A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days

Kobayashi, Eiji; Mizukoshi, Eishiro; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nagai, Torao; Nakagawa, Hidetoshi; Jin, Aishun; Kaneko, Shuichi; Muraguchi, Atsushi

doi:10.1038/nm.3358

Cited by 80 publications

(77 citation statements)

References 24 publications

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“…These reports also showed that T cells modified with an antigen-specific TCR gene have excellent prospects for application in clinical practice. 42 In this report, we found that the TRBV families TRBV5.1, BV11, BV13.1, BV15, BV20 and BV24 were more frequently expanded monoclonally than other TRBV family members in recovered AHI subjects, regardless of the presence of CDR3 in the CD4 1 or CD8 1 T-cell subsets, further demonstrating that the T-cell immune response was polyclonal and polyspecific in the recovered AHI subjects studied. 43 This phenomenon was also discovered in the peripheral blood of patients with chronic …”

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confidence: 49%

Profiling the repertoire of T-cell receptor beta-chain variable genes in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection

Yang

Chen

et al. 2014

Cell Mol Immunol

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The profile of T-cell receptor beta-chain variable (TRBV) genes usually skews in subjects with virus infection or cancer. The gene melting spectral pattern (GMSP) can be used to determine the profile of the TRBV gene family. To explore the portrait of the TRBV family in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection (AHI), peripheral blood mononuclear cells (PBMCs) were separated and further sorted into CD41 and CD8 1T-cell subsets. The molecular features of the TRBV complementary determining region 3 (CDR3) motifs were determined using GMSP analysis. When a GMSP profile showed a single peak, the monoclonally expanded TRBV gene was cloned and sequenced. Skewed expansions of multiple TRBV genes were observed among the CD4 1 and CD8 1 T-cell subsets and the PBMCs. The frequency of monoclonally expanded TRBV genes in the CD8 1 T-cell subset was significantly higher than that of the CD4 1 T-cell subset and the PBMCs. Compared to other members of the TRBV gene family, TRBV11, BV15 and BV20 were predominantly expressed in the repertoire of peripheral blood lymphocytes in recovered AHI subjects. The relatively conserved amino acid motifs of TRBV5.1 and BV20 CDR3 were also detected in the CD4 1 and CD8 1 T-cell subsets. These results demonstrate the presence of multiple biased TRBV families in recovered AHI subjects. TRBV11, BV15 and BV20, especially from the CD8 1 T-cell subset, may be relevant to the pathogenesis of subjects with AHI. The preferentially selected TRBV5.1 and BV20 with the relatively conserved CDR3 motif may be potential targets for personalized treatments of chronic HBV infection.

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confidence: 49%

Profiling the repertoire of T-cell receptor beta-chain variable genes in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection

Yang

Chen

et al. 2014

Cell Mol Immunol

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“…This approach may be further enhanced by the development of techniques such as sorting with specific MHC tetramers to increase the frequency of mutation-reactive T cells from bulk TILs that may contain significant numbers of non-tumor reactive cells (34). Alternatively, TCRs isolated from cells reactive with mutated epitopes can be isolated and introduced into autologous peripheral blood T cells for the subsequent adoptive T-cell transfer (35, 36). …”

Section: Discussionmentioning

confidence: 99%

Efficient Identification of Mutated Cancer Antigens Recognized by T Cells Associated with Durable Tumor Regressions

Yao

Crystal

et al. 2014

Clinical Cancer Research

357

296

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Purpose Cancer immunotherapy with adoptive transfer of tumor infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma, with the objective regressions in up to 72% of patients in three clinical trials. However, the antigen targets recognized by these effective TILs remain largely unclear. Experimental Design Melanoma patients 2359 and 2591 both experienced durable complete regressions of metastases ongoing beyond five years following adoptive TIL transfer. Two conventional screening approaches were carried out to identify the antigens recognized by these clinically effective TILs. In addition, a novel approach was developed in this study to identify mutated T-cell antigens by screening a tandem minigene library, which comprised non-synonymous mutation sequences identified by whole-exome sequencing of autologous tumors. Results The autologous melanoma cDNA library screening led to the identification of previously undescribed non-mutated targets recognized by TIL 2359 or TIL 2591. On the other hand, the screening of tandem minigene libraries resulted in the identification of mutated kinesin family member 2C (KIF2C) antigen as a target of TIL 2359, and mutated DNA polymerase alpha subunit B (POLA2) antigen as a target of TIL 2591. Both KIF2C and POLA2 have been found to play important roles in cell proliferation. Conclusions These findings suggest that the minigene screening approach can facilitate the antigen repertoire analysis of tumor reactive T cells, and lead to the development of new adoptive cell therapies with purified T cells that recognize candidate mutated antigens derived from genes essential for the carcinogenesis.

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“…Not only are we able to study dual TCR expression, using this method we also can delineate the different (productively rearranged) TCR-a chains of a single T cell. Compared with other strategies that aim to validate functional TCRs from single T cells (15,16), our approach includes the directed cloning of amplified variable regions into IVT vectors providing the TCR-a/b constant regions for extremely rapid generation of full-length TCRs for validation assays that include the determination of epitope specificity for any antigen. Although next-generation sequencing-based methods and the use of barcodes allows for identification of potentially hundreds of a/b-TCRs in a single experiment (22), validation of these TCRs still requires subsequent cost-intensive gene synthesis and cloning.…”

Section: Discussionmentioning

confidence: 99%

Functional TCR Retrieval from Single Antigen-Specific Human T Cells Reveals Multiple Novel Epitopes

Simon

Omokoko

Breitkreuz

et al. 2014

Cancer Immunology Research

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The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-a and TCR-b chains directly from single antigen-specific CD8 þ or CD4 þ T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro-transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigenspecific TCRs from human CD8 þ and CD4 þ T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes.

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A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days

Cited by 80 publications

References 24 publications

Profiling the repertoire of T-cell receptor beta-chain variable genes in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection

Profiling the repertoire of T-cell receptor beta-chain variable genes in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection

Efficient Identification of Mutated Cancer Antigens Recognized by T Cells Associated with Durable Tumor Regressions

Functional TCR Retrieval from Single Antigen-Specific Human T Cells Reveals Multiple Novel Epitopes

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