Oncology ltd.wuxiapptec.com

Immuno-oncology Platform

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Oncology & Immunology

• Biomarker » Soluble biomarker – ELISA, ELISPOT » Cell-based biomarker – FACS » Tissue-based biomarker – Multiplex IHC » Gene expression profiling-RNAseq, Nanostring

• Tumor infiltrating lymphocytes (TILs) analysis on human and murine tumor tissues

» CD4, CD8, MDSC, M1/M2, DC cell, NK cell, B cell » PD1, TIM3, LAG3, OX40, 4-1BB » Treg, Th1,2,3,17 ……

Tumor types Cell linesBladder (1) MBT2S ,GT

Breast (6) 4T1S ,GC, 4T1-luc5, JCGC, Eph4 1424GC, EMT6GC, FM3A

Colorectal (3) Colon-26, CT-26S,GC, MC38S,GC/T

Hemangioendothelioma (1) EOMA

Liver (2) MH-22AS/GT, H22

Lung (4) LLC1(LL/2)S,GT, KLN205S/GT, 3LLS,GT, M109

Leukemia (4) L1210S, WEHI-3, C1498, WEHI 3BD

Lymphoma (9) EL4, A20S, P388D1, L5178-R, WR19L, A20-Luc5, L5178-S(LY-S), L5178Y TK+/- clone (3.7.2C), P3/NSI/1-Ag4-1(NS-1)

Myeloma (5) MPC-11, FO, P3X63Ag8, J558L, MOPC31C

Mastocytoma (3) P815S, P815-lucS, P1.HTR

Melanoma (6) B16-F10S,GC/T, B16-F10-luc-G5, B16-F0S, CloudmanS91S,GC/T, B16-F1, C57/B1

Neuroblastoma (3) Neuro-2a, N1E-115, N18(Hamprecht)

Prostate (1) RM-1S,GT

Renal (1) RENCAGT

Sarcoma (2) WEHI164S,GT, K7M2 wt

Testis (1) MLTC-1

ovarian cancer (1) OV3121

Schwannoma (1) TR6Bc1

54 Syngeneic models establishedLargest collection of syngeneic model

Note: All 54 models have growth curve data,19 models with reference drug treatment data (either chemotherapy/target therapy or immunotherapy) were marked as S. 16 models

Outstanding platform to enable cancer immunotherapy with one stop service » Immunological function assessment » Tumor microenvironment analysis » In vivo efficacy evaluation on animal models » NGS to understand TCR/BCR Repertoire » Patient TILs analysis

with genomic profiling data were marked with GC (cell-line profiled), GT (Tumor tissue profiled) or GC/T (both).

• T cell activation / Functions » T cell proliferation / activation• 3H incorporation, SEB stimulation assay

• In vivo efficacy evaluation on animal models » Syngeneic murine models » Immune-checkpoint humanized models » Immune-Avatar humanized models

» PBMC humanized models » HSC humanized models

CD8 T cells activation by immune-checkpoint blockade TILs analysis of syngeneic tumor

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Immuno-oncology platform – immune profiling

Syngeneicmodels

Tumorcellprofiling

Tcell/Bcellprofiling

MutationCNV

ExpressionMHCtype

Neo-antigen…

Genetic/expressionchangebefore

andaftertreatment

TCR/BCRRepertoireanalysis

• Immune profiling capability has been established for both human and murine, including » Tumor cell: exome sequence, RNAseq, neo-antigen prediction, MHC typing » T cell/B cell Repertoire sequencing and analysis

Nature Biotechnology V32, P158–168:(2014)

onco.wuxiapptec.comEmail:info_onco@wuxiapptec.com

Oncology & Immunology

Oncology Oncologyltd.wuxiapptec.com ltd.wuxiapptec.com

3 4 5

FACS analysis of TILs in clinical tumors, a case study

CD

3

CD45

Live/dead gated

CD45+ CD3+ gated

CD8

CD

4

-- Isotype-- Panel

PD-1 Tim-3 Lag-3

CD4+CD8-

CD4-CD8+

FACS analysis

NSCLC

H&N

Ovariancancer

TNBC

Esophageal

Gastriccancer

Cervicalcancer

……..

Tumor types

Freshtumor

Live(7-AAD)

Doubletexclusion

CD45

CD3

PD-L1

PD-L1

CD4 CD8

PD-1,Tim-3,LAG-3

+

-+

+ +

+ +PD-1,Tim-3,LAG-3

Immune-Avatar PBMC humanized models

CRISPR library screening for novel immune checkpoint targets

IsolationTcellsfromtransgenicmice(OT-IorDO11.10)

InfectTcellswithmurineCRISPRlibraryandselection

InfectedTcellsstimulatedwithIL7/IL15/IL2 in vitro

InjectionofinfectedTcellstoB16tumorbearingC57BL/6animals

IsolationofTcellsfromtumor/spleen/lymphnodes

DeepsequencingtoidentifysgRNAenrichedintumor-infiltratingTcells*Nature2014,506(7486):52-57

Methodology of IDOi PD study in LPS-stimulated lung injury model

Immuno-competentmice

-24 hr -12 hr 0 h

LPS stimulation(Intranasal) IDO inhibitor or vehicle treatment

Lung and plasma harvested at different time-points

Biomarker analyzed by LC/MS

PD study of NLG919 and INCB024360Pilot study of LPS stimulation

Assay WuXi Capability (Human/Mouse)Cell activation T cell Yes (H/M)

B cell Yes (H)

NK cell Yes (H)

Cell differentiation M1/M2 macrophage Yes (H/M)

MDSC Yes (H)

DC Yes (H/M)

Co-culture DC with T (MLR) Yes (H)

MDSC suppressive assay Yes (H)

Tumor cell+immune cell co-culture Yes (H)

Treg suppressive assay Yes (H)

Cytotoxic T lymphocyte assay (OT-1) Yes (M)

Treg suppressive assay Yes (H)

T cell polarization Treg Yes (H/M)

TH1 Yes (H/M)

TH2 Yes (H/M)

TH17 Yes (H/M)

Other assays ADCC, CDC, ADCP Yes (H)

Service list of In-vitro and ex-vivo immunology assays

Co-cultureShowcase for DC and T cell (MLR assay)

» MLRassay.Humanmonocyte-derivedDCswereco-culturedwithallogeneicTcellsisolatedfromPBMCFor4days.Afterco-culture,TcellproliferationwasmeasuredbyFACSandIFN-γproductionwasdetectedwithELISA.

Immnue checkpoint humanized models

hPD-1,hCTLA-4andhTIM-3available,hLag3invalidation

YervoyonMC38models

huCTLA-4KIC57BL/6 WildtypeC57BL/6

FACS analysis of TILs and immune checkpoints in Chinese cancer patients IDO-related in vivo model: LPS-stimulated PD model Immunology-Oncology Platform – In-vitro Assays

Oncology & Immunology

Oncology & Immunology

Oncology & Immunology