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

Functional human antigen-specific T cells produced in vitro using retroviral T cell receptor transfer into hematopoietic progenitors

In vitro production of human T cells with known Ag specificity is of major clinical interest for immunotherapy against tumors and infections. We have performed TCRalphabeta gene transfer into human hemopoietic progenitors from postnatal thymus or umbilical cord blood, and subsequently cultured these precursors on OP9 stromal cells expressing the Notch human ligand Delta-like1. We report here that fully mature, functional T cells with controlled Ag specificity are obtained from such cultures. Using vectors encoding TCRalphabeta-chains directed against melanoma (MART-1), viral (CMV), and minor histocompatibility (HA-2) Ags, we show that the obtained Ag-specific T cells exert cytolytic activity against their cognate Ag and expand in vitro upon specific TCR stimulation. Therapeutic applications may arise from these results because they provide a way to produce large numbers of autologous mature Ag-specific T cells in vitro from undifferentiated hemopoietic progenitor

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

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

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

T cell receptor engineering of primary NK cells to therapeutically target tumors and tumor immune evasion

Background T cell receptor (TCR)-engineered cells can be powerful tools in the treatment of malignancies. However, tumor resistance by Human Leukocyte antigen (HLA) class I downregulation can negatively impact the success of any TCR-mediated cell therapy. Allogeneic natural killer (NK) cells have demonstrated efficacy and safety against malignancies without inducing graft-versus-host-disease, highlighting the feasibility for an ‘off the shelf’ cellular therapeutic. Furthermore, primary NK cells can target tumors using a broad array of intrinsic activation mechanisms. In this study, we combined the antitumor effector functions of NK cells with TCR engineering (NK-TCR), creating a novel therapeutic strategy to avoid TCR-associated immune resistance.Methods BOB1, is a transcription factor highly expressed in all healthy and malignant B cell lineages, including multiple myeloma (MM). Expression of an HLA-B*07:02 restricted BOB1-specifc TCR in peripheral blood–derived NK cells was achieved following a two-step retroviral transduction protocol. NK-TCR was then compared with TCR-negative NK cells and CD8-T cells expressing the same TCR for effector function against HLA-B*07:02+ B-cell derived lymphoblastoid cell lines (B-LCL), B-cell acute lymphoblastic leukemia and MM cell lines in vitro and in vivo.Results Firstly, TCR could be reproducibly expressed in NK cells isolated from the peripheral blood of multiple healthy donors generating pure NK-TCR cell products. Secondly, NK-TCR demonstrated antigen-specific effector functions against malignancies which were previously resistant to NK-mediated lysis and enhanced NK efficacy in vivo using a preclinical xenograft model of MM. Moreover, antigen-specific cytotoxicity and cytokine production of NK-TCR was comparable to CD8 T cells expressing the same TCR. Finally, in a model of HLA-class I loss, tumor cells with B2M KO were lysed by NK-TCR in an NK-mediated manner but were resistant to T-cell based killing.Conclusion NK-TCR cell therapy enhances NK cell efficacy against tumors through additional TCR-mediated lysis. Furthermore, the dual efficacy of NK-TCR permits the specific targeting of tumors and the associated TCR-associated immune resistance, making NK-TCR a unique cellular therapeutic

αβ-T Cells Engineered to Express γδ-T Cell Receptors Can Kill Neuroblastoma Organoids Independent of MHC-I Expression

Currently ~50% of patients with a diagnosis of high-risk neuroblastoma will not survive due to relapsing or refractory disease. Recent innovations in immunotherapy for solid tumors are highly promising, but the low MHC-I expression of neuroblastoma represents a major challenge for T cell-mediated immunotherapy. Here, we propose a novel T cell-based immunotherapy approach for neuroblastoma, based on the use of TEG002, αβ-T cells engineered to express a defined γδ-T cell receptor, which can recognize and kill target cells independent of MHC-I. In a co-culture killing assay, we showed that 3 out of 6 neuroblastoma organoids could activate TEG002 as measured by IFNγ production. Transcriptional profiling showed this effect correlates with an increased activity of processes involved in interferon signaling and extracellular matrix organization. Analysis of the dynamics of organoid killing by TEG002 over time confirmed that organoids which induced TEG002 activation were efficiently killed independent of their MHC-I expression. Of note, efficacy of TEG002 treatment was superior to donor-matched untransduced αβ-T cells or endogenous γδ-T cells. Our data suggest that TEG002 may be a promising novel treatment option for a subset of neuroblastoma patients