Driver-Map™

ImmuneNet™: Immuno-Oncology Applications

Gene expression & Molecular Profiling Service

Obtain a Molecular Snapshot of the Tumor Microenvironment

(877) 938-3910 www.driver-map.com

Driver-Map is a novel, comprehensive end-to-end service portfolio for identifying differential gene expression, mapping of clinically actionable mutations in RNA, detecting cellular composition, and profiling immunotherapy targets intended for research applications. It is a quantitative, targeted multiplexed approach that leverages the power of Next-Generation Sequencing (NGS) and PCR technologies. ImmuneNet™ is the first of the panels in the Driver-Map portfolio, developed to generate unique molecular portraits of the tumor microenvironment.

Human carcinomas consist of a mixture of neoplastic epithelial cells, endothelial cells, fibroblasts, myofibroblasts, and immune cells, which collectively form the Tumor Microenvironment. The complex interaction between these cells is implicated in disease progression, metastasis, and drug resistance. Quantitative molecular profiles of tumor-associated cells may provide important insights into tumor biology and facilitate the discovery of new biomarkers and therapeutic targets. To address these issues, Cellecta offers the Driver-Map™ portfolio.

Functionally Validated PrimersAll multiplexing PCR primers are functionally validated. Cellecta’s unique multiplex primer design minimizes primer dimerization and cross-reactivity while maximizing specificity and efficacy.

High-Throughput CapabilitiesProfile thousands of genes in one reaction.

Exquisite Sensitivity & SpecificityAllows low abundance transcripts to be identified, facilitating biomarker discovery.

Large Dynamic RangeAllows characterization of all major immune cell types, and detect infiltrating immune cells with zero background.

FlexibleFully Customizable panel with data analysis.

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Salient Features

ImmuneNet Service for Immuno-Oncology Applications

Profiling Infiltrating Immune Cells

Increasing evidence suggests that the number, type, and location of tumor-infiltrating lymphocytes in primary tumors harbor prognostic & Predictive value. Driver-Map ImmuneNet assay has been built to detect the presence of immune cells by their gene signatures.

Why Driver-Map?

The panel includes a comprehensive set of genes specific for detection and profiling.

Cancer Driver GenesFDA Drug

Targets

Immune mechanisms &activation status

28 Immune cell type specific signatures

Immunotherapy BIOMARKERS from 18 clinical trials

ImmuneNet

Immunerelated

Immunotherapy drug targets in clinical & preclinical studies

GrowthFactor

CytokineAdipocyteMacrophageImmuneCell (T)

ImmuneCell (B)

CSCCancer CellFibroblast MesenchymalCell

Major Cell Types that can be Detected

Basophil, B-cell, CD8 T-cell, Cytotoxic cell, DC, Endothelial, Eosinophil, Erythrocyte, Fibroblast, Hematopoietic stem cell, Macrophages, Megakaryocyte, Neutrophil, NK cell, NK CD56bright cell, NK CD56dim cell, Th1 cell, Th2 cell, Th17 cell, Treg

(877) 938-3910 www.driver-map.com

Reproducibility & Sensitivity

The ImmuneNet assay panel has extreme sensitivity similar to quantitative RT-PCR, high dynamic range (up to 100,000-fold range), with virtually no background (as each specific amplicon is validated by NGS) and great reproducibility as demonstrated in the figure below.

How Does It Work?

Our novel workflow leverages the power of quantitative PCR (upstream) and Next-Gen Sequencing (downstream read-out). Experimentally validated gene-specific primers are used to amplify selected target regions. Synthetic calibration controls act as an effective QC metric. The protocol begins with cDNA synthesis, stranded anchor addition, followed by PCR and NGS.

Assay Validation

The ImmuneNet assay was validated with clinical samples (such as frozen tissue, blood, and FFPE samples). RNA isolated from these clinical samples were run on the ImmuneNet assay panel. FFPE slides from patients with triple negative breast cancer (TNBC) were used in this study. Immunohistochemistry delineates the tumor tissue and immune infiltration, while ImmuneNet can identify immune cells based on their gene signatures and reveal biomarkers associated with these clinical samples.

Validation of assay with TNBC clinical samples. (A) Tumor immune cell infiltration visualized by H&E staining (40X magnification). (B) Heat map showing differentially expressed genes detected by ImmuneNet assay using immune-infiltrated and control tumor samples. (C) Example immune enrichment score for immune infiltrating dendritic cells.

Highly Infiltrated tumor cellsBREAST CANCER PATIENT 166

Immune CellsTumor

RES

1.0

0.5

0.0

-0.5

Immune Enrichment score

Dendritic cell signatures

(A)

(B)

(C)

ADORA2ABATFBTLACCL13CCL17CCL22CCR2CCR3CD19CD1ACD1BCD2CD27CD38CD40LGCD6CD8BCR2CXCR6EWSR1FOXP3FUT5GATA3GNLYGZMBHAVCR2HLA-DOBID01IL21RKIR3DL1LAG3LAIR2LTALTKMAGEA1MAGEA12MAGEA3MAGEA4MAGEB2MAGEC1MAGEC2NCR1PDCD1PRAMEREPS1SEMG1STAT6TBX21TNFRSF4TNFRSF9

NormalTissue

1 2 3 1 2 3 1 2 3

BreastCancer

Patients

166170

Replicate:

n/a + +++Immune Infiltration:

Sample:

(A) (B)

Median of 100 ng MDA231 RNA(triplicate)

25 n

g of

MD

A23

1 RN

A

rerep #1 #2 #3 #1 #2 #3 #1 #2 #3

#1 1.00

#2

#3

#1

#2

#3

#1

#2

#3 0.90

P-Value

100

ng

6.25 ng 25 ng 100 ng

6.25

ng

25 n

g

Correlation Matrix of expression profiles at differing amounts of Human Universal RNA (6.25, 25, and 100 ng) in triplicates.

Scatterplot of expression profiles of three replicates independently run on ImmuneNet assay at 25 ng vs. median of 100 ng of MDA231 RNA showed high reproducibility.

Correlation Analysis

Detect and enumerate immune cell types using

reference cell type-specific signatures

Relative Fraction - and -

Enrichment Score

Classifier

Immune CellEnrichment Score

TargetedRNAseqProfile

Samples Blood(FFPE)

0.0

0.2

0.4

0.6

0.8

1.0

Immune CellsStromal CellsCancer

Cells

Cell Populationsin Tumor MicroenvironmentCancer

CellT Cell

MacrophageB Cell NK CellEndothelial

FibroblastStroma

Tumor

Targeted RNAseq expression profile of ~2000 immune-associated genes

Driver-Map™ ImmuneNet™ Services

Output

Report Details

Input

• Sample Characteristics• Raw data & transformed data• QC Metrics and Analysis Sample report is available on request

• Comprehensive Report

• Total RNA (10-100 ng)• Frozen FNA, Blood, Tumor samples, FFPE, Mouse PDX

Catalog #s DMAP-IN Driver-Map ImmuneNet Profiling ServiceDMAP-QC Driver-Map Sample QC ServiceDMAP-RS Driver-Map RNA-Seq Service

Service Workflow

Sample Submission

Sample QC Service

Driver -Map™ Assay

Analysis Output Report

Email us at driver-map@cellecta.com, visit www.driver-map.com, or call us at (877) 938-3910 or +1 650-938-3910 for more information.

Cellecta, Inc.320 Logue AveMountain View, CA 94043www.cellecta.com

info@cellecta.com+1 (650) 938-3910