Development of T cell receptor-engineered T cells targeting the sarcoma-associated antigen papillomavirus binding factor.

We previously identified papillomavirus binding factor (PBF) as an osteosarcoma antigen recognized by an autologous cytotoxic T lymphocyte clone. Vaccination with PBF-derived peptide presented by HLA-A24 (PBF peptide) elicited strong immune responses. In the present study, we generated T cell receptor-engineered T cells (TCR-T cells) directed against the PBF peptide (PBF TCR-T cells). PBF TCR was successfully transduced into T cells and detected using HLA-A*24:02/PBF peptide tetramer. PBF TCR-T cells generated from a healthy donor were highly expanded and recognized T2-A24 cells pulsed with PBF peptide, HLA-A24+ 293T cells transfected with PBF cDNA, and sarcoma cell lines. To establish an adoptive cell therapy model, we modified the PBF TCR by replacing both α and ß constant regions with those of mice (hybrid PBF TCR). Hybrid PBF TCR-T cells also showed reactivity against T2-A24 cells pulsed with PBF peptide and to HLA-A24+ 293T cells transfected with various lengths of PBF cDNA including the PBF peptide sequence. Subsequently, we generated target cell lines highly expressing PBF (MFH03-PBF [short] epitope [+]) containing PBF peptide with in vivo tumorigenicity. Hybrid PBF TCR-T cells exhibited antitumor effects compared with mock T cells in NSG mice xenografted with MFH03-PBF (short) epitope (+) cells. CD45+ T cells significantly infiltrated xenografted tumors only in the hybrid PBF TCR T cell group and most of these cells were CD8-positive. CD8+ T cells also showed Ki-67 expression and surrounded the CD8-negative tumor cells expressing Ki-67. These findings suggest that PBF TCR-T cell therapy might be a candidate immunotherapy for sarcoma highly expressing PBF.

Development of CAR-T cells specifically targeting cancer stem cell antigen DNAJB8 against solid tumours.

BACKGROUND: As therapy for solid tumours, various tumour antigens have been selected as targets, but CAR-T cells targeting these antigens have shown limited efficacy, in contrast to the effectiveness of CAR-T cells targeting haematological malignancies. In a previous report, we identified a cancer-testis antigen, DNAJB8. DNAJB8 plays a major role in tumorigenicity in cancer stem-like cells/cancer-initiating cells (CSCs/CICs). Here, we report a DNAJB8-reactive CAR yielding anti-tumour effects against renal cell carcinoma (RCC) and osteosarcoma. METHODS: We constructed a second-generation chimeric antigen receptor (CAR) against HLA-A*24:02/DNAJB8-derived peptide (DNAJB_143) complex (B10 CAR). The reactivity of B10-CAR T cells against T2-A24 cells pulsed with the cognate peptide and an RCC and osteosarcoma cell lines were quantified. The effects of adoptive cell transfer (ACT) therapy were assessed using in vivo xenografted mice models. RESULTS: B10 CAR-T cells recognised DNAJB8_143-pulsed T2-A24 cells and HLA-A*24:02(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. Moreover, ACT using B10 CAR-T cells showed anti-tumour effects against RCC and osteosarcoma cells. CONCLUSION: B10 CAR-T cells could show specific cytotoxicity against RCC and osteosarcoma cells in vitro and in vivo. B10 CAR-T cells targeting the CSC/CIC antigen DNAJB8 might be a candidate immunotherapy for carcinoma and sarcoma.

Osteosarcoma-initiating cells show high aerobic glycolysis and attenuation of oxidative phosphorylation mediated by LIN28B.

Osteosarcoma (OS) is a highly malignant bone tumor and the prognosis for non-responders to chemotherapy remains poor. Previous studies have shown that human sarcomas contain sarcoma-initiating cells (SIC), which have the characteristics of high tumorigenesis and resistance to chemotherapy. In the present study, we characterized SIC of a novel OS cell line, screened for SIC-related genes, and tried to regulate the proliferation of OS by metabolic interference. Initially, we established a new human OS cell line (OS13) and isolated clones showing higher tumorigenesis as SIC (OSHIGH ) and counterpart clones. OSHIGH cells showed chemoresistance and their metabolism highly depended on aerobic glycolysis and suppressed oxidative phosphorylation. Using RNA-sequencing, we identified LIN28B as a SIC-related gene highly expressed in OSHIGH cells. mRNA of LIN28B was expressed in sarcoma cell lines including OS13, but its expression was not detectable in normal organs other than the testis and placenta. LIN28B protein was also detected in various sarcoma tissues. Knockdown of LIN28B in OS13 cells reduced tumorigenesis, decreased chemoresistance, and reversed oxidative phosphorylation function. Combination therapy consisting of a glycolysis inhibitor and low-dose chemotherapy had antitumor effects. In conclusion, manipulation of glycolysis combined with chemotherapy might be a good adjuvant treatment for OS. Development of immunotherapy targeting LIN28B, a so-called cancer/testis antigen, might be a good approach.

Comparison of Posterior Lumbar Interbody Fusion and Microendoscopic Muscle-preserving Interlaminar Decompression for Degenerative Lumbar Spondylolisthesis With >5-Year Follow-up.

STUDY DESIGN: Retrospective analysis of prospectively collected observational multicenter data. OBJECTIVE: To compare the clinical results and rates of revision surgery after posterior lumbar interbody fusion (PLIF) and microendoscopic muscle-preserving interlaminar decompression (ME-MILD) in patients with single-level, mild degenerative lumbar spondylolisthesis (DLS) and follow-up of at least 5 years. SUMMARY OF BACKGROUND DATA: Surgery for symptomatic DLS remains controversial. Evaluating long-term results may reveal problems such as adjacent segmental diseases of the PLIF and decreased quality of life because of slippage and restenosis of the ME-MILD. METHODS: We enrolled 116 patients who underwent PLIF (79 patients) or ME-MILD (37 patients). Operative times, blood losses, surgical complications, Short-Form 36 (SF-36), Japanese Orthopedic Association (JOA) score, the JOA Back Pain Questionnaire (JOABPEQ), visual analog scales (VAS), and Zurich Claudication Questionnaire (ZCQ) were evaluated. RESULTS: PLIF was observed to require significantly longer operative times and entailed greater operative blood losses than did ME-MILD (151.1 vs. 119.9 min; 202.2 vs. 6.4 mL, respectively). Surgery-related complications were identified in 3 cases in the PLIF group and 2 cases in the ME-MILD group. Seventy-eight patients (50 and 28 patients in the PLIF and ME-MILD groups, respectively) were successfully followed-up for >5 years. The follow-up rate was 67.2%. No significant differences between the groups were found in terms of preoperative and postoperative JOA scores, postoperative JOABPEQ, VAS, or ZCQ. Significant improvements in JOA scores were observed in both groups. Significant improvements in the SF-36 were observed in all subscales except in role physical, general health, vitality, and mental health in the ME-MILD group. Revision surgical procedures were performed in 2 patients in the ME-MILD group and 4 patients in the PLIF group. CONCLUSIONS: PLIF and ME-MILD resulted in equivalent improvements in SF-36 and JOA scores. There were no differences in revision surgery rates among patients with single-level, mild DLS. LEVEL OF EVIDENCE: Level III-a retrospective analysis.

Comparison of Health-related Quality of Life Between Double-door Laminoplasty and Selective Laminoplasty for Degenerative Cervical Myelopathy, With a Minimum Follow-up of 5 Years.

STUDY DESIGN: A retrospective analysis of prospectively collected multicenter observational data. OBJECTIVE: The aim of this study was to compare the health-related quality of life (HR-QOL) of double-door laminoplasty (DDL) and selective laminoplasty (SL) in patients with degenerative cervical myelopathy (DCM) in two institutions, with a minimum follow-up of 5 years. SUMMARY OF BACKGROUND DATA: No study has compared DDL and SL regarding postoperative HR-QOL with a follow-up of more than 5 years. METHODS: One-hundred ninety patients who underwent DDL (n = 77) or SL (n = 113) participated in this study. Short-form 36 (SF-36), Japanese Orthopedic Association Cervical Myelopathy Evaluation Questionnaire (JOACMEQ), Neck Disability Index (NDI), and visual analog scale (VAS) values were compared between the groups. RESULTS: Thirty-seven DDL and 52 SL patients were evaluated. The mean follow-up period was 8 years and the follow-up rate was 46.8%. No significant differences were found regarding age and JOA score at baseline. At the follow-up, there were no significant differences in SF-36, JOACMEQ, and VAS score, while the NDI score for headache and sleeping were higher in the SL group. After dividing the SL group into short and long SL subgroups, the long SL subgroup showed a significantly lower score in bodily pain in SF-36, lower and bladder function in JOACMEQ, and pain intensity, personal care, headaches, and sleeping in NDI compared with the other groups. CONCLUSION: No significant differences were found in SF-36, JOA score, and NDI, except for the NDI subscale of headache and sleeping. The subgroup analysis showed that the long SL group showed a decreased QOL compared with the short SL and DDL groups. LEVEL OF EVIDENCE: 3.