novel multiplexing technologies for comprehensive ...qiong (chelsea) xue, ph.d. novartis...
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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