Expression Of Recombinant Antibodies Research Articles

Generation of functional scFv intrabodies for triggering anti-tumor immunity

Intracellular expression of recombinant antibodies (intrabodies) allows to interfere with the functions of oncogenic or viral molecules expressed in different cell compartments and has therefore a vast clinical potential in therapy. Although the use of phage-display libraries has made it possible to select Fab or single chain Fv (scFv) antibody fragments usable for intracellular targeting, a major source of recombinant antibodies for therapeutic use still remains hybridoma B cells producing well-characterized monoclonal antibodies (mAbs). However, the cloning and the intracellular expression of antibody fragments derived from mAbs can be markedly hampered by a number of technical difficulties that include failure of cloning functional variable regions as well as lack of binding of the antibody fragments to the targeted molecule in an intracellular environment. We discuss herein various molecular methods that have been developed to generate functional recombinant antibody fragments usable as anti-tumor triggering agents when expressed in tumor cells. Such antibodies can neutralize or modify the activity of oncogenic molecules when addressed in specific subcellular compartments and/or they can be used to trigger anti-tumor immunity when expressed on tumor cell surface.

Prokaryotic expression of antibodies and affibodies.

Recent advances have been made in the development of systems for the display and expression of recombinant antibodies and affibodies in filamentous phages, Escherichia coli and other prokaryotic cells. Emphasis has been placed on improving phage and phagemid vectors, alternative systems for expression in different cellular compartments (e.g. the outer membrane, periplasm, cytoplasm and extracellular secretion) and novel multimerization systems for generating bivalent or multivalent binding molecules.

Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen.

In planta expression of recombinant antibodies recognizing pathogen-specific antigens has been proposed as a strategy for crop protection. We report the expression of fusion proteins comprising a Fusarium-specific recombinant antibody linked to one of three antifungal peptides (AFPs) as a method for protecting plants against fungal diseases. A chicken-derived single-chain antibody specific to antigens displayed on the Fusarium cell surface was isolated from a pooled immunocompetent phage display library. This recombinant antibody inhibited fungal growth in vitro when fused to any of the three AFPs. Expression of the fusion proteins in transgenic Arabidopsis thaliana plants conferred high levels of protection against Fusarium oxysporum f.sp. matthiolae, whereas plants expressing either the fungus-specific antibody or AFPs alone exhibited only moderate resistance. Our results demonstrate that antibody fusion proteins may be used as effective and versatile tools for the protection of crop plants against fungal infection.

ANTIBODY-BASED PATHOGEN RESISTANCE IN PLANTS

Genetic engineering can be used to create plants with enhanced resistance to disease. Several strategies have been developed and tested, including expression of pathogen-derived sequences, the modification and overexpression of endogenous resistance genes, and the expression of heterologous resistance genes from diverse sources. Another promising approach is the expression of recombinant antibodies that bind to and neutralize essential components of the pathogen and therefore interfere with its infection cycle. The impact of this approach depends on appropriate target selection, careful antibody design, subcellular targeting of the recombinant antibodies and antibody accumulation levels and stability. This review describes the current status of antibody-based disease resistance in plants, focussing on optimization and the remaining obstacles to its widespread use.

Expression of recombinant antibodies in mammalian cell lines.

Since the advent of hybridoma technology, mammalian-cell culture has been employed for the expression and high-level production of monoclonal antibodies (MAbs). Recent adaptations in recombinant technology have developed the use of numerous prokaryotic and eukaryotic systems for the expression of heterologous molecules. The major systems used for MAb expression have been reviewed and compared (,), and a number of the key methodologies are detailed within this series. Prokaryotic expression systems offer the potential of high production yields at a substantial reduced cost of goods (see Chapter 14). However, because of the complexity of the protein-folding pathway, bacterial expression has been limited to small antibody fragments, and in some cases may require refolding to produce a biologically active product. Another limitation is that recombinant MAbs expressed in bacteria are aglycosylated and can result in the reduction or loss of biological effector functions (). For large, multidomain molecules such as full-length MAbs or complex recombinant antibody fragments, eukaryotic systems such as mammalian or yeast expression have been utilized. Currently, nine USFDA-licensed Mabs for in vivo human use have been expressed from mammalian-cell culture for commercial production.

Optimization of codon pair use within the (GGGGS) 3 linker sequence results in enhanced protein expression

Here, we report that a significant increase in recombinant fusion antibody expression can be accomplished by adjusting the nucleotide sequence to conform to certain codon pairing rules. We investigated the expression of a protein in which a single chain Fv specific for HER2/ neu with V H and V L joined by a flexible (GGGGS) 3 linker was linked to the C H3 of a human anti-rat transferrin receptor IgG3 heavy chain with the same flexible (GGGGS) 3 linker. In initial experiments we failed to achieve significant expression of this protein. However, when we made a single nucleotide change in each (GGGGS) 3 linker we were able to achieve expression The change of one nucleotide within each linker did not alter either the amino acid sequence or the frequency score of these codon triplets’ usage in mammalian cells. Instead they removed two codon pairs predicted to be detrimental to expression. In a transient transfection assay we find that this change results in an over 30-fold increase in expression that is not the result of an increase in the level of accumulated mRNA. In addition, the changes made it possible to isolate stably transfected mammalian cell clones producing high levels of fusion protein, which had not been possible using the original gene.

Recombinant antibodies against subcellular fractions used to track endogenous Golgi protein dynamics in vivo.

Generation of specific antibodies against enriched subcellular fractions is a powerful strategy to identify and characterize cellular components. We show that recombinant antibodies can be selected in vitro by phage display against complex subcellular fractions, namely microtubule-binding proteins and Golgi stacks. This technique has allowed us to overcome many limitations of the classical animal-based approach and generate cell biology-compliant antibodies. In addition, we show that intracellular expression of GFP-tagged recombinant antibodies can reveal the dynamics of endogenous proteins in vivo. Endogenous Giantin is very static and outlines the Golgi in living cells. It accumulates neither onto Golgi-derived tubules upon Brefeldin A treatment before Golgi disappearance, nor onto de novo formed Golgi mini-stacks upon microtubule depolymerization, and remains instead on the 'old' pericentriolar Golgi. This suggests that, in contrast to other Golgi matrix proteins, endogenous Giantin is very stably associated with the Golgi and does not efficiently recycle to the ER. Altogether, we show that the antibody phage display technique represents an efficient alternative to rapidly generate versatile antibodies that represent new tools to study protein function.

Achieving plant disease resistance by antibody expression

Molecular biotechnology has provided powerful new measures for the control of crop disease. Crops can now be engineered to be resistant through creation of transgenic plants producing recombinant proteins, pathogen-related proteins, or antisense RNAs that block pathogenesis. Antibody engineering is a novel approach to create pathogen-resistant plants, which is based on the expression of recombinant antibodies (rAbs) or rAb fragments that inactivate pathogens and pathogen proteins. The feasibility of this approach has been shown for viral pathogens, where a high degree of resistance has been obtained. The effectiveness of antibody-based resistance is related to the antibody affinity for the target protein, subcellular targeting of the rAb, and the limitations of current rAb expression technology, such as antibody stability in cellular compartments. Antibody-based resistance can create plants that are resistant to pathogens and do not require the undesirable chemical controls currently used in agriculture. Importantly, recombinant-antibody expression can be optimized to overcome any resistance developed by pathogens and ‘pyramided’ to create resistance to several pathogens. As more suitable target proteins are identified in plant pathogens, the use of antibody-based resistance and other traits will help to increase crop yields and reduce agrochemical usage. We anticipate that antibody-based resistance will become a major strategy for the molecular breeding of pathogen-resistant crops and pathogen-resistant plant lines.

Affinity Maturation of Natural Antibody Using a Chain Shuffling Technique and the Expression of Recombinant Antibodies in Escherichia coli

The affinity of natural antibody (Ka = 8 × 106 M−1) recognizing preS1 of hepatitis B virus (HBV) was improved by replacing the heavy (H) chain gene with repertoires of VH genes, obtained from two nonimmunized donors. Two separate clones, 1C2 and 1E4, showed affinities of 2.3 × 107 and 5.2 × 107 M−1, which were increased by factors of 2.8 and 6.5, respectively, compared to the parental clone. Recombinant scFvs (rscFvs) were expressed as fusion protein with minor coat protein, pIII, and secreted into medium after 3 h of induction with 1 mM IPTG. The expression level of functional rscFv capable of binding to preS1 reached a peak after 6–10 h (1C2) and 8–10 h (1E4) of IPTG induction, and afterwards decreased gradually. In order to achieve the overexpression of rscFv in E. coli, gene encoding scFv of 1C2 or 1E4 was inserted into pRSET vector. RscFvs were overexpressed as cytoplasmic inclusion bodies in E. coli BL 21 strain, which were denatured and carefully refolded using a continuous dialysis system. The purified recombinant fragments were pure when analyzed by SDS–PAGE and had the predicted size of 34 kDa. Clone 1E4 used the heavy chain gene belonging to family VII and subgroup III. Chain shuffling offers an alternative to random point mutation for affinity maturation of human antibody in vitro.

Delivery of Immunoprophylactics in Transgenic Plants

The search for economic alternatives for the production of recombinant immunoprophylactics has resulted in the concept of generating them in plants at relatively low cost. Two basic strategies are employed: the expression of recombinant antigens or antibodies in transgenic plants, or the presentation of small antigen epitopes on the surface of genetically modified plant viruses. Both approaches have resulted in the production of biologically active immunoprophylactics that have been shown in animal studies to protect from the development of disease symptoms. An especially interesting feature of producing vaccines in edible plant parts is the potential to use the transgenic plant as a vehicle for oral delivery. Substantial progress has been made over the past few years, and the first clinical trials have demonstrated the potential of this new technology. Future challenges are to express the immunoprophylactics at controlled, high concentrations in plants that could be grown locally where they are needed, and to develop easy and standardised administration procedures.

Ras and p53 intracellular targeting with recombinant single-chain Fv (scFv) fragments: a novel approach for cancer therapy?

Intracellular expression of recombinant antibodies allows one to interfere with the functions of oncogenic molecules expressed in various cell compartments and has therefore a vast clinical potential in cancer therapy. We inhibited the functions of oncogenic Ras mutant forms by intracellular expression of a neutralizing single-chain antibody (scFv). In vitro studies indicated that the scFv is expressed in the cytosol of Xenopus laevis oocytes and of tumor cells, blocks ras-mediated activation processes, and induces tumor cell death. In vivo studies performed using scFv cDNA inserted into an adenoviral vector showed that the scFv dramatically affects tumor growth. Second, intracellular expression of scFvs directed against p53 indicated that these antibody fragments can be successfully targeted to cell nucleus, bind p53, and partially restore the transcriptional activity of p53 mutants in human tumor cells. Thus, intracellular scFvs directed against oncogenic molecules may represent a new class of antitumor agents.

Molecular farming of recombinant antibodies in plants.

'Molecular farming' is the production of recombinant proteins in plants. It is intended to harness the power of agriculture to cultivate and harvest transgenic plants producing recombinant therapeutics. Molecular farming has the potential to provide virtually unlimited quantities of recombinant antibodies for use as diagnostic and therapeutic tools in both health care and the life sciences. Importantly, recombinant antibody expression can be used to modify the inherent properties of plants, for example by using expressed antipathogen antibodies to increase disease resistance. Plant transformation is technically straightforward for model plant species and some cereals, and the functional expression of recombinant proteins can be rapidly analyzed using transient expression systems in intact or virally infected plants. Protein production can then be increased using plant suspension cell production in fermenters, or by the propagation of stably transformed plant lines in the field. Transgenic plants can be exploited to produce organs rich in a recombinant protein for its long-term storage. This demonstrates the promise of using transgenic plants as bioreactors for the 'molecular farming' of recombinant therapeutics, blood substitutes and diagnostics, such as recombinant antibodies.

Cloning, Expression, and Characterization of Recombinant Fab Antibodies against Dioxin

Using two hybridoma cell lines (DD1 and DD3) secreting anti-dioxin monoclonal antibodies as a source for messenger RNA and cDNA, light and heavy chain gene fragments of Fab domains were amplified by the polymerase chain reaction (PCR). The amplified gene fragments were cloned into the pFabUSDAI vector for expression of recombinant Fab antibodies in Escherichia coli. Expression of the soluble and functional recombinant Fab antibodies (designated rFab1-1 and rFab3-3) was confirmed by an indirect immunoassay using dioxin conjugated to rabbit serum albumin. On the basis of these rFabs, two competitive inhibition immunoassays using 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) as a competitor were developed. The concentration of 2,3,7,8-TCDD required to inhibit color development by 50% (IC50) determined from the dose response curves for rFAB1-1 and rFAB3-3 were 10.4 ± 2.4 and 12.2 ± 6.0 ng/mL, respectively. The binding properties of both rFab antibodies for other chemically related compounds were relatively similar to those of their respective monoclonal antibodies and enzymatically derived Fab fragments. Keywords: Dioxin; recombinant antibody; cloning antibody genes; Fab; expression vector; Escherichia coli; immunoassay

Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity.

Expression and stability of immunoglobulins in transgenic plants have been investigated and optimized by accumulation in different cellular compartments as cytosol, apoplastic space and endoplasmic reticulum (ER) as will be discussed in this review. In several cases described the highest accumulation of complete active antibodies was achieved by targeting into the apoplastic space. High-level expression of active recombinant single-chain Fv antibodies (scFv's) was obtained by retention of these proteins in the lumen of the endoplasmic reticulum. This has been shown for leaves and seeds of transgenic tobacco as well as for potato tubers. Transgenic tobacco seeds, potato tubers and tobacco leaves can facilitate stable storage of scFv's accumulated in the ER over an extended (seeds, tubers) or a short (leaves) period of time. The expression of specific scFv's in different plant species, plant organs and cellular compartments offers the possibility of blocking regulatory factors or pathogens specifically. Examples are scFv's expressed in the cytosol and the apoplastic space of transgenic plant cells modulating the infection process of plant viruses and a cytosolically expressed scFv that influenced the activity of phytochrome A protein. The immunomodulation approach has been shown to be also applicable for investigating the action of the phyto-hormone abscisic acid (ABA). High-level accumulation of specific anti-ABA scFv's in the ER of all leaf cells has been used to block the influence of ABA on the stomatal functions. Seed-specific expression of high amounts of anti-ABA-scFv's at a defined time of seed-development induced a developmental switch from seed ripening to vegetative growth. It has been demonstrated that ER retention is essential for the accumulation of sufficient scFv to bind high concentrations of ABA in the transgenic seeds.

Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in shape Nicotiana tabacum

We evaluated the concept for protection of plants against virus infection based on the expression of single-chain Fv (scFv) fragments in the apoplasm or cytosol of transgenic plants. Cloned cDNA of a tobacco mosaic virus (TMV)-specific scFv antibody, which binds to intact virions, was integrated into the plant expression vector pSS and used for Agrobacterium-mediated transformation of Nicotiana tabacum cv. Xanthi-nc. Regenerated transgenic tobacco plants were analysed by northern blot, western blot and ELISA to assess expression and functionality of recombinant antibody (rAb) fragments. A significant increase of scFv levels in T1 progeny was obtained for plants secreting apoplastic scFv antibodies but not for scFvs expressed in the cytosol. Bioassays revealed that T1 progeny producing scFvs in different plant cell compartments showed different levels of resistance upon inoculation with TMV. The most dramatic reduction of necrotic local lesion numbers upon virus infection was observed in T1 plants expressing scFv fragments in the cytosol. Infectivity could be reduced by more than 90%, despite the observation that protein expression levels for functional scFv antibodies were very low. Furthermore, upon inactivation of the N-resistance gene at elevated temperature, a significant portion of the T1 progenies inhibited systemic virus spread, indicating that expression of TMV-specific cytosolic scFvs confers virus resistance in these transgenic plants. Moreover, inoculation of protoplasts isolated from transgenic and non-transgenic tobacco plants with TMV-RNA demonstrated that accumulation of virus particles is affected by cytosolic scFv expression.

Rescue of a neutralizing anti-viral antibody fragment from an intracellular polyclonal repertoire expressed in mammalian cells

The intracellular expression of recombinant antibodies in mammalian cells is an experimental strategy to inhibit in vivo the function of selected molecules. However, one limitation of this technology is represented by the unpredictable behaviour of antibodies, under conditions of intracellular expression. For this reason, it would be desirable to exploit intracellular expression of antibodies to select or rescue more efficient ones from a polyclonal mixture. In this work we have successfully explored this possibility by rescuing a neutralizing anti-viral antibody fragment from an intracellularly expressed anti-reverse transcriptase polyclonal repertoire.

Expression of a recombinant single chain Fv antibody fragment in mammalian cells

Expression of recombinant antibodies and antibody fragments in procaryotic production systems is characterized by low yields and sometimes a reduced affinity for antigen. These problems have been attributed in part to folding constraints in procaryotes, and may be overcome by expressing in eucaryotic production systems. This work is an attempt to produce scFvs of WM65 in mammalian expression system. Since the scFvs of WM65 recognizes a cell surface antigen on normalleukocytes and leukemic cells therefore it CQuld have potential therapeutic application for treatment of chronic lymphocytic leukemia (Ca) and for T-cell depletion of bone marrow to prevent the graft rejection in bone marrow transplantation. The cDNA for scFvs of WM65 in the pHEN1 bacterial expression vector was amplified and modified using polymerase chain reaction, and subcloned into the mammalian expression vector pSVL. The recombinant vector was transfected into the COS cell line by the calcium phosphate technique for transient expression of the scFvs. Immunoaffinity purification (via the C-terminal c-Myc tag) of the scFvs from the cell culture supernatant yielded a protein with molecular weight 28.5 kDa (corresponding to the molecular weight of the scFvs). Two other protein contaminants co-purified with the scFvs, which were tentatively identified by molecular weight as host c-Myc proteins. Purification of the scFvs will allow a comparison of the scFvs produced by mammalian cells and bacteria with respect to the affinity of the scFvs for antigen.

Molecular Cloning and Expression of Recombinant Phage Antibody against Fumonisin B1

Bacteriophage expression systems offer promise for the development of novel antibody reagents applicable to detection of microbial agents and their toxins in foods. In this study, fumonisin B1 (FB1)-specific antibodies, composed of a single chain containing a variable heavy (VH) and light (VL) chain fragments (ScFv), were cloned using mRNA from either spleen cells of mice immunized with FB1-BSA conjugate or from an existing hybridoma cell line that produces anti-FB1 antibody. The approach consisted of (i) reverse transcription of isolated mRNA, (ii) polymerase chain reaction amplification of VH and VL cDNAs, (iii) ligation of VL and VH chains, and (iv) expression of ScFv proteins on the surface of a filamentous bacteriophage or as a soluble fragment. The efficacy of using the recombinant ScFv proteins for the detection of FB1 were evaluated in a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). Compared to polyclonal or monoclonal antibodies for FB1, the recombinant ScFv proteins were 10 to 100 times less sensitive in the CI-ELISA. The secreted ScFv protein did not cross-react with FB2 or FB3. The results indicated that production of a recombinant antibody to a mycotoxin is feasible. However, problems associated with the affinity or avidity of ScFv fragments need to be further addressed before this technology is adaptable to the development of improved immunodiagnostic kits for mycotoxins or other food borne disease hazards.

Lung cancer-reacting human recombinant antibody AE6F4: Potential usefulness in the sputum cytodiagnosis

Human monoclonal antibody (hMAb) AE6F4 has been shown to be potentially useful for immunocytological detection of lung cancer cells in sputum. By recombinant DNA technology, IgM type hMAb AE6F4 was switched to lgG. The IgG mimic recombinant AE6F4 antibody expression plasmid was assembled using the antibody heavy chain gene, which ligated the gene encoding VH and CH1(mu) domains of hMAb AE6F4 heavy chain to the gene encoding CH2(gamma 1) and CH3(gamma 1) domains of human IgG heavy chain, and the antibody light chain gene of hMAb AE6F4. The recombinant antibody expressed by baby hamster kidney (BHK)-21 cells showed molecular size equivalence to IgG, and consisted of human mu-gamma hybrid heavy and kappa light chains. The immunological specificity of the recombinant antibody was the same as that of hMAb AE6F4 by immunoblotting analysis to the 14-3-3 protein, the putative antigen of hMAb AE6F4, and by immunohistochemical and immunocytological analyses using tissue sections and sputa of lung cancer patients. The transfected BHK-21 cells produced the recombinant antibody persistently and the productivity was greater than 20 times that by human-human hybridoma producing hMAb AE6F4.

Design of cassette baculovirus vectors for the production of therapeutic antibodies in insect cells

Background: Various systems have been described for the expression of recombinant monoclonal antibodies for therapeutical applications. Insect cells offer great advantages with respect to post-translational modifications, stability, yields and applications. Objectives: To construct plasmid cassette transfer vectors in order to express chimeric, humanized or human antibodies in insect cells using baculovirus expression system. Study design: Two transfer vectors, pBHUCκ and pBHuγl, were designed. They contain a viral promoter (polyhedrin or p10 promoters, respectively), a signal peptide sequence and a human immunoglobulin light chain Ca gene or heavy chain Cγl sequence, respectively. Restriction sites have been introduced to allow insertion of rearranged variable genes, after amplification by polymerase chain reaction. Results: Recombinant baculoviruses expressing complete immunoglobulins have been generated by a double-recombination event between baculovirus DNA and the loaded cassette transfer vectors. Conclusion: Our genetic cassette approach makes this system a very flexible and convenient one for the rapid production of therapeutic monoclonal antibodies with heavy and light chains of any human isotype. Specific variable regions selected by the antibody phage display technology can be easily transferred in these vectors to obtain a complete antibody.