Antibody Discovery and Bispecific Design: Development ... › › resource › ... · Hybridoma • High affinity antibodies specific to Ag –Cross-reactivity to host Ag difficult!

Antibody Discovery and Bispecific Design:

Development Considerations

Aaron K. Sato, Ph.D.

LakePharma, CSO

My First Chemistry Set

The Chemistry Set

In 1914, American chemist John J. Porter produced the first

line of chemistry sets, “Chemcraft.”

Overview

• LakePharma and the Antibody Center

• Antibody Discovery

• Developability

• Bispecific Design and Testing

• Brief Overview of Expression Systems

Company mission is to provide industry-leading protein

engineering and biomanufacturing services

▪ Biologics company focusing on providing integrated solutions

▪ Founded in 2009 and based in San Francisco Bay Area

▪ Named as an Inc. 5000 Fastest Growing Companies in 2015 and 2016

▪ Named on San Francisco Business Times’ list of 100 Fastest Growing

Private Companies in the Bay Area in 2016

▪ Acquired Blue Sky BioServices (Worcester, MA) in March 2016

▪ ~150 employees, 30% have PhDs

▪ LakePharma is the largest US-based biologics CRO, with most complete

capabilities

LakePharma

Who is LakePharma?

Discovery Development ManufacturingEngineering

LakePharma is a biotech company that enables and supports the discovery and development of the biologics of tomorrow

LakePharma’s Integrated Solutions for

Drug Development and Manufacturing

▪ Our technical capabilities cover the entire spectrum of biologics development and analytics

▪ We can take drug program from idea to lead to stable cell line to a formulated protein

LakePharma Antibody Center

Stable Cell Line

Development

Cell-based

Binding Assays

Functional

Assays

Antibody

DiscoveryAntibody

EngineeringCell Biology

Protein &

Analytical Science

(in vitro focus)

Phage Display

Ribosome Display

DNA Cloning

HT Screening

HT Ab fragment

expression/purific

ation (E. coli)

Affinity Maturation

(in vivo focus)

Hybridoma

Campaign

Assay

Development to

Support

Hybridoma

Tech Transfer

Single B-cell

Technologies

▪ Focus on antibody

▪ No downstream development

BioExpression

Assay & Analytics

Bispecific

Antibody

Developability

Assays

Humanization

Immunogenicity

Assessment (in

silico)

High Throughput

Expression

Hybridoma

Sequencing

Recombinant

Antibody

Production

DNA cloning

DNA scale Up

Multiple shots on goal for antibody

discovery and engineering …

Antibody Structure

Human and humanized antibodies

Abgenix, Medarex, PDL and othersSpecialized Antibodies

Fundamental Breakthroughs in

Therapeutic Antibodies

0

5

10

15

20

25

30

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86

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90

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92

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94

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96

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98

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FDA-approved therapeutic antibodies

Human or humanized mAbs

$37.9B (2012 U.S. sales)

Murine or

chimeric mAbs

New state-of-

the-art for ADCs:

homogeneity,

site-specific

conjugation

ProteinSAR™

bispecifics: optimal

structures for

superior drug-like

properties

Source of Antigen

• Tagged Antigen

– Fc Fusions

– His Tag

– BirA Tag Enzymatic Biotinylation

• Cell-based Antigen

– Stable CHO line over-expressing antigen

– Tumor cell line that expresses antigen

– Primary cells/tissues expressing antigen

Linker-

warhead

Source of Antibodies and

Integration

Engineered

Antibody

Ribosome Display

Phage Display

Cell-Based

Display

B-Cell Based Technologies

Antibody

Engineering

Bispecifics ADCsnnAA

Many

Scaffolds

Phage Display

>109 different antibodies

Library Types

Naïve Immune

Lower affinity to human antigen?

Synthetic

Requires careful design

Higher affinity and broader

epitope space?

Ag Specific Immune

Focused effort

Developability?

Phage Display Selection

Ribosome Display

How & what?

mRNA

Ribosome

Antigen-biotinAntibody

+ Ag No AgCDR H3

Round 1

Round 2

Round 3

Round 4

Streptavidin

capture

PCR amplify Screen output

Affinity Maturation

Naïve Libraries

(Synthetic)

Limited to

recombinant

protein selections

IVTT

Ribosome Display

A Tool for Lead Optimization

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

0 500 1000 1500 2000

KD

(n

M)

Recovered protein (ug/mL)

Parent

New lead

Rd1

Rd2

Rd3

+Ag -Ag

Parent

Improved clones

Cell Binding ELISAKD vs protein recovered

Mouse Hybridoma

(Köhler/Milstein, 1975)

Nobel Prize, 1984

http://en.wikipedia.org/wiki/File:Hybridoma_technology.png

Hybridoma

• High affinity antibodies

specific to Ag

– Cross-reactivity to host

Ag difficult!

• Multiple immunization

approaches

– Recombinant protein

– Cells

– DNA

• Source of diversity

– Mouse, Rat, Rabbit,

Chicken, …

– Humanized equivalents

• Requires humanization

• Good precedence for

developability

– The “in vivo filter”

The Process of Humanization

Example: An Approach to Humanization

MuHC HC-1 HC-2 HC-3 HC-4

MuLC 1 2 3 4 5

LC-1 6 7 8 9 10

LC-2 11 12 13 14 15

LC-3 16 17 18 19 20

LC4 21 22 23 24 25

• DNA Synthesis

– 4 HC and 4 LC grafts

with murine HC/LC

controls

• Express Antibodies

• 5x5 matrix = 25 IgGs

• Test for: (vs. muParent)

– Assembly/Titer

– Affinity: SPR

– Thermal Stability

– FACS Binding

– Functional Activity

Immortalized Human B-cell Technologies

• After immortalized

through various methods,

screening workflow

similar to hybridoma

• Limited exposure to this

technology

• Pro: Human Antibody

• Con: Immunization?

Cell-based Display Technologies

Bacterial

Advantages over phage display?

Yeast

FACS driven selections

Affinity maturation

Mammalian

More difficult, but worth it

Eukaryotic Processing Machinery

Like the “in vivo filter”?

Developability Assessment

In silico DNA Sequence check and Protein Sequence

Hot Spot Analysis, followed by Mass Spec verification

✓ DNA codon preference

✓ Protein sequence hot spot analysis

Productivity Readiness Check

✓ Transient production in HEK293 or CHO system

Integrity and Stability Check (to demonstrate whether

the antibody is stable biochemically)

✓ Intact Mass/Peptide Mapping/ PTM by Mass

Spec

✓ DSF/DLS – Thermostability assessment

✓ Aggregation/Fragmentation/PTM and Glycan

profiling over 2 weeks incubation under stressed

conditions

✓ SE-HPLC/LabChip CE-SDS

PK Readiness Check

✓ Poly-specificity ELISA, Surface hydrophobicity

assay – Specificity requirement

P o ly -S p e c if ic ity E L IS A

OD

45

0

BsA

b

Ref

1

Ref

2

Ref

3

Ref

4P

C

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 5 0 u g /m L

5 0 u g /m L

1 7 u g /m L

2 n d A b o n ly

HPLC

DSF/SLS

DLS

CE-SDS

mass23140 23160 23180 23200 23220 23240 23260 23280 23300 23320 23340 23360

%

0

100

PP9650_DG_R_20170426_SEC 399 (3.577) M1 [Ev-623677,It19] (Gs,0.800,1034:3697,0.20,L33,R33); Sb (15,2.00 ); Cm (395:399) 1: TOF MS ES+ 9.13e623206.4

23185.0

23264.6

23228.2 23245.8

mass48400 48450 48500 48550 48600 48650 48700 48750 48800 48850 48900 48950 49000 49050 49100 49150

%

0

100

PP9650_DG_R_20170426_SEC 345 (3.114) M1 [Ev-383616,It27] (Gs,0.800,1132:2662,0.50,L33,R33); Sb (15,2.00 ); Cm (342:347) 1: TOF MS ES+ 6.15e648600.0

48570.5

48783.5

48658.5

48630.0

48722.048691.0 48754.5

48843.0

48814.5

Heavy ChainLight ChainIntact Mass

Case Study: mAb PPXXXX Ready

to Move Forward

Sample ID %LMWC %(HC+LC) %MMWC %HMWC

mAb reduced N/A 99.3 0.7 N/A

mAb non-reduced 6.5 93.5 N/A N/A

✓– Purity

requirement

rCE>95%

✓– Purity

requirement

nrCE>90%


to Move Forward

3

min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

100

200

300

400

500

21

Peak # Peak Size

(kDa)

Peak Area %

Peak ID Notes

1 ~1500 1.4 Aggregate


3 ~150 96.3 Monomer

✓SE-HPLC (Mab-Pac1) – Monomeric state


to Move Forward

✓ Intact mass measurement – Integrity of the molecule

23639.1

36180.4

48508.9

WangS_i5615_PP6410red_Ldp800_34_01_15896.d: +MS, 12.3-14.2min, Smoothed (0.19,1,GA), Baseline subtracted(0.00), Deconvoluted (MaxEnt)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

4x10

Intens.

10000 20000 30000 40000 50000 60000 70000 80000 90000 m/z

23639.1

23794.4


0.0

0.5

1.0

1.5

2.0

2.5

3.0

4x10

Intens.

23400 23600 23800 24000 24200 m/z

48508.9

48636.9


0.0

0.5

1.0

1.5

2.0

4x10

Intens.

48400 48600 48800 m/z

Heavy

Chain

Heavy Chain Light Chain

Measured Mass 48636.90 Da 23794.40 Da

Predicted Mass 48634.90 Da 23794.53 Da

Delta Mass -2.00 Da 0.13 Da

Light Chain


to Move Forward

Peak # pI Peak Area % Peak ID Notes

1-2 --- 15.38 Acidic Peak Group3 8.28 65.28 Main Peak

4-5 --- 19.34 Basic Peak Group

✓ cIEF – Charge variants profiling


to Move Forward

✓DSC –

Thermostability

ANALYSIS

Peak # Tm (°C) ΔHcal (kJ/mol)

1 71.3 438

2 80.7 1487

3 82.1 808

✓DSF – Thermostability and

Aggregation onset

Tagg266: 70.7 °C

MethodLoading

Sample IDSample ID KD (M) kon(1/Ms) kdis(1/s) Full X^2 Full R^2

Octet Antigen PPXXXX 1.30E-11 1.60E+05 2.00E-06 0.0217 0.9998

✓Affinity measurement against Antigen in BLI or SPR – Affinity requirement


to Move Forward

Peak # Peak Size

(kDa)

Peak

Area %Peak ID


2 ~150 96.8 Monomer

3

Analysis

✓Poly-specificity ELISA, Surface hydrophobicity assay – Specificity requirement

5x uncoated

background

5x

uncoated

background

HEK293 WCL and BVP ELISA showed

consistent results, which serve as good

PK indicators.


to Move Forward

Countless Bispecifics Scaffolds

are Continually Being Developed

Speiss et al 2015 review

• Biology may

dictate the best

format to use

• Is there a one

size fits all

format?

• Ideally, it would

be good to have

a variety of

formats

available

An Example of Specialization

Bispecific T-cell Engager

Many Different Bispecifics are in

Clinical Trials

Kontermann et al 2015 review

Bispecifics by Activity

Dual Targeting

Act directly on multiple target

structures

Coupled with Delivery of X

Bi-Epitopics/Paratopics

Dual binding to non-overlapping target epitopes

Coupled with Delivery of X

X

X

Focus on Dual Targeting

Dual Targeting

Same Cell

Soluble Ligand & Cell Target

Different Cells

Bispecific T-cell Engagers (BiTEs)

Bispecific NK-cell Engagers

All others that do not recruit immune cells

My Bispecific Risk Scorecard

Evaluation Criteria

Criteria Grade

Cost of Goods (0-3, Low to High)

Ease of use (0-3, Easy to Hard)

Time to discovery (0-3, Short to Long)

“Does it look like an antibody?” (0, Yes; 2, No)

Potential for immunogenicity (0-3, Low to High)

Biophysical properties/stability (0-3, Stable to Unstable)

Advantage over cocktail of antibodies (0, Yes; 1, No)

Half-life extension (0, Yes; 3, No)

Total

Case Study:

Lower is

better!


Evaluation Criteria

Criteria Grade

Cost of Goods (0-3, Low to High) 1

Ease of use (0-3, Easy to Hard) 1

Time to discovery (0-3, Short to Long) 1

“Does it look like an antibody?” (0, Yes; 2, No) 2

Potential for immunogenicity (0-3, Low to High) 2

Biophysical properties/stability (0-3, Stable to Unstable) 0

Advantage over cocktail of antibodies (0, Yes; 1, No) 0

Half-life extension (0, Yes; 3, No) 0

Total 7

Case Study: IgG-scFv: EGFR x IGFR, IGFR (Dual Epitopes)Morrison-type Bispecific


Evaluation Criteria

Criteria Grade

Cost of Goods (0-3, Low to High) 1

Ease of use (0-3, Easy to Hard) 1

Time to discovery (0-3, Short to Long) 1

“Does it look like an antibody?” (0, Yes; 2, No) 2

Potential for immunogenicity (0-3, Low to High) 2

Biophysical properties/stability (0-3, Stable to Unstable) 0

Advantage over cocktail of antibodies (0, Yes; 1, No) 0

Half-life extension (0, Yes; 3, No) 0

Total 7

Case Study: FIT-Ig

Case 1: FIT-Ig (Fabs-In-Tandem Ig)

▪ Tetravalent bispecific molecule

▪ Symmetric

▪ Correct pairing of VH/VL

▪ Flexibility allowing dual binding

▪ Linker is optionalCH2CH3

CH2CH3

CH2 CH3VHB CH1VLA CL

VLB CL

VHA CH1

Heavy chain

Light chain

Short chain

N’-

N’-

N’-

-C’

-C’

-C’

Case 1: FIT-Ig Expresses Well Like IgG

FITIG (from HEK293) (NR)FITIG (from HEK293) (R)A generic IgG (NR)A generic IgG (R)

CH2 CH3VHB CH1VLA CL

VLB CL

VHA CH1

Heavy chain

Light chain

Short chain

N’-

N’-

N’-

-C’

-C’

-C’

▪ ProA purification

▪ Transient yield >300 mg/L

Case 1: SE-HPLC Profile Showed

High Monomeric Content

Molecular weight standardFIT-Ig

Case 1: Antigen Binding Arms

Function Independently

Loading Sample ID Sample ID KD (M) kon(1/Ms) kdis(1/s) Full X^2 Full R^2

FIT-Ig Antigen 1 8.7E-11 6.4E+05 5.6E-05 0.008 0.99

FIT-Ig Antigen 2 5.2E-10 2.5E+05 1.3E-04 0.006 0.99

Flow

BsAb

Flow

Antigen 1

Flow

Buffer

Flow

Antigen

2

▪ Binding of antigen 2 remains the same whether

antigen 1 is present or not

▪ Antigen 2 on rate:

– With Antigen 1: 2.4 E5

– Without Antigen 1: 2.5 E5

▪ FIT-Ig Retains Full Functions of Parental IgGs

Neutralization Potency IC50 (pM) mAb1 mAb2 FIT-Ig

Antigen 1 101 102

Antigen 2 50 54

Case 1: FIT-Ig Has Similar

Thermostability as IgG

FIT-Ig

IgG

Case 1: FIT-Ig Displays IgG-like PK

Profile

0.01

0.1

1

10

100

1000

0 7 14 21 28

Se

rum

co

nce

ntr

atio

n

(ug

/mL

)

Time (day)

FIT1-Ig SC

0.01

0.1

1

10

100

1000

0 7 14 21 28Seru

m c

oncentr

ation (

ug/m

L)

Time (day)

FIT1-Ig IVParameter Unit

ELISA method

Antigen 1 Capture

Antigen 2 Capture

Cl ml/day/kg 12.2 11.9

Vss ml/kg 131 126

t1/2 day 10.8 10.8

AUClast day*ug/ml 377 385

AUCINF day*ug/ml 411 419

MRTINF day 10.7 10.6

PK parameters Unit ELISA method

Antigen 1 Capture

Antigen 2 Capture

Tmax day 4.00 4.00

Cmax ug/mL 26.9 23.1

Terminal t1/2 day 10.95 10.40

AUClast day*ug/mL 336 289

AUCINF day*ug/mL 406 350

CL/F mL/day/kg 12.4 14.3

F % 103.7 86.4

Expression Systems

Contact Information

Aaron K. Sato, Ph.D.

LakePharma

CSO

[email protected]

P: 650-489-9125 Ext. 250

F: 888-635-3618

520 Harbor Blvd, Belmont, CA 94002

http://www.linkedin.com/in/aaronsato

Documents

Antibody Discovery and Bispecific Design: Development ... › › resource › ... · Hybridoma • High affinity antibodies specific to Ag –Cross-reactivity to host Ag difficult!