Wei Ren, Ph.D. Sino Biological Inc.

Monoclonal Antibody Generation - Technology Platforms & Case Studies

Hybridoma Technology

Immunized Phage Display Antibody Library

Human Naive Antibody Library

Single B-cell Antibody Technology

Contents

Hybridoma Technology

1975 1985 1986 1993 1997 2002 2009 2011 2014

Phage Display

1st FDA Approved Mouse MAb

Chimeric Antibody

2000

Humanized Antibody

1st FDA Approved ADC

1st FDA Approved Full-human MAb

1st FDA Approved Bispecific Antibody

Immune Checkpoint Antibody

Anti-PD-1 Antibody

Orthoclone OKT3 Muromonab

Reopro Abciximab

Zenanapx Daclizumab

Mylotarg Gemtuzumab Ozogamicin

Humira Adalimumab

Removab Catumaxomab

Yervoy Ipilimumab

Opdivo Nivolumab

A Brief History of Therapeutic MAbs

REOPRO (1993) ADCETRIS (2011)

RITUXAN (1997) SYLVANT (2014)

SIMULECT (1998) UNITUXIN (2016)

REMICADE (1998) ANTHIM (2016)

ERBITUX (2004)

SYNAGIS (1998) EMPLICITI (2015)

HERCEPTIN (1998) PRAXBIND (2015)

MYLOTARG (2000) NUCALA (2015)

CAMPATH (2001) TALTZ (2016)

XOLAIR (2003) CINQAIR (2016)

AVASTIN (2004) TECENTRIQ (2016)

TYSABRI (2004) ZINBRYTA (2016)

ACTEMRA (2005) OCREVUS (2017)

LUCENTIS (2006) BESPONSA (2017)

SOLIRIS (2007) FASENRA (2017)

CIMIZA (2008) HEMLIBRA (2017)

THERACIM (2008) AJOVY (2018)

PERJETA (2012) EMGALITY (2018)

GAZYVA (2013) CABLIVI (2018)

KADCYLA (2013) POTELIGEO (2018)

ALZUMAB (2013) TROGARZO (2018)

KEYTRUDA (2014) ILUMYA (2018)

ENTYVIO (2014) ULTOMIRIS (2018)

LEMTRADA (2014)

HUMIRA (2002) PORTRAZZA (2015)

VECTIBIX (2006) COSENTYX (2015)

ILARIS (2009) LARTRUVO (2016)

SIMPONI (2009) ZINPLAVA (2016)

ARZERRA (2009) SILIQ (2017)

STELARA (2009) BAVENCIO (2017)

XGEVA (2010) DUPIXENT (2017)

BENLYSTA (2011) IMFINZI (2017)

YERVOY (2011) KEVZARA (2017)

ABTHRAX (2012) TREMFYA (2017)

OPDIVO (2014) AIMOVIG (2018)

CYRAMZA (2014) CRYSVITA (2018)

PRALUENT (2015) TAKHZYRO (2018)

DARZALEX (2015) LIBTAYO (2018)

REPATHA (2015) GAMIFANT (2018)

5.6%

9.11%

37.46%

30.37%

鼠源 嵌合 人源化 全人源

Landscape of Current Therapeutic MAbs

Mouse Chimeric Humanized Human MAbs withdrawn from the market are not included

OKT3 (1992)

ROSTASCINT (1996)

ZEVALIN (2002)

BLINCYTO (2014)

LUMOXITI (2018)

Technologies for Antibody Discovery

1st Generation: Mouse-derived MAb

2nd Generation: Chimeric & Humanized MAb

3rd Generation: Full-human MAb

4th Generation: Natural Full-human MAb

Hybridoma Technology

Phage Display

Human Naive Antibody Library

Single B-cell Technology

Hybridoma Technology

1

Immunization Cell Fusion

Clone Screening Antibody Production

Steps in Hybridoma Generation

· Choose an Immunogen Based on

Experimental Design

· Choose the Best Immunization

Strategy

· PEG Fusion

· Electro-fusion

· ELISA, FAC, IHC, Cell-based Assays, etc. · By Mice Ascitic

· By Cell Culture

Type Epitope Advantages Disadvantages

Protein Natural Conformational / Linear

Present on natural specimens Correct conformation

Difficult to purify Low yield

Recombinant Conformational / Linear High yield and large quantity Sequences close to natural proteins

May not have natural confirmation

Polypeptide Linear Easy to synthesis with high purity For PTM specific antibody generation Species specificity / Cross-reactivity

Low immunogenicity Unnatural conformation

DNA Conformational / Linear No need for protein purification In vivo protein expression Proteins with natural conformations

Low immunogenicity

Cells Conformational / Linear Correct protein conformation Epitope present on natural specimens

Complicated cell surface protein profile Low immunogenicity

Features of Immunogens

Case Study

Cell Natural Cells

Cells Over-expressing Targets

Target Plasmid

Helper Plasmid

Insect Virus

Insect Cells

Commercial CD56 antibody CD56 antibody generated via viral immunization

CD56

CD

3

CD56

CD

3

DNA

Virus

CD7 antibody generated via DNA immunization

Control

Commercial CD74 Antibody

CD

3

CD2 CD74

CD

19

CD2 and CD74 antibodies generated via cell immunization

Antibody Development Using Non-protein Immunogens

Immunization Strategies

Immunization routes

Adjuvant Immunization schedule

● Diverse immunization routes

Subcutaneous, abdominal, venous, intrasplenic, lymph nodes

● Flexible immunization schedules

Routine immunization, rapid immunization

● Various adjuvants

Freund's adjuvant, aluminum phosphate adjuvant,

aluminum hydroxide adjuvant, oil-in-water adjuvant

Case Study

Antibody Mouse MAb Rabbit PAb

Duration 20 weeks 8 weeks

Antibody Mouse MAb Rabbit PAb

Duration 13 weeks 6 weeks

Conventional Immunization Schedule

Rapid Immunization Schedule

Optimized immunization

cycle and site Significantly shortened turn-around time

without extra amount of immunogens.

Rapid Immunization

PEG-mediated Cell Fusion

Mechanism: PEG (polyethylene glycol) interferes with cell membrane structure and cause membrane rearrangement, during which adjacent cells merge with each other and result in cell fusion.

Key Factors for Fusion Efficiency ：

✓ Molecular weight of PEG

✓ Concentration

✓ Incubation time

✓ Temperature

Low Cost Easy to Operate

Low Fusion Efficiency Damages to Cells

Electro-fusion

Mechanism: Under an electric field alternating at high-frequencies, the cells are arranged into spherical shapes. Adjacent cells are positioned in a point-contact state and under the stimulation of an electric pulse, the cell plasma membranes are rearranged and fused.

Key Factors for Fusion Efficiency ：

✓Strength of the electric field

✓ Number of pulses

✓ Stimulation time

✓ Intervals

High Fusion Efficiency Little Damage to Cells

Expensive Instrumentations

Target PEG Fusion Electro-fusion

1 <20% >50%

2 <50% >50%

3 <80% >90%

4 <20% >50%

5 <60% 100%

Target PEG Fusion Electro-fusion

1 <100 8 folds

2 <100 2 folds

3 <150 3 folds

4 <100 equal

5 0 40

Same Cell Densities Same Spleen Cell Counts

Cell Viability Number of Positive Clones

PEG Fusion vs. Electro-fusion

Hybridoma Cell Fusion

1st-round ELISA Screening

1st-round Limiting Dilution

2nd-round ELISA Screening

2nd-round Limiting Dilution

3rd-round ELISA Screening

Amplification & Production

10-14 Days

Positive Clones

7-10 Days

Identify Positive Single Clones

7-10 Days

Confirm Clonality

Positive Clone Screening - ELISA

Positive Clone Screening - Advanced Screenings

Applicable to： Screen antibodies for specific applications such as IHC, WB, FC, IF

•Select mouse with highest titer by specific methods (e.g.: FAC, IHC, etc.)

Animal Immunization

•Positive clone screening by a combination of ELISA and specific methods

Clone Screening Phase

•Positive clone identification by a combination of ELISA and specific methods

Subcloning Phase

•Confirm antibody functionalities

Purified Antibody

Significant increase in success rate

Antibody Production

✓Ease of Operation

✓High Concentration and Titer

✓Low Purity Due to Mouse Protein Contaminants (including endogenous Ig)

✓ Prone to Contamination by Animal Viruses

✓ Issues Regarding Animal-welfare

By Cell Culture ✓ High Quality Antibody without Protein and Virus Contaminations

✓ Easy to Purify

✓ Adaptable for Large Scale Production

✓ Animal-free Production

By Mouse Ascites

Immunized Phage Display Antibody Library

2

T4 phage

T7 phage

Lambda phage

Structural Proteins

pIII

pVI

pVIII

pVII

pIX

33aa, 5 copies

32aa, 5 copies

50aa, 2700 copies

112aa, 5 copies

406aa, 5 copies

Non-structural Proteins

pII

pX

pV

pI

pIV

pXI

DNA replication, 410aa DNA replication, 111aa binding ssDNA, 87aa assembly, 348aa assembly, 405aa assembly, 108aa

pIII

pVI

pVIII

pVII pIX

ssDNA

Phage Display Systems

Life Cycle of Filamentous Phage

Line Ledsgaard-2018

A. Double-stranded Replicative

DNA (RFDNA) Formation

B. Incision

C. Replication to Produce

Template DNA

D. Generation of Progeny DNA

E. pV Coat Protein Expression

F. Phage Assembly

5’ 3’ AAAAAA mRNA cDNA TTTTTTT

VL

VH

Linker SfiI

SfiI

scFv

Immunization

RNA Extraction

Electro Transformation

Phage Packaging

scFv Display Library Phagemid

Light Chain and Heavy Chain Amplification

VL VH scFv

Library Construction

scFv Assembly

Specificity

Round1 Round2 Round3

Binding

Washing

Elution

Amplification

Timeline

Day1: Grow library Day2: Collect phage Day3: Round 1 Day4: Regrow Day5: Collect phage Day6: Round 2 Day7: Regrow Day8: Collect phage Day9: Round 3

Round 4 ~ Round 6

ELISA

Panning

Carol M Y Lee-2007

Beads

SA Bio

Liquid-phase Panning Cell-based Panning

Cells

Solid-phase Panning

Panning Methods (I)

Subtraction Panning Sandwich Panning

Cross Panning Competitive Panning

Panning Methods (II)

Selection of ELISA plates Antigen and antibody coating conditions

Proper blocking agent and blocking concentration

Dilution gradient optimization

Concentration of the enzyme-labelled antibody

Use enhanced TMB coloring solution

ELISA Screening

Optimizations

Single Clone Identification and Optimization

Tips for Phage Library Construction and Panning

Ⅱ

Sequence Diversity

Ⅲ

Panning Strategies

• Primer Options ✓ FR1 and FR4 conserved sequences ✓ Familial conserved sequences of antibody germline genes ✓ 5' end primer: leader sequence; 3' end primer: J fragment • PCR conditions ✓ Reaction cycles ✓ Annealing temperature

✓ Quantity of coating antigen ✓ Rounds of washing steps ✓ Temperature ✓ Incubation time ✓ pH

• Transformation ✓ RNA quantity ✓ Ligation ratio ✓ Helper phage

• Optimization ✓ Vector reconstruction ✓ Helper phage reconstruction

Library Capacity

Ⅰ

Low Immunogen Concentration ① Increase coating temperature, 4℃ 37℃; ② Increase panning capacity

Panning Method Optimization Switch from solid-phase panning to liquid-phase panning

Low Serum Titer ① Use more coating antigen; ② Increase panning capacity

Small Molecule and Peptide Coating Use plates with better binding capacity

Case Study

Coating: Antigen Control Antigen-

1

Control Antigen-2

lib dilution OD450 OD450 OD450

scFV-4th

1000x 0.083 0.069 0.079 100x 0.21 0.073 0.18 10x 1.224 0.124 1.178 1x 0.272 0.099 0.447

Coating: Antigen Control Antigen-

1

Control Antigen-2

lib dilution OD450 OD450 OD450

scFV-4th

1000x 0.659 0.136 0.084 100x 2.79 0.129 0.091 10x 3.472 0.163 1.152 1x 3.37 0.397 0.578

Coating: Antigen Control Antigen-

1

Control Antigen-2

lib dilution OD450 OD450 OD450

scFV-2nd

1000x 0.083 0.062 0.081 100x 0.077 0.078 0.119 10x 0.194 0.211 0.372 1x 0.091 0.049 0.110

Coating: Antigen Control Antigen-

1

Control Antigen-2

lib dilution OD450 OD450 OD450

scFV-2nd

(liquid)

1000x 0.897 0.233 0.060 100x 0.884 0.273 0.078 10x 1.727 0.407 0.283 1x 0.546 0.258 0.089

Coating: Antigen Control Antigen-

1

Control Antigen-2

lib dilution OD450 OD450 OD450

scFV-2nd

1000x 0.582 0.295 0.148 100x 0.706 0.326 0.156 10x 0.951 0.668 0.153 1x 0.764 0.266 0.455

Coating: Antigen Control Antigen-

1

Control Antigen-2

lib dilution OD450 OD450 OD450

scFV-2nd

1000x 0.262 0.146 0.156 100x 1.122 0.112 0.148 10x 3.089 0.105 0.153 1x 1.56 0.317 0.455

Coating: Antigen Control Antigen

lib dilution OD450 OD450

scFV-3rd

1000x 0.511 0.395 100x 0.543 0.437 10x 0.83 0.664 1x 0.400 0.260

Coating: Antigen Control Antigen

lib dilution OD450 OD450

scFV-4th

1000x 0.225 0.157 100x 1.364 0.100 10x 2.548 0.198 1x 0.129 0.123

Panning and Screening

Anti-AGT RAb

SKBR3 HepG2

Rabbit MAb Developed by Phage Display Antibody Library

Anti-E-Cad RAb

Colon cancer Liver Skin

Anti-CD71 RAb

Sino Biological Competitor 1 Competitor 2

Features of Rabbit MAb (RAb) High affinity and sensitivity High specificity and low background Applicable for IHC, IF, ELISA, FAC, WB, IP… Ideal for mAb generation against mouse antigens

Human Naive Antibody Library

3

*Adalimumab(CAT) Belimumab(CAT) Raxibacumab(CAT)

Ramucirumab(Dyax) Necitumumab(Dyax) Avelumab(Dyax)

Ranibizumab Durvalumab

Human Antibody Library

Company Library Type Size

Morphosys HuCAL GOLD Fab 1.6 x 1010

HuCAL PLATINUM Fab 4.5 x 1010

Dyax Dyax Library Fab 1.0 x 1010

Cambridge Antibody Technology

CAT 1.0 scFv 1.4 x 1010

CAT 2.0 scFv 1.29 x 1011

*26.7%

73.3%

FDA-approved Fully Human Antibody Drugs

噬菌体展示

转基因小鼠

Phage Display

Transgenic Mice

2.8 8.52 14. 20. 30.

45. 54.89

64.58 79.79

92.65 106.59

125.43 140.12

160.78

184.27 199.

2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018

2003-2018 Global Sales ($/Billion) Antigen Clone Kon M-1S-1 Koff S-1 Kd

TNFα D2E7 4.7x105 4.8x10-5 1.0x10-10M

Adalimumab Fab TNFα

Fully Human Antibody-adalimumab

Library Construction via Cre/loxP Mediated Recombination

Wild-type strain

After recombination Daniele Sblattero and Andrew Bradbury-2000

No recombination

Recombinant strain

Library Construction via Chain Exchange between LoxP Sites

pLac pelB g3p

SfiI NheI XbaI NotI

loxP511 loxPwt (His)6

E-tag

pLac pelB g3p

SfiI NheI XbaI NotI

loxP511 loxPwt (His)6

E-tag

VL

VL

VH

VL

VL

VH

VH

H H H H

L L L L

mouse VH mouse VL

H H H H

L L L L

L L L L

L L L L

L L L L

mouse VH human VL repertoire

H H H H

H H H H

H H H H

H H H H L

L L L

human VH repertoire mouse VL

H H H H

L L L L mouse VH human VL

H H H H

L L L L human VH mouse VL

L L L L

H H H H

human VL human VH

Guided Selection Strategies

Human Naive Antibody Library

Company Type Size

Sino Biological scFv 1.56 x 1011

Diversity

VL: 1.25E+06/1.71E+06, 73.1%

VH: 1.23E+06/1.61E+06, 76.4% (NGS)

VH-CDR3 Length Distribution

Neutralization Antibody Derived from Human Antibody Library

Conc.(μg/ml) Conc.(μg/ml)

H3N2 clone049-IgG1 H3N2 clone287-IgG1 H3N2 clone322-IgG1 H3N2 clone388-IgG1

Inhi

bitio

n ra

tio

H1N1 clone017-IgG1 H1N1 clone033-IgG1 H1N1 clone087-IgG1 H1N1 clone142-IgG1

Inhi

bitio

n ra

tio

High-Affinity Neutralization Antibody

KD (M) Kon (1/Ms) Kdis (1/s)

5.39E-9 7.65E+05 4.12E-03

KD (M) Kon (1/Ms) Kdis (1/s)

3.65E-9 8.88E+05 3.24E-03

KD (M) Kon (1/Ms) Kdis (1/s)

4.25E-8 4.82E+05 2.05E-03

KD (M) Kon (1/Ms) Kdis (1/s)

3.32E-9 9.10E+05 3.03E-03

KD (M) Kon (1/Ms) Kdis (1/s)

9.04E-9 1.36E+05 1.23E-03

KD (M) Kon (1/Ms) Kdis (1/s)

9.12E-9 1.59E+05 1.45E-03

KD (M) Kon (1/Ms) Kdis (1/s)

8.06E-9 1.60E+05 1.29E-03

KD (M) Kon (1/Ms) Kdis (1/s)

1.38E-9 1.47E+05 2.03E-04

Features of Human Naive Antibody Library

Short turn-around time

Versatile antigen formats

Wide range of epitopes

Comprehensive methods

Full human source

No immunization required

Suitable for a variety of antigens, especially ones that elicit relatively weak immune responses

With minimum influence from the extent of immune responses

Comprehensive method sets for library construction and panning

100% human sequence with no need for humanization

Single B-cell Antibody Technology

4

VDJH VJL

Pro-B-Cell Pre-B-Cell Immature B-Cell Mature B-Cell

Plasma cell

Memory B-cell

IgM

Igμ

IgL (K or λ) Igα

Igβ

BCR

Igμ

λ5 VpreB Igα

Igβ

Pre-BCR

IgM IgD

Secreted Ig, any isotype

Membrane Ig, any isotype

Bone marrow Peripheral compartments

B Cell Differentiation and Development

Immunization Splenocytes & PBMC Single B Cell Sorting mRNA Extraction

RT-PCR & Insert Vector Expression ELISA Validation

Workflow of Single B Cell Antibody Discovery

Mouse

IgG FSC Antigen

Antig

en

IgM

SSC

Rabbit

Single B Cell Sorting

CD19 IgG1 IgD Antigen Ig

M

IgG

1

Antig

en

Thomas Tiller-2009

Amplification of Heavy & Light Chain Variable Regions

0%

18%

35%

53%

70% Chain Pairing Success Rate

Up to 60% success rate, higher than reference rate

0%

25%

50%

75%

100% Positive Detection Rate in ELISA Assay

Up to 90%, exceeding the 40% market level

Case Study

Reference 1-PLOS ONE DOI:10.1371/journal.pone.0152282

Reference 2-Virology.2014 June; 458-459: 114–124.

Antibody Chain Pairing and Antigen Binding

Competition Assay

Positive Control: Therapeutic Antibody #2

Positive Control: Therapeutic Antibody #1

Cell-based Binding Assay TUBE NAME Mean: FL1-H

mh75-3Ae0-IgG1(o) 133

Positive control 106

Blank 7.78

Nor

mal

ized

To

Mod

e

Nor

mal

ized

To

Mod

e

TUBE NAME Mean: FL1-H mh17Ae0-IgG1(o) 117

Positive control 106

Blank 7.78

FL1-H FL1-H

Variable Region Gene Amplification

✓Ensure Rnase removal ✓ Lysis condition optimization

✓ Design effective reverse transcription primer

✓ Variable region amplification

Tips for Single B Cell Sorting and Amplification

Single Cell Sorting

✓ Enrich target cells before sorting.

✓ Adjustment of instrument compensation

✓ Perform assay to determine optimal antibody usage

✓ Add 7-AAD and other cell reactive dyes to remove interference from dead cells

✓ Properly reduce the sorting speed to ensure higher sorting efficiency

Methods Advantages Disadvantages

Hybridoma ・well-established processes ・a well-recognized method with low R&D costs ・High specificity

・Long turn-around time ・Genetic rearrangement resulting in non-functional light chains ・Require humanization of the Ab sequences

Phage Display

・Provide a direct linker between phenotype and genotype of the antibody ・Antibodies derived from a wide variety of B cell types ・Flexible and specific screening schemes available according to the experimental design and antibody applications. ・Antibody sequence information to avoid loss of clones ・Wide range of applications (epitope analysis, vaccine development, protein interaction analysis, peptide drugs, CAR-T…)

・Unnatural pairing of VL/VH with preference by the phage system ・Post-translational modification is restricted due to prokaryotic expression ・Only qualitative assays available during panning. Full-length antibody expression required for complicated activity assays.

Single Cell ・Natural pairing of VL/VH ・Antigen-specific B cells sorting ・High specificity, high affinity, rich genetic diversity…

・Fresh sample, such as PBMC, required for best sorting efficiency ・Antigen-specific cells exists at a low level that requires screening with high-quality antigens ・Strict operating environment

Summary

Monoclonal Antibody

Single Cell

Human Library

Phage Display

Antibody Discovery Platforms from Sino Biological Hybridoma

10,000+ Antibodies

6,000+ Proteins

800+ ELISA Kits

400,000+ Genes

Emerging Leader in Biological Reagents & Services

Largest Inventory Worldwide, 100% Made in House

• Recombinant Protein Expression Service

One-stop & 10+ years experience CRO Services

• Recombinant Antibody Production Service

Sino Biological – Accelerates Your Research

5 expression systems Preferred provider for top 10 Pharmas

marketing@sinobiological.com | www.sinobiological.com

Contact Us

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Tel: +86-400-890-9989

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