Credible Leads to Incredible™

Fang Tian, PhD

Principal Scientist

Head of Cell biology R&D group

November 2020

Advanced drug-resistant cell models for cancer therapeutic resistance studies

2

About ATCC

Founded in 1925, ATCC is a non-profit organization with HQ in Manassas, VA, and an R&D and Services center in Gaithersburg, MD

World’s largest, most diverse biological materials and standards development organization. Information resource for microbes

Innovative R&D company featuring gene editing, microbiome, NGS, advanced models

Multiple accreditations, including ISO 9001 and ISO 13485, cGMP biorepository

Partner with government, industry, and academia

Leading global supplier of authenticated cell lines, viral and microbial standards

Global presence. Sales and distribution in 150 countries, 19 international distributors

Talented team of ~500 employees, over one-third with advanced degrees

About ATCC

3

Therapy resistance in cancer

Therapeutic resistance in cancer is multifactorial and heterogeneous.

Spatial and temporal resistance occurs in tumor cells, within the stroma, or in metastasis.

The underlying mechanisms of drug resistance are diverse and complex, often driven by

• Tumor heterogeneity • Genetic and epigenetic alternations • Drug transporters • Lineage plasticity• Adaptive signaling events• Tumor microenvironment (TME)

Wheel of Resistance

About ATCC

4

Overcoming resistance: new approaches

Applying evolutionary principles• Competitive release of drug-resistant subclone• Intermittent therapy: allow drug-sensitive cells to

outcompete drug-resistant subclones

Modulating the TME• Targeting the tumor vasculature, stroma, and

immune cells

Learning from exceptional responders• Extending exceptional responses to broader

patient populations

Many of the models used in the early stages of research don’t capture cancer’s mechanisms of resistance to therapeutics, which impede progress in drug development and clinical trials.

To overcome this roadblock, ATCC is committed to providing the advanced cell models to push the envelope in cancer research

ATCC drug resistant cell models

Resistant to hormonal therapies

Resistant to chemotherapy

drugs

• CPT • FUdR• doxorubicin • etoposide • dactinomycin • bleomycin • mitoxantrone • thioguanine

• Herceptin• Imatinib

Resistant to targeted therapies

• Tamoxifen

• BRAF inhibitors• MEK inhibitors

Resistant to targeted therapies

Long-term drug selection derived cell lines CRISPR engineered cell lines

5

About ATCC

6

Establish drug resistance cell model through gene editing

Drug resistance in melanoma Develop isogenic lines of drug resistance

Ras/Raf/MEK/ERK MAP kinase signaling regulates BRAF inhibitor drug resistance

RAS

Cell survival and proliferation

Wagle N, et al. J Clin Oncol 29(22): 3085-3096, 2011Week 1 Week 15 Week 23

BRAF inhibitor resistance in melanoma patientsAdvanced in vitro cell models that contain defined genetic drug resistance mechanisms are needed to facilitate the development of next-generation therapeutics that can overcome BRAF drug resistance in melanoma.

About ATCC

7

Characterization of A375 isogenic cell lines

Genome sequence

Transcript sequence

Protein expression

Off-target screening

Cell morphology

Cell growth kinetics

Drug response

Cell line stability

Cell line authentication

Sterility test

A375 isogenic lines for 2D drug screening

- 9 . 5 - 9 . 0 - 8 . 5 - 8 . 0 - 7 . 5 - 7 . 0 - 6 . 5 - 6 . 0 - 5 . 5

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

D a b r a f e n i b R e s i s t a n c e i n A 3 7 5

I s o g e n i c M e l a n o m a M o d e l s

l o g [ D a b r a f e n i b ] , M

Su

rv

iva

l r

ate

(%

)

K R A S M u t a n t A 3 7 5 I s o g e n i c

A 3 7 5

N R A S M u t a n t A 3 7 5 I s o g e n i c

M E K 1 M u t a n t A 3 7 5 I s o g e n i c

- 8 . 0 - 7 . 5 - 7 . 0 - 6 . 5 - 6 . 0 - 5 . 5

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

N o E t o p o s i d e R e s i s t a n c e i n A 3 7 5

I s o g e n i c M e l a n o m a M o d e l s

l o g [ E t o p o s i d e ] , M

Su

rviv

al

rate

(%

)

K R A S M u t a n t A 3 7 5 I s o g e n i c

A 3 7 5

M E K 1 M u t a n t A 3 7 5 I s o g e n i c

N R A S M u t a n t A 3 7 5 I s o g e n i c

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A375 isogenic lines for 3D culture drug screening

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About ATCC

10

MEK1 isogenic A375 lines for combination therapy study

2D c

ultu

re

3D c

ultu

re

About ATCC

11

KRASG13D A375 isogenic line highly expressing EGFR and PD-L1

KRASG13D A375

PD-L1

IFNγ +, Isotype Control

IFNγ -, Isotype Control

IFNγ -, PD-L1 staining

IFNγ +, PD-L1 staining

Unique features of KRAS isogenic A375 line

About ATCC

12

0

20

40

60

PD-L1 mAb concentration (ug/mL)

IFNγ

conc

entr

atio

n (p

g/m

L)

Activated CD8+ T cells + A375-KRAS Co-Culture

PD-L1 mAbIgG1 Isotype

1010.10.01

Isogenic A375 lines for IO and combination therapy study

• KRASG13D A375 Isogenic line harbors BRAF600E, KRASG13D

mutations, highly expresses EGFR and IO checkpoint PD-L1

• Together with it’s parental cell line, KRASG13D A375 Isogenic line can be an ideal drug resistant melanoma model for the studies of commination therapy using BRAF inhibitor and IO checkpoint blockade

About ATCC

13

Drug-resistant isogenic A375 lines for in vivo studyGeneration of Luciferase Expressing isogenic A375 Cell Line

Expansion and validation of single clones

Pool evaluation and selectionViral transductionLenti-Luc2 plasmid

80 400

2,00

0

10,0

00

0

1×109

2×109

3×109

Tota

l Flu

x (p

/s)

010

0020

0030

0040

0050

0060

0070

0080

0090

0010

0000

5×104

1×105

1.5×105

2×105

2.5×105

Cell Bioluminescence

Cell Number (cells/well)

Lum

ines

cenc

e (R

LU)

R2 = 0.9997

In vitro Confirmation of Luciferase Expression and Stability

0 10 20 30 400

1×108

2×108

3×108

4×108

5×108

KRAS mutant A-375 Isogenic-Luc2 radiance vs time

Days Post Injection

Rad

ianc

e (p

/s/c

m²/s

r)

KRASG13D A375 Isogenic-Luc2 Tumor Model and in vivo Bioluminescence Imaging

7.0E5

9.3E3

Cou

nts

Day: 3 7 13 21 28

0 1 21×100

1×101

1×102

1×103

1×104

1×105

1×106

KRAS mutant-A375 Isogenic-Luc2

Lum

ines

ence

(RLU

) Blasticidin -Blasticidin +

freezethaw

(months)

Summary

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Cell-based models are critical tools for understanding the mechanisms of drug resistance and developing novel therapeutics.

ATCC has been developing state-of-the-art drug-resistant cancer cell models by using CRISPR-Cas9 gene-editing technology to introduce critical mutations into disease-relevant cell lines.

These novel cell lines have several advantages including cell line homogeneity, stability of the relevant genotype, do not require for continued drug pressure to maintain the cell line, and modeling the acquired resistance to newly developed therapeutics.

Credible Leads to InCredible™

Acknowledgements:

Dedicated R&D and Commercial Teams at ATCC