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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  
TM  
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  
TM  
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.  
TM  
TM  
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  
TM  
ProSpeed Expression  
Various in-chip binding  
assays  
Binding confirmation  
Signaling blockade assay for  
IL-8/CXCR1 by RGA  
by ELISA  
92  
46  
0
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/  
88  
44  
0
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
TM  
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  
TM  
TM  
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)  
TM  
Plasmid based  
Expression  
ProSpeed  
Expression  
Screening Assays with Supernatants of  
Beacon Screening  
TM  
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  
79  
39.5  
0
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
TM  
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  
TM  
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  
TM  
ProSpeed Expression  
TM  
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)  
86  
43  
0
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  
56  
46  
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  
TM  
Beacon Screening  
TM  
Supernatants of ProSpeed  
Expression  
CONCLUSION  
Exported binders for ProSpeed  
Beacon cell  
screening  
TAPsupernatant cell  
binding by FACS  
expression and assays  
TM  
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  
62  
31  
0
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
ProBioCDMO.com