Preclinical Strategies to Identify Off-Target Toxicity of High-Affinity TCRs

Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Extracellular domains of CD8α and CD8ß subunits are sufficient for HLA class I restricted helper functions of TCR-engineered CD4(+) T cells.

By gene transfer of HLA-class I restricted T-cell receptors (TCRs) (HLA-I-TCR) into CD8(+) as well as CD4(+) T-cells, both effector T-cells as well as helper T-cells can be generated. Since most HLA-I-TCRs function best in the presence of the CD8 co-receptor, the CD8αß molecule has to be co-transferred into the CD4(+) T-cells to engineer optimal helper T-cells. In this study, we set out to determine the minimal part of CD8αβ needed for optimal co-receptor function in HLA-I-TCR transduced CD4(+) T-cells. For this purpose, we transduced human peripheral blood derived CD4(+) T-cells with several HLA-class I restricted TCRs either with or without co-transfer of different CD8 subunits. We demonstrate that the co-transduced CD8αβ co-receptor in HLA-I-TCR transduced CD4(+) T-cells behaves as an adhesion molecule, since for optimal antigen-specific HLA class I restricted CD4(+) T-cell reactivity the extracellular domains of the CD8α and ß subunits are sufficient

Optimization of the HA-1-specific T-cell receptor for gene therapy of hematologic malignancies

To broaden the applicability of adoptive T-cell therapy for the treatment of hematologic malignancies, we aim to start a clinical trial using HA-1-TCR transferred virus-specific T cells. TCRs directed against the minor histocompatibility antigen (MiHA) HA-1 are good candidates for TCR gene transfer to treat hematologic malignancies because of the hematopoiesis-restricted expression and favorable frequency of HA-1. For optimal anti-leukemic reactivity, high cell-surface expression of the introduced TCR is important. Previously, however, we have demonstrated that gene transferred HA-1-TCRs are poorly expressed at the cell-surface. In this study several strategies were explored to improve expression of transferred HA-1-TCRs

Improved HLA-class I restricted avidity of CD8αß expressing HA-2-TCR td CD4⁺ T-cells results in improved proliferation.

<p>(A) To study whether co-transfer of CD8 would also improve the peptide sensitivity of CD4⁺ T-cells transduced with a next generation HA-2-TCR, both mock and HA-2-TCR td CMV-specific CD4⁺ T-cells with or without co-transfer of different CD8 subunits as indicated in the figure were purified using flow cytometry based cell sorting and stimulated with unpulsed HLA-A2⁺ HA-2⁻ LCL IZA (white bars; LCL IZA), HLA-A2⁺ HA-2⁻ LCL-IZA pulsed with decreasing concentrations of HA-2 peptide (range 1 µM-10 pM) or HLA-A2⁺ HA-2⁺ LCL JYW (striped bars; LCL JYW). IFN-γ production was measured after 18 h of stimulation in duplicate, and a representative experiment out of 2 is depicted. The IFN-γ production of ΔCD8αß and wtCD8αß expressing HA-2-TCR_CC td CD4⁺ T-cells significantly higher (p-values <0.05) than CD8 negative or CD8αα expressing HA-2-TCR_CC td CD4⁺ T-cells is indicated with an asterisk. (B) To investigate their proliferative capacity, both mock and HA-2-TCR td CD4⁺ T-cells without CD8 or co-transferred with wtCD8α, wtCD8αß, or ΔCD8αß were purified based on markergene expression and CD8 cell surface expression and were either not stimulated (filled histograms) or stimulated with HLA-A2⁺ HA-2⁺ LCL-JYW (thick black line). Histograms depict PKH dilution measured 5 days after stimulation, and a representative example of 2 independent experiments is depicted.</p

In general, co-transfer of the extracellular domains of CD8α and ß is required and sufficient.

<p>To confirm the generality of the previous data, total CD4⁺ T-cells were transduced with codon optimized and cysteine modified HA-1-, HA-2- or PRAME-TCR (transduction efficiency 48%, 48% and 22%, respectively) either with or without co-transfer of different CD8 molecules, as indicated in the figure. One week after transduction, non-purified TCR td CD4⁺ T-cells were stimulated and tested for cytokine production using flow cytometry. HA-1- or HA-2-TCR td CD4⁺ T-cells were stimulated either with HA-1 or HA-2 peptide pulsed or unpulsed HLA-A2⁺ HA-1^- HA-2⁻ LCL-IZA, or HLA-A2⁺ HA-1⁺ HA-2⁺ LCL-MRJ, and PRAME-TCR td CD4⁺ T-cells were stimulated either with PRAME peptide pulsed or unpulsed HLA-A2⁺ PRAME⁻ melanoma cells, or HLA-A2⁺ PRAME⁺ melanoma cells. 5 h After stimulation, T-cells were permeabilized and stained with anti-NGF-R in combination with either anti-IFN-γ (upper panel), anti-IL-2 (middle panel) or anti-TNF-α (lower panel), and analyzed using flow cytometry. The percentage of markergene positive and CD8 positive T-cells producing cytokines after stimulation with antigen-negative cells (white bars; control), peptide pulsed cells (grey bars; pulsed peptide) or antigen-positive cells (black bars; endogenous peptide) is indicated.</p

HLA-I-TCR td CD4⁺ T-cells co-transferred with wtCD8αß or intracellularly modified CD8αß demonstrate equal effector functions.

<p>To study the minimal part of CD8 needed for optimal co-receptor function in HLA-I-TCR td CD4⁺ T-cells, HA-2-TCR td CMV-specific CD4⁺ T-cells (A) co-transferred with wtCD8αα or wtCD8αß co-receptor, or (B) co-transferred with either wtCD8α,ΔCD8α or CD8α Lck in combination with either wtCD8ß or ΔCD8ß were purified and used in a stimulation assay. Td T-cell populations were tested against HLA-DR1⁺ LCL-CBH either unpulsed (grey striped bars) or pulsed with pp65 peptide (grey bars), or against HLA-A2⁺ HA-2⁻ LCL-IZA either unpulsed (white bars) or pulsed with HA-2 peptide (black bars), or against HLA-A2⁺ HA-2⁺ LCL-JYW (black striped bars). IFN-γ production was measured after 18 h of stimulation in duplicate, and a representative experiment out of 3 is depicted. The IFN-γ production of the different CD8αß expressing TCR td T-cells was compared to the IFN-γ production of CD8αα expressing TCR td T-cells within their group using students' t-test. P-values <0.05 are indicated with an asterisk. (C) To study whether co-transfer of CD8 would also result in polyfunctional helper functions of TCR td CMV-specific CD4⁺ T-cells, both mock and HA-2-TCR td CMV-specific CD4⁺ T-cells with or without co-transfer of different CD8 subunits as indicated in the figure were stimulated with HLA-DR1⁺ LCL-CBH pulsed with pp65 peptide (grey bars; pp65 pep), unpulsed HLA-A2⁺ HA-2⁻ LCL-IZA (white bars; control), HA-2 peptide pulsed HLA-A2⁺ HA-2⁻ LCL-IZA (black bars; HA-2 pep) or HLA-A2⁺ HA-2⁺ LCL-JYW (striped bars; HA-2 endogenous). After 5 h of stimulation, T-cells were stained with anti-IFN-γ, anti-TNF-α, anti-CD40L and anti-IL-2 mAbs and were analyzed using flow cytometry. The percentage of IFN-γ, TNF-α and IL-2 producing or CD40L expressing T-cells after stimulation is depicted. The percentages of cytokine producing and CD40L upregulating CD8αß expressing TCR td T-cells that were significantly higher than CD8 negative and CD8αα expressing TCR td T-cells (p-values <0.05) are indicated with an asterisk. (D/E) To study differences in avidity between HLA-I-TCR td CD4⁺ T-cells co-transferred with the different CD8α and CD8ß constructs, HA-2 tetramer staining was analyzed. (D) Mock or (E) HA-2-TCR td CD4⁺ T-cells co-transferred with either wtCD8α-T2A-wtCD8ß (wtCD8 T2A; left dot plots) or ΔCD8α-T2A-ΔCD8ß (ΔCD8 T2A, right dot plots) were stained with anti-CD8α and ß mAbs and HA-2-tetramers and analyzed using flow cytometry. Populations were gated on CD8αß positive expression and HA-2 tetramer staining is depicted for the gated populations. Percentages of HA-2-tetramer positive T-cells are indicated in the upper right and MFI of the HA-2-tetramer staining in the upper left of the dot plots. Data shown are representative for 2 independent experiments.</p

SARS-CoV-2-specific CD4+ and CD8+ T cell responses can originate from cross-reactive CMV-specific T cells

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific CD4+ and CD8+ T cells in SARS-CoV-2-unexposed donors has been explained by the presence of T cells primed by other coronaviruses. However, based on the relatively high frequency and prevalence of cross-reactive T cells, we hypothesized cytomegalovirus (CMV) may induce these cross-reactive T cells. Stimulation of pre-pandemic cryo-preserved peripheral blood mononuclear cells (PBMCs) with SARS-CoV-2 peptides revealed that frequencies of SARS-CoV-2-specific T cells were higher in CMV-seropositive donors. Characterization of these T cells demonstrated that membrane-specific CD4+ and spike-specific CD8+ T cells originate from cross-reactive CMV-specific T cells. Spike-specific CD8+ T cells recognize SARS-CoV-2 spike peptide FVSNGTHWF (FVS) and dissimilar CMV pp65 peptide IPSINVHHY (IPS) presented by HLA-B*35:01. These dual IPS/FVS-reactive CD8+ T cells were found in multiple donors as well as severe COVID-19 patients and shared a common T cell receptor (TCR), illustrating that IPS/FVS-cross-reactivity is caused by a public TCR. In conclusion, CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-existing immunity against SARS-CoV-2

PRAME and HLA Class I expression patterns make synovial sarcoma a suitable target for PRAME specific T-cell receptor gene therapy

Synovial sarcoma expresses multiple cancer testis antigens that could potentially be targeted by T-cell receptor (TCR) gene therapy. In this study we investigated whether PRAME-TCR-gene therapy could be an effective treatment for synovial sarcoma by investigating the potential of PRAME-specific T-cells to recognize sarcoma cells and by evaluating the expression patterns of PRAME and HLA class I (HLA-I) in synovial sarcoma tumor samples. All PRAME expressing sarcoma cell lines, including 2 primary synovial sarcoma cell cultures (passage < 3), were efficiently recognized by PRAME-specific T-cells. mRNA FISH demonstrated that PRAME was expressed in all synovial sarcoma samples, mostly in an homogeneous pattern. Immunohistochemistry demonstrated low HLA-I baseline expression in synovial sarcoma, but its expression was elevated in specific areas of the tumors, especially in biphasic components of biphasic synovial sarcoma. In 5/11 biphasic synovial sarcoma patients and in 1/17 monophasic synovial sarcoma patients, elevated HLA-I on tumor cells was correlated with infiltration of T-cells in these specific areas. In conclusion, low-baseline expression of HLA-I in synovial sarcoma is elevated in biphasic areas and in areas with densely infiltrating T-cells, which, in combination with homogeneous and high PRAME expression, makes synovial sarcoma potentially a suitable candidate for PRAME-specific TCR-gene therapy