTG
VH (nt) CACTGTCTCTGGTGGCTCCATCAGTAATTACT
134
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SEQ
Sequence
ID Sequence
Description
NO.
ACTGGAGCTGGATCCGGCAGCCCCCAGGGAAG
GGACTGGAGTGGATTGGGTATATCTATTACAC
TGGGAGCACCTACTACAACCCCTCCCTCAAGA
GTCGAGTCACCATATCAGTAGACACGTCCAAGA
ACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCG
CTGCGGACACGGCCGTCTATTACTGTGCGAGA
GAAGTTCATAACTGGAACACAGAAAACTGGT
TCGACCCCTGGGGCCAGGGAACCCTGGTCACC
GTCTCCTCAG
CAGCCTGTGCTGACTCAGTCTCCAGGCACCCTG
TCTITGICTCCAGGGGAAAGAGCCACCCICTCC
TGCAGGGCCAGTCAGAGTGTTAGCAGCACCT
ACTTAGCCTGGTACCAGCAGAAACCTGGCCAG
GCTCCCAGGCTCCTCATCTATGGTGCATCCAGC
Antibody 418 21 241 AGGGCCACTGGCATCCCAGACAGGTTCAGTGGC
VL (VK) (nt) AGTGGGTCTGGGACAGACTTCACTCTCACCATC
AGCAGACTGGAGCCTGAGGATTTTGCAGTGTAT
TACTGTCAGCAGTATGGTGGCTCACCTCCGC
TGATCACCTTCGGCCAAGGGACACGACTGGAG
ATTAAAC
QVQLVQSGAEVKKPGA SVKVSCKA SGYTFTSYA
Antibody 418 22 MHWVRQAPGQRLEWMGWINAGSGNTKYSQKF
VH ( ¨ 242 QGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR
aa)
EGTEGVRFLEYLFGTWFDPWGQGTLVTVSS
Antibody 418 22 243 GYTFTSYA
CDRHI (aa)
Antibody 418 22 244 INAGSGNT
CDRH2 (aa)
Antibody 418_22 245 AREGTEGVRFLEYLFGTWFDP
CDRH3 (aa)
QAGLTQSPATLSLSPGERATLSCRASQSVISYLAW
Antibody 418 22 YQQKPGHAPRLLIYDASNRATGIPARFSGSGSGTD
¨ VL(VK) (aa) 246 FTLTISSLEPEDFAVYYCQQRSHWPETFGQGTKV
EIK
Antibody 418 22 247 QSVISY
CDRL I
Antibody 418_22 DAS
248
CDRL2
Antibody 418 22 249 QQRSHWPET
135
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PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
CDRL3
CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTG
AAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGC
AAGGCTTCTGGATACACCTTCACTAGCTATGC
TATGCATTGGGTGCGCCAGGCCCCCGGACAAA
GGCTTGAGTGGATGGGATGGATCAACGCTGGC
AGTGGTAATACAAAATATTCACAGAAGTTCCA
(
Antibody 418-22 250 GGGCAGAGTCACCATTACCAGGGACACATCCG
VH nt)
CGAGCACAGCCTACATGGAGCTGAGCAGCCTG
AGATCTGAAGACACGGCTGTGTATTACTGTGCG
AGAGAGGGGACCGAAGGCGTACGATTTTTG
GAGTACTTATTCGGAACCTGGTTCGACCCCT
GGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
CAGGCAGGGCTGACTCAGTCTCCAGCCACCCTG
TCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCC
TGCAGGGCCAGTCAGAGTGTTATCAGCTACTT
AGCCTGGTACCAACAGAAACCTGGCCACGCTCC
CAGGCTCCTCATCTATGATGCATCCAACAGGG
Antibody 418-22 251 CCA CTGGCA TC CC A GC CAGGTTCAGTGGC AGTG
VL (VK) (nt)
GGTCTGGGACAGACTTCACTCTCACCATCAGCA
GCCTAGAGCCTGAAGATTTTGCAGTTTATTACT
GTCAGCAGCGTAGCCACTGGCCTGAGACGTT
CGGCCAAGGGACCAAGGTGGAAATCAAAC
QVTLRESGPVLVKPTETLTLTCTVSGFSLSNAK1V1
Antibody 418 23 GVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSR
VH ( 252 LTISKDTSKSQVVLTMTNMDPVDTATYYCARID
aa)
WWSSYLVGDYWGQGTLVTVSS
Antibody 418 23 253 GFSLSNAKMG
CDRH1 (aa)
Antibody 418 23 254 IFSNDEK
CDRH2 (aa)
Antibody 418 23 255 ARIDWWSSYLVGDY
CDRH3 (aa)
QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYHV
HWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKS
Antibody 418-23 256 GTSASLAITGLQAEDEADYYCQSYDSSLSVVFGG
VL (aa)
GTKL'TVL
Antibody 418_23 SSNIGAGYH
257
CDRL1
136
CA 03194162 2023- 3- 28
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SEQ
Sequence
ID Sequence
Description
NO.
Antibody 418 23 GNS
258
CDRL2
Antibody 418 23
259 QSYDSSLSVV
CDRL3
CAGGTCACCTTGAGGGAGTCTGGTCCTGTGCTG
GTAAAACCCACAGAGACCCTCACGCTGACCTGC
ACCGTCTCTGGGTTCTCACTCAGCAATGCTAA
AATGGGTGTGAGCTGGATCCGTCAGCCCCCAG
GGAAGGCCCTGGAGTGGCTTGCACACATTTTTT
Antibody 418 23 CGAATGACGAAAAATCCTACAGCACATCTCTG
260 AAGAGCAGGCTCACCATCTCCAAGGACACCTCC
VH (nt)
AAAAGCCAGGTGGTCCTTACTATGACCAACATG
GACCCTGTGGACACAGCCACATATTACTGTGCA
CGGATAGATTGGTGGAGTAGTTATTTAGTTG
GTGACTACTGGGGCCAGGGAACCCTGGTCACC
GTCTCCTCAG
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCT
GGGGCCCCAGGGCAGAGGGTCACCATCTCCTGC
ACTGGGAGCAGCTCCAACATCGGGGCGGGTT
ATCATGTACACTGGTACCAGCAGCTTCCAGGAA
CAGCCCCCAAACTCCTCATCTATGGTAACAGC
Antibody 418 23 61 AATCGGCCCTCAGGGGTCCCTGACCGATTCTCT
2
VL (nt) GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC
ATCACTGGGCTCCAGGCTGAGGATGAGGCTGAT
TATTACTGCCAGTCCTATGACAGCAGTCTGAG
TGTGGTATTCGGCGGAGGGACCAAGCTGACCG
TCC TAG
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
NWVRQAPGKGLEWVSSISSSRGYIYYADSVKGRF
Antibody 418-24
VH ( 262 TISRDNAKNSLYLQMNSLRAEDTAVYYCARWLT
aa)
YYYDSSGYFPSPFDYWGQGTLVTVSS
Antibody 418 24 63 GFTFSSYS
2
CDRH1 (aa)
Antibody 418 24 264 ISSSRGYI
CDRH2 (aa)
Antibody 418_24 265 ARWLTYYYDSSGYFPSPFDY
CDRH3 (aa)
Antibody 418 24 266 EIEMMQSPATLSVSPGERATLSCRASQSVSSNLA
VL(VK) (aa) WYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGT
137
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SEQ
Sequence
ID Sequence
Description
NO.
EFTLTISSLQSEDFAVYYCQQYYNWPPWTFGQGT
KVEIK
Antibody 418_24 267 QSVSSN
CDRL1
Antibody 418_24 GAS
268
CDRL2
Antibody 418_24 269 QQYYNWPPWT
CDRL3
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCT
GGTCAAGCCCGGGGGGTCCCTGAGACTCTCCTG
TGCAGCCTCTGGATTCACCTTCAGTAGCTATA
GTATGAACTGGGTCCGCCAGGCTCCAGGAAAG
GGGCTGGAGTGGGTCTCATCCATTAGTAGTAG
Antibody 418 24 TAGAGGTTACATATACTACGCAGACTCAGTGA
270 AGGGCCGATTCACCATCTCCAGAGACAACGCCA
VH (nt)
AGAACTCACTGTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACGGCTGTGTATTACTGTGCG
AGATGGCTTACATATTACTATGATAGTAGTG
GTTATTTCCCCTCGCCTTTTGACTACTGGGGC
CAGGGAACCCIGGICACCGTCTCCTCAG
GAAATAGAGATGATGCAGTCTCCAGCCACCCTG
TCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCC
TGCAGGGCCAGTCACAC TC TTAC CAC CAACTT
AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCACCAGGG
Antibody 418-24 271 CCACTGGTATCCCAGCCAGGTTCAGTGGCAGTG
VL (VK) (nt)
GGTCTGGGACAGAGTTCACTCTCACCATCAGCA
GCCTGCAGTCTGAAGATTTTGCAGTTTATTACT
GTCAGCAGTATTATAACTGGCCTCCGTGGAC
GTTCGGCCAAGGGACCAAGGTGGAAATCAAAC
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM
Antibody 418 25 NWVRQAPGKGLEWVSSISSSRSFIVYADSVKGRI
¨ VH ( ) 272 TISRDNAKNSLYLQMNSLRAEDTAVYYCARVKIT
aa
NYYDSSGYYPDAFDIWGQGTMVTVSS
Antibody 418_25 273 GFTFSSYS
CDRH1 (aa)
Antibody 418_25 274 ISSSRSFI
CDRH2 (aa)
Antibody 418_25 275 ARVKITNYYDSSGYYPDAFDI
138
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SEQ
Sequence
ID Sequence
Description
NO.
CDRH3 (aa)
DIVMTQSPATLSVSPGERATLSCRASQSVSSNLAW
Antibody 418 25 YQQKPGQAPRLLIYGAS I RATGVPARFTGSGSGT
¨ 276 DFTLTISSMQSEDFAVYYCQQYYNWPPWTFGQG
VL(VK) (aa)
TKVEIK
Antibody 418_25
277 QSVSSN
CDRL1
Antibody 418 25 GAS
278
CDRL2
Antibody 418_25 279
wyyNwppwi-
CDRL3
GAGGTGC A A CTGGTGGAGTCTGGGGGAGGCCT
GGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTG
TGCAGCCTCTGGATTCACCTTCAGTAGCTATA
GCATGAACTGGGTCCGCCAGGCTCCAGGGAAG
GGGCTGGAGTGGGTCTCATCCATTAGTAGTAG
Antibody 418 25 TAGGAGTTTCATATACTACGCAGACTCAGTGA
VH (nt) ¨ 280 AGGGCCGAATCACCATCTCCAGAGACAACGCC
AAGAACTCACTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACGGCTGTGTATTACTGTGC
GAGAGTGAAAATTACGAATTACTATGATAGT
AGTGGTTATTACCCTGATGCTTTTGATATCTG
GGGCCAAGGGACAATGGTCACCGTCTCTTCAG
GATATTGTGATGACTCAGTCTCCAGCCACCCTG
TCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCC
TGCAGGGCCAGTCAGAGTGTTAGCAGCAACTT
AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
Antibody 418-25 281 CAGGCTCCTCATCTATGGTGCATCCACCAGGG
CCACTGGTGTCCCAGCCAGGTTCACTGGCAGTG
VL (VK) (lit)
GGTCTGGGACAGATTTCACTCTCACCATCAGCA
GCATGCAGTCTGAAGATTTTGCAGTTTATTACT
GTCAGCAGTATTATAACTGGCCTCCGTGGAC
GTTCGGCCAAGGGACCAAGGTGGAAATCAAAC
EVHLVQSGAEVKKPGASVKVSCKVSGYTLTELS
Antibody 418 26 MHWVRQAPGKGLEWMGGFDPQDAETIYAQKFQ
VH ( ¨ 282 GRVTMTEDTSTDTAYMELSSLRSEDTAVYYCVT
aa)
ATAVAGTPDLYYYHYGLDVWGQGTTV'TVSS
Antibody 418_26 83 GYTLTELS
2
CDRH1 (aa)
139
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SEQ
Sequence
ID Sequence
Description
NO.
Antibody 418 26 284 FDPQDAET
CDRH2 (aa)
Antibody 418_26 285 VTATAVAGTPDLYYYHYGLDV
CDRH3 (aa)
QTVVTQTPLSSPVTLGQPASISCRSSQSLVHSDGN
TYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGS
Antibody 418-26
( 286 GAGTDFTLQISRVEAEDVGVYYCMQATQFPRTF
VL(VK) aa)
GQGTKVEIK
Antibody 418_26 287 QSLVHSDGNTY
CDRL1
Antibody 418 26 KIS
288
CDRL2
Antibody 418 26 289 MQATQFPRT
CDRL3
GAGGTGCATCTGGTACAATCTGGGGCTGAGGTG
AAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGC
AAGGTTTCCGGATACACCCTCACTGAATTATC
CATGCACTGGGTGCGACAGGCTCCTGGAAAAG
GGCTTGAGTGGATGGGAGGTTTTGATCCTCAA
Antibody 418 26 GATGCTGAAACAATCTACGCACAGAAGTTCCA
¨ 290 GGGCAGAGTCACCATGACCGAGGACACATCTA
VH (nt)
CAGACACAGCCTACATGGAACTGAGCAGCCTG
AGATCTGAGGACACGGCCGTGTATTACTGTGTA
ACAGCGACAGCAGTGGCTGGCACCCCAGAC
CTATACTACTACCACTACGGTTTGGACGTCT
GGGGCCAAGGGACCACGGTCACCGTCTCCTCA
CAGACTGTGGTGACCCAGACTCCACTCTCCTCA
CCGGTCACCCTTGGACAGCCGGCCTCCATCTCC
TGCAGGTCTAGTCAAAGCCTCGTCCACAGTGA
TGGAAACACCTACTTGAGTTGGCTTCAGCAGA
GGCCAGGCCAGCCTCCAAGACTCCTAATTTATA
Antibody 418 26 291 AGATTTCTAACCGGTTCTCTGGGGTCCCAGACA
VL (VK) (nt) GATTCAGTGGCAGTGGGGCAGGGACAGATTTC
ACACTGCAAATCAGCAGGGTGGAAGCTGAGGA
TGTCGGGGTTTATTACTGCATGCAAGCTACAC
AG TTTCCTCG TACG TTCGGCCAAGGGACCAAG
GTGGAAATCAAAC
Antibody 418 27 EVQLVESGGGLVRPGGSLRLSCAASGFTFSSCGM
VH (aa) 292 NWVRQAPGKGLEWVSSISRSSNYIYYADSVKGRF
140
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SEQ
Sequence
ID Sequence
Description
NO.
TISRDNAKNSLYLQMNSLRAEDTAVYYCARIPHT
SLYGDYRDDYYYYYGMDVWGQGTTVTVSS
Antibody 418_27 293 GFTFSSCG
CDRH1 (aa)
Antibody 418_27 294 ISRSSNYI
CDRH2 (aa)
Antibody 418_27 295 ARIPHTSLYGDYRDDYYYYYGMDV
CDRH3 (aa)
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAW
Antibody 418 27 YQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTE
¨ VL(VK) (aa) 296 FTLTISSLQSEDFAVYYCQQYNNWPPLTFGGGTK
VEIK
Antibody 418 27
297 QSVSSN
CDRL1
Antibody 418 27 GAS
298
CDRL2
Antibody 418 27
299 QQYNNWPPLT
CDRL3
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCT
GGTCAGGCCTGGGGGGTCCCTGAGACTCTCCTG
TGCAGCCTCTGGATTCACCTTCAGTAGCTGTG
GCATGAACTGGGTCCGCCAGGCTCCAGGGAAG
GGGCTGGAGTGGGTCTCATCCATTAGTAGGAG
TAGTAATTATATATACTACGCAGACTCAGTGA
Antibody 418 27 300 AGGGCCGATTCACCATCTCCAGAGACAACGCCA
VH (nt) AGAACTCACTGTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACGGCTGTATATTACTGTGCG
AGAATCCCCCACACCTCACTCTACGGTGACT
ACCGGGATGATTACTACTATTACTACGGTAT
GGACGTCTGGGGCCAAGGGACCACGGTCACCG
TCTCCTCA
GAAATAGTGATGACGCAGTCTCCAGCCACCCTG
TCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCC
TGCAGGGCCAGTCAGAGTGTTAGCAGCAACTT
Antibody 418 27 AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
VL (VK) (nt) .. 301CAGACTCCTCATCTATGGTGCATCCACCAGGG
CCACTGGTATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGAGITCACTCTCACCATCAGCA
GCCTGCAGTCTGAAGATTTTGCAGTTTATTACT
141
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SEQ
Sequence
ID Sequence
Description
NO.
GTCAGCAGTATAATAACTGGCCTCCGCTCAC
TTTCGGCGGAGGGACCAAGGTGGAGATCAAAC
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAI
Antibody 418 28 HWVRQAPGKGLEWVAVISYDRINKYYADSVKG
¨ 302 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDE
VH (aa)
LPSPYSGYDGGFLYYFDSWGQGTLVTVSS
Antibody 418 28 303 GFTFSSYA
CDRH1 (aa)
Antibody 418 28 304 ISYDRINK
CDRH2 (aa)
Antibody 418 28 305 ARDELP SPY SGYDGGFLYYFD S
CDRH3 (aa)
NIQMTQSPSSLSASVGDRVTITCRASQGISSALAW
Antibody 418 28 YQQKPGKAPKLLIYDASSLASGVPSRFSGSGSGTD
¨ VL(VK) (aa) 306 FTLTISSLQPEDFATYYCQQFNSYPPTFGQGTKVE
IK
Antibody 418_28 307 QGIS SA
CDRL1
Antibody 418_28 DAS
308
CDRL2
Antibody 418_28 309 QQFNSYPPT
CDRL3
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGT
GGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTG
TGCAGCCTCTGGATTCACCTTCAGTAGCTATG
CTATACACTGGGTCCGCCAGGCTCCAGGCAAG
GGGCTAGAGTGGGTGGCAGTTATATCATATGA
TAGAATTAATAAATACTACGCAGACTCCGTGA
Antibody 418 28 310 AGGGCCGATTCACCATCTCCAGAGACAATTCCA
VH (nt) AGAACACGCTGTATCTGCAAATGAACAGCCTGA
GAGCTGAGGACACGGCTGTGTATTACTGTGCG
AGAGATGAGCTCCCGTCCCCATATAGTGGCT
ACGATGGGGGATTTTTATACTACTTTGACTC
CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC
AG
AACATCCAGATGACCCAGTCTCCATCCTCCCTG
Antibody 418 28 3 TCTGCATCTGTAGGAGACAGAGTCACCATCACT
11
VL (VK) (nt) .. TGCCGGGCAAGTCAGGGCATTAGCAGTGCTTT
AGCCTGGTATCAGCAGAAACCAGGGAAAGCTC
142
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SEQ
Sequence
ID Sequence
Description
NO.
CTAAACTCCTGATCTATGATGCCTCCAGTTTGG
CAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG
GATCTGGGACAGATTTCACTCTCACCATCAGCA
GCCTGCAGCCTGAAGATTTTGCAACTTATTACT
GTCAACAGTTTAATAGTTACCCTCCGACGTTC
GGCCAAGGGACCAAGGTGGAAATCAAAC
QVQLVQSGAEVKKPGASVKVSCKVSGYSLIEVS
Antibody 418 29 MEIWVRQAPGKGLEWMGGFDPENVETIYAQKFQ
VH ( 312 GRVTMTEDTSADTAYMELSSLRSEDTAVYYCAT
aa)
TFAFGATTRNLVDYWGQGTLVTVSS
Antibody 418_29
313 GYSLIEVS
CDRH1 (aa)
Antibody 418_29 314 FDPENVET
CDRH2 (aa)
Antibody 418 29 315 ATTFAFGATTRNLVDY
CDRH3 (aa)
SYELTQPPSASGTPGQRVTISCSGSSSNIGSNYVY
WYQQVPGTAPKLLIFKNYQRPSGVPDRFSGSKSG
VL (
Antibody 418-29 316 TSASLAISGLRSEDEADYYCAAWDDTLSGVLFGG
aa)
GTKLTVL
Antibody 418 29 317 SSNIGSNY
CDRL1
Antibody 418 29 KNY
318
CDRL2
Antibody 418 29 319 AAWDDTLSGVL
CDRL3
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTG
AAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGC
AAGGTTTCCGGATACTCCCTCATTGAAGTATC
CATGCACTGGGTGCGACAGGCTCCTGGAAAAG
GACTTGAGTGGATGGGAGGTTTTGATCCTGAA
Antibody 418 29 AATGTGGAAACAATCTACGCACAGAAGTTCCA
320 GGGCAGAGTCACCATGACCGAGGACACATCTG
VH (nt)
CAGACACAGCCTACATGGAGCTGAGCAGCCTG
AGATCTGAGGACACGGCCGTATATTACTGTGCA
ACAACCTTCGCCTTCGGAGCTACAACGAGGA
ACTTAGTAGACTACTGGGGCCAGGGAACCCTG
GTCACCGTCTCCTCAG
143
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SEQ
Sequence
ID Sequence
Description
NO.
TCCTATGAGCTGACACAGC CAC C CTCAGCGTCT
GGGACCCCCGGGCAGAGGGTCACCATCTCTTGC
TCTGGAAGCAGCTCCAACATCGGAAGTAATTA
TGTATACTGGTACCAGCAGGTCCCAGGAACGGC
CC CCAAACTCCTCATCTTTAA GAATTAT CAGCG
Antibody 418 29 321 GCCCTCAGGGGTCCCTGACCGATTCTCTGGCTC
VL (nt) CAAGTCTGGCACCTCAGCCTCCCTGGCCATCAG
TGGGCTCCGGTCCGAGGATGAGGCTGATTATTA
CTGTGCAGCATGGGATGACACCCTGAGTGGT
GTGCTATTCGGCGGAGGGACCAAGCTGACCGT
CCTAG
QVQLVQSGADVKKPGASVKVSCKASGYTFISYY
Antibody 418 30 MEIWVRQAPGQGLEWMGIINP SS GSTIYAQKFQG
VEI
¨ 322 RVTMTTDTSTSTVYMDLS SLTSEDTAVYYCARD
aa) (
GRPREMIERDSSGPYFDYWGQGTLVT1SS
Antibody 4 I 8 30 323 GYTFISYY
CDRH 1 (aa)
Antibody 418_30
324 INP S SG ST
CDRH2 (aa)
Antibody 418 30 325 ARDGRPREMIERDSSGPYFDY
CDRH3 (aa)
SYELTQPPSVSVSPGQTARITCSGDALPKQYAYW
YQQKPGQAPVLVIYKDSERPSGIPERFSGS SSGTT
VL (
Antibody 418-30 326 VTLTISGVQAEDEADYYCQSTDSSGTHVVFGGGT
aa)
KLTVL
Antibody 418_30
327 ALPKQY
CDRL 1
Antibody 418 30 KDS
328
CDRL2
Antibody 418_30 329 Q STD S SGTHVV
CDRL3
CAGGTACAGCTGGTGCAGTCTGGGGCTGATGTG
AAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGC
AAGGCATCTGGATACACCTTCATCAGTTACTA
Antibody 418 30 30 TATGCATTGGGTGCGACAGGCCCCTGGACAAG
3
VH (nt) GGCTTGAGTGGATGGGAATAATCAACCCTAGT
AGTGGTAGCACAATCTACGCACAGAAGTTC CA
GGGCAGAGTCACCATGACCACGGACACGTCCA
CGAGCACAGTTTACATGGACTTGAGCAGCCTGA
144
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SEQ
Sequence
ID Sequence
Description
NO.
CATCTGAGGACACGGCCGTGTATTACTGTGCGA
GAGATGGGCGACCGCGAGAGATGATC GAAC
GTGATAGTAGTGGGCCTTACTTTGACTACTG
GGGCCAGGGAACCCTGGTCACCATCTCCTCAG
TCCTATGAGCTGACACAGCCACCCTCGGTGTCA
GTGTCCCCAGGACAGACGGCCAGGATCACCTGC
TCTGGAGATGCA TT GCCAAAGCAA TATGCTTA
TTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGT
Antibody 418 30
GCTGGTGATATATAAAGACAGTGAGAGGCCCT
VL (nt) ¨
331 CAGGGATCCCTGAGCGATTCTCTGGCTCCAGCT
CAGGGACAACAGTCACGTTGACCATCAGTGGA
GTCCAGGCAGAAGATGAGGCTGACTATTACTGT
CAATCAACAGACAGCAGTGGTACCCACGTGG
TATTCGGCGGAGGGACCAAGCTGACCGTCCTAG
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSTSYY
Antibody 418 31
WSWIRQPAGKGLEWIGRIYNSGSTNYNPSLKSRV
VH (
332 TISVDTSKNQFSLKLTSVTAADTAVYYCARDLDY
aa)
YDFWSGYSDWYFDLWGRGTQVTVSS
Antibody 418 31
333 GGSISSTSYY
CDRH1 (aa)
Antibody 418 31
334 IYNSGST
CDRH2 (aa)
Antibody 418 31 335 A RDLDYYDFWSGY SDWYFDL
CDRH3 (aa)
SYELTQPPSVSKGLRQTATLTCTGNSNNVGDQGA
Antibody 418 31
AWLQQHQGHPPKLL SYRNNNRP SGI SERF SA SRS
VL ( ¨
336 GNTA SLTITGLQPEDEADYYCSAWDTSLSAWVF
aa )
GGGTKLTVL
Antibody 418 31 SNNVGDQG
33 7
CDRL 1
Antibody 418_31 RNN
338
CDRL2
Antibody 418 31
33, SAWDTSLSAWV
CDRL3
CAGGTACAGCTGCAGGAGTCGGGCCCAGGACT
GGTGAAGCCTTCACAGACCCTGTCCCTCACCTG
Antibody 418 31
340 CACTGTCTCTGGTGGCTCCATCAGTAGTACTA
VH (nt) GTTACTACTGGAGCTGGATCCGGCAGCCCGCC
GGGAAGGGACTGGAGTGGATTGGGCGTATATA
145
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SEQ
Sequence
ID Sequence
Description
NO.
TAACAGTGGGAGCACCAACTACAATCCCTCCC
TCAAGAGTCGAGTCACCATATCAGTAGACACGT
CCAAGAACCAGTTCTCCCTGAAGCTGACCTCTG
TGACCGCCGCCGACACGGCCGTCTATTACTGTG
CGAGAGATCTTGACTACTACGATTTTTGGAG
TGGTTATTCTGACTGGTACTTCGATCTCTGGG
GCCGTGGCACCCAGGTCACTGTCTCCTCAG
TCCTATGAGCTGACTCAGCCACCCTCGGTGTCC
AAGGGCTTGAGACAGACCGCCACACTCACCTGC
ACTGGGAACAGCAACAATGTTGGCGACCAAG
GAGCAGCTTGGCTGCAGCAGCACCAGGGCCAC
CCTCCCAAACTCCTATCCTACAGGAATAACAAC
Antibody 418 31 341 CGGCCCTCAGGGATCTCAGAGAGATTCTCTGCA
VL (nt) TCCAGGTCAGGAAACACAGCCTCCCTGACCATT
ACTGGACTCCAGCCTGAGGACGAGGCTGACTAT
TACTGCTCAGCATGGGACACCAGCCTCAGTG
CTTGGGTGTTCGGCGGAGGGACCAAACTGACC
GTCCTAA
QVQLQESGPGLVKPSETLSLTCTVSDDSISSYYWS
WIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTIS
Antibody 418-33 342 VDTSKNQFSLNLSSVTAADTAVYYCARDRGWD
VH (aa)
GYNLGFDYWGQGTLVTVSS
Antibody 418_33
343 DDSISSYY
CDRH1 (aa)
Antibody 418_33 IYYSGST
344
CDRH2 (aa)
Antibody 418 33 345 ARDRGWDGYNLGFDY
CDRH3 (aa)
SYELTQPPSVSVAPGQTARITCGGNKIGSKSVHW
Antibody 418 33 YQQMPGQAPVLVVYDDSDRPSGIPERFSGSNSGN
¨ 346 TATLTISRVEAGDEADYYCQVVVDNNSDQGVFGG
VL (aa)
GTKLTVL
Antibody 418_33 347 KIGSKS
CDRL1
Antibody 418 33 DDS
348
CDRL2
Antibody 418 33 349 QVWDNNSD QG V
CDRL3
146
CA 03194162 2023- 3- 28
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PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACT
GGTGAAGCCTTCGGAGACCCTGTCCCTCACTTG
CACTGTCTCTGATGACTCCATCAGTAGTTACT
ACTGGAGCTGGATTCGGCAGCCCCCAGGGAAG
GGACTGGAGTGGATTGGGTATATCTATTACAG
Antibody 418 33 TGGGAGCACCAACTACAACCCCTCCCTCAAGA
¨ 350 GTCGAGTCACCATATCAGTAGACACGTCCAAGA
VH (nt)
ACCAGTTCTCCCTGAACCTGAGCTCTGTGACCG
CTGCGGACACGGCCGTATATTACTGTGCGAGA
CATAGAGGATGCGATGGCTACAATTTAGGCT
TTGACTACTGGGGCCAGGGAACCCTGGTCACC
GTCTCCTCAG
TCCTATGAGCTGACTCAGCCACCCTCGGTGTCA
GTGGCCCCAGGACAGACGGCCAGGATAACCTG
TGGGGGAAACAAGATTGGAAGTAAAAGTGTG
CACTGGTACCAGCAGATGCCAGGCCAGGCCCC
GGTGCTGGTCGTCTATGATGATAGCGACCGGC
Antibody 418 33 351 CCTCAGGGATCCCTGAGCGATTCTCTGGCTC CA
VL (nt) ACTCTGGGAACACGGCCACCCTGACCATCAGCA
GGGTCGAAGCCGGGGATGAGGCCGACTATTAC
TGTCAGGTGTGGGATAATAATAGTGACCAGG
GGGTGTTCGGCGGAGGGACCAAGCTGACCGTC
CTAG
QITLKESGPALVKPTQTLTLTCTFSGFSLSTTGMR
Antibody 418 34 VSWIRQPPGKALEWLARIDWDDDKFYSTSLKTRL
VTI
¨ 352 TISKDTSKNQVVLTMTNMDPVDTGTYYCARAYG
(aa)
DHEDYWGQGTLVTVSS
Antibody 418 34 353 GFSLSTTGMR
CDRHI (aa)
Antibody 418_34 354 IDWDDDK
CDRH2 (aa)
Antibody 418_34 355 ARAYGDHEDY
CDRH3 (aa)
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNN
KNYLVWYQQKPGQPPKLLIYWASTRESGVPDRFS
Antibody 418-34 356 GSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPITF
VL(VK) (aa)
GQGTRLEIK
Antibody 418 34
357 QSVLYSSNNKNY
CDRLI
147
CA 03194162 2023- 3- 28
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PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
Antibody 418 34
358 WAS
CDRL2
Antibody 418 34 359 QQYYSTPIT
CDRL3
CAGATCACCTTGAAGGAGTCTGGTCCTGCGCTG
GTGAAACCCACACAGACCCTCACACTGACCTGC
ACCTTCTCGGGGTTCTCACTCAGCACTACTGG
AATGCGTGTGAGCTGGATCCGTCAGCCCCCAG
GGAAGGCCCTGGAGTGGCTTGCACGCATTGAT
Antibody 418 34 60 TGGGATGATGATAAATTCTACAGCACATCTCT
3
VH (nt) GAAGACCAGGCTCACCATCTCCAAGGACACCTC
CAAAAACCAGGTGGTCCTTACAATGACCAACAT
GGACCCTGTGGACACAGGCACGTATTACTGTGC
ACGGGCCTACGGTGATCACGAAGACTACTGG
GGCCAGGGAACCCTGGTCACCGTCTCCTCAG
GATATTGTGATGACTCAGTCTCCAGACTCCCTG
GCTGTGTCTCTGGGCGAGAGGGCCACCATCAAC
TGCAAGTCCAGCCAGAGTGTTTTATACAGCTC
CAACAATAAGAATTACTTAGTTTGGTACCAGC
AGAAACCAGGACAGCCTCCTAAGCTGCTCATTT
Antibody 418 34 361 ACTGGGCATCTACGCGGGAATCCGGGGTCCCT
VL(VK) (nt) GACCGATTCAGTGGCAGCGGGTCTGGGACAGA
TTTCACTCTCACCATCAGCAGCCTGCAGGCTGA
AGATGTGGCAGTTTATTACTGTCAGCAATATTA
TAG TACTCCCATCACCTTCGGCCAAGGGACAC
GACTGGAGATTAAAC
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEM
Antibody 418 35 NWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRF
¨ 362 TISRDNAKNSLYLQMNSLRAEDTAVYYCARGEG
VH (aa)
SGYYIFYYYGMDVWGRGTTVTVSS
Antibody 418_35 363 GFTFSSYE
CDRH1 (aa)
Antibody 418_35 364 ISSSGSTI
CDRH2 (aa)
Antibody 418_35 365 ARGEGSGYYIFYYYGMDV
CDRH3 (aa)
Antibody 418 35 DIVMTQSPLSLSVTPGQPASISCKSSESLLHSDGK
366
VL(VK) (aa) TYLSWYVQKPGQPPRLLIHELSNRFPGVPDRFSGS
148
CA 03194162 2023- 3- 28
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PCT/US2021/052481
SEQ
Sequence
ID Sequence
Description
NO.
GSETDFTLRISRVEAEDVGVYYCMQFGEKFTFGP
GTKVDIK
Antibody 418_35 367 ESLLHSDGKTY
CDRL1
Antibody 418_35 68 ELS
3
CDRL2
Antibody 418_35 369 MQFGEKFT
CDRL3
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTT
GGTACAGCCTGGAGGGTCCCTGAGACTCTCCTG
TGCAGCCTCTGGATTCACCTTCAGTAGTTATG
AAATGAACTGGGTCCGCCAGGCTCCAGGGAAG
GGTCTGGAGTGGGTTTCATACATTAGTAGTAG
Antibody 418 35 TGGTAGTACCATATACTACGCAGACTCTGTGA
370 AGGGCCGATTCACCATCTCCAGAGACAACGCCA
VH (nt)
AGAACTCACTGTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACGGCTGTTTATTACTGTGCGA
GAGGGGAGGGCAGTGGCTACTATATTTTCTA
CTACTACGGTATGGACGTCTGGGGCCGAGGG
ACCACGGTCACCGTCTCCTCA
GATATTGTGATGACTCAGTCTCCACTCTCTCTGT
CCGTCACCCCTGGACAGCCGGCCTCCATCTCCT
GCAAGTCTAGTCACACCCTCCTACATACTCAT
GGAAAGACCTATTTGTCTTGGTACGTGCAGAA
GCCAGGCCAGCCTCCACGGCTCCTGATCCATGA
Antibody 418 35 371 ACTTTCCAACCGGTTCCCTGGAGTGCCAGATAG
VL(VK) (nt) GTTCAGTGGCAGCGGGTCAGAGACAGACTICAC
ACTGAGGATCAGCCGGGTGGAGGCTGAGGATG
TTGGCGTTTATTACTGCATGCAATTCGGGGAG
AAATTCACTTTCGGCCCTGGGACCAAAGTGGA
CATCAAAC
QVQLQESGPGLVKPSGTLSLTCDVSGASISSSNW
Antibody 418 37 WSWVRQPPGKGLEWIGEIYHSGNTNYNPSLKSR
VH
372 VTMSVDKSKNQFSLTVSSVTAADTAVYYCASRV
aa ()
SGRYNWFDPWGQGTLVTVSS
Antibody 418 37 GASISSSNW
373
CDRH1 (aa)
Antibody 418 37 1YHSGNT
374
CDRH2 (aa)
149
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SEQ
Sequence
ID Sequence
Description
NO.
Antibody 418 37 A SRVSGRYNWFDP
375
CDRH3 (aa)
QSVLTQPASVSGSPGQSITISCTGTSSDVGSYNLVS
Antibody 418 37 WYQQHPGKAPKLMIYEGSKRPSGISNRFSGSKSG
376 NTASLTISGLQAEDEADYYCFSYAGFSTWVFGGG
VL (aa)
TKLTVL
Antibody 418 37 SSDVGSYNL
377
CDRL1
Antibody 418 37 EGS
378
CDRL2
Antibody 418_37 FSYAGFSTWV
379
CDRL3
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACT
GGTGAAGCCTTCGGGGACCCTGTCCCTCACCTG
CGATGTCTCTGGTGCCTCCATCAGCAGTAGTA
ACTGC 1G GAM_ I GGG I CCGCCAGCCCCCAGGG
AAGGGGCTGGAGTGGATTGGGGAAATCTATCA
Antibody 418 37 TAGTGGGAACACCAACTACAACCCGTCCCTCA
VH
380 AGAGTCGAGTCAC CATGTCAGTAGACAAGTC CA
(nt)
AGA ACCAGTTCTCCCTGACGGTGAGCTCTGTGA
CCGCCGCGGACACGGCCGTGTACTACTGTGCG
AGCCGAGTTTCAGGGAGGTACAACTGGTTCG
ACCCCTGGGGCCAGGGAACCCTGGTCACCGTC
TCCTCAG
CAGTCTGTGTTGACTCAGCCTGCCTCCGTGTCTG
GGTCTCCTGGACAGTCGATCACCATCTCCTGCA
CTGGAACCAGCAGTGATGTTGGCAGTTATAA
CCTTGTCTCCTGGTACCAACAGCACCCAGGCAA
AGCCCCCAAACTCATGATTTATGAGGGCAGTA
Antibody 418 37 381 AGCGGCCCTCAGGAATTTCTAATCGCTTCTCTG
VL (nt) GCTCCAAGTCTGGCAACACGGCCTCCCTGACAA
TCTCTGGGCTCCAGGCTGAGGACGAGGCTGATT
ATTACTGCTTCTCATATGCAGGTTTTAGCACT
TGGGTGTTCGGCGGAGGGACCAAGCTGACCGT
CCTAG
QVQLVESGGGVVQPGKSLRLSCAASGFTFNNYG
Antibody 418 38 MHWVRQAPGKGLEWVAVIWYDGSNKYYTDSV
( ) 382 KGRFTISRDNSKNTLYLQIVENSLRAEDTAVYYCAR
aa
ETSDYGDYIRLRRNAFDIWGQGTMVTVSS
150
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SEQ
Sequence
ID Sequence
Description
NO.
Antibody 418 38 383 GFTFNNYG
CDRH1 (aa)
Antibody 418 38 384 IWYDGSNK
CDRH2 (aa)
Antibody 418_38 385 ARETSDYGDYIRLRRNAFDI
CDRH3 (aa)
EIVMMQ SP ATLSVSPGA RVTLS CRA SQSISNNLA
WYQQKPGQAPRLLIYGASTRASGIPARFSGSGSGT
Antibody 418-38 386 EFTLTISSLQ SEDFAVYYCQQYDKWPPWTFGQG
VL(VK) (aa)
TKVEIK
Antibody 418_38
387 QSISNN
CDRL 1
Antibody 418 38 GAS
388
CDRL2
Antibody 418 38 389 QQYDKWPPWT
CDRL3
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGT
GGTCCAGCCTGGGAAGTCCCTGAGACTCTCCTG
TGCAGCGTCTGGATTCACCTTTAATAACTATG
GCATGCACTGGGTCCGCCAGGCTCCAGGCAAG
GGGCTGGAGTGGGTGGCAGTTATATGGTAT GA
Antibody 418 38 TGGAAGTAATAAATAC TATA CAGAC TC CGTGA
¨ 390 AGGGCCGATTCAC CATCTCCAGAGACAATTC CA
VH (nt)
AGAACACGCTGTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACGGCTGTCTATTACTGTGCG
AGAGAGACAAGTGACTACGGTGACTACATAC
GCTTGCGCAGGAATGC T TT TGATATCTGGGG
CCAAGGGACAATGGTCACCGTCTCTICAG
GAAATAGTGATGATGCAGTCTCCAGCCACCCTG
TCTGTGTCTCCAGGGGCGAGAGTCACCCTCTCC
TGCAGGGCCAGTCAGAGTATTAGCAACAACTT
AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
Antibody 418 38 CAGGCTCCTCATCTATGGTGCATCCACCAGGG
vL(vi() (nt) 391 CCTCTGGTATCCCAGCCAGGTTCAGTGGCAGTG
GGTCTGGGACAGAGTTCACTCTCACCATCAGCA
GCTTGCAGTCTGAAGATTTTGCAGTTTATTACTG
TCAGCAGTATGATAAGTGGCCTCCGTGGACG
TTCGGCCAAGGGACCAAGGTGGAAATCAAAC
151
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SEQ
Sequence
ID Sequence
Description
NO.
QVQLVQSGAEVKKPGS SVKVSCKASGGTISSYAI
SWVRQAPGQGLEWMGGIMRIFGTPNYAQKFQG
VH (
Antibody 418-39 392 RVTITADESTSTAYMELSSLRSEDTAVYYCAREG
aa)
YC SS SNCYDDALDIWGQGTMVTVS S
Antibody 418_39 393 GTIS SYA
CDRH1 (aa)
Antibody 418_39
394 IMRIFGTP
CDRH2 (aa)
Antibody 418 39 395 AREGYCSS SNCYDDALDI
CDRH3 (aa)
QPVLTQSPSASASLGASVKLTCTLSSGHSSYAIAW
Antibody 418 39 HQ Q QPEKGPRYLMKLNSD GSHSKGDGIPDRFSGS
¨ 396 SSGAERYLTISSLQSEDEADYYCQTWGIGIRVFG
VL (aa)
GGTKLTVL
Antibody 418 39 397 SGHS SYA
CDRL 1
Antibody 418 39 398 LNSDGSH
CDRL2
Antibody 418_39 399 QTWGIGIRV
CDRL3
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTG
AAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGC
AAGGCTTCTGGAGGCACCATCAGCAGCTATG
C TATCAGC TGGGTGC GA CAGGC C CCTG GA CAA
GGGCTTGAGTGGATGGGAGGGATCAT GC GTAT
CTTTGGTACACCAAACTACGCACAGAAGTTCC
VH t
Antibody 418-39 400 AGGGCAGAGTCACGATTACCGCGGACGAATCC
n) (
ACGAGCACAGCCTACATGGAGCTGAGCAGCCT
GAGATCTGAGGACACGGCCGTGTATTACTGTGC
GAGGGAAGGATATTGTAGTAGTAGTAACTGT
TATGAC GAT GC T T TAGATATC TGGGGCCAAGG
GACAATGGTCACCGTCTCTTCAG
CAGCCTGTGCTGACTCAATCGCCCTCTGCCTCT
GCCTCCCTGGGAGCCTCGGTCAAGCTCACCTGC
ACTCTGAGCAGTGGGCACAGCAGCTACGCCA
Antibody 418 39
401 TCGCATGGCATCAGCAGCAGCCAGAGAAGGGC
VL (nt) CCTCGGTACTTGATGAAGCTTAACAGTGATGG
CAGCCACAGCAAGGGGGACGGGATCCCTGATC
GCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGTT
152
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SEQ
Sequence
ID Sequence
Description
NO.
ACCTCACCATCTCCAGCCTCCAGTCTGAGGATG
AGGCTGACTATTACTGTCAGACGTGGGGCATT
GGCATTCGGGTATTCGGCGGAGGGACCAAACT
GACCGTCCTAG
EVQLVQSGAEVKKPGASVKVSCKVSGYTLPELSI
Antibody 418 41 HWVRQAPGKGLEWMGGFDPEDGETIYAQKFQG
¨ 402 RVTMTEDTSTDTAYMELTSLRSDDTAVYYCATSP
VH (aa)
AVVRKNWFDPWGQGTLVTVSS
Antibody 418_41 403 GYTLPELS
CDRH1 (aa)
Antibody 418 41 404 FDPEDGET
CDRH2 (aa)
Antibody 418_41 405 ATSPAVVRKNWFDP
CDRH3 (aa)
SYELTQPP SVSVSPGQTA SITC SGDKL GD KD A CW
Antibody 418 41 YQQKPGQSPVLVIYEDNKRPSGIPERFSGSNSGNT
VL ( ¨ 406 ATLTISGTQAMDEADYYCQAWDSSTHVVFGGGT
aa)
KLTVL
Antibody 418 41 407 KLGDKD
CDRL1
Antibody 418 41 EDN
408
CDRL2
Antibody 418_41 409 QAWDSSTHVV
CDRL3
GAGGTGCAGCTGGTACAGTCTGGGGCTGAGGT
GAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTG
CAAGGTTTCCGGATACACCCTCCCTGAATTAT
CCATACACTGGGTGCGACAGGCTCCTGGAAAA
GGGCTTGAGTGGATGGGAGGTTTTGATCCTGA
Antibody 418 41 AGA TGGTGAAACAATCTATGCACAGAAGTTCC
¨ 410 AGGGCAGAGTCACCATGACCGAGGACACATCT
VH (nt)
ACAGACACAGCCTACATGGAGCTGACCAGCCT
GAGATCTGACGACACGGCCGTCTATTACTGTGC
AACCTCCCCGGCTGTGGTACGAAAGAACTGG
TTCGACCCCTGGGGCCAGGGAACCCTGGTCAC
CGTCTCCTCAG
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCC
Antibody 418 41
411 GTGTCCCCAGGACAGACAGCTAGCATCACCTGC
VL (nt) TCTGGAGATAAATTGGGGGATAAAGATGCCTG
153
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SEQ
Sequence
ID Sequence
Description
NO.
CTGGTATCAGCAGAAGCCAGGCCAGTCCCCTGT
GCTGGTCATCTATGAAGATAACAAGCGGCCCT
CAGGGATCCCTGAGCGATTCTCTGGCTCCAACT
CTGGGAACACAGCCACTCTGACCATCAGCGGG
ACCCAGGCTATGGATGAGGCTGACTATTACTGT
CAGGCGTGGGACAGCAGCACTCATGTGGTAT
TCGGCGGAGGGACCAAGCTGACCGTCCTAG
QVQLQESGPGLVKPSQTLSLTCTVSGDSISSGDHY
Antibody 418 42 WSWIRQPPGKGLEWIGYIYYSGNTYYNPSLKSRL
¨ 412 TISVDTSNNQFSLKLSSVTAADTAVYYCARAIVG
VH (aa)
MVRGVILLWYFDPWGRGTLVTVSS
Antibody 418 42 413 GDSISSGDHY
CDRH1 (aa)
Antibody 418_42 414 IYYSGNT
CDRH2 (aa)
Antibody 418 42 415 ARAIVGMVRGVILLWYFDP
CDRH3 (aa)
QSVLTQPPSVSAAPGQKVTISCSGNRSNIGNNYVS
Antibody 418 42 WYQQFPGTAPKLLIYDINKRPSGIPDRFSGSKSGTS
¨ 416 ATLGITGLQTGDEADYYCGTWDSSLSGPVFGGG
VL (aa)
TKL'TVL
Antibody 418 42 417 RSNIGNNY
CDRL1
Antibody 418 42
418 DIN
CDRL2
Antibody 418 42 419 GTWDSSLSGPV
CDRL3
CAGGTGCAGCTACAGGAGTCGGGCCCAGGACT
GGTGAAGCCTTCACAGACCCTGTCCCTCACCTG
CACTGTCTCTGGTGACTCCATCAGCAGTGGTG
ATCACTACTGGAGTTGGATCCGCCAGCCCCCA
GGGAAGGGCCTGGAGTGGATTGGTTACATCTA
TTACAGTGGCAACACCTACTACAACCCGTCCC
Antibody 418-42 420 TCAAGAGTCGACTTACCATATCAGTAGACACGT
VH (nt)
CCAATAATCAGTTCTCCCTGAAGCTGAGCTCTG
TGACTGCCGCAGACACGGCCGTGTATTACTGTG
CCAGAGCAATCGTGGGTATGGTTCGGGGAGT
TATTCTTCTCTGGTACTTCGATCCCTGGGGCC
GTGGCACCCTGGTCACTGTCTCCTCAG
154
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SEQ
Sequence
ID Sequence
Description
NO.
CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCT
GCGGCCCCAGGACAGAAGGTCACCATCTCCTGC
TCTGGAAACAGATCCAACATAGGGAATAATTA
TGTATCCTGGTACCAGCAGTTCC CAGGAACAGC
CCCCAAACTCCTCATTTATGACATTAATAAGCG
Antibody 418 42 421 ACCCTCAGGGATTCCTGACCGATTCTCTGGCTC
VL (nt) CAAGTCTGGCACGTCAGCCACCCTGGGCATCAC
CGGACTCCAGACTGGGGACGAGGCCGATTATTA
CTGCGGAACATGGGATAGCAGCCTGAGTGGT
CCTGTATTCGGCGGAGGGACCAAGCTGACCGT
CCTAG
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAM
Antibody 418 43 SWVRQAPGKGLEWVSSISGDGGSTYYADSVKGR
VI-I
¨ 422 FTVSRDNSKNTVYLQMNSLRVEDTAVYYCAKGD
a a) (
TFMVPYNWFDPWGQGTLVTVSS
Antibody 418 43 423 GFTFSSYA
CDRHI (aa)
Antibody 418 43 424 ISGDGGST
CDRH2 (aa)
Antibody 418 43 AKGDTFMVPYNVVFDP
425
CDRH3 (aa)
EIVLTQSPATLSLSPGERATLSCRASQSISSRLAWY
QQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDF
Antibody 418-43
VL(VK) (aa) 426 TLTISGLEPEDFAVYYCQQRSNWPGTFGQGTKVE
IK
Antibody 418_43 427 QSISSR
CDRLI
Antibody 418 43 DAS
428
CDRL2
Antibody 418 43 429 QQRSNWPGT
CDRL3
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTG
GTACAGCCTGGGGGGTCCCTGAGACTCTCCTGT
GCAGCCTCTGGATTCACCTTTAGCAGCTATGC
Antibody 418 43 3 CATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG
0 4
VH (nt) GGCTGGAGTGGGTCTCATCTATTAGTGGTGAT
GGTGGTAGCACATATTACGCAGACTCCGTGAA
GGGCCGGTTCACCGTCTCCAGAGACAATTCCAA
GAACACGGTATATCTGCAAATGAACAGCCTGA
155
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SEQ
Sequence
ID Sequence
Description
NO.
GAGTCGAGGACACGGCCGTATATTACTGTGCG
AAAGGGGATACATTTATGGTTCCGTACAACT
GGTTCGACCCCTGGGGCCAGGGAACCCTGGTC
ACCGTCTCCTCAG
GAAATTGTGCTGACTCAGTCTCCAGCCACCCTG
TCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCC
TGCAGGGCCAGTCAGAGTATTAGCAGCCGCTT
AGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
Antibody 418 43 CAGGCTCCTCATCTATGATGCATCCAACAGGG
¨ 431 CCACTGGCATCCCAGCCAGGTTCAGTGGCAGTG
VL(VK) (nt)
GGTCTGGGACAGACTTCACTCTCACCATCAGCG
GCCTAGAGCCTGAAGATTTTGCTGTTTATTACT
GTCAGCAGCGTAGCAACTGGCCGGGGACGTT
CGGCCAAGGGACCAAGGTGGAAATCAAAC
EVQLQESGPGLVKSSETLSLTCTVSGGSISSDYWN
Antibody 418-44 432 WIRQPPGKGPEWIGYIYYSGSTHYNPSLKSRVTIS
VH (
VDTSKSQFSLKLSSVTAADTAVYYCARLLYYYDS
aa)
SGYSIGGAFDIWGQGTMVTVSS
Antibody 418 44
433 GGSISSDY
CDRH1 (aa)
Antibody 418 44 IYYSGST
434
CDRH2 (aa)
Antibody 418 44 435 ARLLYYYDSSGYSIGGAFDI
CDRH3 (aa)
SYELTQPPSVSVSPGQTARITCSGDALAKQYAYW
Antibody 418 44 YQQKPGQAPVLVIYKDTERPSGIPERFSGSSSGTT
VL ( ¨ 436 VTLTISGVQ AEDEADYYCQSADSSSTYVVFGGGT
aa )
RLTVL
Antibody 418 44 437 ALAKQY
CDRL1
Antibody 418_44 KDT
438
CDRL2
Antibody 418_44
43, QSADSSSTYVV
CDRL3
GAGGTGCAGCTGCAAGAGTCGGGCCCAGGACT
GGTGAAGTCTTCGGAGACCCTGTCCCTCACTTG
Antibody 418 44
440 CACTGTCTCTGGTGGCTCCATCAGTAGTGACT
VH (nt) ACTGGAATTGGATTCGGCAGCCCCCAGGGAAG
GGACCGGAGTGGATTGGGTATAT C TAT TACA G
156
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SEQ
Sequence
ID Sequence
Description
NO.
TGGGAGCACCCACTACAACCCCTCCCTCAAGA
GTCGAGTCACCATATCAGTAGACACGTCCAAGA
GCCAGTTCTCCCTAAAGCTGAGCTCTGTGACCG
CTGCGGACACGGCCGTCTATTACTGTGCGAGG
CTTTTATATTACTATGATAGTAGTGGTTATTC
CATAGGAGGTGCTTTTGATATCTGGGGCCAA
GGGACAATGGTCACCGTCTCTTCAG
TCCTATGAGCTGACACAGCCACCCTCGGTGTCA
GTGTCCCCAGGACAGACGGCCAGGATCACCTGC
TCTGGAGATGCATTGGCAAAGCAATATGCTTA
TIGGTACCAACAGAAGCCAGGCCAGGCCCCIGT
Antibody 418 44 GCTGGTGATATATAAAGACACTGAGAGGCCCT
441 CAGGGATCCCTGAGCGATTCTCTGGCTCCAGCT
VL (nt)
CAGGGACAACAGTCACGTTGACCATCAGTGGA
GTCCAGGCAGAAGACGAGGCTGACTATTACTGT
CAATCAGCAGACAGCAGTTCTACTTATGTGG
TATTCGGCGGAGGGACCAGGCTGACCGTCCTAG
QVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGI
SARS-CoV-2 SWVRQAPGQGLEWMGWISTYNGNTNYAQKFQG
S309 mAb VU 442 RVTMTTDTSTTTGYMELRRLRSDDTAVYYCARD
(aa) YTRGAWFGESLIGGEDNWGQGTLVTVSS
SARS-CoV-2
S309 mAb 443 GYPFTSYG
CDRH1 (aa)
SARS-CoV-2
S309 mAb 444 ISTYNGNT
CDRH2 (aa)
SARS-CoV-2
S309 mAb 445 ARDYTRGAWFGESLIGGFDN
CDRH3 (aa)
EIVLTQSPGTLSLSPGERATLSCRASQTVSSTSLAW
SARS-CoV-2 YQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTD
S309 mAb VL 446 FTLTISRLEPEDFAVYYCQQHDTSLTFGGGTKVEI
(VK) (aa)
SARS-CoV-2
S309 mAb CDRL1 447 QTVSSTS
(aa)
SARS-CoV-2
S309 mAb CDRL2 448 GAS
(aa)
157
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SEQ
Sequence
ID Sequence
Description
NO.
SARS-CoV-2
S309 mAb CDRL3 449 QQHDTSLT
(aa)
QVQLVQSGPEVKKPGTSVRVSCKASGFTFTSSAV
QWVRQARGQRLEWVGWIVVGSGNTNYAQKFHE
SARS-CoV-2 RVTITRDMSTSTAYMELSSLRSEDTAVYYCASPY
S2E12 mAb VH 450 CSGGSCSDGFDIWGQGTMVTVSS
(aa)
SARS-CoV-2
S2E12 mAb 451 GFTFTSSA
CDRH1 (aa)
SARS-CoV-2
S2E12 mAb 452 IVVGSGNT
CDRH2 (aa)
SARS-CoV-2
S2E12 mAb 453 ASPYCSGGSCSDGFDI
CDRH3 (aa)
DIVLTQTPGTLSLSPGERATLSCRASQSVSSSYLA
WYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT
SARS-CoV-2 DFTLTISRLEPEDFAVYYCQQYVGLTGWTFGQG
S2E12 mAb 454 TKVEIK
VL(VK) (aa)
SARS-CoV-2
S2E12 mAb 455 QSVSSSY
CDRL I (aa)
SARS-CoV-2
S2E12 mAb 456 GAS
CDRL2 (aa)
SARS-CoV-2
S2E12 mAb 457 QQYVGLTGWT
CDRL3 (aa)
EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYY
MHWVRQAPGQGLEWMGWINPISSGTSYAQTFQ
SARS-CoV-2 GRVTMTSDTSITTAYMELSRLRSDDTAVYYCARA
S2M11 m Ab VH 458 APFYDFWSGYSYFDYWGQGTLVTVSS
(aa)
158
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SEQ
Sequence
ID Sequence
Description
NO.
SARS-CoV-2
S2M11 mAb 459 GYTFTGYY
CDRH1 (aa)
SARS-CoV-2
S2M11 mAb 460 INPISSGT
CDRH2 (aa)
SARS-CoV-2 ARAAPFYDFWSGYSYFDY
S2M11 mAb 461
CDRH3 (aa)
EIVMMQSPGTLSLSPGERATLSCRASQSVSSSYLA
WYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT
SARS-CoV-2 DFTLTISRLEPEDFAVYYCQQYGSSAWTFGQGTK
S2M11 mAb 462 VEIK
VL(VK) (aa)
SARS-CoV-2
S2M11 mAb 463 QSVSSSY
CDRL1 (aa)
SARS-CoV-2
S2M11 mAb 464 GAS
CDRL2 (aa)
SARS-CoV-2 QQYGSSAWT
S2M11 mAb 465
CDRL3 (aa)
QVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGI
SARS-CoV-2 SWVRQAPGQGLEWMGWISTYQGNTNYAQKFQG
S309 N55Q mAb 466 RVTMTTDTSTTTGYMELRRLRSDDTAVYYCARD
VH (aa) YTRGAWFGESLIGGFDNWGQGTLVTVSS
SARS-CoV-2
S309 N55Q mAb 467 ISTYQGNT
CDRH2 (aa)
159
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EXAMPLES
EXAMPLE 1
RECOMBINANT EXPRESSION OF CERTAIN ANTIBODIES
Antibodies were recombinantly expressed in ExpiCHO cells transiently co-
transfected with plasmids expressing the heavy and light chains as previously
described
(Stettler et at. (2016)). Specificity, cross-reactivity, and function of
antibodies elicited
by Zika virus infection. Science, 353(6301), 823-826). The concentration of
antibody
in cell culture supernatant was measured for antibodies as shown in Table 2.
Table 2.
Monoclonal Antibody Expression in CHO cells
(mg/m1)
Antibody 418 1 0.078
Antibody 418 2 0.722
Antibody 418 3 0.541
Antibody 418 4 0.366
Antibody 418j4 0.14
Antibody 418 42 0.17
Antibody 418 43 0.20
160
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EXAMPLE 2
CHARACTERIZATION OF CERTAIN ANTIBODIES
Certain antibodies of the present disclosure were characterized by
identification
of the germline VH and VL genes, their EC50 and KD for binding to SARS-CoV-2
Domain A, and whether they exhibit neutralizing activity against SARS-CoV-2.
The
results are shown in Table 3. The notation "nn" indicates that the antibody
was not
neutralizing by this assay. Blank cells in the table indicate that no
measurement was
made.
Table 3.
mAb IgVH IgVL DomA KD N T
Maximal
gene gene ELISA Domain A (IC50
(1/0 neutr.
(EC50 (M) ng/ml)
ng/ml)
418_7 1-69 K1-9 1468 <1E-12 119.3
95%
418 13 2-70 K2-28 148.7 <1E-12 nn
nn
418_14 3-21 1(3-15 8.943 <1E-12 nn
nn
418 39 1-69 L4-69 507.4 <1E-12 nn
nn
418_40 3-30 K1-33 447.7 <1E-12 2982
98%
418 8 1-24 K1-27 602.3 <1E-12 nn
nn
418_41 1-24 L3-10 47.72 <1E-12 1941.
77%
418 9 3-21 L2-14 34.26 7.10E-11 76.38
77%
418_42 4-30 L1-51 22.1 <1E-12 nn
nn
418 43 3-23 K3-11 26.06 <1E-12 nn
nn
418 44 4-59 L3-25 63.72 <1E-12 24.88
91%
418_6 4-61 K1-39 698.7 <1E-12 nn
nn
418_5 3-33 L3-25 31.66 <1E-12 53.73
88%
418_22 1-24 L1-47 12.3 nn
nn
41S1 3-30 L3-10 337.6 5.52E-10 90
91%
418 23 2-26 L1-40 18.5 nn
nn
418_24 3-21 K3-15 14.2 nn
nn
418_25 3-21 K3-15 19.4 nn
nn
418 37 4-4 L2-23 11.5 nn
nn
418 26 1-24 K2-24 60.5 40.3
87%
418_38 3-21 K3-15 13.4 nn
nn
161
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mAb IgVH IgVL DomA KD N T
Maximal
gene gene ELISA Domain A (IC50
% neutr.
(EC50 (M) ng/ml)
ng/ml)
418_11 3-3 K3-15 8.7 nn
nn
418 27 3-53 K3-15 20 nn
nn
418 21 4-59 K3-20 24.3 nn
nn
418 20 4-59 K3-15 12.4 nn
nn
418_19 4-38 K1-39 69 7.9
65%
418_28 3-30 1(1-13 30 8.9
68%
418 18 3-21 L1-40 24 nn
nn
418_29 1-24 L1-47 18.7 32.2
96%
418 30 1-46 L3-25 16.4 17
83%
418_31 4-61 L10-54 51.8 0.67
98%
418 12 3-23 L1-51 10.9 nn
nn
418_17 3-21 1(3-15 26.7 nn
nn
418 33 4-59 L3-21 52.8 nn
nn
418 16 3-21 K3-15 16.8 nn
nn
418_34 2-70 1(4-1 474.6 nn
nn
418_15 1-2 L2-23 18.3 nn
nn
418 2 2-5 L3-1 14.2 <1E-12 79.1
85%
418_10 4-34 L2-23 8 nn
nn
418 35 3-48 K2D-29 97.8 nn
nn
418_3 3-33 1(1-33 26.9 <1E-12 147.9
42%
418 4 3-33 L3-21 7.6 2.75E-11
43.3 94%
Additional characterization of monoclonal antibodies 418_i, 418 2, 418 3, and
4i8_4 was performed. The kon, kdis, neutralization activity against SARS-CoV-2
with
or without tosyl phenylalanyl chloromethyl ketone (TPCK), ability to block
SARS-
CoV-2 binding to ACE2, ability to induce antibody-dependent cellular
phagocytosis
(ADCP) (i.e., FcyRIIa activation), ability to induce antibody-dependent cell-
mediated
cytotoxicity (ADCC) (i.e., Fc7RIIIa activation), and measurement of antibody-
mediated
shedding of SARS-CoV-2 Si protein from infected cells for each of these
antibodies is
shown in Table 4.
Table 4.
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mAb 418_i 418_2 418_3 418_4
kon (1/Ms) 2.4E+05 4.5E+05 1.7E+05
2.4E+05
kdis (1 /s) 1.34E-04 <1.0E-07 <1.0E-7
6.67E-06
Neutr IgG + TPCK (IC50, 90.0 79.1 147.1 43.3
ng/ml)
Neutr IgG ¨ TPCK (IC50, 139.4 92.2 97.1 239.6
ng/ml)
ACE2 blockade No No No No
ADCP (FcyRIIa activation) + +++
ADCC (FcyRIIIa activation) +++
Si shedding no
Additional characterization was carried out for six antibodies, as shown in
Table
5. EC50 values were measured by ELISA for binding to SARS-CoV-2 Spike protein
Domain A. KD, km, and kdis values were measured by BLI for binding to SARS-CoV-
2
Spike protein Domain A.
Table 5.
mAb EC50 (ng/ml) KD (M) k.. (1/Ms) kdis
(Vs)
418 37 11.5 3.35E-09 1.80E+05 6.05E-
04
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mAb EC50 (ng/ml) KD (M) k0 (1/Ms) kdis
(Vs)
481A 8.7 3.73E-08 2.14E+04 7.97E-
04
418 20 12.4 1.39E-08 5.02E+04 6.96E-
04
418 22 12.3 1.87E-09 1.72E+05 3.22E-
04
418 12 10.9 1.49E-08 3.77E+04 5.63E-
04
418 10 8 5.58E-09 9.58E+04 5.35E-
04
EXAMPLE 3
FURTHER STUDIES USING NTD-SPECIFIC ANTIBODIES
Introduction
The emergence of SARS-CoV-2 coronavirus at the end of 2019 resulted in the
ongoing COVID-19 pandemic. The lack of pre-existing immunity to SARS-CoV-2
combined with its efficient human-to-human transmission has already resulted
in more
than 86 million infections and over 1.85 million fatalities as of January
2021.
Prophylactic and/or therapeutic anti-viral drugs may be helpful for
unvaccinated
individuals or those who respond poorly to vaccination as well as upon waning
of
immunity or emergence of antigenically distinct strains.
SARS-Coli-2 infects host cells through attachment of the viral transinembrane
spike (S) glycoprotein to angiotensin-converting enzyme 2 (ACE) followed by
fusion
of the viral and host membranes (Letko et al., 2020; Walls et al., 2020c;
Wrapp et al.,
2020; Zhou et al., 2020). SARS-CoV-2 S also engages cell-surface heparan-
sulfates
(Clausen et al., 2020), neuropilin-1 (Cantuti-Castelvetri et al., 2020; Daly
et al., 2020)
and L-SIGN/DC-SIGN (Chiodo et al., 2020; Gao et al., 2020; Soh et al., 2020;
Thepaut
et al., 2020) which were proposed to serve as co-receptors, auxiliary
receptors, or
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adsorption factors. SARS-CoV-2 S is the main target of neutralizing Abs in
infected
individuals and the focus of the many nucleic acid, vectored, and protein
subunit
vaccines currently deployed or in development (Corbett et al., 2020a; Corbett
et al.,
2020b, Erasmus et al., 2020; Hassan et al., 2020; Keech et al., 2020; Mercado
et al.,
2020; Walls et al., 2020b). Besides blocking ACE2 attachment (Piccoli et al.,
2020;
Tortorici et al., 2020), some neutralizing Abs may interfere with heparan-
sulfate,
neuropilin-1 or L-SIGN/DC-SIGN interactions.
The SARS-CoV-2 S protein comprises an N-terminal Si subunit responsible for
virus¨receptor binding, and a C-terminal Sz subunit that promotes virus¨cell
membrane
fusion (Walls et al., 2020c; Wrapp et al., 2020). The Si subunit comprises an
N-
terminal domain (NTD) and a receptor-binding domain (RBD), also known as
domain
A and B, respectively (Tortorici and Veesler, 2019) Antibodies targeting the
RBD
account for 90% of the neutralizing activity in COVID-19 convalescent sera
(Piccoli et
al., 2020) and numerous monoclonal antibodies (mAbs) recognizing this domain
have
been isolated and characterized (Barnes et al., 2020a; Barnes et al., 2020b,
Baum et al.,
2020b; Brouwer et al., 2020; Hansen et al., 2020; Ju et al., 2020; Piccoli et
al., 2020;
Pinto et al., 2020; Tortorici et al., 2020; Wang et al., 2020; Wu et al.,
2020). Several
RBD-specific mAbs capable of protecting small animals and non-human primates
from
SARS-CoV-2 challenge are able to neutralize viral infection by targeting
multiple
distinct antigenic sites (Baum et al., 2020a; Hansen et al., 2020; Jones et
al., 2020; Pinto
et al., 2020; Rogers et al., 2020; Tortorici et al., 2020; Zost et al., 2020).
A subset of
these mAbs is currently being evaluated in clinical trials or have recently
received
emergency use authorization from the FDA
The apparent limited immunogenicity of the SARS-CoV-2 NTD in COVID-19
patients (Piccoli et al., 2020; Rogers et al., 2020) has been hypothesized to
result from
its N-linked glycan shielding (Walls et al., 2020c; Watanabe et al., 2020).
However,
some studies have reported on the isolation of NTD-targeted mAbs and their
ability to
neutralize SARS-CoV-2 infection in vitro suggesting they could be useful for
COVID-
19 prophylaxis or treatment (Chi et al., 2020; Liu et al., 2020a). Although
the NTD has
been proposed to interact with auxiliary receptors in cell types that do not
express
ACE2 (e.g. DC-SIGN/L-SIGN), its role and the mechanism of action of NTD
targeted
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neutralizing mAbs remain unknown (Soh et al., 2020). Understanding the
immunogenicity of different S domains and the function of mAbs targeting them,
including the NTD, is important to understanding immunity during the pandemic.
Ab responses in three COVID-19 convalescent individuals were analyzed and
41 NTD-specific human mAbs were identified. Integrating cryo-electron
microscopy
(cryoEM), binding assays, and antibody escape mutants analysis a SARS-CoV-2
NTD
antigenic map was defined, and a supersite recognized by potent neutralizing
mAbs was
identified. These mAbs exhibit neutralization activities on par with potent
RBD-specific
mAbs and efficiently activate Fc-mediated effector functions. Immunologically
important variations of the SARS-CoV-2 NTD were also identified, suggesting
that the
S glycoprotein is under selective pressure from the host humoral immune
response. A
highly potent NTD mAb was shown to provide prophylactic protection against
lethal
SARS-CoV-2 challenge of Syrian hamsters.
NTD-specific mAbs with potent neutralizing activity
To discover mAbs targeting diverse SARS-CoV-2 epitopes, IgG- memory B
cells from peripheral blood mononuclear cells (PBMCs) of three COVID-19
convalescent individuals (L, M, X) were sorted using biotinylated prefusion
SARS-
CoV-2 S as a bait. The percentage of SARS-CoV-2 S-reactive IgG B cells ranged
between Ll - L3 % of IgG+ memory B cells. A total of 278 mAbs were isolated
and
recombinantly produced as human IgG I (Figure 20). Characterization by ELISA
showed that most mAbs isolated from the three donors recognize the RBD (65-
77%),
with a smaller fraction targeting the NTD (6-20%). The remaining mAbs (4-20%)
are
expected to bind to either the Sz subunit or the C-D domains within the Si
subunit
(Figure 20). The low proportion of NTD-specific mAbs isolated from these
donors is in
line with the previously observed limited NTD immunogenicity in SARS-CoV-2
exposed individuals (Piccoli et al., 2020; Rogers et al., 2020). Overall, 41
mAbs
recognizing the SARS-CoV2 NTD were identified, with EC50s ranging between 7.6 -
698 ng/ml and nanomolar binding affinities, as evaluated using ELISA and
biolayer
interferometry, respectively (Figures 21, 24A-24D, and 28A-28F, and Tables 6
and
7). These NTD-specific mAbs use a large repertoire of V genes, with an over-
representation of IGHV3-21 and IGK3-15 genes (Figure 25 and Tables 6 and 7).
These
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mAbs harbor few somatic hypermutations (VH and VL are 97.57% and 97.54%
identical to V germline genes, respectively; (Figure 26, Tables 6 and 7), as
previously
described for most SARS-CoV-2 neutralizing mAbs binding to the RBD (Piccoli et
al.,
2020; Seydoux et al., 2020). Antibody 418_i is also referred to herein as
S2X28.
Antibody 418 2 is also referred to herein as S2X303. Antibody 418 3 is also
referred
to herein as S2X320. Antibody 418 4 is also referred to herein as S2X333.
Antibody
418 5 is also referred to herein as S2M28. Antibody 418_6 is also referred to
herein as
S2M24 or S2M24v2. Antibody 418_7 is also referred to herein as S2L7. Antibody
418 8 is also referred to herein as S2L24. Antibody 4i89 is also referred to
herein as
S2L28. Antibody 418 10 is also referred to herein as S2X310. Antibody 418 11
is
also referred to herein as S2X94. Antibody 418_12 is also referred to herein
as
S2X169. Antibody 418 13 is also referred to herein as S2L11. Antibody 418 14
is
also referred to herein as S2L12. Antibody 418 15 is also referred to herein
as
S2X186. Antibody 418_16 is also referred to herein as S2X175. Antibody 418 17
is
also referred to herein as S2X170. Antibody 418_18 is also referred to herein
as
S2X125. Antibody 418 19 is also referred to herein as S2X107. Antibody 418 20
is
also referred to herein as S2X105. Antibody 418 21 is also referred to herein
as
S2X102. Antibody 418_22 is also referred to herein as S2X15. Antibody 418_23
is
also referred to herein as S2X49. Antibody 418_24 is also referred to herein
as S2X51.
Antibody 418 25 is also referred to herein as S2X72. Antibody 418_26 is also
referred
to herein as S2X91. Antibody 418 27 is also referred to herein as S2X98.
Antibody
418 28 is also referred to herein as S2X124. Antibody 418_29 is also referred
to herein
as S2X158. Antibody 418 30 is also referred to herein as S2X161. Antibody 418
3 I
is also referred to herein as S2X165. Antibody 418_33 is also referred to
herein as
S2X173. Antibody 418 34 is also referred to herein as S2X176. Antibody 418_35
is
also referred to herein as S2X316. Antibody 418 37 is also referred to herein
as
S2X90. Antibody 418_38 is also referred to herein as S2X93. Antibody 418 39 is
also
referred to herein as S2L14. Antibody 418_40 is also referred to herein as
S2L20 or
S2L20v1. Antibody 418 41 is also referred to herein as S2L26. Antibody 4i8_42
is
also referred to herein as S2L35. Antibody 418 43 is also referred to herein
as S2L38.
Antibody 418 44 is also referred to herein as S2L50.
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CDRI-13 lengths of these mAbs range between 10 and 24 amino acid residues
(Figure 26). Collectively, these data indicate that the Ab response to the
SARS-CoV-2
NTD is polyclonal.
Table 6.
NT IgG vs IgVH HCDR3 IgVL ELISA vs NTD Antigenic ND
Mx %
# Donor mAb VH % G L VL ./0 G L
MLV-S2 pp NT
gene length gene (EC50 ng/ml) site
NTD (M)
(IC50 ng/m1)
1 S2L11 2-70 21 98.63 K2-28 100
148.7 ii 7.25E-09 MI nn
2 S2L 12 3-21 19 98.26 K3-15 97.49
8.943 0.1 na nn MI
3 S2L14 1-69 18 98.61 L4-69 98.64
507.4 iii 1.18E-08 66 nn
4 S2L20 3-30 15 97.92 K1-33 96.42
447.7 iv 3.05E-08 2982 98%
S2L24 1-24 14 98.96 K1-27 99.28 602.3 i 1.17E-08
49.1 83.50%
L
is 182L26 1-24 14 97.22 L7-10 9/1.21
47.72 i 4L56E-119 19.41 77%
7 S2L28 3-21 19 96.53 L2-14 97.57
34.26 i 1.20E-07 76.38 77%
8 S2L35 4-30 21 96.9 Li-Si 98.25 22.1
iii 7.63E-09 66 nn
9 S2L38 3-23 17 97.22 K3-11 97.13
26.06 iii 1.62E-09 nn on
S2L50 4-59 20 95.44 L3-25 98.92 63.72 i 5.96E-
09 24.88 91%
11 S2M24 4-61 20 97.25 K1-39 95.34 698.7
vi 6.98E-09 no no
M
12 S2M28 3-33 12 97.57 L3-25 97.85 31.66 i 6.89E-09
53.73 88%
13 S2X15 1-3 21 98.98 K3-11 96.14 12.3
iii 1.87E-09 nn nn
14 S2X28 3-30 18 97.92 L3-10 99.64 337.6 i na
90 91%
S2X19 2-26 11 98.66 Li-10 98.98 18.5 iii 1.97E-09
no no
16 S2X51 3-21 20 98.95 K3-15 98.29
14.2 iii 3.24E-08 no no
17 S2X72 3-21 21 97.21 K3-15 98.93
19.4 iii 2.19E-08 iiii no
18 S2X90 4-4 13 97.61 L2-23 96_94 11.5
iii 3_35E-09 no on
19 S2X9 I 1-24 21 97.28 K2-24 97.64
60.5 i 3.39E-09 40.3 87%
S2X93 3-33 20 97.97 K3-15 96.51 13.4 iii 6.71E-08
66 no
21 S2X94 3-53 14 97.6 K3-15 98.59 8.7
iii 3.73E-08 nn no
X
22 S2X98 3-21 24 97.61 K3-15 99.65 20
iii 6.14E-08 MI no
23 S2X102 4-59 15 97.81 K3-20 96.81 24.3 0.1
8.23E-09 nn MI
24 S2X105 4-59 17 98.29 K3-15 99.3
12.4 iii 1.39E-08 66 no
S2X107 4-38 16 96.95 K1-39 96.83 69 i 4.08E-08 7.9 65%
26 S2X124 3-30 22 98.98 K1-13 98.25 30 i
4.70E-08 8.9 68%
27 S2X125 3-21 17 95.6 L1-40 97.57 24 111
3.39E-09 MI no
28 S2X158 1-24 16 96.25 L1-47 95.91 18.7
i 8.54E-09 32.2 96%
29 S2X161 1-46 21 95.46 L3-25 99.64 16.4
i 8.42E-09 17 83%
S2X165 4-61 20 96.65 L10-54 96.59 51.8 i 3.46E-08 61.07
98%
_
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31 S2X169 3-23 17 97.96 L1-51 98.64
10.9 iii 1.49E-08 nn nn
32 S2X170 3-21 20 96.96 K3-15 97.9
26.7 iii 1.62E-08 nn nn
33 S2X173 4-59 15 97.95 L3-21 97.21 52.8
v 1.22E-08 nn nn
34 S2X175 3-21 20 97.72 K3-15 96.167
16.8 Ill 8.94E-09 nn nn
35 S2X176 2-70 10 98.65 K4-1 98.01 474.6
ii 1.08E-08 nn nn
36 S2X186 1-2 12 98.97 L2-23 98.62 18.3
iii 9.58E-09 nn nn
37 S2X303 2-5 17 95.88 L3-1 95.34 14.2
1 6.30E-09 79.1 85"/o
38 S2X310 4-34 20 97.14 1,2-23 96.53
8 iii 5.5812-09 nn nn
39 S2X316 3-48 18 99.66 K2-29 94.73 97.8
v 1.77E-08 nn nn
40 S2X320 3-33 17 96.53 K1-33 97.85 26.9
i 1.81E-08 147.9 42%
41 S2X333 3-33 17 96.53 L3-21 97.49
7.6 i 2.89E-08 43.3 94%
-
Table 7.
NT IgG vs NT Fab vs
Mx %
# Donor mAb live virus live virus
Mx % NT
NT
IC50 (ng/m1) IC50 (Hg/ml)
1 S2L11
2 S2L12
3 S2L14
4 S2L20
L S2L24
6 S2L26
7 S2L28 26.2 98.20% 928
55.60%
8 S2L35
9 S2L38
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NT IgG vs NT Fab vs
Mx %
# Donor mAb live virus live virus Mx
% NT
NT
IC50 (ng/ml) IC50 (ng/ml)
S2L50
11 S2M24
12 S2M28 5 98.50% 26.3
66%
13 S2X15
14 S2X28 9.1 99.10% 248.9
87.30%
S2X49
16 S2X51
17 S2X72
18 S2X90
19 X S2X91
S2X93
21 S2X94
22 S2X98
23 S2X102
24 S2X105
S2X107
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NT IgG vs NT Fab vs
Mx %
# Donor mAb live virus live virus Mx
% NT
NT
IC50 (ng/ml) IC50 (ng/ml)
26 S2X124
27 S2X125
28 S2X158
29 S2X161
30 S2X165
31 S2X169
32 S2X170
33 S2X173
34 S2X175
35 S2X176
36 S2X186
37 S2X303
38 S2X310
39 S2X316
40 S2X320
41 S2X333 3 98.70% 6.1
82.20%
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In vitro neutralization activity of the NTD-specific mAbs was evaluated using
a
SARS-CoV-2 S pseudotyped murine leukemia virus system (Millet and Whittaker,
2016; Walls et al., 2020c). Out of 41 mAbs, 9 are potent neutralizers (IC0 <
50 ng/mL)
and 6 are moderate neutralizers (IC50 of 50-150 ng/mL) (Figure 21). The
remaining 25
mAbs were non-neutralizing. Most of the mAbs plateaued around 80-90% maximum
neutralization in this assay (Figures 21 and 29A-29F). Evaluation of the
neutralization
potency of a subset of NTD-specific mAbs measured 6 hours post-infection of
Vero E6
cells infected with authentic SARS-CoV-2 virus confirmed that these mAbs did
not
completely block viral entry and instead plateaued at 80-90% neutralization,
as opposed
to the RBD-specific mAbs S309, S2E12 and S2M11 that achieved 100%
neutralization
(Figure 22) (Pinto et al., 2020; Tortorici et al., 2020). When the activity
was measured
at 24 hours post-infection, however, all mAbs tested achieved 95-100%
neutralization
with a marked enhancement of neutralization potency (Figure 23). For instance,
S2X333 neutralized SARS-CoV-2 with an IC50 of 2 ng/ml and an IC90 of 12 ng/ml,
on
par with the potent RBD-targeting mAbs S2E12 and S2M11 (Figure 23).
Previous studies established that SARS-CoV-2 infection of Vero E6 cells
proceeds through cathepsin-activated endosomal fusion, as opposed to TMPRSS2-
dependent entry which is thought to occur at the level of the plasma membrane
and to
be the most relevant route of lung cells infection (Hoffmann et al., 2020a;
Hoffmann et
al., 2020b; Hoffmann et al., 2020c) Antibodies S2L28, S2M28, S2X28 and S2X333
efficiently block cell-cell fusion (Figure 27)
Definition of a SARS-CoV-2 NTD antigenic map
Competition biolayer interferometry binding assays were carried out using
recombinant SARS-CoV-2 S The data indicated that the mAbs recognize six
distinct
antigenic sites, designated i, ii, iii, iv, v and vi. Most mAbs clustered
within antigenic
sites i and iii, whereas sites ii, iv, v and vi each accounted for only one or
a small
number of mAbs from the panel (Figures 30 and 31A-31I). All potently
neutralizing
mAbs tested competed for binding to the NTD site i (Figures 21, 22, and 30)
Mechanism of action of NTD-specific neutralizing mAbs
The ability of these mAbs to block ACE2 binding was evaluated, as this step
correlates with neutralization titers in SARS-CoV-2 exposed individuals
(Piccoli et al.,
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2020). None of the site i-targeting mAbs (S2L28, S2M28, S2X28, and S2X333)
blocked binding of SARS-CoV-2 S to immobilized human recombinant ACE2 as
measured by biolayer interferometry (Figure 32), indicating that interference
with
engagement of the main entry receptor is unlikely as the mechanism of action.
Moreover, these mAbs did not promote shedding of the Si subunit from cell-
surface-
expressed full-length SARS-CoV-2 S (Figure 8C), suggesting that premature S
triggering does not occur, unlike what was previously shown for a SARS-CoV and
several SARS-CoV-2 RBD-specific mAbs (Huo et al., 2020; Piccoli et al., 2020;
Walls
et al., 2019; Wec et al., 2020; Wrobel et al., 2020a).
The neutralization potency of each of 5L28, 52M28, 52X28, and 52X333 was
evaluated, in both Fab and IgG formats, against authentic SARS-CoV-2-Nlue
(Figure
33). NTD-specific Fabs displayed a potency reduction, both in terms of IC50
values and
maximal neutralization plateau reached (Tables 6 and 7), as compared to IgGs,
possibily due to reduced avidity as observed by surface plasmon resonance
(Figure 34).
Since the Fabs could still partially neutralize SARS-CoV-2, at least part of
the observed
neutralization activity may result from direct interaction with their
respective epitopes
It is possible that NTD-specific mAb-mediated neutralization further relies on
steric
hindrance provided by Fc positioning, similar to what was observed for anti-
hcmagglutinin influenza A virus neutralizing mAbs (Xiong et al., 2015).
Potential additive, antagonistic or synergistic effects of NTD- and RBD-
targeting mAbs was examined, as mAb synergy was previously described for SARS-
CoV and SARS-CoV-2 neutralization (Pinto et al., 2020; ter Meulen et al.,
2006).
Cocktails of S2X333 with S309, S2EI2, or S2M11 additively prevented entry of
SARS-
CoV-2 S-MLV pseudotyped virus in Vero E6 cells (Figures 9A-9C). This additive
effect was also observed between 52X333 and S309 using authentic SARS-CoV-2 at
24
hours post-infection in Vero E6 cells (Figure 36). These results are
consistent with
RBD- and NTD-targeting mAbs mediating inhibition by distinct mechanisms and
demonstrate that they could be used as cocktails for prophylaxis or therapy.
Since Fc-mediated effector functions can contribute to protection by promoting
viral clearance and anti-viral immune responses in vivo (Bournazos et al.,
2020;
Bournazos et al., 2016; Schafer et al., 2021; Winkler et al., 2020), the
ability of site i-
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targeting mAbs to trigger activation of FcyRIIa and FcyRIIIa was evaluated as
a proxy
for Ab-dependent cellular phagocytosis (ADCP) and Ab-dependent cellular
cytotoxicity
(ADCC), respectively. S2L28, S2M28, S2X28, and S2X333 promoted dose-dependent
FcyRIIa and FcyRIIIa-mediated signaling to levels comparable to those of the
highly
effective mAb S309 (Pinto et al., 2020) (Figure 35). In contrast, the non-
neutralizing
site vi-targeting S2M24 mAb did not promote FcyR-mediated signaling, possibly
due to
the different orientation relative to the membrane of the effector cells in
comparison to
site i-specific mAbs (Figure 35). These findings suggest that besides their
neutralizing
activity, mAbs recognizing site i can exert a protective activity via
promoting Fc-
mediated effector functions.
NTD neutralizing mAbs protect against SARS-CoV-2 challenge in hamsters
The S2X333 mAb was selected for a prophylactic study in a Syrian hamster
model (Boudewijns et al., 2020). The mAb was administered at 4 and 1 mg/kg via
intraperitoneal injection 48 hours before intranasal SARS-CoV-2 challenge.
Four days
later, lungs were collected for the quantification of viral RNA and infectious
virus
titers. Prophylactic administration of S2X333 decreased the amount of viral
RNA
detected in the lungs by ¨3 orders of magnitude, compared to hamsters
receiving a
control mAb (Figure 37A) and completely abrogated viral replication in the
lungs of
most animals at both doses tested (Figure 37B). Although all animals had
similar scrum
mAb concentrations within each group, no reduction in the amount of viral RNA
or
infectious virus was observed for one hamster at each dose compared to those
administered with a control mAb (Figures 37C-37D). Based on the aforementioned
variability and mutation tolerance of the SARS-CoV-2 NTD, it may be that
S2X333
escape mutants were selected in these animals. Overall, these data suggest
that low
doses of anti-NTD mAbs provide prophylactic activity in vivo, comparable to
RBD-
specific mAbs S2E12 and S2M11 (Tortorici et al., 2020), consistent with their
potent in
vitro neutralizing activity. The protection efficacy of S2X333 (and related
NTD mAbs)
may be further enhanced in humans by engineering to enhance interactions with
with
human Fey receptors.
Discussion
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The data herein suggest that neutralizing NTD-targeting mAbs represent one
aspect of immunity to SARS-CoV-2 and account for 5-20% of SARS-CoV-2 S-
specific
mAbs cloned from memory B cells isolated from the PBMCs of three COVID-19
individuals. Analysis of a large panel of neutralizing and non-neutralizing
mAbs
defined an antigenic map of the heavily glycosylated SARS-CoV-2 NTD, in which
6
antigenic sites (i-vi) were identified. All the neutralizing mAbs from the
three donors
investigated targeted the same antigenic supersite (site i). The neutralizing
mAbs
described here along with the mAbs 4A8 (Chi et al., 2020), FC05 (Zhang et al.,
2020)
and CM25 (Voss et al., 2020), which also target this antigenic supersite, use
various
germline V genes to recognize overlapping epitopes, thereby providing examples
of
convergent solutions to NTD-targeted mAb neutralization. A highly potent NTD
mAb
provides prophylactic protection against SARS-CoV-2 challenge of Syrian
hamsters
demonstrating that this class of mAbs can be a critical barrier to infection.
These data show that site i-targeting NTD neutralizing mAbs efficiently
activate
FcyRIIa and FcyRIIIa in vitro. Fc-mediated effector functions can be affected
by the
epitope specificity of the mAbs (Piccoli et al., 2020), highlighting the
importance of the
orientation of the S-bound Fe fragments for efficient FeyR cross-linking and
engagement. The site vi-targeting NTD mAb S2M24 did not activate either
FcyRIIa or
FcyRIIIa. The contribution of Fe-mediated effector functions could further
enhance the
prophylactic activity of potent NTD-specific mAbs against SARS-CoV-2 in
humans.
As several examples of single amino acid mutations reducing or completely
abrogating neutralization by immune sera have been reported (Li et al., 2020;
Liu et al.,
2020b; Weisblum et al., 2020), combinations of mAbs targeting distinct domains
may
reduce the likelihood of emergence of escape mutants.
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Huang, Y., Allen, J.D., Case, J.B., et al. (2020). Human neutralizing
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Rosenthal, P.B., Skehel, J.J., and Gamblin, S.J. (2020a). Antibody-mediated
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EXAMPLE 4
MATERIALS AND METHODS
Affinity determination using Octet (BLI, biolayer interferometry)
For KD determination of full-length antibodies, protein A biosensors (Pall
ForteBio) were used to immobilize recombinant antibodies at 2.7 ug/m1 for 1
minute,
after a hydration step for 10 minutes with Kinetics Buffer (KB). Association
curves
were recorded for 5min by incubating the antibody-coated sensors with SARS-CoV-
1
Domain A analyte at 10 ug/m1 (66.6 nM) in KB for 5 minutes (association
phase),
followed by dissociation with KB for 9 minutes. Signals were recorded and
analysed
with Octet Systems Software.
ELISA binding
The reactivities of mAbs with SARS-CoV Spike Si Subunit Protein (strain
WH20) protein were determined by enzyme-linked immunosorbent assays (ELISA).
Briefly, 96-well plates were coated with 3 ug/m1 of recombinant SARS-CoV Spike
Si
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Subunit Protein (Sino. Biological). Wells were washed and blocked with PB
S+1%B SA
for 1 h at room temperature and were then incubated with serially diluted mAbs
for 1 h
at room temperature. Bound mAbs were detected by incubating alkaline
phosphatase-
conjugated goat anti-human IgG (Southern Biotechnology: 2040-04) for 1 h at
room
temperature and were developed by 1 mg/ml p-nitrophenylphosphate substrate in
0.1 M
glycine buffer (pH 10.4) for 30 min at room temperature. The optical density
(OD)
values were measured at a wavelength of 405 nm in an ELISA reader (Powerwave
340/96 spectrophotometer, BioTek).
Pseudoparticle neutralization assay
Unless otherwise indicated, Murine leukemia virus (MLV) pseudotyped with
SARS-CoV-2 Spike protein (SARS-CoV-2pp) was used. DBT cells stably transfected
with ACE2 (DBT-ACE2) were used as target cells. SARS-CoV-2pp was activated
with
trypsin TPCK at lOug/ml. Activated SARS-CoV-2pp was added to a dilution series
of
antibodies (starting 5Oug/m1 final concentration per antibody, 3-fold
dilution). DBT-
ACE2 cells were added to the antibody-virus mixtures and incubated for 48h.
Luminescence was measured after aspirating cell culture supernatant and adding
steady-
GLO substrate (Promega).
In some cases, pseudoparticle neutralization assays use a VSV-based luciferase
reporter pscudotyping system (Kcrafast). VSV pseudoparticles and antibody arc
mixed
in DMEM and allowed to incubate for 30 minutes at 37C. The infection mixture
is then
allowed to incubate with Vero E6 cells for lh at 37C, followed by the addition
of
DMEM with Pen-Strep and 10% FBS (infection mixture is not removed). The cells
are
incubated at 37C for 18-24 hours. Luciferase is measured using an Ensight
Plate
Reader (Perkin Elmer) after the addition of Bio-Glo reagent (Promega).
Expression of recombinant antibodies
Recombinant antibodies were expressed in ExpiCHO cells transiently co-
transfected with plasmids expressing the heavy and light chain as previously
described
(Stettler et al. (2016) Specificity, cross-reactivity, and function of
antibodies elicited
by Zika virus infection. Science, 353(6301), 823-826)
Authentic SARS-CoV-2 neutralization assay
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Vero E6 cells cultured in DMEM supplemented with 10% FBS (VWR) and lx
Penicillin/Streptomycin (Thermo Fisher Scientific) were seeded in white 96-
well plates
at 20,000 cells/well and attached overnight. Serial 1:4 dilutions of the
monoclonal
antibodies were incubated with 200 pfu of SARS-CoV-2 (isolate USA-WA1/2020,
passage 3, passaged in Vero E6 cells) for 30 minutes at 37 C in a BSL-3
facility. Cell
supernatant was removed and the virus-antibody mixture was added to the cells.
24
hours post infection, cells were fixed with 4% paraformaldehyde for 30
minutes,
followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-
100
in PBS for 30 minutes. After blocking in 5% milk powder/PBS for 30 minutes,
cells
were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid
protein
(Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1 hour. After
washing and
incubation with a secondary Alexa647-labeled antibody mixed with 1 tg/ml
Hoechst33342 for 1 hour, plates were imaged on an automated cell-imaging
reader
(Cytation 5, Biotek) and nucleocapsid-positive cells were counted using the
manufacturer's supplied software. Data were processed using Prism software
(GraphPad Prism 8.0).
Cell lines
Cell lines were obtained from ATCC (FIEK293T and Vero-E6)or ThermoFisher
Scientific (Expi CHO cells, FreeStyleTm 293-F cells and Expi293FTM cells).
Sample donors
Samples were obtained from three SARS-CoV-2 recovered individuals (L, M
and X) under study protocols approved by the local Institutional Review Boards
(Canton Ticino Ethics Committee, Switzerland, the Ethical committee of Luigi
Sacco
Hospital, Milan, Italy). All donors provided written informed consent for the
use of
blood and blood components (such as PBMCs, sera or plasma).
Samples were collected 14 and 52 days after symptoms onset for donor L and
M, respectively. Blood drawn from donor X was obtained at day 36, 48, 75 and
125
after symptoms onset
Cloning and mutant generation
SARS-CoV-2 NTD was sub-cloned with E. coli DHIOB Competent Cells into
pCMV using primers NTD fwd and NTD rev. The resulting construct was mutated by
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PCR mutagenesis to generate N149Q, D253G/Y, T19A, R246A, L18F, H146Y,
A222V, Y144del, S254F, K147T, C136Y, and the NTD construct with native signal
peptide with and without S12P, using the eponymously named primers (Key
Resources
Table). The genes encoding for the Sarbecovirus S proteins tested were cloned
in the
phCMV1 or pcDNA.3 vectors, and the gene for the C-terminally his-tagged
ectodomain
of P-GD S was cloned into pCMV (Key Resources Table). Plasmid sequences were
verified by Genewiz sequencing facilities (Brooks Life Sciences).
Recombinant ectodomains production
All SARS-CoV-2 S spike ectodomains were produced in 500 mL cultures of
FreeStyleTM 293-F cells (ThermoFisher Scientific) grown in suspension using
FreeStyle
293 expression medium (ThermoFisher Scientific) at 37 C in a humidified 8% CO2
incubator rotating at 130 r.p.m. Cells grown to a density of 2.5 million cells
per mL
were transfected using PEI (9 pg/mL) and pCMV::SARS-CoV-2 S ecto hexapro,
pCMV::SARS-CoV-2 S ecto 2P DS, pCMV::P-GD S ecto, pCMV::SARS-CoV-
2 S ecto avi, pCMV::SARS-CoV-2 S D614G ecto avi and cultivated for 4 days.
The supernatant was harvested and cells were resuspended for another three
days,
yielding two harvests. S ectodomains were purified from clarified supernatants
using a
Cobalt affinity column (Cytiva, HiTrap TALON crude), washing with 20 column
volumes of 20 mM Tris-HC1 pH 8.0 and 150 mM NaCl and eluted with a gradient of
600 mM imidazole. The same protocol was followed for P-GD spike ectodomain
purification, except that 25 mM sodium phosphate pH 7 and 300 mM sodium
chloride
were used instead of 20 mM Tris-HC1 pH 8.0 and 150 mM NaCl. At this stage,
SARS-
CoV-2 S with the avi tag (from pCMV::SARS-CoV-2 S ecto avi) was biotinylated
(BirA biotin-protein ligase standard reaction kit, Avidity) and further
purified by size
exclusion chromatography (Superose6, GE Healthcare). All purified proteins
were then
concentrated using a 100 kDa centrifugal filter (Amicon Ultra 0.5 mL
centrifugal filters,
MilliporeSigma), residual imidazole was washed away by consecutive dilutions
in the
centrifugal filter unit with 20 mM Tris-HC1 pH 8.0 and 150 mM NaC1, and
finally
concentrated to 5 mg/ml and flash frozen.
All SARS-CoV-2 S NTD domain constructs (residues 14-307) with a C-terminal
8XHis-tag were produced in 100 mL culture of Expi293FTM Cells (ThermoFisher
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Scientific) grown in suspension using Expi293TM Expression Medium
(ThermoFisher
Scientific) at 37 C in a humidified 8% CO2 incubator rotating at 130 r.p.m.)
(Walls et
al., 2020) (Walls et al., 2020) (Walls et al., 2020) (Walls et al., 2020)
(Walls et al.,
2020) (Walls et al., 2020) (Walls et al., 2020) (Walls et al., 2020) (Walls et
al., 2020)
(Walls et al., 2020) (Walls et al., 2020). Cells grown to a density of 3
million cells per
mL were transfected using pCMV::SARS-CoV-2 S NTD derivative mutants with the
ExpiFectamineTM 293 Transfection Kit (ThermoFisher Scientific) with and
cultivated
for five days at which point the supernatant was harvested. His-tagged NTD
domain
constructs were purified from clarified supernatants using 2 ml of cobalt
resin (Takara
Bio TALON), washing with 50 column volumes of 20 mM HEPES-HC1 pH 8.0 and
150 mM NaCl and eluted with 600 mM imidazole. Purified protein was
concentrated
using a 30 kDa centrifugal filter (Amicon Ultra 0.5 mL centrifugal filters,
MilliporeSigma), the imidazole was washed away by consecutive dilutions in the
centrifugal filter unit with 20 mM HEPES-HC1 pH 8.0 and 150 mM NaCl, and
finally
concentrated to 20 mg/ml and flash frozen. For crystallization, the purified
NTD was
not frozen but was further purified by size exclusion chromatography (Superdex
Increase 75 10/300 G, GE Healthcare), concentrated using a new 30 kDa
centrifugal
filter, and used immediately.
Intact mass spectrometry analysis of purified NTD constructs
The purpose of intact MS was to verify the n-terminal sequence on four
constructs. N-linked glycans were removed by PNGase F after overnight non-
denaturing reaction at room temperature. 4ug of deglycosylated protein was
used for
each injection on the LC-MS system to acquire intact MS signal after
separation of
protease and protein by LC (Agilent PLRP-S reversed phase column). Thermo MS
(Q
Exactive Plus Orbitrap) was used to acquire intact protein mass under
denaturing
condition. BioPharma Finder 3.2 software was used to deconvolute the raw m/z
data to
protein average mass.
Isolation of peripheral blood mononuclear cells (PBMCs), plasma and sera
PBMCs were isolated from blood draw performed using tubes pre-filled with
heparin, followed by Ficoll density gradient centrifugation. PBMCs were either
used
freshly along SARS-CoV2 Spike protein specific memory B cells sorting or
stored in
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liquid nitrogen for later use. Sera were obtained from blood collected using
tubes
containing clot activator, followed by centrifugation and stored at -80 C.
B-cell isolation and recombinant mAb production
Starting from freshly isolated PBMCs or upon cells thawing, B cells were
enriched by staining with CD19 PE-Cy7 (BD Bioscience 341113) and incubation
with
anti-PE bead (Miltenyi Biotec, cat. 130- 048-801), followed by positive
selection using
LS columns. Enriched B cells were stained with anti-IgM, anti-IgD, anti-CD14
and
anti-IgA, all PE labelled, and prefusion SARS-CoV-2 S with a biotinylated avi
tag
conjugated to Streptavidin Alexa-Fluor 647 (Life Technologies). SARSCoV-2 S-
specific IgG+ memory B cells were sorted by flow cytometry via gating for PE
negative
and Alexa-Fluor 647 positive cells. Cells were cultured for the screening of
positive
supernatants. Antibody VH and VL sequences were obtained by RT-PCR and mAbs
were expressed as recombinant human Fab fragment or as IgG1 (G1m3 allotype)
carrying the half-life extending M428L/N434S (LS) mutation in the Fc region.
ExpiCHO cells were transiently transfected with heavy and light chain
expression
vectors as previously described (Pinto et al., 2020).
Affinity purification was performed on AKTA Xpress FPLC (Cytiva) operated
by UNICORN software version 5.11 (Build 407) using HiTrap Protein A columns
(Cytiva) for full length human and hamster mAbs and CaptureSelect CHI -XL
MiniChrom columns (ThermoFisher Scientific) for Fab fragments, using PBS as
mobile
phase. Buffer exchange to the appropriate formulation buffer was performed
with a
HiTrap Fast desalting column (Cytiva). The final products were sterilized by
filtration
through 0.22 urn filters and stored at 4 C.
Enzyme-linked immunosorbent assay (ELISA)
To determine specificity of recombinantly produced mAbs, 96 half area well-
plates (Corning) were coated over-night at 4 C with of SARS-CoV-2 S, NTD or
RBD
proteins prepared 1 ug/ml, 2 ug/m1 and 5 ug/m1 in PBS pH 7.2, respectively.
Plates
were then blocked with PBS 1% BSA (Sigma) and subsequently incubated with mAbs
serial dilutions for 1 h at room temperature. After 2 washing steps with PBS
0.05%
Tween 20 (PBS-T) (Sigma-Aldrich) goat anti-huma IgG secondary antibody
(Southern
Biotech) was added in incubated for 1 h at room temperature. Plates were then
washed
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again with PBS-T and 4-NitroPhenyl phosphate (pNPP, Sigma-Aldrich) substrate
added. After 30 min incubation, absorbance at 405 nm was measured by a plate
reader
(Biotek) and data plotted using Prism GraphPad.
For all other applications reported, the following ELISA procedure was
followed: 30 ?Al of ectodomains (stabilized prefusion trimer) of S or NTD from
SARS-
CoV-2 were coated on 384 well ELISA plates at 1 ng/til for 16 hours at 4 C.
Plates
were washed with a 405 TS Microplate Washer (BioTek Instruments) then blocked
with
80 ttl SuperBlock (PBS) Blocking Buffer (Thermo Scientific) for 1 hour at 37
C. Plates
were then washed and 30 pi antibodies were added to the plates at
concentrations
between 0.001 and 100,000 ng/ml and incubated for 1 h at 37 C. Plates were
washed
and then incubated with 30 tl of 1/5000 diluted goat anti-human Fc IgG-HRP
(invitrogen A18817). Plates were washed and then 30 ul Substrate T1VIB
microwell
peroxidase (Seracare 5120-0083) was added for 4 min at room temperature. The
colorimetric reaction was stopped by addition of 30 pi of 1 N HC1. A450 was
read on a
Varioskan Lux plate reader (Thermo Scientific).
MLV-based pseudovirus production and neutralization
To generate SARS-CoV-2 S murine leukemia virus pseudotyped virus,
HEK293T cells were seeded in 10-cm dishes in DMEM supplemented with 10% FBS.
The next day cells were transfected with a SARS-CoV-2 S glycoprotein-encoding
plasmid harboring the D19 C-terminal truncation (Ou et al., 2020) using the X-
tremeGENE HP DNA transfection reagent (Roche) according to the manufacturer's
instructions. Cells were then incubated at 37 C with 5% CO2 for 72 h.
Supernatant was
harvested and cleared from cellular debris by centrifugation at 400 X g, and
stored at -
80 C.
For neutralization assays, Vero E6 cells were seeded into white 96-well plates
(PerkinElmer) at 20,000 cells/well and cultured overnight at 37 C with 5 %
CO2 in
100 pi DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The next
day, MLV-SARS-CoV-2 pseudovirus was activated with 10 Fl g/m1 TPCK treated-
Trypsin (Worthington Biochem) for 1 h at 37 C. Then recombinant antibodies at
various concentrations were incubated with activated pseudovirus for 1 h at 37
C. The
Vero E6 cells were then washed with DMEM, and the 50 01 of pseudovirus/mAbs
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mixes were added and incubated for 2 h at 37 C with 5 % CO2. After
incubation, 50 .1
of DMEM containing 20% FBS and 2 % penicillin/streptomycin was added and the
cells were incubated 48 h at 37 C with 5 % CO2. Following these 48 h of
infection,
culture medium was removed from the cells and 50 litl/wellof Bio-Glo (Promega)
(diluted 1:2 with PBS with Ca2+Mg2+ (Thermo Fisher) was added to the cells and
incubated in the dark for 15 min before reading on a Synergy H1 Hybrid Multi-
Mode
plate reader (Biotek). Measurements were done in duplicate and RLU values were
converted to percentage of neutralization and plotted with a nonlinear
regression curve
fit in Graph Prism.
Neutralization of authentic SARS-CoV-2-Nluc virus
Neutralization of authentic SARS-CoV-2 by entry-inhibition assay
Neutralization was determined using SARS-CoV-2-Nluc, an infectious clone of
SARSCoV-2 (based on strain 2019-nCoV/USA WA1/2020) which encodes
nanoluciferase in place of the viral ORF7 and demonstrated comparable growth
kinetics
to wildtype virus (Xie et al., 2020). Vero E6 cells were seeded into black-
walled, clear-
bottom 96-well plates at 2 x 104 cells/well and cultured overnight at 37 C.
The next
day, 9-point 4-fold serial dilutions of mAbs were prepared in infection media
(DIVLEM
+ 10% FBS). SARS-CoV-2-Nluc was diluted in infection media at a final MOI of
0.1 or
0.01 PFU/cell, added to the mAb dilutions and incubated for 30 minutes at 37
C.
Media was removed from the Vero E6 cells, mAb-virus complexes were added and
incubated at 37 C for 6 or 24 hours. Media was removed from the cells, Nano-
Glo
luciferase substrate (Promega) was added according to the manufacturer's
recommendations, incubated for 10 minutes at room temperature and the
luciferase
signal was quantified on a VICTOR Nivo plate reader (Perkin Elmer).
Binding and affinity determination by Biolayer Interferometry (BLI)
BLI measurements were performed using an Octet Red96 (ForteBio). All
reagents were prepared in kinetics buffer (PBS plus 0.01% BSA) at the
indicated
concentrations
BLI was used to assess antibody binding affinity to SARS-CoV-2 NTD. IgG
antibodies were prepared at 2.7 1.tg/m1 and captured on pre-hydrated Protein A
biosensors (Sartorius) for 1 min. The biosensors with immobilized antibodies
were
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moved into kinetics buffer with SARS-CoV-2 NTD (concentrations tested: 333.3,
166.6, 83.3, 41.7, 20.8, 10.4, 5.2 nM) for 5 min (i.e. association). The
dissociation of
the SARS-CoV-2 NTD was then recorded for 9 min in wells containing kinetics
buffer.
Affinity constants were calculated using a global fit model and results were
plotted
using GraphPad Prism.
BLI was also used to assess antibody competition studies to define the NTD
antigenic map. Biotinylated SARS-CoV-2 S protein was prepared at 10 p.g/m1 in
kinetics buffer and loaded on pre-hydrated High Precision Streptavidin SAX
Biosensors
(Sartorius) for 3 min. NTD mAbs at 20 pg/ml in kinetics buffer were then
sequentially
added to observe binding competition and signal recorded for 5 min (or 7 min)
BLI was also used to assess mAb-mediated inhibition of SARS-CoV-2 S
binding to human recombinant ACE2. Before the assay SARS-CoV2 S ectodomain
trimer (5 g/ml) was incubated with tested mAbs (30 p.g/m1) or no mAb for 30
minutes
at 37 C. Biotinylated recombinant human ACE2 protein (2 ug/m1) was immobilized
on
High Precision Streptavidin SAX Biosensors (Sartorius). Next, an association
step with
S/mAb complexes was performed for 10 minutes. Results were plotted using
GraphPad
Prism.
Affinity determination by Surface Plasmon Resonance (SPR)
SPR binding measurements were performed using a Biaeore T200 instrument
where purified avi-tagged SARS-CoV-2 S D614G ectodomain trimer was captured
using anti-AviTag pAb covalently immobilized on a CM5 sensor chip. The running
buffer was Cytiva HBS-EP-F pH 7.4; measurements were performed at 25 C.
Affinity/avidity determinations were run as single-cycle kinetics, with a 3-
fold dilution
series of mAb starting from 300 nM, and each concentration injected for 180
sec.
Double reference-subtracted data were fit to a 1:1 binding model using Biacore
Evaluation software. Fit results for IgG yielded apparent equilibrium
dissociation
constants due to avidity. For dissociation rates that were too slow to fit,
equilibrium
dissociation constants are reported as an upper limit
Transient Expression of Sarbecovirus S protein in ExpiCHO-S Cells.
Immediately before transfection, ExpiCHO-S cells were seeded at 6 x 106 cells
cells/mL in a volume of 5 mL in a 50 mL bioreactor. Spike coding plasmids were
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diluted in cold OptiPRO SFM, mixed with ExpiFectamine CHO Reagent (Life
Technologies) and added to the cells. Transfected cells were then incubated at
37 C
with 8% CO2 with an orbital shaking speed of 120 RPM (orbital diameter of 25
mm)
for 42 hours
Binding to cell surface expressed Sarbecovirus S proteins by Flow Cytometry
Transiently transfected ExpiCHO cells were harvested and washed two times in
wash buffer (PBS 1% BSA, 2 mM EDTA). Cells were counted and distributed into
round bottom 96-well plates (Corning) and incubated with the NTD antibodies at
the
final concentration of 5 mg/mi. Alexa Fluor647-labelled Goat Anti-Human IgG
secondary Ab (Jackson Immunoresearch) was prepared at 1.5 g/ml added onto
cells
after two washing steps. Cells were then washed twice and resuspended in wash
buffer
for data acquisition at ZE5 cytometer (Biorad).
Fusion inhibition assay
Vero E6 cells were seeded in 96 well plates at 15,000 cells per well in 70
1.11
DMEM with high glucose and 2.4% FBS (Hyclone). After 16 h at 37 C with 8 %
CO2,
the cells were transfected with SARS-CoV-2-S-D19_pcDNA3.1 as follows: for 10
wells, 0.57 lig plasmid SARS-CoV-2- S-D19_pcDNA3.1 were mixed with 1.68 tl X-
tremeGENE HP in 30 tl OPTIMEM. After 15 minutes incubation, the mixture was
diluted 1:10 in DMEM medium and 30111 was added per well. A 4-fold serial
dilution
mAbs was prepared and added to the cells, with a starting concentration of 20
[tg/ml.
The following day, 30 [11 5X concentrated DRAQ5 in DMEM was added per well and
incubated for 2 hours at 37 C. Nine images of each well were acquired with a
Cytation
5 equipment for analysis.
Measurement of Fc-effector functions
mAb-dependent activation of human FcyRIIIa was performed with a
bioluminescent reporter assay. ExpiCHO cells stably expressing full-length
wild-type
SARS-CoV-2 S (target cells) were incubated with different amounts of mAbs.
After a
15-minute incubation, .Turkat cells stably expressing FcyRIIIa receptor (V158
variant)
or FcyRIIa receptor (H131 variant) and NFAT-driven luciferase gene (effector
cells)
were added at an effector to target ratio of 6:1 for FcyRIIIa and 5:1 for
FcyRIIa.
Signaling was quantified by the luciferase signal produced as a result of NFAT
pathway
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activation. Luminescence was measured after 20 hours of incubation at 37 C
with 5%
CO2 with a luminometer using the Bio-Glo-TM Luciferase Assay Reagent according
to
the manufacturer's instructions (Promega, Cat. Nr.: G9798, G7018 and G9995).
Cell-surface mAb-mediated Si shedding
CHO cells stably expressing wild-type SARS-CoV-2 S were resuspended in
wash buffer (PBS 1 % BSA, 2 mM EDTA) and treated with 10 l.t.g/mL TPCK-trypsin
(Worthington Biochem) for 30 min at 37 C. Cells were then washed and
distributed
into round bottom 96-well plates (90,000 cells/well). MAbs were added to cells
at 15
mg/mL final concentration for 180 min at 37 C. Cells were collected at
different time
points (5, 30, 60, 120 and 180), washed with wash buffer at 4 C, and
incubated with
1.5 mg/mL secondary goat anti-human IgG, Fc fragment specific (Jackson
ImmunoResearch) on ice for 20 min. Cells were washed and resuspended in wash
buffer and analyzed with ZE5 FACS (Bio-rad).
Generation of stable overexpression cell lines
Lentiviruses were generated by co-transfection of Lenti-X 293T cells (Takara)
with lentiviral expression plasmids encoding DC-SIGN (CD209), L-SIGN (CLEC4M),
SIGLEC1, TMPRSS2 or ACE2 (all obtained from Genecopoeia) and the respective
lentiviral helper plasmids. Forty-eight hours post transfection, lentivirus in
the
supernatant was harvested and concentrated by ultracentrifugation for 2 h at
20,000
rpm. Lenti-X 293T (Takara), Vero E6 (ATCC), MRCS (Sigma-Aldrich), A549 (ATCC)
were transduced in the presence of 6 ug/mL polybrene (Millipore) for 24 h.
Cell lines
overexpressing two transgenes were transduced subsequently. Selection with
puromycin and/or blastici din (Gibco) was started two days after transduction
and
selection reagent was kept in the growth medium for all subsequent culturing.
Single
cell clones were derived from the A549-ACE2-TMPRSS2 cell line, all other cell
lines
represent cell pools.
SAPS-Co V-2 neutralization
Vero E6 or Vero E6-TMPRSS2 cells cultured in DMEM supplemented with
10% FBS (VWR) and lx Penicillin/Streptomycin (Thermo Fisher Scientific) were
seeded in black 96-well plates at 20,000 cells/well. Serial 1:4 dilutions of
the
monoclonal antibodies were incubated with 200 pfu of SARS-CoV-2 (isolate USA-
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WA1/2020, passage 3, passaged in Vero E6 cells) for 30 min at 37 C in a BSL-3
facility. Cell supernatant was removed and the virus-antibody mixture was
added to the
cells. 24 h post infection, cells were fixed with 4% paraformaldehyde for 30
min,
followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-
100
in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were
incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein
(Sino
Biological, cat. 40143-R001) at a 1:2000 dilution for lh. After washing and
incubation
with a secondary Alexa647-labeled antibody mixed with 1 ug/ml Hoechst33342 for
1
hour, plates were imaged on an automated cell-imaging reader (Cytation 5,
Biotek) and
nucleocapsid-positive cells were counted using the manufacturer's supplied
software.
SAPS-CoV-2-Nhic neutralization
Neutralization was determined using SARS-CoV-2-Nluc, an infectious clone of
SARS-CoV-2 (based on strain 2019-nCoV/USA WA1/2020) encoding nanoluciferase
in place of the viral ORF7, which demonstrates comparable growth kinetics to
wild type
virus (Xie et al., Nat Comm, 2020, https://doi.org/10.1038/s41467-020-19055-
7). Cells
were seeded into black-walled, clear-bottom 96-well plates at 20,000
cells/well (293T
cells were seeded into poly-L-lysine-coated wells at 35,000 cells/well) and
cultured
overnight at 37 C. The next day, 9-point 4-fold serial dilutions of antibodies
were
prepared in infection media (DMEM + 10% FBS). SARS-CoV-2-Nluc was diluted in
infection media at the indicated MOT, added to the antibody dilutions and
incubated for
min at 37 C. Media was removed from the cells, mAb-virus complexes were added,
and cells were incubated at 37 C for 24 h. Media was removed from the cells,
Nano-
Glo luciferase substrate (Promega) was added according to the manufacturer's
recommendations, incubated for 10 min at RT and luciferase signal was
quantified on a
25 VICTOR Nivo plate reader (Perkin Elmer).
SAPS-Co V-2 pseudotyped VSV production and neutralization
To generate SARS-CoV-2 pseudotyped vesicular stomatitis virus, Lenti-X 293T
cells (Takara) were seeded in 10-cm dishes for 80% next day confluency The
next day,
cells were transfected with a plasmid encoding for SARS-CoV-2 5-glycoprotein
30 (YP 009724390.1) harboring a C-terminal 19 aa truncation using TransIT-
Lenti (Minis
Bio) according to the manufacturer's instructions. One day post-transfection,
cells were
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infected with VSV(G*AG-luciferase) (Kerafast) at an MOI of 3 infectious
units/cell.
Viral inoculum was washed off after one hour and cells were incubated for
another day
at 37 C. The cell supernatant containing SARS-CoV-2 pseudotyped VSV was
collected
at day 2 post-transfection, centrifuged at 1000 x g for 5 minutes to remove
cellular
debris, aliquoted, and frozen at -80 C.
For viral neutralization, Cells were seeded into black-walled, clear-bottom 96-
well plates at 20,000 cells/well (293T cells were seeded into poly-L-lysine-
coated wells
at 35,000 cells/well) and cultured overnight at 37 C. The next day, 9-point 4-
fold serial
dilutions of antibodies were prepared in media. SARS-CoV-2 pseudotyped VSV was
diluted 1:30 in media in the presence of 100 ng/mL anti-VSV-G antibody (clone
8G5F11, Absolute Antibody) and added 1:1 to each antibody dilution.
Virus:antibody
mixtures were incubated for 1 hour at 37 C. Media was removed from the cells
and 50
pL of virus:antibody mixtures were added to the cells. One hour post-
infection, 100 pL
of media was added to all wells and incubated for 17-20 hours at 37 C. Media
was
removed and 50 pi, of Bio-Glo reagent (Promega) was added to each well. The
plate
was shaken on a plate shaker at 300 RPM at room temperature for 15 minutes and
RLUs were read on an EnSight plate reader (Perkin-Elmer).
Transfection-based attachment receptor screen
Lenti-X 293T cells (Takara) were transfected with plasmids encoding the
following receptor candidates (all purchased from Genecopoeia): ACE2 (NM
021804),
DC-SIGN (NM 021155), L-SIGN (BC110614), LGALS3 (NM 002306), SIGLEC1
(NM 023068), SIGLEC3 (XM 057602), SIGLEC9 (BC035365), SIGLEC10
(NM 033130), MGL (NM 182906), MINCLE (NIVI 014358), CD 147 (NMI 98589),
ASGR1 (NM 001671.4), ASGR2 (NM 080913), NRP1 (NM 003873). One day post
transfection, cells were infected with SARS-CoV-2 pseudotyped VSV at 1:20
dilution
in the presence of 100 ng/mL anti-VSV-G antibody (clone 8G5F11, Absolute
Antibody) at 37 C. One hour post-infection, 100 pL of media was added to all
wells
and incubated for 17-20 hours at 37 C Media was removed and 50 pL of Bio-Glo
reagent (Promega) was added to each well. The plate was shaken on a plate
shaker at
300 RPM at room temperature for 15 minutes and RLUs were read on an EnSight
plate
reader (Perkin-Elmer).
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Trans-infection
Parental HeLa cells or HeLa cells stably expressing DC-SIGN, L-SIGN or
SIGLEC1 were seeded at 5,000 cells per well in black-walled clear-bottom 96-
well
plates. One day later, cells reached about 50% confluency and were inoculated
with
SARS-CoV-2 pseudotyped VSV at 1:10 dilution in the presence of 100 ng/mL anti-
VSV-G antibody (clone 8G5F11, Absolute Antibody) at 37 C for 2 h. For antibody-
mediated inhibition of trans-infection, cells were pre-incubated with 10 ug/mL
anti-
SIGLEC1 antibody (Biolegend, clone 7-239) for 30 min. After 2 h inoculation,
cells
were washed four times with complete medium and 10,000 VeroE6-TIVIPRSS2 cells
per
well were added and incubated 17-20 h at 37 C for trans-infection. Media was
removed
and 50 [it of Bio-Glo reagent (Promega) was added to each well. The plate was
shaken
on a plate shaker at 300 RPM at room temperature for 15 minutes and RLUs were
read
on an EnSight plate reader (Perkin-Elmer).
Cell-cell fusion of CHO-S cells
CHO cells stably expressing SARS-CoV-2 S-glycoprotein were seeded in 96
well plates for microscopy (Thermo Fisher Scientific) at 12'500 cells/well and
the
following day, different concentrations of mAbs and nuclei marker Hoechst
(final
dilution 1:1000) were added to the cells and incubated for additional 24h
hours. Fusion
degree was established using the Cytation 5 Imager (BioTek) and an object
detection
protocol was used to detect nuclei as objects and measure their size. The
nuclei of fused
cells (i.e., syncytia) are found aggregated at the center of the syncitia and
are
recognized as a unique large object that is gated according to its size. The
area of the
objects in fused cells divided by the total area of all the object multiplied
by 100
provides the percentage of fused cells
Immunofluorescence analysis
HEK 293T cells were seeded onto poly-D-Lysine-coated 96-well plates (Sigma-
Aldrich) and fixed 24 h after seeding with 4% paraformaldehyde for 30 min,
followed
by two PBS (pH 7.4) washes and permeabilizati on with 0.25% Triton X-100 in
PBS for
min. Cells were incubated with primary antibodies anti-DC-SIGN/L- SIGN
30 (Biolegend, cat. 845002, 1:500 dilution), anti-DC-SIGN (Cell Signaling,
cat. 13193S,
1:500 dilution), anti-SIGLEC1 (Biolegend, cat. 346002, 1:500 dilution) or anti-
ACE2
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(R&D Systems, cat. AF933, 1:200 dilution) diluted in 3% milk powder/PBS for 2
h at
room temperature. After washing and incubation with a secondary Alexa647-
labeled
antibody mixed with 1 ug/ml Hoechst33342 for 1 hour, plates were imaged on an
inverted fluorescence microscope (Echo Revolve).
ACE2/TIVIPRSS2 RT-qPCR
RNA was extracted from the cells using the NucleoSpin RNA Plus kit
(Macherey-Nagel) according to the manufacturer's protocol. RNA was reverse
transcribed using the High Capacity cDNA Reverse Transcription kit (Applied
Biosystems) according to the manufacturer's instructions. Intracellular levels
of ACE2
(Forward Primer: CAAGAGCAAACGGTTGAACAC, Reverse Primer:
CCAGAGCCTCTCATTGTAGTCT), HPRT (Forward Primer:
CCTGGCGTCGTGATTAGTG, Reverse Primer: ACACCCTTTCCAAATCCTCAG),
and TMPRSS2 (Forward Primer: CAAGTGCTCCRACTCTGGGAT, Reverse Primer:
AACACACCGRTTCTCGTCCTC) were quantified using the Luna Universal qPCR
Master Mix (New England Biolabs) according to the manufacturer's protocol.
Levels of
ACE2 and TMPRSS2 were normalized to HPRT. Hela cells were used as the
reference
sample. All qPCRs were run on a QuantStudio 3 Real-Time PCR System (Applied
Biosystems).
SARS2 D614G Spike Production and biotinylation
Prefusion-stabilized SARS2 D614G spike (comprising amino acid sequence
Q14 to K1211) with a C-terminal TEV cleavage site, T4 bacteriophage fibritin
foldon,
8x His-, Avi- and EPEA-tag was transfected into HEK293 Freestyle cells, using
293fectin as a transfection reagent. Cells were left to produce protein for
three days at
37 C. Afterwards, supernatant was harvested by centrifuging cells for 30
minutes at 500
xg, followed by another spin for 30 minutes at 4000 xg. Cell culture
supernatant was
filtered through a 0.2 um filter and loaded onto a 5 mL C-tag affinity matrix
column,
pre-equilibrated with 50 mM Tris pH 8 and 200 mM NaCl. SARS2 D614G spike was
eluted, using 10 column volumes of 100 mM Tris, 200 mM NaCl and 3.8 mM SEPEA
peptide. Elution peak was concentrated and injected on a Superose 6 increase
10/300
GL gel filtration column, using 50 mM Tris pH 8 and 200 mM NaCl as a running
buffer. SEC fractions corresponding to monodisperse SARS2 D614G spike were
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collected and flash frozen in liquid nitrogen for storage at -80 C. Purified
SARS2
D614G spike protein was biotinylated using BirA500 biotinylation kit from
Avidity. To
50 ug of spike protein, 5 ug of BirA, and 11 uL of BiomixA and BiomixB was
added.
Final spike protein concentration during the biotinylation reaction was ¨1 uM.
The
reaction was left to proceed for 16 hours at 4 C. Then, protein was desalted
using two
Zeba spin columns pre-equilibrated with lx PBS pH 7.4.
_How cytometry analysis for DC-,S'IGN, L-SIGN, ,SYGLEC1 and ACE-2
HEK 293T cells expressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2 were
resuspended at 4x106 cells/mL and 100 pL per well were seeded onto V-bottom 96-
well
plates (Corning, 3894). The plate was centrifuged at 2,000 rpm for 5 minutes
and
washed with PBS (pH 7.4). The cells were resuspended in 200 p1_, of PBS
containing
Ghost violet 510 viability dye (Cell Signaling, cat. 13-0870-T100, 1:1,000
dilution),
incubated for 15 minutes on ice and then washed. The cells were resuspended in
100 pL
of FACS buffer prepared with 0.5% BSA (Sigma-Aldrich) in PBS containing the
primary antibodies at a 1:100 dilution: mouse anti-DC/L-SIGN (Biolegend, cat.
845002), rabbit anti-DC-SIGN (Cell Signaling, cat. 13193), mouse anti-SIGLEC1
(Biologend, cat. 346002) or goat anti-ACE2 (R&D Systems, cat. AF933). After 1
h
incubation on ice, the cells were washed two times and resuspended in FACS
buffer
containing the Alexa Fluor-488-labeled secondary antibodies at a 1:200
dilution: goat
anti-mouse (Invitrogen cat. A11001), goat anti-rabbit (Invitrogen cat. Al1008)
or
donkey anti-goat (Invitrogen cat. A11055). After incubation for 45 min on ice,
the cells
were washed three times with 200 L of FACS buffer and fixed with 2004, of 4%
PFA
(Alfa Aesar) for 15 mins at room temperature. Cells were washed three times,
resuspended in 200pL of FACS buffer and analyzed by flow cytometry using the
CytoFLEX flow cytometer (Beckman Coulter).
Flow cytometry of SARS-CoV-2 Spike and RBD binding to cells
Biotinylated SARS-CoV-2 Spike D6 14G protein (Spikebiotin, in-house
generated) or the biotinylated SARS-CoV-2 Spike receptor-binding domain
(RBDbiotin, Sino Biological, 40592-V08B) were incubated with Alexa Fluor 647
streptavidin (AF647-strep, Invitrogen, S21374) at a 1:20 ratio by volume for
20 min at
room temperature. The labeled proteins were then stored at 4 C until further
use. Cells
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were dissociated with TrpLE Express (Gibco, 12605-010) and 105 cells were
transferred
to each well of a 96-well V bottom plate (Corning, 3894). Cells were washed
twice in
flow cytometry buffer (2% FBS in PBS (w/o Ca/Mg)) and stained with Spikebiotin-
AF647-strep at a final concentration of 20 [tg/m1 or RBDbiotin-AF647-strep at
a final
concentration of 7.5 g/ml for lh on ice. Stained cells were washed twice with
flow
cytometry buffer, resuspended in I% PFA (Electron Microscopy Sciences, 15714-
S)
and analyzed with the Cytoflex LX (Beckman Coulter).
Recombinant expression of SARS-CoV-2-specific mAbs.
Human mAbs were isolated from plasma cells or memory B cells of SARS-
CoV-2 immune donors, as previously described. Recombinant antibodies were
expressed in ExpiCHO cells at 37 C and 8% CO2. Cells were transfected using
ExpiFectamine. Transfected cells were supplemented 1 day after transfection
with
ExpiCHO Feed and ExpiFectamine CHO Enhancer. Cell culture supernatant was
collected eight days after transfection and filtered through a 0.2 p.m filter.
Recombinant
antibodies were affinity purified on an AKTA xpress FPLC device using 5 mL
HiTrapTm MabSelectTM PrismA columns followed by buffer exchange to Histidine
buffer (20 mM Histidine, 8% sucrose, pH 6) using HiPrep 26/10 desalting
columns
SARS-CoV-2 infection model in hamster
Virus preparation
The SARS-CoV-2 strain used in this study, BetaCov/Belgium/GTM-03021/2020
(EPI ISL 109 40797612020-02-03), was recovered from a nasopharyngeal swab
taken
from an RT-qPCR confirmed asymptomatic patient who returned from Wuhan, China
in February 2020. A close relation with the prototypic Wuhan-Hu-1 201 9-nCoV
(GenBank accession 112 number MN908947.3) strain was confirmed by phylogenetic
analysis. Infectious virus was isolated by serial passaging on HuH7 and Vero
E6 cells,
passage 6 virus was used for the study described here. The titer of the virus
stock was
determined by end-point dilution on Vero E6 cells by the Reed and Muench
method.
Cells
Vero E6 cells (African green monkey kidney, ATCC CRL-1586) were cultured
in minimal essential medium (Gibco) supplemented with 10% fetal bovine serum
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(Integro), 1% L- glutamine (Gibco) and 1% bicarbonate (Gibco). End-point
titrations
were performed with medium containing 2% fetal bovine serum instead of 10%.
SARS-Co V-2 infection model in hamsters
The hamster infection model of SARS-CoV-2 has been described before. The
specific study design is shown in the schematic below. In brief, wild-type
Syrian
Golden hamsters (Mesocricetus auratus) were purchased from Janvier
Laboratories and
were housed per two in ventilated isolator cages (IsoCage N Biocontainment
System,
Tecniplast) with ad libitum access to food and water and cage enrichment (wood
block).
The animals were acclimated for 4 days prior to study start. Housing
conditions and
experimental procedures were approved by the ethics committee of animal
experimentation of KU Leuven (license P065- 2020). Female 6-8 week old
hamsters
were anesthetized with ketamine/xylazine/atropine and inoculated intranasally
with 50
pL containing 2x106 TCID50 SARS-CoV-2 (day 0).
Treatment regimen
Animals were prophylactically treated 48h before infection by intraperitoneal
administration (i.p.) and monitored for appearance, behavior, and weight. At
day 4 post
infection (p.i.), hamsters were euthanized by i.p. injection of 500 uL
Dolethal (200
mg/mL sodium pentobarbital, Vetoquinol SA). Lungs were collected and viral RNA
and infectious virus were quantified by RT-qPCR and end-point virus titration,
respectively. Blood samples were collected before infection for PK analysis.
SARS-CoV-2 RT-qPCR
Collected lung tissues were homogenized using bead disruption (Precellys) in
RLT buffer (RNeasyMinikit, Qiagen)and centrifuged (10.000 rpm, 5 min) to
pellet the cell debris. RNA was extracted according to the manufacturer's
instructions.
Of 50 uL eluate, 4 uL was used as a template in RT-qPCR reactions. RT-qPCR was
performed on a LightCycler96 platform (Roche) using the iTaq Universal Probes
One-
Step RT-qPCR kit (BioRad) with N2 primers and probes targeting the
nucleocapsid.
Standards of SARS-CoV-2 cDNA (IDT) were used to express viral genome copies
per
mg tissue or per mL serum.
End-point virus titrations
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Lung tissues were homogenized using bead disruption (Precellys) in 350 uL
minimal essential medium and centrifuged (10,000 rpm, 5min, 4 C) to pellet the
cell
debris. To quantify infectious SARS-CoV-2 particles, endpoint titrations were
performed on confluent Vero E6 cells in 96- well plates. Viral titers were
calculated by
the Reed and Muench method using the Lindenbach calculator and were expressed
as
50% tissue culture infectious dose (TCID50) per mg tissue.
Histology
For histological examination, the lungs were fixed overnight in 4%
formaldehyde and embedded in paraffin. Tissue sections (5 prn) were analyzed
after
staining with hematoxylin and eosin and scored blindly for lung damage by an
expert
pathologist. The scored parameters, to which a cumulative score of 1 to 3 was
attributed, were the following: congestion, intra-alveolar hemorrhagic,
apoptotic bodies
in bronchus wall, necrotizing bronchiolitis, perivascular edema,
bronchopneumonia,
perivascular inflammation, peribronchial inflammation and vasculitis.
Binding of immunocomplexes to hamster monocytes
Immunocomplexes (IC) were generated by complexing S309 mAb (hamster
IgG, either wt or N297A) with a biotinylated anti-idiotype fab fragment and
Alexa-488-
streptavidin, using a precise molar ratio (4:8:1, respectively). Pre-generated
fluorescent
IC were serially diluted incubated at 4 C for 3 hrs with freshly revitalized
hamster
splenocytes, obtained from a naïve animal. Cellular binding was then evaluated
by
cytometry upon exclusion of dead cells and physical gating on monocyte
population.
Results are expressed as Alexa-488 mean florescent intensity of the entire
monocyte
population.
Bionyarmatic analyses
Processed Human Lung Cell Atlas (HLCA) data and cell-type annotations were
downloaded from Github (github.com/krasnowlab/HLCA). Processed single-cell
transcriptome data and annotation of lung epithelial and immune cells from
SARS-
CoV-2 infected individuals were downloaded from NCBI GEO database (ID:
GSE158055) and Github (github.com/zhangzlab/covid balf). Available sequence
data
from the second single-cell transcriptomics study by Liao et al. were
downloaded from
NCBI SRA (ID: PRJNA608742) for inspection of reads corresponding to viral RNA.
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The proportion of sgRNA relative to genomic RNA was estimated by counting TRS-
containing reads supporting a leader-TRS junction. Criteria and methods for
detection
of leader-TRS junction reads were adapted from Alexandersen et al. The viral
genome
reference and TRS annotation was based on Wuhan-Hu-1 NC 045512.2/MN908947.
Only 2 samples from individuals with severe COVID-19 had detectable leader-TRS
junction reads (SRR11181958, SRR11181959).
EXAMPLE 5
ACE2-INDEPENDENT MECHANISM OF SARS-CoV2 NEUTRALIZATION
In the following experiments, unless otherwise indicated, S309 antibody (VH of
SEQ ID NO.442, VL of SEQ ID NO. :446) was expressed as recombinant IgG1 with
M428L and N434S Fe mutations. In certain experiments, antibody S2X333 (VH of
SEQ ID NO.52, VL of SEQ ID NO. :56) and/or 52E12 (VH of SEQ ID NO.:450, VL of
SFQ TD NO .454) (also expressed as rTgG1) were tested Other tested antibodies
included S2M11 (VH of SEQ ID NO.:458, VL of SEQ ID NO.462), S2D106, and
52X58 (Starr et al, Nature 597:97-102 (2021), which antibodies are
incorporated
herein by reference).
The effect of ACE2 overexpression on S309 antibody neutralization of infection
was investigated. Vero E6 or Vero E6-TMPRSS2 cells were infected with SARS-CoV-
2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of S309 (10 g/ml). Cells
were fixed 24h post infection, viral nucleocapsid protein was immunostained
and
quantified. Nucleocapsid staining was effectively absent in antibody-treated
cells.
S309 had an IC50 (ng/mL) in Vero E6 cells of 65 and in Vero E6-TMPRSS2 of 91
(data not shown).
A panel of 7 cell lines (HeLa, 293T (wt), Vero E6, Huh7, 293T ACE2, MRC 5-
ACE2-IMPRSS2, A549-ACE2-IMPRSS2 clone 5, A549-ACE2-IMPRSS2 clone 10)
were infected with SARS-CoV-2-Nluc or VSV pseudotyped with the SARS-CoV-2
spike protein in the presence of S309. Luciferase signal was quantified 24h
post
infection. S309 maximum neutralization values were as shown in Table 8.
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Table 8. Maximum Neutralization Values of S309
Virus/Pseudotype
Cell Type SARS- VSV Pseudotype
CoV-2-Nlue
Vero E6 >99% >99%
Vero E6-TMPRSS2 >99% 96%
Huh7 98% 78%
293T ACE2 26% 34%
MRC5-ACE2-TMF'RSS2 87% 45%
A549-ACE2-TMPRSS2 89% 65%
clone 5
A549-ACE2-TMPRS S2 81% 42%
clone 10
Binding of purified, fluorescently-labeled SARS-CoV-2 spike protein binding to
these cell lines was quantified by flow cytometry. HeLa and 239T WT cells had
he
lowest MFIs, followed by Huh7 and VeroE6 cells. 293T ACE2 cells (highest), MRC
5-
ACE2-TMPRSS2 (third-highest), A549-ACE2-TMPRSS2 clone 5 (fourth-highest), and
A549-ACE2-TMPRSS2 clone 10 (second-highest) had higher MFIs. Correlation
analysis between spike binding maximum neutralization potential of S309 was
determined; S309 Spearman correlation values were: r = -0.94 for both viral
models. p
= 0.017.
To further characterize SARS-CoV-2-susceptible cell lines, the seven cell
lines
described above were incubated with purified, fluorescently-labeled SARS-CoV-2
spike protein or RBD protein and protein binding was quantified by flow
cytometry. In
descending order of MFI, the cell lines were: A549-ACE2-TMPRSS2 clone 10; 293T
ACE2; MRC 5-ACE2-TMPRSS2; A549-ACE2-TMPRSS2 clone 5; Vero E6; Huh7;
293T (wt), and HeLa.
Selected lectins and published receptor candidates were screened using
HEK293T cells infected with SARS-CoV-2 VSV pseudoviruses. ACE2, DC-SIGN, L-
SIGN, and SIGLEC-1 gave the highest signals. ACE2 provided a signal of
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approximately 105 relative luminescence units (RLUs), and DC-SIGN, SIGLEC-1,
and
L-SIGN had signals of approximately 104RLUs. All other lectins/candidates
tested
gave signals of approximately 102¨ 103 RLUs.
HEK 293T, HeLa and MRCS cells were transiently transduced to overexpress
DC-SIGN, L-SIGN, SIGLEC1 or ACE2 and infected with SARS-CoV-2 VSV
pseudoviruses. Uninfected cells and untransduced cells were included as
controls. In
1-1EK2931 cells, ACE2, DC-SIGN, SIGLEC-1, and L-SIGN all provided substantial
increases in infection. In HeLa and MRC5 cells, only ACE2 increased infection.
Stable HEK293T cell lines overexpressing DC-SIGN, L-SIGN, SIGLEC-1 or
ACE2 were infected with authentic SARS-CoV-2 (MOI 0.1), fixed and
immunostained
at 24 hours for the SARS-CoV-2 nucleoprotein. Wild-type cells (infected and
uninfected) were used as controls. Increased staining was observed in cells
overexpressing DC-SIGN, L-SIGN, or SIGLEC-1, and staining was significantly
increased in cells overexpressing ACE2.
Stable cell lines were infected with SARS-CoV-2-Nluc and luciferase levels
were quantified at 24 hours. In ascending order of RLUs. uninfected (approx.
102-103
RLUs); parental 293T (approx. 104RLUs); DC-SIGN (approx. 105RLUs); L-SIGN
(approx. 105RLUs), SIGLEC-1 (approx. 105-106RLUs), ACE2 (>107 RLUs).
Stable cell lines were incubated with different concentration of anti-SIGLEC1
mAb (clone 7-239) and infected with SARS-CoV-2-Nluc. Infection as a percentage
of
untreated cells remained near to exceeded 100% in 293T cells expressing DC-
SIGN, L-
SIGN, or ACE2, but dropped to below 50% (0.2 1.1g/mL anti-SIGLEC) to close to
0 (1
Kg/mL or 5 Kg/mL anti-SIGLEC) in 293T cells expressing SIGLEC- I .
Single cell expression levels of selected potential SARS-CoV-2 (co)receptor
candidates were determined in different lung cell types derived from the Human
Lung
Cell Atlas (nature.com/articles/s41586-020-2922-4). DC-SIGN, L-SIGN and SIGLEC-
1 are expressed in a variety of cell types in the lung at levels similar to or
even higher
than ACE2.
Binding of antibodies targeting DC-/L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on
HEK293T cells stably over-expressing the respective attachment receptor was
analyzed
by flow cytometry and immunofluorescence analysis. HEK 293T cells over-
expressing
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the respective attachment receptors were infected with VSV pseudotyped with
SARS-
COV-2 wildtype spike or spike bearing mutations of the B1.1.7 lineage.
Luminescence
was analyzed one day post infection. Infection was increased in cells
expressing the
attachment receptors. Infection by VSV pseudotyped with either spike was
similar for
each test group. Cells expressing ACE2 gave the highest luminescence signal.
Vero E6 cells, in vitro differentiated moDCs or PBMCs were infected with
SARS-CoV-2 at MOI 0.01. At 24h post infection, cells were fixed, immunostained
for
viral nucleocapsid protein and infected cells were quantified. Only VeroE6
cells
showed infection (approximately 7% of cells). Supernatant of the infected
cells was
taken at 24, 48 and 72h and infectious viral titer was quantified by FFU assay
on Vero
E6 cells.
Major cell types with detectable SARS-CoV-2 genome in bronchoalveolar
lavage fluid (BALF) and sputum of severe COVID-19 patients were assessed. A t-
SNE
plot was generated, and the count of each SARS-CoV-2+ cell type was determined
(total n=3,085 cells from 8 subjects in Ken et al. Cell 2021). Cell types were
T, NK,
plasma, neutrophil, macrophage, ciliated, squamous, and secretory. Expression
of
ACE2, DC-SIGN, L-SIGN, SIGLEC-1, and combinations of these was assessed for
each cell type.
ACE2, DC-SIGN (CD209), L-SIGN (CLEC4M), SIGLEC1 transcript counts
were correlated with SARS-CoV-2 RNA counts in macrophages and in secretory
cells.
Correlation was based on counts (before log transformation), from Ren et al.
Cell 2021.
Representative data showing expression of receptors in stable HEK293T cell
lines are shown in Figure 40. Cell lines were generated to overexpress DC-
SIGN, L-
SIGN or ACE2 by transducing HEK293T cells with lentivirus encoding the
transgene,
and immunofluorescence assays were performed to assess transgene expression.
Representative data showing the ability of VSV pseudovirus expressing SARS-
CoV-2 S protein with luciferase reporter to infect the HEK293T cells (using a
luminescence assay) are shown in Figure 41; expression of DC-SIGN or L-SIGN
increased pseudovirus infection levels by over 10-fold compared to infection
of WT
HEK293T cells, and expression of ACE2 increased pseudovirus infection levels
by over
100-fold compared to infection of WT HEK293T cells.
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Neutralizing activity of mAb S309 against the VSV pseudovirus was assessed in
the engineered HEK293T cells. S309 fully neutralized infection via DC-SIGN and
L-
SIGN, and to a lesser extent, ACE2.
The ability of live SARS-CoV-2 with luciferase reporter to infect the HEK293T
cells was examined using a luminescence assay. expression of DC-SIGN or L-SIGN
increased live virus infection levels by over 3-fold compared to infection of
WT
1-1EK2931 cells, and expression of ACE2 increased live virus infection levels
by over
100-fold compared to infection of WT HEK293T cells.
Neutralizing activity of mAb S309 against the VSV pseudovirus was assessed in
the engineered HEK293T cells. S309 fully neutralized infection via DC-SIGN and
L-
SIGN, and neutralized infection via ACE2 to a lesser extent.
Experiments were performed to investigate whether S309 antibody can
neutralize entry of SARS-CoV-2 via SIGLEC-1. Briefly, stable cell HEK293T
lines
were generated as described above to overexpress DC-SIGN/L-SIGN, DC-SIGN,
SIGLEC-1, or ACE2. Expression of DC-SIGN, L-SIGN, or SIGLEC increased live
virus infection levels by over 10-fold compared to infection of WT HEK293T
cells, and
expression of ACE2 increased pseudovirus infection levels by over 100-fold
compared
to infection of WT HEK293T cells. S309 fully neutralized infection via DC-
SIGN, L-
SIGN, and SIGLEC-1.
Expression of DC-SIGN (CD209) and other cell surface receptor proteins
including SIGLEC-1 and other SIGLECs was determined on a variety of cell
types.
Data are summarized in Figure 45.
Further experiments were performed to investigate the function(s) of DC-SIGN,
L-SIGN, and SIGLEC-1 in SARS-CoV-2 infection. In one set of experiments,
HEK293T cells stably expressing DC-SIGN, L-SIGN, SIGLEC-1 or ACE2 were
infected with live SARS-CoV-2 Nluc at three different multiplicities of
infection
(MOI): 0.01, 0.1, and 1). Infection was determined using relative luminescence
units
and compared to infection in HEK293T cells (parental) At the lowest MOT
tested, an
increase of infection in cells expressing DC-SIGN, L-SIGN, or SIGLEC was
observed.
At the highest MOI tested, infection was not further increased versus parental
by
expression of DC-SIGN, L-SIGN, or SIGLEC. These data indicate that the
parental
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293T cells are susceptible to infection by SARS-CoV-2 and L-SIGN, DC-SIGN, and
SIGLEC-1 enhance infection levels but do not function as primary receptors for
infection.
In another set of experiments, 293T cells, HeLa cells, and MRCS cells were
transiently transduced with lentivirus encoding DC-SIGN, L-SIGN, SIGLEC-1 or
ACE2 and infected with VSV pseudovirus three days after transduction. While
the
293T cells showed a low level of susceptibility (compare uninfected with
untransduced), HeLa and MRCS cells were completely refractory to the virus.
The low
level of infection in 293T cells can be increased by expression of L-SIGN, DC-
SIGN,
or SIGLEC-1, consistent with a role for these proteins as as attachment
factors. The
HeLa and MRCS cells remained refractory to infection even after expression of
L-
SIGN, DC-SIGN, or SIGLEC-1, and only become susceptible after expression of
ACE2. These data indicate that L-SIGN, DC-SIGN, and SIGLEC-1 are not primary
receptors for SARS-CoV-2.
The various embodiments described above can be combined to provide further
embodiments. All of the U.S. patents, U.S. patent application publications,
U.S. patent
applications, foreign patents, foreign patent applications and non-patent
publications
referred to in this specification and/or listed in the Application Data Sheet,
including
U.S. Provisional Application No. 63/084,501, filed September 28, 2020; U.S.
Provisional Application No. 63/111,435, filed November 9, 2020; U.S.
Provisional
Application No. 63/112,505, filed November 11, 2020; U.S. Provisional
Application
No. 63/119,545, filed November 30, 2020; U.S. Provisional Application No.
63/137,112 filed January 13, 2021; and U.S. Provisional Application No.
63/170,356,
filed April 2, 2021, are incorporated herein by reference, in their entirety.
Aspects of
the embodiments can be modified, if necessary to employ concepts of the
various
patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-
detailed description. In general, in the following claims, the terms used
should not be
construed to limit the claims to the specific embodiments disclosed in the
specification
and the claims, but should be construed to include all possible embodiments
along with
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the full scope of equivalents to which such claims are entitled. Accordingly,
the claims
are not limited by the disclosure.
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