Telitacicept in patients with active systemic lupus erythematosus: results of a phase 2b, randomised, double-blind, placebo-controlled trial.

OBJECTIVES: This phase 2b, randomised, double-blind, placebo-controlled trial evaluated the efficacy and safety of telitacicept, a novel fusion protein that neutralises signals of B lymphocyte stimulator and a proliferation-inducing ligand, in active systemic lupus erythematosus (SLE). METHODS: Adult patients with active SLE (n=249) were recruited from 29 hospitals in China and randomised 1:1:1:1 to receive subcutaneous telitacicept at 80 mg (n=62), 160 mg (n=63), 240 mg (n=62) or placebo (n=62) once weekly in addition to standard therapy. The primary endpoint was the proportion of patients achieving an SLE Responder Index 4 (SRI-4) response at week 48. Missing data were imputed using the last observation carried forward method. RESULTS: At week 48, the proportion of patients achieving an SRI-4 response was 75.8% in the 240 mg telitacicept group, 68.3% in the 160 mg group, 71.0% in the 80 mg group and 33.9% in the placebo group (all p<0.001). Significant treatment responses were observed in secondary endpoints, including a ≥4-point reduction on the Systemic Lupus Erythematosus Disease Activity Index, a lack of Physician's Global Assessment score worsening and a glucocorticoid dose reduction in the 240 mg group. Telitacicept was well tolerated, and the incidence of adverse events and serious adverse events was similar between the telitacicept and placebo groups. CONCLUSIONS: This phase 2b clinical trial met the primary endpoint. All telitacicept groups showed a significantly higher proportion of patients achieving an SRI-4 response than the placebo group at week 48, and all doses were well tolerated. These results support further investigations of telitacicept in clinical trials involving more diverse populations and larger sample sizes. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT02885610).

Efficacy and safety of telitacicept in primary Sjögren's syndrome: a randomized, double-blind, placebo-controlled, phase 2 trial.

OBJECTIVE: To evaluate the efficacy and safety of telitacicept in adult patients with primary SS (pSS) in a phase II randomized double-blind placebo-controlled trial. METHODS: Patients with pSS with positive anti-SSA antibody and ESSDAI ≥ 5 were randomly assigned, in a 1:1:1 ratio, to receive weekly subcutaneous telitacicept 240 mg, 160 mg, or placebo for 24 weeks. The primary end point was the change from baseline in the ESSDAI at week 24. Safety was monitored. RESULTS: A total of 42 patients were enrolled and randomized (n = 14 per group). Administration of telitacicept 160 mg resulted in a significant reduction in ESSDAI score from baseline to week 24 compared with placebo (P < 0.05). The placebo-adjusted least-squares mean change from baseline was -4.3 (95% CI -7.0, -1.6; P = 0.002). While, mean change of ESSDAI in telitacicept 240 mg was -2.7(-5.6-0.1) with no statistical difference when compared that in placebo group (P = 0.056). In addition, MFI-20 and serum immunoglobulins decreased significantly (P < 0.05) at week 24 in both telitacicept groups compared with placebo. No serious adverse events were observed in the telitacicept treating group. CONCLUSION: Telitacicept showed clinical benefits and good tolerance and safety in the treatment of pSS. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT04078386.

A multicenter, randomized, open-label, phase 2 clinical study of telitacicept in adult patients with generalized myasthenia gravis.

BACKGROUND AND PURPOSE: This study aimed to investigate the clinical efficacy and safety of telitacicept in patients with generalized myasthenia gravis (gMG) who tested positive for acetylcholine receptor antibodies or muscle-specific kinase antibodies and were receiving standard-of-care therapy. METHODS: Patients meeting the eligibility criteria were randomly assigned to receive telitacicept subcutaneously once a week for 24 weeks in addition to standard-of-care treatment. The primary efficacy endpoint was the mean change in the quantitative myasthenia gravis (QMG) score from baseline to week 24. Secondary efficacy endpoints included mean change in QMG score from baseline to week 12 and gMG clinical absolute score from baseline to week 24. Additionally, safety, tolerability and pharmacodynamics were assessed. RESULTS: Twenty-nine of the 41 patients screened were randomly selected and enrolled. The mean (± standard deviation [SD]) reduction in QMG score from baseline to week 24 was 7.7 (± 5.34) and 9.6 (± 4.29) in the 160 mg and 240 mg groups, respectively. At week 12, mean reductions in QMG scores for these two groups were 5.8 (± 5.85) and 9.5 (± 5.03), respectively, indicating rapid clinical improvement. Safety analysis revealed no adverse events leading to discontinuation or mortalities. All patients showed consistent reductions in serum immunoglobulin (Ig) A, IgG and IgM levels throughout the study. CONCLUSION: Telitacicept demonstrated safety, good tolerability and reduced clinical severity throughout the study period. Further validation of the clinical efficacy of telitacicept in gMG will be conducted in an upcoming phase 3 clinical trial.

N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global public health crisis. The causative agent, the SARS-CoV-2 virus, enters host cells via molecular interactions between the viral spike protein and the host cell ACE2 surface protein. The SARS-CoV-2 spike protein is extensively decorated with up to 66 N-linked glycans. Glycosylation of viral proteins is known to function in immune evasion strategies but may also function in the molecular events of viral entry into host cells. Here, we show that N-glycosylation at Asn331 and Asn343 of SARS-CoV-2 spike protein is required for it to bind to ACE2 and for the entry of pseudovirus harboring the SARS-CoV-2 spike protein into cells. Interestingly, high-content glycan binding screening data have shown that N-glycosylation of Asn331 and Asn343 of the RBD is important for binding to the specific glycan molecule G4GN (Galß-1,4 GlcNAc), which is critical for spike-RBD-ACE2 binding. Furthermore, IL-6 was identified through antibody array analysis of conditioned media of the corresponding pseudovirus assay. Mutation of N-glycosylation of Asn331 and Asn343 sites of the spike receptor-binding domain (RBD) significantly reduced the transcriptional upregulation of pro-inflammatory signaling molecule IL-6. In addition, IL-6 levels correlated with spike protein levels in COVID-19 patients' serum. These findings establish the importance of RBD glycosylation in SARS-CoV-2 pathogenesis, which can be exploited for the development of novel therapeutics for COVID-19.

A HER2 target antibody drug conjugate combined with anti-PD-(L)1 treatment eliminates hHER2+ tumors in hPD-1 transgenic mouse model and contributes immune memory formation.

PURPOSE: Disitamab vedotin (RC48) is an HER2-directed antibody-drug conjugate, emerging as an effective strategy for cancer therapy, which not only enhances antitumor immunity in previous animal models but also improves clinical outcomes for patients such as with gastric cancer, urothelium carcinoma, and HER2 low-expressing breast cancer. Here, we explore the combination therapeutic efficacy of this novel HER2-targeting ADC with immune checkpoint inhibitors in a human HER2-expressing syngeneic breast cancer model. METHODS: The human HER2+ cancer cell line is constructed by stable transfection and individual clones were isolated by single-cell sorting. Flow cytometry was performed to determine its binding activity. Cytotoxic effect was determined using an MTT assay with the supplement of RC48. Human PD-1 transgenic mice were used to analyze the in vivo antitumor effects of the ADC and its combination therapy with PD-1/PD-L1 antibody. RESULTS: The combination of RC48 and PD-1/PD-L1 immune checkpoint inhibition significantly enhanced tumor suppression and antitumor immunity. Tumor rejection in the synergistic groups was accompanied by massive T cell infiltration and immune marker activation. Furthermore, the combination therapy promoted immunological memory formation in the tumor eradication animals, protecting them from tumor rechallenge. CONCLUSION: A novel HER2-targeting ADC combined with immune checkpoint inhibitors can achieve remarkable effects in mice and elicit long-lasting immune protection in a hHER2+ murine breast cancer model. This study provides insights into the efficacy of RC48 therapeutic activity and a rationale for potential therapeutic combination strategies with immunotherapy.

Development and evaluation of time-resolved fluorescent immunochromatographic assay for quantitative detection of SARS-CoV-2 spike antigen.

BACKGROUND: The spread of COVID-19 worldwide caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has necessitated efficient, sensitive diagnostic methods to identify infected people. We report on the development of a rapid 15-minute time-resolved fluorescent (TRF) lateral flow immunochromatographic assay for the quantitative detection of the SARS-CoV-2 spike protein receptor-binding domain (S1-RBD). OBJECTIVES: Our objective was to develop an efficient method of detecting SARS-CoV-2 within 15 min of sample collection. METHODS: We constructed and evaluated a portable, disposable lateral flow device, which detected the S1-RBD protein directly in nasopharyngeal swab samples. The device emits a fluorescent signal in the presence of S1-RBD, which can be captured by an automated TRF instrument. RESULTS: The TRF lateral flow assay signal was linear from 0 to 20 ng/ml and demonstrated high accuracy and reproducibility. When evaluated with clinical nasopharyngeal swabs, the assay was performed at >80% sensitivity, >84% specificity, and > 82% accuracy for detection of the S1-RBD antigen. CONCLUSION: The new S1-RBD antigen test is a rapid (15 min), sensitive, and specific assay that requires minimal sample preparation. Critically, the assay correlated closely with PCR-based methodology in nasopharyngeal swab samples, showing that the detected S1-RBD antigen levels correlate with SARS-CoV-2 virus load. Therefore, the new TRF lateral flow test for S1-RBD has potential application in point-of-care settings.

Phase I study of the recombinant humanized anti-HER2 monoclonal antibody-MMAE conjugate RC48-ADC in patients with HER2-positive advanced solid tumors.

PURPOSE: RC48 contains the novel humanized anti-HER2 antibody hertuzumab conjugated to MMAE via a cleavable linker. A phase I study was initiated to evaluate the toxicity, MTD, PK, and antitumor activity of RC48 in patients with HER2-overexpressing locally advanced or metastatic solid carcinomas, particularly gastric cancer. PATIENTS AND METHODS: This was a 2-part phase I study. Successive cohorts of patients received escalating doses of RC48 (0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, and 3.0 mg/kg). Dose expansion proceeded at the dose of 2.0 mg/kg Q2W. The efficacy and safety set included all patients who received at least one dose of RC48. RESULTS: Fifty-seven patients were enrolled, the MTD was unavailable due to termination of 3.0 mg/kg cohort; 2.5 mg/kg Q2W was declared the RP2D. RC48 was well tolerated, the most frequent grade 3 or worse TRAEs included neutropenia (19.3%), leukopenia (17.5%), hypoesthesia (14.0%), and increased conjugated blood bilirubin (8.8%). Four deaths occurred during the whole study, three of which were believed to be related to RC48. Overall, ORR and DCR were 21.0% (12/57) and 49.1% (28/57). Notably, patients who were HER2 IHC2+/FISH- responded similarly to those who were IHC2+/FISH+ and IHC3+, with ORRs of 35.7% (5/14), 20% (2/10), and 13.6% (3/22), respectively. In patients who were pretreated with HER2-targeted drugs, RC48 also showed promising efficacy, with ORR of 15.0% (3/20) and DCR of 45.0% (9/20). CONCLUSION: RC48 was well tolerated and showed promising antitumor activity in HER2-positive solid tumors, including gastric cancer with HER2 IHC 2+/FISH- status. CLINICAL TRIAL INFORMATION: NCT02881190.

Improved recombinant protein production by regulation of transcription and protein transport in Chinese hamster ovary cells.

OBJECTIVES: To identify genes that affected protein expression in Chinese hamster ovary (CHO) cells was significant, and we identified the changes in the transcriptome and the functional gene sets that would contribute to increase expression of recombinant protein. RESULTS: Here two sub-clones from a methotrexate-treated parental recombinant CHO cell line were selected. The two sub-clones, with different expression levels (qp were 42.8 pg/cell/day and 14.0 pg/cell/day), were analyzed through RNA-seq. More than 600 genes were identified as differently expressed, and we found that the differentially expressed genes were involved in processes such as RNA processing, transcription, protein catabolism, and protein transport. Among these, we cloned genes encoding proteins that were involved in transcription and protein transport to investigate their effect on protein production. CONCLUSIONS: We found that some genes involved in transcription and protein transport would improve recombinant protein production in CHO cells.

Brain Transforming Growth Factor-ß Resists Hypertension Via Regulating Microglial Activation.

BACKGROUND AND PURPOSE: Hypertension is the major risk factor for stroke. Recent work unveiled that hypertension is associated with chronic neuroinflammation; microglia are the major players in neuroinflammation, and the activated microglia elevate sympathetic nerve activity and blood pressure. This study is to understand how brain homeostasis is kept from hypertensive disturbance and microglial activation at the onset of hypertension. METHODS: Hypertension was induced by subcutaneous delivery of angiotensin II, and blood pressure was monitored in conscious animals. Microglial activity was analyzed by flow cytometry and immunohistochemistry. Antibody, pharmacological chemical, and recombinant cytokine were administered to the brain through intracerebroventricular infusion. Microglial depletion was performed by intracerebroventricular delivering diphtheria toxin to CD11b-diphtheria toxin receptor mice. Gene expression profile in sympathetic controlling nucleus was analyzed by customized qRT-PCR array. RESULTS: Transforming growth factor-ß (TGF-ß) is constitutively expressed in the brains of normotensive mice. Removal of TGF-ß or blocking its signaling before hypertension induction accelerated hypertension progression, whereas supplementation of TGF-ß1 substantially suppressed neuroinflammation, kidney norepinephrine level, and blood pressure. By means of microglial depletion and adoptive transfer, we showed that the effects of TGF-ß on hypertension are mediated through microglia. In contrast to the activated microglia in established hypertension, the resting microglia are immunosuppressive and important in maintaining neural homeostasis at the onset of hypertension. Further, we profiled the signature molecules of neuroinflammation and neuroplasticity associated with hypertension and TGF-ß by qRT-PCR array. CONCLUSIONS: Our results identify that TGF-ß-modulated microglia are critical to keeping brain homeostasis responding to hypertensive disturbance.

Angiotensin-converting enzyme affects the presentation of MHC class II antigens.

Antigen processing and presentation through the MHC class II pathway is critical for activating T helper cells. Angiotensin-converting enzyme (ACE) is a carboxyl peptidase expressed by antigen-presenting cells. By analysis of ACE null (knockout), wild-type, and ACE-overexpressing (ACE10) mice and the antigen-presenting cells derived from these mice, we found that ACE has a physiological role in the processing of peptides for MHC class II presentation. The efficiency of presenting MHC class II epitopes from ovalbumin (OVA) and hen egg lysosome is markedly affected by cellular ACE levels. Mice overexpressing ACE in myeloid cells have a much more vigorous CD4+ T-cell and antibody response when immunized with OVA. ACE is present in the endosomal pathway where MHC class II peptide processing and loading occur. The efficiency of MHC class II antigen presentation can be altered by ACE overexpression or ACE pharmacological inhibition. Thus, ACE is a dynamic participant in processing MHC class II peptides. Manipulation of ACE expression by antigen-presenting cells may prove to be a novel strategy to alter the immune response.

Genetic analysis of heterogeneous sub-clones in recombinant Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells have been widely used for production of recombinant proteins and therapeutic antibodies. However, owing to the instability and heterogeneity of CHO cells, the development of stable and high-expression recombinant CHO cell lines is often time-consuming. To investigate the mechanisms associated with heterogeneity in protein productivity, we performed transcriptome analysis on the subclones derived from a stable parental CHO clone. Two high-expression subclones and one low-expression subclone were selected based on their similar genomic background and subjected to RNA-seq analysis. Over 100 differentially expressed genes were identified between the subclones with high and low productivity. The molecular functions of the differentially expressed genes were enriched for translational elongation, sterol biosynthetic process, and regulation of secretion. In addition, analyses of the two subclones with high protein expression levels identified over 300 differentially expressed genes involved in DNA metabolic processes, cellular macromolecule catabolic processes, cell cycle, protein catabolic processes, and RNA processing and transcription. A subset of the differentially expressed genes was overexpressed in CHO cells to identify their effects on protein production. Together, these results indicate that transcriptome variation can cause significant inter-cellular heterogeneity in CHO cells and a better understanding of the molecular mechanism underlying heterogeneity might help to improve the production of recombinant proteins by CHO cells.

Pharmacokinetics, pharmacodynamics, short term efficacy and safety of RCT-18, a novel BLyS/APRIL fusion protein, in patients with rheumatoid arthritis.

AIM: RCT-18 is a recombinant fusion protein that interferes with the selection and survival of mature B-lymphocytes by inhibiting B-lymphocyte stimulator and a proliferation-inducing ligand. METHODS: This single blind, randomized, placebo controlled, clinical pharmacological study explored the short term efficacy and safety of RCT-18 in 21 rheumatoid arthritis (RA) patients with three different dosing regimens. The pharmacological behaviour of RCT-18 was also characterized through a six level biomarker cascade approach to identify potential predictors for clinical responses. RESULTS: Nine out of 10 patients (>80%) experienced moderate to good EULAR response at the end of 3 months with once or twice weekly doses of 180 mg RCT-18, whereas weekly administration of 360 mg RCT-18 or placebo, however, only resulted in moderate improvement in one patient in each group. Absence of IgM-type rheumatoid factor reduction, recovery of IgM 2 weeks after drug cessation, lack of decrease in the count of CD27(+) B-lymphocytes and a DAS28 change from baseline <6 in 4-6 weeks after the treatment initiation may indicate poor clinical response. No anti-drug antibody of RCT-18 was detected. The active treatments were well tolerated, although more mild to moderate infections were reported in patients receiving RCT-18. CONCLUSION: The study results support further development of RCT-18 in RA patients and provide important information for future dose selection.

Mesencephalic astrocyte-derived neurotrophic factor reduces cell apoptosis via upregulating GRP78 in SH-SY5Y cells.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects dopaminergic neurons from damage. In this study, we used MTT, immunohistochemistry, and TUNEL staining to investigate the protective effect of MANF in SH-SY5Y cells treated with 6-OHDA or overexpressed α-synuclein. Cleaved caspase-3 levels significantly increased in cells treated with 6-OHDA or overexpressed α-synuclein. 6-OHDA or α-synuclein overexpression that induced cleaved caspase-3 levels to increase was reduced by MANF treatment. In addition, MANF treatment upregulated GRP78 expressions in cells treated with 6-OHDA or overexpressed α-synuclein, and RNAi knockdown for GRP78 could block the MANF induced cell survival from 6-OHDA treatment. Furthermore, GRP78 overexpression inhibited 6-OHDA-induced apoptosis. Our data suggest that MANF inhibits apoptosis induced by 6-OHDA and overexpressed α-synuclein in SH-SY5Y cells via upregulating GRP78 in the transcriptional pattern.

Detection of Human Epididymis Protein 4 (HE4) in Human Serum Samples Using a Specific Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay (ELISA).

BACKGROUND: The human epididymis protein 4 (HE4) may have high specificity in the detection of malignant diseases, making the development of an immunoassay for HE4 essential. METHODS: In our study, a fusion gene was constructed encoded with the HE4 protein. This protein was then produced in the bacterial cells (Escherichia coli) and used to immunize mice in order to eventually generate hybridomas specific to HE4. The hybridoma supernatants were then screened, and four positive anti-HE4 cell lines were selected. These cell lines produce monoclonal antibodies against HE4 epitopes, as demonstrated in the Western blot as well as by direct enzyme-linked immunosorbent assay (ELISA). Using the developed antibodies, we successfully identified several good antibody pairs from the hybridomas, which allowed for the development of a sandwich ELISA to measure HE4 levels. By using the HE4 ELISA, we measured HE4 levels of 60 clinical human serum samples. RESULTS: Compared with the Food and Drug Administration (FDA) approved kit (Roche), our results showed a strong positive correlation to those of the FDA-approved kit. CONCLUSIONS: In summary, highly sensitive antibody pairs were screened against HE4, and a sandwich ELISA was developed as an accurate analytical tool for the detection of HE4 in human serum, which could be especially valuable for diagnosing ovarian carcinomas.

Identification and characterization of monoclonal antibodies against GP73 for use as a potential biomarker in liver cancer screening and diagnosis.

Golgi membrane protein 1, or GP73, is recently being evaluated as a novel cancer biomarker against prostate cancer, lung adenocarcinoma, and hepatocellular carcinoma (HCC). In the microenvironment of HCC, GP73 expression levels are significantly elevated. It is this elevation that may prove more specific and sensitive for HCC detection than that of the traditional biomarker, alpha-fetoprotein (AFP). This may be especially true if it can be measured and identified earlier in the diagnostic process. We sought to develop a testing platform to measure GP73 levels for the purposes of earlier diagnostic screening of at risk patients. We expressed recombinant GP73 protein to use as an immunogen in order to develop several monoclonal anti-GP73 antibodies. Three clones, 1D7, 2B2, and 5B4, were identified with all three having a higher than 1:5,000,000 titer. These clones were then isotyped and validated to bind the immunogen protein. Different combinations of antibody pairs were then tested in order to create a functional sandwich antibody pair. Using this pair on liver disease patient serum samples, we found that GP73 was significantly elevated when compared to healthy control patient serum (P < 0.0001). Average GP73 levels in HCC patients was 284.0 ng/mL, slightly higher than liver disease patients (265.6 ng/mL), and significantly elevated over normal serum levels is (74.86 ng/mL). The area under the receiver-operating characteristic curve (ROC) for GP73 to detect liver cancer was 0.98 (95% CI, 0.95 to 1.00; P < 0.0001), and GP73 levels had a sensitivity of 97%, a specificity of 87% for detecting liver cancer. By contrast, the sensitivity and specificity of liver disease detection was 76% and 97%, respectively. We then tested detection of 74 serum samples (n control = 46, n liver disease = 7, n liver cancer = 21) by our ELISA testing methodology and commercial kit simultaneously. The results found that our kit and the commercial kit had a good linear correlation coefficient, r(2) = 0.932. Together these clones and our ELISA pair may prove extremely useful in the detection and monitoring of GP73 in HCC and other at risk patients.

Mesencephalic astrocyte-derived neurotrophic factor attenuates inflammatory responses in lipopolysaccharide-induced neural stem cells by regulating NF-κB and phosphorylation of p38-MAPKs pathways.

Mesencephalic astrocyte-derived neurotrophic factor (MANF), a new evolutionary conserved neurotrophic factor (NTF), has been reported to protect midbrain dopaminergic neurons of neurodegenerative diseases such as Parkinson's disease (PD) model. Neural stem cells (NSCs) can play a role as the therapeutic tool in neurodegenerative diseases, but the inflammatory responses of central nervous system (CNS) appear to harm this function. Although studies have previously demonstrated the protective effect of MANF on neurons of CNS, it is lacking in making great efforts on the function of MANF on NSCs. The aim of this study was to investigate the antiinflammatory responses and signaling mechanisms of MANF on lipopolysaccharide (LPS)-induced NSCs. In the results, MANF decreased the proinflammatory cytokines of IL-1ß, TNF-α, and IFN-γ induced by LPS by regulating NF-κB and phosphorylation of p38-mitogen-activated protein kinases (MAPKs) pathways, neither p-JNK nor p-ERK signaling. These findings suggest that MANF can facilitate to protect the inflammatory responses of NSCs, and provide beneficial function for the application of NSCs in the therapy.

A novel humanized anti-HER2 antibody conjugated with MMAE exerts potent anti-tumor activity.

Human epidermal growth factor receptor-2 (HER2) is a validated therapeutic target for breast cancer and trastuzumab (Herceptin), a humanized anti-HER2 antibody, has significant anti-cancer effects in the clinic. However, breast cancer patients often experience disease progression after prolonged Herceptin treatment. To develop a more effective therapy, we generated humanized monoclonal antibody hertuzumab and hertuzumab-drug conjugates as novel breast cancer therapies. The hertuzumab was conjugated with small molecule cytotoxic agents monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF) with various linkers to generate antibody-drug conjugates (ADCs), which were evaluated for their in vitro and in vivo anti-cancer activities. Among these ADCs, hertuzumab-vc-MMAE can be effectively internalized and potently kill HER2 over-expressing tumor cells. In xenograft tumor models, hertuzumab-vc-MMAE showed a more potent anti-tumor activity than T-DM1, a FDA-approved ADC drug. More importantly, this novel ADC drug also showed superior anti-tumor activity than T-DM1 in trastuzumab- and lapatinib-resistant xenograft tumor models, suggesting its potential as an improved therapy for HER2-positive breast cancers. The novel ADC, hertuzumab-vc-MMAE, is an effective and selective agent for the treatment of HER2-positive breast tumors.

IMPACT OF IMMUNE RESPONSE OF A PARASITIC BEETLE Dastarcus helophoroides ON ITS HOST BEETLE Monochamus alternatus.

Dastarcus helophoroides is an ectoparasitoid beetle of Monochamus alternatus, and the parasitism by D. helophoroides larvae remarkably influenced on the immune responses of M. alternatus larvae in many aspects. The hemolymph melanization reactions in the hosts were inhibited 1 h and 24 h postparasitization. The phenoloxidase activities of hemolymph were significantly stimulated 4 h postparasitization and inhibited 12 h postparasitization, and back to control level. The antibacterial activities of hemolymph in the parasitized hosts were significantly lower than that in the unparasitized ones 1 h postparasitization. By 72 h postparasitism, the total hemocyte numbers of the parasitized larvae declined to not more than one-seconds of the number collected from the unparasitized larvae. All sampled hemolymph held the capability of nodulation, and there were fluctuations in the number of nodules the hemocytes made. However, there were no significant differences between unparasitized and parasitized larvae at each time point in the hemagglutination activity and the ratios of spreading hemocytes. In conclusion, D. helophoroides larvae could regulate M. alternatus immune system and resulted in the changes in host immune responses.

[Construction of Lentiviral Expression Vector Containing Extracellular Domain of Human Hepatocyte Growth Factor Receptor and Its Expression in 293T Cell].

This research aims to construct a lentiviral expression vector carrying the extracelluar domain (ED) of human hepatocyte growth factor receptor (C-Met), and to express it in transfected 293T cells. The extracellular domain of C-Met was amplified by RT-PCR, ligated with lentiviral expression vector p RRL-CMV-ED, and then expressed in 293T cell line. The expressed protein was purified and identified by RT-PCR and Western blot. The enzyme digestion and sequence analysis showed that the lentiviral expression vector p RRL-CMV-ED was constructed correctly. The size of amplified genes was about 2 700 bp. The purified protein with Ni-affinity column was about 105 kD analyzed by SDS-PAGE. The Western blot and ELISA results showed that the expressed protein which could bind to HGF specifically was the extracelluar domain of human hepatocyte growth factor receptor. This research may lay a foundation for further study of anti-C-MET monoclonal antibody and neutralizing antibody.

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence.

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PPi ) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF-hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PPi ) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP-OE) resulted in a significant decrease in cytosolic PPi , and short and long-chain polyP levels. Additionally, the TcVSP-OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PPi and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.