Molecular mechanisms of action of anti-TNF-α agents - Comparison among therapeutic TNF-α antagonists.

Tumor necrosis factor (TNF)-α is a potent pro-inflammatory and pathological cytokines in inflammatory diseases such as rheumatoid arthritis and inflammatory bowel diseases. Anti-TNF-α therapy has been established as an efficacious therapeutic strategy in these diseases. In clinical settings, three monoclonal anti-TNF-α full IgG1 antibodies infliximab, adalimumab, and golimumab, PEGylated Fab' fragment of anti-TNF-α antibody certolizumab pegol, extracellular domain of TNF receptor 2/IgG1-Fc fusion protein etanercept, are almost equally effective for rheumatoid arthritis. Although monoclonal full IgG1 antibodies are able to induce clinical and endoscopic remission in inflammatory bowel diseases, certolizumab pegol without Fc portion has been shown to be less effective for inflammatory bowel diseases compared to full IgG1 antibodies. In addition, there are no evidences that etanercept leads clinical remission in inflammatory bowel diseases. Besides the common effect of anti-TNF-α agents on neutralization of soluble TNF-α, each anti-TNF-α agent has its own distinctive pharmacological properties which cause the difference in clinical efficacies. Here we focus on the distinctions of action of anti-TNF-α agents especially in following points; (1) blocking ability against ligands, transmembrane TNF-α and lymphotoxin, (2) effects toward transmembrane TNF-α-expressing cells, (3) effects toward Fcγ receptor-expressing cells, (4) degradation and distribution in inflamed tissue. Accumulating evidence will give us the idea how to modify anti-TNF-α agents to enhance the clinical efficacy in inflammatory diseases.

Ultrafast Separation and Analysis of Monoclonal Antibody Aggregates Using Membrane Chromatography.

We discuss a method for rapid and cost-effective analysis of monoclonal antibody (mAb) aggregates. Hydrophobic interaction membrane chromatography, which was previously shown to be highly suitable for such separation and analysis, was used in a recently developed format referred to as laterally fed membrane chromatography (or LFMC). A stack of rectangular polyvinylidene fluoride (or PVDF) membranes having 0.22 µm pores housed within a modified analytical-scale LFMC device was used for analyzing aggregate types and content in different monoclonal antibody samples. High-resolution separations could be achieved in less than 1.5 min, this being faster than other currently available techniques such as size exclusion ultraperformance liquid chromatography (SE-UPLC). Moreover, the operating pressure was less than 200 kPa, which eliminated the need for an expensive high-pressure pump and chromatography system. The resolution obtained using the LFMC was comparable to that obtained using SE-UPLC. The effect of design variations such as change in dead volume and pillar size within the lateral channels within the LFMC device was also examined.

A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment.

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS-KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293.

Comparative study of recent wide-pore materials of different stationary phase morphology, applied for the reversed-phase analysis of recombinant monoclonal antibodies.

Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ~250 Å and macropores of ~ 1.1 µm, a column packed with 3.6 µm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 µm particles having pore size of ~300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene-divinyl benzene) organic monolithic column, with a macropore size of approximately 1 µm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 µm and fully porous 1.7 µm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 µm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 µm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.

Role of HIV membrane in neutralization by two broadly neutralizing antibodies.

Induction of effective antibody responses against HIV-1 infection remains an elusive goal for vaccine development. Progress may require in-depth understanding of the molecular mechanisms of neutralization by monoclonal antibodies. We have analyzed the molecular actions of two rare, broadly neutralizing, human monoclonal antibodies, 4E10 and 2F5, which target the transiently exposed epitopes in the membrane proximal external region (MPER) of HIV-1 gp41 envelope during viral entry. Both have long CDR H3 loops with a hydrophobic surface facing away from the peptide epitope. We find that the hydrophobic residues of 4E10 mediate a reversible attachment to the viral membrane and that they are essential for neutralization, but not for interaction with gp41. We propose that these antibodies associate with the viral membrane in a required first step and are thereby poised to capture the transient gp41 fusion intermediate. These results bear directly on strategies for rational design of HIV-1 envelope immunogens.

Echinoderms as blueprints for biocalcification: regulation of skeletogenic genes and matrices.

Echinoderms have an extensive endoskeleton composed of magnesian calcite, a form of calcium carbonate that contains small amounts of magnesium carbonate and occluded matrix proteins. Adult sea urchins have several calcified structures, including test, teeth, and spines, composed of numerous ossicles which form a three-dimensional meshwork of mineral trabeculae, the stereom. The biomineral development begins in 24-hour-old embryos within the primary mesenchyme cells (PMCs), the only cells producing a set of necessary matrix proteins. The deposition of the biomineral occurs in a privileged extracellular space produced by the fused filopodial processes of the PMCs. We showed for the first time that signals from ectoderm cells overlying PMCs play an important role in the regulation of biomineralization-related genes. It is believed that growth factors are produced by ectoderm cells and released into the blastocoel where they interact with cognate receptor tyrosine kinases restricted to PMCs, which activate signaling cascades regulating the expression of biomineralization-related genes. We demonstrated the implication of a TGF-beta family factor by a perturbation model in which skeleton elongation was indirectly blocked by monoclonal antibodies to an extracellular matrix (ECM) protein located on the apical surface of ectoderm. Thus, it was inferred that interfering with the binding of the ECM ligand, a member of the discoidin family, to its cell surface receptor, a ßC integrin, disrupts the ectodermal cell signaling cascade, resulting in reduced or aberrant skeletons. During the last few years, we analyzed the expression of biomineralization-related genes in other examples of experimentally induced skeleton malformations, produced by the exposure to toxic metals, such as Cd and Mn or ionizing radiations, such as UV-B and X-rays. Besides the obvious toxicological implication, since the mis-expression of spicule matrix genes paralleled skeleton defects, we believe that by means of these studies we can dissect the molecular steps taking place and possibly understand the physiological events regulating embryonic biomineralization.

The epitope for the polyol-responsive monoclonal antibody 8RB13 is in the flap-domain of the beta-subunit of bacterial RNA polymerase and can be used as an epitope tag for immunoaffinity chromatography.

Polyol-responsive monoclonal antibodies (PR-mAbs) are useful for the purification of proteins in an easy, one step immunoaffinity step. These antibodies allow for gentle purification of proteins and protein complexes using a combination of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotrophic salt in the eluting buffer. mAb 8RB13 has been characterized as one of these PR-mAbs and has been used to purify RNA polymerase from five species of bacteria. Here the epitope for 8RB13 has been identified as PEEKLLRAIFGEKAS, a sequence that is highly conserved in the ß-subunit of bacterial RNA polymerase. This sequence is located in the "beta-flap" domain of RNA polymerase (and essentially comprises the "flap-tip helix"), an important binding site for sigma70. This location explains why only the core RNAP is purified using this mAb. This amino acid sequence has been developed into an epitope tag that can be used to purify a target protein from either bacterial or eukaryotic cells when genetically fused to a protein of interest.

Coiled coil peptides as universal linkers for the attachment of recombinant proteins to polymer therapeutics.

We have designed, synthesized, and characterized peptides containing four repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide K). While the peptides alone adopt in aqueous solutions a random coil conformation, their equimolar mixture forms heterodimeric coiled coils as confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected to the N-terminus of peptide E via a short poly(ethylene glycol) spacer. The terminal azide group enabled conjugation of the peptide with a synthetic drug carrier based on the N-(2-hydroxypropyl)methacrylamide copolymer containing propargyl groups using "click" chemistry. When incorporated into the polymer drug carrier, peptide E formed a stable noncovalent complex with peptide K belonging to a recombinant single-chain fragment (scFv) of the M75 antibody. The complex thereby mediates a noncovalent linkage between the polymer drug carrier and the protein. The recombinant scFv antibody fragment was selected as a targeting ligand against carbonic anhydrase IX-a marker overexpressed by tumor cells of various human carcinomas. The antigen binding affinity of the polymer-scFv complex was confirmed by ELISA. This approach offers a well-defined, specific, and nondestructive universal method for the preparation of protein (antibody)-targeted polymer drug and gene carriers designed for cell-specific delivery.

Cloning and characterization of heavy and light chain genes encoding the FimA-specific monoclonal antibodies that inhibit Porphyromonas gingivalis adhesion.

FimA of Porphyromonas gingivalis, a major pathogen in periodontitis, is known to be closely related to the virulence of these bacteria and has been suggested as a candidate for development of a vaccine against periodontal disease. In order to develop a passive immunization method for inhibiting the establishment of periodontal disease, B hybridoma clones 123-123-10 and 256-265-9, which produce monoclonal antibodies (Mabs) specific to purified fimbriae, were established. Both mAbs reacted with the conformational epitopes displayed by partially dissociated oligomers of FimA, but not with the 43 kDa FimA monomer. Gene sequence analyses of full-length cDNAs encoding heavy and light chain immunoglobulins enabled classification of the genes of mAb 123-123-10 as members of the mVh II (A) and mVκ I subgroups, and those of mAb 256-265-9 as members of the mVh III (D) and mVκ I subgroups. More importantly, 50 ng/mL of antibodies purified from the culture supernatant of antibody gene-transfected CHO cells inhibited, by approximately 50%, binding of P. gingivalis to saliva-coated hydroxyapatite bead surfaces. It is expected that these mAbs could be used as a basis for passive immunization against P. gingivalis-mediated periodontitis.

Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans.

A functional comparison was made between a monoclonal secretory antibody generated in transgenic plants and its parent murine IgG antibody.The affinity constants of both antibodies for a Streptococcus mutans adhesion protein were similar. However the secretory antibody had a higher functional affinity due to its dimeric structure. In the human oral cavity, the secretory antibody survived for up to three days, compared with one day for the IgG antibody. The plant secretory antibody afforded specific protection in humans against oral streptococcal colonization for at least four months. We demonstrate that transgenic plants can be used to produce high affinity, monoclonal secretory antibodies that can prevent specific microbial colonization in humans. These findings could be extended to the immunotherapeutic prevention of other mucosal infections in humans and animals.

V kappa gene family in (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies that express CGAT (or pGAT) public idiotypic specificities. Protein and mRNA sequencing of eight monoclonal V kappa chains.

A large proportion of (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies expresses public idiotypic specificities, termed CGAT (or pGAT), that require the presence of both the heavy and the light chains in order to be expressed. We report in this paper the complete sequence of eight V kappa regions pertaining to eight anti-GAT monoclonal antibodies derived from three strains of mice: BALB/c, DBA/2, and C57BL/6. The methodology used a combination of NH2-terminal amino acid and mRNA nucleotide sequencing. All eight sequences analyzed, although highly homologous and all pertaining to the same V kappa 1 subgroup, allowed definition of three germline genes that are likely to be present in all three strains of mice and also in NZB. It seems likely, however, that any given strain may not necessarily use all three genes for making anti-GAT antibodies. The search for structural correlates of idiotypes could not be framed in a simple picture, but our data suggest that similar idiotopes may result from different interacting primary structures, leading to structural homologies that should be visualized at three-dimensional level.

Generation and characterization of genetically stable heterohybridomas producing foot-and-mouth disease virus-specific porcine monoclonal antibodies.

This report covers the methodology for generation of stable heterohybridoma clones producing Foot-and-mouth disease virus (FMDV) reactive porcine monoclonal antibodies (mAbs). Swine received five inoculations of an inactivated O1 Manisa FMDV vaccine prior to the harvest of splenocytes. Due to the lack of a species-specific hybridoma fusion partner, the Sp2/0 murine myeloma cell line was utilized for the formation of porcine-murine heterohybridoma clones. Twenty-nine FMDV-reactive parental clones were generated. Following sub-cloning and monitoring of reactivity over 20 serial passages, eleven subclones derived from unique parental origins were characterized and are reported herein. This methodology demonstrated the production of porcine mAbs by fusion of porcine splenocytes from immunized pigs with murine myeloma cells to generate heterohybridomas. The porcine immune response may differ from the murine immune response in relation to recognized epitopes. Therefore, application of this methodology may provide valuable resources for swine immunology and enhance the understanding of the mechanisms for antibody based protection from diseases in swine.

Immunotoxin Screening System: A Rapid and Direct Approach to Obtain Functional Antibodies with Internalization Capacities.

Toxins, while harmful and potentially lethal, have been engineered to develop potent therapeutics including cytotoxins and immunotoxins (ITs), which are modalities with highly selective targeting capabilities. Currently, three cytotoxins and IT are FDA-approved for treatment of multiple forms of hematological cancer, and additional ITs are tested in the clinical trials or at the preclinical level. For next generation of ITs, as well as antibody-mediated drug delivery systems, specific targeting by monoclonal antibodies is critical to enhance efficacies and reduce side effects, and this methodological field remains open to discover potent therapeutic monoclonal antibodies. Here, we describe our application of engineered toxin termed a cell-based IT screening system. This unique screening strategy offers the following advantages: (1) identification of monoclonal antibodies that recognize cell-surface molecules, (2) selection of the antibodies that are internalized into the cells, (3) selection of the antibodies that induce cytotoxicity since they are linked with toxins, and (4) determination of state-specific activities of the antibodies by differential screening under multiple experimental conditions. Since the functional monoclonal antibodies with internalization capacities have been identified successfully, we have pursued their subsequent modifications beyond antibody drug conjugates, resulting in development of immunoliposomes. Collectively, this screening system by using engineered toxin is a versatile platform, which enables straight-forward and rapid selection for discovery of novel functional antibodies.

Coincident exposure of phosphatidylethanolamine and anionic phospholipids on the surface of irradiated cells.

The major anionic phospholipid, phosphatidylserine (PS), and the neutral phospholipid, phosphatidylethanolamine (PE), are largely confined to the inner leaflet of the plasma membrane bilayer in mammalian cells under normal conditions. This asymmetry is lost when cells undergo apoptosis, become activated, or are exposed to irradiation, reactive oxygen species or certain drugs. It is not known whether exposure of anionic phospholipids (APLs) and PE occurs simultaneously or in the same region of the plasma membrane. Here we examined the coincidence of exposure of APLs and PE on the surface of bovine aortic endothelial cells and NS0 myeloma cells after irradiation. The cells were irradiated (5 Gy) and stained for APLs and PE using liposomes coated with either an Fab' fragment of a PS-binding antibody (bavituximab) or a PE-binding peptide (duramycin). Using live cell imaging and flow cytometry, we showed that irradiation leads to synchronous externalization of APLs and PE. The time course of appearance of APLs and PE on the cell surface was the same and the two phospholipid types remained colocalized over time. Distinct patches double positive for APLs and PE were visible. Larger areas of APLs and PE appeared to have detached from the cytoskeleton to form membrane blebs which protruded and drifted on the cell surface. We conclude that APLs and PE coincidently appear on the external leaflet of the plasma membrane of cells after irradiation. Probably, this is because PE and the major APL, PS, share common regulatory mechanisms of translocation.

Gene delivery into ischemic myocardium by double-targeted lipoplexes with anti-myosin antibody and TAT peptide.

The treatment of myocardial ischemia using gene therapy is a rather novel but promising approach. Gene delivery to target cells may be enhanced by using double-targeted delivery systems simultaneously capable of extracellular accumulation and intracellular penetration. With this in mind, we have used low cationic liposomes-plasmid DNA complexes (lipoplexes) modified with cell-penetrating transactivating transcriptional activator (TAT) peptide (TATp) and/or with monoclonal anti-myosin monoclonal antibody 2G4 (mAb 2G4) specific toward cardiac myosin, for targeted gene delivery to ischemic myocardium. In vitro transfection of both normoxic and hypoxic cardiomyocytes was enhanced by the presence of TATp as determined by fluorescence microscopy and ELISA. The in vitro transfection was further enhanced by the additional modification with mAb 2G4 antibody in the case of hypoxic, but not normoxic cardiomyocytes. However, we did not observe a synergism between TATp and mAb 2G4 ligands under our experimental condition. In in vivo experiments, we have clearly demonstrated an increased accumulation of mAb 2G4-modified TATp lipoplexes in the ischemic rat myocardium and significantly enhanced transfection of cardiomyocytes in the ischemic zone. Thus, the genetic transformation of normoxic and hypoxic cardiomyocytes can be enhanced by using lipoplexes modified with TATp and/or mAb 2G4. Such complexes also demonstrate an increased accumulation in the ischemic myocardium and effective transfection of hypoxic cardiomyocytes in vivo.

High-level protein expression in scalable CHO transient transfection.

Chinese hamster ovary cells (CHO) have been extensively utilized as the production platform for therapeutic proteins including monoclonal antibodies in pharmaceutical industry. For early development, it would be advantageous to rapidly produce large amounts of protein in the same cell line; therefore, development of a CHO transient transfection platform with high protein expression level is highly desirable. Here, we describe the development of such a platform in CHO cells. Polyethylenimine (PEI) was used as the transfection reagent. Different media were screened for the best transfection and expression performance, and UltraCHO was chosen as the best performer. DMSO and lithium acetate (LiAc) were discovered to improve CHO transient transfection expression levels significantly. A 14-day fed-batch process was successfully developed to further increase production yield. With an optimized transient transfection process, we were able to express monoclonal antibody (Mab) in CHO cells at a high level, averaging 80 mg/L. The process was successfully scaled up to 10 L working volume in a 20 L wave bioreactor. As expected, the Mabs had similar glycosylation patterns in comparison to the Mabs produced from a stably transfected CHO cell line, while in contrast Mabs expressed transiently from HEK293EBNA cells differed.

In silico site-directed mutagenesis of neutralizing mAb 4C4 and analysis of its interaction with G-H loop of VP1 to explore its therapeutic applications against FMD.

Investigating the behaviour of bio-molecules through computational mutagenesis is gaining interest to facilitate the development of new therapeutic solutions for infectious diseases. The antigenetically variant genotypes of foot and mouth disease virus (FMDV) and their subsequent infections are challenging to tackle with traditional vaccination. In such scenario, neutralizing antibodies might provide an alternate solution to manage the FMDV infection. Thus, we have analysed the interaction of the mAb 4C4 with a synthetic G-H loop of FMDV-VP1 through in silico mutagenesis and molecular modelling. Initially, a set of 25,434 mutants were designed and the mutants having better energetic stability than 4C4 were clustered based on sequence identity. The best mutant representing each cluster was selected and evaluated for its binding affinity with the antigen in terms of docking scores, interaction energy and binding energy. Six mutants have confirmed better binding affinities towards the antigen than 4C4. Further, interaction of these mutants with the natural G-H loop that is bound to mAb SD6 was also evaluated. One 4C4 variant having mutations at the positions 2034(N→L), 2096(N→C), 2098(D→Y), 2532(T→K) and 2599(A→G) has revealed better binding affinities towards both the synthetic and natural G-H loops than 4C4 and SD6, respectively. A molecular dynamic simulation for 50 ns was conducted for mutant and wild-type antibody structures which supported the pre-simulation results. Therefore, these mutations on mAb 4C4 are believed to provide a better antibody-based therapeutic option for FMD. Communicated by Ramaswamy H. Sarma.

Expression and purification of two anti-CD19 single chain Fv fragments for targeting of liposomes to CD19-expressing cells.

Antibody-targeted liposomal anticancer drugs combine the specificity of antibodies with large payloads of entrapped drugs. We previously showed that liposomal doxorubicin (DXR) targeted via anti-CD19 monoclonal antibodies (mAb) or their Fab' fragments against the B-cell antigen CD19 led to improved therapeutic effects in murine B-cell lymphoma models relative to non-targeted liposomal DXR. We now are examining the use of anti-CD19 single chain fragments of the antibody variable region (scFv) as a targeting moiety, to test the hypothesis that scFv have advantages over full-sized mAb or Fab' fragments. We expressed two different anti-CD19 scFv constructs, HD37-C and HD37-CCH in E. coli, and purified the scFvs using two different methods. The HD37-CCH construct was selected for coupling studies due to its relative stability and activity in comparison to HD37-C. When coupled to liposomes, the HD37-CCH scFv showed increased binding in vitro to CD19-positive Raji cells, compared to non-targeted liposomes. Cytotoxicity data showed that HD37-CCH scFv-targeted liposomes loaded with DXR were more cytotoxic than non-targeted liposomal DXR. Our results suggest that anti-CD19 scFv constructs should be explored further for their potential in treating B-lymphoid leukemias and lymphomas.

Characterization of mechanical properties of transgenic tobacco roots expressing a recombinant monoclonal antibody against tooth decay.

In this article, we describe a new approach that allows the determination of the magnitude of force required to break single plant roots. Roots were taken from transgenic tobacco plants, expressing a secreted monoclonal antibody. They were divided into four key developmental stages. A novel micromanipulation technique was used to pull to breakage, single tobacco roots in buffer in order to determine their breaking force. A characteristic uniform step-wise increase in the force up to a peak force for breakage was observed. The mean breaking force and mean work done were 101mN and 97microJ per root respectively. However, there was a significant increase in breaking force from the youngest white roots to the oldest, dark red-brown roots. We speculate that this was due to increasing lignin deposition with root stage of development (shown by phloroglucinol staining). No significant differences between fresh root mass, original root length, or mean root diameter for any of the root categories were found, displaying their uniformity, which would be beneficial for bioprocessing. In addition, no significant difference in antibody yield from the different root categories was found. These data show that it is possible to characterise the force requirements for root breakage and should assist in the optimisation of recombinant protein extraction from these roots.

Stable antibody expression at therapeutic levels using the 2A peptide.

Therapeutic monoclonal antibodies (mAbs) are currently being developed for the treatment of cancer and other diseases. Despite clinical success, widespread application of mAb therapies may be limited by manufacturing capabilities. In this paper, we describe a mAb delivery system that allows continuous production of a full-length antibody at high-concentrations in vivo after gene transfer. The mAb is expressed from a single open reading frame by linking the heavy and light chains with a 2A self-processing peptide derived from the foot-and-mouth disease virus. Using this expression system, we generated a recombinant adeno-associated virus vector encoding the VEGFR2-neutralizing mAb DC101 (rAAV8-DC101). A single dose of rAAV8-DC101 resulted in long-term expression of >1,000 microg/ml of DC101 in mice, demonstrating significant anti-tumor efficacy. This report describes the first feasible gene therapy approach for stable delivery of mAbs at therapeutic levels, which may serve as an attractive alternative to direct injection of mAbs.