Engineered CD4 TCR T cells with conserved high-affinity TCRs targeting NY-ESO-1 for advanced cellular therapies in cancer

Abstract

While cancer immunotherapy has primarily focused on CD8 T cells, CD4 T cells are increasingly recognized for their role in antitumor immunity. The HLA-DRB3*02:02 allele is found in 50% of Caucasians. In this study, we screened HLA-DRB3*02:02 patients with melanoma for tumor-specific CD4 T cells and identified robust New York esophageal squamous cell carcinoma 1 (NY-ESO-1)_123-137/HLA-DRB3*02:02 CD4 T cell activity in both peripheral blood and tumor tissue. By analyzing NY-ESO-1_123-137/HLA-DRB3*02:02-restricted CD4 T cell clones, we uncovered an unexpectedly high cytotoxicity, strong T helper 1 polarization, and recurrent αβ T cell receptor (TCRαβ) usage across patients and anatomical sites. These responses were also present in other NY-ESO-1-expressing cancers. TCRs from these clones, when transduced into primary CD4 T cells, showed direct antitumor efficacy both in vitro and in vivo. Our findings suggest that these TCRs are promising for adoptive T cell transfer therapy, enabling broader targeting of NY-ESO-1-expressing adult and pediatric cancers in clinical settings.

Fig. 1.. Detection of antigen-specific CD4 T cells using the pMHCII multimer technology.

(A) Schematic of the experimental strategy to detect antigen-specific CD4 T cells by pMHCII multimer staining. (B) Representative dot plots of pMHCII multimer staining of CD4 T cells specific for NY-ESO-1_123–137, hTERT_916–930, and Melan-A_94–108 in PBMCs and TILs/TILNs of HLA-DRB3*02:02^+/− patients’ and HDs’ samples. (C) Summarizing graph of antigen-specific CD4 T cell frequencies in PBMCs and TILs/TILNs from patients with melanoma (n = 8) and HDs (n = 3).

Fig. 2.. Cytotoxic potency of diverse tumor antigen–specific CD4 T cells.

(A) Schematic of the experimental strategy to generate tumor antigen–specific CD4 T cell clones and representative dot plots of NY-ESO-1_123–137–, hTERT_916–930–, and Melan-A_94–108–specific CD4 T cell clones using the pMHCII multimer technology. (B) Representative example of the specific lysis using lactate dehydrogenase (LDH) cytotoxic assay conducted with the CIITA-transduced HLA-DRB3*02:02⁺ T333A tumor cell line (in blue), HLA-DRB3*02:02⁺ Me252 tumor cell line (in brown), or HLA-DRB3*02:02⁻ GEFI tumor cell line (in gray) and NY-ESO-1_123–137–, hTERT_916–930–, and Melan-A_94–108–specific CD4 T cell clones. (C) Left: Cumulative analysis of LDH cytotoxic assays conducted with tumor cell lines transduced with CIITA and cocultured with NY-ESO-1_123–137–specific (n = 33), hTERT_916–930–specific (n = 11), and Melan-A_94–108–specific (n = 14) CD4 T cell clones (E:T ratio, 30:1). Right: Cumulative analysis of LDH cytotoxic assays conducted with tumor cell lines transduced with CIITA and cocultured with NY-ESO-1_123–137–specific CD4 T cell clones from PBMCs (n = 22), TILs (n = 7), and TILNs (n = 4) (E:T ratio, 30:1). Statistical power was assessed using one-way analysis of variance (ANOVA). (D) Cumulative analysis of LDH cytotoxic assays conducted with tumor cell lines, transduced or not with CIITA, and cocultured with NY-ESO-1_123–137–specific CD4 T cell clones (n = 16) (E:T ratio, 30:1). Statistical power was assessed using t test. (E) Cumulative data of the maximum values of IFN-γ, TNF-α, IL-4, IL-5, IL-6, IL-9, IL-17A, IL-17F, IL-10, IL-22, and IL-13 secretion obtained in peptide titration experiments using NY-ESO-1_123–137/DRB3*02:02 CD4 T cell clones isolated from PBMCs/TILs/TILNs and analyzed by LEGENDplex. (F) Cumulative data of the maximum values of IFN-γ, TNF-α, and IL-13 secretion obtained in peptide titration experiment using Melan-A_94–108–specific CD4 T cell clones (n = 3) and hTERT_916–930-specific CD4 T cell clones (n = 3) (right) isolated from PBMCs and analyzed by LEGENDplex.

Fig. 3.. Phenotypic characterization of diverse tumor antigen–specific CD4 T cells.

(A) Graphs summarizing PD-1, TIGIT, CD57, and VISTA expression in NY-ESO-1_123–137 PBMC–specific (n = 16), TIL/TILN–specific (n = 11), hTERT_916–930 PBMC–specific (n = 8), and Melan-A_94–108 PBMC–specific (n = 4) CD4 T cell clones. Statistical power was assessed using one-way ANOVA. (B) Graphs summarizing GZMB, SLAMF7, OX40, 41BB, and CTLA-4 expression in NY-ESO-1_123–137 PBMC–specific (n = 16), TIL/TILN–specific (n = 11), hTERT_916–930 PBMC–specific (n = 8), and Melan-A_94–108 PBMC–specific (n = 4) CD4 T cell clones. Statistical power was assessed using one-way ANOVA. (C) Graphs summarizing the correlation between the percentage of specific lysis of NY-ESO-1_123–137–specific CD4 T cell clones and GZMB, SLAMF7, 41BB, OX40, and CTLA-4 (n = 27). Statistical power was assessed using simple linear regression.

Fig. 4.. Highly conserved TCR Vα and TCR Vβ usage in NY-ESO-1_123–137/DRB3*02:02–specific CD4 T cells.

(A) Pies illustrating the relative abundance of each clonotype among all NY-ESO-1_123–137/DRB3*02:02 CD4 T cell clones in the PMBCs (n = 20) and TILs/TILNs (n = 19) of patients with melanoma and HDs’ PBMCs (n = 7), Melan-A_94–108 (n = 9), and hTERT_916–930/DRB3*02:02 (n = 9) CD4 T cell clones. (B) Pies summarizing TCR sequencing of the alpha and beta chains of multimer-positive (left pies) or multimer-negative (right pies) CD4 T cells sorted from in vitro–expanded PBMCs and TILs/TILNs from patients (n = 9). (C) Pies summarizing TCR sequencing of the alpha and beta chains of memory CD4 T cells isolated from adult blood (n = 4). (D) Pies summarizing TCR sequencing of the alpha and beta chains of naïve CD4 T cells isolated from cord blood (n = 3).

Fig. 5.. 3D structural model obtained using TCRmodel2 for the complex TCR1/NY-ESO-1/HLA-DRA + HLA-DRB3*02:02.

TCR1 is colored gray (TCRα) and blue (TCRβ), HLA-DRA and HLA-DRB3*02:02 are colored brown, and NY-ESO-1 is colored orange. Residues of the peptide are displayed in ball-and-stick representation, while those of the MHC and TCR are shown in stick representation. Hydrogen bonds are shown as thin green lines. Nonpolar hydrogens are hidden for clarity.

Fig. 6.. NY-ESO-1_123–137–specific CD4 T cells in other tumor types.

Representative dot plots and pies summarizing TCR alpha and beta chains of multimer-positive CD4 T cells after in vitro stimulation with the NY-ESO-1_123–137 peptide in (A) samples from patients with lung cancer (n = 5), (B) samples from patients with ovarian cancer (n = 3), (C) samples from patients with neuroblastoma pediatric cancer (n = 3), and (D) healthy donor samples.

Fig. 7.. TCR transduction in human primary CD4 T cells.

(A) Schematic of experimental strategy to generate NY-ESO-1/TCR–transduced human CD4 T cells. KO, knockout. (B) Cumulative analysis of TCR transduction in CD4 (n = 12) and CD8 (n = 4) T cells, with or without CRISPR editing. Statistical power was assessed using one-way ANOVA. (C) Cumulative analysis of percentage of live cells in TCR-transduced CD4 (n = 12) or CD8 (n = 4) T cells, with or without CRISPR editing. Statistical power was assessed using one-way ANOVA. (D) Cytotoxic capacity of TCR-transduced CD4 T cells against different HLA-matched and HLA-mismatched tumor cell lines (E:T ratio, 30:1) (n = 4). (E) Cytokine secretion analysis (TNF-α, IFN-γ, IL-2, IL-5, and IL-13) by LEGENDplex in supernatants of NY-ESO-1_123–137/TCR–transduced CD4 T cells for the higher ratio E:T (30:1) (n = 6).

Fig. 8.. Adoptive transfer of NY-ESO-1/TCR T cells in tumor-bearing IL-2–NOG mice.

(A) Schematic of experimental strategy. (B) In vivo efficacy of adoptively transferred NY-ESO-1/TCR–transduced T cells against HLA-matched tumor xenografts (six mice per group). Statistical power was assessed using Kruskal-Wallis test. (C) Representative dot plots of human CD3 staining of the PBMCs of untreated mice and mice receiving NY-ESO-1/TCR–transduced CD4 T cells or NY-ESO-1/TCR–transduced CD8 T cells. (D) Monitoring of the frequency of persisting human T cells by flow cytometry (six mice per group).