Qiong (Chelsea) Xue, Ph.D.

Novartis Pharmaceuticals

13-Sepember-2016

BioMAN Workshop

Novel multiplexing technologies for comprehensive functional characterizations of CAR T final products

| Comprehensive functional characterization of CAR T | Qiong Xue | 2016.09.13 | BioMAN | Business Use Only 2

Published in: Yao Lu; Jonathan J. Chen; et. al.; Anal. Chem. 2013, 85, 2548-2556.

DOI: 10.1021/ac400082e

Copyright © 2013 American Chemical Society

The principle of the Technology

| Comprehensive functional characterization of CAR T | Qiong Xue | 2016.09.13 | BioMAN | Business Use Only 3

An overview of the assay

1. Bind CAR T Cells

to anti-CAR

beads/IgG control

beads: stimulation

2. CAR T cells

stained &

delivered to system

3. Assay ~1000 single cells on 16 plex multi-

functional panel

4. Function results per cell across

1000’s of cells

5. Correlate donor data & markers using

automated algorithmic and analytic software

| Comprehensive functional characterization of CAR T | Qiong Xue | 2016.09.13 | BioMAN | Business Use Only 4

Significant cytokine upregulation in response to antigen were measured across 4 different donors

Donor 1 Donor 2 Donor 3 Donor 4

Cytokines that were secreted by at least 20 cells or 2% of cells and with a signal-to-noise ratio (SNR) of at

least 2 (relative to zero-cell noise) were labeled as significantly secreted cytokines of the sample.

| Comprehensive functional characterization of CAR T | Qiong Xue | 2016.09.13 | BioMAN | Business Use Only 5

Polyfunctional CAR T cells were observed across all 4 donors

2 cytokines 3 cytokines 4 cytokines 5+ cytokines

| Comprehensive functional characterization of CAR T | Qiong Xue | 2016.09.13 | BioMAN | Business Use Only 6

Polyfunctional CAR T cells predominately possess anti-tumor effector functions

| Comprehensive functional characterization of CAR T | Qiong Xue | 2016.09.13 | BioMAN | Business Use Only 7

Polyfunctional heat map reveals functional subsets across donors

| Comprehensive functional characterization of CAR T | Qiong Xue | 2016.09.13 | BioMAN | Business Use Only 8

Overview of PAT PCA for polyfunctionality visualization and comparison

| Comprehensive functional characterization of CAR T | Qiong Xue | 2016.09.13 | BioMAN | Business Use Only 9

Distinct polyfunctional profiles across four donors revealed by PAT PCA analysis

| Comprehensive functional characterization of CAR T | Qiong Xue | 2016.09.13 | BioMAN | Business Use Only 10

Summary

We used a single-cell 16-plex cytokine assay microdevice to delineate the

response of CD19 CAR T cells to antigen-specific stimulation.

The results confirmed that CD19 CAR T cells are polyfunctional with a

dominant profile of anti-tumor effector response at a single-cell level from 4

donors, albeit mixed with a range of other functions such as stimulatory,

immunosuppressive, and immunotoxic/proinflammatory.

A computational tool modified from principal component analysis further

distinguishes various polyfunctional subsets within the same populations of

CAR-T cells, yielding a landscape of immune effector response in CD19 CAR

T cells to antigen-specific challenge.

The ability to dissect polyfunctional subsets in correlation to therapeutic

response may lead to a new route toward more efficacious and less

immunotoxic CAR T cell therapies.

11

Acknowledgements

IsoPlexis Emily Bettini

Patrick Paczkowski

Colin Ng

Alaina Kaiser

Tim McConnell

Jing Zhou

Sean Mackay

California Institue of

Technology James Heath

Novartis Sadik Kassim

Mark Dudley

Olja Kodrasi

Maire Quigley

Nebojsa Milovic

Lana Parent

Amy Shaw

Piotr Pierog

Iulian Pruteanu

Junxia Wang

Arnaud Colantonio

Bassel Ghadder

Michael Greene

Sean Garrity

Poonam Vaidya

Yale University Rong Fan

| Comprehensive functional characterization of CAR T | Qiong Xue | 2016.09.13 | BioMAN | Business Use Only