Increased proportions

of pdcd1-deleted CAR-
T cells promotes tumor
relapse
R Y AN H A H N
Introduction
• CAR-T cells are effective against B-cell leukemias and
lymphomas while current research is investigates use for other
liquid and solid tumors1.
• PD-L1 expression on tumor cells inhibit CAR-T cell facilitated cell-
lysis and tumor degradation in mice1.
• PD-1 prevents overstimulation of CD8+ T cells and subsequent
excessive cell death during chronic viral infection2.
• It remains unclear if changing the proportion of Pdcd1 deficient
CAR-T cells transferred enhances or inhibits anti-tumor efficacy.
• Subjects that receive higher proportions of Pdcd1 edited CAR-T
cells may experience a relapse in tumor.
Figure 1: Pdcd1 gene deleted through
Cas9-RPN protocol

PD-1 deletion accomplished in primary human T-cells. PD-1 ablation and

was measured via flow cytometry 2–4 days following nucleofection in the
Cas9-RPN assay . There’s a reduction greater than 50% of CAR+ PD-1+ cells
48 hours after editing1.
Figure 2: PD-1 edited anti-CD19 CAR-T
cells anti-tumor efficacy in vivo

PD-1 deficient anti-CD19 CAR-T cells demonstrate increased anti-tumor

functioning and reduce subcutaneous CD19+ PD-L1+ tumor xenografts in
mice. Mice were injected with 5×106 CD19+ PD-L1+ K562 cells
subcutaneously to promote tumor growth and were later treated with
intravenous 4×106 CD4+ CAR+ and 4×106 CD8+ CAR+ control T cells or PD-
1 edited cells to measure tumor clearance1.
Figure 3: CD8+ T-cells develop reduce
immune function in the absence of PD-1

PD-1 KO cells produced significantly less IFNγ than WT cells. CD8+ cells
were isolated from peripheral mouse blood and stained intracellularly for
cytokine IFNγ after LCMV clone 13 infection2.
Figure 4: Reduced survival of PD-1 KO
P14 cells during T cell contraction

WT and PD-1 KO P14 cells were mixed at a 1:1 ratio, transferred into
recipient mice, and infected with LCMV clone 13. Percent decrease in the
frequency of WT and PD-1 KO P14 cells measured from peak of T cell
response to chronic phase of infection. PD-1 knockouts showed a
significant decrease in cell survival compared to WT cells2.
Important Findings
• CD8+ T cell deficient in PD-1 experience a loss of cytokine
production and elevated inhibitory receptor co-expression
• PD-1 prevents overstimulation of CD8+ T cells and subsequent
excessive cell death during chronic viral infection.
• The mouse lymphocytic choriomeningitis virus (LCMV) model of
chronic infection demonstrates that genetic deletion of Pdcd1 T
cells leads to increased exhaustion and impaired CD8 T cell
survival and function.
• Deletion of Pdcd1 may increase short-term functions of CAR-T
cells but make edited cells more susceptible to impaired
functioning long-term allowing for tumor relapse.
Research Focus
Given that Pdcd1 deletion increases CAR-T cell anti-tumor
efficacy but decreases T-cell functioning long-term, I propose the
following study:
Specific aim 1: Test CAR-T cell functionality across different Pdcd1
deletion edited cell proportions for PDL1+ tumors.
I hypothesize that CAR-T cell functioning will peak shortly after the
initial dosage and will decrease in time
Specific aim 2: Test tumor burden (and relapse) across different
Pdcd1 deletion proportions in PDl1+ tumors.
I hypothesize that tumor burden will decrease in small Pdcd1
deletion proportions of edited cells and will experience tumor
relapse in larger edited cell proportions.
 Research Plan
Testing CAR-T cell functionality across different Pdcd1
deletion edited cell proportions for PDL1+ tumors.
• Produce edited anti-CD19 CAR-T cells for Pdcd1 using
Cas9-RPN assay.
• Inject mice with CD19+ PD-L1+ tumor cells to promote
tumor growth.
• Sample mice blood and treat with different proportions
of edited CAR-T cells (once tumor is established).
• Sample mice blood initially versus post-treatment and
assay for cytokines and inhibitory receptor co-
expression.
• Expected observations: larger proportions of edited
CAR-T cells show lower levels of cytokine production
and increased levels of inhibitory receptor co-
expression.
 Research Plan
Testing tumor burden (and relapse) across different
Pdcd1 deletion proportions in PDl1+ tumors. Produce
anti-CD19 CAR-T cells from primary human T-cells.
• Use Cas9-RPN assay to edit CAR-T cells for Pdcd1
gene.
• Inject mice with CD19+ PD-L1+ tumor cells to
promote tumor growth.
• Inject mice with different proportions of edited CAR-
T cells (once tumor is established) and measure
tumor burden via weekly bioluminescence imaging.
• Expected observations: larger proportions of
edited CAR-T cells will see increased anti-tumor
activity initially compared to controls but will
cause a relapse in tumor burden long-term.
 Significance
• Increase CAR-T cell anti-tumor efficacy with Pdcd1
deletion
• Identifies cell proportion for optimal effectiveness
that balances anti-tumor activity and T-cell
viability
• Highlights the role PD-1 has in preventing long-term
T-cell impairment
• Provides robust anti-tumor therapy protocol using
primary human T-cells
• Offers possible avenues for new research in
identifying the mechanism behind PD-1 and other
protein binders in T-cell functioning
References
• 1. Rupp, L. J. , Schumann, K., Roybal, K. T., Gate, R. E.,Ye, C. J.,
Lim, W. A., and Marson, A. 2017. CRISPR/Cas9-mediated PD-1
disruption enhances anti-tumor efficacy of human chimeric
antigen receptor T cells. Scientific Reports. 737(7): 1-10.
• 2. Odorizzi, P. M., Pauken, K. E., Paley, M. A., Sharpe, A. and
Wherry, E. J. 2015. Genetic absence of PD-1 promotes
accumulation of terminally differentiated exhausted CD8+ T
cells. J. of Experimental Med. 212(7): 1125–1137.