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

Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

Comparing CAR and TCR engineered T cell performance as a function of tumor cell exposure

Chimeric antigen receptor (CAR) T cell therapies have resulted in profound clinical responses in the treatment of CD19-positive hematological malignancies, but a significant proportion of patients do not respond or relapse eventually. As an alternative to CAR T cells, T cells can be engineered to express a tumor-targeting T cell receptor (TCR). Due to HLA restriction of TCRs, CARs have emerged as a preferred treatment moiety when targeting surface antigens, despite the fact that functional differences between engineered TCR (eTCR) T and CAR T cells remain ill-defined. Here, we compared the activity of CAR T cells versus engineered TCR T cells in targeting the B cell malignancy-associated antigen CD20 as a function of antigen exposure. We found CAR T cells to be more potent effector cells, producing higher levels of cytokines and killing more efficiently than eTCR T cells in a short time frame. However, we revealed that the increase of antigen exposure significantly impaired CAR T cell expansion, a phenotype defined by high expression of coinhibitory molecules and effector differentiation. In contrast, eTCR T cells expanded better than CAR T cells under high antigenic pressure, with lower expression of coinhibitory molecules and maintenance of an early differentiation phenotype, and comparable clearance of tumor cells

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