1. Renier Brentjens, MD, PhD
Using a PD-1-Blocking scFv Along with a Chimeric Antigen Receptor to Prevent T cell Inhibition in the Tumor Microenvironment
Daniel Plaisance
Renier Brentjens, MD, PhD
Hollie Pegram, PhD
Terence Purdon

2. Introduction
Chronic Lymphocytic Leukemia (CLL) and Acute Lymphoblastic Leukemia (ALL) are two B cell blood cancers
The immune system does not naturally recognize or fight B cell cancers
Current treatments for these therapies include chemotherapy and stem cell transplants
High rate of relapse

3. Introduction
T cells are white blood cells that play an important role in activating immune responses
Lytic Function
Proliferation
Cytokine Production
Antigen receptors are proteins on the surface of the T cell that allow it to recognize and fight diseases

4. Introduction
Chimeric Antigen Receptors (CARs) are receptors that are created through genetic modification
CARs can be created that bind to a specific “target antigen” on a disease that the body does not normally fight
T cells can be genetically modified to express a CAR

5. Introduction
PD-1 is an immune regulatory molecule found on the surface of T cells
PD-L1 binds to PD-1 and is found on most human cells
The PD-1 PD-L1 pathway regulates the potency of an immune response
Smartpatients.com

6. Review of Literature
T cells that have been modified with a CAR targeted to the CD19 protein (on B cells) can effectively fight ALL
Brentjens et al., 2011
Di Stasi et al., 2011
Porter et al., 2011
Louis et al., 2011
Kalos et al., 2012

7. Review of Literature PD-1 can be engaged by PD-L1
Ramsay et. al, 2013
The engagement of PD-1 significantly decreases T cell function by disrupting many important processes
Sznol et. al, 2013
The expression of PD-L1 is upregulated (increased) on CLL cells
Francisco et. al, 2010

8. Macmillan Cancer Support
Review of Literature
The RMPI-14 antibody binds to the PD-1 receptor
Yamazaki et. al, 2005
Macmillan Cancer Support

9. Purpose
To create a genetic construct that codes for an scFv that will be secreted from the T cell and bind to the PD-1 receptor
To create a single construct combining the CAR gene and the scFv gene
To transduce cells with the construct

10. Methodology CD19-targeted CAR consists of:
CD19-specific scFv
Mouse CD28 molecule
Mouse zeta chain
Gene that codes for this CAR is denoted as “19m28mζ”
19m28mζ gene

11. Methodology RMPI scFv consists of:
P2A element
mIgk leader sequence
RMPI VL chain
GS Linker
RMPI VH chain
Myctag
Gene that codes for this scFv is denoted as “P2ARMPI”
P2ARMPI gene

12. Methodology: Part 1 CAR Gene scFv Gene Combined Construct
19m28mζ gene from previous studies used
Digested with XhoI restriction enzyme and SAP
scFv Gene
P2ARMPI Gene was created
Isolated by PCR
Combined Construct
The CAR gene and scFv gene were used
Combined through ligation

13. Final Construct: 19m28mζP2ARMPI gene

14. Methodology: Part 2 Construct transformed into E. Coli cells
Colony PCR performed
PCR results evaluated by gel electrophoresis; successful colonies chose

15. Methodology: Part 2 (cont’d)
H29 cells transfected with the construct
Phoenix cells transduced with the construct
Gene expression levels in phoenix cells evaluated

16. Methodology
Three phoenix cell samples were stained with 19e3 antibody and evaluated for FL6 intensity
Empty phoenix cells (negative control)
1928ζIREShIL12 (positive control)
19m28mζP2ARMPI (experimental)

17. Results Gel electrophoresis analysis of colony PCR products
Confirms that colony 6 contains the construct

18. Results Gel electrophoresis analysis of colony 6 digestion
7,575 base pairs
842 base pairs
268 base pairs
Confirms that the orientation of the genes was correct

19. Results
0.58%
59.11%
96.36%

20. Results
Successful creation of the 19m28mζ construct, with all parts in the correct orientation
Successful transduction of phoenix cells with the construct
Overwhelming expression of the gene on transduced phoenix cells

21. Discussion Future Steps:
Verification of scFv production (flow on golgi plug)
Confirmation of mIgK leader sequence function (staining supernatant with anti-myctag antibody)
Transduction of mouse T cells with construct
In vitro culture of cells with PD-L1+ tumor cells (and controls)
In vivo tests in mice with PD-L1+ cancers (and controls)
Repeat with human genes and human patients

22. Discussion
The creation of a PD-1-blocking scFv could have major implications for the future of CLL treatment
As the field of CAR T cell therapy expands, the RMPI scFv may be applicable to treatments of other PD-L1+ cancers

23. Acknowledgements
My Teachers: Marybeth Foisy Kirsten Kleinman Deirdre O’Hagan Heather Lijoi Keith Green
My Mentors:
Renier Brentjens, MD, PhD
Hollie Pegram, PhD
Terence Purdon
Dayenne Van Leeuwen
Brian Osborne, PhD
My Family:
Ric Plaisance
Elizabeth McGrory
Adrian Plaisance
Bob McGrory
Carolyn McGrory

24. Renier Brentjens, MD, PhD
Using a PD-1-Blocking scFv Along with a Chimeric Antigen Receptor to Prevent T cell Inhibition in the Tumor Microenvironment
Daniel Plaisance
Renier Brentjens, MD, PhD
Hollie Pegram, PhD
Terence Purdon