NY-ESO-1-specific redirected T cells with endogenous TCR knockdown mediate tumor response and cytokine release syndrome

Abstract

Background: Because of the shortage of ideal cell surface antigens, the development of T-cell receptor (TCR)-engineered T cells (TCR-T) that target intracellular antigens such as NY-ESO-1 is a promising approach for treating patients with solid tumors. However, endogenous TCRs in vector-transduced T cells have been suggested to impair cell-surface expression of transduced TCR while generating mispaired TCRs that can become self-reactive.

Methods: We conducted a first-in-human phase I clinical trial with the TCR-transduced T-cell product (TBI-1301) in patients with NY-ESO-1-expressing solid tumors. In manufacturing TCR-T cells, we used a novel affinity-enhanced NY-ESO-1-specific TCR that was transduced by a retroviral vector that enables siRNA (small interfering RNA)-mediated silencing of endogenous TCR. The patients were divided into two cohorts. Cohort 1 was given a dose of 5×108 cells (whole cells including TCR-T cells) preconditioned with 1500 mg/m2 cyclophosphamide. Cohort 2 was given 5× 109 cells preconditioned with 1500 mg/m2 cyclophosphamide.

Results: In vitro study showed that both the CD8+ and CD4+ T fractions of TCR-T cells exhibited cytotoxic effects against NY-ESO-1-expressing tumor cells. Three patients and six patients were allocated to cohort 1 and cohort 2, respectively. Three of the six patients who received 5×109 cells showed tumor response, while three patients developed early-onset cytokine release syndrome (CRS). One of the patients developed a grade 3 lung injury associated with the infiltration of the TCR-T cells. No siRNA-related adverse events other than CRS were observed. Cytokines including interleukin 6 I and monocyte chemotactic protein-1/chemokine (C-C motif) ligand (CCL2)increased in the sera of patients with CRS. In vitro analysis showed these cytokines were not secreted from the T cells infused. A significant fraction of the manufactured T cells in patients with CRS was found to express either CD244, CD39, or both at high levels.

Conclusions: The trial showed that endogenous TCR-silenced and affinity-enhanced NY-ESO-1 TCR-T cells were safely administered except for grade 3 lung injury. The TCR-T cell infusion exhibited significant tumor response and early-onset CRS in patients with tumors that express NY-ESO-1 at high levels. The differentiation properties of the manufactured T cells may be prognostic for TCR-T-related CRS.

Trial registration number: NCT02366546.

Keywords: cell engineering; clinical trials as topic; cytokines; immunotherapy, adoptive.

Conflict of interest statement

Competing interests: SO, DT, IN, HC, and JM are employees of Takara Bio. The Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, to which SKa, YM, TW, and HS belonged, was funded by Takara Bio. MFK, PDHN, YS, AN, EN, and MF are employees or consultants of ImmunoScape. MI received honoraria from Chugai, Eisai, MSD, Ono Pharmaceutical, Daiichi Sankyo, and Eli Lilly. As a potential conflict of interest, SKi and NY received research grants from Takara Bio.

© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Preclinical study of G50A+A51E T cell receptor (TCR)-transduced T cells. (A) Construct of retroviral vector, pMS3-NY-ESO-1-siTCR. siRNAs, siRNA sequences for endogenous TCR-α and TCR-β; EU, untranslated region 5 of human EF1a; coTCRβ, codon-optimized TCR-β gene sequences for NY-ESO-1; 2A, 2A peptide; coTCRα, codon-optimized TCR-α gene sequences for NY-ESO-1; pA, polyA signal addition; Ψ+, packaging signal; 5'-LTR and R region of 3'-LTR are Moloney murine sarcoma virus-derived, U3 region of 3’-LTR is PCC4-cell-passaged myeloproliferative sarcoma virus-derived. The coTCRα and coTCRβ are formed by codon optimization and are not affected by small interfering RNAs (siRNAs). (B) Transduction of TCR gene to CD8+ T cells and CD4+ T cells. Using the vector of pMS3-NY-ESO-1-siTCR, the TCR gene was transduced to peripheral blood of a healthy volunteer and cultured for 10 days. CD8+ or CD4+ T cells were stained with the NY-ESO-1 peptidemajor histocompatibity complex (MHC) tetramer. 87.6% of CD8+ T cells and 89.0% of CD4+ T cells were positive for the tetramer. (C) Suppression of endogenous TCR-α and TCR-β chain expression by siTCR. RNAs were extracted from 5×106 cells of TCR-gene modified T cells (GMCs) and the T cells that were not gene-transduced (NGMCs). Reverse transcription PCR for endogenous TCR-α and TCR-β chains was performed. 35.3% (±7.9%) and 33.8% (±6.2%) reductions in TCR-α and TCR-β chain expression, respectively. Three experiments using T cells from healthy volunteers were performed in each TCR chain. (D) Interferon gamma (IFN-γ) ELISPOT assay of the NY-ESO-1-TCR-gene transduced CD8+ and CD4+ T cells from healthy volunteers. After TCR-gene transduction and 10-day culture, CD8+ T cells and CD4+ T cells were separated using microbeads. SK-MEL37 is a NY-ESO-1(+) and HLA-A*02:01(+) melanoma cell line. 397mel is a NY-ESO-1(+) and HLA-A*02:01(−) melanoma cell line. HLA-A*02:01-gene or HLA-A*02:06-gene transduced 397mel cells were used as target cells. (E) IFN-γ ELISPOT assay of NY-ESO-1-TCR (G50A+A51E)-gene transduced T cells targeting tumor cells. 1G4-clone is a wild-type (WT) TCR whose affinity is not modified, and G50A+A51E is the affinity-enhanced TCR that was used in the clinical trial. SK-MEL37 cells, 397mel cells, and HLA-A*02:01-transfected 397mel cells were used as target cells. (F) Cytotoxicity assay of G50A+A51E TCR-gene transduced CD8+ and CD4+ T cells. Cytotoxicity was determined by 51Cr-release assay. T cells were transduced with the NY-ESO-1-TCR(G50A+A51E) transgene, and CD8+ and CD4+ T cells were enriched after sorting with NY-ESO-1/HLA-A*02:01-tetramer. As target cells, T2 cells pulsed with NY-ESO-1 peptide (ESO1-T2), T2 cells pulsed with MAGE-A4 peptide (MAGE-T2), melanoma cell line (NW-MEL-38, NY-ESO-1+/HLA-A*02:01+) and colon tumor cell line (HCT116, NY-ESO-1−/HLA-A*02:01+) were used. 51Cr-labeled target cells were cocultured with CD8+ and CD4+ T cells for 12 hours. (G) Cross-reactivity of the NY-ESO-1-TCR with other peptides with amino acid sequences similar to that of the NY-ESO-1 peptide. WT and nine similar amino acid sequences. The WT NY-ESO-1/HLA*02:01-restricted peptide, SLLMWITQC, is listed at the top. Nine peptides are prepared by replacing each amino acid with alanine. (H) Nine peptides were synthesized, S1A, L2A, L3A, M4A, W5A, I6A, T7A, Q8A, C9A, by replacing each amino acid with alanine. TCR-T cells that responded to WT peptide and the synthesized nine peptides were analyzed by intracellular IFN-γ staining. The response to WT peptide was a positive control. Four amino acids (M, W, I, Q) were evaluated to be essential to TCR recognition. (I) Intracellular IFN-γ staining of TCR-T cells responding to the 11 analogous peptide-pulsed target cells. The 11 peptides are derived from known human proteins. YLNMWITTC, taste receptor type 2 member 8; GMDMWSTQD, claudin-18 isoform 1 precursor; QYLMWLTQK, phosphatidylinositol 3-kinase regulatory subunit alpha; AMVAWVTQE, serine/threonine-protein phosphatase 6 regulatory subunit 1; FQDMWITAA, actin-related protein T3; FQQMWITKQ, actin, alpha skeletal muscle; GQKMWITNG, medium-chain specific acyl-CoA dehydrogenase, mitochondrial; GQPLWITAT, ectonucleotide pyrophosphatase/phosphodiesterase family; YVIMWITYM, Niemann-Pick C1 protein precursor; LVGLWITQW, phosphatidylcholine:ceramide cholinephosphotransferase 2; KIALWITYG, gasdermin-C. The response to WT peptide was a positive control (figure 1H).

Figure 2

Clinical course of patient no 8. (A) Clinical course of patient no 8, who developed grade 3 lung injury after grade 2 cytokine release syndrome. (B) Chest X-ray (days 2, 9, and 14) and chest CT scan (days 5 and 9) of patient no 8. (C) Flow cytometry analysis of NY-ESO-1 tetramer–positive T cells in peripheral blood (PB) and bronchoalveolar lavage fluid (BALF) on day 10. Mononuclear cells were separated and were ex vivo stained with anti-CD8 antibody and NY-ESO-1 peptide/MHC tetramer. 25.0% of CD8+ T cells in PB were tetramer positive, and 53.6% of CD8+ T cells in BALF were also positive. TCR-T, T-cell receptor-engineered T cells.

Figure 3

Tumor responses after TBI-1301 infusion. (A) Waterfall plot of seven patients. Seven of the nine patients had measurable lesions that could be evaluated for tumor response. Patient nos 2 and 14 had no measurable tumor lesions. Patient nos 7, 8, and 16 had synovial sarcoma. Patient nos 8, 9, and 16 developed CRS. (B) Swimmer plot for the nine patients treated with TBI-1301. Patient no 2 had no measurable lesion. CRS, cytokine release syndrome; TBI-1301, TCR-transduced T cell product; TCR-T, T-cell receptor-engineered T cells.

Figure 4

Chronological serum cytokine level in six patients who received 5×109 TBI-1301 cells. (A) Serum IL-6, IFN-γ, TNF-α, and IL-2 levels in three patients with CRS. (B) Cytokine levels in three patients without CRS. (C) Multiple cytokines levels measured by Bio-Plex Pro human cytokine screening 48-Plex panel in three patients with CRS. (D) Cytokine levels in two patients without CRS. The serum sample from patient no 7 was unavailable. (E) Cytokine levels in the supernatants of TBI-1301 products from six patients after culturing with NY-ESO-1 peptide–pulsed target cells. Data from patients with CRS are shown on the left and those from patients without CRS are on the right. (F) Cytokine profiles responding to NY-ESO-1-peptide stimulation of TBI-1301 manufactured products in patients with and without CRS. CRS, cytokine release syndrome; IFN-γ, interferon gamma; IL, interleukin; TBI-1301, TCR-transduced T cell product; TNF-α, tumor necrosis factor alpha.

Figure 5

T cell phenotypes and high-dimensional immune profiles of TBI-1301 manufactured products. (A) T cell phenotypes of TBI-1301 manufactured products. Three patients, nos 8, 9, and 16, with CRS had significantly more frequent CD8+ T cells with effector memory phenotypes (p=0.008). Stem cell-like memory T cells, CD45RA+/CCR7+; central memory T cells, CD45RA−/CCR7+; effector memory T cells, CD45RA−/CCR7−; terminally differentiated T cells, CD45RA+/CCR7−. (B and C) High-dimensional immune profiles of NY-ESO-1 tetramer+ CD8+ T cells in the manufactured T cell product. CD244 and CD39 levels in NY-ESO-1-specific CD8+ T cells among the manufactured T cell products. Nos 8, 9, and 16 developed CRS, whereas nos 7, 14, and 15 did not. (D) Cells located in either cluster A (area enclosed with red line), cluster B (area enclosed with orange line), or both in infusion products of patients with CRS are not seen in infusion products of patients without CRS. No statistical difference was found between the two groups. (E) Clusters A and B are characterized by high levels of CD244 and CD39 and a lack of CCR7. CRS, cytokine release syndrome; TBI-1301, TCR-transduced T cell product; UMAP, uniform manifold approximation and projection.