Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen

To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC₅₀: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (K_D: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.     

Selection of tumor-specific internalizing human antibodies from phage libraries

Antibody internalization into the cell is required for many targeted therapeutics, such as immunotoxins, immunoliposomes, antibody-drug conjugates and for targeted delivery of genes or viral DNA into cells. To generate directly tumor-specific internalizing antibodies, a non-immune single chain Fv (scFv) phage antibody library was selected on the breast tumor cell line SKBR3. Internalized phage were recovered from within the cell and used for the next round of selection. After three rounds of selection, 40 % of clones analyzed bound SKBR3 and other tumor cells but did not bind normal human cells. Of the internalizing scFv identified, two (F5 and C1) were identified as binding to ErbB2, and one (H7) to the transferrin receptor. Both F5 and H7 scFv were efficiently endocytosed into SKBR3 cells, both as phage antibodies and as native monomeric scFv. Both antibodies were able to induce additional functional effects besides triggering endocytosis: F5 scFv induces downstream signaling through the ErbB2 receptor and H7 prevents transferrin binding to the transferrin receptor and inhibits cell growth. The results demonstrate the feasibility of selecting internalizing receptor-specific antibodies directly from phage libraries by panning on cells. Such antibodies can be used to target a variety of molecules into the cell to achieve a therapeutic effect. Furthermore, in some instances endocytosis serves as a surrogate marker for other therapeutic biologic effects, such as growth inhibition. Thus, a subset of selected antibodies will have a direct therapeutic effect.     

Fluorescence characterisation of multiply-loaded anti-HER2 single chain Fv-photosensitizer conjugates suitable for photodynamic therapy.

We report the synthesis, spectroscopic properties and intracellular imaging of recombinant antibody single chain fragment (scFv) conjugates with photosensitizers used for photodynamic therapy of cancer (PDT). Two widely-studied photosensitizers have been selected: preclinical pyropheophorbide-a (PPa) and verteporfin (VP), which has been clinically approved for the treatment of acute macular degeneration (Visudyne). Pyropheophorbide-a and verteporfin have been conjugated to an anti-HER2 scFv containing on average ten photosensitizer molecules per scFv with a small contribution (相似文献   

抗EGFRvⅢ噬菌体抗体库的构建和筛选

目的:应用噬菌体展示技术筛选针对表皮生长因子受体突变体Ш(epidermal growth factorreceptor variant typeⅢ,EGFRvIII)的单链抗体(single chain Fv,scFv)。方法:利用原核表达纯化的人EGFRvIIIex蛋白和高表达EGFRvIIIex的小鼠成纤维细胞系NIH3T3免疫小鼠,扩增VH和VL片段并拼装成scFv基因,连接至噬菌粒pCANTAB5E,电击转化Hpd3cells,构建噬菌体单链抗体库,并进行3轮富集筛选。在第4轮筛选时,采用了降低抗原浓度的方法。然后将筛选得到的阳性克隆测序分析,转化E.coliHB2151,IPTG诱导可溶性scFv的表达。结果:构建了库容为7.9×107的噬菌体单链抗体库。经过第4轮低浓度抗原筛选,得到了较高亲和力的克隆。取单个阳性克隆测序分析结果表明,该抗EGFRvIII scFv基因序列长807bp,编码268个氨基酸。IPTG诱导后表达的可溶性scFv可分别与纯化的EGFRvⅢex抗原以及细胞表面的EGFRvⅢex结合。结论:利用噬菌体抗体库筛选得到了高亲和力的抗EGFRvⅢ scFv,为开发针对EGFRvⅢ...     

Multi-parametric analysis reveals enhanced G2-phase arrest of an optimized anti-HER2 antibody to inhibit breast cancer

Objectives

To find a “me-better” antibody by epitope-specific antibody optimization and multi-parametric analysis.

Results

Using epitope-specific library based on the commercial drug, Pertuzumab/2C4, we screened a novel human anti-HER2 antibody, MIL5, which has slightly higher affinity than the drug. MIL5 and 2C4 share the same epitope to bind HER2; however, MIL5 bound to HER2 His235–His245 more tightly than 2C4, which could be the main reason of its enhanced affinity. In vivo experiments also showed MIL5 had stronger anti-cancer activity than 2C4; however, the classical flow cytometry assays to detect cell apoptosis or cycling did not show convincing evidence of the advantages of MIL5. Thus we introduced the multi-parameter in-cell analysis method to evaluate the superiority of MIL5 to 2C4 in arresting cancer cells in G2-phase to inhibit cell growth and/or proliferation.

Conclusion

Multi-parametric method confirmed stronger arrest of G2 by MIL5 to show better anti-cancer function both in vitro and in vivo than 2C4.

     

抗EGFRvIII噬菌体抗体库的构建和筛选

目的：应用噬菌体展示技术筛选针对表皮生长因子受体突变体Ш (epidermal growth factor receptor variant type Ⅲ, EGFRvIII)的单链抗体 (single chain Fv, scFv)。方法：利用原核表达纯化的人EGFRvIIIex蛋白和高表达EGFRvIIIex的小鼠成纤维细胞系NIH3T3免疫小鼠,扩增VH和VL片段并拼装成scFv 基因,连接至噬菌粒pCANTAB 5E,电击转化Hpd3cells,构建噬菌体单链抗体库,并进行3轮富集筛选。在第4轮筛选时,采用了降低抗原浓度的方法。然后将筛选得到的阳性克隆测序分析,转化E.coli HB2151,IPTG 诱导可溶性scFv 的表达。结果：构建了库容为7.9×107 的噬菌体单链抗体库。经过第4轮低浓度抗原筛选,得到了较高亲和力的克隆。取单个阳性克隆测序分析结果表明,该抗EGFRvIII scFv 基因序列长807 bp,编码268个氨基酸。IPTG诱导后表达的可溶性scFv 可分别与纯化的EGFRvIIIex抗原以及细胞表面的EGFRvIIIex结合。结论：利用噬菌体抗体库筛选得到了高亲和力的抗EGFRvIII scFv,为开发针对EGFRvIII的抗体药物提供了靶向载体分子。     

Targeting HER2/neu with a fully human IgE to harness the allergic reaction against cancer cells

Breast and ovarian cancer are two of the leading causes of cancer deaths among women in the United States. Overexpression of the HER2/neu oncoprotein has been reported in patients affected with breast and ovarian cancers, and is associated with poor prognosis. To develop a novel targeted therapy for HER2/neu expressing tumors, we have constructed a fully human IgE with the variable regions of the scFv C6MH3-B1 specific for HER2/neu. This antibody was expressed in murine myeloma cells and was properly assembled and secreted. The Fc region of this antibody triggers in vitro degranulation of rat basophilic cells expressing human FcεRI (RBL SX-38) in the presence of murine mammary carcinoma cells that express human HER2/neu (D2F2/E2), but not the shed (soluble) antigen (ECD(HER2)) alone. This IgE is also capable of inducing passive cutaneous anaphylaxis in a human FcεRIα transgenic mouse model, in the presence of a cross-linking antibody, but not in the presence of soluble ECD(HER2). Additionally, IgE enhances antigen presentation in human dendritic cells and facilitates cross-priming, suggesting that the antibody is able to stimulate a secondary T-cell anti-tumor response. Furthermore, we show that this IgE significantly prolongs survival of human FcεRIα transgenic mice bearing D2F2/E2 tumors. We also report that the anti-HER2/neu IgE is well tolerated in a preliminary study conducted in Macaca fascicularis (cynomolgus) monkeys. In summary, our results suggest that this IgE should be further explored as a potential therapeutic against HER2/neu overexpressing tumors, such as breast and ovarian cancers.     

Affinity maturation of antiHER2 monoclonal antibody MIL5 using an epitope-specific synthetic phage library by computational design

Increased affinities mainly equal to improved biological efficacy in many cases. By now, display methods including phage library are widely exploited to obtain higher affinity antibodies. Traditional library methods mainly focus on complementary determining region mutagenesis, in which extensive screening of variant combinations as well as large library capacity is required to find higher affinity clones. In this study, based on the modeling 3D complex structure of antigen (HER2)–antibody (MIL5) complex, the key residues of contact surface were predicted and identified to guide the synthetic phage library design. Then, epitope-specific site-directed mutagenesis phage library comprised of MIL5_scFv mutants was constructed, from which a higher affinity single chain antibody (M5scFv_ph) was screened out. Following experimental results showed that the novel antibody M5scFv_ph retained superimposed epitope to the parent antibody MIL5_scFv, and possessed similar tumor growth inhibitory activity in vivo on ovarian carcinoma xenografts.     

The FGF‐1‐specific single‐chain antibody scFv1C9 effectively inhibits breast cancer tumour growth and metastasis

Immunotherapy mediated by recombinant antibodies is an effective therapeutic strategy for a variety of cancers. In a previous study, we demonstrated that the fibroblast growth factor 1 (FGF‐1)‐specific recombinant antibody scFv1C9 arrests the cell cycle at the G0/G1 transition by blocking the intracrine FGF‐1 pathway in breast cancer cells. Here, we further show that the overexpression of scFv1C9 in MCF‐7 and MDA‐MB‐231 breast cancer cells by lentiviral infection resulted in decreased tumourigenicity, tumour growth and lung metastasis through FGF‐1 neutralization. We found that scFv1C9 resulted in the up‐regulation of p21, which in turn inhibited the expression of CDK2 and blocked cell cycle progression. To explore the potential role of scFv1C9 in vivo, we delivered the gene into solid tumours by electroporation, which resulted in significant inhibition of tumour growth. In tumour tissue sections, immunohistochemical staining of the cellular proliferation marker Ki‐67 and the microvessel marker CD31 showed a reduction in the proliferative index and microvessel density, respectively, upon expression of scFv1C9 compared with the appropriate controls. Thus, our data indicate a central role for scFv1C9 in blocking the intracrine pathway of FGF‐1, therefore, scFv1C9 could be developed in an effective therapeutic for breast cancer.     

Anti HER2 ScFv-GFP融合抗体靶向检测HER2的表达

为验证真核表达的携带绿色荧光的抗HER2单链抗体应用于临床诊断HER2阳性肿瘤细胞和病理组织的可靠性,构建融合基因Anti HER2 ScFv-GFP,重组入pFAST Bac HT A载体,在昆虫细胞Sf9中表达,以Ni2+-NTA亲和层析法纯化Anti HER2 ScFv-GFP融合蛋白,测定其浓度与纯度,将同浓度的纯化蛋白分别与3种乳腺癌细胞BT474、SKBR3和MCF7各混合12 h、24 h和48 h,分析其在不同时间段结合HER2阳性肿瘤细胞的稳定性。用纯化蛋白直接检测经抗原修复的乳腺癌病理组织,与免疫组织化学法检测结果对比。结果在昆虫细胞Sf9中可观察到明显绿色荧光,纯化的融合蛋白相对分子量约60 kDa,浓度为115.5μg/mL,纯度约97%,SKBR3和BT474鉴定为HER2阳性。结合12 h、24 h、48 h后其细胞表面均有明显绿色荧光,而HER2阴性MCF7被洗脱后无荧光,该抗体滴度为1:64,48 h内该荧光抗体仍具有稳定性。携带绿色荧光的融合抗体检测病理组织与IHC法的结果完全一致。表明成功表达的携带绿色荧光的抗HER2单链抗体可特异性检测HER2阳性乳腺癌细胞BT474和SKBR3,在HER2阳性肿瘤细胞和临床病理组织检测上具有应用前景。     

An anti-leukemic single chain Fv antibody selected from a synthetic human phage antibody library

The display of human antibody repertoire on the cell surface of the filamentous bacteriophage has offered a novel strategy for selecting antibodies to a diverse range of purified targets. However, the selection of antibodies with biological functions has not yet been fully investigated. To select phage antibodies with therapeutic potential, a synthetic human single chain Fv (scFv) phage antibody library was panned on whole premyelocytic leukemia cell line (HL60). Phages binding to common receptors and undesirable phages were subtracted by incubating the library with human glioma cells. High affinity binding phages to HL60 cells were enriched by fluorescence-activated cell sorting. After the 6th round of selection, 50% of the selected phage antibodies showed significant binding to HL60 cells, whereas none of the analyzed phage antibodies bound to human pre-B cells (Nalm-6). In addition to binding, one scFv antibody inhibited HL60 cell proliferation by 90% compared to irrelevant scFv antibodies. Taken together the data demonstrate that specific scFv antibodies with biological functions can be isolated by using whole cells as affinity matrix.     

Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4

The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.     

Phosphodiesterase Type 5 Inhibitors Increase Herceptin Transport and Treatment Efficacy in Mouse Metastatic Brain Tumor Models

Background

Chemotherapeutic drugs and newly developed therapeutic monoclonal antibodies are adequately delivered to most solid and systemic tumors. However, drug delivery into primary brain tumors and metastases is impeded by the blood-brain tumor barrier (BTB), significantly limiting drug use in brain cancer treatment.

Methodology/Principal Findings

We examined the effect of phosphodiesterase 5 (PDE5) inhibitors in nude mice on drug delivery to intracranially implanted human lung and breast tumors as the most common primary tumors forming brain metastases, and studied underlying mechanisms of drug transport. In vitro assays demonstrated that PDE5 inhibitors enhanced the uptake of [¹⁴C]dextran and trastuzumab (Herceptin®, a humanized monoclonal antibody against HER2/neu) by cultured mouse brain endothelial cells (MBEC). The mechanism of drug delivery was examined using inhibitors for caveolae-mediated endocytosis, macropinocytosis and coated pit/clathrin endocytosis. Inhibitor analysis strongly implicated caveolae and macropinocytosis endocytic pathways involvement in the PDE5 inhibitor-enhanced Herceptin uptake by MBEC. Oral administration of PDE5 inhibitor, vardenafil, to mice with HER2-positive intracranial lung tumors led to an increased tumor permeability to high molecular weight [¹⁴C]dextran (2.6-fold increase) and to Herceptin (2-fold increase). Survival time of intracranial lung cancer-bearing mice treated with Herceptin in combination with vardenafil was significantly increased as compared to the untreated, vardenafil- or Herceptin-treated mice (p<0.01). Log-rank survival analysis of mice bearing HER2-positive intracranial breast tumor also showed a significant survival increase (p<0.02) in the group treated with Herceptin plus vardenafil as compared to other groups. However, vardenafil did not exert any beneficial effect on survival of mice bearing intracranial breast tumor with low HER2 expression and co-treated with Herceptin (p>0.05).

Conclusions/Significance

These findings suggest that PDE5 inhibitors may effectively modulate BTB permeability, and enhance delivery and therapeutic efficacy of monoclonal antibodies in hard-to-treat brain metastases from different primary tumors that had metastasized to the brain.     

Cloning,expression, and identification of anti‐carbofuran single chain Fv gene

Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly₄Ser)₃. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC₅₀ value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A herpes simplex virus recombinant that exhibits a single-chain antibody to HER2/neu enters cells through the mammary tumor receptor, independently of the gD receptors

The human epidermal growth factor receptor 2/neuregulin (HER2/neu) receptor is overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis. It is a target for therapy; humanized monoclonal antibodies to HER2 have led to increased survival of patients with HER2/neu-positive breast cancer. As a first step in the design of an oncolytic herpes simplex virus able to selectively infect HER2/neu-positive cells, we constructed two recombinants, R-LM11 and R-LM11L, that carry a single-chain antibody (scFv) against HER2 inserted at residue 24 of gD. The inserts were 247 or 256 amino acids long, and the size of the gD ectodomain was almost doubled by the insertion. We report the following. R-LM11 and R-LM11L infected derivatives of receptor-negative J or CHO cells that expressed HER2/neu as the sole receptor. Entry was dependent on HER2/neu, since it was inhibited in a dose-dependent manner by monoclonal antibodies to HER2/neu and by a soluble form of the receptor. The scFv insertion in gD disrupted the ability of the virus to enter cells through HVEM but maintained the ability to enter through nectin1. This report provides proof of principle that gD can tolerate fusion to a heterologous protein almost as large as the gD ectodomain itself without loss of profusion activity. Because the number of scFv's to a variety of receptors is continually increasing, this report makes possible the specific targeting of herpes simplex virus to a large collection of cell surface molecules for both oncolytic activity and visualization of tumor cells.     

Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo

Monoclonal antibodies specific for the p185HER2/neu growth factor receptor represent a significant advance in receptor-based therapy for p185HER2/neu-expressing human cancers. We have used a structure-based approach to develop a small (1.5 kDa) exocyclic anti-HER2/neu peptide mimic (AHNP) functionally similar to an anti-p185HER2/neu monoclonal antibody, 4D5 (Herceptin). The AHNP mimetic specifically binds to p185HER2/neu with high affinity (KD=300 nM). This results in inhibition of proliferation of p185HER2/neu-overexpressing tumor cells, and inhibition of colony formation in vitro and growth of p185HER2/neu-expressing tumors in athymic mice. In addition, the mimetic sensitizes the tumor cells to apoptosis when used in conjunction with ionizing radiation or chemotherapeutic agents. A comparison of the molar quantities of the Herceptin antibody and the AHNP mimetic required for inhibiting cell growth and anchorage-independent growth showed generally similar activities. The structure-based derivation of the AHNP represents a novel strategy for the design of receptor-specific tumor therapies.     

Neural Stem Cells as a Novel Platform for Tumor-Specific Delivery of Therapeutic Antibodies

Background

Recombinant monoclonal antibodies have emerged as important tools for cancer therapy. Despite the promise shown by antibody-based therapies, the large molecular size of antibodies limits their ability to efficiently penetrate solid tumors and precludes efficient crossing of the blood-brain-barrier into the central nervous system (CNS). Consequently, poorly vascularized solid tumors and CNS metastases cannot be effectively treated by intravenously-injected antibodies. The inherent tumor-tropic properties of human neural stem cells (NSCs) can potentially be harnessed to overcome these obstacles and significantly improve cancer immunotherapy. Intravenously-delivered NSCs preferentially migrate to primary and metastatic tumor sites within and outside the CNS. Therefore, we hypothesized that NSCs could serve as an ideal cellular delivery platform for targeting antibodies to malignant tumors.

Methods and Findings

As proof-of-concept, we selected Herceptin™ (trastuzumab), a monoclonal antibody widely used to treat HER2-overexpressing breast cancer. HER2 overexpression in breast cancer is highly correlated with CNS metastases, which are inaccessible to trastuzumab therapy. Therefore, NSC-mediated delivery of trastuzumab may improve its therapeutic efficacy. Here we report, for the first time, that human NSCs can be genetically modified to secrete anti-HER2 immunoglobulin molecules. These NSC-secreted antibodies assemble properly, possess tumor cell-binding affinity and specificity, and can effectively inhibit the proliferation of HER2-overexpressing breast cancer cells in vitro. We also demonstrate that immunoglobulin-secreting NSCs exhibit preferential tropism to tumor cells in vivo, and can deliver antibodies to human breast cancer xenografts in mice.

Conclusions

Taken together, these results suggest that NSCs modified to secrete HER2-targeting antibodies constitute a promising novel platform for targeted cancer immunotherapy. Specifically, this NSC-mediated antibody delivery system has the potential to significantly improve clinical outcome for patients with HER2-overexpressing breast cancer.     

Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury

Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots.     

Application of fusion protein 4D5 scFv-dibarnase:barstar-gold complex for studying P185HER2 receptor distribution in human cancer cells

Overexpression of the P185^HER2 protein determines the malignancy and unfavorable prognosis of ovarian and breast tumors. In this work, the distribution of P185^HER2 in human cancer cells was studied by electron microscopy, using a novel approach. It is based on the interaction between barnase (a ribonuclease from Bacillus amyloliquefaciens) and its specific inhibitor barstar. The monoclonal antibody 4D5 scFv to extracellular P185^HER2 domain fused with two molecules of barnase was used as a recognizing agent, and the conjugate of colloidal gold with barstar, as an electron dense label for electron microscopic visualization. For labeling, we used supramolecular complexes 4D5 scFv-dibarnase:barstar-Au.     

Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis

Many targeted cancer therapies require endocytosis of the targeting molecule and delivery of the therapeutic agent to the interior of the tumor cell. To generate single chain Fv (scFv) antibodies capable of triggering receptor-mediated endocytosis, we previously developed a method to directly select phage antibodies for internalization by recovering infectious phage from the cytoplasm of the target cell. Using this methodology, we reported the selection of a panel of scFv that were internalized into breast cancer cells from a nonimmune phage library. For this work, an immunotherapeutic was generated from one of these scFv (F5), which bound to ErbB2 (HER2/neu). The F5 scFv was reengineered with a C-terminal cysteine, expressed at high levels in Escherichia coli, and coupled to sterically stabilized liposomes. F5 anti-ErbB2 immunoliposomes were immunoreactive as determined by surface plasmon resonance (SPR) and were avidly internalized by ErbB2-expressing tumor cell lines in proportion to the levels of ErbB2 expression. F5-scFv targeted liposomes containing doxorubicin had antitumor activity and produced significant reduction in tumor size in xenografted mice compared to nontargeted liposomes containing doxorubicin. This strategy should be applicable to generate immunotherapeutics for other malignancies by selecting phage antibodies for internalization into other tumor types and using the scFv to target liposomes or other nanoparticles.