Poster
Utilization of ProSpeed™ Expression System To Empower Single B Cell based Antibody Discovery Platform
Utilization of ProSpeed™ Expression System
To Empower Single B Cell based Antibody
Discovery Platform
Derek Chen, Yu Liang, Lindi Wang, Wenwan Fang, Jie Ma
ABSTRACT
APPLICATION SCENARIOS
In the last four years, as a newly emerged technology featuring shorter timeline, better
diversity and better developability, single B cell approach contributed to the discovery
of almost 20% of FDA approved Ab drugs. However, this new technology platform also
faces some limitations, including difficulty in functional screening, limited assay sensitivity
and poor cost-effectiveness when the hit rate is low. To address these limitations, here
1. Functional Screening with Recombinant Supernatants
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Fig 3. Functional screening assays were applied using ProSpeed expression supernatants
to identify IL-8 mAbs that block the IL-8/CXCR1 signaling. (a) 116 positive B cells were
identified by Beacon on-chip screening with human and cyno IL-8 proteins, followed by
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we integrated a ProSpeed linear expression system to the Beacon Single B cell platform,
allowing a fast and cost-effective discovery of functional Ab leads.
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ProSpeed linear expression. The supernatant of ProSpeed expression were tested by
ELISA for binding to human and cyno IL-8, confirming 85 hits were dual binders (b),
among which 16 candidates were shown to block IL-8/CXCR1 signaling by
reporter gene assay (c)
CHALLENGES & SOLUTIONS
Screening Assays with Supernatants of
Due to the extremely low cell density, antibody concentrations in Beacon on-chip screening
tend to be low and fluctuate substantially, which may lead to a number of limitations.
Beacon Screening
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ProSpeed Expression
Various in-chip binding
assays
Binding confirmation
Signaling blockade assay for
IL-8/CXCR1 by RGA
by ELISA
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Difficulty in on-chip
Reduced success
rate for
multi-round (>3) of
on-chip screening
Limited assay
sensitivity
leading to false
negative/positive
Poor cost-
effectiveness when
hit rate is low
functional Assays
(RGA/ Enzymatic/
internalization/
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epitope binning, etc.)
Binders
Fig 1. Limitations in Beacon-based single B cell screening
Functional binders
Confirmed
exported for
(IL-8 signaling
116
85
16
binders
linear
blockers)
expression
To confirm the binding & function of Ab sequences derived from exported single B cells,
in the workflow of conventional Beacon screening (left panel), recombinant expression
based on costly and time-consuming gene syntheses/plasmid preparation is used. In
a
b
c
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contrast, in a ProSpeed Expression workflow (right panel), a PCR based linear expression
cassette is first constructed and used for transient expression, to allow the recombinant
production of mAbs derived from the exported single B cells in the supernatant, and
their functional characterization. Since not all exported B cells but only those secreting
confirmed binding Abs with desirable function profile will be sequenced and pursued,
the turnaround time needed from exported B cell to sequence of confirmed hits is
greatly reduced, from 5-6 wks to 2-3 weeks, and with a substantial reduction of cost as
well.
2. Multiple Rounds of Screening Beyond Beacon’s Limits
Fig 4. Identification of specific binders against a matrix of positive and negative antigens
by multiple rounds of screening. (a) 156 positive B cells were identified by Beacon on-chip
screening with binding to human, cyno ECD proteins and human antigen cell line, followed
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by ProSpeed linear expression. Using this ProSpeed expression supernatant, 56 candidates
were confirmed to be binders to hu and cyno Ag proteins by ELISA (b), and 46 candidates
to be specific binder to human Ag cell line by FACS (c), among which only 14 showed low
nonspecific binding to 3 undesired Ags (with high homology to the target Ag) in additional
counter-screen assays (d)
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Plasmid based
Expression
ProSpeed
Expression
Screening Assays with Supernatants of
Beacon Screening
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7 days
1 days
ProSpeed Expression
Single B cell
VH/VL PCR
Transient
Single B cell
Screening
5-6 vs 2-3
expression
VH/VL PCR
assays
Various in-chip
binding assays
Species
cross-reactivity by
ELISA
Cell binding
by FACS
Counter screening
for non-specific
binding
Weeks
10 days
1 days
3 days
5 days
Linear
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From exported B
cell to sequence
of confirmed hits
Screening
assays
expression
cassette
Sequencing
Sequencing
construction
Human Ag Cyno Ag Human Ag
ECD
ECD
Cell
7 days
5 days
HTP
Plasmid
Binders
transient
expression
Hits
Hits
Confirmed
Confirmed
Specific
preparation
exported for
linear
ELISA
FACS
FACS
156
56
46
14
binders
binders
binders
expression
a
b
c
d
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Fig 2. Workflow of plasmid-based and ProSpeed expression
ProBioCDMO.com
Utilization of ProSpeed™ Expression System
To Empower Single B Cell based Antibody
Discovery Platform
Derek Chen, Yu Liang, Lindi Wang, Wenwan Fang, Jie Ma
APPLICATION SCENARIOS
APPLICATION SCENARIOS
3. Improved Assay Sensitivity to Reduce False Negatives
5. “Rescuing” Missed Hits Due to Unsuccessful RT-PCR
Fig 5. (a) The initial on-chip Beacon screening identified 86 positive B cells binding to
Fig 7. (a) In the plasmid-based approach, the overall outcome heavily relies on the success
of RT-PCR. If this is not successful, no further sequencing and expression of a certain B cell
clone will be pursued and such a potential hit will be lost. (b) In a ProSpeed expression
workflow, a nested-PCR with improved sensitivity was employed. Thus even trace amount
of cDNA below the detection limit of a conventional PCR may still be effectively amplified
and incorporated into the linear expression system, allowing further characterization and
sequencing of such hits that may be missed in a conventional Beacon workflow.
human and cyno antigen proteins, among which only 38 hits shown undesirable binding to
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cross-famility Ag F1. However, in the follow-up screening using supernatant of ProSpeed
expression with improved assay sensitivity, 76 of them were shown to non-specifically bind
to cross family Ag F1, and only 10 hits were confirmed to be the desirable low binders to all
3 cross-famility Ags F1, F2 and F3.
Screening Assays with Supernatants of
Beacon Screening
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ProSpeed Expression
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Plasmid Based Expression
ProSpeed Expression
Various in-chip
binding assays
Species cross-
Cell binding
Counter screening for
cross-family
reactivity by ELISA
by FACS
（Human & cyno cell ratio>50）
non-specific binding
HTP liner expression of 100+
binders
(clone A included)
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Single B cell
exporting
Single B cell
exporting
Single B cell
VH/VL PCR
Positive binding
detected by
ELISA
Single B cell
VH/VL PCR
No band visible in a gel for
Linear
the RT-PCR product of clone A
Binders
exported for
linear
expression
Expression
Desired
binders w/o
cross-family
binding
Confirmed
ELISA
binders
Confirmed
FACS
binders
86
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10
Proceeding with
sequencing
Screening
Assay
No further sequencing and
expression for clone A
Sequencing
a
b
c
d
Sequencing
Clone A Cross-Reactivity by ELISA
Specific binding
confirmed!
Lost hits
rescued!
Binding
confirmation
by IgG
Ab
Potential hits lost!
4. Improved Assay Sensitivity to Identify True Cell Binders
expression
conversion
Fig 6. Due to the low and fluctuating Ab concentration in a Beacon on-chip screening,
FACS binding data may not be conclusive especially when the cell surface Ag level is low.
(a) initially 121 hits were identified by Beacon on-chip screening to be potential binders to
Hu Ag cell. However, in a follow-up FACS assay using ProSpeedTM expression supernatant
with improved assay sensitivity, only 24 of them were confirmed to be true cell binders (b).
a
b
Screening Assays with
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Beacon Screening
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Supernatants of ProSpeed
Expression
CONCLUSION
Exported binders for ProSpeed
Beacon cell
screening
TAPsupernatant cell
binding by FACS
expression and assays
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In summary, the ProSpeed workflow addressed the bottlenecks of current single B cell
platform based on Beacon, by the integration of a linear expression technology to Beacon
platform. This upgrade of the Beacon platform allows high thruput functional screenings
based on recombinant supernatant without the costly gene synthesis/plasmid preparation,
makes it feasible for multiple rounds of screening beyond Beacon’s limits, increases the
assay sensitivity to reduce false negative/positive results, improves the cost-effectiveness
of single B cell screening especially when the hit rate is low, and as a bonus may potentially
“rescue” missed hits due to unsuccessful gene amplification by RT-PCR. Overall, with these
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human cell line
human cell line
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inviting new features, the ProSpeed Single B Cell Ab Discovery Platform, coupled with our
substantial experience of over 100 projects, may offer a favorable solution for antibody
discovery campaigns with expedited timeline, good Ab sequence diversity and better
cost-effectiveness.
Confirmed
cell binders
Binders exported for linear expression
(a confidence value >0.3)
24
121
a
b
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