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How to Screen a Billion Drug Candidates?
Antibody Engineering 2017
Single Cell Functional Assays: Ultra-sensitive, Ultra-fast, Ultra-high-throughput Next-Generation Drug Discovery
The advantages of de novo protein engineering for the generation of functional fully human antibodies to GPCRs
Michael GalloPresidentInnovative Targeting Solutions
No GPCR Targeting Antibody has been Approved by the United States Food and Drug Administration and few are in Clinical Development
The GPCR Functional Challenge
Innovative Targeting Solutions Inc
1. Soluble antigen
Problem Solution
3. Challenging epitope (rare specificities)
2. Tolerance/immunogenicity
Use full length functional receptor in its native confirmation on the cell surface
In vitro system required
Large CDR3 diversity
4. Potency Maturation* De novo targeted diversity to CDRs 100s of millions of variants
Ultra-high throughput functional screens* Primary binders will not have the potency required for a drug development
De Novo Protein Engineering
1. Large repertoires (>1 billion unique CDR3s)
2. Single cell functional screens (>1 billion cells)
V(D)J Recombination
V segments D segments J segments
V(D)J Recombination via the proteins RAG1, RAG2 and TdT
*Imprecise joining of segments adds to diversity
*
Innovative Targeting Solutions Inc
Nature’s most powerful diversification system
Each cell expresses a unique antibody
Pre-recombination Engineered HEK293 based HuTARG™ cell
De Novo Protein Engineering: Expand, Induce, Screen
Scale up >109 cells
Tetracycline Induction of V(D)J recombination
No cloning of librariesNo transformation of libraries
Functional assays at the single cell level
A billion cells screened for function in a day
De novo engineering: the cell does all the work
Innovative Targeting Solutions Inc
High Homogeneous Antibody Expression with HuTARG™
7
• Very high surface expression allows efficient selection of antigen-specific cells
• Libraries consist of all human V, (D) and J segments
• Antibodies expressed as full-length IgG (no reformatting)
• Complex screens can be built into the cell line for functional screening up front (receptor neutralization, species cross-reactivity)
Innovative Targeting Solutions Inc
Identical positions: 75Identity: 44%
XX---XXXXXXXXXXXX--XXXX--------XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX------** ::*. *** *.* * *.* *: : .** .
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX::*:* * * . . * : .******* . **:**** : :*:**.******:*:
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX* ********* **:**: * **:***:**:********::***
Signal peptide
Rare Specificities:Generation of Fully Human Antibodies to Two Proteins with Minimal Homology
Innovative Targeting Solutions Inc
Dr. Song TanPenn State
Ultra-rare Specificities:Targeting MHC-Peptide Complexes
• Specificity determined by a limited number of amino acids• The majority of the epitopes represent the MHC heterodimer
Innovative Targeting Solutions Inc
Innovative Targeting Solutions Inc.
De Novo Engineering: The Cell does all the Work
1. Large repertoires (>1 billion unique CDR3s)
2. Single cell functional screens (>1 billion cells)
Innovative Targeting Solutions Inc
Functional GPCR Agonists Directly from De Novo Libraries
Cells expressing an agonist antibody will activate the reporter allowing isolation of the cell with the agonist
Cell expressing non-agonist antibody will not activate cell surface reporter marker
Un-recombined cell line Integration GPCR and cAMP reporter gene Induction of recombination to generate > 10^9 diversity
Innovative Targeting Solutions Inc
Library Screen to Identify GPCR Agonists
• Cells producing agonist peptide grafted antibodies are visible as reporter positive cells following magnetic bead enrichment
• Isolation of reporter positive cells identified fully human antibodies that activate the GLP1 receptor
Antibody Expression
Rep
orte
r Act
ivat
ion
Agonists
Negative Control Library
Peptide Graft Library #1 Peptide Graft Library #2
Characterization of Agonist Antibody to the GLP-1 Receptor
0
2
4
6
8
10
12
14
GLP-1R negative GLP-1R positive
Cell line
ITS007-V129 (inactive mutant)
ITS007-V125
0
25
50
75
100
125
150
175
0%
5%
10%
15%
20%
0.001 0.1 10
Bind
ing
(AU
)
cAM
P (p
mol
/ml)
Cells
Ex
pres
sing
Su
rfac
e R
epor
ter
1. Agonist activity of antibody is GLP-1 Receptor dependent2. Agonist activity correlates with binding to GLP-1 receptor in a dose
dependent manner
ITS007-V125 (ug/ml)
Innovative Targeting Solutions Inc
GLP-1R and GCGR Agonist Assay0.5ug/ml agonist
X axis = IgG (binding)Y axis = Cell Surface Reporter
Glucagon-Fc Dulaglutide ITS007-V129 ITS007-V130 ITS007-V222
GLP1-R agonists specifically activate the GLP-1 receptor and not the Glucagon receptor
Glu
cago
n Re
cept
orG
LP-1
Rec
epto
r
Specificity of Agonists for the GLP1 Receptor
Innovative Targeting Solutions Inc
3.0 x 10^6 cells per ml
300 ml = approx. 1 billion cells = 1 day of FACS sorting 3000 ml = approx. 10 billion cells = 10 days of FACS sorting
Innovative Targeting Solutions Inc
Scalable
Innovative Targeting Solutions Inc
Functional Activation of a Drug Selectable Marker
Cells expressing an agonist antibody will activate the reporter allowing selection with puromycin
Cell expressing non-agonist antibody will not express the gene for puromycin resistance
Un-recombined cell line Integration GPCR and cAMP reporter gene Induction of recombination to generate > 10^9 diversity
Puromycin
Functional Selection Utilizing a Puromycin Resistance Marker
• Puromycin selection greatly improves the ease of library enrichment
• Repertoires can be processed at a much larger scale
Add Puromycin to Culture
Cell alone Cell with ligand Cell with agonist antibody
Puromycin ResistanceMinimal Promoter
TF binding site
Y
Y Y
YInactive Receptor
No TranscriptionSensitive to Puromycin
Minimal Promoter
TF binding site
Activated ReceptorY Y
Y
Y
Puromycin Resistance
Minimal Promoter
TF binding site
Activated Receptor
Puromycin Resistance
Active Transcription Resistant to Puromycin
Active Transcription Resistant to Puromycin
Innovative Targeting Solutions Inc
Y Antibody
GPCRTranscription activeTranscription inactive
Day 1
Day 3
Agonist AntibodyNo Agonist
Puromycin sensitive cells do not adhere
Puromycin resistant cells expand and pack in the plate
Puromycin Selection of Agonist Antibodies
Day 0: Cells trypsinized and plated 1:3 with or without agonist antibody (100ng/ml) and Puromycin 4ug/ml
Innovative Targeting Solutions Inc
De Novo In Situ Mutagenesis
Potency Maturation
Innovative Targeting Solutions Inc
GATCGATCttt cGATCGATCCTAGCTAGaaa gCTAGCTAG
Deletions
GATCXXXX XXXXXXTCCTAGXXXX XXXXXXAG
• Tdt recruits factors which have 3’5’ exonuclease activity to the RAG synapse• Increased Tdt levels results in increased deletions• There is a linkage between deletion and Tdt insertion (N nucleotide addition)
• Large deletion have correspondingly large N insertions• Small deletions have correspondingly smaller N insertions
• Reaction can be tuned so that the end result is that final length of the products are approximately equivalent to original size
Tdt: Primary MoA is Intimately Coupled to Deletion/Replacement
CDR1 Optimization Cassette Structure
RSS Insertion
QASQD ISNYLN
QASQDISNYLNExample: CDR1 Sequence
~90% will be composed of variants within 3AA each side of the junction diversified
12bp RSS 23bp RSS
Innovative Targeting Solutions Inc
QAXXXISNYLNQASXXISNYLNQASQDXXXYLNQAXQDIXXYLNQASXDISXYLNQASQXIXNYLN
Affinity Maturation of an Antibody Diversified at Light Chain CDR1 using RAG/TdT Mediated Recombination
Innovative Targeting Solutions Inc
• Light chain diversified at CDR1 using RAG/TdT
• Enrichment with antigen linked to magnetic beads
• Cells expressing antibodies of similar affinity cluster along diagonal lines
Antibody expression
Antig
en b
indi
ng
Antibody expression
Antig
en b
indi
ng
Red: Original antibodyBlue: Library of antibodies diversified at light chain CDR1 using RAG/TdT and then antigen enrichedGreen: High affinity variant isolated from library by flow cytometry
CDR Mutagenesis Design
CDR1 CDR3CDR2
FR1 FR2 FR3 JK
Constant
CDR1 CDR3CDR2
FR1 FR2 FR3 JK
Constant
CDR1 CDR3CDR2
FR1 FR2 FR3 JK
Constant
All 6 CDRs in parallel
A RSS cassette is placed every other amino acid Each variant antibody has amino acid changes in only 1 CDR Mutations from different CDRs can be combined Heavy chain and light chain CDRs can be combined
Innovative Targeting Solutions Inc
• Approximately 1 billion variants are screened for affinity and expression• 100 to1000 fold affinity improvement has been observed in a single round
HuTARG Clone 150pM High Affinity
15nM Parental
Light Chain Optimization
Heavy Chain Optimization
Bind
ing
to T
arge
t
Surface IgG ExpressionSurface IgG Expression
Targ
et B
indi
ng
Clone 1 Clone 2
Targ
et B
indi
ng
Clone 1 Clone 2
In Situ Mutagenesis/Maturation: Optimization of 6 CDRs in Parallel
Innovative Targeting Solutions Inc
CDR Optimization to Increase Potency Directly
TET
V(D)J Variants
Reporter GeneCRE
• Antibody under the control of a Tetracylcine induced promoter
• “Tune” promoter activity such that current lead does not activate reporter gene
• Generate a library of variants and sort out cells able to signal at this new potency threshold
Innovative Targeting Solutions Inc
Innovative Targeting Solutions Inc
Functional GPCR Agonists Directly from De Novo Libraries
Cells expressing an agonist antibody will activate the reporter allowing isolation of the cell with the agonist
Cell expressing non-agonist antibody will not activate cell surface reporter marker
Un-recombined cell line Integration GPCR and cAMP reporter gene Induction of recombination to generate > 10^9 diversity
Scale up >109 cells
Tetracycline Induction of V(D)J recombination
Each cell expresses a unique antibody
Pre-recombination Engineered HEK293 based HuTARG™ cell
De Novo Protein Engineering: Expand, Induce, Screen
No cloning of librariesNo transformation of libraries
Functional assays at the single cell level
A billion cells screened for function in a day
De novo engineering: the cell does all the work
Innovative Targeting Solutions Inc
Direct Potency Enrichments
Post - Sort 1 Post - Sort 2 Post - Sort 3
cAMP Responsive Reporter Gene Expression
• After 3 rounds of sorting variants able to deliver an agonist signal at new threshold are clearly visible
• Initial sort was with >500 million variants
Innovative Targeting Solutions Inc
Potency of Assessment of Isolated Leads from CDR3 Potency Maturation
Innovative Targeting Solutions Inc
• Isolated clones from potency maturation libraries exhibit very large increases in activity
Traditional Screening Paradigm
Scale 384 well
Number screened 400K (1000 plates)
Isolate Antigen Specific Binders
Develop Functional
Assay
Screen for Function
Antibody reformatting/production
Innovative Targeting Solutions Inc
The HuTARG™ Way
Develop Reporter Based Functional
Assay WithinHuTARG Line
• Scale single-cell
• Ultra high-throughput (up to a billion variants)
• Ability to directly mature based on function
• Ability to screen for increased potency
INDUCE
EXPAND
SORT
Innovative Targeting Solutions Inc
HuTARG™:Generation and Engineering of Future Biologics
Fully Human mAbs
Single Domains (HCAb), Bites, Darts
Single Chain mAbs(CARs)
Peptide-grafted mAbs
Alternative Scaffolds(Adnectins, Darpins,
Avimers, etc)
Fully Human TCRs
Affinity MaturationPotency Maturation
Innovative Targeting Solutions Inc
Function First Screening
GPCRs, Ion Channels
Innovative Targeting Solutions Inc
Acknowledgements
Paul KangCraig PigottAbby LinFalene ChaiChristine YaoZenlynn TsangMarie-Christine PerryKatie Baillie