Human Immune System (HIS) mouse models for translational research

Barbara Joyce-Shaikh

Humanized Immune System (HIS) Mouse Models

Goals– Enable clinically relevant in vivo studies of human cells, tissues, and immune

function– Allow early functional readout to develop treatment signatures and biomarker

discovery plan

Work at MRL PA– Study Targets where no rodent orthologue exists– Study cellular mechanism and profile of clinical IMR targets- PD-1, CTLA4– Study novel combination and sequential therapies – Testing hypotheses driven by clinical study data- Reverse Translation

Limitations of model system– Lack of HLA molecules to facilitate PKPD studies– Limited lymph node development– Residual murine innate immunity

Tumor cell lines Compare tumor growth kinetics between Melanoma and Pancreatic tumor

cells in NSG mice Compare treatment response in models with Keytruda

Tissue Doubling Time (Days)

HLA-A Class I

Melanoma 12 A*02

Pancreas 7 A*02:01

Tumor progression- Melanoma vs Pancreatic tumors with Keytruda

αPD1

Pancreatic tumor Melanoma tumor

Metastatic melanoma tumor promotes the development and trafficking of human myeloid cells

5

•1x106 melanoma cell line sc

Tumor presence :•Myeloid cell survival and expansion•Conversion to a tolerogenic microenvironment•Factors detected in plasma from tumor-bearing NSG mice:TNF, IL1b, IL6, IL8, IL10

NSG Naïve

mC

D45

hCD45

hCD

11b

hCD3hC

D11

bhCD3

Melanoma

Cell populations in humanized mice with different tumors types

CD33

Pancreatic Melanoma

CD

66b

Tumor

Spleen

1.29

9.21

32.3

1.54

0.24

6.86

0.84

14.2

• CD33+ Mo-MDSC % similar in spleen with

both tumor types

• CD33+ Mo-MDSC much lower % in

tumor Panco8 model

• CD66b+ Gr-MDSC not present in

Panco8 but is a large % in SKMEL5

Cells are gated on lymphocytes- hu CD45+ Live

T-cell cell populations in HIS mice in tumors microenvironments with PD1 treatment

CD8

Pancreatic Melanoma

CD

3

Isotype

Anti-PD18.69

6.5227.4

52.3

Cells are gated on live hu CD45+

Pancreatic

MelanomaC

D66

b

1.29

32.3

0.24

14.2

CD33

Immune profile comparison in TILs of implanted tumor lines

T-cell signature MDSC signature

ARG1 0 1092CCR2 16 222

CD11B 53 539CD11C 95 322

CD33 39 414CD66B 0 1117

CD68 510 951CLEC5A 30 234

IL10 4 17MMP9 479 2862

MPO 4 9865S100A8 258 117566S100A9 459 57648

SIRPA 119 1167

a co8 S 5 CD40 927 124CD69 450 118CD8A 555 151CD8B 108 15

CTLA4 123 16ICOS 75 12IFNG 97 13

IL2RA 42 14IL17A 27 1

IL6 215 8PD1 94 9

PDL1 1446 151PDL2 150 40

Perforin 133 28

Conclusions• We have established tumor cell line models in NSG mice

that replicate different aspects of human tumors• Tumor produced factors shape initial development of tumor

immune microenvironment• SKMEL5 tumor line induces the egress and expansion of

myeloid cell populations in the periphery and tumor of humanized mice

• Models that demonstrate in vivo response to anti-PD1 correlate with activated T-cell infiltration into tumor

• Tumor resistance mechanism can be explored utilizing specific “cold” tumor models

• Understanding engrafted cell interactions with specific tumor types can guide model selection and combination therapy strategies

High GITR expression on Treg in spleen and SKMEL-5 tumor of humanized mice

•GITR expression on human Treg (blue line),• non-Treg CD4+ T cells (dashed line), and •CD8 T cells (shaded grey) in spleen or tumor

•GITR

•Cou

nts

•Spleen •Tumor

Anti-hu GITR mimics many features of mDTA-1 in Hu-CD34+ NSG HIS tumor model

Hu-CD34+ humanized mice dosed with 10 mg/kg MK-4166 (humanized IgG1 anti-huGITR) when SKMEL-5 tumors reached 130 mm3.

0 7 1 4 2 1 2 8 3 5 4 2 4 90

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

D a y s fo llo w in g s ta r t o f t re a tm e n t

Tu

mo

r V

olu

me

mm

3 Is o ty p e C o n tro lM K -4 1 6 6

Anti-huGITR slows tumor growth,

TILS from Anti-hu GITR treated mice make more IL-2 and IFNγ after overnight culture

Is o ty p e C o n tro l

M K -4 1 6 60

1 0 0

2 0 0

3 0 0

4 0 0

IL -2

IL-2

pg

/mL

**

Is o ty p e C o n tro l

M K -4 1 6 60

5 0

1 0 0

1 5 0

IF N γ

IFNγ

pg

/mL

0.0849 •TILS isolated and cultured overnight

S p le e n T IL s0

1 0

2 0

3 0

C D 8 :T re g ra tioC

D8

+ T

cel

l:T

reg

rat

io ***

S p le e n T IL s0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

IC O S M F I o n T re g

MF

I

****

•Flow cytometry for CD8+:Treg ratio• and ICOS MFI on day 4 post-dose.

Anti-hu GITR increases CD8:Treg ratio in spleen, and decreases activation markers on tumor Treg

Summary

• In Mice DTA-1 reduces tumor Treg number and activation status by depleting highly activated Treg

• GITR+ Foxp3+ Tregs are present in humanized mice and can be modulated with anti-GITR treatment

• Anti-hu GITR mimics many features of mDTA-1 in humanized mice

• Decrease in Treg, though primarily in spleen• Reduction in activated Treg in tumor

Dual roles for regulatory T cell depletion and co-stimulatory signaling in agonistic GITR targeting for tumor immunotherapy.Ashley Mahne, Smita Mauze, Barbara Joyce-Shaikh, Jane Xia, Edward Bowman,Amy Beebe, Daniel Cua,and Renu Jain. DOI: 10.1158/0008-5472.CAN-16-0797 Published 20 October 2016

Anti-Ceacam-1 study in HIS mice

13

Ceacam1 NSG- Human Tumor Expression

14

• CEACAM1 is highly expressed on the surface of granulocytic myeloid in human tumors.

• Representative FACS data NSCLC (n=6), RCC (n=11), CRC (n=4), and melanoma (n=4)

Ceacam1 Background

• Carcinoembryonic antigen-related cell adhesion molecules 1 (CEACAM1)is a transmembrane glycoprotein that belongs to the Ig superfamily.

• It is implicated in the regulation of various cellular functions includinggrowth, differentiation, and immune modulation.

• CEACAM1 expression in vitro stimulation studies have implicatedCeacam1 expression on T-cell subsets and has been implicated as apossible modulated by check-point blockadeAtsushi Nakajima et al. J Immunol February 1, 2002,168(3) 1028-1035; DOI: https://doi.org/10.4049/jimmunol.168.3.1028

• CEACAM1 on tumor cells has been well characterized, the expressionpattern of CEACAM1 on immune cells within the tumor microenvironmenthave been incompletely characterized.

• .

16

CEACAM1 CD8

DAPI Merge

CEACAM1 MPO

DAPI Merge

0

2 0

4 0

6 0

8 0

1 0 0

Pe

rce

nt

Po

sit

ive

CD8+CD4+

No Culture IL-2Only

IL-2 +anti-CD3

No Culture IL-2Only

IL-2 +anti-CD3

Ceacam1 -Tumor Expression

CEACAM1 expression co-localizes with MPO

CEACAM1 is not expressed on CD8+ cells

Ceacam-1 expression is greatly up-regulated with In vitro stimulation

Ceacam1 NSG-Tumor Expression

17

CEACAM1 expression profiles in tumor infiltrating cells from human and humanized mice are different than those frommouse syngeneic models.Cell surface expression of CEACAM1 on various cell types for human (clone MRG1) and mouse (clone CC1) were determined byflow cytometry. Representative FACS histograms from human renal cell carcinoma (RCC; n=11), SKMEL5 model of humanizedmice (n=3), and MC38 mouse model (n=1) are shown here. MB49, B16F10, and CT26 mouse models show similar pattern asMC38 (n=1 each, data not shown).

HIS mouse tumor model

•Monitor for weight loss and tumor growth

End of study read-outFACS profiling- spleen, TILs

Plasma- drug levels/Gene expression

Melanoma melanoma cell

line sc

d20

Ave Tumor: 130 mm3 n=9 / groupWeight and tumor size measured weekly Blood sampling for PK (alternate groups 1 sample / 2wks

d50d0

Group TreatmentDose mg/k

gDosing

Schedule1ROA

Group

Size2

#Donors

1 Isotype (hIgG4) 12 q d3.5 sc. 9 3

2 MK-6018 10 q d3.5 sc. 9 3

3 Keytruda 2 q d3.5 sc. 9 3

4 MK-6018Keytruda

102 q d3.5 sc. 9 3

M-MDSC Gr-MDSC

FACS Iso

Ceacam1

huIgG4 Iso

Ceacam-1 binding on G-MDSC cells

Study 16-M320-7929- In life tumor growth

19

Ceacam1 NSG-FACS –T cells SPL

20

Spleen

Live Cells Single Cells huCD45+

CD20+

CD4+

CD8+

CD3+

Live Cells Single Cells huCD66b+huCD45+

huCD14+

Cell subsets CD4+ CD8+ cells in HIS mouse spleen

21

Tumor resistance mechanisms-myeloid cells

•

CD14+

CD66b+

Myeloid Cells

Sorted cell profile

•Genes expressed equally by both cell populations

ANGPT1 22233 18414CLDN3 7339 4704CD66B 362 258

ARG1 235 14399149MMP9 168 293

CEACAM1 33 76CLEC5A 9 43CXCR2 7 8

COX2 5 11MPO 3 4

CXCR4 3 4CD47 3 4

S100A8 3 3CD11B 2 3

S100A9 2 3VEGFA 2 4

HIF1A 2 2IL4RA 2 2

IL8 2 3SIRPA 2 2TGFB1 1 2CD45 1 1

CD11C 1 1ITGA4 1 1NFKB1 1 1

CD33 2 2IL1B 2 3

ICAM1 3 2LGALS9 3 4

TIMP1 4 6IRF8 5 4CD4 7 6

PDL1 7 3CD68 7 5CD14 9 9

HLA-DRA 11 11ITGB7 33 13CCR2 39 59CD86 79 58

CXCL2 108030 0TIM3 549618 94

RNA-seq data show distinct populationsClusters based on monocytes vs. granulocytes

M-MDSC 1

M-MDSC 2

M-MDSC 3

Gr-MDSC 1

Gr-MDSC 2

Gr-MDSC 3

Gr-MDSC 4

25

Graphic View of Immune Checkpoint Blocker Combinations

Conclusions

• Ceacam-1 expression is similar between human tumors and HIS mouse tumors

• Anti-human Ceacam1 did not demonstrate efficacy in HIS mouse models as a monotherapy or in combination with Keytruda or Ipilimumab

• HIS mouse data in conjunction with preclinical and clinical data helped define program path decision

• HIS systems can help gain insights into target expression profiles that can support biomarker discovery

Acknowledgments

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MRL HistopathologyJennifer YearleyLakshmanan Annamalai

MRL Protein Engineering Laurence Fayadat-DilmanDaniel CiprianoHai Ling Li

PA Animal FacilityPriscilla LaprescaJoann DominguezRavi Tolwani

JAX LabsDwayne DexterRick HuntressLewis VannJames Keck

Special ThanksJohn Mudgett (Genesis)Michael Brehm (UMASS)

MRL IOI DiscoveryDewan HossainAlissa ChackerianDan CuaRobbie McleodJoann O'ConnorJuha PunnonenRob Kastelein

MRL Profiling & ExpressionTerri McClanahanJeff GreinWendy BlumenscheinSvetlana SadekovaMike LeeJerelyn WongDoug WilsonVanessa PetersonSarah JavaidEric Gustafson