Expression of humanized anti-Her2/neu single-chain IgG1-like antibody in mammary glands of transgenic mice

A system for production of single-chain antibody in mammary glands of mice was developed on the basis of a hybrid gene constructed from the coding sequence of anti-Her2/neu single-chain antibody inserted into the first exon of the sheep beta-lactoglobulin gene. Lines of transgenic mice were obtained that expressed humanized single-chain anti-Her2/neu IgG1-like antibody in their milk. These antibodies interact with Her2/neu antigen with high affinity (K_d = 0.4 nM). The expression level of the transgene depended on its integration site in the genome but not on the copy number. The transgene had no toxic effect on the mice and was stably inherited, at least for two generations. The results reveal new opportunities of producing single-chain antibodies in the milk of animals.     

Expression, purification, and in vitro refolding of a humanized single-chain Fv antibody against human CTLA4 (CD152)

A human-derived single-chain Fv (scFv) antibody fragment specific against human CTLA4 (CD152) was produced at high level in Escherichia coli. The scFv gene was cloned from a phagemid to the expression vector pQE30 with a N-terminal 6His tag fused in-frame, and expressed as a 29 kDa protein in E. coli as inclusion bodies. The inclusion body of scFv was isolated from E. coli lysate, solubilized in 8M urea with 10mM dithiothreitol, and purified by ion-exchange chromatography. Method for in vitro refolding of the scFv was established. The effects of refolding buffer composition, protein concentration and temperature on the refolding yield were investigated. The protein was renatured finally by dialyzing against 3mM GSH, 1mM GSSG, 150 mM NaCl, 1M urea, and 50 mM Tris-Cl (pH 8.0) for 48 h at 4 degrees C, and then dialyzed against phosphate-buffered saline (pH 7.4) to remove remaining denaturant. This refolding protocol generated up to a 70% yield of soluble protein. Soluble scFv was characterized for its specific antigen-binding activity by indirect cellular ELISA. The refolded scFv was functionally active and was able to bind specifically to CTLA4 (CD152). The epitopes recognized by refolded anti-CTLA4 scFv do not coincide with those epitopes recognized by CD80/CD86.     

Development of single-chain Fv of antibody to DNA as intracellular delivery vehicle

Application of antibodies in most therapeutic area is limited to extracellular or membranous targets because of their impermeability of membrane. For the purpose of biotechnological and therapeutic application, developing intracellular localizing antibody is the invaluable research field. A new recombinant single-chain variable fragment of an anti-dsDNA monoclonal antibody G2-6, IgG of which has been previously shown to have a cell-penetrating activity, was engineered and produced for the use as a delivery vehicle of biomolecule(s). The penetrating capacity of single-chain variable fragment in three mammalian cell lines, L929, NIH/3T3, and COS-7 was analyzed using flow cytometry and confocal microscopy. The results demonstrated that the single-chain variable fragment can effectively internalize all three cell lines, although the internalization level varied. It was also shown that the internalization was time- and concentration-dependent. Moreover, the single-chain variable fragment was located in nuclei as well as cytoplasm of L929 cells. Overall, the G2-6 single-chain variable fragment might be a candidate vehicle which could be used to deliver specific genes or biomolecules for therapy or diagnosis into the cytoplasm or cell nucleus.     

Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures

A synthetic gene encoding an anti-phytochrome single-chain Fv (scFv) antibody bearing an N-terminal signal peptide has been used to transform tobacco plants. Immunoblot analysis showed that transformed plants accumulate high levels of scFv protein, accounting for up to 0.5% of the total soluble protein fraction, which could be extracted by simple infiltration and centrifugation of leaf tissue. A substantial proportion of the scFv protein extracted in this way was found to possess antigen-binding activity. Callus cell suspension cultures derived from transformed plants secrete functional scFv protein into the surrounding medium. Compared with the levels of scFv protein observed in plants expressing the native scFv gene, the incorporation of an N-terminal signal peptide, to target the scFv to the apoplast, results in elevated accumulation of the protein.     

Anti-herbicide single-chain antibody expression confers herbicide tolerance in transgenic plants

An anti-chlorpropham single-chain variable-fragment (scFv) gene was introduced into Arabidopsis in a manner to express the antibody fragment in each of four different subcellular compartments. The accumulation of scFv in transgenic plants was detected by targeting the fragment in the endoplasmic reticulum or apoplastic space, or by expressing the fragment as a glycosylphosphatidylinositol-anchored protein, while no accumulation could be detected by targeting the fragment in the cytosol. Transgenic plants accumulating the scFv gene at a high level in the endoplasmic reticulum had enhanced tolerance to chlorpropham in comparison with the non-transformants.     

Biosynthetic antibody binding sites: Development of a single-chain Fv model based on antidinitrophenol IgA myeloma MOPC 315

The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein inE. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values forK _a,app of 1.5 to 2.2×10⁶ M^–1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv³¹⁵ fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins.     

转铁蛋白受体单链抗体与BDNF融合蛋白的表达及活性鉴定

脑源性神经营养因子（BDNF）对中枢神经系统的多种神经元具有营养,修复和保护功能,但因无法通过血脑屏障限制了其应用。本文利用抗转铁蛋白受体（TfR）的单链抗体（ox26-scFv）作为脑转运载体,分别扩增单链抗体和BDNF基因,插入pTIG-Trx载体,构建融合基因表达载体pTIG-Trx/scFv-BDNF,在大肠杆菌BL21（DE3）中实现了高效表达。经Ni-NTA金属鏊合层析柱纯化后,在41Kd处可见目的纯化条带。大鼠GH3细胞免疫酶染色显示,ScFv-BDNF融合蛋白能与转铁蛋白受体特异性结合。同时能够促进鸡胚背根节神经突起的生长,具备了BDNF的生物学活性。为使BDNF能够跨越血脑屏障成为中枢神经系统的治疗药物打下了实验基础。     

Production and validation of the pharmacokinetics of a single-chain Fv fragment of the MGR6 antibody for targeting of tumors expressing HER-2

The HER-2 antigen, which is overexpressed in many breast carcinomas, is an ideal target for monoclonal antibodies due to its low expression in normal tissue and its homogeneous distribution in the tumor mass. We have developed and characterized the murine MAb MGR6 against HER-2, which is able to inhibit proliferation of tumor cells overexpressing HER-2. On the basis of these preclinical results, phase I studies in breast carcinoma patients were conducted and radiolocalization data indicated an antibody half life which directly paralleled that of other whole antibodies and thus resulting in a limited in vivo diagnostic capacity. To obtain a smaller reagent with possibly improved in vivo properties, a single chain variable fragment (scFv) of the original MGR6-producing hybridoma was generated by phage display technology. Biologically active MGR6 scFv was purified rapidly and at high yield by metal affinity chromatography. Competition FACS and ELISA analyses identified an epitope on the HER-2 extracellular domain that was shared by the scFv and the parental MAb. BIAcore analysis indicated a K_off of 9.3 × 10⁻⁴ s⁻¹, similar to that of the intact MGR6 MAb. Distribution and elimination half-lives of MGR6 scFv, calculated from in vivo preclinical evaluations, were much faster (13 min and 6.2 h, respectively) than previously published results for the intact MAb (mean t1/2β of 46 h). This represents a theoretical improvement in pharmacokinetics with respect to the parental murine MAb and points to the potential for utilizing this fragment in redirecting therapeutic agents, such as radioisotopes, to different human carcinomas overexpressing HER-2. Received: 10 August 2000 / Accepted: 19 October 2000     

Benefits and risks of antibody and vaccine production in transgenic plants

Phytopharming, the production of protein biologicals in recombinant plant systems, has shown great promise in studies performed over the past 13 years. A secretory antibody purified from transgenic tobacco was tested successfully in humans, and prevented bacterial re-colonization after topical application in the mouth. Rapid production of patient-tailored anti-lymphoma antibodies in recombinant Tobamovirus-infected tobacco may provide effective cancer therapy. Many different candidate vaccines from bacterial and viral sources have been expressed in transgenic plants, and three human clinical trials with oral delivery of transgenic plant tissues have shown exciting results. The use of crop plants with agricultural practice could allow cheap production of valuable proteins, while providing enhanced safety by avoidance of animal viruses or other contaminants. However development of this technology must carefully consider the means to ensure the separation of food and medicinal products when crop plants are used for phytopharming.     

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.     

Pharmacokinetics and biodistribution of radioimmunoconjugates of anti-CD19 antibody and single-chain Fv for treatment of human B-cell Malignancy

The comparative advantages and disadvantages of intact antibodies and single-chain Fv as immunotoxins and radioimmunoconjugates have been widely discussed but not directly compared. In this study, the in vivo properties of anti-CD19 B43 monoclonal antibody and its derived single-chain Fv (FVS191) were studied in athymic nude mice bearing CD19-positive human lymphomas. B43 mab and FVS191 were labeled with iodine-125 using iodine-beads, and immunoreactivities were determined to be 57% and 72%, respectively. Scatchard analysis showed a similar high affinity for both. The results of pharmacokinetic studies revealed that FVS191 had a rapid biphasic clearance from the circulation (T1/2α = 2.5 min, T1/2β = 3.7 h); The T1/2α and T1/2β phases of B43 mab were determined to be 0.72 h and 57 h respectively. Biodistribution studies compared the uptake of labeled antibodies by CD19-positive and by CD19-negative tumors. The peak percentages of injected dose were 5.7% at 12 h for B43 and 2.45% at 1 h for FVS191. Radiolocalization indices (RI) demonstrated tumor-specific uptake for both, but higher uptake for B43. The optimal RI was seen at 15 min for FVS191 and 6 h for B43. FVS191 was unstable in vivo, approximately 50% of the injected dose being degraded in blood in 100 min. Radioactivity detected in the urine was present mainly as the deiodinized form of FVS191. The results suggest that B43 mab is favored over FVS191 in biodistribution properties and in vivo stability. Because B43 Mab showed early tumor-specific uptake, high RI values, and favorable tissue-to-blood ratios, it is a potential candidate for radioimmunotherapy and immunotoxin therapy of B-cell leukemia and lymphoma. Received: 17 June 1997 / Accepted: 17 June 1998     

Expression of Barstar as a selectable marker in yeast mitochondria

We describe a new and potentially universal selection system for mitochondrial transformation based on bacterial genes, and demonstrate its feasibility in Saccharomyces cerevisiae. We first found that cytoplasmically synthesized Barnase, an RNase, interferes with mitochondrial gene expression when targeted to the organelle, without causing lethality when expressed at appropriate levels. Next, we synthesized a gene that uses the yeast mitochondrial genetic code to direct the synthesis of the specific Barnase inhibitor Barstar, and demonstrated that expression of this gene, BARSTM, integrated in mtDNA protects respiratory function from imported barnase. Finally, we showed that screening for resistance to mitochondrially targeted barnase can be used to identify rare mitochondrial transformants that had incorporated BARSTM in their mitochondrial DNA. The possibility of employing this strategy in other organisms is discussed.Communicated by R. G. Herrmann     

Nuclear male sterility induced by pollen-specific expression of a ribonuclease

The promoter of a rice pollen-specific gene, PS1, has been fused to the Bacillus amyloliquefaciens Barnase gene which encodes a secreted ribonuclease. The PS1-Barnase chimeric gene has been introduced into tobacco. These transgenic tobacco plants show normal vegetative and floral development, but they display a range of reproductive properties from slightly reduced in fertility to completely sterile. Barnase mRNAs are detectable in the pollen from transgenic plants which do not show an obvious fertility-related phenotype, and in a few plants which have a mildly reduced-fertile phenotype. However, transgenic plants with a severely reduced-fertile or sterile phenotype do not accumulate detectable amounts of Barnase mRNA in their pollen, and the quality of their RNA is poor, presumably because of extensive RNA degradation. Reciprocal crosses between these transgenic plants and wild-type controls showed that the reduced-fertile phenotype is associated only with the transgenic pollen. When used as the female parent, these PS1-Barnase transgenic plants are fully fertile. Anthers in the severely sterile transgenic plants develop normally, but the majority of their pollen grains have abnormal morphology and they fail to germinate. These results indicate that expression of a pollen-specific cytotoxic gene induces lethality in pollen and may lead to severely reduced male fertility.     

Production and characterization of a bacterial single-chain antibody fragment specific to B-cell-activating factor of the TNF family

An active form of a single-chain antibody fragment (scFv) from the murine monoclonal antibody ABL-1, which is specific for B-cell-activating factor of the TNF family, was produced in Escherichia coli. The complementary DNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker, using an assembly polymerase chain reaction. The construct VH-linker-VL was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli BL21(DE3) as inclusion bodies. After extraction from the E. coli cells, the inclusion bodies were solubilized and denatured in the presence of 8M urea. The expressed scFv fusion proteins were purified by Ni(2+)-IDA His-bind resin and finally renatured by dialysis. The purity and activity of the purified scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and enzyme-linked immunosorbent assay. The result revealed that the ABL-1 scFv retains the specific binding activity to BAFF with an affinity constant of 0.9x10(-8)molL(-1).     

Expression of anti human IL-4 and IL-6 scFvs in transgenic tobacco plants

The two murine single-chain Fv (scFv) genes against human interleukin IL-4 and IL-6 cytokines were cloned in a plant expression vector (pGEJAE1) and mobilized to Agrobacterium tumefaciens. Tobacco leaf discs were co-cultured with Agrobacterium and transferred to selective media for regeneration. The tobacco in vitro plants produced scFvs against human IL-4 and IL-6. Only 8% of transformed plants expressing anti-IL-4 scFv were obtained versus 76% of transformed plants expressing anti-IL-6 scFv. In addition, some plants producing anti-IL-4 and anti-IL-6 scFvs aged more rapidly in in vitro conditions and in greenhouse pots than did control plants. Western blot analysis showed that the transformed Nicotiana tabacum plants contained proteins with an apparent molecular mass on electrophoresis of ca. 32 kDa, corresponding to the predicted size of the scFvs. As entire plant root seemed to accumulate more scFv than did leaves, we decided to continue working with isolated roots. Anti-IL-6 scFvs were detected in cultivated roots and their culture media. Functional anti-IL-6 scFv accounted for 0.16–0.18% of total soluble proteins. The affinity of the anti-IL-6 scFv produced in plants and measured by Biacore was similar to that of scFv produced in Escherichia coli. The high levels of antibody accumulation in isolated roots and secretion into the medium demonstrate the potential for producing recombinant protein in bioreactor systems.these authors contributed equally to this workthese authors contributed equally to this work     

Bacterial aspects associated with the expression of a single-chain antibody fragment in Escherichia coli

The bacterial expression of a single-chain antibody fragment, designated L6 sFv, was examined. Periplasmic targeting resulted in the production of a correctly folded protein that bound tumor antigen. However, immediately after induction at either 30°C or 37°C there was a significant loss in bacterial viability, which was followed by a loss in absorbance. The loss in absorbance correlated with cell lysis and release of the L6 sFv into the culture supernatant. The kinetics of appearance of L6 sFv in the supernatant paralleled that of periplasmic \-lactamase and confirmed an initial loss of cell-wall integrity prior to cell lysis. Bacteria incubated at 30°C produced approximately threefold more correctly folded antibody fragment because of an increase in the number of cells/A ₆₆₀ at the lower incubation temperature. More than 95% of the L6 sFv, made at either incubation temperature, was incorrectly folded. Osmotic-shock procedures did not release L6 sFv. However, in situ subtilisin susceptibility experiments with bacterial spheroplasts confirmed a periplasmic location. French press disruption resulted in the release of correctly but not incorrectly folded material. Membrane fractionation revealed that the incorrectly folded L6 sFv remained associated with both the inner and outer membrane. These results demonstrate that, in this system, antibody fragment expression resulted initially in cell death, which was followed by release of protein into the culture supernatant and eventually cell lysis. It is also suggested that membrane association in the periplasmic space may impede proper folding.     

Expression of the rolC gene and nicotine production in transgenic roots and their regenerated plants

Transformation of Nicotiana tabacum cv. Xanthi leaf sections with the pPCV002-ABC (rol genes A, B and C together under the control of their own promoter) or pPCV002-CaMVC (rol gene C alone under the control of the CaMV 35S promoter) construction present in trans-acting Agrobacterium tumefaciens vectors yielded several transgenic root lines. The two types (rolABC and rolC) of transgenic root lines were examined for their nicotine productivity in relation to growth rate and the amount of rolC gene product measured with specific antibodies. In all cases, the changes in the amount of this polypeptide were positively correlated with the capacity of the transgenic roots to grow and produce nicotine. Both capacities were greatly increased when the rolA, rolB and rolC genes were present together, which demonstrates that the activity of the three rol-gene-encoded functions is synergistic. Consistent observations were also made in the corresponding regenerated plants. Received: 22 February 1997 / Revision received: 22 April 1997 / Accepted: 1 June 1997     

Optimization of in vivo tumor targeting in SCID mice with divalent forms of 741F8 anti-c-erbB-2 single-chain Fv: effects of dose escalation and repeated i.v. administration

Single-chain Fv molecules in monovalent (sFv) and divalent [(sFv')₂] forms exhibit highly specific tumor targeting in mice as a result of their small size and rapid systemic clearance. As a consequence, there is a rapid reversal of the sFv blood/tumor gradient, resulting in diminished retention of sFv species in tumors. In this report we investigate two distinct strategies, dose escalation and repetitive intravenous (i.v.) dosing, aiming to increase the absolute selective retention of radiolabeled anti-c-erbB-2¹²⁵I-741F8 (sFv')₂ in c-erbB-2-overexpressing SK-OV-3 tumors in mice with severe combined immunodeficiency (SCID). A doseescalation strategy was applied to single i.v. injections of¹²⁵I-741F8 (sFv')₂. Doses from 50 g to 1000 g were administered without a significant decrease in tumor targeting or specificity. High doses resulted in large increases in the absolute retention of¹²⁵I-741F8 (sFv')₂. For example, raising the administered dose from 50 g to 1000 g increased the tumor retention 24 h after injection from 0.46 g/g to 9.5 g/g, and resulted in a net increase of greater than 9 /g. Over the same dose range, the liver retention rose from 0.06 g/g to 1 g/g, and resulted in a net increase of less than 1 g/g. The retention of 9.5 g/g in tumor 24 h fllowing the 1000-g dose of (sFv')₂ was comparable to that seen 24 h after a 50-g dose of¹²⁵I-741F8 IgG, indicating that the use of large doses of (sFv')₂ may partially offset their rapid clearance. When two doses were administered by i.v. injection 24 h apart, the specificity of delivery to tumor observed after the first dose was maintained following the second injection. Tumor retention of¹²⁵I-741F8 (sFv')₂ was 0.32 g/g at 24 h and 0.22 g/g at 48 h following a single injection of 20 g/g, while 0.04 g/ml and 0.03 g/ml were retained in blood at the same assay times. After a second 20-g injection at the 24-h assay time, tumor retention increased to 0.49 g/g, and blood retention was 0.06 g/ml, at the 48-h point. These results suggest that multiple high-dose administrations of radiolabeled 741F8 (sFv')₂ may lead to the selective tumor localization of therapeutic radiation doses.Supported by National Cancer Institute (NCI) National Cooperative Drug Discovery Group grant U01 CA51880, CA06927, an appropriation from the Commonwealth of Pennsylvania, and the Bernard A. and Rebecca S. Bernard Foundation     

Expression of recombinant human acetylcholinesterase in transgenic tomato plants

Enzyme therapy for the prevention and treatment of organophosphate poisoning depends on the availability of large amounts of cholinesterases. Transgenic plants are being evaluated for their efficiency and cost-effectiveness as a system for the bioproduction of therapeutically valuable proteins. Here we report production of a recombinant isoform of human acetylcholinesterase in transgenic tomato plants. Active and stable acetylcholinesterase, which retains the kinetic characteristics of the human enzyme, accumulated in tomato plants. High levels of specific activity were registered in leaves (up to 25 nmol min(-1) mg protein(-1)) and fruits (up to 250 nmol min(-1) mg protein(-1)).     

Expression of active hBMP2 in transgenic tobacco plants

Bone morphogenetic protein 2 (BMP2) is important for bone tissue repair. The goal of this research is to construct a high level human BMP2 (hBMP2) expression system using transgenic tobacco plants as a bioreactor. Cauliflower mosaic virus (CaMV) 35S promoter, alfalfa mosaic virus (AMV) enhancer, tobacco mosaic virus (TMV) enhancer, matrix attachment regions (MARs) sequence, and “Kozak” sequence were used to construct recombinant expression vectors and the high-expression vectors were screened out through GUS-fusions assay. The promoter is the most important factor; double-CaMV 35S promoter is more effective than single promoter. The AMV or TMV enhancer is able to promote the foreign protein expression. After four-step purification, the activated hBMP2 (0.02% total soluble protein) was obtained. Our results suggested that the transgenic tobacco has great potential to be used as a bioreactor to produce hBMP2.