Application Note

Boosting the Efficacy of Tumor Infiltrating Lymphocytes via PD-1 Gene Editing


Adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) has shown clinical success as a treatment for metastatic melanoma with 20% of patients achieving complete and durable tumor regression and a 50% overall response rate.1 While promising, avenues to improve TIL therapeutic efficacy are needed.

PD-1, a receptor implicated in suppressing T cell activity, is expressed by the tumor reactive fraction of TIL. Conversely, metastatic melanoma tumor cells express PD-1 ligands, PD-L1 and PD-L2, potentially creating an immunosuppressive tumor micro-environment that would negatively impact therapeutic efficacy.2, 3 Clinical treatment of melanoma patients with anti-PD-1 antibodies has led to modest tumor regression, validating the importance of the PD-1:PD-L1/L2 interaction.4

Disrupting the PD-1 gene in autologous TIL prior to adoptive transfer offers an alternative and promising approach to improving the efficacy of TIL, while avoiding the toxicities and cost associated with long-term anti-PD-1 antibody treatment.



Determine the feasibility and safety of disrupting the PD-1 gene in human TIL via zinc finger nuclease (ZFN)-mediated gene editing using the MaxCyte GT, a clinically-approved platform for scalable T cell engineering.

TIL Electroporation

  • TIL isolated from three melanoma patients were induced to rapidly expand on day 0 using a previously reported expansion protocol.5
  • On day 7 of the rapid expansion protocol, TIL were harvested, washed, and resuspended in MaxCyte® Electroporation Buffer at a concentration of 1 × 108 cells/mL. Cells were mixed with 120 μg/mL of PD-1 ZFN RNA and transferred to a 3 mL CL1.1 (small-scale) or 100 mL CL-2 (large-scale) processing assembly for electroporation on the MaxCyte GT.
  • Electroporation was performed using MaxCyte recommended protocol. Up to 3x109 cells were electroporated per donor.
  • Post electroporation, cells were resuspended in AIM-V media and incubated overnight at 30°C followed by two days at 37°C.6

Full methods for in vitro assays and in vivo safety studies are detailed in Mol. Ther., 23(8), 1380-1390, 2015.


75% PD-1 Gene Disruption

Electroporation of TIL from three human melanoma patient donors with mRNA encoding a PD-1-targeted ZFN resulted in 75% PD-1 gene disruption as determined by deep sequencing using the Illumina platform with a 44% bi-allelic disruption frequency (Table 1). Cells electroporated using small-scale versus large-scale MaxCyte electroporation had similar gene disruption frequencies.7 Additionally, no negative impacts on cell proliferative capacity or changes to T cell phenotype were observed.7

PD-1 Knockout TIL Exhibit Enhanced In Vitro Effector Functions

PD-1 gene disruption resulted in a 76% reduction (electroporated vs mock electroporated) of PD-1 expression on CD3+ TIL following anti-CD3/CD28 bead stimulation (Figure 1). These edited TIL had significantly enhanced in vitro T cell effector functions such as secretion of TNFα, GM-CSF, and IFN-γ following co-culture with antigen-specific tumor target cells. Importantly, enhanced cytokine secretion was observed even when the target cells expressed PD-L1, suggesting that PD-1 knock out allows TIL to overcome immunosuppression.7

In Vivo Toxicity Studies Establish Safety

PD-1 edited or mock electroporated TIL were administered to NOD SCID gamma (NSG) mice to assess whether PD-1 gene knock out results in proliferative abnormalities or tumors upon engraftment. No carcinogenic effects were noted supporting the safety of PD-1-ZFN-mediated engineering of T cells prior to adoptive transfer.7

Surface Expression of PD-1 on CD3+ Cells


Figure 1. 7 days post electroporation, TIL were restimulated with anti-CD3/CD28 beads for 48 hours. Cells were then assessed for surface expression of CD3+ and PD-1 via FACS.


Combining high efficiency gene disruption and high primary T cell viability post engineering with consistent performance enabled by MaxCyte’s regulatory-compliant GT electroporation instrument resulted in extremely effective gene editing at clinical-scale. Using this approach, the efficacy of TIL and other T cell adoptive cell therapies against a wide variety of cancers can be enhanced, enabling developers to attain the required therapeutic index and safety profiles while simplifying manufacturing.

PD-1 Gene Disruption Determined Using Deep Sequencing

% PD-1 Indelsa Bi-allelic PD-1 Disruption
Donor 1 84.1 48%
Donor 2 69.9 N.D.
Donor 3 70.4 40%
Mean 74.8 44%

Table 1. TIL from three donors were electroporated with mRNA encoding PD-1-specific ZFN.