Identification of peptide-specific TCR genes by in vitro peptide stimulation and CDR3 length polymorphism analysis.

Identification of TCR genes specific for tumor-associated antigens (TAAs) is necessary for TCR gene modification of T cells, which is applied in anti-tumor adoptive T cell therapy (ACT). The usual identification methods are based on isolating single peptide-responding T cells and cloning the TCR gene by in vitro expansion or by single-cell RT-PCR. However, the long and exacting in vitro culture period and demanding operational requirements restrict the application of these methods. Immunoscope is an effective tool that profiles a repertoire of TCRs and identifies significantly expanded clones through CDR3 length analysis. In this study, a survivin-derived mutant peptide optimized for HLA-A2 binding was selected to load DCs and activate T cells. The monoclonal expansion of TCRA and TCRB genes was separately identified by Immunoscope analysis and following sequence identification, the properly paired TCR genes were transferred into T cells. Peptide recognition and cytotoxicity assays indicated that TCR-modified PBMCs could respond to both the mutant and wild type peptides and lyse target cells. These results show that combining Immunoscope with in vitro peptide stimulation provides an alternative and superior method for identifying specific TCR genes, which represents a significant advance for the application of TCR gene-modified T cells.

Shao H ，Lin Y ，Wang T ，Ou Y ，Shen H ，Tao C ，Wu F ，Zhang W ，Bo H ，Wang H ，Huang S ... -  《-》

T Cells Engineered to Express a T-Cell Receptor Specific for Glypican-3 to Recognize and Kill Hepatoma Cells In Vitro and in Mice.

Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican-3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC), but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice. We used mass spectrometry to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of human leukocyte antigen (HLA)-A2 and bioinformatics to identify immunodominant peptides. To circumvent GPC3 tolerance resulting from fetal expression, dendritic cells from HLA-A2-negative donors were cotransfected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells. Peptide GPC3367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3367 multimer and secreted interferon-γ when cultured with GPC3367, but not with control peptide-loaded cells. By genomic sequencing of these T-cell clones, we identified a gene encoding a dominant T-cell receptor. The gene was cloned and the sequence was codon optimized and expressed from a retroviral vector. Primary CD8(+) T cells that expressed the transgenic T-cell receptor specifically bound GPC3367 on HLA-A2. These T cells killed GPC3-expressing hepatoma cells in culture and slowed growth of HCC xenograft tumors in mice. We identified a GPC3367-specific T-cell receptor. Expression of this receptor by T cells allows them to recognize and kill GPC3-positive hepatoma cells. This finding could be used to advance development of adoptive T-cell therapy for HCC.

Dargel C ，Bassani-Sternberg M ，Hasreiter J ，Zani F ，Bockmann JH ，Thiele F ，Bohne F ，Wisskirchen K ，Wilde S ，Sprinzl MF ，Schendel DJ ，Krackhardt AM ，Uckert W ，Wohlleber D ，Schiemann M ，Stemmer K ，Heikenwälder M ，Busch DH ，Richter G ，Mann M ，Protzer U ... -  《-》

A fast and robust method to clone and functionally validate T-cell receptors.

Birkholz K ，Hofmann C ，Hoyer S ，Schulz B ，Harrer T ，Kämpgen E ，Schuler G ，Dörrie J ，Schaft N ... -  《-》

Peptide-pulsed dendritic cells induce tumoricidal cytotoxic T lymphocytes from healthy donors against stably HLA-A*0201-binding peptides from the Melan-A/MART-1 self antigen.

van Elsas A ，van der Burg SH ，van der Minne CE ，Borghi M ，Mourer JS ，Melief CJ ，Schrier PI ... -  《EUROPEAN JOURNAL OF IMMUNOLOGY》

Development of a CD8 co-receptor independent T-cell receptor specific for tumor-associated antigen MAGE-A4 for next generation T-cell-based immunotherapy.

The cancer-testis antigen MAGE-A4 is an attractive target for T-cell-based immunotherapy, especially for indications with unmet clinical need like non-small cell lung or triple-negative breast cancer. An unbiased CD137-based sorting approach was first used to identify an immunogenic MAGE-A4-derived epitope (GVYDGREHTV) that was properly processed and presented on human leukocyte antigen (HLA)-A2 molecules encoded by the HLA-A*02:01 allele. To isolate high-avidity T cells via subsequent multimer sorting, an in vitro priming approach using HLA-A2-negative donors was conducted to bypass central tolerance to this self-antigen. Pre-clinical parameters of safety and activity were assessed in a comprehensive set of in vitro and in vivo studies. A MAGE-A4-reactive, HLA-A2-restricted T-cell receptor (TCR) was isolated from primed T cells of an HLA-A2-negative donor. The respective TCR-T-cell (TCR-T) product bbT485 was demonstrated pre-clinically to have a favorable safety profile and superior in vivo potency compared with TCR-Ts expressing a TCR derived from a tolerized T-cell repertoire to self-antigens. This natural high-avidity TCR was found to be CD8 co-receptor independent, allowing effector functions to be elicited in transgenic CD4+ T helper cells. These CD4+ TCR-Ts supported an anti-tumor response by direct killing of MAGE-A4-positive tumor cells and upregulated hallmarks associated with helper function, such as CD154 expression and release of key cytokines on tumor-specific stimulation. The extensive pre-clinical assessment of safety and in vivo potency of bbT485 provide the basis for its use in TCR-T immunotherapy studies. The ability of this non-mutated high-avidity, co-receptor-independent TCR to activate CD8+ and CD4+ T cells could potentially provide enhanced cellular responses in the clinical setting through the induction of functionally diverse T-cell subsets that goes beyond what is currently tested in the clinic.

Davari K ，Holland T ，Prassmayer L ，Longinotti G ，Ganley KP ，Pechilis LJ ，Diaconu I ，Nambiar PR ，Magee MS ，Schendel DJ ，Sommermeyer D ，Ellinger C ... -  《Journal for ImmunoTherapy of Cancer》