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 Pancreatic tumor Melanoma tumor αpd1
Metastatic melanoma tumor promotes the development and trafficking of human myeloid cells 1x10 6 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 Melanoma mcd45 hcd11b hcd11b 5 hcd45 hcd3 hcd3
Cell populations in humanized mice with different tumors types Pancreatic Melanoma 0.24 32.3 Tumor CD33+ Mo-MDSC % similar in spleen with both tumor types CD66b 1.29 0.84 14.2 1.54 Spleen CD33+ Mo-MDSC much lower % in tumor Panco8 model CD66b+ Gr-MDSC not present in 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 a co8 S 5 CD40 927 124 CD69 450 118 CD8A 555 151 CD8B 108 15 CTLA4 123 16 ICOS 75 12 IFNG 97 13 IL2RA 42 14 IL17A 27 1 IL6 215 8 PD1 94 9 PDL1 1446 151 PDL2 150 40 Perforin 133 28 CD66b 0.24 1.29 Melanoma 32.3 14.2 MDSC signature ARG1 0 1092 CCR2 16 222 CD11B 53 539 CD11C 95 322 CD33 39 414 CD66B 0 1117 CD68 510 951 CLEC5A 30 234 IL10 4 17 MMP9 479 2862 MPO 4 9865 S100A8 258 117566 S100A9 459 57648 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 Counts 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 Anti-huGITR slows tumor growth, Tumor Volume mm 3 1000 Isotype Control MK-4166 800 600 400 200 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 mm 3.
Anti-hu GITR increases CD8:Treg ratio in spleen, and decreases activation markers on tumor Treg CD8:Treg ratio ICOS MFI on Treg CD8 + T cell:treg ratio 30 20 10 0 *** Spleen TILs MFI 25000 20000 15000 10000 5000 0 Spleen **** TILs Flow cytometry for CD8+:Treg ratio and ICOS MFI on day 4 post-dose. 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 IL-2 pg/m L 300 200 100 IFN γ pg/ml 100 50 0 Isotype Control MK-4166 0 Isotype Control MK-4166
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 CEACAM1 expression co-localizes with MPO DAPI Merge CEACAM1 is not expressed on CD8+ cells CD4+ Percent Positive 100 80 60 40 20 CD8+ No Culture IL-2 Only IL-2 + anti-cd3 0 No Culture IL-2 Only IL-2 + 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 d0 Monitor for weight loss and tumor growth d20 d50 Melanoma melanoma cell line sc Ave Tumor: 130 mm 3 n=9 / group Weight and tumor size measured weekly Blood sampling for PK (alternate groups 1 sample / 2wks End of study read-out FACS profiling- spleen, TILs Plasma- drug levels/ Gene expression Group 1 Treatment Isotype (higg4) Dose mg/k g Dosing RO Grou # Schedule 1 A p Don Size 2 ors 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-6018 Keytruda 10 2 q d3.5 sc. 9 3 Ceacam-1 binding on G-MDSC cells M-MDSC Gr-MDSC huigg4 Iso Ceacam1 FACS Iso
19 Study 16-M320-7929- In life tumor growth
Ceacam1 NSG-FACS T cells SPL Spleen Live Cells Single Cells hucd45+ CD3+ CD4+ CD8+ CD20+ Live Cells Single Cells hucd45+ hucd66b+ hucd14+ 20
21 Cell subsets CD4+ CD8+ cells in HIS mouse spleen
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 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 Genes expressed equally by both cell populations
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
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 MRL IOI Discovery Dewan Hossain Alissa Chackerian Dan Cua Robbie Mcleod Joann O'Connor Juha Punnonen Rob Kastelein MRL Histopathology Jennifer Yearley Lakshmanan Annamalai MRL Protein Engineering Laurence Fayadat-Dilman Daniel Cipriano Hai Ling Li MRL Profiling & Expression Terri McClanahan Jeff Grein Wendy Blumenschein Svetlana Sadekova Mike Lee Jerelyn Wong Doug Wilson Vanessa Peterson Sarah Javaid Eric Gustafson PA Animal Facility Priscilla Lapresca Joann Dominguez Ravi Tolwani JAX Labs Dwayne Dexter Rick Huntress Lewis Vann James Keck Special Thanks John Mudgett (Genesis) Michael Brehm (UMASS) 27