Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use

Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity. It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. Isothermal titration calorimetry potency studies showed that METH binding by ch-mAb7F9 is efficient. Pharmacokinetic studies of METH given after ch-mAb7F9 doses in rats demonstrated the in vivo application of these in vitro METH-binding characteristics. While METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition, dramatically reducing the volume of distribution and clearance of METH. The elimination half-life of METH was increased by ch-mAb7F9, but it was still very fast compared with the elimination of ch-mAb7F9. Importantly, the rapid elimination of unbound METH combined with previous knowledge of mAb:target ligand binding dynamics suggested that ch-mAb7F9 binding capacity regenerates over time. This finding has substantial therapeutic implications regarding the METH doses against which ch-mAb7F9 will be effective, on the duration of ch-mAb7F9 effects, and on the safety of ch-mAb7F9 in METH users who use METH while taking ch-mAb7F9. These results helped to support initiation of a Phase 1a study of ch-mAb7F9.     

Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use

Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity. It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. Isothermal titration calorimetry potency studies showed that METH binding by ch-mAb7F9 is efficient. Pharmacokinetic studies of METH given after ch-mAb7F9 doses in rats demonstrated the in vivo application of these in vitro METH-binding characteristics. While METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition, dramatically reducing the volume of distribution and clearance of METH. The elimination half-life of METH was increased by ch-mAb7F9, but it was still very fast compared with the elimination of ch-mAb7F9. Importantly, the rapid elimination of unbound METH combined with previous knowledge of mAb:target ligand binding dynamics suggested that ch-mAb7F9 binding capacity regenerates over time. This finding has substantial therapeutic implications regarding the METH doses against which ch-mAb7F9 will be effective, on the duration of ch-mAb7F9 effects, and on the safety of ch-mAb7F9 in METH users who use METH while taking ch-mAb7F9. These results helped to support initiation of a Phase 1a study of ch-mAb7F9.     

Actions of the Klenow fragment of DNA polymerase I and some DNA glycosylases on chemically stable analogues of N7-methyl-2′-deoxyguanosine

N7-methyl-9-deaza-dG was synthesized and incorporated into oligonucleotides. Thermal melting studies showed that replacement of dG by N7-methyl-9-deaza-dG only slightly decreased DNA duplex stability. Replication of DNA templates containing N7-methyl-9-deaza-dG and the related 7-methyl-7-deaza-dG and 7-deaza-dG by the Klenow fragment of Escherichia coli DNA polymerase I was examined. The dNTP misinsertion frequencies on all three templates were comparably low, although the 7-methyl group significantly slowed down the turnover rates of the polymerase when dCTP was incorporated. The stabilities of N7-methyl-9-deaza-dG and 7-methyl-7-deaza-dG against the actions of formamidopyrimidine DNA glycosylase (Fpg) and human alkyladenine DNA glycosylase (hAAG) were also examined. N7-methyl-9-deaza-dG was stable in the presence of both enzymes. In contrast, 7-methyl-7-deaza-dG was cleaved by Fpg, and possibly by hAAG but at an extremely slow rate. This study suggests that N7-alkyl-9-deaza-dG is a better analogue than 7-alkyl-7-deaza-dG for cellular studies.     

Functional significance of the GAG trinucleotide-repeat polymorphism in the gene for the catalytic subunit of gamma-glutamylcysteine ligase

gamma-Glutamylcysteine ligase (GCL) is the rate-limiting enzyme in glutathione (GSH) synthesis. A GAG-repeat polymorphism in the 5' UTR of the gene coding for the catalytic subunit of GCL (GCLC) has been associated with altered GSH levels in vitro. Thus, we hypothesized that this polymorphism is associated with altered GCL activity and blood GSH levels in vivo. A total of 256 healthy United States black and white adults were genotyped for the GAG polymorphism and blood GSH levels were measured. In a subset of 107 individuals, blood GCL activity was determined. Five alleles with 4, 7, 8, 9, and 10 GAG repeats were observed. The most prevalent genotype was 7/9 (40%) followed by 7/7 (32%) and 9/9 (11%). GSH levels were 15% lower in 9/9 individuals than 7/9 individuals (P=0.05). GCL activity was 21% lower in 9/9 individuals than 7/7 individuals (P=0.04). A decreasing trend of GCL activity was observed in the order of 7/7>7/9>9/9 (P=0.04). These findings show that 9/9 individuals have lower blood GSH levels, which is likely due to a decrease in GCL activity. Such individuals might be more susceptible to oxidative stress-related diseases than individuals with other genotypes.     

The methyltransferase Set7/9 (Setd7) is dispensable for the p53-mediated DNA damage response in vivo

p53 is the central regulator of cell fate following genotoxic stress and oncogene activation. Its activity is controlled by several posttranslational modifications. Originally defined as a critical layer of p53 regulation in human cell lines, p53 lysine methylation by Set7/9 (also called Setd7) was proposed to fulfill a similar function in?vivo in the mouse, promoting p53 acetylation, stabilization, and activation upon DNA damage (Kurash et?al., 2008). We tested the physiological relevance of this circuit in an independent Set7/9 knockout mouse strain. Deletion of Set7/9 had no effect on p53-dependent cell-cycle arrest or apoptosis following sublethal or lethal DNA damage induced by radiation or genotoxic agents. Set7/9 was also dispensable for p53 acetylation following irradiation. c-myc oncogene-induced apoptosis was also independent of Set7/9, and analysis of p53 target genes showed that Set7/9 is not required for the p53-dependent gene expression program. Our data indicate that Set7/9 is dispensable for p53 function in the mouse.     

对粉纹夜蛾高毒力cry9Eα基因的克隆及表达

[目的]从本实验室分离的Bt4菌株中克隆cry9Eα基因,并研究其表达和杀虫活性.[方法]以PCR-RFLP方法鉴定Bt4菌株含有cry9基因,然后以菌株Bt4的质粒为模板,利用全长引物F9EA/R9EA进行PCR扩增全长基因.[结果]将目的片段插入到表达载体pET21b,得到大肠杆菌重组表达质粒pETcrygEa.转化E.coli BL21(DE3),诱导后表达130 kDa的蛋白,再将cry9Eα7基因连接到穿梭载体pSXY422b,电激转化HD73-(cry-),得到工程菌BioHD9Ea7,提取Cry9Ea7晶体蛋白,并进行生物活性测定.生物活性测定结果显示CrygEa7蛋白对粉纹夜蛾(Trichoplusia ni)初孵幼虫具有高毒力,LC_(50)为0.044 μg/mL,而对甜菜夜蛾(Spodoptera exigua)和棉铃虫(Helicoverpa armigera)初孵幼虫未显示活性.[结论]克隆和表达了一个对粉纹夜蛾高毒力的基因cry9Eα7,并成功构建了工程菌BioHD9Ea7.     

Proteolytic action of kallikrein-related peptidase 7 produces unique active matrix metalloproteinase-9 lacking the C-terminal hemopexin domains

The gelatinases, matrix metalloproteinase (MMP)-9 and -2, are produced as latent, inactive enzymes that can be proteolytically activated by a number of proteases. In many normal and pathological conditions, where the expression of MMPs is deregulated, changes in the expression of other proteases have also been reported. Human kallikrein-related peptidase 7 (KLK7), a chymotryptic-like serine protease, is overexpressed in many different types of neoplastic conditions, which have also been shown to express high levels of both MMP-9 and -2. Since the activation of MMPs by KLK7 has never been examined, we sought to determine whether KLK7 can activate these MMPs. To test this hypothesis KLK7 was incubated with the recombinant MMPs and the products of the reaction were analyzed for their activity. Incubation of proMMP-9 with KLK7 resulted in the production of a novel truncated, active MMP-9 lacking the C-terminal hemopexin domains. In contrast, KLK7 degraded, but did not activate, proMMP-2. The novel activation of proMMP-9 by KLK7 was further confirmed using conditioned medium prepared from an MMP-9-expressing cell line, MDA-MMP-9. Our results clearly establish that KLK7 activates proMMP-9 to produce a novel truncated, active MMP-9 product not generated by other proteases. These findings suggest that KLK7 may play an important role in the activation of MMP-9 in tumors that express high levels of both these proteases and the resulting truncated MMP may possess altered substrate specificities compared with full-length MMP-9 activated by other proteases.     

Continued antigenic variation of highly pathogenic avian influenza A (H7N9) virus in laying hens in China, 2020–2021

Highlights
1. 13 strains of H7N9 viruses from laying hens in 2020 and 2021 were identified.
2. H7N9 viruses in China comprised at least 11 genotypes.
3. H7N9 viruses are high pathogenic in chickens, not in ducks.
4. The most H7N9 viruses cross-reacted poorly with H7-Re3 antiserum.
5. The H7-Re3 vaccine was unable to prevent H7N9 infection.     

Assembly of the membrane attack complex of complement on small unilamellar phospholipid vesicles

Light-scattering intensity was shown to be a reliable, direct, and quantitative technique for monitoring the assembly of the membrane attack complex of complement (proteins C5b-6, C7, C8, and C9) on small unilamellar phosphatidylcholine vesicles. The assembly on vesicles occurred in a simple fashion; complexes of C5b-7 bound noncooperatively to the vesicles, and final assembly of C5b-9 did not induce vesicle aggregation or fragmentation. When C5b-6 and C7 were mixed in the presence of vesicles but at molar protein/vesicle ratios of less than 1, there was quantitative binding of C5b-7 to the vesicles with no concomitant aggregation of C5b-7. If C7 was added at a slower rate, quantitative binding was obtained at molar C5b-7/vesicle ratios of up to 5. The latter observations (a) were consistent with the proposal that C5b-7 aggregation and membrane binding were competitive events and (b) defined conditions under which light-scattering intensity measurements could monitor C5b-9 assembly on vesicles without contribution from the fluid-phase assembly. The C8/C5b-7 ratio in the phospholipid-C5b-8 complex was 0.97 +/- 0.12, and the maximum ratio of C9/C5b-8 in the final complex was 16.2 +/- 2.0. One C9 molecule associated rapidly with each phospholipid-C5b-8, followed by slower incorporation of the remaining C9 molecules. The initial velocity of the slow phase of C9 addition was easily saturated with C9 and gave an activation energy of 37 kcal/mol. This was identical with the value measured for the analogous process in the fluid-phase assembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Entry Properties and Entry Inhibitors of a Human H7N9 Influenza Virus

The recently identified human infections with a novel avian influenza H7N9 virus in China raise important questions regarding possible risk to humans. However, the entry properties and tropism of this H7N9 virus were poorly understood. Moreover, neuraminidase inhibitor resistant H7N9 isolates were recently observed in two patients and correlated with poor clinical outcomes. In this study, we aimed to elucidate the entry properties of H7N9 virus, design and evaluate inhibitors for H7N9 virus entry. We optimized and developed an H7N9-pseudotyped particle system (H7N9pp) that could be neutralized by anti-H7 antibodies and closely mimicked the entry process of the H7N9 virus. Avian, human and mouse-derived cultured cells showed high, moderate and low permissiveness to H7N9pp, respectively. Based on influenza virus membrane fusion mechanisms, a potent anti-H7N9 peptide (P155-185-chol) corresponding to the C-terminal ectodomain of the H7N9 hemagglutinin protein was successfully identified. P155-185-chol demonstrated H7N9pp-specific inhibition of infection with IC₅₀ of 0.19 µM. Importantly, P155-185-chol showed significant suppression of A/Anhui/1/2013 H7N9 live virus propagation in MDCK cells and additive effects with NA inhibitors Oseltamivir and Zanamivir. These findings expand our knowledge of the entry properties of the novel H7N9 viruses, and they highlight the potential for developing a new class of inhibitors targeting viral entry for use in the next pandemic.     

对粉纹夜蛾高毒力cry9Ea基因的克隆及表达

【目的】从本实验室分离的Bt4菌株中克隆cry9Ea基因,并研究其表达和杀虫活性。【方法】以PCR-RFLP方法鉴定Bt4菌株含有cry9基因,然后以菌株Bt4的质粒为模板,利用全长引物F9EA/R9EA进行PCR扩增全长基因。【结果】将目的片段插入到表达载体pET21b,得到大肠杆菌重组表达质粒pETcry9Ea。转化E.coli BL21(DE3),诱导后表达130kDa的蛋白,再将cry9Ea7基因连接到穿梭载体pSXY422b,电激转化HD73-(cry-),得到工程菌BioHD9Ea7,提取Cry9Ea7晶体蛋白,并进行生物活性测定。生物活性测定结果显示Cry9Ea7蛋白对粉纹夜蛾(Trichoplusia ni)初孵幼虫具有高毒力,LC50为0.044μg/mL,而对甜菜夜蛾(Spodoptera exigua)和棉铃虫(Helicoverpa armigera)初孵幼虫未显示活性。【结论】克隆和表达了一个对粉纹夜蛾高毒力的基因cry9Ea7,并成功构建了工程菌BioHD9Ea7。     

Apparent re-emergence of serotype G9 in 1995 among rotaviruses recovered from Japanese children hospitalized with acute gastroenteritis

Serotype G9 rotaviruses have been detected in about 0.5% of the circulating strains worldwide. However, G9 strains emerged globally in the middle of the 90s and thereafter. A rotavirus, contained in stool specimen 95H115, possessing a G9 VP7 emerged in Japan in the 1994-1995 season for the first time after a 9-year interval since prototype G9 strains AU32 and F45 were discovered in the 1985-1986 season. In comparison with other G9 VP7 genes thus far published, the sequencing of the VP7 genes of AU32 and 95H115 revealed that the 95H115 VP7 gene did not directly evolve from the AU32 VP7 gene but was much more closely related to the contemporary G9 VP7 genes found in the United States of America. Thus, recently emerging G9 VP7 genes were not direct descendants of the VP7 genes of the prototype strains in the 80s, rather they evolved independently into 4 phylogenetic clusters from a common ancestor.     

Physical conditions of propagation media and their influence on the rooting of cuttings

Summary The air content in three types of propagation media, Jiffy-7 and Jiffy-9 which are Sphagnum peat and Grodan which is rockwool, were investigated when they were held at moisture tensions of 0,6 and 12 cm measured from the base of the media. At 0 cm tension the air content (vol. %) was highest in Jiffy-9 and lowest in Jiffy-7. At 12 cm tension the air content was higher in Grodan than in Jiffy-9 and Jiffy-7. Oxygen diffusion coefficients (ODC) and oxygen diffusion rates (ODR) were measured at the different air contents. At air contents below 20 vol. % ODC was about the same for Jiffy-9 and Grodan but at air contents above 20 vol.% it was larger for Jiffy-9 than for Grodan. The oxygen diffusion rate was measured at 0, 4 and 8 cm moisture tension. At all tensions it was approximately 20% higher in Jiffy-9 than in Grodan and Jiffy-7. The ODR in Jiffy-7 and Grodan were affected equally at the same tension, although Grodan contained more air. Report no 253     

H7N9禽流感病毒与其他流感病毒对雪貂致病性与传播力的比较

目的针对2013年3月中国爆发的人感染H7N9禽流感病毒,在雪貂体内进行致病性及传播力的研究,并与甲型H1N1流感病毒、H5N1禽流感病毒进行比较。方法对新发H7N9毒株、甲型H1N1流感病毒、H5N1禽流感病毒感染雪貂后的临床症状、体征,呼吸道排毒情况,组织病理学变化等进行评价和比较,并对H7N9毒株在雪貂群体中的传播力进行研究。结果雪貂模型的临床症状、死亡率、病毒传播以及组织病理学分析显示:H7N9病毒的致病性低于H5N1,与2009年起源于北美的甲型H1N1流感病毒相当。新发H7N9禽流感病毒可以在雪貂的呼吸道、心脏、肝脏以及嗅球进行复制。值得注意的是H7N9禽流感可以通过飞沫在雪貂间进行低水平的传播,并且在传播过程中,病毒基因组内有多个位点的氨基酸发生了替换。结论 H7N9禽流感病毒对雪貂的致病性较H5N1禽流感病毒低,与甲型H1N1流感病毒对雪貂的致病性相当,H7N9禽流感病毒可在雪貂间进行传播。     

Genetic and biological properties of H7N9 avian influenza viruses detected after application of the H7N9 poultry vaccine in China

The H7N9 avian influenza virus (AIV) that emerged in China have caused five waves of human infection. Further human cases have been successfully prevented since September 2017 through the use of an H7N9 vaccine in poultry. However, the H7N9 AIV has not been eradicated from poultry in China, and its evolution remains largely unexplored. In this study, we isolated 19 H7N9 AIVs during surveillance and diagnosis from February 2018 to December 2019, and genetic analysis showed that these viruses have formed two different genotypes. Animal studies indicated that the H7N9 viruses are highly lethal to chicken, cause mild infection in ducks, but have distinct pathotypes in mice. The viruses bound to avian-type receptors with high affinity, but gradually lost their ability to bind to human-type receptors. Importantly, we found that H7N9 AIVs isolated in 2019 were antigenically different from the H7N9 vaccine strain that was used for H7N9 influenza control in poultry, and that replication of these viruses cannot, therefore, be completely prevented in vaccinated chickens. We further revealed that two amino acid mutations at positions 135 and 160 in the HA protein added two glycosylation sites and facilitated the escape of the H7N9 viruses from the vaccine-induced immunity. Our study provides important insights into H7N9 virus evolution and control.     

Antitumorigenic effect of Wnt 7a and Fzd 9 in non-small cell lung cancer cells is mediated through ERK-5-dependent activation of peroxisome proliferator-activated receptor gamma

The Wnt pathway is critical for normal development, and mutation of specific components is seen in carcinomas of diverse origins. The role of this pathway in lung tumorigenesis has not been clearly established. Recent studies from our laboratory indicate that combined expression of the combination of Wnt 7a and Frizzled 9 (Fzd 9) in Non-small Cell Lung Cancer (NSCLC) cell lines inhibits transformed growth. We have also shown that increased expression of peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits transformed growth of NSCLC and promotes epithelial differentiation of these cells. The goal of this study was to determine whether the effects of Wnt 7a/Fzd 9 were mediated through PPARgamma. We found that Wnt 7a and Fzd 9 expression led to increased PPARgamma activity. This effect was not mediated by altered expression of the protein. Wnt 7a and Fzd 9 expression resulted in activation of ERK5, which was required for PPARgamma activation in NSCLC. SR 202, a known PPARgamma inhibitor, blocked the increase in PPARgamma activity and restored anchorage-independent growth in NSCLC expressing Wnt 7a and Fzd 9. SR 202 also reversed the increase in E-cadherin expression mediated by Wnt 7a and Fzd 9. These data suggest that ERK5-dependent activation of PPARgamma represents a major effector pathway mediating the anti-tumorigenic effects of Wnt 7a and Fzd 9 in NSCLC.