Immuno-Modulatory Drug and Biomarker Discovery Using Onco-Hu Mice

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1 Immuno-Modulatory Drug and Biomarker Discovery Using Onco-Hu Mice Brian W. Soper, PhD Senior Technical Information Scientist Manager, Technical Information Services Dec, 2017

2 Presentation Overview wks NSG and propagation of patient derived xenografts Humanization of NSG and NSG -SGM3 mice Onco-Hu : Immuno-oncology efficacy data In Vivo Pharmacology Services 2

3 Presentation Overview wks NSG and propagation of patient derived xenografts Humanization of NSG and NSG -SGM3 mice Onco-Hu : Immuno-oncology efficacy data In Vivo Pharmacology Services 3

4 What Makes NSG Such a Good Host? NSG, NOD scid gamma NOD background contributes: Absence of hemolytic complement Reduced dendritic cell function Defective macrophages Optimal human hematopoietic stem cell engraftment (Sirpα allele) Scid mutation prevents development of mature T and B cells Il2rg deficiency Eliminates signaling from 6 distinct interleukins (IL-2, IL-4, IL-7, IL-9, IL-15, & IL-21) Blocks NK cell development Considerations: scid side effects: radiation sensitivity; genotoxic drugs may have higher toxicity JAX Mice 4

5 Mouse Tumor Database: PDX Model Resource Search by: o Tumor ID o Primary tumor site o Diagnosis o Gene variant In Vivo Pharmacology Services 5

6 NSCLC Adenocarcinoma: Drug Susceptibility Based on Gene Expression TM00186-PT TM00186-P0 Gene Expression KRAS Gly12Cys EGFR wild-type ERCC1 low TM00186-P1 TM00186-P2 Tumor fidelity through P2 In Vivo Pharmacology Services 6

7 NSCLC Adenocarcinoma: Gene Expression Predicts Drug Susceptibility Cisplatin: platinum-based DNA replication inhibitor Erlotinib: tyrosine kinase inhibitor acting on the epidermal growth factor receptor (EGFR) In Vivo Pharmacology Services 7

8 Presentation Overview wks NSG and propagation of patient derived xenografts Humanization of NSG and NSG -SGM3 mice Onco-Hu : Immuno-oncology efficacy data In Vivo Pharmacology Services 8

9 NSG vs. NSG -SGM3 Mice NOD scid gamma (NSG ) NOD.Cg-Prkdc scid Il2rg tm1wjl /SzJ (005557) The current gold standard for reconstitution of the human immune system Excellent functional T cell responses Limited human myeloid lineage development NSG -SGM3 NOD.Cg-Prkdc scid Il2rg tm1wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (013062) Promotes improved AML engraftment efficiency Improves normal human myeloid cell development after HSC transplantation Wunderlich M. et al Leukemia 24(10): PMID: Billerbeck E. et al Blood 117(11): PMID:

10 Humanization of NSG vs. NSG -SGM3 Direct Comparison Experimental Design Mice o o Female NSG, 3 weeks of age, 140 cgy (N=20) Female NSG -SGM3, 4 weeks of age, 100 cgy (N=9) Donor Cells o o Hu CD34+ HSC, ~130,000 cells/mouse All mice transplanted with cells from the same cord blood donor Blood Collection o o PBL collected 4, 6, 9, 12, 15 & 18 weeks post Tx Flow for hucd45, hucd33, hucd19, hucd3, hucd4, hucd8, hut reg In Vivo Pharmacology Services 10

11 Human Immune Cells in Peripheral Blood of Hu- NSG vs. Hu-NSG -SGM3: Absolute Counts Total Human Donor (cells/µl) Greater total cell numbers of hucd45 in NSG -SGM3 Greater numbers of myeloid cells in NSG -SGM3 Earlier B cell development and higher numbers of T cells in NSG -SGM3 HuCD33 Myeloid Cells (cells/µl) HuCD19 B Cells (cells/µl) HuCD3 T Cells (cells/µl) In Vivo Pharmacology Services 11

12 Human Immune Cells in Peripheral Blood of Hu- NSG vs. Hu-NSG -SGM3: Absolute Counts HuCD3 T Cells (cells/µl) Greater numbers of hucd3 T cells in NSG -SGM3 Greater expansion of hucd4 T cells in NSG -SGM3 Greater expansion of hucd8 T cells in NSG -SGM3 HuCD4 Helper T Cells (cells/µl) HuCD8 Cytotoxic T Cells (cells/µl) In Vivo Pharmacology Services 12

13 Onco-Hu TM Are Humanized Tumor-Bearing NSG TM and NSG TM -SGM3 Mice: The next step in cancer modeling Irradiation hucd34 HSC NSG (005557) NOD.Cg-Prkdc scid Il2rg tm1wjl /SzJ NSG -SGM3 (013062) NOD.Cg-Prkdc scid Il2rg tm1wjl Tg(CMV- IL3,CSF2,KITLG)1Eav/MloySzJ NSG Humanized NSG PDX Tumor or Cell Line Humanized NSG with Tumor (When tumors reach mm 3 mice are treated with therapeutics for 21 to 28 days) 13

14 Humanization of NSG Mice Has No Significant Impact on PDX Growth Kinetics Breast Lung Soft Tissue Carcinoma No difference between NSG and Hu-NSG mice on tumor growth curve No HLA match testing performed Fresh tumor tissue engraftment 100% take rate in NSG or Hu-NSG mice HuCD45+ more than 20% Wang et al., 2017 FASEB PMID:

15 Presentation Overview wks NSG and propagation of patient derived xenografts Humanization of NSG and NSG -SGM3 mice Onco-Hu : Immuno-oncology efficacy data In Vivo Pharmacology Services 15

16 Humanized Mice Are a Platform for Preclinical Immuno-Oncology Drug Discovery Merck & Co. o Anti-PD1 mab Pembrolizumab (Keytruda) o Anti-GITR mab Bristol-Meyers Squibb o Anti-CTLA-4 mab ImaginAb, Inc. Ipilimumab (Yervoy) o Anti-OX40 mab Pfizer, Inc. o IDO1 inhibition o Anti-PD-L1 Avelumab 16

17 Immune Cell Modulation in Humanized Mice, Recognition is Likely Allogeneic Yervoy (Ipilimumab); anti-ctla-4 Keytruda (Pembrolizumab); anti-pd-1 Ribas, 2012 N Engl J Med PMID:

18 Hu-NSG MDA-MB-231 Mice: Suppression of Breast Tumor Growth by Pembrolizumab Engrafted with 5x10 6 cells/mouse s.c. with matrigel MDA-MB-231 cell surface expression of PD-L1: 49.2% 18

19 Hu-NSG MDA-MB-231 Mice: Characterization of Human Tumor Infiltrating Cells & PD-1 Levels % HuCD45 Cells in Blood Prembolizumab treatment; o o Does not increase human cell percentages in peripheral blood Does not typically lead to greater tumor infiltration Prembolizumab is targeting human immune cells in the tumor and blocks binding of anti-pd-1 FACS reagent % HuCD45 Cells in Tumor % PD-1 on HuCD45 Cells in Tumor 19

20 Hu-NSG MDA-MB-231 Mice: In Situ Characterization of CD8 + T Cell Infiltration Vehicle Pembrolizumab DAPI CD8 Wang et al., 2017 FASEB PMID:

21 Hu-NSG MDA-MB-231 Mice: Efficacy Of Pembrolizumab Is CD8 + T Cell Dependent MDA-MB-231 Tumor Response in Non-Humanized Mice Tumor volume(mm 3 )+SEM MDA-MB-231 Tumor Response in Hu-NSG Mice Vehicle (Q5DX6)+isotype (Q7DX5) Pembrolizumab (Q5Dx6)+isotype (Q7DX5) Pembrolizumab (Q5DX6)+anti-CD8 (Q7DX5) Days Wang et al., 2017 FASEB PMID:

22 Hu-NSG -SGM3 MDA-MB-231 Mice: Suppression of Breast Tumor Growth by Pembrolizumab Engrafted with 5x10 6 cells/mouse s.c. with matrigel MDA-MB-231 cell surface expression of PD-L1: 95.1% In Vivo Pharmacology Services 22

23 Hu-NSG BR1126 TNBC PDX Mice: Pembrolizumab Inhibits Tumor Growth Pembrolizumab (Keytruda); anti-pd-1 mab Tumor PD-L1 surface expression: 56.9% Hu-CD45 engraftment in Hu-NSG >25% HLA match CD34 + HPC donor Tumor BR1126 HLA-C, DPA1 HLA-A,DQA1, DPB1, DPA1 HLA-C, DPA1 In Vivo Pharmacology Services 23

24 Hu-NSG LG1208 NSCLC Lung PDX Mice: No Inhibition of Tumor Growth By Pembrolizumab HLA match Tumor LG1208 CD34+ HPC Donor HLA-DRB4, DPA1 24

25 Hu-NSG LG1208 NSCLC Lung PDX Mice: PD-1 and PD-L1 Expression in Tumor Tissue Pembrolizumab entered tumors and bound to CD45+ PD-1+ leukocytes PD-1 Expression: Activated T cells, Tregs, B cells, NK cells and monocytes; occasionally on tumor cells PD-L1 Expression: Mainly on tumor cells and normal tissues; also on T cell, B cells, macrophages, DCs 25

26 Hu-NSG LG1306 Lung PDX Mice: Variability in Drug Response Provides Diagnostic Opportunity Unique tumor populations in individual mice Opportunity to explore differences in gene expression and immune cell infiltration Pembro Treated Arm In Vivo Pharmacology Services 26

27 Hu-NSG -SGM3 LG1306 Lung PDX Mice: Pembrolizumab & Ipilimumab Inhibit Tumor Growth HuCD45+ in whole blood: 36-81% HuCD3+/HuCD45: average 14.3% LG1306 PD-L1 surface expression: 89.1% In Vivo Pharmacology Services 27

28 Human CD45+ & CD33+ Infiltration into PDX Tumors in Humanized NSG TM -SGM3 Mice PDX model % hcd45+ % CD33+ of CD45+ BR1126 (TNB) ~15 J99327 (TNB) LG1306 (Lung) H460 cell line (Lung) SK1260 (Melanoma) MDA-MB-231 (TNB cell line) ~20 ~5 CN1572 (Colon) ~3 ~35 (mdcs) SA0209 (Sarcoma) PS4050 (Melanoma) (TAMs) TM1149 (Melanoma) (TAMs) 28

29 OX40 in Anti-Tumor Immunity CD4+ T Cells CD4+ T cell engages tumor peptide through MHC II, OX40 upregulated OX40-specific mab binds and induces an activation signal Cytokine production CD8+ T Cells Cytokine production by CD4 cell provides help to CD8+ CTL CD8 CTL produces perforin and granzyme, killing tumor STIMULATORY+MOLECULE+OX40 29

30 Hu-NSG MDA-MB-231 Mice: Suppression of Breast Tumor Growth by Anti-OX40 Jean Gudas VP, Research & Development ImaginAb, Inc. M ean Tum or Volum e (m m 3 ) +/- SEM Tw o Donors (#5038, #5040) ** *** * * * G roup 1 (Vehicle) G roup 3 (anti-o X40) * p <0.05 ** p <0.01 *** p <0.005 **** p < tailed unpaired t -te s t 30 Study Days

31 ADCC & ADCP Mediated Depletion of Treg s by mdta-1 (anti-gitr mab) Mouse Syngeneic Tumor Studies DTA-1 (mouse anti-gitr mab) Decreased Suppression of CTL s Tumor Cell Killing Adapted from: Targeting regulatory T cells in tumor immunotherapy 31

32 Hu-NSG SK-MEL-5 Mice: Suppression of Human Melanoma by MK-4166 (anti-gitr mab) Depletion of Treg s in Tumor Suppression of Tumor Growth Increased Effector Cytokines in Tumor Merck Mahne et al., 2016 Cancer Res PMID:

33 Hu-NSG MDA-MB-231 Mice: Suppression of Breast Tumor Growth by IDO1 Inhibition & Avelumab IDO1; immunomodulatory enzyme, inhibition improves T cell response Avelumab; anti-pd-l1 mab Pfizer Manfred Kraus Stephanie Shi Xianxian Zheng Martin Wythes In Vivo Pharmacology Services 33

34 Onco-Hu Platform Summary PDX growth in humanized mice is not grossly effected by HLAtype matching o Tumor growth kinetics are similar in humanized and non-humanized hosts o ~15% of PDX tumors fail to grow in humanized mice Some tumor cell/donor immune cell combinations are resistant to therapy, mimicking clinical observations Human immune effector and modulatory cells infiltrate human tumors at different frequencies based on tumor type o Some tumors have high T cell infiltration o Some tumors have high myeloid cell infiltration PDX engrafted Hu-CD34-NSG and Hu-CD34-SGM3 respond to immuno-modulators in vivo; anti-pd-1, anti-pd-l1, anti-ctla4, anti-ox40, anti-gitr, IDO1 inhibition In Vivo Pharmacology Services 34

35 Acknowledgements JAX In Vivo Pharmacology Services o James Keck, Minan Wang, Li-Chin Yao, Mingshan Cheng and Danying Cai JAX Genomic Medicine o Karolina Palucka JAX Mammalian Genetics o Lenny Shultz, Rick Huntress, Carol Bult, Susie Airhart and Ed Liu UMASS o Dale Greiner and Mike Brehm 35

36 Thank You! wks Contact JAX Technical Support JAX Mice, Clinical & Research Services

37 Hu-NSG -SGM3 PS4050 Melanoma PDX: Pembrolizumab Inhibits Tumor Growth In Vivo Pharmacology Services 37

38 Hu-NSG -SGM3 PS4050 Melanoma PDX: Human Immune Cell Infiltration In Vivo Pharmacology Services 38

39 Hu-NSG -SGM3 PS4050 Melanoma PDX: Characterization of Infiltrating T Cells PS4050 T cells in PS4050/Hu-SGM3 Markers Levels CD3T CD3 20% of CD45+ Treg CD25high D127 lowfoxp3+ 5% of CD45+ Exhausted CD4T PD1+ CD4+ 10% of CD4+ Exhausted CD8T PD1+ CD8+ 55% of CD8+ Naïve CD8T CCR7+CD45RA+CD8+ 3% of CD8+ Central memory CD8T CCR7+CD45RA-CD8+ 2% of CD8+ Effector memory CD8T CCR7-CD45RA- CD8+ 95% of CD8+ Naïve CD4T CCR7+CD45RA+CD4+ 74% of CD4+ Central memory CD4T CCR7+CD45RA-CD4+ 1% of CD4+ Effector memory CD4T CCR7-CD45RA- CD4 + 25% of CD4+ In Vivo Pharmacology Services 39