Human Immune System (HIS) mouse models for translational ...€¦ · Humanized Immune System (HIS)...
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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
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•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
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Ceacam1 NSG- Human Tumor Expression
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• 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.
• .
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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
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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
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Ceacam1 NSG-FACS –T cells SPL
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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
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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
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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