Engineered T Cells with Novel PD-L1-Specific TCR Target Both Cancer and Immunosuppressive Immune Cells

A team of researchers has demonstrated that CD8+ T cells can be engineered with a novel PD-L1 (search)Search target-specific T cell receptor (PDL101-TCR (search)Search drug) to simultaneously attack PD-L1-bearing cancer cells and immunosuppressive myeloid cells within the tumor microenvironment. The findings, published in Signal Transduction and Targeted Therapy, represent an early in vitro proof-of-concept for a physiological, MHC-restricted approach to targeting PD-L1 that may carry lower on-target, off-tumor toxicity risk compared to engineered PD-L1 CAR-T therapies.

Engineering Strategy and Gene Editing Efficiency

The research team isolated CD8+ T cells from peripheral blood mononuclear cells of healthy donors and stimulated them with anti-CD3/CD28 beads, IL-2, and IL-7. Using non-viral CRISPR-Cas9 editing, they first assessed endogenous TCR knockout efficiency by transfecting cells with TRAC- and TRBC-targeting ribonucleoprotein complexes across four healthy donors. Subsequently, the transfections were repeated in seven healthy donors with a double-stranded DNA homology-directed repair template, enabling insertion of the PDL101-TCR (search)Search drug into the TRAC locus while simultaneously knocking out endogenous TRBC loci to prevent receptor mispairing.

Engineered T cells were purified as CD3+PDL101-tetramer+ cells using fluorescence-activated cell sorting and expanded with a rapid expansion protocol. Antigen specificity before and after enrichment was assessed by staining with control (HLTV-1 Tax11-19) or PDL101 peptide-loaded HLA-A2 tetramers.

Functional Activity and Cytotoxicity

Antigen-dependent functional responses were quantified using intracellular cytokine staining following PDL101 peptide stimulation at 5 µM. The engineered cells produced IFN-γ and TNF-α upon antigen encounter, with each data point representing a separate donor and showing the fraction of PDL101-tetramer+ cells, IFN-γ+/TNF-α+ cells, or CD107a+ cells within the CD8+ population.

Antigen-specific cytotoxicity was assessed by 51Cr-release assays using radioactively labeled T2 cells loaded with either DMSO, PDL101, or PDL1Long1 peptides (4 hours, 20 µM) as target cells for enriched PDL101-TCR (search)Search drug-T cells from five donors. The engineered T cells lysed peptide-loaded targets into the low picomolar range. Statistical analysis at discrete effector-to-target ratios was performed using paired, two-tailed t-tests comparing peptide-loaded target cells to DMSO controls.

Critically, the PDL101-TCR (search)Search drug-T cells preferentially killed PD-L1 (search)Search target-positive cutaneous T-cell lymphoma (search)Search disease cell lines, including MAC-1 and MAC-2a, demonstrating tumor-directed cytotoxicity.

Targeting the Immunosuppressive Microenvironment

Beyond direct tumor killing, the researchers investigated whether the engineered T cells could target immunosuppressive myeloid populations. Autologous CD14+ myeloid cells were purified from three healthy donors and differentiated toward either a tumor-associated macrophage (TAM)-like phenotype using M-CSF, IL-4, IL-10, and tumor-conditioned medium from MDA-MB-231 cells, or an MDSC/M2-like phenotype using GM-CSF and IL-6.

Surface expression of CD163, CD206, CD86, HLA-DR, and PD-L1 (search)Search target was quantified by flow cytometry on undifferentiated control cells and the differentiated TAM-like and M2/MDSC-like myeloid subsets. Between 0.75 and 1.0 × 10⁵ enriched PDL101-TCR (search)Search drug-T cells were either plated alone, with PDL101 peptide, or co-cultured with each of the three myeloid subsets at an effector-to-target ratio of 2:1 in IFN-γ ELISPOT assays. The engineered T cells responded to both TAM-like and MDSC-like myeloid subsets, both of which contribute to local immunosuppression. Statistical significance was determined using the DFR rule (*p ≤ 0.05).

Potential for In Vivo Boosting via Vaccination

The team explored whether peptide-loaded dendritic cells could expand the engineered T cell population. Autologous monocyte-derived dendritic cells were generated by differentiating CD14-purified cells with IL-4 and GM-CSF and subsequently maturing them with IL-6, IL-1β, TNF-α, and prostaglandin E2. Peptide-loaded (50 µg/mL, 4 hours), irradiated dendritic cells were used to expand CD8+ T cells over two rounds performed one week apart.

The proportion of PDL101-TCR (search)Search drug+ T cells was determined before DC stimulation and one week after each DC stimulation using tetramer staining. Stimulation with peptide-loaded dendritic cells expanded the modest initial editing yield approximately five- to six-fold, pointing to scope for in vivo boosting through vaccination.

In a further experiment, autologous dendritic cells were used as target cells for expanded PDL101-TCR (search)Search drug-T cells from a single donor in IFN-γ ELISPOT at an effector-to-target ratio of 2:1. Dendritic cells were either mock transfected or transfected with PD-L1 (search)Search target-targeting siRNA (0.375 µM) prior to use as target cells, with PD-L1 expression confirmed by flow cytometry.

A Physiological Alternative to CAR-T

The authors argue that a physiological, MHC-restricted receptor may carry a lower on-target, off-tumor toxicity risk than engineered PD-L1 (search)Search target CARs, though this remains to be confirmed in future studies. The work is framed as an early, in vitro proof-of-concept rather than a clinical candidate. Tumors frequently evade immune attack by upregulating PD-L1, which dampens T-cell activity and sustains an immunosuppressive microenvironment. Building on their earlier identification of naturally occurring PD-L1-specific T cells, the team reconstructed the PD-L1-directed receptor from a patient-derived clone.