Human Immune System (HIS) mouse models for translational research. Barbara Joyce-Shaikh

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1 Human Immune System (HIS) mouse models for translational research Barbara Joyce-Shaikh

2 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

3 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

4 Tumor progression- Melanoma vs Pancreatic tumors with Keytruda Pancreatic tumor Melanoma tumor αpd1

5 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

6 Cell populations in humanized mice with different tumors types Pancreatic Melanoma Tumor CD33+ Mo-MDSC % similar in spleen with both tumor types CD66b Spleen CD33+ Mo-MDSC much lower % in tumor Panco8 model CD66b+ Gr-MDSC not present in Panco8 but is a large % in SKMEL CD33 Cells are gated on lymphocytes- hu CD45+ Live

7 T-cell cell populations in HIS mice in tumors microenvironments with PD1 treatment Pancreatic Melanoma Isotype CD Anti-PD1 CD8 Cells are gated on live hu CD45+

8 Immune profile comparison in TILs of implanted tumor lines Pancreatic T-cell signature a co8 S 5 CD CD CD8A CD8B CTLA ICOS IFNG IL2RA IL17A 27 1 IL PD PDL PDL Perforin CD66b Melanoma MDSC signature ARG CCR CD11B CD11C CD CD66B CD CLEC5A IL MMP MPO S100A S100A SIRPA CD33

9 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

10 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 Isotype Control MK 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.

11 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 *** Spleen TILs MFI 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 ** TILS isolated and cultured overnight IL-2 pg/m L IFN γ pg/ml Isotype Control MK Isotype Control MK-4166

12 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: / CAN Published 20 October 2016

13 Anti-Ceacam-1 study in HIS mice 13

14 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

15 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) ; DOI: CEACAM1 on tumor cells has been well characterized, the expression pattern of CEACAM1 on immune cells within the tumor microenvironment have been incompletely characterized..

16 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 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

17 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

18 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 MK q d3.5 sc Keytruda 2 q d3.5 sc 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 19 Study 16-M In life tumor growth

20 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 21 Cell subsets CD4+ CD8+ cells in HIS mouse spleen

22 Tumor resistance mechanisms-myeloid cells Myeloid Cells CD66b+ CD14+

23 Sorted cell profile ANGPT CLDN CD66B ARG MMP CEACAM CLEC5A 9 43 CXCR2 7 8 COX MPO 3 4 CXCR4 3 4 CD 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 CD CD11C 1 1 ITGA4 1 1 NFKB1 1 1 CD IL1B 2 3 ICAM1 3 2 LGALS9 3 4 TIMP1 4 6 IRF8 5 4 CD4 7 6 PDL1 7 3 CD CD HLA-DRA ITGB CCR CD CXCL TIM Genes expressed equally by both cell populations

24 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 25 Graphic View of Immune Checkpoint Blocker Combinations

26 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

27 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