Cutting Edge Human Disease Modeling Using The NSG Advanced and future disease modeling Additional information and resources JAX ... Cutting Edge Human Disease Modeling The NSG Mouse

Cutting Edge Human Disease Modeling Using The NSG Mouse

Dominique Kagele, Ph.D. Technical Information Services

Cutting Edge Human Disease Modeling The NSG Mouse

NSG Basics

Enhanced disease modeling

o Solid and hematological cancers o Human immunity and inflammation

Advanced and future disease modeling

Additional information and resources

JAX® Mice | 2


NSG Basics





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The Immune System in Mice

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NOD scid gamma (NSG)

Strain nomenclature: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ

Stock Number: 005557

Additional resources: NSG Web Resources

www.jax.org/jaxmice/research/immunology/005557.html

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NSG A Highly Immunodeficient Mouse NOD background

Absence of hemolytic complement Reduced dendritic and NK cell function Defective macrophages Optimal human hematopoietic stem cell engraftment (Sirpa allele)

scid mutation prevents development of mature T and B cells

gamma chain (Il2rg knockout) Eliminates signaling from 6 distinct interleukins and blocks NK cell development

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NSG A Highly Immunodeficient Mouse

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Gamma Chain Knockout (Il2rgtm1Wjl)

Complete deficiency of the interleukin 2 receptor, gamma chain

Common subunit found in multiple cytokine cell signaling receptors

IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 are key signaling molecules in function and maturation of T, B, NK, neutrophils and dendritic cells

Rochman Y et al. 2009. Nat Rev Immunol 9(7):480-90. PMID:19543225

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Longer lifespan (> 16 months) than NOD-scid (~ 9 months)

scid side effects: Radiation sensitivity; genotoxic drugs can have higher toxicity

A platform for developing refined models (transgenic and knockout)

NSG A Highly Immunodeficient Mouse

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The Most Useful and Versatile Immunodeficient Mouse Research applications:

Primary tumor engraftment

Cancer stem cells

Human hematopoiesis

Humanized mice

Infectious disease

Transplantation research

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NSG Basics





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Before treatment After treatment

Classical Cancer Xenograft Modeling Nude Mice

Gulliya et al. 1994. Cancer 74:1725-1732. PMID:8082074

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NSG

C.B-17 scid

H&E Human cells (anti-HLA)

Mouse blood cells (anti-mCD45)

H&E

Cancer Patient’s Lung

Tumor Fragment

Patient Tumor Characteristics NSG vs scid

Simpson-Abelson MR et al. 2008.J Immunol 180(10):7009-18. PMID:18453623

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JAX® Patient-Derived Xenograft (PDX) Program

Patient-derived tumor bank in NSG mice Clinical information o Tumor type, grade and markers (if known) o Treatment history Histology Gene expression and CNV analysis

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JAX® Patient-Derived Xenograft (PDX) Program


JAX® PDX Tumor Model Bank (>350)

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Tumor type Number of models

Appendiceal 1

Bladder 15

Brain 33

Breast 16

Colorectal 32

Endometrial 4

Gastric/GIST 2

Hematology 4

Hepatocellular 1

Kidney 9

Lung 49

Ovary 10

Pancreas 18

Prostate 2

Sarcoma 14

Skin 8

To see all of our PDX models, visit www.tumor.informatics.jax.org In Vivo Pharmacology Services |

PDX modeling in NSG Estrogen Receptor-Positive Breast Carcinoma

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TM00386-PT

PT= Patient Tumor; P0 = first patient derived xenograft (PDX) generation in mouse; P1 = 2nd generation

• NSG supports growth of ER+ breast tumors • Tumors retain “organoid” growth pattern

TM00386 (ER+/PR+/Her2-)

TM00386-P0 TM00386-P1

Fidelity during tumor passaging (subcutaneous)

Orthotopic model

Orthotopic PDX Modeling in NSG Bladder Cancer

Lin TY et al. 2012. Nanomedicine (Lond).[Epub ahead of print] PMID:23199207


Tumor-Targeting Micelles Prevent Tumor Growth…

…and Improve Survival

PDX Modeling in NSG mice Bladder Cancer

Lin TY et al. 2012. Nanomedicine (Lond).[Epub ahead of print] PMID:23199207


Cell line: A375 (RhoC, Luciferase) injected I.V.

NSG Improved Melanoma Metastasis Modeling

Carreno et al. 2008. Clin Cancer Res 15(10):3277-86. PMID:19447870


*NSG also better platform for cell line engraftment (data not shown)

PDX Live™ NSG Mice Readily Available from Inventory and Pre-Engrafted with

Patient-Derived Tumors

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Fast-track PDX Studies of 12 Human Tumor Types Triple negative breast carcinoma (2), acute myeloid leukemia (4), non-small cell lung cancer, B-cell lymphomas, metastatic colon adenocarcinoma, metastatic rectal carcinoma, serous adenocarcinoma, squamous cell carcinoma Save 6-12 Weeks of Valuable Research Time Eliminate the need to cryo-recover & passage tumors between donor and study mice Make Decisions Earlier during Drug Development JAX® PDX Mice are available at lower passages than any other patient-derived xenograft model provider. Low passage tumors…

o Preserve the heterogeneity of the original human cancers and the closest possible response to primary human tumors

o Improve accuracy in assessing clinical efficacy of novel therapeutics

PDX Live™

NSG M

NEW


NSG Basics





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Cells: Unsorted patient-derived T or B cell ALL Route: Tail vein Age of mice: 6-8 weeks

*Patient samples engrafted at lower doses and with faster kinetics in NSG

Effective Modeling of Patient-Derived ALL (T & B Cell)

Diamanti P et al. 2011. Leukemia Feb;26(2):376-80. PMID:21860430

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Samples from different patients showed improved engraftment versus other models (NSB)

NSG Efficiently Models Patient AML

Sanchez PV et al. 2009. Leukemia 23(11):2109-2117. PMID:19626050

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NSG Basics





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NSG Supports a Human Immune System

Most efficient engraftment of human HSCs to date

Permits differentiation of all major cell types:

o Myeloid lineage Erythrocytes, megakaryocytes, platelets Monocytes, macrophages, dendritic cells, granulocytes

o Lymphoid lineage B cells T cells (CD4+ and CD8+) NK cells

Some mucosal and adaptive immune function

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NSG Supports a Human Immune System

NSG is a superior host for human immune cells when compared to other strains: 1. Any strain expressing the scid mutation alone (NOD-scid,

B6-scid, C.B17-scid)

2. “scid beige”

3. The same mutations—scid (or a Rag1 or Rag2 knockout) and “gamma”—on other backgrounds (for example, BALB/c)

Shultz LD et al. 2005. J Immunol 174(10):6477-89. PMID:15879151 Lepus CM et al. 2009. Hum Immunol Oct;70(10):790-802. PMID:19524633

Brehm MA et al. 2010. Clin Immunol 135(1):84-98. PMID:20096637

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Improved Engraftment of CD34+ HSCs and PBMCs

King M et al. 2008. Clin Immunol 126(3):303-14. PMID:18096436

Brehm MA et al. 2010. Clin Immunol 135(1):84-98 PMID:20096637


*NSG also steadily engrafts human immune cell populations over time (data not shown)

Engraftment Efficiency of Bone Marrow

Strain Exp 1 Exp 2 Combined Percentage

NOD/Lt-scid 9/11 5/7 14/18 77.8

NOD/Shi-scid 12/12 3/11 15/23 65.2

NSG 12/12 11/11 23/23 100

NOG 10/12 9/10 19/22 86.4

*NSG also had the greatest efficiency in spleen & thymus

NSG The Most Efficient Humanized Model

Adapted from McDermott SP et al. 2010. Blood. Jul 15;116(2):193-200. PMID:20404133

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Experimental Timeline for JAX® Hu-NSG

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CD34+ cells engrafted in 3 week old female mice

Human T cells appear

Human B cells appear

Whole body irradiation Tail vein injection

3 weeks 12 weeks 15 weeks Mouse Age

In Vivo Pharmacology Services |

Human Mouse Engrafted NSG H

uman

CD

45

Mouse CD45

Hum

an C

D3

Human CD20

Human white blood cells abundant in circulation

Mature human T (CD3+) and B (CD20+) cells differentiated in the NSG mouse

Mostly neutrophils, monocytes

Successful Engraftment in Peripheral Blood


mCD45 hC

D45

88.6%

FSC

SS

C

86.1% 0.13% 11.2%

hCD38

hCD

34

Human vs Mouse Human HSC

Human Erythroid Human B cells Human APCs

Multi-lineage Differentiation in Bone Marrow

Sensitization Sensitization Challenge +/- treatmentDay -1 Day 1 Day 2 Day 7 Day 8 Day 9 Day 14Ear thickness Ear thickness Ear thickness Ear thickness Ear thickness Ear thickness

Sensitized with DNFB

Naive

Sensitized with Olive Oil

DNFB + Hydrocortisone

DTH Response in Humanized NSG is Evidence for Intact T Cell Functions


H&

E

hCD

45

Insu

lin

+ PBMC 32 days post-engraft. No PBMC

Transplant Rejection Studies: Diabetes Diabetic mice: NSG treated with STZ Transplanted with human islets, +/- PBMCs

King M et al. 2008. Clin Immunol 126(3):303-14. PMID:18096436

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Infectivity correlates with infiltration of human T cells into vaginal mucosa

NSG is Superior to NOD scid for Humanized HIV Models

Stoddart CA et al. 2011. Virology Aug 15;417(1):154-60. PMID:21684569

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Research Ready Humanized NSG

Routinely engraft NSG mice with human CD34+ hematopoietic stem cells

BLT & PBMC engrafted mice available

Mice are delivered 12 weeks after engraftment with at least 25% hCD45+ cells in the peripheral blood



NSG Basics





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Humanized tumor-bearing NSG mice The next step in cancer modeling

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Human T cells appear

Human B cells appear

Whole body irradiation Tail vein injection of

human CD34+ HSCs and tumors

3 weeks 12 weeks 15 weeks Mouse Age:


Humanized tumor-bearing NSG mice Proof-of-concept validation

Study design:

o Human CD34+ HSCs were co-transplanted with human breast cancer cell lines (BT474 or SK-BR-3)

o Tumor growth and metastasis were measured o Human immune cell engraftment and function were assessed

Goal:

o Validate a preclinical model that supports both human tumors and immune cells for the development of novel cancer immunotherapies

39 In Vivo Pharmacology Services |

Wege et.al., mAbs. 2014. PMID: 24870377

Humanized NSG mice Engraftment of human tumors

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Injected tumor cells grew, metastasized (data not shown), and retained Her2- positivity



Humanized tumor-bearing NSG mice Human immune cell engraftment and tumor infiltration

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T cells T cells Macrophages B cells



Humanized tumor-bearing NSG mice

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Humanized NSG

Solid tumor fragment or tumor cell line

NSG

Irradiation

Humanized tumor-bearing NSG mice

CD34+ HSC


NOD Rag1 Gamma (NRG) A Radiation Resistant Alternative to NSG Nomenclature: NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ Stock Number: (007799) Webpage: www.jax.org/jaxmice/strain/007799

Severe defects in both innate and adaptive immunity

Hematopoietic stem cell engraftment comparable to NSG

Radiation resistant

Emerging model

Pearson T et al. 2008. Clin Exp Immunol 154(2): 270-84. PMID:18785974

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New NSG Model Innovations HLA Class I Transgenic

NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA/H2-D/B2M)1Dvs/SzJ (014570) o HLA Class I heavy and light chains (A2.1 haplotype) NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A2.1)1Enge/SzJ (009617) o Expresses HLA Class I transgene (A2.1 haplotype)

HLA Class II Transgenic NOD.Cg-Prkdcscid Il2rgtm1Wjl H2-Ab1tm1Gru Tg(HLA-DRB1)31Dmz/SzJ (017637) o Lacks mouse MHC class II, expresses chimeric HLA class II DR4 transgene NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-DRA*0101,HLA DRB1*0101)1Dmz/GckRoly (012479)o NSG strain expresses HLA class II transgene (DR1 haplotype) NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito/ScasJ (017914)o “DRAG” strain with HLA class II transgene (DR4; HLA-DRA/HLA-DRB1*0401) For all of our NSG-derived strains, please visit our NSG mouse model portfolio.

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New NSG Model Innovations PBMC Engraftment

NOD.Cg-Prkdcscid B2mtm1Unc Il2rgtm1Wjl/SzJ (010636) o MHC class I deficient NSG strain with enhanced PBMC engraftment

Cytokine Transgenic NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(PGK1-KITLG*220)441Daw/SzJ (017830) o Kit ligand (SCF) transgenic that does not require preconditioning

irradiation NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (013062) o Expresses human IL-3, CSF2, Kit ligand o Supports human myeloid cells (CD34-humanized immune system;

acute myeloid leukemia)

Cancer Research NOD.Cg-Prkdcscid Il2rgtm1Wjl Hprtb-m3/EshJ (012480) o Hprt-null NSG mouse o Addition of HAT medium depletes mouse cells to enable isolation of

human cells JAX® Mice | 45


NSG Basics





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Online NSG Resources

www.jax.org/jaxmice/research/immunology/005557

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Categorized & Annotated NSG References List

www.jax.org/jaxmice./research/immunology/005557

IMMUNOLOGY Hematopoiesis and stem cell engraftment T cell function / B cell function / Myeloerythroid lineages NSG as a superior host for human immune system components NSG strains expressing human cytokine transgenes NSG strains expressing HLA transgenes Reviews and protocols Transplantation research / graft-versus-host disease (GVHD) / PBMC engraftmentVaccines Autoimmunity INFECTIOUS DISEASE Malaria Cytomegalovirus Dengue virus Epstein-Barr HIV modeling & therapeutics Measles Sepsis Typhoid feverVaricella-zoster virus

STEM CELLS / REGENERATIVE MEDICINE DIABETES SOLID TUMORS Breast cancer Brain cancer Cancer stem cells Colon cancer Esophageal cancer Head and neck cancer Hepatoblastoma Hepatocellular carcinoma Lung cancer Immunotherapy Melanoma / metastasis Ovarian cancer Prostate cancer Sarcoma HEMATOLOGICAL CANCERS AML Lymphoma ALL Mastocytoma CML Multiple myeloma CLL Chronic lymphocytic leukemia

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Cutting Edge Human Disease Modeling Using The NSG Advanced and future disease modeling Additional information and resources JAX ... Cutting Edge Human Disease Modeling The NSG Mouse