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

Application Note


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/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 3×109 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 Knock Out 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


Combining high efficiency gene disruption and high primary T cell viability post engineering with consistent performance enabled by MaxCyte’s regulatory-compliant GT electroporation platform 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.

Table 1: PD-1 Gene Disruption Determined Using Deep Sequencing

TIL from three donors were electroporated with mRNA encoding PD-1- specific ZFN.
a Percent indels was calculated by dividing the indel containing sequence countby the total sequence count.

Figure 1: Surface Expression of PD-1 on CD3+ Cells

7 days post electroporation, TIL were restimulated with anti-CD3/CD28beads for 48 hours. Cells were then assessed for surface expression of CD3+ and PD-1 via FACS.


1. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. (2011) Clin Cancer Res 17: 4550–4557.
2. Tumor antigen-specific CD8+ T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. (2009) Blood 114: 1537–1544.
3. PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors. (2014) J Clin Invest 124: 2246–2259.
4. Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. (2013) N Engl J Med 369:134–144.
5. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. (2002) Science 298: 850–854.
6.Transient cold shock enhances zinc-finger nuclease-mediated gene disruption. (2010) Nat Methods 7: 459–460.
7. Clinical scale zinc finger nuclease-mediated gene editing of PD-1 in tumor infiltrating lymphocytes for the treatment of metastatic melanoma (2015) Mol Ther 23(8): 1380-1390.