Secretion of proteins and antibody fragments from transiently transfected endothelial progenitor cells.

In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-ß-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-ß-amyloid Fab protein functions in ß-amyloid aggregate solubilization.

A novel excision selection method for isolation of antibodies binding antigens expressed specifically by rare cells in tissue sections.

There is a growing appreciation of single cell technologies to provide increased biological insight and allow development of improved therapeutics. The central dogma explains why single cell technologies is further advanced in studies targeting nucleic acids compared to proteins, as nucleic acid amplification makes experimental detection possible. Here we describe a novel method for single round phage display selection of antibody fragments from genetic libraries targeting antigens expressed by rare cells in tissue sections. We present and discuss the results of two selections of antibodies recognizing antigens expressed by perivascular cells surrounding capillaries located in a human brain section; with the aim of identifying biomarkers expressed by pericytes. The area targeted for selection was identified by a known biomarker and morphological appearance, however in situ hybridizations to nucleic acids can also be used for the identification of target cells. The antibody selections were performed directly on the tissue sections followed by excision of the target cells using a glass capillary attached to micromanipulation equipment. Antibodies bound to the target cells were characterized using ELISA, immunocytochemistry and immunohistochemistry. The described method will provide a valuable tool for the discovery of novel biomarkers on rare cells in all types of tissues.

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.

BACKGROUND: The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. RESULTS: Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. CONCLUSIONS: Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody.

To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor-like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor-like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application.

Recombinant antibody production evolves into multiple options aimed at yielding reagents suitable for application-specific needs.

BACKGROUND: Antibodies have been a pillar of basic research, while their relevance in clinical diagnostics and therapy is constantly growing. Consequently, the production of both conventional and fragment antibodies constantly faces more demanding challenges for the improvement of their quantity and quality. The answer to such an increasing need has been the development of a wide array of formats and alternative production platforms. This review offers a critical comparison and evaluation of the different options to help the researchers interested in expressing recombinant antibodies in their choice. RESULTS: Rather than the compilation of an exhaustive list of the recent publications in the field, this review intendeds to analyze the development of the most innovative or fast-growing strategies. These have been illustrated with some significant examples and, when possible, compared with the existing alternatives. Space has also been given to those solutions that might represent interesting opportunities or that investigate critical aspects of the production optimization but for which the available data as yet do not allow for a definitive judgment. CONCLUSIONS: The take-home message is that there is a clear process of progressive diversification concerning the antibody expression platforms and an effort to yield directly application-adapted immune-reagents rather than generic naked antibodies that need further in vitro modification steps before becoming usable.

Expression of bioactive anti-CD20 antibody fragments and induction of ER stress response in Arabidopsis seeds.

Seed-based expression system is an attractive platform for the production of recombinant proteins in molecular farming. Despite the many advantages of molecular farming, little is known about the effect of the different subcellular accumulation of recombinant proteins on the endoplasmic reticulum (ER) quality control system in host plants. In this study, we analyzed the expression of anti-CD20 antibody fragments in seeds of Arabidopsis thaliana (ecotype Columbia) and corresponding glycosylation mutants, and evaluated the influence of three different signal sequences on the expression levels of scFv-Fc of C2B8. The highest protein accumulation level, with a maximum of 6.12 % total soluble proteins, was observed upon fusing proteins to the signal peptide of Arabidopsis seed storage albumin 2. The ER stress responses in developing seeds at 13 days post-anthesis were also compared across different transgenic lines under normal and heat shock conditions. Based on the gene expression profiles of ER stress transducers, our results suggest that accumulation of antibody fragments in the ER exerts more stress on ER homeostasis. In addition, quantitative PCR results also implicate enhanced activation of ER-associated degradation in transgenic lines. Last but not the least, we also demonstrate the anti-tumor potency of plant-derived proteins by showing the anti-tumor activity of purified scFv-Fc proteins against Daudi cells. Together, our data implies that better understanding of the interaction between exogenous protein production and the cellular quality control system of the host plant is necessary for the development of an optimal expression strategy that will be especially beneficial to commercial protein manufacturing.

Targeting high affinity and epitope-distinct oligoclonal nanobodies to HER2 over-expressing tumor cells.

Modern anti-HER2 antibody therapy tends to exploit a panel of different antibodies against different epitopes on the antigen. For this aim, nanobodies are very striking targeting agents and can be easily produced against any cell-specific membrane antigen. The oligoclonal nanobodies can be used to block more than one functional epitope on a target antigen and inhibit the generation of escape variants associated with cancer therapy. In this study, 12 nanobody clones selected from an immune camel library were examined for their ability to differ between tumor markers. These oligoclonal nanobodies targeted breast cancer cells better than each individual nanobody. In epitope mapping, several nanobodies overlapped in the epitope recognized by trastuzumab and some of the non-overlapping nanobodies could affect the binding of trastuzumab to HER2. This study demonstrates that the oligoclonal nanobodies are potential therapeutic tools that can be used instead of, or in combination with trastuzumab to assess tumor viability during treatment.

Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis.

BACKGROUND: Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY) is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance. RESULTS: Single-chain variable fragments of immunoglobulins (scFvs) were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G4S)4 were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells. CONCLUSIONS: Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis.

Transgenic expression of single-chain anti-CTLA-4 Fv on beta cells protects nonobese diabetic mice from autoimmune diabetes.

T cell-mediated immunodestruction of pancreatic beta cells is the key process responsible for both the development of autoimmune diabetes and the induction of rejection during islet transplantation. In this study, we investigate the hypothesis that transgenic expression of an agonistic, membrane-bound single-chain anti-CTLA-4 Fv (anti-CTLA-4 scFv) on pancreatic beta cells can inhibit autoimmune processes by selectively targeting CTLA-4 on pathogenic T cells. Strikingly, transgenic expression of anti-CTLA-4 scFv on pancreatic beta cells significantly protected NOD mice from spontaneous autoimmune diabetes. Interestingly, local expression of this CTLA-4 agonist did not alter the diabetogenic properties of systemic lymphocytes, because splenocytes from transgenic mice or their nontransgenic littermates equally transferred diabetes in NOD/SCID recipients. By analyzing the T cell development in anti-CTLA-4 scFv/Th1/Th2 triple transgenic mice, we found that beta cell-specific expression of CTLA-4 agonist did not affect the development of Th1/Th2 or CD4(+)CD25(+) regulatory T cells. Most strikingly, islets from transgenic mice inhibited T cell response to immobilized anti-CD3 in a T cell-islet coculture system, suggesting a trans-mediated inhibition provided by transgenic islets. Finally, transgenic islets implanted in diabetic recipients survived much longer than did wild-type islets, indicating a therapeutic potential of this genetically modified islet graft in autoimmune diabetes.

The Intervening Removable Affinity Tag (iRAT) System for the Production of Recombinant Antibody Fragments.

Fv and Fab antibody fragments are versatile co-crystallization partners that aid in the structural determination of otherwise "uncrystallizable" proteins, including human/mammalian membrane proteins. Accessible methods for the rapid and reliable production of recombinant antibody fragments have been long sought. In this chapter, we describe the concept and protocols of the intervening removable affinity tag (iRAT) system for the efficient production of Fv and Fab fragments in milligram quantities, which are sufficient for structural studies. As an extension of the iRAT system, we also provide a new method for the creation of genetically encoded fluorescent Fab fragments, which are potentially useful as molecular devices in various basic biomedical and clinical procedures, such as immunofluorescence cytometry, bioimaging, and immunodiagnosis.

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.

Independent synthesis of light and heavy chains: quantitation of light-chain production by mouse myeloma variants.

The rates of immunoglobulin synthesis have been examined in two MFC-11 cell lines which were independently adapted to tissue culture and in light-chain-producing variants derived from each of them. One cell line synthesized 2.5 pg immunoglobulin/cell/h, while the other synthesized 3.6 pg immunoglobulin/cell/h. The ratio of heavy and light chains in the two cell lines was approximately the same, and the size of the intracellular pool of immunoglobulin was proportioned to the rate of synthesis. Variants which had spontaneously lost the ability to produce heavy chains continued to synthesize light chains at approximately the same rate as their parent cell line.

Identification of secretory component as an IgA receptor on rat hepatocytes.

Secretory component (SC) was found to be synthesized by isolated rat hepatocytes. SC was detected by radioimmunoassay and cultured hepatocytes were found to synthesize 0.078 microgram SC/10(6) hepatocytes in a 48-h period. SC was also present on the surface of hepatocytes as detected by the specific binding of radiolabeled anti-SC antibodies as well as by the detection of specific membrane staining in indirect immunofluorescence tests using specifically purified anti-SC antibodies. Rat SC was detected on hepatocytes and intestinal epithelial cells but not on peripheral blood lymphocytes, unfractionated spleen cells, or erythrocytes. Specific binding of radiolabeled rat dimeric IgA to rat hepatocytes was also observed and evidence was obtained to indicate that such binding was mediated by SC. Thus, prior incubation of hepatocytes with anti-SC prevented binding of radiolabeled IgA. Moreover, prior incubation of radiolabeled IgA with rat SC prevented binding of the IgA to isolated hepatocytes. Cells treated with 0.25% trypsin lost their ability to bind to radiolabeled dimeric IgA.

In vivo evaluation of anticoccidial effect of antibody fragments expressed in pea (Pasum sativum) on Eimeria tenella sporozoites.

Coccidiosis in chicken causes great economic losses. The increasing resistance of Eimeria species to anticoccidials has induced the search for alternative methods of control. In vivo antibody neutralization assay was conducted to study the inhibitory effect of nine antibody fragments (Ab1-Ab9) on Eimeria tenella sporozoites. These anti-E. tenella antibody fragments were expressed in pea plant (Pasum sativum). To assess the inhibitory effect on parasite reproduction, the in vivo antibody neutralization assay was done by retrograde infection of chicken with sporozoites previously incubated with antibody fragments. The pre-incubated sporozoites with the examined antibody fragments displayed a reduced ability to reproduce following intracloacal application to chicken (especially Ab1, Ab3, Ab5, and Ab9). Other antibody fragments (Ab2, Ab4, Ab6, Ab7, and Ab8) were less capable to reduce sporozoite infectivity and reproduction.

"Cleavable" hapten-biotin conjugates: preparation and use for the generation of anti-steroid single-domain antibody fragments.

Antibody engineering technology has the potential to provide artificial antibodies with higher performance than conventional antibodies. Filamentous phage particles are often used to express a vast diversity of mutated antibody fragments from which clones displaying improved fragments can be isolated. We recently showed that hapten-biotin conjugates, combined via a linker involving a reductively cleavable disulfide bond, are useful for isolating phage clones displaying high-affinity anti-hapten antibody fragments. Here we prepare cleavable hapten-biotin conjugates and use them to isolate anti-hapten antibody fragments with relatively low affinities. Three diagnostically important steroids (estradiol-17beta [E(2)], cortisol, and 17alpha-hydroxyprogesterone) were each coupled with a biotin derivative containing a disulfide bond. These conjugates could be bound simultaneously by their relevant anti-steroid antibody and NeutrAvidin, and their linkers were easily cleaved by dithiothreitol (DTT) treatment. The E(2)-biotin conjugate was used to generate anti-E(2) single-domain antibody fragments (sdAbs). Random point mutations were introduced by error-prone PCR into the gene fragment encoding the V(H) domain of a mouse anti-E(2) antibody, and these products were expressed as phagemid particles that were reacted with the E(2)-biotin conjugates that had already been immobilized on a solid-phase via NeutrAvidin. Thorough washing off of nonspecific phages and subsequent DTT treatment provided a phagemid clone that displayed a mutated sdAb with improved binding properties.

Cleavage of membrane secretory component to soluble secretory component occurs on the cell surface of rat hepatocyte monolayers.

Rat liver secretory component is synthesized as an integral membrane protein (mSC) and cleaved to an 80-kD soluble form (fSC) sometime during transcellular transport from the sinusoidal to the bile canalicular plasma membrane domain of hepatocytes. We have used 24-h monolayer cultures of rat hepatocytes to characterize the conversion of mSC to fSC. Cleavage of mSC in cultured hepatocytes is inhibited by the thiol protease inhibitors leupeptin, antipain, and E-64, but not by other inhibitors, including disopropylfluorophosphate, pepstatin, N-ethylmalemide, p-chloromercuribenzoic acid, and chloroquine. Leupeptin-mediated inhibition of cleavage is concentration dependent and reversible. In the presence or absence of leupeptin, only 10-20% of mSC is accessible at the cell surface. To characterize the behavior of surface as opposed to intracellular mSC, cell surface mSC was labeled with 125I by lactoperoxidase-catalyzed iodination at 4 degrees C. Cell surface 125I-mSC was converted to extracellular fSC at 4 degrees C in the absence of detectable internalization. Cleavage was inhibited by leupeptin and by anti-secretory component antiserum. Cleavage also occurred at 4 degrees C after cell disruption. In contrast, 125I-mSC that had been internalized from the cell surface was not converted to fSC at 4 degrees C in either intact or disrupted cells. Hepatocytes metabolically labeled with [35S]cys also released small quantities of fSC into the medium at 4 degrees C. The properties of fSC production indicate that cleavage occurs on the surface of cultured rat hepatocytes and not intracellularly. Other features of the cleavage reaction suggest that the mSC-cleaving protease is segregated from the majority of cell surface mSC, possibly within a specialized plasma membrane domain.

Mutations in immunoglobulin-producing mouse myeloma cells.

Three mouse myeloma cell lines were cloned in soft agar and screened by an antiserum overlay method for variants defective in secretion of the myeloma protein. Variants that had lost the capacity to synthesize heavy chains arose spontaneously at a high rate of about 10(-3) per cell per generation. Such variants lost the capacity to produce light chains at a similarly high rate. After cells were treated with the acridine half mustard ICR-191, variants occurred with an even higher incidence, and some of these synthesized heavy chains differing from that of the parent.

Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda.

A novel bacteriophage lambda vector system was used to express in Escherichia coli a combinatorial library of Fab fragments of the mouse antibody repertoire. The system allows rapid and easy identification of monoclonal Fab fragments in a form suitable for genetic manipulation. It was possible to generate, in 2 weeks, large numbers of monoclonal Fab fragments against a transition state analog hapten. The methods described may supersede present-day hybridoma technology and facilitate the production of catalytic and other antibodies.

Generation and assembly of secretory antibodies in plants.

Four transgenic Nicotiana tabacum plants were generated that expressed a murine monoclonal antibody kappa chain, a hybrid immunoglobulin A-G heavy chain, a murine joining chain, and a rabbit secretory component, respectively. Successive sexual crosses between these plants and filial recombinants resulted in plants that expressed all four protein chains simultaneously. These chains were assembled into a functional, high molecular weight secretory immunoglobulin that recognized the native streptococcal antigen I/II cell surface adhesion molecule. In plants, single cells are able to assemble secretory antibodies, whereas two different cell types are required in mammals. Transgenic plants may be suitable for large-scale production of recombinant secretory immunoglobulin A for passive mucosal immunotherapy. Plant cells also possess the requisite mechanisms for assembly and expression of other complex recombinant protein molecules.

Production of antibody fragments in Arabidopsis seeds.

Plants offer a number of attractive benefits over conventional mammalian or bacterial cell culture systems for the production of valuable pharmaceutical and industrial proteins. Currently, antibodies and their derived fragments represent the largest and most important group of biotechnological products in clinical trials. In particular, single-chain antibodies are an interesting class of biopharmaceuticals because they are able to overcome specific problems associated with full-length antibodies. Another valuable antibody format is the scFv-Fc: fusion of the Fc domain to a single-chain variable fragment restores antibody effector functions, allows purification, and mimics, despite being a 'single-gene' product, the bivalent properties of a full-length IgG. Although many different plant-based production platforms have been evaluated for antibody production, seeds are especially attractive because, as natural storage organs, they provide an optimal biochemical environment for the accumulation and long-term storage of large amounts of functional proteins. This chapter describes how to achieve high-level seed-specific expression of antibody fragments, how to select the best transgenic lines, and how to evaluate the accumulation level in the seed stocks from the selected lines.