Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs

We have used phage display to isolate a range of human domain antibodies (dAbs) that bind to mouse, rat and/or human serum albumin (SA) and can be expressed at very high levels in bacterial, yeast or mammalian cell culture. In contrast to non-SA-binding dAbs, which have terminal half-lives of less than 45 min, the half-lives of these 12 kDa 'AlbudAbs' can match the half-life of SA itself. To demonstrate the use of AlbudAbs for extending the half-lives of therapeutic drugs, we created a fusion of the interleukin-1 receptor antagonist (IL-1ra) with an AlbudAb. Soluble IL-1ra is potent inhibitor of IL-1 signalling that is approved for the treatment of rheumatoid arthritis but has a relatively short in vivo half-life. Here we show that although the AlbudAb/IL-1ra fusion has a similar in vitro potency, its in vivo efficacy can be dramatically improved due to its extended serum half-life. AlbudAbs could potentially be used to generate a range of long half-life versions of many different drugs in order to improve their dosing regimen and/or clinical effect.     

表达IFNα2b-HSA融合蛋白的CHO细胞培养基优化及发酵放大

毕赤酵母表达的干扰素与白蛋白融合蛋白虽然避开了干扰素单体半衰期短的缺陷,但毕赤酵母对药物的糖基化修饰与人体的糖基化修饰差异性导致了药物的毒副作用。目前药物在CHO系统的表达得到了广泛应用。本研究室首次构建了能表达干扰素α2b和人血清白蛋白融合蛋白（IFNα2b-HSA）的CHO细胞株。在此基础上,本研究通过对12种国产商业化基础无血清培养基和5种流加培养基进行优化筛选,获得最适培养方案：基础培养基选择最适于生长的5号培养基（M2：M4=1：1）,流加培养基选择最有适于表达的F4培养基。在此基础上,进行5L生物反应器的发酵放大,pH为6.9～7.4,DO为40%～60%,细胞密度达到7.0×10⁶cells/mL时,温度由37℃降温至34℃,细胞活率降至80%时停止发酵,最终融合蛋白表达量达到137mg/L。初步实现了IFNα2b-HSA融合蛋白在CHO细胞中的高密度发酵。     

Dual‐Crosslinked Human Serum Albumin‐Polymer Hydrogels for Affinity‐Based Drug Delivery

A dual‐crosslinked in situ gelling drug delivery scaffold based on dextran (DEX), thiolated serum albumin, and poly(ethylene glycol) (PEG) is presented. Dextran–vinyl sulfone conjugates with varied molecular weight and degrees of substitution are synthesized by controlling the reaction time and temperature with divinyl sulfone. Dextran–human serum albumin (sHSA) hydrogels are prepared using a thiol‐vinyl sulfone Michael addition reaction with thiolated albumin as the crosslinker. Poly(ethylene glycol) dithiol is added as a third component to the crosslinked dextran–human serum albumin hydrogel to facilitate additional crosslinking, and reduce gelation time, while modulating the physicochemical properties of the Dex–sHSA–PEG network. The onset of gelation of the modular three‐component dual‐crosslinked hydrogel network ranges from 45 min to 1.5 h depending on gel constituent concentrations and the gelation temperature (25 or 37 °C). All gels remain stable for over a 25 d period under physiological conditions. In vitro drug release assays show that dual‐crosslinked Dex–sHSA–PEG hydrogels can deliver doxorubicin in a sustained manner over 7 d. Finally, a Tetrazolium‐based assay shows the biocompatible nature of the Dex–sHSA–PEG hydrogels and capacity to deliver doxorubicin successfully to MCF‐7 breast cancer cells.     

人乳腺癌相关成纤维细胞的原代培养及其生物学特性

目的原代培养人乳腺癌相关成纤维细胞(Cancer-associated fibroblast,CAF),并检测其生物学特性。方法采用胶原酶消化培养法对26份乳腺癌患者标本进行原代细胞分离培养,倒置显微镜下观察人乳腺癌CAF的形态学特性,免疫荧光染色法鉴定所分离培养的人乳腺癌CAF纤维连结蛋白(Fibronectin,FN)和成纤维细胞活化蛋白(Fibroblast activated protein,FAP)的表达,并通过MTT法和细胞计数法绘制细胞生长曲线。结果胶原酶消化法的最适酶浓度为0.12%,最适消化时间为10 h。原代培养23份成功,3份失败。培养成功的细胞贴壁生长,形态完整,生长状态良好,背景清晰,有明显的对数生长期,并可传代培养。培养的细胞FN和FAP表达阳性,表明原代培养的CAF为乳腺癌CAF。结论胶原酶消化培养法适合人乳腺癌CAF的原代培养,通过该方法可建立理想的人乳腺癌CAF体外实验模型。     

A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G

The therapeutic application of small recombinant antibody molecules is often limited by a short serum half-life. In order to improve the pharmacokinetic properties, we have investigated a strategy utilizing fusion with an albumin-binding domain (ABD) from streptococcal protein G. This strategy was applied to a bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. This novel tri-functional fusion protein (scDb-ABD) was expressed in mammalian cells and recognized both antigens as well as human and mouse serum albumin. scDb-ABD was capable to retarget T cells to CEA-expressing target cells in vitro and to activate the effector cells as measured by stimulation of IL-2 release. Although activity was reduced 3-fold compared with scDb and further reduced 4-fold in the presences of human serum albumin, this assay demonstrated that scDb-ABD is active when exposed to all three antigens. Compared with scDb, the circulation time of scDb-ABD in mice was prolonged 5- to 6-fold similar to a previously described scDb-HSA fusion protein. This strategy, which adds only a small protein domain (46 amino acids) and which utilizes high-affinity, non-covalent albumin interaction, should be broadly applicable to improve serum half-lives of small recombinant antibody molecules.     

Albumin Suppresses Human Hepatocellular Carcinoma Proliferation and the Cell Cycle

Many investigations have revealed that a low recurrence rate of hepatocellular carcinoma (HCC) is associated with high serum albumin levels in patients; therefore, high levels of serum albumin are a major indicator of a favorable prognosis. However, the mechanism inhibiting the proliferation of HCC has not yet been elucidated, so we investigated the effect of serum albumin on HCC cell proliferation. Hep3B was cultured in MEM with no serum or containing 5 g/dL human albumin. As control samples, Prionex was added to generate the same osmotic pressure as albumin. After 24-h incubation, the expressions of α-fetoprotein (AFP), p53, p21, and p57 were evaluated with real-time PCR using total RNA extracted from the liver. Protein expressions and the phosphorylation of Rb (retinoblastoma) were determined by Western blot analysis using total protein extracted from the liver. For flow cytometric analysis of the cell cycle, FACS analysis was performed. The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACScalibur (BD) with appropriate software (ModFit LT; BD). The cell proliferation assay was performed by counting cells with using a Scepter handy automated cell counter (Millipore). The mRNA levels of AFP relative to Alb(−): Alb(−), Alb(+), and Prionex, were 1, 0.7 ± 0.2 (p < 0.001 for Alb(−)), and 1 ± 0.3, respectively. The mRNA levels of p21 were 1, 1.58 ± 0.4 (p = 0.007 for Alb(−) and p = 0.004 for Prionex), and 0.8 ± 0.2, respectively. The mRNA levels of p57 were 1, 4.4 ± 1.4 (p = 0.002 for Alb(−) and Prionex), and 1.0 ± 0.1, respectively. The protein expression levels of Rb were similar in all culture media. The phosphorylation of P807/811 and P780 of Rb protein was reduced in Alb(+). More cells in the G0/G1 phase and fewer cells in S and G2/M phases were obtained in Alb(+) than in Alb(−) (G0/G1: 60.9%, 67.7%, 61.5%; G2/M: 16.5%, 13.1%, 15.6%; S: 22.6%, 19.2%, 23.0%, Alb(−), Alb(+), Prionex, respectively). The same results were obtained in HepG2. Cell proliferation was inhibited in 5 g/dL albumin medium in both HepG2 cells and Hep3B cells in 24 h culture by counting cell numbers. The presence of albumin in serum reduces the phosphorylation of Rb proteins and enhances the expression of p21 and p57, following an increase in the G0/G1 cell population, and suppresses cell proliferation. These results suggest that albumin itself suppresses the proliferation of hepatocellular carcinoma.     

Controlled release of recombinant human nerve growth factor (rhNGF) from poly[(lactic acid)‐co‐(glycolic acid)] microspheres for the treatment of neurodegenerative disorders

Recombinant human nerve growth factor (rhNGF)/bovine serum albumin‐loaded microspheres were prepared by a water/oil/water emulsion and solvent evaporation technique with some modifications. The microspheres were characterized with respect to encapsulation efficiency, morphological properties and drug release. Using higher protein/polymer ratios in the primary emulsion resulted in higher protein content in the microspheres. The protein encapsulation efficiency increased from 89.1% to 97.5% on adding poly(ethylene glycol) to the inner aqueous phase. The in vitro rhNGF release lasted for more than 5 weeks. The biological activity of released rhNGF was confirmed by PC12 cell culture. The microspheres maintained a sustained release of rhNGF for at least 4 weeks in the basal forebrain as detected by fluorescence‐labeled and indirect immunofluorescent techniques. These results demonstrated the rhNGF‐containing microspheres are an effective means for delivering this molecule into the brain and their use may be a promising strategy in the treatment of neurodegenerative disorders such as Alzheimer's disease. Copyright © 2007 Society of Chemical Industry     

C50株抗CEA杂交瘤细胞抗体生成动力学及体外培养条件的优化

目的　研究C5 0株抗CEA杂交瘤细胞的抗体生成动力学 ,确定优化体外培养条件。方法　体外静态培养C5 0细胞 ,采用ELISA和细胞计数的方法检测培养上清抗体浓度和细胞数量 ,分析细胞生长与抗体分泌的关联性。采用体内复壮和添加新型免疫增强剂CpGODN的方法 ,尝试恢复长期体外培养的C5 0细胞的抗体生成能力。结果　C5 0细胞的抗体生成动力学为非生长关联型 ,经过体内复壮 ,C5 0细胞体外培养上清中抗体浓度显著提高 ,但活细胞密度或细胞活力均未发生明显变化。在培养基中添加一定浓度的CpGODN ,可以恢复长期体外培养的C5 0细胞的抗体生成能力。结论　根据细胞的抗体生成动力学特征 ,通过调节细胞的生长状态 ,可提高上清抗体浓度。根据抗体基因的组织特异性表达的机制 ,通过优化杂交瘤细胞的体外培养条件 ,能保持细胞正常的抗体生成能力 ,维持细胞高且稳定的上清抗体浓度     

Neuroprotection Mediated by Human Blood Plasma in Mouse Hippocampal Slice Cultures and in Oxidatively Stressed Human Neurons

Neuroprotection from oxidative stress is critical during neuronal development and maintenance but also plays a major role in the pathogenesis and potential treatment of various neurological disorders and neurodegenerative diseases. Emerging evidence in the murine system suggests neuroprotective effects of blood plasma on the aged or diseased brain. However, little is known about plasma-mediated effects on human neurons. In the present study, we demonstrate the neuroprotective effect mediated by human plasma and the most abundant plasma–protein human serum albumin against oxidative stress in glutamatergic neurons differentiated from human neural crest-derived inferior turbinate stem cells. We observed a strong neuroprotective effect of human plasma and human serum albumin against oxidative stress-induced neuronal death on the single cell level, similar to the one mediated by tumor necrosis factor alpha. Moreover, we detected neuroprotection of plasma and human serum albumin against kainic acid-induced excitatory stress in ex vivo cultured mouse hippocampal tissue slices. The present study provides deeper insights into plasma-mediated neuroprotection ultimately resulting in the development of novel therapies for a variety of neurological and, in particular, neurodegenerative diseases.     

Canola Oil Inhibits Breast Cancer Cell Growth in Cultures and In Vivo and Acts Synergistically with Chemotherapeutic Drugs

Certain fatty acids in canola oil (CAN) have been associated with a reduced risk of breast cancer. This study assessed the effects of CAN on proliferation and death of human breast cancer cells in vitro and in vivo in chemically induced mammary carcinogenesis. We hypothesize that CAN reduces breast cancer cell growth by inducing cell death. In a series of in vitro experiments, human breast cancer T47D and MCF-7 cells were cultured and treated with CAN and two chemotherapeutic drugs, tamoxifen and cerulenin. Cell proliferation and caspase-3 and p53 activities were measured. Reduced cancer cell growth and increased expression of caspase-3 and p53 were seen in T47D and MCF-7 cells treated with CAN. Moreover, CAN showed synergistic cancer cell growth inhibition effects with tamoxifen and cerulenin. In a subsequent live animal experiment, 42 female Sprague–Dawley rats were randomly assigned to corn oil (CORN) or CAN diets, and mammary tumors were chemically induced by N-nitroso-N-methylurea. CAN-dieted rats had reduced tumor volumes and showed an increased survival rate as compared to CORN-dieted rats. We demonstrated that CAN has suppressive effects on cancer growth, and reduces tumor volumes. The results suggest that CAN may have inhibitory effects on breast cancer cell growth, and warrants further investigation of the synergistic effects of CAN with anti-cancer drugs.     

Pretreatment serum albumin as a predictor of cancer survival: A systematic review of the epidemiological literature

Background  

There are several methods of assessing nutritional status in cancer of which serum albumin is one of the most commonly used. In recent years, the role of malnutrition as a predictor of survival in cancer has received considerable attention. As a result, it is reasonable to investigate whether serum albumin has utility as a prognostic indicator of cancer survival in cancer. This review summarizes all available epidemiological literature on the association between pretreatment serum albumin levels and survival in different types of cancer.     

Bio-Orthogonal Conjugation of a Cationic Metalloporphyrin to BSA and HSA via “Click” Chemistry

In this study, we present a convenient method for the labelling of tyrosine residues on bovine serum albumin (BSA) and human serum albumin (HSA) and report for the first time their subsequent bio-orthogonal conjugation with porphyrins via “click” chemistry. We demonstrate that these serum proteins can be labelled with an alkyne-diazonium heterobifunctional linker and can then undergo chemo-selective bio-orthogonal conjugation with a water-soluble azido metalloporphyrin via “click” chemistry to yield protein-conjugates that retain their photodynamic properties. In our hands, this method was found to be highly reproducible, scalable, and tuneable which allows for the production of bioconjugates where the porphyrin-protein conjugate not only retains an ability to generate singlet oxygen but possess an enhanced relative singlet oxygen quantum yields relative to the porphyrin alone. Furthermore, we have investigated the photochemical properties of these conjugates through photospectrometric techniques and have determined that the porphyrin macrocycles remain appreciably photostable under light irradiation. Our phototoxic protein-photosensitizer-conjugates show excellent photodynamic activity against a human colorectal adenocarcinoma cancer cell line (HT-29) with cell viabilities of 7.7±0.5 % (IC50 8.76±2.14 μM) and 1.7±1.9 % (IC50 8.48±5.11 μM) for BSA and HAS, respectively, when irradiated with 20 J cm⁻² of white-light. Importantly, neither of the conjugates was found to possess any significant “dark” toxicity even at concentrations of 100 μM. Furthermore, the natural fluorescent properties of the bioconjugates allowed for the determination of cellular uptake in vitro via fluorescence microscopy thus highlighting the potential theranostic applications of these unique protein-drug-conjugates.     

Effect of mesenchymal cells on human hair growth and death

Animals have typically been used in efficacy tests, but there are a number of dissimilarities between humans and animals. To overcome the problems associated with animal testing, a model which is reproduced in vitro with longterm culture with cell growth with in vivo activity must be developed. We made a gel-type dermal equivalent (DE) that contained dermal papilla cells (DPCs) or dermal sheath cells (DSCs) isolated from human hair bulbs in order to mimic human scalp tissue. Hair follicles were organ-cultured on DE containing DPCs or DSCs. The DE used for organ culture was a reconstructed 3-dimensional contraction of collagen gel, and the cell density of the DE did not affect the increase in hair length. We tested the effects of cell types in DE on increases in hair length, and the results showed a large increase in hair length and long-term viability in the air-liquid interface culture on DE containing DSCs. We compared the submerged culture with the hair air-liquid interface culture on DE using immunohistochemical staining, and found that the hair follicles that were air-liquid interface cultured on DE maintained the growth phase (anagen) for a longer period of time than the hair follicles that were submerged. Since the hair follicles were cultured under an air-liquid interface condition, the increase in hair length was a reflection of the epithelial cell growth that resulted from the improved oxygen supply and paracrine factors secreted from hair origin cells.     

A Novel Method Facilitating the Simple and Low-Cost Preparation of Human Osteochondral Slice Explants for Large-Scale Native Tissue Analysis

For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-β3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-β3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.     

Interaction of Conazole Pesticides Epoxiconazole and Prothioconazole with Human and Bovine Serum Albumin Studied Using Spectroscopic Methods and Molecular Modeling

The interactions of epoxiconazole and prothioconazole with human serum albumin and bovine serum albumin were investigated using spectroscopic methods complemented with molecular modeling. Spectroscopic techniques showed the formation of pesticide/serum albumin complexes with the static type as the dominant mechanism. The association constants ranged from 3.80 × 10⁴–6.45 × 10⁵ L/mol depending on the pesticide molecule (epoxiconazole, prothioconazole) and albumin type (human or bovine serum albumin). The calculated thermodynamic parameters revealed that the binding of pesticides into serum albumin macromolecules mainly depended on hydrogen bonds and van der Waals interactions. Synchronous fluorescence spectroscopy and the competitive experiments method showed that pesticides bind to subdomain IIA, near tryptophan; in the case of bovine serum albumin also on the macromolecule surface. Concerning prothioconazole, we observed the existence of an additional binding site at the junction of domains I and III of serum albumin macromolecules. These observations were corroborated well by molecular modeling predictions. The conformation changes in secondary structure were characterized by circular dichroism, three-dimensional fluorescence, and UV/VIS absorption methods.     

Conductive Supramolecular Polymer Nanocomposites with Tunable Properties to Manipulate Cell Growth and Functions

Synthetic bioactive nanocomposites show great promise in biomedicine for use in tissue growth, wound healing and the potential for bioengineered skin substitutes. Hydrogen-bonded supramolecular polymers (3A-PCL) can be combined with graphite crystals to form graphite/3A-PCL composites with tunable physical properties. When used as a bioactive substrate for cell culture, graphite/3A-PCL composites have an extremely low cytotoxic activity on normal cells and a high structural stability in a medium with red blood cells. A series of in vitro studies demonstrated that the resulting composite substrates can efficiently interact with cell surfaces to promote the adhesion, migration, and proliferation of adherent cells, as well as rapid wound healing ability at the damaged cellular surface. Importantly, placing these substrates under an indirect current electric field at only 0.1 V leads to a marked acceleration in cell growth, a significant increase in total cell numbers, and a remarkable alteration in cell morphology. These results reveal a newly created system with great potential to provide an efficient route for the development of multifunctional bioactive substrates with unique electro-responsiveness to manipulate cell growth and functions.     

重组人酸性成纤维细胞生长因子滴眼液的制备及其稳定性

目的制备重组人酸性成纤维细胞生长因子(rhaFGF)滴眼液,并进行稳定性检测。方法在rhaFGF中添加复方保护剂,并以透明质酸钠作为保湿剂,制备rhaFGF滴眼液,对其进行鉴定和体外药效学检测,并进行稳定性试验。结果制备的3批滴眼液各项质量指标均合格,可促进碱烧伤家兔角膜细胞和鸡胚绒毛膜血管增殖。在4℃保存12个月,-20℃保存18个月,(25±2)℃保存6个月,滴眼液各项质量指标与0月基本一致。结论制备的rhaFGF滴眼液稳定性良好,可促进损伤角膜细胞修复。     

30L生物反应器培养重组CHO细胞生产重组人组织型纤溶酶原激活剂

目的应用30L填充床生物反应器培养重组CHO细胞生产重组人组织型纤溶酶原激活剂(rht-PA)。方法将表达rht-PA的CHO细胞株用含10%胎牛血清的IMDM复苏并放大培养,接种至30L生物反应器中,并采用BiocommandPlus软件系统实时监控。先用含血清的培养基生长培养,再更换为无血清培养基进行表达培养。在整个培养过程中,采用灌流培养方式,每日采样测定培养上清中葡萄糖浓度,隔日测定rht-PA的表达水平及生物学活性。采用Lysine-Sepharose4B和Zn2+-Sepharose4B两步亲和层析法纯化rht-PA,并检测纯化产物的比活、产率及纯度。结果整个培养过程持续51d,包括生长培养6d,表达培养45d,平均日灌流量为46.7L,最高达60L,共收获表达培养液约2100L;rht-PA的平均表达水平为15.15mg/L,最高可达19.25mg/L,生物学活性平均约为8000IU/ml;表达培养至第13天时,葡萄糖消耗量达最高水平(15.97g/L·d);纯化的rht-PA比活达6×105IU/mg,产率为63%,纯度达99%以上。结论应用30L填充床生物反应器可实现重组CHO细胞的长时间连续培养及产物rht-PA的高效表达。     

The Human Induced Pluripotent Stem Cell Test as an Alternative Method for Embryotoxicity Testing

The evaluation of substances for their potency to induce embryotoxicity is controlled by safety regulations. Test guidelines for reproductive and developmental toxicity rely mainly on animal studies, which make up the majority of animal usage in regulatory toxicology. Therefore, there is an urgent need for alternative in vitro methods to follow the 3R principles. To improve human safety, cell models based on human cells are of great interest to overcome species differences. Here, human induced pluripotent stem cells (hiPSCs) are an ideal cell source as they largely recapitulate embryonic stem cells without bearing ethical concerns and they are able to differentiate into most cell types of the human body. Here, we set up and characterized a fetal bovine serum (FBS)-free hiPSC-based in vitro test method, called the human induced pluripotent stem cell test (hiPS Test), to evaluate the embryotoxic potential of substances. After 10 days in culture, hiPSCs develop into beating cardiomyocytes. As terminal endpoint evaluations, cell viability, qPCR analyses as well as beating frequency and area of beating cardiomyocytes by video analyses are measured. The embryotoxic positive and non-embryotoxic negative controls, 5-Fluorouracil (5-FU) and Penicillin G (PenG), respectively, were correctly assessed in the hiPS Test. More compounds need to be screened in the future for defining the assay’s applicability domain, which will inform us of the suitability of the hiPS Test for detecting adverse effects of substances on embryonic development.