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

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

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

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

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

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

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

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

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

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

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

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

Final Construct: 19m28mζP2ARMPI gene

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

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

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)

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

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

Results 0.58% 59.11% 96.36%

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

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

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

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

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