Utilizing Humanized NSG Mice to Evaluate Drug Efficacy...

Utilizing Humanized NSG Mice to Evaluate Drug Efficacy in Immuno-Oncology

Rick Huntress

Director Business Development

March 15, 2017

2

Onco-Hu™: Humanized Mice for Evaluation

of Immuno-Oncology Therapeutics

Characteristics of humanized

NSG™ and NSG™-SGM3 mice

PDX Tumor Growth

Mechanism of Action

Efficacy data

CD34+ Donor Variability

In Vivo Pharmacology Services | 2

http://jaxservices.jax.org/invivo/index.html

NSG™ vs. NSG™-SGM3 Mice

NOD scid gamma (NSG™)

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (005557)

The current gold standard for reconstitution of the human immune system

Excellent functional T cell responses

Limited human myeloid lineage development

NSG™-SGM3

NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (013062)

Promotes improved AML engraftment efficiency

Improves normal human myeloid cell development after HSC transplantation

Wunderlich M. et al. 2010. Leukemia 24(10):1785-8. PMID: 20686503

Billerbeck E. et al. 2011. Blood 117(11):3076-86. PMID: 21252091

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https://www.jax.org/strain/005557

https://www.jax.org/strain/013062

http://www.ncbi.nlm.nih.gov/pubmed/20686503

http://www.ncbi.nlm.nih.gov/pubmed/21252091

Experimental Timeline for Humanization

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Human cord blood derived CD34+ cells engrafted in female mice

Mature T cell

development

Whole body irradiation

Tail vein injection

3 weeks

4 weeks 12 weeks ~18 weeks

~18 weeks

NSG

NSG-SGM3

In Vivo Pharmacology Services |

Validation of

Engraftment by

FACS

16 weeks

B cells mature ahead of T cells

Myeloid cells peak at 15 weeks in

NSG-SGM3



Human Immune Cells in Peripheral Blood of Hu-

NSG™ vs. Hu-NSG™-SGM3: Absolute Counts

5

Total Human Donor (cells/µl)

HuCD33 Myeloid Cells (cells/µl)






6

HuCD19 B Cells (cells/µl)

HuCD3 T Cells (cells/µl)






7

HuCD8 Cytotoxic T Cells (cells/µl)

HuCD4 Helper T Cells (cells/µl)




8





PDX Tumor Growth

Mechanism of Action

Efficacy data

CD34+ Donor variability



Humanization of NSG™ Mice Has No

Significant Impact on PDX Growth Kinetics

9

No difference between NSG™ and

Hu-NSG™ mice on tumor growth curve

No HLA match testing performed

Fresh tumor tissue engraftment

100% take rate in NSG™ or Hu-NSG™ mice

HuCD45+ more than 20%

PDX Tumors Are Infiltrated With

Human Immune Cells

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HuCD45+ in BR0744 Tumor

Hu-N

SG

NSG

0.0

0.5

1.0

1.5

2.0

% o

f h

CD

45+

cells

No passenger leukocytes in PDX tumors

Tumors infiltrated with engrafted human cells

MDA-MB-231 Tumor Grows Slower in

Hu-NSG™-SGM3 Than in Hu-NSG™

11

12





PDX Tumor Growth

Mechanism of Action

Efficacy data




Hu-NSG™ MDA-MB-231 Mice: Efficacy

Requires the engrafted immune system

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MDA-MB-231 Tumor Response in Non-Humanized Mice

Hu-NSG™ MDA-MB-231 Mice: Suppression of

Breast Tumor Growth by Pembrolizumab

MDA-MB-231 in Hu-NSGTM Mice: Flow Data (T Cells) Tumor Infiltration Levels are Variable

16

DAPI CD8

Pembrolizumab Vehicle

Hu-NSG™ MDA-MB-231 Mice: Pembrolizumab Initiates CD8+ T Cell Infiltration

Hu-NSG™ MDA-MB-231 Mice: Efficacy Of

Pembrolizumab Is CD8+ T Cell Dependent

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MDA-MB-231 Tumor Response inHu-NSG Mice

0 10 20 300

200

400

600

800Vehicle (Q5DX6)+isotype (Q7DX5)

Pembrolizumab (Q5Dx6)+isotype (Q7DX5)

Pembrolizumab (Q5DX6)+anti-CD8 (Q7DX5)

Days

Tu

mo

r vo

lum

e(m

m3)+

SE

M

Hu-NSG™ MDA-MB-231 Mice: Characterization

of Human Tumor Infiltrating Cells & PD-1 Levels

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% PD-1 on HuCD45 Cells in Tumor

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Efficacy with a checkpoint inhibitor requires an

engrafted immune system

CD8+ T cell infiltration is increased with anti PD-1

CD8 Depletion blocks checkpoint efficacy

Drug is binding the intended target (PD-1)



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PDX Tumor Growth

Mechanism of Action

Efficacy data




21

Hu-NSG™-SGM3 MDA-MB-231 Mice:

Suppression of Breast Tumor Growth by

Pembrolizumab




MDA-MB-231 in Hu-SGM3 Mice: Flow Data (T Cells)

MDA-MB-231 in Hu-SGM3 Mice: Flow Data (PD-1)

24 In Vivo Pharmacology Services |

Hu-NSG™ BR1126 TNBC PDX Mice:

Pembrolizumab Inhibits Tumor Growth

HLA match

Tumor 1 2 3

BR1126 HLA-C, DPA1 HLA-A,DQA1, DPB1, DPA1 HLA-C, DPA1

CD34+HPC donor



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BR1126 in Hu-NSGTM Mice: Flow Data (T Cells) No Correlation of Human Immune Cell

Infiltration with Tumor Growth Rate Reduction

26

Hu-NSG™-SGM3 BR1126 TNBC PDX Mice:

Pembrolizumab & Doxorubicin Inhibit

Tumor Growth




BR1126 in Hu-SGM3 Mice: Flow Data (T Cells)

27

BR1126 in Hu-SGM3 Mice: Flow Data (PD-1)

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Evaluating the effects of PD-1 inhibitors on

MDA-MB-231 tumor model in Hu-NSGTM

• Nivolumab and Pembrolizumab – matched efficacy


Hu-NSG™-SGM3 LG1306 Lung PDX Mice:

Pembrolizumab & Ipilimumab Inhibit

Tumor Growth

HuCD45+ in whole blood: 36-81%

HuCD3+/HuCD45: average 14.3%

LG1306 PD-L1 surface expression: 89.1%



Hu-NSG™ MDA-MB-231 Mice: Suppression

of Breast Tumor Growth by Anti-OX40

31

0 5 1 0 1 5 2 0 2 5 3 0 3 5

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

T w o D o n o r s ( # 5 0 3 8 , # 5 0 4 0 )

S t u d y D a y s

Me

an

T

um

or

Vo

lum

e (

mm

3)

+/-

SE

M

G r o u p 1 ( V e h i c l e )

G r o u p 3 ( a n t i - O X 4 0 )

*

* p < 0 . 0 5

* * p < 0 . 0 1

* * * p < 0 . 0 0 5

* * * * p < 0 . 0 0 0 1

2 - t a i l e d u n p a i r e d t - t e s t

* *

*

* * *

*

Jean Gudas VP, Research & Development ImaginAb, Inc.

32

*p≤0.009 21MR ± PD1 vs Control, D11 **p≤0.0006 21MR ± PD1 vs Control, D19

Hu-NSG™-SGM3 OMP-LU121 NSCLC Mice:

Demcizumab (21MR) Inhibits Tumor Growth

Data aquired in collaboration with Chris

Murriel &Tim Hoey, OncoMed

Pharmaceuticals

OMP-LU121 NSCLC did not respond to

Pembrolizumab in Hu-NSG-SGM3 mice

Demcizumab (21MR) alone suppressed

progression of tumor growth

Anti-DLL4 Significantly Inhibits OMP-LU121 NSCLC AdC Growth in Hu-SGM3 Mice

34





PDX Tumor Growth

Mechanism of Action

Efficacy data




35

HLA match

Tumor CD34+ HPC Donor

LG1208 HLA-DRB4, DPA1

Hu-NSG™ LG1208 NSCLC Lung PDX Mice: No Inhibition of Tumor Growth By Pembrolizumab

36

PD-1 Expression: Activated T cells, Tregs, B cells, NK cells and monocytes;

occasionally on tumor cells

PD-L1 Expression: Mainly on tumor cells and normal tissues; also on T cell, B cells,

macrophages, DCs

Hu-NSG™ LG1208 NSCLC Lung PDX Mice: PD-1 and PD-L1 Expression in Tumor Tissue

Pembrolizumab entered tumors and bound to CD45+ PD-1+ leukocytes

Immuno-Oncology Summary

NSG™ and NSG™-SGM3 are a proven platform for

engraftment of the human immune system

PDX growth is not grossly effected by HLA-type matching

o Tumor growth kinetics are similar in humanized and non-humanized hosts

o ~15% of PDX tumors fail to grow in humanized mice

Human immune effector cells infiltrate human tumors in both

Hu-CD34-NSG™ and Hu-CD34-SGM3

o CD4 and CD8 T cells and CD19 B cells

Hu-CD34-NSG™ and Hu-CD34-SGM3™ PDX respond to anti-

tumor agents; anti-PD-1, anti-CTLA4, anti-OX40, and anti-DLL4




Acknowledgements

JAX – In Vivo Pharmacology Services

o James Keck, Minan Wang, Li-Chin Yao, Mingshan Cheng and Danying Cai

JAX – Genomic Medicine

o Karolina Palucka

JAX – Mammalian Genetics

o Lenny Shultz, Brian Soper, Carol Bult, Susie Airhart and Ed Liu

UMASS

o Dale Greiner and Mike Brehm

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In need of humanized

mice to advance your

research?

39

Contact JAX Technical Support

https://www.jax.org/jax-mice-and-services/customer-

support/technical-support

JAX® Mice, Clinical & Research Services

1-800-422-6423 • 1-207-288-5845

[email protected] • www.jax.org/jaxmice

12-16 wks

https://www.jax.org/jax-mice-and-services/customer-support/technical-support











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