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Confidential
Immuno-Oncology Solutions
Confidential
• Our I-O Technology Platforms and Service Portfolio
• Highlighted Services:
• I-O Biomarker Discovery & Clinical Applications
• Neoantigen Identification & Clinical Applications
• Regulatable CAR-T Development
Confidential
ICI Efficacy Prediction
Cancer Vaccine
Checkpoint Inhibitor & drug targeting
Regulatable CAR-T
Neoantigen Identification
Immune Repertoire
• Tumor Mutational Burden• Microbiome• dMMR, MSI-H• Checkpoint Inhibitor
Expression
• Neoantigen• Immune Repertoire• MHC Binding/Prediction• Epigenetic Analysis
• Transcriptome Seq• Exome Seq• Epigenetic Analysis• Single-cell profiling• MHC Binding• scRNA-seq
AptaNxTM RegCAR-TTM
Biomarker Discovery
• Exome Seq• Transcriptome Seq• Epigenetic Analysis • HLA Typing
• Exome Seq• Transcriptome Seq• Epigenetic Analysis• AptaNxTM
• Immune Repertoire• MHC Binding• Checkpoint Inhibitor
Discovery• Exome Seq• Transcriptome Seq• scRNA-seq
Solution
Technology
DiscoveryClinical
Translation
Therapeutics
Immuno-OncologySolutions
Legend
Stem cell Transplantation
• HLA Matching• Transcriptome Seq
Minimal Residual Disease • Transcriptome Seq
• Exome Seq
Tumor Escape & Resistance
Confidential
Confidential
Distribution and Growth of Cumulative Immuno-Oncology Biomarker
Mentions by Test Purpose 2014 – 17Growth of Cumulative Mentions of Top 30 Immuno-Oncology Biomarkers; 2014 – ’17
https://www.decibio.com
Increasing Importance of Biomarkers in I-O
Confidential
J Yuan et al. Journal for ImmunoTherapy of Cancer20164:3
Biomarker Solutions for Personalized Immunotherapy
Technology
Whole Exome Sequencing
Gene signature/RNA Seq
Epigenetic Analysis
Antigen/Neoantigen Identification
B/T-cell receptor repertoire
Flow cytometry/WES/RNA Seq
Multicolor IHC
Therapeutic strategy
Confidential
Putative I-O Biomarkers in the TME
• PD-L1 expression
• Tumor-infiltrating lymphocytes (TILs)
• Mutational load and neoantigens
• Immunosuppressive cell types
• Macrophage and DC polarization
• Immunosuppressive molecules
• Cytokine signatures
Tumor cell Dead tumor cell MDSC CD8+ T cells CD4+ T cells Immature
dendritic cell
Primed
dendritic cell
M1
macrophage
M2
macrophage
PD-L1 PD-1 MHC I CTLA-4 TIM-3 LAG-3 Tumor Neoantigens IDO IFNγ M-CSF T-regulatory cell
antigens
© 2017 American Association for Cancer Research
M1
M2
IFNγ
M-CSF
iDC
CD8+
CD8+
CD8+
MDSC
CD8+
MDSC
MDSC
TIL
TIL
TIL
TIL
IDO
M2
IDO
iDC
Mutational
load
pDC
CD4+
Treg Treg
Multiple Biomarkers Needed for Understanding TME
Confidential
Clinical Application Examples
Efficacy Biomarkers of ICI (Immunotherapy Checkpoint Inhibitors)
Confidential
Barnhart C. J Adv Pract Oncol. 2015 May-Jun;6(3):234-8.
PD-1 inhibitors:• Pembrolizumab (Keytruda)
• Nivolumab (Opdivo)
PD-L1 inhibitors:• Atezolizumab (Tecentriq)
• Avelumab (Bavencio)
• Durvalumab (Imfinzi)
CTLA-4 inhibitors:• Ipilimumab (Yervoy)
Confidential
28%
18%
27%
Melanoma NSCLC Renal-cell cancer
Response rate (Nivolumab)
33%
27%
13%
Melanoma NSCLC PD-L1–positive endometrial
cancer
Response rate (Pembrolizumab)
10.90%
Melanoma
Response Rate (Ipilimumab)
SL Topalian, FS Hodi, JR Brahmer , etal N Engl J Med 366: 2443– 2454,2012
A Ribas, et al. JAMA. 2016 Apr 19. doi: 10.1001/jama.2016.4059
R Hui, EB Garon, et al. Ann Oncol. 2017 Apr 1;28(4):874-881. doi: 10.1093/annonc/mdx008.
Ott et al. Journal of Clinical Oncology 35, no. 22 (August 2017) 2535-2541.
F Stephen Hodi et al. N Engl J Med. 2010 Aug 19;363(8):711-23. doi: 10.1056/NEJMoa1003466.
Confidential
• TMB (Tumor Mutational Burden)
• RNA signature
• PD-1, PD-L1 expression
• dMMR, MSI-H
• Microbiome
• Neoantigen
Hugo W. et al. Cell. 2017;168:542. doi: 10.1016/j.cell.2017.01.010.
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Snyder, A. et al. N. Engl. J. Med. 371, 2189–2199, doi:10.1056/NEJMoa1406498 (2014).
There was a significant difference in mutational load between patients with a long-term clinical
benefit and those with a minimal benefit or no benefit.
Our Solution:
TMB analysis by WES (Whole Exome Sequencing) or our OncoGx gene panel
Confidential
Hugo W. et al. Cell. 2017;168:542. doi: 10.1016/j.cell.2017.01.010.
Identification of transcriptomic features (IPRES:
innate anti-PD-1 resistance) associated with anti-
PD-1 resistance
Our Solution:
RNA expression signature analysis by RNA-Seq
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Sunshine, J. & Taube, J. M. 23, 32–38, doi:10.1016/j.coph.2015.05.011 (2015).
Association of PD-L1 expression in pre-
treatment tumor specimens with
objective response to anti-PD-1/PD-L1
therapy
Our Solution:
Expression analysis of PD-1 and PD-L1
in tumor tissue by IHC
Confidential
Le DT, et al. N Engl J Med. 2015;372:2509–2520. doi: 10.1056/NEJMoa1500596.
Mismatch repair–deficient tumors are more responsive to
PD-1 blockade than are mismatch repair–proficient tumors
Our Solution:
MSI-H and dMMR status testing by our MSI-H/dMMR or
OncoGx gene panels.
Confidential
Metagenomics of cancer
patient stools revealed
correlations between clinical
responses to ICI.
Routy B. et al. Science. 2017 Nov 2. pii: eaan3706. doi: 10.1126/science.aan3706.
Our Solution:
Gut microbiome analysis by metagenomics
or our FloraCheck™ assay.
Confidential
A peptide signature from the candidate neoepitopes is generated. This set of neoepitopes
defines a signature linked to a benefit from CTLA-4 blockade.
Snyder, A. et al. N. Engl. J. Med. 371, 2189–2199, doi:10.1056/NEJMoa1406498 (2014).
Our Solution:
Neoantigen signature analysis by WES (whole exome sequencing),
RNA seq, MHC binding prediction and bioinformatics analysis
Confidential
Confidential
• Solid tumor test panel designed to provide comprehensive genomic analysis for
cancer therapy
• Provides clinically actionable genomic aberrations as well as HLA-typing and
MMR (mismatch repair) information
• 333 genes
• point mutations, small insertions/deletions, fusions, copy number variations
• Covers all coding exons and UTRs, as well as select intronic regions
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Matched
normal
Tumor
Sample
WES data from 376 TCGA COAD tumor samples
Somatic
variant data
from TCGA
TCGA
pipeline
OncoGxOne
Plus
pipeline
All variant data
without matched
normal
● # of potential somatic variants (n_somatic)
● # of deleterious variants (n_deleterious)
● # of CADD-score high variants (n_CADDphred20)
● # of COSMIC variants (n_COSMIC)
● # of MMR damaging variants (n_MMR)
● existence of BRAF V600E (V600E)
333 genes
w/o matched normal
WES
w/ matched normal
# of somatic variants
/ MB
prediction
Filter out low confident variants
(DP, AD, QUAL, Allele Freq.)
Filter out common variants
(Pop. Freq.: ExAC, 1000GP, ESP6500)
Filter out variants in intronic regions
Filter out homozygous germline variants
(Allele Freq. >0.9)
Confidential
TMB-H (73)
TMB-L (303)
10 somatic mutations / MB
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● # of potential somatic variants (n_somatic)
● # of deleterious variants (n_deleterious)
● # of CADD-score high variants (n_CADDphred20)
● # of COSMIC variants (n_COSMIC)
● # of MMR damaging variants (n_MMR)
● existence of BRAF V600E (V600E)
# of somatic
variants
/ MB
SVM classifier
15
61
15
61
15
61
14
60
14
60
15
61
15
61
15
61
14
60
14
60
15
61
15
61
15
61
14
60
14
60
15
61
15
61
15
61
14
60
14
60
15
61
15
61
15
61
14
60
14
60
Fold 0
Fold 1
Fold 2
Fold 3
Fold 4
Training Validation
# of TMB-H samples
# of TMB-L samples
Cross validation design
Accuracy:
0.95 ±0.04
Confidential
• High accuracy (95% vs 90% F1CDx)
• Cost effective (targeted panel vs WES)
• Fast TAT (7-10 Days)
• Without matched normal
Confidential
Highlighted Service II
Neoantigen Identification & Clinical Applications
Confidential
Neoantigen in Cancer Immunotherapy
Ton N. Schumacher, Robert D. Schreiber Science 03 Apr 2015: Vol. 348, Issue 6230, pp. 69-74
Confidential
Solution for Neoantigen Discovery
H. Hackl, et al. Nature Reviews Genetics 17, 441–458 (2016)
Confidential
• Whole-exome sequencing (WES) - identified neoantigen
• RNA-seq - Validate and assess the expression of neoantigen
• HLA binding - predict
• Vaccine synthesize and administration
At a median of 25 months after vaccination
4 patients: no disease recurrence
2 patients with lung metastases: disease recurrence
ICI treatment
complete responses
Ott PA et al. Nature. 2017 Jul 13;547(7662):217-221..
Cancer vaccineClinical Application
ConfidentialHinrichs CS. et al. Immunol Rev. 2014 Jan; 257(1): 56–71
Clinical Application
Tumor
Normal TissueWES
RNA Seq
Non-synonymous Mutation
Expression ConfirmHLA Typing
MHC Binding Neoantigen
T-cell Activation
Reintroduce to Patient
TCR Clone/Construct
T-cell Expression
Transfect to T-cell
Confidential
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DMM classification analysis of 35 cancer samples based
on SNV, Indel, fusion, CNV, total mutation loading for
each mutated gene and overall mutated genes.
OncoGxOne PlusTM panel for mutation analysis
Tang et al. Chin Med Biotechnol, April 2016, Vol. 11, No. 2
Confidential
333 oncogene heatmap with clustering from 35
cancer samples (labelled with cancer clinical
type, MMR deficiency type and DMM groups)
Barplot of HLA-I genes mutation loading of each
samples (Line indicated the HLA-I mutation
loading was 6 mutations)
Confidential
HLA-I genes mutation loading statistical analysis in DMM groups from 35 cancer samples
Confidential
ID LQL SJS HJL LKS
Cancer type Lung adenocarcinoma Esophageal cancer Renal pelvic carcinoma Lung adenocarcinoma
MMR Mutation
MLH1 5 0 0 0
MSH2 4 0 0 0
MSH6 14 0 0 0
PMS2 7 0 0 0
MMR result* 1 0 0 0
Mutation loading 3243 86 130 141
Neoantigen prediction
Point mutation improved MHC-I - 17 19 24
Neoantigen improved TCR affinity - 3 6 6
HLA-I gene mutation loading
HLA-A 20 4 1 1
HLA-B 14 1 0 0
HLA-C 21 3 1 1
B2M 2 0 0 0
DMM 1 1 0 0
Clinical data
PD-1(X/times) 3X - 5X 5X
MASCT(X/times) 4X 3X 5X -
Response# PD PD PR PR
Confidential
• Neoantigen prediction
• HLA-I genes mutation loading evaluation
• Patients predicted as for their response for MHC-I
restricted immunotherapy
• Precise immunotherapy through NGS for cancer
associated mutation
Confidential
Confidential 37
CAR generation 1st 2nd 3rd
Chronology 1989 2002 2009
Li H et al,2017, PMID: 28434147
Confidential
Clinical trial 1
63 r/r B-cell ALL
(3-21 yrs old)
Clinical trial 2
101 NHL
(77 DLBCL + 24 TFL/PMBCL)]
CR: complete remission; ORR: objective response rate; NHL: Non-Hodgkin's Lymphoma;
DLBCL: Diffuse Large B-Cell Lymphoma; TFL: transformed follicular lymphoma;
PMBCL: Primary Mediastinal Large B-Cell Lymphoma
Data from public news release
Confidential
Clinical Trial 1
63 patients with r/r B-cell ALL
(3-21 yrs old)
Clinical Trial 2
101 patients with NHL
(77 DLBCL + 24 TFL/PMBCL)
CR: complete remission; ORR: objective response rate; CRS: cytokine release syndrome
ALL: Acute Lymphoblastic Leukemia; NHL: Non-Hodgkin's Lymphoma;
DLBCL: Diffuse Large B-Cell Lymphoma; TFL: transformed follicular lymphoma;
PMBCL: Primary Mediastinal Large B-Cell Lymphoma;
Data from public news release
Confidential
ON-switch OFF-switch
Li H et al,2017, PMID: 28434147
Dose tuning to reduce CRS and avoid long-term B-cell aplasia while maintaining CAR-T
efficacy (PMID: 26759369; 26759368).
Tagged Ab
Tumor cell
Y
Confidential
• Limited options of the split proteins with the capability of
chemical induced dimerization (CID)
• The function of tagged antibodies may be affected by the
tagging position, tagging efficiency and tissue penetration
• The construct is too large to allow multi-layer T-cell
engineering
Confidential
ON-Switch ( Turn on CAR by a ligand)
OFF-Switch ( Turn off CAR by a ligand)
AAACAR
AAACAR
AAACAR
AAACAR
Aptazyme-ligand as a switch Potential Advantages
• Control CAR expression at mRNA level, no cell
stress due to constitutive overexpression of
CAR components.
• Aptazymes may be developed against
intracellular and external ligands, allowing
multiple layers of control.
• Aptazyme is small (~100 nt), allowing multiple
layer engineering of CAR constructs.
Aptazyme = Aptamer + Ribozyme
↑
Ligand
Confidential
Combinatory
Library (1015)
Ligand
Selection (n=10-30)
Partition
NGS Aptazymes
Nn
Our AptaNxTM Technology
ON-Switch ( Turn on CAR by a ligand)
AAACAR
AAACAR
Aptazyme-ligand as a switch
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Aptazyme Enrichment in Selection Pools
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Decrease IC50 of individual aptazymes with selection progress
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Ligand-dependent inhibition of aptazyme cleavage
0%
20%
40%
60%
0 200 400 600 800 1000
R18
Cle
ava
ge
(%
)
Drug (µM)
0N
Drug (µM)
0%
10%
20%
30%
40%
0 20 40 60 80 100
R26
Cle
ava
ge
(%
)
Drug (µM)
0N
Drug (µM)
0N
Drug (µM)
0%
20%
40%
60%
0 2000 4000
R9
Cle
ava
ge
(%
)
Drug (µM)
AAACAR
AAACARON-Switch ( Turn on CAR by a ligand)
Full length
Cleaved
aptazyme
Confidential
CAR-T AAACAR
RegCAR-TTM
Efficacy/toxicity
uncontrollable
AAACAR controllable
AptaNxTM