Development of a Novel CD26-Targeted Chimeric Antigen Receptor T-Cell Therapy for CD26-Expressing T-Cell Malignancies.

Chimeric-antigen-receptor (CAR) T-cell therapy for CD19-expressing B-cell malignancies is already widely adopted in clinical practice. On the other hand, the development of CAR-T-cell therapy for T-cell malignancies is in its nascent stage. One of the potential targets is CD26, to which we have developed and evaluated the efficacy and safety of the humanized monoclonal antibody YS110. We generated second (CD28) and third (CD28/4-1BB) generation CD26-targeted CAR-T-cells (CD26-2G/3G) using YS110 as the single-chain variable fragment. When co-cultured with CD26-overexpressing target cells, CD26-2G/3G strongly expressed the activation marker CD69 and secreted IFNgamma. In vitro studies targeting the T-cell leukemia cell line HSB2 showed that CD26-2G/3G exhibited significant anti-leukemia effects with the secretion of granzymeB, TNFα, and IL-8, with 3G being superior to 2G. CD26-2G/3G was also highly effective against T-cell lymphoma cells derived from patients. In an in vivo mouse model in which a T-cell lymphoma cell line, KARPAS299, was transplanted subcutaneously, CD26-3G inhibited tumor growth, whereas 2G had no effect. Furthermore, in a systemic dissemination model in which HSB2 was administered intravenously, CD26-3G inhibited tumor growth more potently than 2G, resulting in greater survival benefit. The third-generation CD26-targeted CAR-T-cell therapy may be a promising treatment modality for T-cell malignancies.

GATS tag system is compatible with biotin labelling methods for protein analysis.

Polypeptide tags and biotin labelling technologies are widely used for protein analyses in biochemistry and cell biology. However, many peptide tag epitopes contain lysine residues (or amino acids) that are masked after biotinylation. Here, we propose the GATS tag system without a lysine residue and with high sensitivity and low non-specific binding using a rabbit monoclonal antibody against Plasmodium falciparum glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (PfGAMA). From 14 monoclonal clones, an Ra3 clone was selected as it recognized an epitope-TLSVGVQNTF-without a lysine residue; this antibody and epitope tag set was called the GATS tag system. Surface plasmon resonance analysis showed that the tag system had a high affinity of 8.71 × 10-9 M. GATS tag indicated a very low background with remarkably high sensitivity and specificity in immunoblotting using the lysates of mammalian cells. It also showed a high sensitivity for immunoprecipitation and immunostaining of cultured human cells. The tag system was highly sensitive in both biotin labelling methods for proteins using NHS-Sulfo-biotin and BioID (proximity-dependent biotin identification) in the human cells, as opposed to a commercially available tag system having lysine residues, which showed reduced sensitivity. These results showed that the GATS tag system is suitable for methods such as BioID involving labelling lysine residues.

DPP8 Selective Inhibitor Tominostat as a Novel and Broad-Spectrum Anticancer Agent against Hematological Malignancies.

DPP8/9 inhibition induces either pyroptotic or apoptotic cell death in hematological malignancies. We previously reported that treatment with the DPP8/9 inhibitor 1G244 resulted in apoptotic cell death in myeloma, and our current study further evaluates the mechanism of action of 1G244 in different blood cancer cell lines. Specifically, 1G244 inhibited DPP9 to induce GSDMD-mediated-pyroptosis at low concentrations and inhibited DPP8 to cause caspase-3-mediated-apoptosis at high concentrations. HCK expression is necessary to induce susceptibility to pyroptosis but does not participate in the induction of apoptosis. To further characterize this DPP8-dependent broad-spectrum apoptosis induction effect, we evaluated the potential antineoplastic role for an analog of 1G244 with higher DPP8 selectivity, tominostat (also known as 12 m). In vitro studies demonstrated that the cytotoxic effect of 1G244 at high concentrations was enhanced in tominostat. Meanwhile, in vivo work showed tominostat exhibited antitumor activity that was more effective on a cell line sensitive to 1G244, and at higher doses, it was also effective on a cell line resistant to 1G244. Importantly, the weight loss morbidity associated with increasing doses of 1G244 was not observed with tominostat. These results suggest the possible development of novel drugs with antineoplastic activity against selected hematological malignancies by refining and increasing the DPP8 selectivity of tominostat.

Selection of highly responsive T cell receptors by an analysis combining the expression of multiple markers.

The clinical success of T cell receptor (TCR) gene-transduced T (TCR-T) cell therapy is expected as one of the next-generation immunotherapies for cancer, in which the selection of TCRs with high functional avidity (high-functional TCRs) is important. One widely used approach to select high-functional TCRs is a comparison of the EC50 values of TCRs, which involves laborious experiments. Therefore, the establishment of a simpler method to select high-functional TCRs is desired. We herein attempted to establish a simple method to select high-functional TCRs based on the expression of T cell activation markers using the mouse T cell line BW5147.3 (BW). We examined relationships between the EC50 values of TCRs in interleukin-2 production and the expression levels of TCR activation markers on BW cells. In TCR-expressing BW cells stimulated with antigenic peptides, the CD69, CD137, and PD-1 expression was differentially induced by various doses of peptides. An analysis of TCRs derived from the tumor-infiltrating lymphocytes of murine melanoma and peripheral blood T cells of hepatocellular carcinoma patients treated with a peptide vaccination revealed that an analysis combining CD69, CD137, and PD-1 expression levels in BW cells stimulated with a single dose of an antigenic peptide selected high-functional TCRs with functional avidity assessed by EC50 values. Our method facilitates the section of high-functional TCRs among tumor-reacting TCRs, which will promote TCR-T cell therapy. The stimulation of BW cells expressing objective TCRs with a single dose of antigenic peptides and analysis combining the expression of CD69, CD137, and PD-1 allows us to select highly responsive TCRs.

Gene modified NK cell line as a powerful tool for evaluation of cloned TCRs for TCR-T cell therapy.

T cell receptor-engineered T cell (TCR-T) therapy is anticipated as a next generation-immunotherapy for cancer and recent advances of TCR isolation technology have enabled patient's T cells to express TCRs recognizing multiple combinations of specific peptides and human leukocyte antigens (HLA). However, evaluation processes for the TCR-induced cytotoxicity activity using primary T cells are laborious and time-consuming. In this study, we established a cell line that do not express endogenous TCRs, enabling to generate large numbers of homogeneous cells, and can measure the cytotoxic activity of the isolated TCRs. To this end, we transduced a Natural Killer (NK) cell line with human CD3 molecules and interleukin (IL)-2. The TCR expressing NK cells killed target cells as similarly to TCR-transduced primary T cells and secreted various cytokines/chemokines including IL-2. Thus, the gene-modified NK cell can be a powerful tool to rapidly and efficiently evaluate the functions of isolated TCRs.

Hematopoietic stem progenitor cells with malignancy-related gene mutations in patients with acquired aplastic anemia are characterized by the increased expression of CXCR4.

The phenotypic changes in hematopoietic stem progenitor cells (HSPCs) with somatic mutations of malignancy-related genes in patients with acquired aplastic anemia (AA) are poorly understood. As our initial study showed increased CXCR4 expression on HLA allele-lacking (HLA[-]) HSPCs that solely support hematopoiesis in comparison to redundant HLA(+) HSPCs in AA patients, we screened the HSPCs of patients with various types of bone marrow (BM) failure to investigate their CXCR4 expression. In comparison to healthy individuals (n = 15, 12.3%-49.9%, median 43.2%), the median CXCR4+ cell percentages in the HSPCs of patients without somatic mutations were low: 29.3% (14.3%-37.3%) in the eight patients without HLA(-) granulocytes, 8.8% (4.1%-9.8%) in the five patients with HLA(-) cells accounting for >90% of granulocytes, and 7.8 (2.1%-8.7%) in the six patients with paroxysmal nocturnal hemoglobinuria. In contrast, the median percentage was much higher (78% [61.4%-88.7%]) in the five AA patients without HLA(-) granulocytes possessing somatic mutations (c-kit, t[8;21], monosomy 7 [one for each], ASXL1 [n = 2]), findings that were comparable to those (66.5%, 63.1%-88.9%) in the four patients with advanced myelodysplastic syndromes. The increased expression of CXCR4 may therefore reflect intrinsic abnormalities of HSPCs caused by somatic mutations that allow them to evade restriction by BM stromal cells.

Evaluation of chimeric antigen receptor of humanized rabbit-derived T cell receptor-like antibody.

T-cell receptor (TCR)-like Abs that specifically recognize antigenic peptides presented on MHC molecules have been developed for next-generation cancer immunotherapy. Recently, we reported a rapid and efficient method to generate TCR-like Abs using a rabbit system. We humanized previously generated rabbit-derived TCR-like Abs reacting Epstein-Barr virus peptide (BRLF1p, TYPVLEEMF) in the context of HLA-A24 molecules, produced chimeric antigen receptor (CAR)-T cells, and evaluated their antitumor effects using in vitro and in vivo tumor models. Humanization of the rabbit-derived TCR-like Abs using the complementarity-determining region grafting technology maintained their specificity and affinity. We prepared a second-generation CAR using single-chain variable fragment of the humanized TCR-like Abs and then transduced them into human T cells. The CAR-T cells specifically recognized BRLF1p/MHC molecules and lysed the target cells in an antigen-specific manner in vitro. They also demonstrated antitumor activity in a mouse xenograft model. We report the generation of CAR-T cells using humanized rabbit-derived TCR-like Abs. Together with our established and efficient generation procedure for TCR-like Abs using rabbits, our platform for the clinical application of humanized rabbit-derived TCR-like Abs to CAR-T cells will help improve next-generation cancer immunotherapy.

Rapid cloning of antigen-specific T-cell receptors by leveraging the cis activation of T cells.

It is commonly understood that T cells are activated via trans interactions between antigen-specific T-cell receptors (TCRs) and antigenic peptides presented on major histocompatibility complex (MHC) molecules on antigen-presenting cells. By analysing a large number of T cells at the single-cell level on a microwell array, we show that T-cell activation can occur via cis interactions (where TCRs on the T cell interact with the antigenic peptides presented on MHC class-I molecules on the same cell), and that such cis activation can be used to detect antigen-specific T cells and clone their TCR within 4 d. We used the detection-and-cloning system to clone a tumour-antigen-specific TCR from peripheral blood mononuclear cells of healthy donors. TCR cloning by leveraging the cis activation of T cells may facilitate the development of TCR-engineered T cells for cancer therapy.

Frequent HLA-DR loss on hematopoietic stem progenitor cells in patients with cyclosporine-dependent aplastic anemia carrying HLA-DR15.

To determine whether antigen presentation by HLA-DR on hematopoietic stem progenitor cells (HSPCs) is involved in the development of acquired aplastic anemia (AA), we studied the HLA-DR expression on CD45dimCD34+CD38+ cells in the peripheral blood of 61 AA patients including 23 patients possessing HLA-class I allele-lacking (HLA-class I[-]) leukocytes. HLA-DR-lacking (DR[-]) cells accounted for 13.0-57.1% of the total HSPCs in seven (11.5%) patients with HLA-DR15 who did not possess HLA-class I(-) leukocytes. The incubation of sorted DR(-) HSPCs in the presence of IFN-γ for 72 h resulted in the full restoration of the DR expression. A comparison of the transcriptome profile between DR(-) and DR(+) HSPCs revealed the lower expression of immune response-related genes including co-stimulatory molecules (e.g., CD48, CD74, and CD86) in DR(-) cells, which was not evident in HLA-class I(-) HSPCs. DR(-) cells were exclusively detected in GPI(+) HSPCs in four patients whose HSPCs could be analyzed separately for GPI(+) and GPI(-) HSPCs. These findings suggest that CD4+ T cells specific to antigens presented by HLA-DR15 on HSPCs may contribute to the development of AA as well as the immune escape of GPI(-) HSPCs in a distinct way from CD8+ T cells recognizing HLA-class I-restricted antigens.

O-glycosylated HBsAg peptide can induce specific antibody neutralizing HBV infection.

BACKGROUND: Hepatitis B virus (HBV), which causes hepatitis, liver cirrhosis, and hepatocellular carcinoma, is a global human health problem. HBV contains three envelope proteins, S-, M-, and L-hepatitis B surface antigen (HBsAg). We recently found that O-glycosylated M-HBsAg, reactive with jacalin lectin, is one of the primary components of HBV DNA-containing virus particles. Thus, we aimed to analyze and target the glycosylation of HBsAg. METHODS: HBsAg prepared from the serum of Japanese patients with HBV were analyzed using mass spectrometry. The glycopeptide modified with O-glycan was generated and used for immunization. The specificity of the generated antibody and the HBV infection inhibition activity was examined. RESULTS: Mass spectrometry analysis revealed that T37 and/or T38 on M-HBsAg of genotype C were modulated by ±NeuAc(α2,3)Gal(ß1,3)GalNAc. Chemically and enzymatically synthesized O-glycosylated peptide (Glyco-PS2) induced antibodies that recognize mainly PreS2 in M-HBsAg not in L-HBsAg, whereas the non-glycosylated peptide (PS2) induced antisera recognizing L-HBsAg but not O-glycosylated M-HBsAg. The removal of O-glycan from M-HBsAg partly decreased the reactivity of the Glyco-PS2 antibody, suggesting that peptide part was also recognized by the antibody. The antibody further demonstrated the inhibition of HBV infection in human hepatic cells in vitro. CONCLUSIONS: Glycosylation of HBsAg occurs differently in different HBsAgs in a site-specific manner. The new Glyco-PS2 antibody, recognizing O-glycosylated M-HBsAg of genotype C, could inhibit HBV infection. GENERAL SIGNIFICANCE: The detailed analysis of HBsAg identified different glycosylations of HBV surface. The glycosylated peptide based on mass spectrometry analysis showed higher potential to induce functional antibody against HBV.

TCR function analysis using a novel system reveals the multiple unconventional tumor-reactive T cells in human breast cancer-infiltrating lymphocytes.

Tumor-infiltrating lymphocytes (TILs) are a potent source for obtaining tumor-reactive T cell receptors (TCRs). Although comprehensive methods to analyze the TCR repertoire in TILs have been reported, the evaluation system for TCR-reactivity to endogenously expressed antigen in tumor cells remains laborious and time consuming. Consequently, very limited numbers of TCRs in TILs have been analyzed for their reactivity to tumor cells. In this study, we developed an efficient evaluation system for TCR function designated c-FIT (comprehensive functional investigation of TCRs) to analyze TCR reactivity. The c-FIT system enabled us to analyze up to 90 TCRs for their reactivity to tumor cells by a single assay within a month. Using c-FIT, we analyzed 70 TCRs of CD8+ TILs derived from two breast cancer patients and obtained 23 TCRs that reacted to tumor cells. Surprisingly, although two TCRs were HLA class I-restricted, the remaining 21 TCRs were non-HLA-restricted. Thus, c-FIT can be applied for monitoring multiple conventional and unconventional antigen-specific killer T cells in TILs, leading to the development of new designs for more effective T-cell-based immunotherapies.

Rapid and efficient generation of T-cell receptor-like antibodies using chip-based single-cell analysis.

Generation of TCR-like monoclonal antibodies using conventional methods is markedly laborious and inefficient. We have proposed improvements of ISAAC (chip-based Ab-secreting cell [ASC] screening method), allows comprehensive analysis of ASCs at the single-cell level to obtain TCR-like antibodies; blocking procedure enables us to avoid the detection of non-TCR-like antibodies.

Characterization of pseudotyped vesicular stomatitis virus bearing the heartland virus envelope glycoprotein.

The heartland virus (HRTV) is a novel phlebovirus that causes severe infections in the USA and closely related to the severe fever thrombocytopenia syndrome virus (SFTSV), a causative agent for SFTS in Asia. The entry mechanisms of HRTV remain unclear. Here, we developed the pseudotyped vesicular stomatitis virus bearing the HRTV glycoprotein (GP) (HRTVpv), and the antigenicity and the entry mechanisms of HRTV were analyzed. HRTVpv was neutralized by anti-SFTSV Gc antibody, but not the anti-SFTSV Gn antibodies. Entry of HRTVpv to cells was inhibited by bafilomycin A1 and dynasore, and but it was enhanced in cells overexpressed with C-type lectins. Production of infectious HRTVpv and SFTSVpv was reduced by Nn-DNJ, α-glucosidase inhibitor. The entry of HRTV occurs via pH- and dynamin-dependent endocytosis. Furthermore, Nn-DNJ may be a possible therapeutic agent against HRTV and SFTSV.

Relationship between T cell receptor clonotype and PD-1 expression of tumor-infiltrating lymphocytes in colorectal cancer.

Adoptive T cell therapy using tumor-specific T cells or TCR-modified T cells is a promising next-generation immunotherapy. The major source of tumor-reactive T cells is PD-1+ tumor-infiltrating lymphocytes (TILs). In contrast, PD-1- TILs have received little attention. Here, we analyzed the TCR-ß repertoires of PD-1- and PD-1+ CD8+ TILs derived from colorectal cancer and breast cancer. Approximately 40-60% of the PD-1+ population consisted of oligoclonal populations in both colorectal cancer and breast cancer. In contrast, approximately 37% of the PD-1- population consisted of an oligoclonal population in colorectal cancer, whereas 14% of them were oligoclonal in breast cancer. In colorectal cancer, the TCR repertoires of PD-1- CD8+ TILs and PD-1+ CD8+ TILs hardly overlapped. Interestingly, clonally expanded CD8+ TILs in primary tumors and the metastases expressing the same clonotypic TCR showed the same phenotype regarding the PD-1-expression. These results suggest that the intrinsic properties of TCRs determine the fate of TILs in terms of whether they become PD-1+ or PD-1- in the tumor microenvironment. Further functional analysis of TCRs in TILs will allow us to better understand the regulatory mechanisms for PD-1 expression on TILs and may contribute to tumor immunotherapy.

A novel and simple method to produce large amounts of recombinant soluble peptide/major histocompatibility complex monomers for analysis of antigen-specific human T cell receptors.

Soluble peptide/major histocompatibility complex (p/MHC) tetramers that directly bind to T cell receptors (TCRs) allow the direct quantification, phenotypic characterization and isolation of antigen-specific T cells. Conventionally, soluble p/MHC tetramers have been produced using Escherichia coli, but this method requires refolding of the recombinant proteins. Here, a novel and technically simple method that does not require protein refolding in vitro has been developed for the high-throughput generation of soluble and functional p/MHC-single chain trimer (SCT) monomers and tetramers in a mammalian cell system. The p/MHC-SCT tetramers generated by this method bound to the corresponding antigen-specific TCRs. Moreover, the immobilized p/MHC-SCT monomers effectively activated antigen-specific T cell lines as well as primary T cells in an antigen-specific manner. This technique provides a robust improvement in the technology, such that recombinant soluble p/MHC monomers and tetramers can be produced more readily and which enables their use in analysis of antigen-specific T cells in basic and clinical studies.

Isolation of Antigen-Specific, Antibody-Secreting Cells Using a Chip-Based Immunospot Array.

Antigen-specific monoclonal antibodies are useful tools to detect very small amounts of antigenic materials and are applicable for antibody therapeutics. To produce mouse monoclonal antibodies, a hybridoma between B lymphocytes and myeloma cells is used to produce antigen-specific monoclonal antibodies. However, a good hybridoma system is not available to obtain human monoclonal antibodies. To produce antigen-specific human monoclonal antibodies, transformation of B lymphocytes with Epstein-Barr viruses or a phage-display system is used. Here, we describe the screening of antigen-specific, antibody-secreting cells using microwell array chips to obtain antigen-specific human monoclonal antibodies. The system can be applied to screen antigen-specific, antibody-secreting cells from any animal species.

Analysis of the clinical significance of DCLK1+ colorectal cancer using novel monoclonal antibodies against DCLK1.

INTRODUCTION: Doublecortin-like kinase 1 (DCLK1) is considered a putative tumor stem cell (TSC) marker and a promising therapeutic target, as DCLK1+ progeny cells exhibit high expression in tumors. However, the biological function of DCLK1+ cells in tumorigenesis and tumor progression remains unclear. MATERIALS AND METHODS: We generated rabbit monoclonal antibodies (mAbs) against DCLK1, DCLK1-42, and DCLK1-87 mAbs, using a novel chip-based immunospot array assay on a chip system. First, the specificity of two mAbs to DCLK1 was confirmed by Western blot, which were bound to DCLK1-long in normal colon cells and to DCLK1-short in a cancer cell line as well as colorectal cancer (CRC) cells. RESULTS: Precise localization analysis using immunofluorescence revealed that both mAbs had cytoplasmic signal and exhibited a high degree of overlap with microtubules. Furthermore, bacterial display technology indicated that the antigenic epitope region of DCLK1-87 mAb was consistent with that of a commercial anti-DCLK1 polyclonal antibody. In addition, DCLK1-42 mAb has the common polyclonal antibody characteristic of binding to more than one site on DCLK1. By immunohistochemistry, it was found that DCLK1-87 mAb was more specific for DCLK1+ cell labeling than a commercial anti-DCLK1 polyclonal antibody. DCLK1 labeled with DCLK1-87 mAb might be a potential TSC marker because the tissue expression site covers the ALDH1 area in CRC tissues. Finally, we analyzed 100 pairs of cancer tissues and matching paracancerous tissue samples from patients with CRC who received 100 months of follow-up with the DCLK1-87 mAb. The results showed that patients with high DCLK1 expression exhibited a longer survival time than that of patients with low DCLK1 expression (P=0.0029). DISCUSSION: Our results indicated that we successfully generated an efficient tool for the precise detection of DCLK1+ cells in cancer tissues. Moreover, we found that high DCLK1 expression in CRC patients appears to play a protective role against tumor progression.

Autoantibodies reactive to PEP08 are clinically related with morbidity and severity of interstitial lung disease in connective tissue diseases.

Anti-Ro52 autoantibodies (Ro52-autoAbs) appear in the sera of connective tissue disease (CTD) patients with interstitial lung disease (ILD). Studies using patient sera have shown a correlation between the generation of Ro52-autoAbs and the clinical morbidity and severity of CTD with ILD. In this study, we used a single B-cell manipulating technology and obtained 12 different monoclonal Ro52-autoAbs (mRo52-autoAbs) from the selected four patients suffering from severe ILD with a high titer of Ro52-autoAbs in their sera. Western blot analysis revealed that 11 of 12 mRo52-autoAbs bound to the coiled-coil domain of Ro52. Competitive ELISA demonstrated that mRo52-autoAbs competed with each other to bind to Ro52. Epitope mapping showed that two of them specifically bound to a peptide (PEP08) in the coiled-coil domain. We then examined the titer of Ro52-autoAbs in the sera of 192 CTD patients and assessed the relationship between the serum levels of Ro52-autoAbs that were reactive to PEP08 peptide and the clinical morbidity and severity of ILD. Statistical analysis revealed that the production of PEP08-reactive Ro52-autoAbs correlated with the morbidity and severity of ILD in CTD. Assessment of the production of PEP08-reactive Ro52-autoAbs in autoimmune diseases is useful for predicting the clinical morbidity of ILD.

Cisplatin-induced non-canonical endocytosis of EGFR via p38 phosphorylation of the C-terminal region containing Ser-1015 in non-small cell lung cancer cells.

The aberrant activation of receptor tyrosine kinases (RTKs) is associated with tumor initiation in various types of human cancer, including non-small cell lung cancers (NSCLCs). Tyrosine kinase-independent non-canonical RTK regulation has also been investigated in tumor malignant alterations, including cellular stress responses. It was recently reported that the phosphorylation of epidermal growth factor receptor (EGFR) at C-terminal Ser-1015 serves a critical role in growth factor and cytokine signaling. In the present study, the role of non-canonical EGFR regulation has been investigated in NSCLC cells treated with cisplatin, a common chemotherapeutic agent. Cisplatin-induced p38 activation triggered the Ser-1015 phosphorylation of EGFR, with similar kinetics to previously reported Ser-1047 phosphorylation, in a tyrosine kinase-independent manner. In addition, phosphorylation around Ser-1015 triggered endocytosis of a dimer deficient mutant of EGFR. The non-canonical endocytosis of EGFR monomers was primarily controlled by the region around Ser-1015 only; however, Ser-1047 on internalized EGFR was equally phosphorylated. The results of the present study provide mechanistic evidence for the cisplatin-induced non-canonical regulation of EGFR.