Structural basis for the therapeutic efficiency of optimal-affinity T cell receptors

Información del proyecto

TCRabX

Identificador del acuerdo de subvención: 794852

DOI

10.3030/794852

Proyecto cerrado

Fecha de la firma de la CE 3 Agosto 2018

Fecha de inicio 1 Agosto 2019

Fecha de finalización 31 Julio 2022

Financiado con arreglo a

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coste total

€ 264 110,40

Aportación de la UE

€ 264 110,40

Coordinado por

CHARITE - UNIVERSITAETSMEDIZIN BERLIN
Germany

Periodic Reporting for period 2 - TCRabX (Structural basis for the therapeutic efficiency of optimal-affinity T cell receptors)

Período documentado: 2021-08-01 hasta 2022-07-31

Resumen del contexto y de los objetivos generales del proyecto

A malignant solid tumour is a life-threatening disease that is difficult to cure. Currently, solid tumours are treated with classical therapies such as surgery, chemotherapy and radiation. Adoptive T cell therapy represents an alternative treatment method that can attack the tumours very specifically. Many tumour patients already benefit from adoptive T cell therapy, but a large proportion of patients also experience severe side effects and the therapy does not work. The prerequisite for the high specificity of the treatment is that the T cells used are equipped with high-affinity T cell receptors (TCRs). These should only recognize the tumour but not recognize other tissue, and they should eliminate the tumour efficiently. Since tumour antigens are mostly endogenous and are therefore self-antigens, it is difficult to isolate high-affinity TCRs from patients or healthy volunteers. In a new approach, human TCRs have been isolated from humanized mice in the last decade. The mice have a human T cell repertoire and are antigen-negative with respect to human tumour antigens. Under these circumstances, it is possible to obtain tumour-specific TCRs with high peptide sensitivity, suitable for therapy. During the isolation of tumour-specific TCRs, the immunological aspects of the TCRs are investigated. The main objective of such initial tests is to determine high effectiveness of the TCRs and to avoid detection of non-malignant somatic cells. There is little information available to date on how a mouse-derived TCR with high peptide sensitivity differs from a human-derived TCR with lower peptide sensitivity with regard to biochemical parameters. Experimental data are required to relate the immunological parameter of peptide sensitivity to biochemical parameters such as affinity or to structural information regarding the interaction of the TCR with its target, the peptide-human leukocyte antigen complexes (pHLA). The objective of this project was to compare the biochemical properties, in particular the parameters of binding of mouse-derived TCRs or human-derived TCRs to the respective pHLA. The methods applied were surface plasmon resonance measurements of TCR-pHLA binding as well as X-ray structural analysis of pHLA molecules and TCR-pHLA complexes. The mouse-derived TCRs and human-derived TCRs showed different affinities for the pHLA. The TCRs also differed in their kinetics of binding to pHLA. In the future, the findings will help to better understand the mode of action of optimal affinity therapeutic TCRs. This information is of interest not only for basic research but also for partners in industry. The data will support both the future development and optimization of existing adoptive T cell therapies.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

T cell receptor (TCR) development in the presence of the tumour antigen, as in the case of human-derived TCRs, or in the absence of the tumour antigen, as in the case of mouse-derived TCRs, results in differences in peptide sensitivity. Such human leukocyte antigen (HLA) class I-restricted TCRs and HLA class II-restricted TCRs each selected in the presence or absence of the human tumour antigen were subject of the project. During the outgoing phase, biochemical experiments and structural analyses were conducted at Monash University in Clayton, Australia. The alpha- and beta-chains of fourteen TCRs were expressed in bacteria, and the TCRs were folded and purified by hydrophobic-interaction chromatography, anion-exchange chromatography, and S200 size-exclusion chromatography. HLA class I molecules and HLA class II molecules were expressed in bacteria and mammalian cells, respectively, and also purified. X-ray diffraction patterns of the crystals for three pHLA classs I molecules and one pHLA class II molecule were recorded at the MX2 beamline of the Australian Synchrotron. Surface plasmon resonance (SPR) binding analysis was performed and analysed for ten TCRs in total, where the peptide-HLA (pHLA) was the ligand and the TCRs were the analyte. During the return phase the data were analysed and were described in two research articles. The above-mentioned differences in peptide sensitivity can now be evidenced and explained by the binding parameters such as association kinetics, dissociation kinetics, and affinity. Evaluation of the data showed that mouse-derived TCRs and human-derived TCRs show different affinities for the pHLA and the TCRs differ in their kinetics of binding to the pHLA.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

T cell receptors (TCRs) usually have a high affinity for neo-antigens and are low affinity for self-antigens. Consequently, TCRs isolated from mice should inherently have a high binding strength to human tumour antigens because they were selected in the absence of their target. Through the data collected in the course of this project it can now be confirmed that TCRs selected in mice differ from TCRs selected in humans with respect to the biochemical parameters of binding to a human tumour antigen presented by human leukocyte antigen complexes (HLA) class I or HLA class II. Comprehensive data regarding the binding affinity and association and dissociation kinetics are now available for mouse-derived and human-derived TCRs binding to the same peptide-HLA (pHLA). Such information will help to better understand the mode of action of therapeutic TCRs and could be the basis for the future development and optimization of anti-cancer therapies that are highly specific to the solid tumour and have few side effects.

Crystals of a pHLA-II in a sitting drop formed during screening at the MMCF, Monash University.