Very rapid cloning, expression and identifying specificity of T-cell receptors for T-cell engineering

Abstract

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αβ T-cell receptors (TCRαβ) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαβs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαβ genes as Sleeping Beauty transposons and the determination of the introduced TCRαβs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.immunotherapy is that ex vivo culture and numeric expansion typically leads to the clonal and/ or oligoclonal expansion of terminally differentiated T cells. Together, these clinical data suggest that the administration of "young" T cells that are sourced from peripheral blood and genetically modified to be neoantigen-specific offers an advantage over TIL-based immunotherapy.The bioengineering of neoantigen-specific T cells requires identifying individual TCRαβs and determining their antigen specificity. Next-generation sequencing (NGS) was used to identify non-synonymous tumor-specific mutations and single-cell RNA sequencing (scRNAseq) to identify paired full-length TCRαβ sequences [18]. This enabled us to reconstruct tumor-specific TCRs and evaluate their antigen specificity to engineer clinical-grade T cells. This was undertaken by very rapidly constructing a library of TCRαβ genes expressed in DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system and then inducing the expression of cloned TCRαβs in a reporter cell line to determine their antigen specificity and avidity. These reporter cells were co-cultured with genetically edited HLA null HEK293 cells and genetically modified with monoallelic HLA and the putative neoantigen as a minigene construct to serve as artificial antigen-presenting cells. This suite of technologies could be used to determine the antigen specificity of TCRs retrieved from primary tumors. In summary, this platform serves as a resource for the very rapid, robust, and high-throughput identification of immunogenic neoantigens and their cognate antigen-specific TCRs. Materials and methods Ethical statementPeripheral blood mononuclear cells (PBMCs) were obtained from patients who had provided written informed consent in accordance with a protocol established and approved by MD Anderson's Institutional Review Board (#LAB07-0296, Acquisition of Peripheral Blood from Healthy Donors). The identities of all patients were kept private. Animals were handled in accordance with strict guidelines established by MD Anderson's Institutional Animal Care and Use Committee (IACUC), which specifically approved this study (#00001131-RN02, Adoptive Immunotherapy w...

A T‐cell reporter platform for high‐throughput and reliable investigation of TCR function and biology

“…Hence, a cell line that provides TCR function close to primary T cells would enable more standardised testing as well as simplify the whole process because of cell lines’ easy handling and almost unlimited proliferative capacity. The urgent need for such a cell line is highlighted by various publications that proposed different cellular platforms for TCR testing 43–47 …”

“…The urgent need for such a cell line is highlighted by various publications that proposed different cellular platforms for TCR testing. [43][44][45][46][47] Here, we propose an advanced Jurkat E6.1based TCR signal reporter system that is unbiased by endogenous TCR expression. Our study, which analysed 59 different human TCRs, isto our knowledgethe first to comprehensively compare TCR function in a cell line with primary human T cells.…”

Evolution of CD8+ T Cell Receptor (TCR) Engineered Therapies for the Treatment of Cancer

“…The combination of IL-2, IL-7, and IL-15 promotes the long-term in vitro survival of memory and naïve T lymphocytes, and improves lentiviral transduction efficiency in the absence of TCR activation [51]. Various techniques, such as Golden Gate cloning and LR cloning are often used to construct the vectors for inserting the TCR α/β genes, and the recently introduced Gibson assembly appears to be very rapid and promising high-throughput method [52]. Adeno-associated virus (AAV) is another widely used viral vector [53,54].…”

“…In a more unbiased approach, NeoAgs can also be expressed as mini-genes in autologous antigen-presenting cells to assess panels of TCRs for antigen reactivity [63,72]. The introduction of DNA libraries to facilitate the quick assembly of TCRαβ genes using a Sleeping Beauty system is another approach featuring tetramerguided isolation of antigen-specific TCRs by paired single-cell TCRαβ cloning and the rapid assembly of TCRαβ genes [52,73]. Various reporter systems measuring luciferase activity or cytokine production utilize flow cytometry or fluorescence microscopy for signal detection.…”

Latest Advances for the Sleeping Beauty Transposon System: 23 Years of Insomnia but Prettier than Ever

“…These neoantigens represent ideal immunotherapy targets, which is being explored successfully in preclinical models and clinical trials. [149] The recent development of a platform to screen for tumorspecific neoantigens by whole transcriptome sequencing and identifying the respective, specifically matching TCRs via single cell RNA-sequencing [150] was significantly aided by SB, as expression cassettes encoding tumor-reactive TCRs could quickly be cloned into transposons and expressed in T cells to manufacture therapeutic cell products. Accordingly, SB will be used to generate genetically modified T cells expressing neoantigen-specific TCRs in an upcoming phase II clinical trial (NCT04102436) set out to treat patients with metastatic cancer, highlighting SB's value as a gene transfer tool, as viral vectors are outclassed when it comes to cost and manufacturing speed, which are essential criteria in truly personalized therapies.…”