VHH Discovery via
Single B Cell Screening
Industry Leading Timelines with Function & On-CellScreening Abilities
April 2022

ABVERIS VHH ANTIBODY DISCOVERY – BENEFITS
Industry-leading platform for generating VHHs against cell surface receptors

Abveris In Vivo VHH Discovery Differentiators•

Superior VHH discovery against cell surface targets• Upfront on-cell screens for specificity/function• Overcoming known limits of display technologies foraddressing cell surface targets•High-affinity antibodies• Natural in vivo affinity maturation• Beacon-based affinity screening•

Rapid timeline• Direct screening on IgG2/3 secreting camelid B cells• No need to build immune-specific libraries•

Royalty free for campaigns initiated in 2022

IgG1IgG2IgG3

VHH / sdAb
In camelids, VHHs are derived from IgG subclasses of IgG2 and IgG3.
Twist Bioscience 2022 Non-confidential | 2

OVERVIEW OF ABVERIS VHH VIA SINGLE B CELL SCREENING
Industry-leading platform for generating VHHs against cell surface receptors
ScheduleRun 1 ~ Day 70
Run 2 ~ Day 90
Run 3 ~ Day 110……
2 – 3 months
Immunization& Assay
Optimization
Multi-strategy Alpaca
Immunization, Reagent
production/labeling,
Assay Scouting

Immunized alpacas maintained & reserved for future screens if necessary

4 – 6 weeks

Beacon-BasedSingle B Cell
Screening

Sequence
RecoveryRecombinant
Expression &Validation

Humanization& Optimization

DevelopabilityAssessment

Sequential, multiplex assays
Bead/cell-based options
Blocking & function

VHH Sequence
recovered from single
cell sequencing

Expression as VHH-Fc
Validation performed forfunction and cell-binding

Bulk, rapid humanizationof multiple VHHs at thesame time

A suite of biophysicalassessments ofhumanized VHHSingle B Cell Screening ( 1 - 2 Days)
Antibody Secreting
B Cells

PBMCIsolation

Production Bleed &PBMC Isolation

Engineering &Developability Assessment(6 – 8 weeks)

1 – 2 weeks

Repeat boost,
bleed & screen

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ABVERIS SINGLE B CELL SCREENING PLATFORM
Optimized, Beacon-based screening for the rapid identification of ideal, rare antibodies•

THROUGHPUT•••RESOLUTION•CUSTOMIZATION•

Tens of thousands of mAbs simultaneouslyscreened~100K of B cells screened in a day
1,000+ specific hits assayed in many cases
Multiple sequential screens including oncell, multiplexed, and functional screensIndustry-leading resolution among all single
B cell platforms

Highly flexible assay setups with assay
development services offered

Photo credit: www.berkeleylights.com

SPEED

•

Project start to paired HC/LC sequencedelivery in as few as 29 days
Twist Bioscience 2022 Non-confidential | 4

SINGLE B CELL DISCOVERY WORKFLOW IN ONE DAY
A quick look at the Beacon-based screening process
Screening

Loading of B Cells•••

14K pens per chip
1 chip = 145 x 96-well plates
10k+ IgG expressing single B cells loaded
into individual nanopens per chip01••

ExportExport hits for single cell sequencing
Downstream expression andvalidation off-Beacon

Sequential, multiplex assays performed toidentify hits
Fluorescent readout detected as blooms inthe channels

•
•02

03

No bloom (-)(No binding)Bloom (+)
(Binding)

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SINGLE B CELL ON-CHIP SCREENING CAPABILITIESUnique techniques constructed for traditionally difficult antigens

Cell Binding••Multi-pass transmembraneproteins – GPCRs, ion channels
Use of adherent cell lines

Multiplexed Assay

Ligand blocking•••

Receptor blockers
Competition
Neutralization•••

Up to 4 sequential 3-color
multiplexed assays
Species cross-reactivity
High homology specificity

FUNCTIONAL ASSAY•••

Antibody internalization
Apoptosis
Custom assays withfluorescent readouts

Ab Internalization

Ultimate flexibility to run up to 4 sequential, multiplexed assays of choice in the same run
Twist Bioscience 2022 Non-confidential | 6SINGLE B CELL ON-CHIP SCREENING CAPABILITIESExamples of Beacon-based on-chip assay deployed at Abveris

Bead-Based Assays
Protein binding

Multiplexed, Blocking Assay

Multiplexed, Cross-Reactivity

Anti-Ig capture beadAnti-Ig capture beadAnti-Ig capture bead

Antigen with fluor
Antigen with fluor

Human Antigen with fluor
Ligand with fluor
Cyno Antigen with fluor

IgG from B cell

Cell-Based Assays
On-Cell Binding
Antigen on cell surface

Functional Assay

Multiplexed, Cross-Reactivity
Target 2 expressing cell
Antigen 1 on cell surface

Antigen on cell surface

Target expressing cellTarget expressing
reporter cell

Target 1 expressing cell

GFP-tag

Antigen 2 on cell surface
Detection antibody with fluor

Detection antibody with fluor

Reporter cell output

Twist Bioscience 2022 Non-confidential | 7

CASE STUDY: ANTI-PSMA VHH DISCOVERY WITH IMMUNIZED ALPACA
Validating Beacon-based VHH discovery workflow targeting prostate-specific membrane antigen (PSMA)POC Study

Phase 1

Phase 2Phase 3Phase 4

Assay Optimization,Immunization & Titer Analysis

Beacon-Based Single B Cell
Screening

Sequence Recovery &Expression

Binding Profile Validation

Goals of POC StudyResults

1Validate the immunization protocol and confirm class switchof alpaca B cells to IgG2/3 isotype

Successfully elicited robust antigen-specific immune responses inalpaca with class switch to IgG2/3 confirmed in as few as 70 days

2

Optimize Beacon screening to identify IgG2/3 antigenspecific binders secreted by alpaca B cells

Successfully screened and identified antigen-specific IgG2/3 onBeacon to guide hit identification and export

3

Establish the sequencing protocol
expression productivity of VHH
characterizationand subsequentfor downstream

Successfully demonstrated the recovery of VHH sequences andrecombinant expression of VHH (as VHH-Fc)
Binding profiles of VHH to the target were validated off-Beacon by
ELISA, Octet BLI and Carterra SPR to show single-digit andsubnanomolar affinities
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POC STUDY: ABVERIS SINGLE B CELL VHH DISCOVERY
Successful class switch was observed at Day 70 of immunization
Phase 1

Phase 2Phase 3Phase 4

Assay Optimization,Immunization & Titer Analysis

Beacon-based Single B Cell
Screening

Sequence Recovery &Expression

Binding Profile Validation

Figure 1A. Anti-human PSMA-His Titer of Alpaca Sera

Immunization Results & Discussion:•Titer analyses from alpaca sera
collected on Day 41, 87, and 97
showed robust immune
responses (Figure 1A)
Subsequent Beacon-based
single B cell screens confirmed
detection of IgG2/3 (Figure 1B)

Day 0

2.5
2.0

Day 41

1.5Day 87

1.0Day 97

0.50.0Anti-sera dilutions% IgG2/3+ Secretion(mean ± SD)•Figure 1B. IgG2/3 Secretion Assay by Beacon

Anti-human PSMA-His Titer

An alpaca was immunized byrecombinant protein of thePSMA ECDOD 450 nm•POC Study

3 51 .03 00 .82 50 .62 00 .41 50 .21 00 .073

12997153

Day

Twist Bioscience 2022 Non-confidential | 9POC STUDY: ABVERIS SINGLE B CELL VHH DISCOVERY
Custom assays by Abveris enabled the identification of antigen-specific IgG2/3 on-Beacon

POC Study

Phase 1

Phase 2Phase 3Phase 4

Assay Optimization,
Immunization & Titer Analysis

Beacon-based Single B Cell
Screening

Sequence Recovery &Expression

Binding Profile Validation

Figure 2A. Assay #1: IgG2/3 Secretion Confirmation

Sequential single B cell assay results:
Assay #1 – IgG2/3 secretion assay using acustom bead that was developed at Abveris(Figure 2A).

Anti-alpaca IgG2/3-AF488

PSMA-AF647

Custom anti-alpaca
capture beads

Custom anti-alpaca
capture beads

IgG2/3IgG2/3

Assay #2 – PSMA binding was confirmed by acustom bead-based assay (Figure 2B).
Combining the results of both assays guides
the identification of PSMA-binding, IgG2/3+
candidates to export the hits for single cell
sequencing, downstream expression andvalidation.

Figure 2B. Assay #2: PSMA Protein Binding Assay

FITC Channel
T0Single IgG2/3-secreting plasma B cellTdetection

CY5 Channel
T0Tdetection

Single IgG2/3-secreting plasma B cell

Twist Bioscience 2022 Non-confidential | 10

POC STUDY: ABVERIS SINGLE B CELL VHH DISCOVERY
Sequencing protocol by Abveris led to successful VHH sequence recovery from clones exported from Beacon

POC Study

Phase 1

Phase 2Phase 3Phase 4

Assay Optimization,Immunization & Titer Analysis

Beacon-based Single B Cell
Screening

Sequence Recovery &Expression

Binding Profile Validation

VHH-FcFigure 3. Representative VHH recovered from Beacon Screening

Hit Identification and Sequence Recovery:• Selected hits identified on-Beacon were
exported for single cell sequencing• VHH sequences were successfully
recovered from IgG2/3 secreting B cells• VHHs were expressed in the format of VHHFc with an average yield of 0.6mg at a highthroughput scale using a transient HEK
expression system

Twist Bioscience 2022 Non-confidential | 11POC STUDY: ABVERIS SINGLE B CELL VHH DISCOVERY
Validation of recombinantly expressed VHH-Fc warrants the quality of Beacon workflow to generate high-quality hits

POC Study

Phase 1

Phase 2Phase 3Phase 4

Assay Optimization,Immunization & Titer Analysis

Beacon-based Single B Cell
Screening

Sequence Recovery &Expression

Binding Profile Validation

Figure 4A. Validation #1: Recombinantly expressed VHH-Fc werevalidated to bind recombinant PSMA ECD protein
1.80

Anti-PSMA ELISA

1.60
human PSMA-His (+)

1.20
1.00

Carterra SPR Validation Results:• Clones of single-digit andsubnanomolar affinities wereidentified (left)

Irrelevant His-Tagged Protein (-)• Representative kinetics
sensograms showing mAbbinding to PSMA recombinant
protein are shown (right)

0.80
0.60
0.40
0.20
0.00

Clone 1
Clone 2
Clone 3
Clone 4
Clone 5Clone 6
Clone 7
Clone 8Clone 9Clone 10
Clone 11
Clone 12
Clone 13
Clone 14
Clone 15
Clone 16
Clone 17
Clone 18
Clone 19
Clone 20
Clone 21
Clone 22
Clone 23

OD450nm

1.40

Figure 4B. Validation #2: Recombinantly expressed VHH-Fc validated by Carterra SPR show single-digit
nanomolar and subnanomolar affinities to recombinant PSMA ECD protein

Representative binding• Validated route for high-affinity
VHH discovery

Twist Bioscience 2022 Non-confidential | 12

successful mAb discovery campaigns+
100 against surface receptorsof our partners return for%
80 additional campaigns
mAb candidates discovered by Abveris+
7 have proceeded to clinical trials

29

days required from immunization start toantibody sequence for a single B cell campaign

10weeks required from immunization start toantibody sequence for a hybridoma campaign

Twist Bioscience 2022 Non-confidential | 13

PARTNERSHIP MODELS DESIGNED TO EMPOWER EVERY TEAM
Flexible and dynamic partnership structures for diverse antibody discovery teams

FEE-FOR-SERVICE

SUCCESS-BASEDMILESTONEEQUITY-BEARING

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