single cell functional assays: ultra- sensitive, ultra ... · michael gallo president nnoi vativet...

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

*


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


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)


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


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.

De Novo Engineering: The Cell does all the Work

1. Large repertoires (>1 billion unique CDR3s)

2. Single cell functional screens (>1 billion cells)


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


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)


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


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


Scalable


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


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


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


De Novo In Situ Mutagenesis

Potency Maturation


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


QAXXXISNYLNQASXXISNYLNQASQDXXXYLNQAXQDIXXYLNQASXDISXYLNQASQXIXNYLN

Affinity Maturation of an Antibody Diversified at Light Chain CDR1 using RAG/TdT Mediated Recombination


• 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


• 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


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



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


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


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


Potency of Assessment of Isolated Leads from CDR3 Potency Maturation


• 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


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


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


Function First Screening

GPCRs, Ion Channels


Acknowledgements

Paul KangCraig PigottAbby LinFalene ChaiChristine YaoZenlynn TsangMarie-Christine PerryKatie Baillie

single cell functional assays: ultra- sensitive, ultra ... · michael gallo president nnoi vativet...

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