Outline. Case studies & Tumor Immunology platform overview. In vitro assay panel. In vivo models. Clinical and pre-clinical sample analyses

Transcription

1 Immuno-Oncology May, 217

2 Outline Case studies & Tumor Immunology platform overview In vitro assay panel In vivo models Ex vivo analyses Clinical and pre-clinical sample analyses 2

3 Case 1: an IS Project Kick off 1 Early lead Cpd Month Month 1 Month 2 Month 9 Goal Chemistry Protein production Biochemistry Assays Cellular Assays hpbmc Assays Syngeneic tumor models ADME Assays PK/PD Crystal Structures Chemistry: ~5 cpds Protein: active protein delivered BioChemistry Assays: on line Cellular Assays: basic assay on line, build monoclonal hpbmc Assays: Optimization Efficacy & PK/PD: 1 st studies w/ ref. ADME Assays Crystal Structures Crystal Structures: Crystal structure with Ref Chemistry: ~5 cpds; grams of scale ups Protein: supply for all biochemical assays BioChemistry Assays: near 1, cpds; MOA Cellular Assays: near 5 cpds Efficacy & PK/PD: multiple cpds; combination ADME Assays: multiple cpds tested Crystal Structures: 3 solved, more crystalized Initial safety Key in vivo assays 3

4 Case 2: Pharmacology for a Lead Compound A lead cpd w/ good PK, efficacy in 1 CDX model Efficacy in orthotopic 4T1 model, MOA: immune Combination efficacy Immune memory & 1 o tumor, metastasis response Specificity 1 st syngeneic More syngeneic Combination i Re challenge hll model & nude mice Tumor volume (mm 3 ) Orthotopic 4T1 Model 2 Vehicle CPD X,5mg/kg *** 15 CPD X,1mg/kg *** CPD X,2mg/kg *** Days post administration Tumor volume(mm3) Tumor growth in nude mice (4T1 cell line) vehicle control CPD X, 5mg/kg CPD X, 1mg/kg CPD X, 2mg/kg Days post administration Tumor volume (mm 3 ) 25 IgG control Tumor growth (CT26) Compound X PD-L1 PD-L1+Cpnd X Days post administration Tumor volume (mm 3 ) CT26 Tumor growth- 118 days after 1st inoculation of CT26 15 Naive mouse Rechallenge mouse Days post inoculation g) Lung weight(g vehicle * CPD X, 5mg/kg Lung Weight ### *** *** CPD X, 1mg/kg CPD DX, 2mg/kg naive Tumor volume (mm m 3 ) Tumor growth (CT26) Vehicle Control CPD X, 25mg/kg CPD X, 5mg/kg CPD X, 1mg/kg CPD X, 2mg/kg Days post administration al Percent surviva 1 CT26 model 5 Vehicle Control Normal IgG PD-L1 Compound X *** PD-L1+Compound X 2 4 Days post administration ** Tumor volume (mm m 3 ) A2 Tumor growth- 3 months after 2nd challenge of CT Naive Rechallenged mice NS Days post inoculation NS, not statistically significant; Repeated measurement Two-way ANOVA 4

5 Immuno-Oncology Platform Overview Immune Modulating Target Ab generation Hybridoma Phage display higtransgenic mice Chemical synthesis MedChem Compound generation Lead identification Binding assays Target specific assays Target specific assays: enzyme, cell based In vitro assays Compound pool Lead optimization Humanization Affinity maturation Syngeneic models Combination therapies Ex vivo analyses Early leads Lead characterization MLR& other hpbmc assays In vivo efficacy (humanized mouse model, htg KI mice) hpbmc assays Antigen specificity it Lead(s) () Candidate Candidate 5

6 In Vitro Immuno-Oncology Assay Panel ID Assay Type Cell Type In CP Panel 1 Biochemical Assays Cell free 2 Mixed Lymphocyte Reaction (MLR) Primary human DC + T cells 3 T cell activation assay (anti CD3/CD28) Primary human T cells 4 T cell activation assay (SEB superantigen) Primary human T cells 5 T cell activation assay (engineered tumor cells) Primary human T cells + engineered tumor morcells 6 CMV antigen recall assay Primary human T cells 7 Target specific reporter gene assay Engineered cell lines 8 ELISpot Primary human PBMC 9 T cell cytotoxicity assay Primary human T cells + tumor cells In development 1 Antibody Dependent Cellular Cytotoxicity (ADCC) Primary human NK cells 11 Complement Dependent Cytotoxicity (CDC) Complement 12 Treg differentiation assay Primary human T cells 13 Treg differentiation assay Primary mouse T cells 14 Th1 polarization assay Primary human T cells 15 Th2 polarization assay Primary human T cells 16 Th17 polarization assay Primary mouse T cells 17 Macrophage differentiation/ polarization assay Primary mouse macrophages 6

7 BioChemistry Assays for Cancer Immunology Targets Signaling/metabolism pathway Amino acid catabolism Signalling of tumour derived extracellular ATP Adenosine signalling Adenosine production Target IDO, TDO, ARG1/2, inos, PDE5, P2X7, P2Y11 A2A, A2B CD39, CD73 Elevation of cyclic AMP Chemokines and chemokine receptors Recognition of foreign organisms to activate the immune response Signal transduction: kinase inhibitors DNA sensor pathways COX2, EP2R, EP4R, CXCR1, CXCR2, CXCR4, CCR2, CCR5 TLR4, TLR7, TLR8, TLR9 ALK4, ALK5, BRAF V6E, RON, CSF1, PI3Kδ, PI3Kγ STING Biochemical i assays ready for green marked kdtargets 7

8 Mixed Lymphocyte Reaction (MLR) MLR IL-2 Dose-Response MLR IFN Dose-Response /ml IL-2 pg/ IFN- pg g/ml Antibody Conc..1 ug/ml.3 ug/ml.1 ug/ml.3 ug/ml 1 ug/ml 3 ug/ml 1 ug/ml Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 Ab1 Ab2 Ab3 Ab4 Ab5 Ab6 Dendritic cells derived from primary human monocytes are co cultured with allogeneic CD3 T cells for 5 days. IFN and IL 2 production were measured to evaluate the effect of immune checkpoint inhibitors on MLR. 8

9 CMV Antigen-Recall Assay /ml) IFN- (pg anti PD-1 antibody stimulation ug//ml.1 ug//ml 1 ug//ml 1 ug//ml IFN- pg g/ml CMV-recall IFN- ug/ml.1 ug/ml.32 ug/ml.1 ug/ml.317 ug/ml 1.1 ug/ml ug/ml 1. ug/ml 5 days 6 days 7 days Ab1 Ab2 Ab3 IgG positive antibody hpbmcs from donors who were previously infected by CMV are re challenged with CMV antigen in vitro, to evaluate the effect of anti PD1 antibody on antigen specific T cell response. 9

10 T Cell-Tumor Coculture Assay IFN, IL 2 TCR PD 1 Primary human T cell Tumor/OS 8/ PD L1 cell OS 8 PD L1 1

11 Representative Luciferase Reporter Assay 4 1BB signal pathway 293F hcd137 NF kb RE luc2p Anti 4 1BB antibodies Urelumab can promote NF kb activation through 4 1BB signal pathway 11

12 Antibody-Dependent Cell-mediated Cytotoxicity 12

13 Complement-Dependent Cytotoxicity 13

14 Treg Differentiation Human naïve T cells +IL 2 +IL 2+TGFβ +IL 2+TGFβ+ treatment Mouse Splenocytes 14

15 Mouse M1 Macrophage Differentiation FCM gating strategy: Single cells F4/8 + cells inos + cells LPS/IFN r Control LPS/IFN r Dexamethasone SSC inos FITC 15

16 Mouse M2 Macrophage Differentiation FCM gating strategy: Single cells F4/8 + cells CD26 + cells IL 4/13 Control IL 4/13 Compound X SSC CD26 BV65 16

17 In vivo Immuno-Oncology Models ChemPartner Humanized Mouse Models Model Name Readiness hpd1 Knockin mice hctla4 Knockin mice h4 1 BB Knockin mice Xenografts with hpbmc adoptive transfer Under development Under development ChemPartner Syngeneic Models Model Name Cell Type SOC Data CT26.WT mouse colon carcinoma (fibroblast) PD1, PDL1, CTLA4 MC 38 mouse colon carcinoma PD1, CTLA 4, cisplatin 4T1 mouse mammary gland carcinoma PD1, Cisplatin A2 B lymphocyte reticulum cell sarcoma B16 F1 mouse melanoma carcinoma tissue PD1 EL4 Lymphoma tumor Doxorubicin EMT6 mammary carcinoma mouse Hepa 1 6 Hepatoma Sorafenib, Cisplatin H22 liver cancer J558 plasmacytoma, myeloma J774A.1 Reticulum Cell Sarcoma; monocyte; macrophage LL/2 (LLC1) Lewis lung carcinoma Cisplatin M 7 Fibrosarcoma MPC 11 B lymphocyte Renca Renal adenocarcinoma mouse Sunitinib, PD1 WEHI 3 leukemia B16 F mouse melanoma carcinoma cell B16 F1 mouse melanoma carcinoma cell JC mammary gland P815 mastocytoma L121 lymphocytic leukemia Clone M3 [Cloudman S91 melanoma] melanoma Most frequently used 17

18 Human Molecule Knock-in Mouse Models ) Tumor volume (mm 3 ) Tumor growth (MC38 model in hu-pd-1 KI mice) higg 1mg/kg x4 anti-pd-1 1mg/kg x4 * *** *** Days post administration *, P<.5; ***, P<.1; Repeated-measurement Two-way ANOVA 3 ) volume (mm 3 Tumor 15 1 Tumor growth (MC38 model in hu-ctla4 KI mice) higg 1mg/kg x4 anti-ctla4 1mg/kg x4 5 *** Days post administration i ti ***, P<.1; Repeated-measurement Two-way ANOVA. 18

19 Under Development: Humanized Animal Models Human Tumor cells Expressing target Target can be inhibitory molecules such as PD L1 (PD1), GAL 9 (TIM 3) Target can be expressed by tumor naturally or transfected Surrogate Ab or mouse cross reactive Ab are suitable for testing in syngeneic tumor models 19

20 Adoptive Transfer of hpbmc hcd45+ cell numbers in mice spleen (day 2) hcd45+ cell numbers in 6x1 4 tumor cells (day 2) % of hcd45+ cell SCID-beige ** SCID-beige+tumor NCG ** NCG+tumor hc CD45+ cell numbe er ** NCG with tumor SCID with tumor +PBMC SCID with tumor r +PBMC NCG with tumor hcd45+ cells in spleen and infiltrating in tumors in NCG mice is higher than in SCID mice 2

21 4T1 Non-Resection Model: Efficacy Study Tumor volume(mm3 3) Tumor growth (4T1 model) 8 PBS 6 Biologics X 4 2 *** Days post administration Lung nodule count (4T1 model) Nod dule Counts 2 15 *** 1 5 PBS Biologics X en weight(g) Sple Spleen weight (4T1 model).7.6 * PBS Biologics X In this model setting, drug efficacy on primary tumor, metastasis (lung tumor nodules) and biomarker (myeloid expansion) can be studied simultaneously. 21

22 CT26 Model: SOC & Combination Immunotherapy Murine colon carcinoma model CT26 is one of the most used models testing immunotherapy Tumor volume (m mm 3 ) 25 IgG control Compound X PD-L1 Tumor growth (CT26) PD-L1+Cpnd X Days post administration 22

23 CT26 Model: SOC & Combination Immunotherapy CT26 model Pe ercent sur rvival 1 5 Vehicle Control Normal IgG PD-L1 Compound X *** PD-L1+Compound X 2 4 Days post administration ** Vehicle Control Normal IgG PD-L1 Compound X PD-L1+Compound X Median survival Undefined ***, p<.1 w hen compared w ith Vehicle control group, Log-Rank Test. **, p<.1 w hen compared w ith PD-L1 or Compound X groups, Log-Rank Test. 23

24 Ex vivo Analyses Phenotype: Flow cytometry: tumor, tumor draining lymph nodes, blood, splenocytes, others Profiling: soluble factors; expression; Functional assay: Proliferations/Cytokine secretion Co-cultures 24

25 Flow Cytometry Identification of Tregs Tumor Tumor Draining Lymph Node Total Lymphocytes CD4 T cells CD4 T cells CD4 CD4 Total CD4+ cells Tregs Tregs FoxP3 FoxP3 25

26 A Myeloid Subpopulation Correlates with Efficacy Tumor Weight (RENCA sc Model) *** Tumor weight(g) CD45 Vehicl cle Sutent,4mg/kg CD11b CD45 + CD11b + Ly-6G low CD11b + Ly-6G + CD11b ** % of total events % of WBC % of WBC % of WBC Vehicle Sutent Vehicle Sutent Vehicle Sutent Vehicle Sutent M s, MDSC subsets 26

27 Ex Vivo Testing: Splenocyte Proliferation Stimulation index anti-cd3 Stimulation (stimulated/control) *** Control Tumor Model 1 index Stimulation anti-cd3 Stimulation stimulated/control Control Tumor Model 2 Splenocyte Proliferation can be used to evaluate systemic immune suppression. 27

28 Inhibitory Function of Cells: Co-cultures MDSCs from tumor bearing mice inhibit anti CD3 induced i d mouse splenocyte proliferation CD3 CD3+CT26 model splenocytes Unstimulated CD3+4T1 model splenocytes Note: 4T1 model splentocytes contain large amount of myeloid derived suppressor cells (MDSCs). This assay can be used for co culturing of tumor effector cells, Treg effector cells, MDSC effector cells and screening of compounds. 28

29 Flow Cytometry Analyses of Patient Tumor Samples Blood Tumor Grey line: FMO control Blue line: Tim 3 staining 29

30 Flow Cytometry Analyses of Patient Tumor Samples Blood Tumor Grey shade: FMO control Blue line: PD L1 or Tim 3 staining i 3

31 PKPD Correlation in Cynomolgus Monkey B cells SSC FSC 1 Lymphocytes SSC Isotype.53% CD19 PE CD19 B cells 14.2% 8 Use CD19 to identify B cell. CD19 PE Beckman Coulter A7769 B cells accounted for 14.2% of the total number of lymphocytes in control monkey. 7 Rituxan (μg g/ml serum) B cell ( 1 4 ce ells/ml blood) B cell decreased dramatically at first 2 hr post i.v. dose and slightly recovered after 21 days post treatment.1.5 h 2 h 8 h 24 h Rituxan-Cyno#1 Time Rituxan-Cyno#2 (day) Rituxan-Mean B cell-cyno#1 B cell-cyno#2 B cell-mean 31

32 Histopathology Expertise Tissue Archive and Histology Animal necropsy & Ex vivo organ perfusion and fixation Tissues processing and Embedding Paraffin section cutting Frozen section cutting Vibratome section cutting Cytospin section preparation HE staining & Special staining Immunohistochemistry(IHC) & Immunofluorescence (IF) IHCand IFstaining on conventional FFPE/Frozen tissue section IHC and IF staining on tissue microarray (TMA) IHC and IF double staining on conventional FFPE/Frozen tissue section and TMA High throughput screening for hybridoma supernatants and monoclonal antibodies by IHC staining In Situ Hybridization Fluorescent InSitu Hybridization (FISH) RNAscope: RNA In Situ Hybridization (RNA ISH) 32

33 Representative Images of IHC Staining CD3 Mouse original tumor CD8 Human tonsil FoxP3 Human colon cancer PD L1 Mouse original tumor EGFR PDX tumor p EGFR PDX tumor 33

34 Thank you! 34