携带Herceptin与MICA胞外结构域融合基因的重组腺病毒的构建及鉴定

通过构建能表达抗体Herceptin与MICA胞外结构域的融合蛋白的重组腺病毒,为进一步联合NK细胞的治疗研究奠定基础.采用Overlap-PCR的方法获得Herceptin与MICA胞外结构域的融合基因,并将融合基因连接到载体pDC339上.接着再利用酶切及PCR方法鉴定载体及重组病毒基因的正确性,并通过ELISA和Westem Blot检测融合蛋白的表达情况,最后经间接免疫荧光分析(IFA)检测融合蛋白的结合特性.构建的载体酶切后经电泳鉴定证实构建成功,重组病毒DNA的PCR显示病毒携带了Herceptin的轻链基因、重链基因及MICA胞外结构域的基因;ELISA和Western Blot证实重组腺病毒成功地表达出了目的融合蛋白;IFA实验表明融合蛋白能与SK-OV-3细胞表面的Her-2受体特异性地结合.最后成功地构建了能表达具有正常结合特性的抗体Herceptin与MICA胞外结构域的融合蛋白的重组腺病毒,为之后与NK细胞的联合治疗研究奠定基础.     

The C-neu mammary carcinoma in Oncomice; characterization and monitoring response to treatment with herceptin by magnetic resonance methods

To characterize spontaneously occurring c-neu/HER2 overexpressing tumours in oncomice and their response to herceptin by non-invasive magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). Oncomice were monitored by localized ³¹P MRS during unperturbed growth and before and after treatment with 10 mg/kg herceptin (Hoffman La Roche) intraperitoneally for up to 21 days post-treatment. Vascular morphology and function was assessed by quantitation of tumour magnetic resonance (MR) relaxation rates R₂* and R₂ prior to and either during carbogen (95% O₂/5% CO₂) breathing or following administration of the blood-pool contrast agent NC100150 (Clariscan, Amersham Health). Immunohistochemistry showed strong membrane staining for HER2 protein overexpression. The ³¹P MRS showed only a significant (p<0.01) increase of phosphomonoester / total phosphate ratio over 21 days of growth. Herceptin increased the tumour volume doubling time compared to untreated tumours and significantly increased the phosphomonoester / -nucleoside triphosphate ratio 2 days after treatment (p=0.01). Tumours showed a highly heterogeneous yet significant (p<0.01) decrease or increase in R₂* in response to carbogen or NC100150 respectively. The absence of a decline in tumour bioenergetics with growth, commonly seen in ³¹P MRS studies of transplanted rodent tumour models, coupled with the heterogeneous blood volume revealed by ¹H MRI, suggest a metabolic and vascular phenotype similar to that found in human tumours.     

携带Herceptin-MICB融合蛋白基因的腺病毒载体的构建

为了构建携带Herceptin-MICB融合蛋白基因的腺病毒载体,筛选出能表达该融合蛋白的重组腺病毒,检测其表达融合蛋白的情况及抗肿瘤特征.首先,通过PCR将NKG2D的配体MICB的基因片段插入含有全长抗体Herceptin基因的5型腺病毒穿梭载体pDC339-Her中,获得载体pDC339-Her-MICB与腺病毒骨架载体pBHGlox△E1,3Cr重组,然后在293细胞内进行病毒包装得到非增殖型腺病毒Ad-Her-MICB,纯化后测病毒滴度.双抗体夹心ELISA和Western blot检测融合蛋白的表达情况;间接免疫荧光实验(IFA)检测融合蛋白的特异性.酶切后DNA电泳鉴定证实载体构建成功,重组病毒经PCR鉴定携带融合蛋白基因.融合蛋白的表达量为(623.25±38.62)ng/mL;Westhern blot显示:该融合蛋白与商品化的Herceptin抗体轻链重链比较,大小一致,浓度匹配;间接免疫荧光检测(IFA)显示了融合蛋白与HER-2过表达的卵巢癌细胞株SK-OV-3结合的特异性.结果表明:腺病毒Ad-Her-MICB携带Herceptin-NKG2D配体融合蛋白基因能在体外正常表达且与商品化的Herceptin生物学特性相似,为融合蛋白的抗肿瘤治疗奠定基础.     

Surface‐functionalized polyurethane nanoparticles for targeted cancer therapy

Polymer nanoparticles (nps) have gained growing interest as carriers for anticancer drugs as they can target tumour tissues by both passive and active pathways. While the passive targeting mechanisms mainly rely on the small size of the carriers, active targeting requires surface modifications of the polymer core in order to introduce specific functionalities to actively recognize cancer cells. The present work proposes an innovative method for the preparation of surface‐functionalized nps based on the use of biodegradable polyester‐ and polyester/ether‐urethanes (PURs) embedding amino functionalities. Two polyurethanes were prepared, one based on just poly(?‐caprolactone) diol (PCL‐PUR) and the other based on both PCL diol and poly(ethylene glycol) (PEG) (70/30 ratio, PCL‐PEG‐PUR). Nanoparticles of small size ranging between 150 and 200 nm and negative ζ potential (ranging from ?18 mV to ?27 mV) were obtained. Functional groups were exposed post nps preparation as confirmed by X‐ray photoelectron spectroscopy, ninhydrin assay and ¹H NMR, which evidenced a 24% tert‐butyloxycarbonyl cleavage for PCL‐PUR‐NH₂ nps and 29% for PCL‐PEG‐PUR‐NH₂ nps. The monoclonal antibody Herceptin (HER), which targets HER‐2 receptors, was coupled through ethyl(dimethylaminopropyl) carbodiimide/N‐hydroxysuccinimide (EDC/NHS) mediated chemistry. The optimal HER:NH₂ ratio was determined to be 1:16 for the PEG‐containing PUR and 1:8 for PCL‐PUR. HER‐nps maintained the intrinsic cytotoxicity of the antibody, as shown by the ca 50% decrease of HER‐2‐expressing HeLa cell viability. The results indicate that our protocol for surface functionalization of PUR nps, based on surface exposure of previously inserted functional groups followed by covalent coupling of biomolecules, is suitable for the preparation of nps for active recognition of target cells. © 2016 Society of Chemical Industry     

Polyhedral Oligomeric Silsesquioxanes‐Containing Conjugated Polymer Loaded PLGA Nanoparticles with Trastuzumab (Herceptin) Functionalization for HER2‐Positive Cancer Cell Detection

The synthesis of polyhedral oligomeric silsesquioxanes (POSS)‐containing conjugated polymer (CP) and the polymer loaded poly(lactic‐co‐glycolic‐acid) (PLGA) nanoparticles (NPs) with surface antibody functionalization for human epidermal growth factor receptor 2 (HER2)‐positive cancer cell detection are reported. Due to the steric hindrance of POSS, NPs prepared from POSS‐containing CP show improved photoluminescence quantum yield as compared to that for the corresponding linear CP encapsulated NPs. In addition, the amount of ‐NH₂ groups on NP surface is well‐controlled by changing the molar ratio of poly(lactic‐co‐glycolic‐acid)‐b‐poly(ethylene glycol) (PLGA‐b‐PEG‐NH₂) to PLGA‐OCH₃ during NP formulation. Further conjugation of the NH₂‐functionalized CP NPs with trastuzumab (Herceptin) yields NPs with fine‐tuned protein density. These NPs are able to discriminate SKBR‐3 breast cancer cells from MCF‐7 breast cancer cells and NIH/3T3 fibroblast cells both on substrate and in suspension by taking advantage of the specific binding affinity between trastuzumab and HER2 overexpressed in SKBR‐3 breast cancer cell membrane. The high quantum yield and fine‐tuned surface specific protein functionalization make the POSS‐containing CP loaded NPs a good candidate for targeted biological imaging and detection.     

Cancer Detection: Polyhedral Oligomeric Silsesquioxanes‐Containing Conjugated Polymer Loaded PLGA Nanoparticles with Trastuzumab (Herceptin) Functionalization for HER2‐Positive Cancer Cell Detection (Adv. Funct. Mater. 2/2011)

The synthesis of polyhedral oligomeric silsesquioxanes (POSS)‐containing conjugated polymer (CP) and the polymer loaded poly(lactic‐co‐glycolic‐acid) (PLGA) nanoparticles (NPs) with surface antibody functionalization for human epidermal growth factor receptor 2 (HER2)‐positive cancer cell detection are reported. Due to the steric hindrance of POSS, NPs prepared from POSS‐containing CP show improved photoluminescence quantum yield as compared to that for the corresponding linear CP encapsulated NPs. In addition, the amount of ‐NH₂ groups on NP surface is well‐controlled by changing the molar ratio of poly(lactic‐co‐glycolic‐acid)‐b‐poly(ethylene glycol) (PLGA‐b‐PEG‐NH₂) to PLGA‐OCH₃ during NP formulation. Further conjugation of the NH₂‐functionalized CP NPs with trastuzumab (Herceptin) yields NPs with fine‐tuned protein density. These NPs are able to discriminate SKBR‐3 breast cancer cells from MCF‐7 breast cancer cells and NIH/3T3 fibroblast cells both on substrate and in suspension by taking advantage of the specific binding affinity between trastuzumab and HER2 overexpressed in SKBR‐3 breast cancer cell membrane. The high quantum yield and fine‐tuned surface specific protein functionalization make the POSS‐containing CP loaded NPs a good candidate for targeted biological imaging and detection.