Evaluation of anticancer activity of PD-1/CTLA-4 bispecific antibody and its lgGl isotype based on target humanized mice / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：构建基于靶点人源化小鼠的程序性死亡受体-1（PD-1）/细胞毒性T淋巴细胞抗原-4（CTLA-4）双特异性抗体（BsAb）并对BsAb及其IgG1亚型进行抗癌活性评价和探讨其潜在的作用机制。方法：构建和扩增并纯化不同结构及抗体亚型的PD-1/CTLA-4抗体BsAb1、BsAb2和BsAb3，对纯化BsAb进行靶点亲和力检测，采用荧光素酶报告基因实验和FCM检测抗体的生物学活性。基于B-hPD-1-hPD-L1-hCTLA-4人源化小鼠的MC38-hPD-L1结肠癌细胞移植瘤模型对BsAb进行体内药效评估，并通过移植瘤组织中肿瘤浸润淋巴细胞（TIL）分析PD-1/CTLA-4抗体的作用机制。结果：成功制备的BsAb1、BsAb2及BsAb3对靶点PD-1和CTLA-4均有较强的特异性亲和力、对靶点通路均有不同程度的阻滞活性，均明显抑制移植瘤的生长（P<0.05或P<0.01）。IgG1亚型BsAb体内药效更优（P<0.01），TIL分析发现BsAb2-IgG1明显增加了CTL百分率（P<0.05），显著降低了肿瘤浸润Treg细胞百分率（P<0.01），使肿瘤免疫微环境更有利于杀伤肿瘤细胞；增强ADCC活性的Fc突变体亚型BsAb2-SI则不能进一步提高抗肿瘤活性。结论：具有Fc效应功能的IgG1亚型的PD-1/CTLA-4抗体体内抗癌药效更优，因其可以更好地清除TIL中的Treg细胞。

Enhancement of the catalytic efficiency and thermostability of Stenotrophomonas sp. keratinase KerSMD by domain exchange with KerSMF.

In this study, we enhanced the catalytic efficiency and thermostability of keratinase KerSMD by replacing its N/C-terminal domains with those from a homologous protease, KerSMF, to degrade feather waste. Replacement of the N-terminal domain generated a mutant protein with more than twofold increased catalytic activity towards casein. Replacement of the C-terminal domain obviously improved keratinolytic activity and increased the k(cat)/K(m) value on a synthetic peptide, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, by 54.5%. Replacement of both the N- and C-terminal domains generated a more stable mutant protein, with a Tm value of 64.60 ± 0.65°C and a half-life of 244.6 ± 2 min at 60°C, while deletion of the C-terminal domain from KerSMD or KerSMF resulted in mutant proteins exhibiting high activity under mesophilic conditions. These findings indicate that the pre-peptidase C-terminal domain and N-propeptide are not only important for substrate specificity, correct folding and thermostability but also support the ability of the enzyme to convert feather waste into feed additives.

High-level extracellular production of glucose oxidase by recombinant Pichia pastoris using a combined strategy.

In this work, a combined strategy was developed to improve the production of glucose oxidase (GOD) (EC 1.1.3.4) in Pichia pastoris. One of the main challenges facing protein production by the high-density fermentation of P. pastoris is the high demand for oxygen. Another challenge is how to balance a reduction in oxygen consumption and its effects on protein production. Herein, a combined strategy involving mannitol co-feeding, two-stage methanol induction, and the co-expression of the transcriptional activator general control non-derepressible 4 (GCN4) from P. pastoris was used. A two-stage, co-feeding strategy, based on a mannitol/methanol mixture in a 3-L fermentor was used to enhance cell viability and protein production. This resulted in an increased GOD yield of 1208.2 U/mL compared with a control strain (427.6 U/mL). An increase in the copy number of the GCN4 gene enhanced the GOD yield (1634.7 U/mL) by 2.8-fold and the protein concentration (19.55 g/L) by 1.58-fold compared with the control (7.59 g/L). This strategy illustrates a way to overcome the high oxygen requirement during high-density fermentation of P. pastoris and balances the reduction of oxygen consumption and protein production. Moreover, the series of strategies presented in this work provide valuable and novel information for the industrial production of GOD.

Comparative analysis of bacterial expression systems for keratinase production.

To explore a better expression system for the production of keratinase, the keratinase gene from Bacillus licheniformis BBE11-1 was expressed in Escherichia coli, Bacillus subtilis, and Pichia pastoris. The corresponding recombinant keratinases were named ker E, ker B, and ker P, respectively. All recombinant keratinases had an optimal pH at 10 although the pH stability of ker E and ker P was higher than that of ker E. The optimal temperature and thermostability of ker P were enhanced compared with those of ker E and ker B. The recombinant keratinases were inhibited by Mn2+ but experienced little influence from other metal ions. Furthermore, all recombinant keratinases could retain almost 80 % activity after treatment with 1 M hydrogen peroxide for 5 h. Under optimized conditions in a 3-L fermenter, the maximum keratinase activities obtained from recombinant B. subtilis and P. pastoris were 3,010 and 1,050 U/mL, respectively. This maximum keratinase activity from B. subtilis is the highest activity ever reported for any bacterial strain. These results indicate that B. subtilis is the ideal host for keratinase production, with potential applications in textile processing and feed supplements.

High-level extracellular production of D-Psicose-3-epimerase with recombinant Escherichia coli by a two-stage glycerol feeding approach.

The aim of this study is to achieve high-level extracellular production of D-Psicose-3-epimerase (DPE) with recombinant Escherichia coli. High-level production of DPE is one of the key factors in D-Psicose production. In the present study, the gene AAL45544.1 from Agrobacterium tumefaciens str. C58 was modified by artificial synthesis for overexpression in E. coli. The total DPE activity reached 3.96 U mL(-1) after optimization of the media composition, induction temperature, and concentration of inducer. Furthermore, it was found that addition of glycine had a positive effect on the extracellular production of DPE, which reached 3.5 U mL(-1). Finally, a two-stage glycerol feeding strategy based on both the specific growth rate before induction and the amount of glycerol residues after induction was applied in a 3-L fermenter. After a series of optimal strategies in the 3-L fermenter, the total and extracellular DPE activity were 5.08- and 3.11-fold higher than that noted in the shake flask. The extracellular and intracellular DPE activity reached 10.9 and 13.2 U mL(-1), achieving 25.5 and 31.1 % conversion of D-fructose to D-psicose, respectively. The systemic strategies presented in this study provide valuable novel information for the industrial application of DPE.

Biodegradation of wool waste and keratinase production in scale-up fermenter with different strategies by Stenotrophomonas maltophilia BBE11-1.

A keratin-degrading strain Stenotrophomonas maltophilia BBE11-1 was grown in a 3-L batch fermenter containing wool waste as the main medium and cell growth rate was determined as the key factor to affect keratinase yield. Three strategies of temperature-shift procedure, two-stage DO control and fed-batch process were used to change growth rate. And a 62.2% improvement of keratinase yield was achieved. With the glucose fed-batch procedure in 30-L fermenter, keratinase production was significantly improved up to 117.7% (1728 U/ml) as compared with initial data (793.8 U/ml) in a 3-L fermenter and with much shortened fermentation time within 18 h. Significant structure changes and high levels of free amino acids from wool decomposition indicated the possible applications for wool waste management and fertilizer industry. The remarkable digestion of wool cuticle also suggested its potential utilization in textile industry.

Enhanced thermostability of keratinase by computational design and empirical mutation.

Keratinases are proteolytic enzymes capable of degrading insoluble keratins. The importance of these enzymes is being increasingly recognized in fields as diverse as animal feed production, textile processing, detergent formulation, leather manufacture, and medicine. To enhance the thermostability of Bacillus licheniformis BBE11-1 keratinase, the PoPMuSiC algorithm was applied to predict the folding free energy change (ΔΔG) of amino acid substitutions. Use of the algorithm in combination with molecular modification of homologous subtilisin allowed the introduction of four amino acid substitutions (N122Y, N217S, A193P, N160C) into the enzyme by site-directed mutagenesis, and the mutant genes were expressed in Bacillus subtilis WB600. The quadruple mutant displayed synergistic or additive effects with an 8.6-fold increase in the t 1/2 value at 60 °C. The N122Y substitution also led to an approximately 5.6-fold increase in catalytic efficiency compared to that of the wild-type keratinase. These results provide further insight into the thermostability of keratinase and suggest further potential industrial applications.

Expression and characterization of extreme alkaline, oxidation-resistant keratinase from Bacillus licheniformis in recombinant Bacillus subtilis WB600 expression system and its application in wool fiber processing.

A keratin-degrading bacterium of Bacillus licheniformis BBE11-1 was isolated and its ker gene encoding keratinase with native signal peptide was cloned and expressed in Bacillus subtilis WB600 under the strong P HpaII promoter of the pMA0911 vector. In the 3-L fermenter, the recombinant keratinase was secreted with 323 units/mL when non-induced after 24 h at 37 °C. And then, keratinase was concentrated and purified by hydrophobic interaction chromatography using HiTrap Phenyl-Sepharose Fast Flow. The recombinant keratinase had an optimal temperature and the pH at 40 °C and 10.5, respectively, and was stable at 10-50 °C and pH 7-11.5. We found this enzyme can retained 80 % activity after treated 5 h with 1 M H2O2, it was activated by Mg(2+), Co(2+) and could degraded broad substrates such as degraded feather, bovine serum albumin, casein, gelatin, the keratinase was considered to be a serine protease. Coordinate with Savinase, the keratinase could efficient prevent shrinkage and eliminate fibres of wool, which showed its potential in textile industries and detergent industries.

[Operative treatment and curative effects of the deltoid ligament injuries of the ankle joint].

OBJECTIVE: To explore the operative methods and curative effects of the deltoid ligament injuries. METHODS: From 2002 to 2008, all 61 patients with ankle fractures complicated with deltoid ligament injuries were treated with open reduction and firm internal fixation. Among the patients, 39 patients were male and 22 patients were female, ranging in age from 14 to 71 years, with an average of 41 years. During the operation, the deltoid ligament was reconstructed to restore the medial and lateral stability of ankle joint. RESULTS: All the patients were followed up ranged from 5 to 30 months, with an average of 17 months. Fifty-nine patients had incision healed at the first stage; 2 patients had superficial infections at lateral malleolus, and healed at the 3rd week after changing dressings. The incisions at the internal medial malleolus were all healed at the first stage. According to Qi evaluation criteria, 35 patients got an excellent result, 13 good and 13 fair. CONCLUSION: The deltoid ligament should be treated properly in the treatment of ankle joint fractures when open reduction and firm internal fixation were emphasized.