Reprogramming of Virus-specific T Cells into Leukemia-reactive T Cells Using T Cell Receptor Gene Transfer

T cells directed against minor histocompatibility antigens (mHags) might be responsible for eradication of hematological malignancies after allogeneic stem cell transplantation. We investigated whether transfer of T cell receptors (TCRs) directed against mHags, exclusively expressed on hematopoietic cells, could redirect virus-specific T cells toward antileukemic reactivity, without the loss of their original specificity. Generation of T cells with dual specificity may lead to survival of these TCR-transferred T cells for prolonged periods of time in vivo due to transactivation of the endogenous TCR of the tumor-reactive T cells by the latent presence of viral antigens. Furthermore, TCR transfer into restricted T cell populations, which are nonself reactive, will minimize the risk of autoimmunity. We demonstrate that cytomegalovirus (CMV)-specific T cells can be efficiently reprogrammed into leukemia-reactive T cells by transfer of TCRs directed against the mHag HA-2. HA-2-TCR–transferred CMV-specific T cells derived from human histocompatibility leukocyte antigen (HLA)-A2+ or HLA-A2− individuals exerted potent antileukemic as well as CMV reactivity, without signs of anti–HLA-A2 alloreactivity. The dual specificity of these mHag-specific, TCR-redirected virus-specific T cells opens new possibilities for the treatment of hematological malignancies of HLA-A2+ HA-2–expressing patients transplanted with HLA-A2–matched or –mismatched donors

Performance Testing of a Homemade Aerosol Generator for Pulmonary Administration of Dry Powder Formulations to Mice

A challenge in the development of dry powder formulations for inhalation is the poor reproducibility of their administration to small laboratory animals. The currently used devices for the pulmonary administration of dry powder formulations to small rodents often function sub-optimally as they use the same puff of air for both powder dispersion and aerosol delivery. As a result, either the air volume and flow rate are too low for complete powder deagglomeration or they are too high for effective aerosol delivery to the lungs of the animal. Therefore, novel and better devices are desired. We here present an aerosol generator designed to administer a pre-generated aerosol to the lungs of mice. By mapping the complex relationship between the airflow rate, delivery time and emitted dose, we were able to control the amount of powder being delivered from the aerosol generator. The emitted aerosol had a size range favorable for lung deposition and could be measured reproducibly. Nevertheless, in vivo fluorescent imaging still revealed considerable differences between the mice in terms of the dose deposited and the distribution of powder over the lungs, suggesting that a certain biological variation in lung deposition is inevitable.</p

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

Starch Digestion and Applications of Slowly Available Starch

Frequent high postprandial glucose concentrations could be a risk factor for the development of type 2 diabetes and/or cardiovascular disease. Starchy products can play a role in the prevention of these hyperglycemic events, if starch-derived glucose is released into the circulation in a more slow and attenuated manner (slowly available starch). In this chapter, we describe the mechanisms that operate to maintain normal plasma glucose levels and the causes and consequences of hyperglycemia. Secondly, possible targets to slow down the rate at which starch-derived glucose becomes available for absorption in the gastrointestinal tract are discussed in more detail. Thirdly, we give an overview over the methods to monitor starch digestion in vitro and in vivo and the current (nutritional) strategies to prevent hyperglycemia. We conclude by describing promising approaches to better characterize starchy food products and to extend the choice of products with slowly available starch

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