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
Doubling HLA-A Tissue Time (Days) Class I
Melanoma 12 A*02
Pancreas 7 A*02:01 Tumor progression- Melanoma vs Pancreatic tumors with Keytruda
Pancreatic tumor Melanoma tumor
αPD1 Metastatic melanoma tumor promotes the development and trafficking of human myeloid cells
Tumor presence : •Myeloid cell survival and expansion •Conversion to a tolerogenic microenvironment •1x106 melanoma •Factors detected in plasma from tumor-bearing NSG mice: cell line sc TNF, IL1b, IL6, IL8, IL10
NSG Naïve Melanoma mCD45 hCD11b hCD11b
hCD45 hCD3 hCD3 5 Cell populations in humanized mice with different tumors types
Pancreatic Melanoma
• CD33+ Mo-MDSC % 0.24 32.3 similar in spleen with Tumor both tumor types
• CD33+ Mo-MDSC 1.29 14.2 much lower % in tumor Panco8 model
0.84 1.54 • CD66b+ Gr-MDSC CD66b not present in Spleen Panco8 but is a large % in SKMEL5 6.86 9.21
CD33 Cells are gated on lymphocytes- hu CD45+ Live T-cell cell populations in HIS mice in tumors microenvironments with PD1 treatment
Pancreatic Melanoma
27.4 6.52 Isotype CD3
52.3 8.69 Anti-PD1
CD8 Cells are gated on live hu CD45+ Immune profile comparison in TILs of implanted tumor lines Pancreatic T-cell signature MDSC signature a co8 S 5 0.24 CD40 927 124 ARG1 0 1092 CD69 450 118 CCR2 16 222 CD8A 555 151 CD11B 53 539 CD11C 95 322 CD8B 108 15 1.29 CTLA4 123 16 CD33 39 414 ICOS 75 12 CD66B 0 1117 IFNG 97 13 Melanoma CD68 510 951 CLEC5A 30 234 IL2RA 42 14 CD66b IL17A 27 1 IL10 4 17 32.3 IL6 215 8 MMP9 479 2862 PD1 94 9 MPO 4 9865 PDL1 1446 151 S100A8 258 117566 PDL2 S100A9 459 57648 150 40 14.2 Perforin 133 28 SIRPA 119 1167
CD33 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 Anti-hu GITR mimics many features of mDTA-1 in Hu-CD34+ NSG HIS tumor model
High GITR expression on Treg in spleen and SKMEL-5 tumor of humanized mice
•Spleen •Tumor
•GITR expression on human Treg (blue line), • non-Treg CD4+ T cells (dashed line), and •CD8 T cells (shaded grey) in spleen or tumor • Counts
•GITR
Anti-huGITR slows tumor growth,
Isotype Control 3 1000 MK-4166 800
600
400
200
Tumor Volume mm 0 0 7 14 21 28 35 42 49 Days following start of treatment
Hu-CD34+ humanized mice dosed with 10 mg/kg MK-4166 (humanized IgG1 anti-huGITR) when SKMEL-5 tumors reached 130 mm3. Anti-hu GITR increases CD8:Treg ratio in spleen, and decreases activation markers on tumor Treg
CD8:Treg ratio ICOS MFI on Treg 30 *** 25000 ****
20000
20 15000 •Flow cytometry for CD8+:Treg ratio
MFI • and ICOS MFI on day 4 post-dose. 10000 10 T cell:Treg ratio T cell:Treg
+ 5000
CD8 0 0 Spleen TILs Spleen TILs
TILS from Anti-hu GITR treated mice make more IL-2 and IFNγ after overnight culture
IL-2 IFN γ
400 ** 150 0.0849 •TILS isolated and cultured overnight 300 100
200 pg/mL γ 50 IL-2 pg/m L
100 IFN
0 0 Isotype MK-4166 Isotype MK-4166 Control Control 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
• 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)
14 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 including growth, differentiation, and immune modulation.
• CEACAM1 expression in vitro stimulation studies have implicated Ceacam1 expression on T-cell subsets and has been implicated as a possible modulated by check-point blockade Atsushi 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 expression pattern of CEACAM1 on immune cells within the tumor microenvironment have been incompletely characterized. • . Ceacam1 -Tumor Expression
CEACAM1 MPO CEACAM1 CD8
DAPI Merge DAPI Merge CEACAM1 expression CEACAM1 is not co-localizes with MPO expressed on CD8+ cells
CD4+ 100 CD8+ 80
60
40
20 Percent Positive Percent
0 No Culture IL-2 IL-2 + No Culture IL-2 IL-2 + Only anti-CD3 Only anti-CD3 Ceacam-1 expression is greatly up-regulated with In vitro stimulation
16 Ceacam1 NSG-Tumor Expression
CEACAM1 expression profiles in tumor infiltrating cells from human and humanized mice are different than those from mouse syngeneic models. Cell surface expression of CEACAM1 on various cell types for human (clone MRG1) and mouse (clone CC1) were determined by flow cytometry. Representative FACS histograms from human renal cell carcinoma (RCC; n=11), SKMEL5 model of humanized mice (n=3), and MC38 mouse model (n=1) are shown here. MB49, B16F10, and CT26 mouse models show similar pattern as MC38 (n=1 each, data not shown).
17 HIS mouse tumor model
•Monitor for weight loss and tumor growth d0 d20 d50
3 Melanoma Ave Tumor: 130 mm n=9 / group End of study read-out Weight and tumor size measured weekly melanoma cell FACS profiling- spleen, TILs Blood sampling for PK (alternate groups 1 line sc sample / 2wks Plasma- drug levels/ Gene expression
Ceacam-1 binding on G-MDSC cells M-MDSC Gr-MDSC Dose Dosing RO Grou # Group Treatment mg/k Schedule1 A p Don g Size2 ors huIgG4 Iso 1 Isotype 12 9 3 (hIgG4) q d3.5 sc. Ceacam1 2 MK-6018 10 q d3.5 sc. 9 3
3 Keytruda sc. 9 3 2 q d3.5 FACS Iso MK-6018 10 4 q d3.5 9 3 Keytruda 2 sc. Study 16-M320-7929- In life tumor growth
19 Ceacam1 NSG-FACS –T cells SPL
Spleen
Live Cells Single Cells huCD45+ CD4+ CD3+ CD8+
CD20+
Live Cells Single Cells huCD45+ huCD66b+
huCD14+
20 Cell subsets CD4+ CD8+ cells in HIS mouse spleen
21 Tumor resistance mechanisms-myeloid cells
Myeloid Cells
CD66b+
CD14+
• Sorted cell profile
ANGPT1 22233 18414 CLDN3 7339 4704 CD66B 362 258 ARG1 235 14399149 MMP9 168 293 CEACAM1 33 76 CLEC5A 9 43 CXCR2 7 8 COX2 5 11 MPO 3 4 CXCR4 3 4 CD47 3 4 S100A8 3 3 CD11B 2 3 S100A9 2 3 VEGFA 2 4 HIF1A 2 2 IL4RA 2 2 IL8 2 3 SIRPA 2 2 TGFB1 1 2 CD45 1 1 CD11C 1 1 ITGA4 1 1 •Genes expressed equally by both cell populations NFKB1 1 1 CD33 2 2 IL1B 2 3 ICAM1 3 2 LGALS9 3 4 TIMP1 4 6 IRF8 5 4 CD4 7 6 PDL1 7 3 CD68 7 5 CD14 9 9 HLA-DRA 11 11 ITGB7 33 13 CCR2 39 59 CD86 79 58 CXCL2 108030 0 TIM3 549618 94 RNA-seq data show distinct populations Clusters 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 Graphic View of Immune Checkpoint Blocker Combinations
25 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 MRL IOI Discovery MRL Histopathology Dewan Hossain Jennifer Yearley Alissa Chackerian Lakshmanan Annamalai Dan Cua Robbie Mcleod MRL Protein Engineering Joann O'Connor Laurence Fayadat-Dilman Juha Punnonen Daniel Cipriano Rob Kastelein Hai Ling Li
MRL Profiling & Expression PA Animal Facility Terri McClanahan Priscilla Lapresca Jeff Grein Joann Dominguez Wendy Blumenschein Ravi Tolwani Svetlana Sadekova Mike Lee JAX Labs Jerelyn Wong Dwayne Dexter Doug Wilson Rick Huntress Vanessa Peterson Lewis Vann Sarah Javaid James Keck Eric Gustafson Special Thanks John Mudgett (Genesis) Michael Brehm (UMASS)
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