Elisa Kieback

Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy

The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy–induced autoimmune pathology.

Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette

The transfer of T cell receptor (TCR) genes allows to endow T cells with a new antigen specificity. For clinical applications of TCR-redirected T cells, efficient functional expression of the transgenic TCR is a key prerequisite. Here, we compared the influence of the transgene cassette on the expression and function of the murine TCR P14 (recognizing a LCMV gp33 epitope) and the human TCR WT-1 (recognizing an epitope of the tumor-associated antigen WT-1). We constructed different vectors, in which TCRα- and β-chain genes were either (a) linked by an internal ribosomal entry site (IRES), (b) combined by a 2A peptide, or (c) introduced into two individual retroviral constructs. While in a TCR-deficient T cell line TCR P14 was expressed equally well by all constructs, we found that IRES- but not 2A-employing TCR expression is hampered in a TCR-bearing cell line and in primary murine T cells where the transgenic TCR has to compete with endogenous TCR chains. Similarly, 2A-linked TCR WT-1 genes yielded highest expression and function as measured by tetramer binding and peptide-specific IFN-γ secretion. Differences in expression were independent of copy number integration as shown by real-time PCR. Thus, linking TCRα- and β-chain genes by a 2A peptide is superior to an IRES for TCR expression and T cell function.

A safeguard eliminates T cell receptor gene-modified autoreactive T cells after adoptive transfer

By transfer of T cell receptor (TCR) genes, antigen specificity of T cells can be redirected to target any antigen. Adoptive transfer of TCR-redirected T cells into patients has shown promising results. However, this immunotherapy bears the risk of autoreactive side effects if the TCR recognizes antigens on self-tissue. Here, we introduce a safeguard based on a TCR-intrinsic depletion mechanism to eliminate autoreactive TCR-redirected T cells in vivo. By the introduction of a 10-aa tag of the human c-myc protein into murine (OT-I, P14) and human (gp100) TCR sequences, we were able to deplete T cells that were transduced with these myc-tagged TCRs with a tag-specific antibody in vitro. T cells transduced with the modified TCR maintained equal properties compared with cells transduced with the wild-type receptor concerning antigen binding and effector function. More importantly, therapeutic in vivo depletion of adoptively transferred T cells rescued mice showing severe signs of autoimmune insulitis from lethal diabetes. This safeguard allows termination of adoptive therapy in case of severe side effects.

Enhanced T cell receptor gene therapy for cancer

Importance of the field: Adoptive therapy with T cell receptor- (TCR-) redirected T cells has shown efficacy in mouse tumor models and first responses in cancer patients. One prerequisite to elicit effective anti-tumor reactivity is the transfer of high-avidity T cells. Their generation, however, faces several technical difficulties. Target antigens are often expressed at low levels and their recognition requires the use of high-affine receptors. Yet, mainly low-affine TCRs have been isolated from tumor-infiltrating lymphocytes. Furthermore, upon transfer into a T cell the introduced receptor has to compete with the endogenous TCR. Areas covered in this review: This review discusses how the functional avidity of TCR-modified T cells can be enhanced by i) increasing the amount of introduced TCR heterodimers on the cell surface; and ii) generating receptors with high affinity. Risks of TCR gene therapy and possible safety mechanisms are discussed. What the reader will gain: The reader will gain an overview of the technical developments in TCR and T cell engineering. Take home message: Despite technical obstacles, many advances have been made in the generation of high-avidity T cells expressing enhanced TCRs. Mouse studies and clinical trials will evaluate the effect of these improvements.

Isolation and functional characterization of hepatitis B virus-specific T-cell receptors as new tools for experimental and clinical use.

T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma.

Targeting high‐grade B cell lymphoma with CD19‐specific T cells

Adoptive T cell therapy is an important additional treatment option for malignant diseases resistant to chemotherapy. Using a murine high‐grade B cell lymphoma model, we have addressed the question whether the B cell differentiation antigen CD19 can act as rejection antigen. CD19−/− mice inoculated with CD19+ B cell lymphoma cells showed higher survival rates than WT mice and were protected against additional tumor challenge. T cell depletion prior to tumor transfer completely abolished the protective response. By heterotypic vaccination of CD19−/− mice against murine CD19, survival after tumor challenge was significantly increased. To define protective epitopes within the CD19 molecule, T cells collected from mice that had survived the tumor transfer were analyzed for IFNγ secretion in response to CD19‐derived peptides. The majority of mice exhibited a CD4+ T cell response to CD19 peptide 27, which was the most dominant epitope after CD19 vaccination. A peptide 27‐specific CD4+ T cell line protected CD19−/− mice against challenge with CD19+ lymphoma and also cured a significant proportion of WT mice from recurrent disease in a model of minimal residual disease after chemotherapy. In conclusion, our data highlight CD19‐specific CD4+ T cells for adoptive T cell therapy of B cell lymphomas.

Safety Modality for X-linked Severe Combined Immunodeficiency Gene Therapy

X-linked severe combined immunodeficiency (SCID-X1), caused by a defect of the cytokine receptor common gamma chain (γc), has been successfully treated by gene therapy in the clinic. However, the occurrence of leukemia in several patients preceded by loss of oligoclonality revealed that treatment is associated with a risk inherent to the genetic modification of hematopoietic stem cells. In this study, we developed a safety approach that allows the specific elimination of gene-modified cells. For this, a small peptide sequence (myc-tag) was introduced into the murine γc protein. Cells expressing the modified chain can be detected with a myc-specific antibody by flow cytometry and are effectively depleted in vitro in the presence of complement factors. Further, thymic-derived T cells from mice reconstituted with myc-tagged γc-transduced bone marrow stem cells can be depleted by antibody administration in vivo. Similarly, specific complement-mediated lysis was observed for human T cells expressing the human myc-tagged γc. In a cell proliferation assay, the modified cytokine receptor chain showed no functional impairment compared to the wild-type chain. In sum, we show proof-of-principle of a safety mechanism for SCID-X1 gene therapy that would allow elimination of gene-corrected cells in a patient upon observation of monoclonal outgrowth.

P73. Functional characterisation of HBV-specific T cell receptors for redirection of T cells against HBV infected hepatocytes

Chronic HBV infection, which is accompanied by a weak and oligoclonal T cell response, is the most common cause of hepatocellular carcinoma (HCC). Current antiviral therapies do not eliminate the virus, but T cell therapy will very likely do so. From PBMCs of two HLA-A2+ acutely infected patients and a donor who cleared HBV infection we have established several HBV-specific monoclonal T cell lines. Thereof we isolated 11 different T cell receptors (TCR) that are specific for the HBV S-protein derived peptides S20 (FLLTRILTI) and S172 (WLSLLVPFV) or for the C18 core-peptide (FLPSDFFPSV). The aim of this study was a functional comparison of our set of HBV-specific TCRs in order to identify TCRs with optimal recognition of HBV peptides presented on HLA-A2. By murinization and codon-optimisation of gene sequences of TCR a and b chains, fused by a P2A element for polycystronic expression, TCR expression after retroviral transduction was increased 2-fold to 60% of PBMCs expressing an HBV-specific TCR. PBMCs transduced with the 11 optimised HBV-specific TCRs were compared in killing assays using peptide-pulsed T2 cells, LCLs and HBV-replicating HepG2.2.15 cells as targets. CD8+ T cells transduced with the core-specific TCRs killed target cells loaded with 0.01 nM of peptide. Cells specific for the S20 and S172 peptide were less sensitive with a specific lysis as low as 0.1 nM. Expression of most of the HLA-A2 restricted HBV-specific TCRs in CD4+ T cells also led to specific cytotoxicity, which was 10-fold reduced in sensitivity compared to CD8+ T cells and independent of CD8 co-receptor binding. Notably, our HBV-specific TCRs recognised peptide presented on various different HLA-A2 subtypes. CD8+ T cells transduced with HBV-specific TCRs were also able to recognise endogenously processed peptides and specifically kill HBV-replicating hepatoma cells and strongly reduce cccDNA levels in HBV-infected HepaRG cells. In addition, intracellular cytokine staining after stimulation showed that the TCR-transduced CD8+ T cells were polyfunctional, secreting INF-γ, TNF-α and IL-2, whereas CD4+ T cells produced mainly TNF-a and/or IL-2. We will further analyse our HBV-specific TCRs in HBV/HLA-A2 transgenic mice in order to identify the TCR that confers best antiviral activity. Our HBV-specific TCRs may be used for elucidating specific anti-HBV mechanisms exerted by T cells, and most importantly, for adoptive T cell therapy of chronic hepatitis B and HBV-induced HCC.