Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study.

BACKGROUND: Commercial anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are efficacious against advanced B-cell non-Hodgkin lymphoma (NHL); however, most patients ultimately relapse. Several mechanisms contribute to this failure, including CD19-negative escape and CAR T dysfunction. All four commercial CART19 products utilize the FMC63 single-chain variable fragment (scFv) specific to a CD19 membrane-distal epitope and characterized by slow association (on) and dissociation (off) rates. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 and that is characterized by faster on- and off-rates could mitigate CART19 failure and improve clinical efficacy. METHODS: We developed an autologous CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218). This antibody is specific to a membrane-proximal CD19 epitope and harbors faster on/off rates compared to FMC63. We tested h1218-CART19 in vitro and in vivo using FMC63-CART19-resistant models. We conducted a first-in-human multi-center phase I clinical trial to test AT101 (clinical-grade h1218-CART19) in patients with relapsed or refractory (r/r) NHL. RESULTS: Preclinically, h1218- but not FMC63-CART19 were able to effectively eradicate lymphomas expressing CD19 point mutations (L174V and R163L) or co-expressing FMC63-CAR19 as found in patients relapsing after FMC63-CART19. Furthermore, h1218-CART19 exhibited enhanced killing of B-cell malignancies in vitro and in vivo compared with FMC63-CART19. Mechanistically, we found that h1218-CART19 had reduced activation-induced cell death (AICD) and enhanced expansion compared to FMC63-CART19 owing to faster on- and off-rates. Based on these preclinical results, we performed a phase I dose-escalation trial, testing three dose levels (DL) of AT101 (the GMP version of h1218) using a 3 + 3 design. In 12 treated patients (7 DLBCL, 3 FL, 1 MCL, and 1 MZL), AT101 showed a promising safety profile with 8.3% grade 3 CRS (n = 1) and 8.3% grade 4 ICANS (n = 1). In the whole cohort, the overall response rate was 91.7%, with a complete response rate of 75.0%, which improved to 100% in DL-2 and -3. AT101 expansion correlates with CR and B-cell aplasia. CONCLUSIONS: We developed a novel, safe, and potent CART19 product that recognizes a membrane-proximal domain of CD19 with fast on- and off-rates and showed significant efficacy and promising safety in patients with relapsed B-cell NHL. TRIAL REGISTRATION: NCT05338931; Date: 2022-04-01.

Diagnostic application of clinical exome sequencing in Leber congenital amaurosis.

PURPOSE: Leber congenital amaurosis (LCA) is a hereditary retinal dystrophy with wide genetic heterogeneity. Next-generation sequencing (NGS) targeting multiple genes can be a good option for the diagnosis of LCA, and we tested a clinical exome panel in patients with LCA. METHODS: A total of nine unrelated Korean patients with LCA were sequenced using the Illumina TruSight One panel, which targets 4,813 clinically associated genes, followed by confirmation using Sanger sequencing. Patients' clinical information and familial study results were obtained and used for comprehensive interpretation. RESULTS: In all nine patients, we identified pathogenic variations in LCA-associated genes: NMNAT1 (n=3), GUCY2D (n=2), RPGRIP1 (n=2), CRX (n=1), and CEP290 or SPATA7. Six patients had one or two mutations in accordance with inheritance patterns, all consistent with clinical phenotypes. Two patients had only one pathogenic mutation in recessive genes (NMNAT1 and RPGRIP1), and the clinical features were specific to disorders associated with those genes. Six patients were solved for genetic causes, and it remains unclear for three patients with the clinical exome panel. With subsequent targeted panel sequencing with 113 genes associated with infantile nystagmus syndrome, a likely pathogenic allele in CEP290 was detected in one patient. Interestingly, one pathogenic variant (p.Arg237Cys) in NMNAT1 was present in three patients, and it had a high allele frequency (0.24%) in the general Korean population, suggesting that NMNAT1 could be a major gene responsible for LCA in Koreans. CONCLUSIONS: We confirmed that a commercial clinical exome panel can be effectively used in the diagnosis of LCA. Careful interpretation and clinical correlation could promote the successful implementation of clinical exome panels in routine diagnoses of retinal dystrophies, including LCA.

Evaluation of an amplicon-based next-generation sequencing panel for detection of BRCA1 and BRCA2 genetic variants.

The recent advances in the next-generation sequencing (NGS) technology have enabled fast, accurate, and cost-effective genetic testing. Here, we evaluated the performance of a targeted NGS panel for BRCA1/2 sequencing and confirmed its applicability in routine clinical diagnostics. We tested samples from 88 patients using the TruSeq custom panel (Illumina Inc, USA) and a MiSeq sequencer (Illumina) and compared the results to the outcomes of conventional Sanger sequencing. All 1015 sequence variations identified by Sanger sequencing were detected by NGS, except for one missense variant that might have been missed due to a rare mutation on a primer-binding site. One deletion variation, c.1909 + 12delT of BRCA2, was falsely called in all samples due to a homopolymer error. In addition, seven different single-nucleotide substitutions with low variant frequencies (range: 16.2-33.3 %) were falsely called by NGS. In a separate batch, 10 different false-positive variations were found in five samples. The overall sensitivity and positive predictive value of NGS were estimated to be 99.9 and 87.5 %, respectively. The false-positive results could be excluded by setting quality and alternative allele ratio filters and/or by visual inspection using the IGV software. Targeted NGS panel for BRCA1 and BRCA2 showed an excellent agreement with Sanger sequencing results. We therefore conclude that this NGS panel can be used for routine diagnostic method in a clinical genetic laboratory.

Proteomic analysis of kidneys from selenoprotein M transgenic rats in response to increased bioability of selenium.

BACKGROUND: To characterize changes in global protein expression in kidneys of transgenic rats overexpressing human selenoprotein M (SelM) in response to increased bioabivility of selenium (Sel), total proteins extracted from kidneys of 10-week-old CMV/hSelM Tg and wild-type rats were separated by 2-dimensional gel electrophoresis and measured for changes in expression. RESULTS: Ten and three proteins showing high antioxidant enzymatic activity were up- and down-regulated, respectively, in SelM-overexpressing CMV/hSelM Tg rats compared to controls based on an arbitrary 2-fold difference. Up-regulated proteins included LAP3, BAIAP2L1, CRP2, CD73 antigen, PDGF D, KIAA143 homolog, PRPPS-AP2, ZFP313, HSP-60, and N-WASP, whereas down-regulated proteins included ALKDH3, rMCP-3, and STC-1. After Sel treatment, five of the up-regulated proteins were significantly increased in expression in wild-type rats, whereas there were no changes in CMV/hSelM Tg rats. Only two of the down-regulated proteins showed reduced expression in wild-type and Tg rats after Sel treatment. CONCLUSIONS: These results show the primary novel biological evidences that new functional protein groups and individual proteins in kidneys of Tg rats relate to Sel biology including the response to Sel treatment and SelM expression.

Prostate specific antigen half-time and prostate specific antigen doubling time as predictors of response to androgen deprivation therapy for metastatic prostate cancer.

PURPOSE: We determined the clinical significance of prostate specific antigen half-time and prostate specific antigen doubling time after the prostate specific antigen nadir as predictors of the response to androgen deprivation therapy for metastatic prostate cancer. MATERIALS AND METHODS: A total of 131 patients with metastatic prostate cancer treated with androgen deprivation were included in this analysis. Clinicopathological features and cancer specific survival were compared among the patients who were divided according to prostate specific antigen half-time and prostate specific antigen doubling time after the prostate specific antigen nadir. RESULTS: Median followup was 53.0 months. Baseline and nadir prostate specific antigen did not differ between the patients with a short prostate specific antigen half-time (1 month or less) and those with a long prostate specific antigen half-time (longer than 1 month). Patients with a short prostate specific antigen half-time had a higher Gleason score, shorter nadir duration and shorter cancer specific survival. No differences were found between the patients with a short (6 months or less) and those with a long (longer than 6 months) prostate specific antigen doubling time after the prostate specific antigen nadir in terms of baseline prostate specific antigen, nadir prostate specific antigen, biopsy Gleason score and prostate specific antigen half-time. A short prostate specific antigen doubling time after the prostate specific antigen nadir was associated with shorter nadir duration and poorer median cancer specific survival. On multivariate analysis Gleason score, nadir prostate specific antigen and prostate specific antigen half-time remained independent predictors of an increase in prostate specific antigen after androgen deprivation therapy. Nadir prostate specific antigen, prostate specific antigen half-time and prostate specific antigen doubling time after the prostate specific antigen nadir were prognostic factors for cancer specific survival. CONCLUSIONS: The results of our study suggest that prostate specific antigen half-time and prostate specific antigen doubling time after the prostate specific antigen nadir are independent prognostic indicators for an increase in prostate specific antigen after androgen deprivation therapy and cancer related death in patients with metastatic prostate cancer treated with androgen deprivation.

Effect of bony pelvic dimensions measured by preoperative magnetic resonance imaging on performing robot-assisted laparoscopic prostatectomy.

OBJECTIVE: To evaluate the effect of bony pelvic dimensions measured by preoperative magnetic resonance imaging (MRI) on performing robot-assisted laparoscopic prostatectomy (RALP). PATIENTS AND METHODS: In this exploratory study, we analysed the data of 141 patients who underwent RALP for clinically localized prostate cancer after undergoing MRI at our institution. Associations of various clinicopathological factors were analysed, including pelvic dimensions measured by preoperative MRI, with operative duration, estimated blood loss (EBL), surgical margin status, and postoperative urinary continence and erectile function. RESULTS: For operative duration, no pelvic dimension had a significant association on univariate analysis, with only the newly developed variable of pelvic cavity index approaching significance (P=0.071). Only prostate volume had a significant association with operative duration on multivariate analysis (P=0.015). For EBL, no bony pelvic dimension had a significant association on univariate analysis, with only intertuberous distance and interspinous distance approaching significance (P=0.087 and P=0.072, respectively). Again, only prostate volume was significantly associated with EBL on univariate analysis (P=0.045). No pelvic dimension had any significant effect on surgical margin status, recovery of urinary continence or erectile function at 6 months after RALP. CONCLUSION: Bony pelvic dimensions may not be a significant factor contributing to the technical difficulty of RALP among Korean patients compared with other patient-related factors such as prostate volume.

Perineural invasion is a prognostic factor for biochemical failure after radical prostatectomy.

OBJECTIVES: To identify the prognostic significance of lymphovascular invasion (LVI) and perineural invasion (PNI) in patients undergoing radical prostatectomy for prostate cancer. METHODS: Overall, 237 patients who had undergone radical prostatectomy for prostate cancer between 1995 and 2004 were analyzed for all clinical and pathological factors. The influence of these two pathological features on biochemical failure-free survival was evaluated by univariate and multivariate analysis. RESULTS: Lymphovascular and perineural invasion were identified in 41 (17.2%) and 100 (42.0%) patients, respectively. LVI and PNI were significantly associated with the preoperative prostate-specific antigen (PSA) level, a higher PSA density, a higher pathological stage, a higher Gleason score, a higher frequency of extracapsular extension, a higher frequency of seminal vesicle invasion, and a higher frequency of a positive resection margin. Positive resection margins (P = 0.001) and perineural invasion (P = 0.011) were identified as independent factors associated with biochemical failure-free survival by the multivariate analysis. CONCLUSIONS: In this series, PNI was associated with established parameters of biologically aggressive disease, and was an important prognostic factor for biochemical failure-free survival in patients undergoing radical prostatectomy. This finding supports routine evaluation of the PNI status in radical prostatectomy specimens and suggests that patients with PNI should be more closely followed after surgery.

Efficient strategy for the molecular diagnosis of intractable early-onset epilepsy using targeted gene sequencing.

BACKGROUND: We intended to evaluate diagnostic utility of a targeted gene sequencing by using next generation sequencing (NGS) panel in patients with intractable early-onset epilepsy (EOE) and find the efficient analytical step for increasing the diagnosis rate. METHODS: We assessed 74 patients with EOE whose seizures started before 3 years of age using a customized NGS panel that included 172 genes. Single nucleotide variants (SNVs) and exonic and chromosomal copy number variations (CNVs) were intensively examined with our customized pipeline and crosschecked with commercial or pre-built software. Variants were filtered and prioritized by in-depth clinical review, and finally classified according to the American College of Medical Genetics and Genomics guidelines. Each case was further discussed in a monthly consensus meeting that included the participation of all laboratory personnel, bioinformaticians, geneticists, and clinicians. RESULTS: The NGS panel identified 28 patients (37.8%) with genetic abnormalities; 25 patients had pathogenic or likely pathogenic SNVs in 17 genes including SXTBP1 (n = 3), CDKL5 (n = 2), KCNQ2 (n = 2), SCN1A (n = 2), SYNGAP1 (n = 2), GNAO1 (n = 2), KCNT1 (n = 2), BRAT1, WWOX, ZEB2, CHD2, PRICKLE2, COL4A1, DNM1, SCN8A, MECP2, SLC9A6 (n = 1). The other 3 patients had pathogenic CNVs (2 duplications and 1 deletion) with varying sizes (from 2.5 Mb to 12 Mb). The overall diagnostic yield was 37.8% after following our step-by-step approach for clinical consensus. CONCLUSIONS: NGS is a useful diagnostic tool with great utility for patients with EOE. Diagnostic yields can be maximized with a standardized and team-based approach.

Detection of Immunoglobulin Heavy Chain Gene Clonality by Next-Generation Sequencing for Minimal Residual Disease Monitoring in B-Lymphoblastic Leukemia.

Minimal residual disease (MRD) following B-lymphoblastic leukemia (B-ALL) treatment has gained prognostic importance. Clonal immunoglobulin heavy chain (IGH) gene rearrangement is a useful follow-up marker in B-ALL owing to its high positivity rate. We evaluated the performance and clinical applicability of a next-generation sequencing (NGS) assay for IGH rearrangement in B-ALL MRD monitoring. IGH rearrangement was tested by using fluorescence PCR-fragment analysis and the NGS assay in eight B-ALL patients. The NGS assay was run on two platforms: the Ion Torrent PGM (Thermo Fisher Scientific, USA) (18 samples from 1st to 7th patients) and the MiSeq system (Illumina, USA) (four samples from 8th patient). All initial diagnostic samples and four follow-up samples were positive for clonal IGH rearrangement with fluorescence PCR-fragment analysis and the NGS assay, and six follow-up samples were positive only with NGS. In one case with BCR-ABL1 translocation, BCR-ABL1 quantitative PCR was negative but the NGS IGH assay was positive just prior to full-blown relapse, suggesting the high sensitivity and clinical utility of the NGS assay. The NGS assay is proposed for MRD monitoring in B-ALL Additional studies are needed to confirm the clinical implications of cases showing positive results only in NGS.

Effects of Neutralization by Soluble ABH Antigens Produced by Transplanted Kidneys From ABO-Incompatible Secretor Donors.

BACKGROUND: Grafts survive despite blood group antigens on the transplant being continuously exposed to antibodies in the blood of recipients in ABO-incompatible kidney transplantation (ABOi KT), owing to the mechanism of accommodation. We analyzed the immunodynamics of soluble ABH antigens in allografts from secretor donors and the influence of such immunodynamics on accommodation and subsequent graft survival in ABOi KT. METHODS: The genotype of a known human ß-galactoside α-1,2-fucosyltransferase gene (FUT2), which determines soluble ABH antigen secretor status, was established in 32 donors for ABOi KT at the Severance Hospital, from June 2010 to July 2015. Clinical outcomes of recipients, such as anti-A/B antibody titer change, renal function, and graft survival, were evaluated. RESULTS: Twenty-five donors were secretors (78.1%), and seven were nonsecretors (21.9%). The frequency of anti-A/B IgG or IgM antibody titer elevation or reduction post-transplantation was not significantly related to donor secretor status. However, IgM titer was rapidly reduced in recipients transplanted from nonsecretor donors (P=0.01), which could be explained by the lack of absorption effect of soluble antigens, enhancing the binding of antibodies to antigens in the allografts. Interestingly, soluble ABH antigens did not affect rejection-free graft survival, which may be due to the nature of ß-galactoside α-1,2-fucosyltransferase. CONCLUSIONS: Soluble ABH antigens produced by transplanted kidneys from secretor donors played a role in inducing accommodation within three months of KT through neutralization; however, major graft outcomes were not affected.

Whole genome and transcriptome analysis reveal MALDI-TOF MS and SDS-PAGE have limited performance for the detection of the key outer membrane protein in carbapenem-resistant Klebsiella pneumoniae isolates.

To detect the outer membrane protein (OMP), which plays a key role in carbapenem resistance, whole-genome and transcriptome analysis of the clinical carbapenem-resistant Klebsiella pneumoniae was carried out. The index strain lacked both OmpK35 and OmpK36, whereas the other strains lacked only OmpK35. After SDS-PAGE, the putative OMP bands were excised and identified as OmpA and OmpK36. MALDI-TOF MS showed peaks at ∼36 and ∼38 kDa that corresponded to OmpA and OmpK36, respectively. In all the strains except YMC2014/03/P345, the ∼38 kDa peaks were present. The K. pneumoniae ATCC 13883 isolate showed three bands on SDS-PAGE and three corresponding peaks on MALDI-TOF MS. The additional third peak at ∼37 kDa corresponding to OmpK35 was observed. To verify OmpK35 peak detection in other K. pneumoniae isolates by MALDI-TOF MS, we analyzed six strains from our laboratory's strain bank. Whole genome sequence indicated that only two isolates had intact OmpK35. Both MALDI-TOF MS and SDS-PAGE did not show a ∼37 kDa peak or an OmpK35 band as observed in the K. pneumoniae ATCC 13883 isolate. Separation using SDS-PAGE showed a single peak representing OmpA. Therefore, both SDS-PAGE and MALDI-TOF MS were not completely reliable for OMP detection because they fail to detect OmpK35. To the best of our knowledge, this is the first report on the performance of SDS-PAGE and MALDI-TOF MS for the detection of OMP's using whole-genome and RNA sequencing analyses.

Telomere length in alcohol dependence: A role for impulsive choice and childhood maltreatment.

Telomere shortening, a marker of cellular aging, has been considered to be linked with psychosocial stress as well as with chronic alcohol consumption, possibly mediated by oxidative stress and inflammatory response. Recent findings suggested that early life adversity on telomere dynamics may be related to impulsive choice. To further our understanding of the association of impulsive choice and childhood trauma on telomere length, we examined whether delayed discounting and childhood trauma or their interaction is related to leukocyte telomere length, while controlling for multiple potential confounding variables, in patients with alcohol dependence who are considered to have higher impulsive choice and shorter telomere length. We recruited 253 male patients with chronic alcohol dependence. All participants performed the delay discounting task, and the area under curve was used as a measure of delay discounting. Steeper delay discounting represents more impulsive choices. The modified Parent-Child Conflict Tactics Scale was used to measure childhood maltreatment. In addition, confounding factors, including socio-demographic characteristics, the Alcohol Use Disorders Identification Test, the Buss-Perry Aggression Questionnaire, the Resilience Quotient, the Beck Depression Inventory, and the Beck Anxiety Inventory, were also assessed. Hierarchical regression analyses showed a significant main effect of delay discounting (ß=0.161, t=2.640, p=0.009), and an interaction effect between delay discounting and childhood maltreatment on leukocyte telomere length (ß=0.173, t=2.138, p=0.034). In subsequent analyses stratified by childhood maltreatment, patients with alcohol dependence and high childhood trauma showed a significant relationship between delay discounting and leukocyte telomere length (ß=0.279, t=3.183, p=0.002), while those with low trauma showed no association between them. Our findings suggest that higher impulsive choice is associated with shorter telomere length, and childhood trauma may exert a moderating effect in the relationship between impulsive choice and telomere length.

Next-generation sequencing of BRCA1/2 in breast cancer patients: potential effects on clinical decision-making using rapid, high-accuracy genetic results.

PURPOSE: We evaluated the clinical role of rapid next-generation sequencing (NGS) for identifying BRCA1/2 mutations compared to traditional Sanger sequencing. METHODS: Twenty-four paired samples from 12 patients were analyzed in this prospective study to compare the performance of NGS to the Sanger method. Both NGS and Sanger sequencing were performed in 2 different laboratories using blood samples from patients with breast cancer. We then analyzed the accuracy of NGS in terms of variant calling and determining concordance rates of BRCA1/2 mutation detection. RESULTS: The overall concordance rate of BRCA1/2 mutation identification was 100%. Variants of unknown significance (VUS) were reported in two cases of BRCA1 and 3 cases of BRCA2 after Sanger sequencing, whereas NGS reported only 1 case of BRCA1 VUS, likely due to differences in reference databases used for mutation identification. The median turnaround time of Sanger sequencing was 22 days (range, 14-26 days), while the median time of NGS was only 6 days (range, 3-21 days). CONCLUSION: NGS yielded comparably accurate results to Sanger sequencing and in a much shorter time with respect to BRCA1/2 mutation identification. The shorter turnaround time and higher accuracy of NGS may help clinicians make more timely and informed decisions regarding surgery or neoadjuvant chemotherapy in patients with breast cancer.

The effect of trauma and PTSD on telomere length: An exploratory study in people exposed to combat trauma.

Telomere length has been suggested to be a cellular marker for age-related diseases as well as psychosocial stress. The present study investigated whether telomere length is associated with post-traumatic stress disorder (PTSD) among veterans exposed to combat trauma in the Vietnam War. The potentially associated factors on cellular aging were considered. Korean male veterans with (n = 122) and without (n = 120) PTSD were included and leukocyte telomere length was measured with a quantitative PCR-based technique. As a whole, no significant difference in telomere length was found between PTSD and non-PTSD groups. In linear regression analysis stratified by trauma levels, among veterans exposed to severe combat (n = 45), PTSD status (B = -1.176, t = -2.259, p = 0.029), antidepressant use (B = 0.168, t = 2.528, p = 0.015), and education level (B = 0.019, t = 2.369, p = 0.023) affected telomere length. However, among veterans with light-to-moderate combat exposure (n = 197), only age (B = -0.007, t = -2.434, p = 0.016) and education level (B = 0.010, t = 2.295, p = 0.023) were associated with telomere length. In the Post-hoc analysis, antidepressant use was associated with longer telomere length in subjects exposed to severe combat. Our exploratory results suggest that PTSD status in combination with severe trauma may be associated with accelerated telomere shortening, and that antidepressant use may have a protective effect on telomere dynamics.

Affinity Maturation of Monoclonal Antibody 1E11 by Targeted Randomization in CDR3 Regions Optimizes Therapeutic Antibody Targeting of HER2-Positive Gastric Cancer.

Anti-HER2 murine monoclonal antibody 1E11 has strong and synergistic anti-tumor activity in HER2-overexpressing gastric cancer cells when used in combination with trastuzumab. We presently optimized this antibody for human therapeutics. First, the complementarity determining regions (CDRs) of the murine antibody were grafted onto human germline immunoglobulin variable genes. No difference in affinity and biological activity was observed between chimeric 1E11 (ch1E11) and humanized 1E11 (hz1E11). Next, affinity maturation of hz1E11 was performed by the randomization of CDR-L3 and H3 residues followed by stringent biopanning selection. Milder selection pressure favored the selection of more diverse clones, whereas higher selection stringency resulted in the convergence of the panning output to a smaller number of clones with improved affinity. Clone 1A12 had four amino acid substitutions in CDR-L3, and showed a 10-fold increase in affinity compared to the parental clone and increased potency in an in vitro anti-proliferative activity assay with HER2-overepxressing gastric cancer cells. Clone 1A12 inhibited tumor growth of NCI-N87 xenograft model with similar efficacy to trastuzumab alone, and the combination treatment of 1A12 and trastuzumab completely removed the established tumors. These results suggest that humanized and affinity matured monoclonal antibody 1A12 is a highly optimized molecule for future therapeutic development against HER2-positive tumors.

Combination of novel HER2-targeting antibody 1E11 with trastuzumab shows synergistic antitumor activity in HER2-positive gastric cancer.

The synergistic interaction of two antibodies targeting the same protein could be developed as an effective anti-cancer therapy. Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-25% of breast and gastric cancer patients, and HER2-targeted antibody therapy using trastuzumab is effective in many of these patients. Nonetheless, improving therapeutic efficacy and patient survival is important, particularly in patients with HER2-positive gastric cancer. Here, we describe the development of 1E11, a HER2-targeted humanized monoclonal antibody showing increased efficacy in a highly synergistic manner in combination with trastuzumab in the HER2-overexpressing gastric cancer cell lines NCI-N87 and OE-19. The two antibodies bind to sub-domain IV of the receptor, but have non-overlapping epitopes, allowing them to simultaneously bind HER2. Treatment with 1E11 alone induced apoptosis in HER2-positive cancer cells, and this effect was enhanced by combination treatment with trastuzumab. Combination treatment with 1E11 and trastuzumab reduced the levels of total HER2 protein and those of aberrant HER2 signaling molecules including phosphorylated HER3 and EGFR. The synergistic antitumor activity of 1E11 in combination with trastuzumab indicates that it could be a novel potent therapeutic antibody for the treatment of HER2-overexpressing gastric cancers.

Interaction of ALG-2 with ASK1 influences ASK1 localization and subsequent JNK activation.

ALG-2 (apoptosis linked gene-2) is an essential protein for the execution of apoptosis whose function is largely unknown. Here, we demonstrate that ALG-2 could interact with the C-terminus (amino acids 941-1375) of the apoptosis signal-regulating kinase 1 (ASK1) in BOSC23 cells as well as in vitro. ASK1 failed to bind to an isotype of ALG-2 found in the liver, ALG-2,1, in which two amino acids (Gly-121 and Phe-122) are deleted. This implies that the interaction is very specific. Cotransfection with ALG-2 resulted in the nuclear presence of ASK1 and inhibited the activation of c-Jun N-terminal kinase (JNK) by ASK1 in BOSC23 cells. This study reports that ALG-2 could regulate the subcellular localization and the JNK activity modulation of ASK1 by direct interaction.

Growth sensitivity in the epiphyseal growth plate, liver and muscle of SD rats is significantly enhanced by treatment with a fermented soybean product (cheonggukjang) through stimulation of growth hormone secretion.

Cheonggukjang (CKJ), a fermented soybean product, has been reported to have beneficial effects on various chronic diseases, including cardiovascular disease, cancer and immune diseases. To investigate whether CKJ induces growth sensitivity in mammals, alterations of key parameters related to their growth were analyzed. Sprague­Dawley (SD) rats were treated with a high concentration of CKJ (H­CKJ) or a low concentration of CKJ (L­CKJ) for 10 days, and compared with vehicle-treated rats. The CKJ contained a high concentration of total flavonoids, phenolic compounds, daidzein and genistein, compared with the non-fermented soybean product. Body weight was higher in the H­CKJ­treated group compared with that in the vehicle­ and L­CKJ­treated groups, whereas the weights of three organs (the brain, liver and kidney) were higher in the L­CKJ­treated group compared with the remaining two groups. However, no significant differences in femur length and weight were detected between the CKJ­ and vehicle­treated groups. The thickness of the epiphyseal growth plate in proximal femoral epiphysis was broadest in the H­CKJ­treated group compared with the vehicle- and L­CKJ­treated groups. Furthermore, the level of growth hormone (GH) was highest in the serum of the L­CKJ­treated group, although that of the H­CKJ­treated group was lower compared with that in the L­CKJ group. Moreover, the expression levels of the GH receptor increased in the liver tissue, but not in the muscle tissue, of the L­CKJ­ and H­CKJ­treated groups. In the downstream signaling pathway of the GH receptor, the phosphorylation levels of Akt and Erk were differentially regulated between the liver and muscle. These results suggest that CKJ extract may enhance the sensitivity of the femur, liver and muscle epiphyseal growth plate in SD rats, through the upregulation of GH secretion.