Antigen-specific T-cell Receptor Gene Research Articles

Detection of mutant antigen-specific T cell receptors against multiple myeloma for T cell engineering

Multiple myeloma (MM) remains an incurable hematological neoplasm. Neoantigen-specific Tcell receptor (TCR)-engineered T(TCR-T) cell therapy is a potential alternative treatment. Particularly, TCRs derived from a third-party donor may cover broad ranges of neoantigens, whereas TCRs in patients suffering from immune disorders are limited. However, the efficacy and feasibility of treating MM have not been evaluated thoroughly. In this study, we established a system for identifying immunogenic mutant antigens on MM cells and their corresponding TCRs using healthy donor-derived peripheral blood mononuclear cells (PBMCs). Initially, the immune responses to 35 candidate peptides predicted by the immunogenomic analysis were investigated. Peptide-reactive T lymphocytes were enriched, and subsequently, TCR repertoires were determined by single-cell TCR sequencing. Eleven reconstituted TCRs showed mutation-specific responses against 4 peptides. Particularly, we verified the HLA-A∗24:02-binding QYSPVQATF peptide derived from COASY S55Y as the naturally processed epitope across MM cells, making it a promising immune target. Corresponding TCRs specifically recognized COASY S55Y+HLA-A∗24:02+ MM cells and augmented tumoricidal activity. Finally, adoptive cell transfer of TCR-T cells showed objective responses in the xenograft model. We initiatively proposed the utility of tumor mutated antigen-specific TCR genes to suppress MM. Our unique strategy will facilitate further identification of neoantigen-specific TCRs.

Chimeric adenoviral vector Ad5F35L containing the Ad5 natural long-shaft exhibits efficient gene transfer into human T lymphocytes

Adoptive therapy using T cells modified with tumour antigen-specific T cell receptor (TCR) genes has become a popular area of research in tumour biotherapy research. However, the efficiency of this treatment is low. To increase the efficiency of this therapy, the antigen specific TCR expression in the T cells needs to be improved. Adenoviral vector-mediated gene expression is an attractive approach to bypass the issue of TCR gene modification. The efficiency of adenovirus vector serotype 5 (Ad5) infection is low due to the absence of coxsackievirus B-adenovirus receptor (CAR) expression in T cells. In the present study, a chimeric adenoviral vector (Ad5F35L) was generated; this construct contained both the natural long-shaft of Ad5 and the Ad35 knob. A transduction study showed that the Ad5F35L vector exhibited a higher transduction efficiency in human primary T lymphocytes than the Ad5 vector and the Ad5F35S vector, which contained the Ad35 natural short-shaft and the Ad35 knob. Similar transduction efficiencies were observed for both CD4+ T lymphocytes and CD8+ T lymphocytes and the transfection was independent of the expression of cell surface receptors. The activation of T lymphocytes resulted in an improvement of the Ad5F35L transduction efficiency in CD4+ T cells and a decrease in Ad5F35L transduction efficiency in CD8+ T cells. The results demonstrate that Ad5F35L is a promising viral vector and will facilitate the clinical application of tumour antigen-specific TCR gene therapy.

Requisite considerations for successful adoptive immunotherapy with engineered T-lymphocytes using tumor antigen-specific T-cell receptor gene transfer

Introduction: Although engineered T-cell-based antitumor immunotherapy using tumor-antigen-specific T-cell receptor (TCR) gene transfer is undoubtedly a promising strategy, a number of studies have revealed that it has several drawbacks.Areas covered: This review covers selected articles detailing recent progress in this field, not only for solid tumors, but also for leukemias. In terms of achieving uniform therapeutic quality of TCR gene-modified T cells as an ‘off-the-shelf’ product, the authors abstract and discuss the requisite conditions for successful outcome, including: i) the optimal target choice reflecting the specificity of the introduced TCR, ii) the quality and quantity of expressed TCRs in gene-modified T cells, and additional genetic modification reflecting enhanced antitumor functionality, and iii) ‘on-’ and ‘off-target’ adverse events caused by the quality of the introduced TCRs and other adverse events related to genetic modification itself. Readers will be able to readily abstract recent advances in TCR gene-transferred T-cell therapy, centering notably on efforts to obtain uniformity in the therapeutic functionality of engineered T cellsExpert opinion: Harmonizing the functionality and target specificity of TCR will allow the establishment of clinically useful adoptive immunotherapy in the near future.

Dual Engineering of Human CD8+ T-Cells with WT1-Specific TCR Gene and Chemokine Receptor Gene Transfer Confers Functionally Strengthened Anti-Cancer Reactivity

AbstractAbstract 4295 Background & Purpose:Currently a novel adoptive therapy with engineered T-cells using cancer antigen-specific T-cell receptor (TCR) gene transfer has attracted the attention as a challenging option for cancer treatment. However, reported efficacy from clinical trials using TCR-gene transfer was generally less than expected. In concurrent with the major issue of generation of mispaired TCRs between introduced and endogenous TCR α/β chains, the less accumulation of adoptively infused engineered T-cells at local tumor site in number could also impede the clinical efficacy. Therefore, for the purpose of sufficient accumulation of adoptively transferred tumor-specific T cells into local tumor sites, we set out to examine the feasibility of dual transduction of cancer antigen-specific TCR gene and chemokine receptor gene into human T-cells. Methods:HLA-A*2402-restricted and WT1235-243-specific TCR α/β genes were cloned into a novel GaLV-pseudotyped retroviral vector carrying silencers for endogenous TCR-a/b chains. With retronectin (TakaraBio, Inc.)-coated plate, WT1-specific TCR-α/β genes were introduced into normal CD8+ T-cells with upto more than 50% of WT1-tetramer positivity. On the other hand, using QRT-PCR, we comprehensively screened the chemokine expression profiles of pre-determined HLA-A*2402+ human leukemia cell lines (n=13) and lung cancer cell lines (n=4) which were all WT1-expressing and sensitive to WT1235-243-specific TCR-transduced CD8+ T-cells. Then, the receptor gene for selected chemokine was cloned and inserted into retroviral vector. Expression and function of induced chemokine receptor into Jurkat cells was examined by flowcytometer and transwell experiment. Next, the chemokine receptor gene was introduced into WT1-specific TCR introduced CD8+ T-cells. Migration ability toward target chemokine, and cytotoxicity against the target tumor cells of double-gene transfectans were examined by transwell experiment and 51Cr-releasing assay, respectively. Finally, the combined anti-cancer effect of double-gene transfectants was examined in a novel assay consisted of transwell part and cytotoxicity assay determined by concentration of LDH released from target tumor cells in the bottom well, which were killed by migrated double-gene transfectants from the upper well towards the chemokine produced by target tumor cells. Results:CCL2 chemokine was abundantly produced by some of human lung cancer cell lines and leukemia cell lines. Additionally, receptor for CCL2 (CCR2) was not expressed in activated CD8+ T-cells. Thus, we selected CCL2/CCR2 interaction for this system. CCR2 gene was successfully transduced into Jurkat cells and conferred migration activity towards CCL2 and CCL2 producing lung cnacer cell line LK79 and leukemia cell line KH88. CCR2 gene was also successfully introduced into WT1-specific TCR gene trnasduced CD8+ T-cells. The double-gene transfectants successfully migrated towards CCL2-producing LK79 and KH88 cells. Eventually, in our novel assay system, only double genes-transduced CD8+ T cells in the upper-well, but not single WT1-specific TCR gene-transduced CD8+ T-cells, could migrate towards LK79 cells in the bottom-well and efficiently killed LK79 cells. Background:Although further investigations are warranted, the dual T-cell engineering of human T-cells with chemokine receptor gene and our novel avidity-enhanced tumor-specific TCR gene may be able to more efficiently accumulate engineered T-cells at local tumor sites, which may enhance the immediate anti-tumor effect of adoptively transferred such engineered T-cells. Disclosures:No relevant conflicts of interest to declare.

Application of Adoptive T-Cell Therapy Using Tumor Antigen-Specific T-Cell Receptor Gene Transfer for the Treatment of Human Leukemia

The last decade has seen great strides in the field of cancer immunotherapy, especially the treatment of melanoma. Beginning with the identification of cancer antigens, followed by the clinical application of anti-cancer peptide vaccination, it has now been proven that adoptive T-cell therapy (ACT) using cancer antigen-specific T cells is the most effective option. Despite the apparent clinical efficacy of ACT, the timely preparation of a sufficient number of cancer antigen-specific T cells for each patient has been recognized as its biggest limitation. Currently, therefore, attention is being focused on ACT with engineered T cells produced using cancer antigen-specific T-cell receptor (TCR) gene transfer. With regard to human leukemia, ACT using engineered T cells bearing the leukemia antigen-specific TCR gene still remains in its infancy. However, several reports have provided preclinical data on TCR gene transfer using Wilms' tumor gene product 1 (WT1), and also preclinical and clinical data on TCR gene transfer involving minor histocompatibility antigen, both of which have been suggested to provide additional clinical benefit. In this review, we examine the current status of anti-leukemia ACT with engineered T cells carrying the leukemia antigen-specific TCR gene, and discuss the existing barriers to progress in this area.