Evaluation of immunomodulatory agents in syngeneic models and immune cell phenotyping of humanized mouse models carrying patient-derived tumors

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1 Evaluation of immunomodulatory agents in syngeneic models and immune cell phenotyping of humanized mouse models carrying patient-derived tumors Philippe Slos 2 nd Annual ICI March 15-17, 2016 Boston

2 Introduction Syngeneic models at Oncodesign Case studies Humanized mouse models PDX panels Conclusions & Perspectives page 2

3 Introduction page 3

4 The needs: to improve predictivity of the models Adapted From Wartha et al. Pharmacology & Therapeutics, 2014 Suitable models for immuno-oncology? Two key components Immune System Tumor Immune system and tumor could be from mouse : standard syngeneic models, GEMM from human : standard xenografts or PDXs on humanized mouse Fit-for purpose use of mouse models to improve predictivity of cancer therapeutics evaluation page 4

5 Quite all is immunomodulation From Galluzzi et al. Oncotarget, 2014, Vol. 5, No. 24, page 5

6 SYNGENEIC MODELS AT ONCODESIGN page 6

7 Syngeneic Models at Oncodesign (1) Good characterization Characterization not complete Limited characterization Model Organ Host rodent species Injection site Tested drugs Metastatic Dissemination 4T1 Breast carcinoma BALB/C mouse OT Radiotherapy, Doxorubicin, PD-1 mab, CTLA- 4 mab A20 B cell lymphoma BALB/C mouse SC IV CTLA-4 mab, PD-1 mab AB12 Mesothelioma BALB/C mouse SC AY27 Bladder transitional carcinoma B16-F10 Malignant melanoma C57BL/6 mouse SC IV IC Ficher rat OT BCG, CDDP Paclitaxel, Doxorubicin, TMZ, PD-1 mab, Cyclophosphamide, CTLA-4 mab, PD-L1 mab C1498 AML C57BL/6 mouse SC IV C26 Colon adenocarcinoma BALB/C mouse IP SC CDDP C38 Colon carcinoma C57BL/6 mouse SC Radiotherapy, CPT-11, L-OHP, CTLA-4 C-51 Colon carcinoma BALB/C mouse SC CPT-11, LOHP C6 Glioma Wistar rat OT SC BCNU CT26 Colon carcinoma BALB/C mouse OT SC *DHD/K12/TRb = PROb Colon adenocarcinoma BDIX rat IP SC *DHD/K12/TSb = REGb Colon adenocarcinoma BDIX rat SC EMT6 Breast carcinoma BALB/C mouse OT SC 5-FU, CPT-11, PD-1 mab, CTLA-4 mab, PD-L1 mab CDDP, Doxorubicin, BCG, 5-FU, L-OHP, Mitoxantrone Doxorubicin, Paclitaxel, Radiotherapy, PD-1 mab, CTLA-4 mab GS-9L Gliosarcoma Ficher rat OT SC BCNU, TMZ page 7 * Ask for rat availability

8 Syngeneic Models at Oncodesign (2) Good characterization Characterization not complete Limited characterization Model Organ Host rodent species Injection site Tested drugs Metastatic Dissemination *GV1A1 Glioma BDIX rat OT SC Hepa1-6 Hepatocarcinoma C57BL/6 mouse OT Sorafenib, CTLA-4 mab L1210 Lymphocytic leukemia DBA/2 mouse IP BCNU, CDDP, Doxorubicin, 5-FU L-OHP, Mitoxantrone L1210/CDDP Lymphocytic leukemia DBA/2 mouse IP CDDP, L-OHP LLC1 Lewis lung carcinoma C57BL/6 mouse IV SC Vinorelbine, Cyclophosphamide CTLA-4 mab, PD-1 mab MAT-LyLu Prostate adenocarcinoma Cop rat SC MBT-2 Bladder carcinoma C3H mouse OT SC BCG, Mitomycin C, PD-1 MPC-11 Plasmacytoma BALB/C mouse SC NBT-II Bladder carcinoma Wistar rat OT P388 Leukemia DBA/2 mouse IP P388/ADR Leukemia DBA/2 mouse IP BCNU, Doxorubicin, Mitoxantrone BCNU, Doxorubicin, Vinblastine R3327-AT3 Prostate adenocarcinoma Cop rat SC R3327H Prostate adenocarcinoma Cop rat OT SC RenCa Kidney carcinoma BALB/C mouse OT SC Paclitaxel, Mitoxantrone, Trastuzumab, Leuroprolin, Castration, Decapeptyl, Bicalutamide, Radiotherapy CDDP, Paclitaxel, Radiotherapy, Gemcitabine, Carboplatin, Sorafenib page 8 * Ask for rat availability

9 CASE STUDIES page 9

10 Tumor volume (mm 3 ) Tumor volume (mm 3 ) Tumor volume (mm 3 ) 4T1 Breast Carcinoma Orthotopic Model Immune checkpoint inhibitors Mice were OT injected with 4T1 murine breast tumor cells at D0. Mice were randomized based on tumor volume at D13 and treated IP with mab against CTLA-4 (clone 9H10) or PD-1 (clone RMP1-14) at 10 mg/kg/inj (Q5Dx3). T/C % (D21) Control 100 CTLA-4 mab 66 PD-1 mab 68 2,000 1,800 1,600 1,400 1,200 1, ,000 1,800 1,600 1,400 1,200 1, ,000 1,800 1,600 1,400 1,200 1, Control Time (Days) CTLA-4 mab Time (Days) PD-1 mab Time (Days) page 10

11 Tumor volume (mm 3 ) Tumor volume (mm 3 ) Tumor volume (mm 3 ) 4T1 Breast Carcinoma Orthotopic Model Correlation of anti-tumoral activity of ICI against tumor and metastasic devolopment Mice were OT injected with 4T1 murine breast tumor cells at D0. Mice were randomized based on tumor volume at D13 and treated IP with mab against CTLA-4 (clone 9H10, Syrian Hamster IgG1) or PD-1 (clone RMP1-14, Rat IgG2a) at 10 mg/kg/inj (Q5Dx3). 2,000 1,800 1,600 1,400 1,200 1, ,000 1,800 1,600 1,400 1,200 1, ,000 1,800 1,600 1,400 1,200 1, Control Time (Days) CTLA-4 mab Time (Days) PD-1 mab Time (Days) page 11

12 Tumor volume (mm 3 ) Tumor volume (mm 3 ) Tumor volume (mm 3 ) Tumor volume (mm 3 ) EMT6 Breast Carcinoma Ectotopic Model Immune checkpoint inhibitors 1,800 1,600 1,400 1,200 1, Control CTLA4 mab PD1 mab Time (Days) Mice were SC injected with EMT6 murine breast tumor cells at D0. Mice were randomized based on tumor volume at D7 and treated IP with mab against CTLA-4 (clone 9H10) or PD-1 (clone RMP1-14) at 10 mg/kg/inj (Q5Dx3). T/C % (D27) Control 100 CTLA-4 mab 4 PD-1 mab 63 2,000 1,800 1,600 1,400 1,200 1, ,000 1,800 1,600 1,400 1,200 1, ,000 1,800 1,600 1,400 1,200 1, Control Time (Days) CTLA-4 mab Time (Days) PD-1 mab Time (Days) page 12

13 EMT6 Breast Carcinoma Ectotopic Model Immune checkpoint inhibitors : Flow Cytometry analysis of FoxP3+ CD4+ T cells TDLN Tumor Control ID#8923 TV 597 mm 3 CTLA-4 mab ID#8901 TV 56 mm 3 Median value Median value without non-responding tumors ( ) CTLA4 mab Non-responding tumors page 13

14 Syngeneic models with immune checkpoint inhibitors : Teff/Treg ratio increase correlated with anti-tumor activity 4T1 Control CT26 Control EMT6 Control CTLA-4 mab CTLA-4 mab CTLA-4 mab Tumor page 14

15 CT26 Colon Carcinoma Ectotopic Model: Teff/Treg ratio increase correlated with anti-tumor activity Combined Immune checkpoint inhibitors Tumor Mice were SC injected with CT26 murine colon tumor cells at D0. Mice were randomized based on tumor volume at D9 and treated IP with mab against CTLA-4 (clone 9H10) at 5 mg/kg/inj, PD-1 (clone RMP1-14) at 10 mg/kg/inj (TWx2) or combination of both. Mice were terminated at D20 and tumors analyzed by Flow Cytometry for T cells content. page 15

16 Checkpoint inhibitor summary table Model Checkpoint Inhibitors NGS Exome Seq. Name Type Site Strain CTLA-4 PD-1 PD-L1 4T1 Breast OT BALB/C X A20 BCL SC BALB/C X B16-F10 Melanoma SC C57BL/6 X C38 Colon SC C57BL/6 CT26 Colon SC BALB/C X EMT6 Breast SC BALB/C X Hepa1-6 Liver OT C57BL/6 LLC Lung SC C57BL/6 X MBT-2 Bladder OT SC C3H RenCa Kidney OT BALB/C X X TC < 42% 42% < TC < 80% TC > 80% page 16

17 Exome sequencing and sensitivity to ICIs Total number of mutations PD-1 / PD-L1 Targeting mabs T/C (%) < 80 is used as cut-off criteria for responder and non-responder populations CTLA-4 Targeting mab Champiat S et al OncoImmunology. Exomics and immunogenics. Bridging mutational load and immune checkpoints efficacy. Rizvi NA et al Science 348:6230 page 17

18 Immunological endpoints Cytokines release quantification ELISA / CBA / Luminex Immune cells phenotyping and quantification Flow Cytometry, IHC Immune cells functionality Cell preparation and/or sorting using FACS or magnetic beads Cytotoxic activity based on Cr 51 release assay Proliferation ability based on H 3 -Thd incorporation assay Specific cytokine release based on ELISpot assay CFU page 18

19 Immune infiltrate phenotyping Immune cell phenotyping using Flow Cytometry page 19

20 Humanized mouse models page 20

21 Recipient mice IL2rg impaired Mouse NOG & upgraded NOG Nude Mouse No T cells Humoral immunity intact NK cell activity ++ Hemolytic complement + CB17-SCID Mouse No T cells or B cells Radiosensitive NK cell activity + Hemolytic complement + NOD-SCID Mouse Defects in innate immunity Impaired macrophage activation NK cell activity +/- Hemolytic complement - Defects in innate & adaptive immunity Impaired macrophage activation NK cell function - Hemolytic complement - BRGS Mouse Defects in innate & adaptive immunity Impaired macrophage activation NK cell function - Hemolytic complement + IMMUNODEFICIENCY ENGRAFTMENT WITH HUMAN IMMUNE CELLS page 21

22 PDX panels page 22

23 PDX models: The IMODI consortium Innovative MODels Initiative Agaisnt Cancer Through collaborative partnerships with Clinicians and scientific experts we are building a collection of > 200 PDX models 4 pharma companies 6 CROs 13 academic groups Total budget 41 M Grant 13.4 M 7 years Oncodesign - 20, rue Jean Mazen - BP Dijon Cedex - France Tel. : +33(0) Fax : +33(0)

24 PDX Collection PDX Available for Services Colon 61 Breast 20 Lung NSCLC 17 Pancreas 13 Brain 5 Ovarian 3 Bladder 3 Head & Neck 3 Lung SCLC 3 Kidney 2 Liver 2 Not only numbers, but an extensive characterization and knowledge of our models (Tumor/Gut microbiota of the host/biomarker identification). PDX Ongoing Development Lung 30 Liver 30 Ovarian 30 Breast 20 Pancreas 20 AML 20 Myeloma 10 Lymphoma 10 Prostate 10 AML 2 Prostate 1 Oncodesign - 20, rue Jean Mazen - BP Dijon Cedex - France Tel. : +33(0) Fax : +33(0)

25 Tumor volume (mm 3 ) Tumor volume (mm 3 ) BRGS mice humanized with HSCs and bearing PDX tumors Growth curves are similar if PDX injected in SWISS-Nude mice 1, LUN-NIC , IM-OVA Time (Days) Time (Days) BRGS mice: BALB/C Rag2 -/- IL-2Rγ c -/- SIRPa NOD (Legrand N et al. PNAS : ) page 25

26 Flow Cytometry analyses General comments Blood, spleen, TDLN, tumor, bone marrow collected Identified immune populations Circulating and central human lymphoid and myeloid cells Lymphoid cells: T cells: CD8, CD4 and Treg evidenced B cells NK cells Myeloid cells: Monocytes others page 26

27 Flow Cytometry analyses : blood Blood : non-tumor bearing mice Lymphoid cells ~90% vs Myeloid cells ~10% T cells: total CD3 ~49% with CD8 (~5%), CD4 (~80%) and Treg (~11%) B cells: ~48% NK cells: ~2% Monocytes: ~5% Other myeloid cells: ~5% Proportion dictated by the murine host! page 27

28 Flow Cytometry analyses : blood Blood : non-tumor bearing mice vs tumor bearing mice Change induced by tumor! increase in Treg change in T/B ratio page 28

29 Flow Cytometry analyses : spleen Spleen: non-tumor bearing mice Lymphoid cells ~98% vs Myeloid cells ~2% T cells: total CD3 ~40% with CD8 (~4%), CD4 (~76%) and Treg (~13%) B cells: ~40% NK cells: ~1% Monocytes: ~0.5% Other myeloid cells: ~1.5% page 29

30 Flow Cytometry analyses : spleen Spleen : non-tumor bearing mice vs tumor bearing mice Change induced by tumor! increase in Treg change in T/B ratio page 30

31 Foxp3 hcd20 Flow Cytometry analyses : spleen Spleen : non-tumor bearing mice vs tumor bearing mice Healthy Tumor-bearing ID20 ID36 ID11 ID37 T/B= 3.0 T/B= 1.3 T/B= 0.1 T/B =0.6 hcd hcd4 page 31

32 Foxp3 Flow Cytometry analyses : Tumor Tumor Lymphoid cells & Myeloid cells T cells: ~55% B cells: ~4% NK cells: ~6% Monocytes: ~3% Other myeloid cells: ~5% hcd4 ID9 Treg identified within the tumor (n=2) page 32

33 BRGS mice humanized with HSCs and bearing PDX tumors Conclusions Growth of SC PDX tumors (ovary and lung) Presence of circulating and central lymphoid and myeloid cells Changes induced by tumor evidenced in blood and spleen increase in Treg change in T/B ratio Presence of Tregs within the tumor Model of interest for further evaluation of compounds targeting Tregs or interfering with CD8/CD4 populations (ipilimumab, nivolumab ) page 33

34 Conclusions & Perspectives page 34

35 Summary table Tumor model HSCs NKs PBMCs T cells Name Histology Injection IV IH IP IV IP IV SC IV 786-O Kidney clear cell adenocarcinoma SC 3 BT-474/matrigel Her2+ Breast ductal carcinoma SC Daudi Burkitt's lymphoma, B cells IV 1 x FaDu Head and Neck carcinoma SC 1 HCT116 Colorectal carcinoma SC x IM-OVA-503 PDX: Ovary SC 1 Jeko-1 Mantle Cell Lymphoma, B cells IV x KARPAS-299 Human T-cell non-hodgkin lymphoma IV 4 LoVo Colorectal adenocarcinoma SC 1 2 LUN-NIC-0084 PDX: NSCLC SC 1 MCF-7 Breast adenocarcinoma SC 2 MKN74/matrigel Gastric carcinoma (stomach) SC 1 MOLM13 acute myeloid leukemia IV x NA (no tumor) NA (no tumor) NCI-H929/matrigel Plasma cell myeloma SC 2 NCI-N87 Gastric carcinoma (stomach) SC 1 NIH:OVCAR-3 Ovarian adenocarcinoma SC 2 Raji* B cell lymphoma IV 1 Ramos B cell lymphoma IV 1 3 RPMI 8226* Multiple myeloma SC 1 * Model not growing properly in humanized condition page 35

36 Immunological endpoints Cytokines release quantification ELISA / CBA / Luminex Immune cells phenotyping and quantification 10-color Flow Cytometry Immune cells functionality Cell preparation and/or sorting using FACS or magnetic beads Cytotoxic activity based on 51 Cr release assay Proliferation ability based on CFSE / 3 H-Thd incorporation assay Specific cytokine release based on ELISpot assay Co-culture assay CFU page 36

37 Ongoing & Future Development To be able to evaluate human specific I-O related compounds Humanization with HSCs Mouse strain : BRGS To validate efficacy of approved immune checkpoint inhibitors Humanization with PBMCs CD4+ and CD8+ T cells from PBMCs To characterize the tumor immune infiltrate To validate efficacy of approved immune checkpoint inhibitors In vitro assays Treg suppresor assay Teff tumor cell killing efficacy page 37

38 Special thanks to : Jean-François Mirjolet - Caroline Mignard Jean-Marie Charpin