---
_id: '12252'
abstract:
- lang: eng
text: The COVID−19 pandemic not only resulted in a global crisis, but also accelerated
vaccine development and antibody discovery. Herein we report a synthetic humanized
VHH library development pipeline for nanomolar-range affinity VHH binders to SARS-CoV-2
variants of concern (VoC) receptor binding domains (RBD) isolation. Trinucleotide-based
randomization of CDRs by Kunkel mutagenesis with the subsequent rolling-cycle
amplification resulted in more than 1011 diverse phage display
library in a manageable for a single person number of electroporation reactions.
We identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants
of concern (VoC) receptor binding domains (RBD) by screening a novel synthetic
humanized antibody library. In order to explore the most robust and fast method
for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling
and avidity engineering by multivalent trimeric VHH fusion protein construction.
As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in
nM and pM range respectively. Importantly, these affinities are weakly influenced
by most of SARS-CoV-2 VoC mutations and they retain moderate binding to BA.4\5.
The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\ml and
9.6 ng\ml for H7-Fc and G12x3-Fc antibodies, respectively, for the emerging Omicron
BA.1 variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2
neutralization binders with the therapeutic potential for present and future SARS-CoV-2
variants.
acknowledgement: The authors declare that this study received funding from Immunofusion.
The funder was not involved in the study design, collection, analysis, interpretation
of data, the writing of this article or the decision to submit it for publication.
article_number: '965446'
article_processing_charge: No
article_type: original
author:
- first_name: Dmitri
full_name: Dormeshkin, Dmitri
last_name: Dormeshkin
- first_name: Michail
full_name: Shapira, Michail
last_name: Shapira
- first_name: Simon
full_name: Dubovik, Simon
last_name: Dubovik
- first_name: Anton
full_name: Kavaleuski, Anton
id: 4968f7ad-eb97-11eb-a6c2-8ed382e8912c
last_name: Kavaleuski
orcid: 0000-0003-2091-526X
- first_name: Mikalai
full_name: Katsin, Mikalai
last_name: Katsin
- first_name: Alexandr
full_name: Migas, Alexandr
last_name: Migas
- first_name: Alexander
full_name: Meleshko, Alexander
last_name: Meleshko
- first_name: Sergei
full_name: Semyonov, Sergei
last_name: Semyonov
citation:
ama: Dormeshkin D, Shapira M, Dubovik S, et al. Isolation of an escape-resistant
SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library. Frontiers
in Immunology. 2022;13. doi:10.3389/fimmu.2022.965446
apa: Dormeshkin, D., Shapira, M., Dubovik, S., Kavaleuski, A., Katsin, M., Migas,
A., … Semyonov, S. (2022). Isolation of an escape-resistant SARS-CoV-2 neutralizing
nanobody from a novel synthetic nanobody library. Frontiers in Immunology.
Frontiers Media. https://doi.org/10.3389/fimmu.2022.965446
chicago: Dormeshkin, Dmitri, Michail Shapira, Simon Dubovik, Anton Kavaleuski, Mikalai
Katsin, Alexandr Migas, Alexander Meleshko, and Sergei Semyonov. “Isolation of
an Escape-Resistant SARS-CoV-2 Neutralizing Nanobody from a Novel Synthetic Nanobody
Library.” Frontiers in Immunology. Frontiers Media, 2022. https://doi.org/10.3389/fimmu.2022.965446.
ieee: D. Dormeshkin et al., “Isolation of an escape-resistant SARS-CoV-2
neutralizing nanobody from a novel synthetic nanobody library,” Frontiers in
Immunology, vol. 13. Frontiers Media, 2022.
ista: Dormeshkin D, Shapira M, Dubovik S, Kavaleuski A, Katsin M, Migas A, Meleshko
A, Semyonov S. 2022. Isolation of an escape-resistant SARS-CoV-2 neutralizing
nanobody from a novel synthetic nanobody library. Frontiers in Immunology. 13,
965446.
mla: Dormeshkin, Dmitri, et al. “Isolation of an Escape-Resistant SARS-CoV-2 Neutralizing
Nanobody from a Novel Synthetic Nanobody Library.” Frontiers in Immunology,
vol. 13, 965446, Frontiers Media, 2022, doi:10.3389/fimmu.2022.965446.
short: D. Dormeshkin, M. Shapira, S. Dubovik, A. Kavaleuski, M. Katsin, A. Migas,
A. Meleshko, S. Semyonov, Frontiers in Immunology 13 (2022).
date_created: 2023-01-16T09:56:57Z
date_published: 2022-09-16T00:00:00Z
date_updated: 2023-08-04T09:49:24Z
day: '16'
ddc:
- '570'
department:
- _id: LeSa
doi: 10.3389/fimmu.2022.965446
external_id:
isi:
- '000862479100001'
file:
- access_level: open_access
checksum: f8f5d8110710033d0532e7e08bf9dad4
content_type: application/pdf
creator: dernst
date_created: 2023-01-30T09:22:26Z
date_updated: 2023-01-30T09:22:26Z
file_id: '12443'
file_name: 2022_FrontiersImmunology_Dormeshkin.pdf
file_size: 5695892
relation: main_file
success: 1
file_date_updated: 2023-01-30T09:22:26Z
has_accepted_license: '1'
intvolume: ' 13'
isi: 1
keyword:
- Immunology
- Immunology and Allergy
- COVID-19
- SARS-CoV-2
- synthetic library
- RBD
- neutralization nanobody
- VHH
language:
- iso: eng
month: '09'
oa: 1
oa_version: Published Version
publication: Frontiers in Immunology
publication_identifier:
issn:
- 1664-3224
publication_status: published
publisher: Frontiers Media
quality_controlled: '1'
scopus_import: '1'
status: public
title: Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel
synthetic nanobody library
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 13
year: '2022'
...
---
_id: '6983'
abstract:
- lang: eng
text: Malaria, a disease caused by parasites of the Plasmodium genus, begins when
Plasmodium-infected mosquitoes inject malaria sporozoites while searching for
blood. Sporozoites migrate from the skin via blood to the liver, infect hepatocytes,
and form liver stages which in mice 48 h later escape into blood and cause clinical
malaria. Vaccine-induced activated or memory CD8 T cells are capable of locating
and eliminating all liver stages in 48 h, thus preventing the blood-stage disease.
However, the rules of how CD8 T cells are able to locate all liver stages within
a relatively short time period remains poorly understood. We recently reported
formation of clusters consisting of variable numbers of activated CD8 T cells
around Plasmodium yoelii (Py)-infected hepatocytes. Using a combination of experimental
data and mathematical models we now provide additional insights into mechanisms
of formation of these clusters. First, we show that a model in which cluster formation
is driven exclusively by T-cell-extrinsic factors, such as variability in “attractiveness”
of different liver stages, cannot explain distribution of cluster sizes in different
experimental conditions. In contrast, the model in which cluster formation is
driven by the positive feedback loop (i.e., larger clusters attract more CD8 T
cells) can accurately explain the available data. Second, while both Py-specific
CD8 T cells and T cells of irrelevant specificity (non-specific CD8 T cells) are
attracted to the clusters, we found no evidence that non-specific CD8 T cells
play a role in cluster formation. Third and finally, mathematical modeling suggested
that formation of clusters occurs rapidly, within few hours after adoptive transfer
of CD8 T cells, thus illustrating high efficiency of CD8 T cells in locating their
targets in complex peripheral organs, such as the liver. Taken together, our analysis
provides novel insights into and attempts to discriminate between alternative
mechanisms driving the formation of clusters of antigen-specific CD8 T cells in
the liver.
article_number: '2153'
article_processing_charge: No
article_type: original
author:
- first_name: Réka K
full_name: Kelemen, Réka K
id: 48D3F8DE-F248-11E8-B48F-1D18A9856A87
last_name: Kelemen
orcid: 0000-0002-8489-9281
- first_name: H
full_name: Rajakaruna, H
last_name: Rajakaruna
- first_name: IA
full_name: Cockburn, IA
last_name: Cockburn
- first_name: VV
full_name: Ganusov, VV
last_name: Ganusov
citation:
ama: Kelemen RK, Rajakaruna H, Cockburn I, Ganusov V. Clustering of activated CD8
T cells around Malaria-infected hepatocytes is rapid and is driven by antigen-specific
cells. Frontiers in Immunology. 2019;10. doi:10.3389/fimmu.2019.02153
apa: Kelemen, R. K., Rajakaruna, H., Cockburn, I., & Ganusov, V. (2019). Clustering
of activated CD8 T cells around Malaria-infected hepatocytes is rapid and is driven
by antigen-specific cells. Frontiers in Immunology. Frontiers. https://doi.org/10.3389/fimmu.2019.02153
chicago: Kelemen, Réka K, H Rajakaruna, IA Cockburn, and VV Ganusov. “Clustering
of Activated CD8 T Cells around Malaria-Infected Hepatocytes Is Rapid and Is Driven
by Antigen-Specific Cells.” Frontiers in Immunology. Frontiers, 2019. https://doi.org/10.3389/fimmu.2019.02153.
ieee: R. K. Kelemen, H. Rajakaruna, I. Cockburn, and V. Ganusov, “Clustering of
activated CD8 T cells around Malaria-infected hepatocytes is rapid and is driven
by antigen-specific cells,” Frontiers in Immunology, vol. 10. Frontiers,
2019.
ista: Kelemen RK, Rajakaruna H, Cockburn I, Ganusov V. 2019. Clustering of activated
CD8 T cells around Malaria-infected hepatocytes is rapid and is driven by antigen-specific
cells. Frontiers in Immunology. 10, 2153.
mla: Kelemen, Réka K., et al. “Clustering of Activated CD8 T Cells around Malaria-Infected
Hepatocytes Is Rapid and Is Driven by Antigen-Specific Cells.” Frontiers in
Immunology, vol. 10, 2153, Frontiers, 2019, doi:10.3389/fimmu.2019.02153.
short: R.K. Kelemen, H. Rajakaruna, I. Cockburn, V. Ganusov, Frontiers in Immunology
10 (2019).
date_created: 2019-11-04T15:50:06Z
date_published: 2019-09-20T00:00:00Z
date_updated: 2023-08-30T07:18:23Z
day: '20'
ddc:
- '570'
department:
- _id: BeVi
doi: 10.3389/fimmu.2019.02153
external_id:
isi:
- '000487187000001'
pmid:
- '31616407'
file:
- access_level: open_access
checksum: 68d1708f7aa412544159b498ef17a6b9
content_type: application/pdf
creator: dernst
date_created: 2019-11-04T15:54:00Z
date_updated: 2020-07-14T12:47:46Z
file_id: '6984'
file_name: 2019_FrontiersImmonology_Kelemen.pdf
file_size: 2083061
relation: main_file
file_date_updated: 2020-07-14T12:47:46Z
has_accepted_license: '1'
intvolume: ' 10'
isi: 1
language:
- iso: eng
month: '09'
oa: 1
oa_version: Published Version
pmid: 1
publication: Frontiers in Immunology
publication_identifier:
issn:
- 1664-3224
publication_status: published
publisher: Frontiers
quality_controlled: '1'
scopus_import: '1'
status: public
title: Clustering of activated CD8 T cells around Malaria-infected hepatocytes is
rapid and is driven by antigen-specific cells
tmp:
image: /images/cc_by.png
legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
short: CC BY (4.0)
type: journal_article
user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8
volume: 10
year: '2019'
...
---
_id: '8237'
abstract:
- lang: eng
text: Monoclonal antibodies find broad application as therapy for various types
of cancer by employing multiple mechanisms of action against tumors. Manipulating
the Fc-mediated functions of antibodies that engage immune effector cells, such
as NK cells, represents a strategy to influence effector cell activation and to
enhance antibody potency and potentially efficacy. We developed a novel approach
to generate and ascertain the functional attributes of Fc mutant monoclonal antibodies.
This entailed coupling single expression vector (pVitro1) antibody cloning, using
polymerase incomplete primer extension (PIPE) polymerase chain reaction, together
with simultaneous Fc region point mutagenesis and high yield transient expression
in human mammalian cells. Employing this, we engineered wild type, low (N297Q,
NQ), and high (S239D/I332E, DE) FcR-binding Fc mutant monoclonal antibody panels
recognizing two cancer antigens, HER2/neu and chondroitin sulfate proteoglycan
4. Antibodies were generated with universal mutagenic primers applicable to any
IgG1 pVitro1 constructs, with high mutagenesis and transfection efficiency, in
small culture volumes, at high yields and within 12 days from design to purified
material. Antibody variants conserved their Fab-mediated recognition of target
antigens and their direct anti-proliferative effects against cancer cells. Fc
mutations had a significant impact on antibody interactions with Fc receptors
(FcRs) on human NK cells, and consequently on the potency of NK cell activation,
quantified by immune complex-mediated calcium mobilization and by antibody-dependent
cellular cytotoxicity (ADCC) of tumor cells. This strategy for manipulation and
testing of Fc region engagement with cognate FcRs can facilitate the design of
antibodies with defined effector functions and potentially enhanced efficacy against
tumor cells.
article_number: '1112'
article_processing_charge: No
article_type: original
author:
- first_name: Kristina M.
full_name: Ilieva, Kristina M.
last_name: Ilieva
- first_name: Judit
full_name: Fazekas-Singer, Judit
id: 36432834-F248-11E8-B48F-1D18A9856A87
last_name: Fazekas-Singer
orcid: 0000-0002-8777-3502
- first_name: Daniela Y.
full_name: Achkova, Daniela Y.
last_name: Achkova
- first_name: Tihomir S.
full_name: Dodev, Tihomir S.
last_name: Dodev
- first_name: Silvia
full_name: Mele, Silvia
last_name: Mele
- first_name: Silvia
full_name: Crescioli, Silvia
last_name: Crescioli
- first_name: Heather J.
full_name: Bax, Heather J.
last_name: Bax
- first_name: Anthony
full_name: Cheung, Anthony
last_name: Cheung
- first_name: Panagiotis
full_name: Karagiannis, Panagiotis
last_name: Karagiannis
- first_name: Isabel
full_name: Correa, Isabel
last_name: Correa
- first_name: Mariangela
full_name: Figini, Mariangela
last_name: Figini
- first_name: Rebecca
full_name: Marlow, Rebecca
last_name: Marlow
- first_name: Debra H.
full_name: Josephs, Debra H.
last_name: Josephs
- first_name: Andrew J.
full_name: Beavil, Andrew J.
last_name: Beavil
- first_name: John
full_name: Maher, John
last_name: Maher
- first_name: James F.
full_name: Spicer, James F.
last_name: Spicer
- first_name: Erika
full_name: Jensen-Jarolim, Erika
last_name: Jensen-Jarolim
- first_name: Andrew N.
full_name: Tutt, Andrew N.
last_name: Tutt
- first_name: Sophia N.
full_name: Karagiannis, Sophia N.
last_name: Karagiannis
citation:
ama: Ilieva KM, Singer J, Achkova DY, et al. Functionally active Fc mutant antibodies
recognizing cancer antigens generated rapidly at high yields. Frontiers in
Immunology. 2017;8. doi:10.3389/fimmu.2017.01112
apa: Ilieva, K. M., Singer, J., Achkova, D. Y., Dodev, T. S., Mele, S., Crescioli,
S., … Karagiannis, S. N. (2017). Functionally active Fc mutant antibodies recognizing
cancer antigens generated rapidly at high yields. Frontiers in Immunology.
Frontiers. https://doi.org/10.3389/fimmu.2017.01112
chicago: Ilieva, Kristina M., Judit Singer, Daniela Y. Achkova, Tihomir S. Dodev,
Silvia Mele, Silvia Crescioli, Heather J. Bax, et al. “Functionally Active Fc
Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields.”
Frontiers in Immunology. Frontiers, 2017. https://doi.org/10.3389/fimmu.2017.01112.
ieee: K. M. Ilieva et al., “Functionally active Fc mutant antibodies recognizing
cancer antigens generated rapidly at high yields,” Frontiers in Immunology,
vol. 8. Frontiers, 2017.
ista: Ilieva KM, Singer J, Achkova DY, Dodev TS, Mele S, Crescioli S, Bax HJ, Cheung
A, Karagiannis P, Correa I, Figini M, Marlow R, Josephs DH, Beavil AJ, Maher J,
Spicer JF, Jensen-Jarolim E, Tutt AN, Karagiannis SN. 2017. Functionally active
Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields.
Frontiers in Immunology. 8, 1112.
mla: Ilieva, Kristina M., et al. “Functionally Active Fc Mutant Antibodies Recognizing
Cancer Antigens Generated Rapidly at High Yields.” Frontiers in Immunology,
vol. 8, 1112, Frontiers, 2017, doi:10.3389/fimmu.2017.01112.
short: K.M. Ilieva, J. Singer, D.Y. Achkova, T.S. Dodev, S. Mele, S. Crescioli,
H.J. Bax, A. Cheung, P. Karagiannis, I. Correa, M. Figini, R. Marlow, D.H. Josephs,
A.J. Beavil, J. Maher, J.F. Spicer, E. Jensen-Jarolim, A.N. Tutt, S.N. Karagiannis,
Frontiers in Immunology 8 (2017).
date_created: 2020-08-10T11:53:32Z
date_published: 2017-09-11T00:00:00Z
date_updated: 2021-01-12T08:17:39Z
day: '11'
doi: 10.3389/fimmu.2017.01112
extern: '1'
intvolume: ' 8'
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://doi.org/10.3389/fimmu.2017.01112
month: '09'
oa: 1
oa_version: Published Version
publication: Frontiers in Immunology
publication_identifier:
issn:
- 1664-3224
publication_status: published
publisher: Frontiers
quality_controlled: '1'
status: public
title: Functionally active Fc mutant antibodies recognizing cancer antigens generated
rapidly at high yields
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 8
year: '2017'
...