POSTER Over last three decades we have developed antibody therapies against a host of diseases including autoimmune, infectious diseases and cancer. Traditional technologies are unable to screen the entire B cell repertoire. As a result antibody therapies have only been developed against simple targets with limited success for more difficult targets such as GPCRs and ion channels. To access these new antibodies against more difficult targets, the high-hanging fruit, will require technology that can functionally screen the entire B cell repertoire. Here we demonstrate how Plasma B Discovery on the Beacon ® optofluidic platform can be used to access and functionally screen broad B cell diversity to discover thousands of hits and recover >650 unique, functional antigen-specific antibody sequences in a single workflow that takes less than 1 week. One of the key challenges of developing antibody therapeutics is finding antibodies with the desired function. Many of the “low-hanging fruit” have already been picked using traditional technologies like hybridoma and phage display. As the market now turns to more difficult targets, such as antibodies that elicit weak immune reactions or targets with rare epitopes, it has become clear that progress is severely hampered by a lack of technologies that allow access to greater diversity and enable functional screening of the entire B cell repertoire. The Beacon bypasses time-consuming and inefficient hybridoma fusion and enables discovery of thousands of hits in a single workflow and down-selection of lead candidates using multiple assays for antigen specificity and function (Figure 1 ). Tens of thousands of individual plasma B cells are manipulated by light and cloned into nanoliter-sized NanoPen™ chambers on a microfluidic chip using Berkeley Lights OptoSelect™ technology in an automated fashion (Figure 2). The small volume of these chambers enables rapid, precise and highly sensitive assays that can be completed in less than 1 hour because secreted antibody levels reach detectable levels within minutes (Figure 3). Chips can then be reset to enable assays for deeper interrogation of the same antibodies. This thorough upfront characterization reduces the expense of sequencing or cloning irrelevant non-functional hits. Following functional characterization and automated assay scoring, OptoSeq BCR enables on-chip genomics integration to enable simple and efficient recovery of paired heavy/light chain sequences (Figure 2 and 4). Plasma B cells are lysed in their respective NanoPen chambers and mRNA is captured on beads for on-chip cDNA synthesis. Sequences associated with desired function can then be recovered for several days without sequence degradation due to death of fragile plasma B cells. Using this approach, we were able to identify thousands of antigen-specific hits (Figure 5) and recover >650 unique antigen-specific, functionally characterized heavy/light chain antibody sequences from a single automated workflow in less than one week (Figure 6 and 7). Antigen-specific antibody sequences recovered over multiple days exhibited rich CDR3 diversity (Figure 7). This access to greater diversity increases the chances of discovering better lead candidates. Plasma B cells were screened cloned and screened in OptoSelect™ 11k chips using the Beacon Plasma B Discovery workflow. Multiple assays were performed on the secreted antibodies from these plasma B cells in order to characterize their antigen specificity and cross-reactivity. Using this approach >5000 antigen-specific hits were discovered from single plasma B cell samples (Figure 5). Following functional characterization, mRNA capture beads were delivered to NanoPens containing antigen-specific plasma B cells followed by cell lysis and reverse transcription for on-chip cDNA synthesis using the OptoSeq BCRseq Kit. By stabilizing cDNA sequences on beads within NanoPen chambers, sequences were recovered for several days without sequence degradation due to death of plasma B cells (Figure 6). >650 antigen-specific antibody sequences were recovered in a single experiment (Figure 7). We have shown that the Beacon Plasma B Discovery workflow enables: • Screening of broad B cell diversity • Discovery of >5000 antigen-specific hits from single plasma B cell samples • Down-selection of lead candidates • Recovery of >650 unique sequences in under one week ABSTRACT INTRODUCTION RESULTS Figure 1. Identify hits and recover functionally characterized lead candidates in 1 day. Plasma B Discovery on the Beacon platform bypasses inefficient hybridoma fusion by directly screening plasma B cells from immunized animals. Plasma B cells are harvested and purified from diverse B cell compartments, including spleen, bone marrow and lymph nodes. These cells are functionally screened on the Beacon platform and antigen-specific sequences are recovered in less than a day. Figure 3. Down-selection of lead candidates through multiplexed assays. Setup of a single assay (A) and multiplexing of 3 assays (B) to screen tens of thousands of individual antibodies for antigen specificity and cross-reactivity. Figure 4. OptoSeq BCR integrates downstream genomics for automated sequence recovery. mRNA capture beads are loaded into specific pens and plasma B cells are lysed to capture their mRNA. Reverse transcription is then performed on-chip in order to generate stable cDNA. The capture beads are then recovered for downstream sequencing. Figure 7. Rich CDR3 diversity obtained by recovery of antibody sequences over multiple days using OptoSeq BCR. >650 unique, antigen-specific sequences were recovered in a single workflow over 5 days. Day 0 (blue), day 1 (red), day 2 (green), day 3 (orange), day 4 (dark blue). Figure 6. OptoSeq BCR enables efficient recovery of >650 antigen-specific antibody sequences in one workflow. By stabilizing cDNA sequences on beads within NanoPen chambers, sequences can be recovered for several days without sequence degradation due to cell death. Figure 5. >5000 antigen-specific hits discovered from single plasma B cell sample. By extending the survival of plasma B cells in tissue culture, multiple screening workflows were performed on single plasma B cell samples. 5858 Horton Street | Suite 320 | Emeryville, CA 94608 [email protected] | +1-510-858-2855 | berkeleylights.com For Research Use Only. Not for use in diagnostic procedures. “Berkeley Lights”, “BLI”, “Beacon”, “NanoPen”, “OptoSelect” and the Berkeley Lights logo are trademarks of Berkeley Lights, Inc. All other marks are the property of their owners. © 2020 Berkeley Lights, Inc. 0112 RevA Reaching the High-Hanging Fruit: Accessing broad B cell diversity to select better lead candidates in under 1 week Yogesh Dhande, Patrick Ingram, Minha Park, Maryam Shansab, Po-Yuan Tung, Jason McEwen, Ravi Ramenani, Matthew Kubit, Catalina Martinez, Joshua Mast, Maaz Hussaini, Jimena Loureiro, Angel Angeles, Chris Shing, James Porter, Dan Bernhard, Darshan Thaker, John Tenney, Zhen Hu, Debjit Ray, Jason Stein, Adrienne Higa, Anupam Singhal Berkeley Lights, Inc. 5858 Horton Street, Suite 320, Emeryville, CA 94608 IMMUNIZATION HARVEST FUSION CULTURE PRELIMINARY SCREEN SINGLE CELL CLONING CULTURE ASSAY SEQUENCE FUNCTIONAL LEADS TYPICAL HYBRIDOMA WORKFLOW 12 WEEKS 1 WEEK PLASMA B CELL WORKFLOW ON THE BEACON ® PLATFORM BEACON WORKFLOW IMMUNIZATION HARVEST SEQUENCE FUNCTIONAL LEADS Cell Lysis + RT Bead Unload cDNA to Sequence Unique CDR3 from Single Cell CARNWDGFDFW CQQGSSLPRTF Plasma Cell + mRNA Capture Bead 0 500 1000 1500 2000 2500 3000 3500 4000 Screen 1 Screen 2 Hits per Screen 0 100 200 300 400 500 600 700 Day 0 Day 1 Day 2 Day 3 Day 4 Total # Unique VH/VL Sequences CONCLUSIONS A Figure 2. Link sequence and function in just 2 days. Day 0 Day 1 Organ harvest from immunized mice Plasma B cell enrichment VH/VL amplification Sequencing Automated on the Beacon platform Single cell cloning Functional screening (e.g. antigen specificity) On-chip cDNA synthesis IMMUNIZATION HARVEST SEQUENCE Brightfield image of cells in pens Antigen X Antigen Y IgG Brightfield image of individual cells in NanoPen chambers Antigen coated beads outside the pens Single Plasma B-cell B
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
POSTER
Over last three decades we have developed antibody therapies against a host of diseases including autoimmune, infectious diseases and cancer. Traditional technologies are unable to screen the entire B cell repertoire. As a result antibody therapies have only been developed against simple targets with limited success for more difficult targets such as GPCRs and ion channels. To access these new antibodies against more difficult targets, the high-hanging fruit, will require technology that can functionally screen the entire B cell repertoire. Here we demonstrate how Plasma B Discovery on the Beacon® optofluidic platform can be used to access and functionally screen broad B cell diversity to discover thousands of hits and recover >650 unique, functional antigen-specific antibody sequences in a single workflow that takes less than 1 week.
One of the key challenges of developing antibody therapeutics is finding antibodies with the desired function. Many of the “low-hanging fruit” have already been picked using traditional technologies like hybridoma and phage display. As the market now turns to more difficult targets, such as antibodies that elicit weak immune reactions or targets with rare epitopes, it has become clear that progress is severely hampered by a lack of technologies that allow access to greater diversity and enable functional screening of the entire B cell repertoire. The Beacon bypasses time-consuming and inefficient hybridoma fusion and enables discovery of thousands of hits in a single workflow and down-selection of lead candidates using multiple assays for antigen specificity and function (Figure 1).
Tens of thousands of individual plasma B cells are manipulated by light and cloned into nanoliter-sized NanoPen™ chambers on a microfluidic chip using Berkeley Lights OptoSelect™ technology in an automated fashion (Figure 2). The small volume of these chambers enables rapid, precise and highly sensitive assays that can be completed in less than 1 hour because secreted antibody levels reach detectable levels within minutes (Figure 3). Chips can then be reset to enable assays for deeper interrogation of the same antibodies. This thorough upfront characterization reduces the expense of sequencing or cloning irrelevant non-functional hits.
Following functional characterization and automated assay scoring, OptoSeq BCR enables on-chip genomics integration to enable simple and efficient recovery of paired heavy/light chain sequences (Figure 2 and 4). Plasma B cells are lysed in their respective NanoPen chambers and mRNA is captured on beads for on-chip cDNA synthesis. Sequences associated with desired function can then be recovered for several days without sequence degradation due to death of fragile plasma B cells. Using this approach, we were able to identify thousands of antigen-specific hits (Figure 5) and recover >650 unique antigen-specific, functionally characterized heavy/light chain antibody sequences from a single automated workflow in less than one week (Figure 6 and 7).
Antigen-specific antibody sequences recovered over multiple days exhibited rich CDR3 diversity (Figure 7). This access to greater diversity increases the chances of discovering better lead candidates.
Plasma B cells were screened cloned and screened in OptoSelect™ 11k chips using the Beacon Plasma B Discovery workflow. Multiple assays were performed on the secreted antibodies from these plasma B cells in order to characterize their antigen specificity and cross-reactivity. Using this approach >5000 antigen-specific hits were discovered from single plasma B cell samples (Figure 5). Following functional characterization, mRNA capture beads were delivered to NanoPens containing antigen-specific plasma B cells followed by cell lysis and reverse transcription for on-chip cDNA synthesis using the OptoSeq BCRseq Kit. By stabilizing cDNA sequences on beads within NanoPen chambers, sequences were recovered for several days without sequence degradation due to death of plasma B cells (Figure 6). >650 antigen-specific antibody sequences were recovered in a single experiment (Figure 7).
We have shown that the Beacon Plasma B Discovery workflow enables:
• Screening of broad B cell diversity
• Discovery of >5000 antigen-specific hits from single plasma B cell samples
• Down-selection of lead candidates
• Recovery of >650 unique sequences in under one week
ABSTRACT
INTRODUCTION
RESULTS
Figure 1. Identify hits and recover functionally characterized lead candidates in 1 day. Plasma B Discovery on the Beacon platform bypasses inefficient hybridoma fusion by directly screening plasma B cells from immunized animals. Plasma B cells are harvested and purified from diverse B cell compartments, including spleen, bone marrow and lymph nodes. These cells are functionally screened on the Beacon platform and antigen-specific sequences are recovered in less than a day.
Figure 3. Down-selection of lead candidates through multiplexed assays. Setup of a single assay (A) and multiplexing of 3 assays (B) to screen tens of thousands of individual antibodies for antigen specificity and cross-reactivity.
Figure 4. OptoSeq BCR integrates downstream genomics for automated sequence recovery. mRNA capture beads are loaded into specific pens and plasma B cells are lysed to capture their mRNA. Reverse transcription is then performed on-chip in order to generate stable cDNA. The capture beads are then recovered for downstream sequencing.
Figure 7. Rich CDR3 diversity obtained by recovery of antibody sequences over multiple days using OptoSeq BCR. >650 unique, antigen-specific sequences were recovered in a single workflow over 5 days. Day 0 (blue), day 1 (red), day 2 (green), day 3 (orange), day 4 (dark blue).
Figure 6. OptoSeq BCR enables efficient recovery of >650 antigen-specific antibody sequences in one workflow. By stabilizing cDNA sequences on beads within NanoPen chambers, sequences can be recovered for several days without sequence degradation due to cell death.
Figure 5. >5000 antigen-specific hits discovered from single plasma B cell sample. By extending the survival of plasma B cells in tissue culture, multiple screening workflows were performed on single plasma B cell samples.
5858 Horton Street | Suite 320 | Emeryville, CA 94608 [email protected] | +1-510-858-2855 | berkeleylights.com
For Research Use Only. Not for use in diagnostic procedures. “Berkeley Lights”, “BLI”, “Beacon”, “NanoPen”, “OptoSelect” and the Berkeley Lights logo are trademarks of Berkeley Lights, Inc. All other marks are the property of their owners. © 2020 Berkeley Lights, Inc. 0112 RevA
Reaching the High-Hanging Fruit: Accessing broad B cell diversity to select better lead candidates in under 1 weekYogesh Dhande, Patrick Ingram, Minha Park, Maryam Shansab, Po-Yuan Tung, Jason McEwen, Ravi Ramenani, Matthew Kubit, Catalina Martinez, Joshua Mast, Maaz Hussaini, Jimena Loureiro, Angel Angeles, Chris Shing, James Porter, Dan Bernhard, Darshan Thaker, John Tenney, Zhen Hu, Debjit Ray, Jason Stein, Adrienne Higa, Anupam SinghalBerkeley Lights, Inc. 5858 Horton Street, Suite 320, Emeryville, CA 94608
IMMUNIZATION HARVEST FUSION CULTURE PRELIMINARY SCREEN
SINGLE CELL CLONING
CULTURE ASSAY SEQUENCE FUNCTIONAL LEADS
TYPICAL HYBRIDOMA WORKFLOW
12 WEEKS
1 WEEK
PLASMA B CELL WORKFLOW ON THE BEACON® PLATFORM
BEACON WORKFLOW
IMMUNIZATION HARVEST SEQUENCE FUNCTIONAL LEADS
Cell Lysis +RT Bead
Unload
cDNA to Sequence
Unique CDR3 from Single CellCARNWDGFDFWCQQGSSLPRTF
Plasma Cell + mRNA Capture Bead
0
500
10001500
2000
2500
3000
35004000
Screen 1 Screen 2
Hits
per
Scr
een
0
100
200
300
400
500
600
700
Day 0 Day 1 Day 2 Day 3 Day 4
Tota
l # U
niqu
e VH
/VL
Sequ
ence
s CONCLUSIONS
A
Figure 2. Link sequence and function in just 2 days.
Day 0
Day 1
Organ harvest from immunized mice
Plasma B cellenrichment
VH/VL amplification Sequencing
Automated on the Beacon platform
Single cell cloning Functional screening (e.g. antigen specificity)
On-chip cDNA synthesis
IMMUNIZATION HARVEST
SEQUENCE
Brightfield image of cells in pens
Antigen X
Antigen Y
IgG
Brightfield image of individual cells in NanoPenchambers
Antigen coated beads outside the pens
Single Plasma B
-cell
B
Related Documents
