introduction to humanized mice for cancer...

INTRODUCTION TO

HUMANIZED MICE FOR

CANCER IMMUNOTHERAPY

Technical Information Services

Upcoming JAX Webinars™

2

Achieving Reproducible Mouse Studies

o March 3, 2016, 1:00 pm ET USA

Comparing Immunodeficient Mice for Cancer, Immunity, and

Transplant Research


Cre-lox Basics – Generating Knockout Mice


Humanized NSG™ Mice for Innovative Preclinical Research


o March 29, 2016, 9:00 am ET USA

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The Jackson Laboratory’s Mission

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Investigating genetics and biology of human disease

Providing Resources

JAX® Mice Clinical & Research Services, bioinformatics data,

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JAX® Mice The Gold Standard for Biomedical Research

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NIH funded resource

>8,000 strains and growing

Unsurpassed genetic quality & animal health

Best characterized & referenced ~100 new pubs/week

Common inbred strains (C57BL/6J, BALB/cJ, DBA/2J) support

development/collection of specialty strains and other valuable

community research resources

JAX® Mice |

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Online Resources to Expedite

Research

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JAX® Mice Database

www.jax.org/jaxmice/query

Mouse Genome Informatics

www.informatics.jax.org

Mouse Phenome Database

www.jax.org/phenome

And many more unique

resources

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Cancer Immunotherapy: Learning Goals

Explain current approaches for

cancer immunotherapy

Distinguish between and choose

appropriate mouse models

o Classic

o Cutting-edge

Successfully find JAX models for

immuno-oncology research

7 JAX® Mice |










o Classic

o Cutting-edge



8 JAX® Mice |





Cancer Antigens: Recognized by several immune cell types

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Tumors express antigens that can be recognized by immune cells:

o Normal proteins at supra-physiological concentrations and/or conditions

o Aberrant proteins (misfolded, mutated, or gene-fusion)

Can be specific or nonspecific:

o Tumor-associated antigens (TAA)

o Tumor-specific antigens (TSA)

Displayed intracellularly or on cell surface

JAX® Mice |





Immune System Components

10 JAX® Mice |





Cancer: Presentation and recognition by several immune cell

types

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Natural Killer cells

(NK)

Cytotoxic

CD8+ T cells Macrophages

Dendritic cells (DC)

B cells

CD4+ T “Helper”

cells

JAX® Mice |





Mechanisms of Tumorigenesis: Immune cell evasion and pathway dysregulation

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CD8+ T cell

JAX® Mice |

MHC TCR

Peptide

PD-1 PD-L1

Cancer cell





Mechanisms of Tumorigenesis: Immune cell evasion and pathway dysregulation

13 JAX® Mice |

Cell

adhesion

EpCAM

CD38

TSLC1

DNA

Repair/Apoptosis

p53

BRCA 1 & 2

Immune Evasion

PD-1

CTLA-4

HLA Class I & II

Proliferation

Flt3

PI3K/AKT

Her2

c-Myc

Angiogenesis

VEGF

Ang-2

PDGF

MHC TCR

Peptide

PD-1 PD-L1

Cancer cell CD8+ T cell





Cancer Immunotherapy: Approaches

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Antibody-based Complement-dependent cytotoxicity (CDC)

Antibody-dependent cytotoxicity (ADCC)

Antibody-drug conjugates (ADC)

Cell-mediated Cytotoxic T cells

NK cells

Dendritic cells

Strategies to target tumor cells include:

GM-

CSF

EPO

Cytokines

IFNα

G-CSF IL-11

IL-2

JAX® Mice |





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Mechanisms include:

Antibody-dependent phagocytosis (ADPh)

Complement-dependent cytotoxicity (CDC)

Antibody-dependent cell mediated cytotoxicity (ADCC)

Antibody neutralization (receptor or ligand)

DC antigen peptide priming

BiTE; bispecific T cell engager

CAR-T; chimeric antigen receptor T cell

Cancer Immunotherapy: Antibody therapeutics

JAX® Mice |

Scott et al. 2012. Nat Rev Cancer 12(4):278-87. PMID: 22437872





http://www.ncbi.nlm.nih.gov/pubmed/22437872

Cancer Immunotherapy: Antibody-based

16 JAX® Mice |

Antibody-based immuno-oncology compounds

approved for clinical use, alone and in combination

with chemotherapeutic compounds:

o Keytruda

o Yervoy

o Avastin

o Adcetris

o Herceptin

o Kadycla

o Many more










o Classic

o Cutting-edge



17 JAX® Mice |





Mouse Models for Immuno-oncology

Immunocompetent and immunodeficient mouse models used for

development and validation of biologic therapies for cancer

o Immunocompetent

BALB/cJ (000651)

C57BL/6J (000664)

DBA2/J (000671)

FVB/NJ (001800)

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o Immunodeficient

Nude (002019)

BALB/c scid (001803)

NOD scid (001303)

NSGTM (005557)

JAX® Mice |

http://jaxmice.jax.org/strain/000651.html












Mouse Models for Immuno-oncology: Immunocompetent models

Benefits:

Inexpensive

Intact immune cell function

Syngeneic tumor hosts

Excellent proof-of-concep

for preclinical studies

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Limitations:

Only permit engraftment of

mouse tumor cell lines

Cannot engraft human tumors

Mechanisms are not always

translatable to humans

Compounds are mouse

specific

JAX® Mice |





Immunocompetent mouse models: ADCC-mediated tumor killing in BALB/c mice

BALB/c-derived cancer cell line

(EGFR/Her2-positive TUBO cells)

injected into BALB/c recipient

Anti-EGFR mAb administered +/-

anti-CD8 depleting mAbs

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Yang et.al. 2013. Mol.Ther 21(1):91-100. PMID: : 22990672

JAX® Mice |

http://www.ncbi.nlm.nih.gov/pubmed/?term=22990672





Immunocompetent mouse models: Syngeneic therapeutic antibody validation

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B16F10 syngeneic tumor cells injected

into C57BL/6 mice +/- mAb (TA99)

Therapeutic antibody treatment

enhances macrophage-mediated

tumor phagocytosis

Gul et. al. 2014. J. Clin. Invest 124(2):812-23. PMID: 24430180

Macrophage

Cancer cell

JAX® Mice |






Mouse Models for Immuno-oncology: Immunodeficient models

Benefits

Support the engraftment of

human tumors and/or cell lines

Orthotopic cancer modeling

Adoptively transfer functional

immune cells

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Limitations

No endogenous immune

cell function

More susceptible to

pathogenic infection

JAX® Mice |





Immune System Components

23 JAX® Mice |





Cancer Therapy Modeling: Blocking homing and proliferation in NOD scid mice

Vaisitti, T. et.al. 2010. Leukemia 24(5):958-69. PMID: 20220774

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IV injection of primary leukemic cells

(CD19+ CD5+, CD38+) isolated

from patient peripheral blood into

NOD scid mice

CD38: required for homing and

proliferation

SUN-4B7: blocking anti-CD38 mAb

OKT10: non-blocking anti-CD38

mAbs

*Downregulates ERK1/2

phosphorylation (data not

shown)

JAX® Mice |

Human CLL in NOD scid






Immune System Components:

25 JAX® Mice |





Cancer Therapy Modeling: ADCC-mediated killing reduces tumor load in NSGTM

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WT1 (Wilm’s Tumor 1); intracellular

oncoprotein upregulated in many

leukemias and solid cancers

ESK1 is a “TCR-like” anti-WT1 mAb;

recognizes WT1 epitope in context of

HLA-A2

Primary WT1-positive AML blasts

from HLA-A2 +/- patients

Dao, T. et.al. 2013. Science Transl Med (5)176:176ra33. PMID: 23486779

JAX® Mice |

LDH; lactate dehydrogenase release






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Cancer Therapy Modeling: ESK1 mAb enhances survival of NSGTM mice

BV173 luciferase tagged cell line

o WT1-positive human pre-B cell leukemia

ESK1 (anti-WT1) mAb alone

ESK1 (anti-WT1) mAb + effectors

(hu-PBMC)

Dao, T. et.al. 2013. Science Transl Med (5)176:176ra33. PMID: 23486779

JAX® Mice |






BiTE; Bispecific T Cell Engager

Frankel, S. et.al. 2013. Cur Opin Chem Bio 17(3). Science Direct

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BiTE Chimera

CD3α single chain fragment variable (scFv) domain binds CD3ε of TCR

Tumor associated antigen scFv

Activation of CD4 and CD8 T cells toward tumor cells

http://www.sciencedirect.com/science/article/pii/S1367593113000549

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Dao, T. et.al. 2015. Nature Biotechnology 33(10). 1079-86. 1079-86. PMID: 26389576

Cancer Therapy Modeling: BiTE targeted WT1 diminishes tumors in NSGTM

WT1 Expressing SET-2 AML Cells Labeled with Luciferase

Alone Hu T Cells

Hu T Cells +

Control BiTE

Hu T Cells +

ESK1 BiTE

T cells alone have minimal impact on tumor growth

WT1 specific BiTE significantly diminished AML growth

Similar response to multiple WT1+ tumor cell lines (not shown)


CAR-T; Chimeric Antigen Receptor-

Engineered T cells

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Lentiviral transduction of

human T cells

Target binding domain

(scFv)

CD8α hinge

transmembrane domain

CD28 (B-1BB)

costimulatory domain

CD3ζ activation domain

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Cancer Therapy Modeling: CAR-T targeted ErbB2 in NSGTM (affinity tuning)

SK-OV3 Left Flank and PC3-CBG Right Flank

(High Dose, High Affinity CAR-T)

(High Dose, Low Affinity CAR-T)

(Low Dose, High Affinity CAR-T)

(Low Dose, Low Affinity CAR-T)

SK-OV3: Ovarian cancer, high ErbB2

expression, luciferase tagged

PC3-CBG: Prostate cancer, normal

ErbB2 expression, luciferase tagged

Liu, X. et.al. 2015. Cancer Research 75(17). 3596-607. PMID: 26330166


Liu, X. et.al. 2015. Cancer Research 75(17). 3596-607. PMID: 26330166

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Cancer Therapy Modeling: CAR-T targeted ErbB2 in NSGTM (affinity tuning)

High Affinity CAR-T:

In vitro activation (increased IFN, CD107a, CD137)

following exposure to cell lines with low, medium, and

high ErbB2 expression

In vivo recognition and killing of both SK-OV3 and PC3

Low Affinity CAR-T:

In vitro activation primarily to cell lines with high ErbB2

expression and not low expression

In vivo recognition and killing of SK-OV3 and reduced

killing PC3 cells

SK-OV3 Cells, Left Flank

PC3 Cells, Right Flank

*Low affinity, high & low dose

*

*


Mouse Models for Immuno-oncology: Hu-CD34 NSGTM

Benefits

Multi-lineage differentiation

from HSCs

Establishment of human

immune function

Co-engraftment of human

cancer cells lines and patient

derived xenografts possible

(emerging models)

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Limitations

Not all human immune cell

populations represented

Not all immune cell function

retained

In Vivo Pharmacology Services |

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

34

Humanized NSGTM mice: A new way to study human immune cell function



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JAX® CD34 hu-NSGTM Model Creation

Human

T cells

appear

Human

B cells

appear

Whole body irradiation

Tail vein injection

3 weeks 12 weeks 15 weeks Mouse Age:



Human Immune Cell Engraftment in

Hu-CD34 NSG™ Mice Bone Marrow

mCD45

hC

D4

5

88.6%

Human vs Mouse APCs B cells


Peripheral Blood

1 10 100 1000 10000

1

10

100

1000

10000

44.7

mCD45 hC

D4

5

44.7%

Human vs Mouse

1 10 100 1000 10000

1

10

100

1000

100002.52 0.99

19.876.6

hCD14

hC

D5

6

20%

2.5%

NK cells & Mono

1 10 100 1000 10000

1

10

100

1000

1000024.9 0.092

46.328.7

hCD20

hC

D3

T & B cells

46.3%

24.9%

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Immune Cell Function

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Innate Immunity

Myeloid phagocytosis

NK cell activity

Adaptive Immunity

T cell cytotoxicity

B cell Immunoglobulins

Delayed Type Hypersensitivity (DTH)



JAX® Hu-CD34 NSGTM : Preclinical model of DTH response

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Sensitization Sensitization Challenge +/- treatment

Day -1 Day 1 Day 2 Day 7 Day 8 Day 9 Day 14

Ear thickness Ear thickness Ear thickness Ear thickness Ear thickness Ear thickness

Sensitization Sensitization Challenge+/- treatment


Delayed-Type Hypersensitivity (DTH)

Immune sensitization and challenge with dinitrofluorobenzene (DNFB)








o Classic

o Cutting-edge



39 In Vivo Pharmacology Services |


JAX Patient-Derived Xenograft (PDX)

Program

Clinical information

o Tumor type, grade and markers (if known)

o Treatment history

Histology

Gene expression array and CNV array analysis

In Vivo Pharmacology Services | 40

Archived

PDX

tumors

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

PDX Development Process:

41

Archived

PDX

tumors

Cohort of mice Donor mice

Multi-step approach for the development of cohorts of

patient derived xenograft mouse models

Study



Established PDX Models


>400 clinically relevant PDX tumors, with

orthotopic engraftment capabilities


43

Lung Adenocarcinoma EGFR L858R: Acquired TKI resistance



44 In Vivo Pharmacology Services | In Vivo Pharmacology Services |

Lung Adenocarcinoma EGFR L858R: PDX tumor drug response in NSGTM



45

huCD34 HSC NSG

Humanized NSG

PDX Tumor or Cell Line

Humanized NSG with Tumor (When tumors reach 70-120 mm3 mice are treated

with therapeutics for 21 to 28 days)

Irradiation

Humanized Tumor-Bearing NSGTM Mice: The next step in cancer modeling

Schilbach, et al. 2015. OncoImmunology. Mar 19;4(7):e1014760. eCollection PMID: 26140238

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Tumor-Bearing Humanized Mice: IL-12/IL-2 stimulated innate & adaptive immunity

Rhabdomyosarcoma cell line A204

http://www.ncbi.nlm.nih.gov/pubmed/26140238

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Innate & Adaptive Immune Cells

Infiltrate Tumors

What is the influence of HLA matching between humanized

mouse and patient PDX material

o Important?

Response to Standard of Care therapy

o Different in the humanized and non-humanized NSGTM?

Immuno-modulation via PD-1 and CTLA-4

o Do immuno-modulators impact tumor growth in hu-CD34 NSGTM?

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Humanized Tumor-Bearing NSGTM Mice: JAX® proof-of-concept validation and questions



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JAX® Data: Humanization of NSGTM mice has no significant impact on PDX growth kinetics

Breast Lung

Soft Tissue Carcinoma

Fresh tumor tissue engraftment

Not HLA-matched

100% take-rate in NSGTM or hu-CD34

NSGTM

Hu-CD45+ in hu-CD34 NSGTM mice:

>25%


JAX® Data: Infiltrating hu-CD45+ cells in three hu-PDX tumors

No hu-CD45 cells in tumors from

non-humanized NSGTM

All three PDX tumor types

contained infiltrates in hu-CD34

NSGTM

Breast Lung

Soft Tissue Carcinoma


51

Humanized Tumor-Bearing NSGTM Mice: Response to standard chemotherapy & Avastin*


Not HLA matched

Hu-CD45+ in hu-CD34 NSGTM mice: >20%

Tumor volume of Colon

Adenocarcinoma PDX in hu-hu-NSGTM

mice

Mean Tumor Volume of Colon

Adenocarcinoma PDX in hu-NSGTM

mice on Day 21


*Avastin; anti-VEGF-A angiogenesis inhibitor

5-Fu; 5-fluorouracil


52

Humanized Tumor-Bearing NSGTM Mice: Keytruda* (anti-PD1) checkpoint inhibitor response


Hu-CD45+ in hu-CD34 NSGTM mice: >25%

PD-L1 surface expression on tumor: 56.9%


*Keytruda; AKA pembrolizumab

Tumor volume of invasive ductal breast

carcinoma (BR1126) PDX in hu-NSGTM mice



Humanized Tumor-Bearing NSGTM Mice: Human T & B cells infiltrate BR1126 PDX tumors of hu-CD34 NSGTM mice

Total Human CD45+ Cells

% Human CD4+ T Cells % Human CD8+ T Cells

% Human CD19+ B Cells



Humanized Tumor-Bearing NSGTM Mice: Keytruda +/- Docetaxel inhibit lung LG1306 PDX in hu-CD34 NSGTM mice

Tumor PD-L1 surface

expression: 89.1%

Hu-CD45 engraftment in

hu-CD34 NSGTM >20%

Tumor volume of lung non-small cell carcinoma

(LG1306P5) PDX in hu-NSGTM mice



Humanized Tumor-Bearing NSGTM Mice: Human T cells infiltrate PDX LG1306 tumors of hu-CD34 NSGTM Mice

Total Human CD45+ Cells

% Human CD4+ T Cells % Human CD8+ T Cells

% of infiltrating T cells within

the tumor huCD45 cells

Similar cell percentages

between treatment groups


56

Lung PDX LG1306-Vehicle Treated: Minimal CD8 T cell infiltration in tumor tissue

CD45 CD8 Cytokeratin

57 CD45 CD8 Cytokeratin

Lung PDX LG1306-Keytruda Treated: Increased CD8 T cell infiltration in tumor tissue

NSGTM Mice: State-of-the-art platform for human cancer studies

Co-engraftment of human HSCs and tumor cells

Co-engraftment of human PBMCs and tumor cells

Widespread applications for NSGTM

Mouse of choice for cancer studies








o Classic

o Cutting-edge





NSG™-Based Models for Cancer and

Efficacy Studies

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.

JAX® Mice | 60






http://jaxmice.jax.org/nod-scid-gamma/nsg-mouse-portfolio.html





PDX Offerings

Offering Value Driver

Efficacy Testing Standard of Care/Experimental Therapeutics

Early passage cohorts P0 includes patient ECM

Includes CNV/mRNA expression; histology

Cohorts of Tumor Bearing Mice Ready for efficacy testing

Supporting Materials: • Snap frozen tumors

• Tissue sections and slides

Immunohistochemistry; RNA FISH,

histology

Tumor Bearing Mice (non-profits) Distribution to your core for expansion



Research Ready Humanized NSGTM


NSG engrafted with human CD34+ hematopoietic stem cells

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

Availability of PBMC model by request


JAX® In Vivo Services

63

Acknowledgements:

Jim Keck, Ph.D.

Mingshan Chen, Ph.D.

Sacramento, California




Common inbred & specialty JAX®

Mice strains

Basic, custom & complex

breeding capabilities and speed

congenics

Genome Scanning

Cryopreservation & recovery

Compound efficacy testing


http://www.jax.org/mouse-search




https://www.jax.org/jax-mice-and-services/breeding-and-rederivation-services



https://www.jax.org/jax-mice-and-services/breeding-and-rederivation-services/genome-scanning

https://www.jax.org/jax-mice-and-services/breeding-and-rederivation-services/genome-scanning

https://www.jax.org/jax-mice-and-services/cryo-and-strain-donation/cryopreservation

https://www.jax.org/jax-mice-and-services/in-vivo-pharmacology

In need of humanized NSGTM mice

to advance your research?

Contact your regional representative today:

www.jax.org/jaxmice/support/regionalcontacts

Contact technical support:

www.jax.org/jaxmice/support/techsupport-index

THANK YOU

http://www.jax.org/jaxmice/support/regionalcontacts




introduction to humanized mice for cancer...

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