Microbial Cell Factories BioMed Central Review Open Access The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi Vivi Joosten*, Christien Lokman, Cees AMJJ van den Hondel and Peter J Punt Address: TNO Nutrition and Food Research, Department of Applied Microbiology and Gene Technology, P.O. Box 360, 3700 AJ Zeist, The Netherlands Email: ViviJoosten*[email protected]; [email protected]; CeesAMJJvan den [email protected]; [email protected] * Corresponding author Published: 30 January 2003 Received: 9 December 2002 Accepted: 30 January 2003 Microbial Cell Factories 2003, 2:1 This article is available from: http://www.microbialcellfactories.com/content/2/1/1 © 2003 Joosten et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. Abstract In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single- chain antibody fragment production (scFv and V ) by these lower eukaryotes and the possible HH applications of these proteins. Also the coupling of fragments to relevant enzymes or other components will be discussed. As an example of the fusion protein strategy, the 'magic bullet' approach for industrial applications, will be highlighted. Introduction tics (e.g. pregnancy tests; [2]), human therapeutics and as Antibodies (also called immunoglobulins) are glycopro- fundamental research tools. teins, which specifically recognise foreign molecules. These recognised foreign molecules are called antigens. More applications outside research and medicine can be When antigens invade humans or animals, an immuno- considered, such as consumer applications. Examples are logical response is triggered which involves the produc- the use of antibodies in shampoos to prevent the forma- tion of antibodies by B-lymphocytes. By this tion of dandruff [3] or in toothpaste to protect against immunological response, microorganisms, larger para- tooth decay caused by caries [4]. For these purposes large sites, viruses and bacterial toxins can be rendered harm- quantities of antibodies are required. However, for these less. The unique ability of antibodies to specifically applications on a larger scale there were some major prob- recognise and bind with high affinity to virtually any type lems concerning the expensive production system based of antigen, made them interesting molecules for medical on mammalian expression, the difficulty of producing an- and scientific research. tibodies in bulk amounts and the low stability and solu- bility of some antibodies under specific (harsh) In 1975 Köhler and Milstein developed the monoclonal conditions. antibody technology [1] by immortalising mouse cell lines that secreted only one single type of antibody with In this review we will discuss the possibilities of large- unique antigen specificity, called monoclonal antibodies scale production of antibodies and fragments thereof by (mAbs). With this technology, isolation and production relevant expression systems. Requirements are that the of mAbs against protein, carbohydrate, nucleic acids and system used for production is cheap, accessible for genetic hapten antigens was achieved. The technology resulted in modifications, easily scaled up for greater demands and a rapid development of the use of antibodies in diagnos- safe for use in consumer applications. Page 1 of 15 (page number not for citation purposes) Microbial Cell Factories 2003, 2 http://www.microbialcellfactories.com/content/2/1/1 Fab region C 1 V C 1 V V C 1 H H H H H H C V C V V C L L L L L L Fab fragment Fv region V V V H H H C 2 C 2 H H V V V L L L Fc region Fv fragment scFv fragment C 3 C 3 H H Conventional IgG Figure 1 Schematical representation of the structure of a conventional IgG and fragments that can be generated thereof. The constant heavy-chain domains C 1, C 2 and C 3 are shown in yellow, the constant light-chain domain (C ) in green and the variable H H H L heavy-chain (V ) or light-chain (V ) domains in red and orange, respectively. The antigen binding domains of a conventional H L antibody are Fabs and Fv fragments. Fab fragments can be generated by papain digestion. Fvs are the smallest fragments with an intact antigen-binding domain. They can be generated by enzymatic approaches or expression of the relevant gene fragments (the recombinant version). In the recombinant single-chain Fv fragment, the variable domains are joined by a peptide linker. Both possible configurations of the variable domains are shown, i.e. the carboxyl terminus of V fused to the N-terminus of V H L and vice versa. First, structure and characteristics of antibodies and anti- and filamentous fungi as suitable expression systems for body fragments generated thereof will be discussed, fol- antibody fragments and antibody fusion proteins. lowed by the impact of recombinant DNA technology and antibody engineering techniques on the generation and Antibodies and their unique antigen binding modification of antibodies and antibody fragments. The domains modification of antibodies is of major interest since Whole antibodies changes in their functionality and physico-chemical prop- In vertebrates five immunoglobulin classes are described erties will broaden their application area. For most appli- (IgG, IgM, IgA, IgD and IgE), which differ in their function cations only the antigen-binding site of the native in the immune system. IgGs are the most abundant im- antibody molecule is required and even preferred. By the munoglobulins in the blood and these molecules have a development of recombinant DNA technology and the in- molecular weight of approximately 160 kDa. They have a creasing knowledge on the structure of antibody mole- basic structure of two identical heavy (H) chain polypep- cules created the opportunity to clone and engineer tides and two identical light (L) chain polypeptides (Fig- smaller fragments of antibody genes [5,6] and subsequent ure 1). The H and L chains, which are all β-barrels, are kept alter their functions, for example improve the affinity for together by disulfide bridges and non-covalent bonds (for their antigen. Besides that, recombinant DNA technology a review about antibody structure see [7]). The chains provides the possibility to generate fusion proteins or themselves can be divided in variable and constant do- 'Magic bullets', consisting of an antibody fragment fused mains. The variable domains of the heavy and light chain to an effector molecule. (V and V ) which are extremely variable in amino acid H L sequences are located at the N-terminal part of the anti- In this review the various expression systems for these type body molecule. V and V together form the unique anti- H L of protein will be outlined. We will detail on using yeasts gen-recognition site. The amino acid sequences of the Page 2 of 15 (page number not for citation purposes) Microbial Cell Factories 2003, 2 http://www.microbialcellfactories.com/content/2/1/1 remaining C-terminal domains are much less variable and It turned out to be difficult to produce them in soluble are called C 1, C 2, C 3 and C . form, although replacement of certain amino acids in- H H H L creased solubility of these single domains (see also Llama Fc fragment Heavy-chain antibody fragments) Besides that, their af- The non-antigen binding part of an antibody molecule, finity for the antigen was much less compared with other the constant domain Fc mediates several immunological antibody fragments [12]. functions, such as binding to receptors on target cells and complement fixation (triggering effector functions that Heavy-chain antibodies in Camelidae eliminate the antigen). The Fc domain is not essential for In 1993 Hamers-Casterman et al. [13] discovered a novel most biotechnical applications, relying on antigen bind- class of IgG antibodies in Camelidae (camels, dromedaries ing. The Fc fragment, which is glycosylated, can have dif- and llamas). These antibodies are devoid of light chains ferent effector functions in the different classes of and therefore called 'heavy-chain' IgGs or HCAb (for immunoglobulins. heavy-chain antibody; Figure 2). HCAbs have a molecular weight of ~95 kDa instead of the ~160 kDa for conven- Antigen binding region tional IgG antibodies. Their binding domains consist only The unique antigen-binding site of an antibody consists of of the heavy-chain variable domains, referred to as V s HH the heavy and light chain variable domains (V and V ). [14] to distinguish it from conventional V s. Since the H L H Each domain contains four conserved framework regions first constant domain (C 1) is absent (spliced out during H (FR) and three regions called CDRs (complementarity de- mRNA processing due to loss of a splice consensus signal; termining regions) or hypervariable regions. The CDRs [15,16]), the variable domain (V ) is immediately fol- HH strongly vary in sequence and determine the specificity of lowed by the hinge region, the C 2 and the C 3 domains. H H the antibody. V and V domains together form a binding Although the HCAbs are devoid of light chains, they have L H site, which binds a specific antigen. an authentic antigen-binding repertoire. The current knowledge about the genetic generation mechanism of Antibody fragments generated thereof HCAbs is reviewed by Nguyen et al. [17,18]. Several functional antigen-binding antibody fragments could be engineered by proteolysis of antibodies (papain Recombinant antibodies, antibody fragments digestion, pepsin digestions or other enzymatic approach- and antibody fusion proteins es), yielding Fab, Fv or single domains (Figure 1). The development and applications of recombinant DNA technology led to the design of several new antibodies and Fab fragments antibody fragments. Firstly, functionalities of these pro- Fab fragments (fragment antigen binding) are the antigen- teins may be altered resulting in novel and improved binding domains of an antibody molecule, containing V functions. One of the possible applications of recom- H + C 1 and C + V . Between C and C 1 an interchain di- binant whole antibodies is the use in human therapeutics H L L L H sulfide bond is present. The molecular weight of the het- (see also Recombinant whole antibodies). Secondly, erodimer is usually around 50 kDa [8]. Fab fragments can smaller antibody fragments may be synthesised having be prepared by papain digestions of whole antibodies. the advantage over whole antibodies in applications re- quiring tissue penetration and rapid clearance from the Fv fragments blood or kidney. Moreover, the use of recombinant ex- The minimal fragment (~30 kDa) that still contains the pression systems could also be the solution for large-scale whole antigen-binding site of a whole IgG antibody is production of antibody (fragments). composed of both the variable heavy chain (V ) and var- H iable light chain (V ) domains. This heterodimer, called Recombinant whole antibodies L Fv fragment (for fragment variable) is still capable of The development of human(ised) antibody molecules is binding the antigen [9]. Normally, native Fv fragments are mostly aimed at reduction of unwanted immunological unstable since the non-covalently associated V and V properties in medical applications [19]. Repeated doses of L H domains tend to dissociate from one another at low pro- foreign (murine) antibody molecules could lead to an im- tein concentrations. mune response in patients recognising the mouse anti- body as foreign. This so-called HAMA (human anti- Single domains mouse antibody) response can lead to severe health Single domain antigen binding fragments (dAbs) or V s problems. H were generated in the past [10,11]. They have good anti- gen-binding affinities, but exposure of the hydrophobic Two strategies are developed to reduce the antigenicity of surface of the V to the solvent, which normally interacts therapeutic antibodies (see also [20]). One of these strat- H with the V , causes a sticky behaviour of the isolated V s. egies is chimerisation. In this case the constant murine do- L H Page 3 of 15 (page number not for citation purposes) Microbial Cell Factories 2003, 2 http://www.microbialcellfactories.com/content/2/1/1 C 1 V V C 1 H H H H V V C V HH HH V C L L L L V HH CH2 CH2 CH2 CH2 V fragment HH CH3 CH3 CH3 CH3 Conventional IgG Heavy Chain IgG Figure 2 Schematical representation of the structure of a conventional IgG, a heavy-chain IgG antibody and the variable heavy-chain anti- body fragment (V ) that can be generated of the latter. Heavy-chain antibodies found in llama and camel are only composed HH of heavy-chains and lack the light chain completely, as shown in this Figure. The antigen-binding domain consists of only the V H domain, which is referred to as V (variable heavy-chain antibody fragment), to distinguish it from a normal V . The constant HH H heavy-chain domains C 1, C 2 and C 3 are shown in yellow, the constant light-chain domain (C ) in green and the variable H H H L heavy-chain (V or V ) or light-chain (V ) domains in red and orange, respectively. H HH L mains are replaced by human constant domains [21,22]. Production of recombinant antibody fragments by Es- The second strategy is grafting of only the murine CDRs cherichia coli onto existing human antibody framework regions, which Much work on antibody fragment production has been is called humanisation [22]. focussed on Escherichia coli as an expression system (re- viewed in [25]). The advantage of this system is the ability At present there are more than 10 recombinant antibodies to produce proteins in relative large amounts. Besides approved by the US Food and Drug Administration (FDA) that, E. coli is easily accessible for genetic modifications, for use in medicine and many more are in a late stage of requires simple inexpensive media for rapid growth and clinical trials. FDA approved recombinant mAbs are e.g. they can easily be cultured in fermentors permitting large- Herceptin™ (Genetech, San Francisco, CA), which targets scale production of proteins of interest. Several antibody and blocks the growth factor Her2 on the surface of breast fragments have been produced in functional form (e.g. cancer cells and Rituxan™ (IDEC Pharmaceuticals Inc., [8,9,26,27]) and expression of relevant gene segments San Diego, CA) used against non-Hodgkin's lymphoma also permitted the production of the recombinant anti- (see for more examples [23,24]). The use of recombinant body fragments. The problem of stability has been tackled antibodies for medical purposes does not require a cheap by generation of single-chain Fv (scFv) or disulfide stabi- large-scale production process per se, since only a limited lised Fv (dsFv) fragments. amount of pure preparations is needed. Selection of antibody fragments with improved functionalities In 1990 McCafferty et al. [28] showed that antibody frag- ments could be displayed on the surface of filamentous Page 4 of 15 (page number not for citation purposes) Microbial Cell Factories 2003, 2 http://www.microbialcellfactories.com/content/2/1/1 phages, called phage-display. This technology is based on Llama Heavy-chain antibody fragments (V s) HH the fusion of the antibody variable genes to a phage coat The other type of interesting antibody fragments are V s HH protein gene (e.g. [20]). After displaying an antibody frag- (see Figure 2) comprising the smallest available intact an- ment on the protein surface of the phage, antigen specific tigen-binding fragment (~15 kDa, 118–136 residues phages can be selected and enriched by multiple rounds [48,49]). The affinities found for V s were in the na- HH of affinity panning (e.g. reviewed in [29,30]). This tech- nomolecular range and comparable with those of Fab and nique makes it possible to select phages that bind almost single chain Fv (scFv) fragments [50,51]. Besides that any antigen, including those previously considered to be V s are highly soluble and more stable than the corre- HH difficult, such as self-antigens or cell surface proteins. sponding derivatives of scFv and Fab fragments [50,52]. V s carry amino acid substitutions that make them more HH Libraries can be prepared from variable genes isolated hydrophilic and prevent the prolonged interaction with from immunised animals, non-immunised sources (naïve BiP (Immunoglobulin heavy-chain binding protein), libraries, thus avoiding the need for immunisation) or which normally binds to the H-chain in the Endoplasmic even (semi-) synthetic libraries can be constructed. The V Reticulum (ER) during folding and assembly, until it is genes can be subjected to random mutagenesis, chain or displaced by the L-chain [53]. There are indications that DNA shuffling methods [31], mimicking the natural hy- this increased hydrophilicity improves secretion of the permutation mechanism. V s from the ER. Hence, production of V s in com- HH HH mercially attractive microorganisms may be favourable. Single-chain Fv fragments and multimers An attractive recombinant antibody fragment is the single- Several ways are described to obtain functional V s: HH chain Fv (scFv) fragment (reviewed in [32,33]). It has a from proteolysed HCAb of an immunised camelid, direct high affinity for its antigen and can be expressed in a vari- cloning of V genes from B-cells of an immunised HH ety of hosts [34]. These and other properties make scFv camelid resulting in recombinant V s or from naïve or HH fragments not only applicable in medicine (reviewed in synthetic libraries [49]. V s with desired antigen HH [35]), but also of potential for biotechnological applica- specificity could be selected by phage display (see Selec- tions. In the scFv fragment the V and V domains are tion of antibody fragments with improved functionali- H L joined with a hydrophilic and flexible peptide linker, ties). Using V s in phage display is much simpler and HH which improves expression and folding efficiency [36,37]. more efficient as compared with Fabs or scFvs, since only Usually linkers of about 15 amino acids are used, of which one domain needs to be cloned and expressed to obtain a the (Gly Ser) linker has been used most frequently [35]. functional antigen-binding fragment [52,54]. 4 3 Unfortunately, some scFv molecules have a reduced affin- ity compared to the parental whole antibody or Fab mol- As already noted before (see Antibody fragments gener- ecule [12,38,39]. Besides that, scFv molecules can be ated thereof), classical V s were difficult to produce in H easily proteolytically degraded, depending on the linker soluble form. To improve their solubility and prevent used [40]. With the development of genetic engineering non-specific binding, residues located on the V side of L techniques these limitations could be practically over- V s were replaced by 'V -like' residues, mimicking the H HH come by research focussed on improvement of function more soluble V fragments. This process has been HH and stability, as discussed in [32]. An example is the gen- termed camelisation [55–57] and these camelised V frag- H eration of disulfide-stabilised Fv fragments where the V - ments, particularly those based on the human framework, H V dimer is stabilised by an interchain disulfide bond are expected to have significant advantages for therapeuti- L [38,41,42]. Cysteïnes are introduced at the interface be- cal purposes in humans (reviewed in [58]). tween the V and V domains, forming a disulfide bridge, L H which holds the two domains together (reviewed in [43]). Fusion proteins ('Magic bullets') A completely new use of the binding capacity of antibody Dissociation of scFvs results in monomeric scFvs, which fragments is the design of a fusion approach, in which an can be complexed into dimers (diabodies), trimers (tria- effector protein is coupled to an antigen recognising anti- bodies) or larger aggregates ([44], reviewed in [45]). The body fragment. In human medicine this approach is re- simplest designs are diabodies that have two functional ferred to 'Magic bullet'. All kinds of molecules can be used antigen-binding domains that can be either similar (biva- as effector molecule only limited by the imagination. The lent diabodies) or have specificity for distinct antigens gene encoding the effector may be directly fused to the (bispecific diabodies). These bispecific antibodies allow gene of the antibody fragment of interest, resulting in nov- for example the recruitment of novel effector functions el bifunctional proteins [59]. Examples of the use of this (such as cytotoxic T cells) to the target cells, which make approach will be given in the section Antibody fragments them very useful for applications in medicine (reviewed and antibody fusion proteins for large-scale applica- in [46,47]). tions and consumer products. Page 5 of 15 (page number not for citation purposes) Microbial Cell Factories 2003, 2 http://www.microbialcellfactories.com/content/2/1/1 Table 1: Extracellular production of antibody fragments in prokaryotic expression systems. Strains Characteristics Food grade Production yields References Lactobacillus zeae Gram+ yes scFv, ND* (secreted/cell-bound) [91] Bacillus subtilis Gram+ yes 10 to 15 mg/L scFv (secreted) [143,144] Streptomyces lividans Gram+ yes 1 mg/L Fv fragment (secreted) [145,146] Staphylococcus carnosus Gram+ yes 5–10 mg/L V domain (secreted) [147] H Proteus mirabilis Gram- no 40 to 200 mg/L scFv (secreted) [148,149] Escherichia coli Gram- no Several fragments (inclusion bod- [25,34] ies/periplasmic space) *ND = not determined Applications of antibody fragments and antibody V s as drug carriers HH fusion proteins It is expected that V s are also applicable in diagnosis HH Applications of antibody fragments in human medicine and therapy in human medicine, especially when an eco- The smaller the better nomically feasible production, small size and stability are Most applications of recombinant antibody fragments are required (reviewed in [49]). Cortez-Retamozo et al. [64] related to diagnosis and therapy in human medicine, recently showed that V s specifically could be targeted HH which is especially focussed on the use of antibodies as to tumour cells, which together with the possibility of the ideal cancer-targeting reagent (reviewed in [19,60– generation of bispecific V constructs [69] is of major in- HH 62]). For some clinical applications small antibody frag- terest for cancer therapy. ments have advantages over whole antibodies. The small size permits them to penetrate tissues and solid tumours V s as delivery carriers in the brain HH more rapidly than whole antibodies [63] which recently Antibodies and many other water soluble compounds are also was shown for V s [64]. Smaller antibody frag- excluded from the brain by the blood-brain barrier (BBB), HH ments have also a much faster clearance rate in the blood thus making treatment of brain-related disease very diffi- circulation, which leads to differences of selectivity [63]. cult. Recently, Muruganandam et al. [70] showed that V HH Nowadays there are also promising pre-clinical and clini- were able to selectively bind to and transmigrate across cal trials with antibody fragments as diagnostic or thera- the BBB in a human in vitro BBB model and partly in vivo peutical agents [61,65]. Another application of antibody in mice. This property can be exploited for the develop- fragments is to treat viral infections with so-called intra- ment of efficient antibody carriers suitable for delivery of bodies, which are intracellular antibodies synthesised by macromolecules across the human BBB and subsequently the cell and targeted to inactivate specific proteins within for treatment of neurological diseases. the cell [66]. V s as potent enzyme inhibitors HH 'Magic bullets' in medicine Hypervariable regions in V s are on average longer than HH The use of bi-functional molecules in medicine is aimed those of V s [71,72]. The extended hypervariable regions H at delivery of a protein drug, which is only active where it of V s are capable of penetrating deep into the cleft of HH is required. It thereby limits the dose of the drug, resulting active sites of enzymes, binding to novel epitopes that are in less side effects of the drug towards healthy tissue and/ not recognised by conventional antibodies [51,73,74]. Be- or less immunogenic response to the protein drug itself. cause of this property V s may act as better potent en- HH Also the physical interaction between the target and the zyme inhibitors [51,75,76]. effector molecule increases the potency of the effector. Fu- sion proteins are ideal immuno agents for cancer diagno- V s in consumer products HH sis [67] and cancer therapeutics. An example is the use of Since llama V s are very stable, even at high tempera- HH cancer-specific bi-functional antibodies targeting potent ture, applications can be envisaged in which a high tem- cytotoxic molecules to tumour cells and subsequently perature step is involved (e.g. pasteurisation), without eliminate these tumour cells without harming healthy losing antigen-binding properties [50]. Recently it was cells [68]. shown that V s could be used to prevent phage infection HH in cheese production processes [77], by recognising a Potential applications of V s structural protein of the phage, which is involved in recog- HH Specific applications of V s are foreseen in the following nition of the host Lactococcus lactis. HH direction: Page 6 of 15 (page number not for citation purposes) Microbial Cell Factories 2003, 2 http://www.microbialcellfactories.com/content/2/1/1 Table 2: Higher eukaryotic expression systems for heterologous protein production and possible advantages and disadvantages of the expression system. Expression systems Ease of molecular upscaling Economic Pathogenic References3 cloning feasibility1 contaminants2 Mammalian cells + +/- + + [59,150–154] Insect cells ++ + + + [155–160] Plants ++ +++ ++ ++ [92,93,161,162] Transgenic animals* +/- +++ +/- +/- [163–166] Yeasts +++ +++ +++ +++ See references in section Production of antibody fragments by lower eukaryotes. Filamentous fungi +++ +++ +++ +++ [4,123,128] +++ = excellent, ++ = good, + = sufficient, +/- = poor. * With transgenic animals in this context is mentioned the production of antibodies or anti- body fragments in the milk of transgenic animals, for example rabbits, sheep, goats or cows 1 With economical feasibility is mentioned the time and cost of molecular cloning, upscaling and downstream processing (purification). 2 Pathogenic contaminants like viruses or pyrogens. 3 Articles dealing with production of antibodies, antibody fragments and antibody fusion proteins. Antibody fragments and antibody fusion proteins for large- amount of proteins into the culture medium. Several of scale applications and consumer products these systems can be considered as suitable (both from Many additional applications can be envisaged if an inex- prokaryotic and eukaryotic origin). Hereafter several of pensive and simple production system is available, yield- these systems will be discussed, with an emphasis on yeast ing large amounts of antibody fragments that can be and fungal systems. purified easily. The highly specific antigen-binding ability could be used for inactivating bacteria or specific enzymes Drawbacks using E. coli as a host for antibody fragment that can cause spoilage of food. Other suggested applica- production tions are the use in biosensors, treatment of wastewater As described in the section Production of recombinant [78], industrial scale separation processes such as separa- antibody fragments by Escherichia coli, this micro-organ- tion of chiral molecules [79], purification of specific com- ism has shown to be a potential expression host for anti- ponents (proteins) from biological materials or the use as body fragments and fusion proteins. Although the general abzymes [80,81]. They have also been considered as com- production yields in shake-flask cultures are low (several ponents of novel consumer goods with new improved mg/L), in fermentation processes several g/L could be functionalities, in oral care and personal hygiene (e.g. in obtained (reviewed in [86]). There are two possibilities of toothpaste or mouthwashes [82]). For dental applications antibody fragment production in E. coli, either by secre- antibody fragments can be coupled to enzymes to increase tion of the fragments into the culture medium and/or the concentration of antimicrobials like hypothiocyanate periplasmic space (the compartment between the inner and hypohalites, for example glucose oxidase (GOX; and outer membrane) or preparation of inclusion bodies [83]), galactose oxidase (GaOX; [84]) or lactate oxidase with subsequent in vitro folding. However, both strategies (LOX; [85]). Other examples are targeted bleach in laun- have disadvantages that make the use of this prokaryote dry washing (e.g. detergents containing antibodies cou- not attractive for the large-scale production of antibody pled to molecules that specifically remove difficult stains) fragments and antibody fusion proteins. Firstly, the secre- or the use in shampoos where antibodies act to prevent tion of folded and fully assembled fragments in the medi- dandruff by inhibiting growth of specific microorganisms um or periplasmic space is often accompanied with cell causing this [3]. lysis and subsequent product loss. Secondly, 'toxicity' of the antibody sequence and concomitant plasmid loss is Suitable expression systems for the large-scale frequently observed, which hamper high production lev- production of antibody fragments and antibody els (reviewed in [25]). Thirdly, expression of the frag- fusion proteins ments in inclusion bodies, which often results in To be able to use antibody fragments and antibody fusion insoluble protein aggregates [87], demands laborious and proteins in these large scale applications, a suitable ex- cost-intensive in vitro refolding (denaturation and rena- pression system has to be chosen. Several expression sys- turation) and purification steps. Hence, the final yield of tems are available, both from prokaryotic (Table 1) and fragments is only a small percentage of the protein that eukaryotic (Table 2) origin. Our main interest goes out to was initially present in the inclusion bodies even though these systems that are able to economically produce large purification steps are nowadays facilitated by affinity Page 7 of 15 (page number not for citation purposes) Microbial Cell Factories 2003, 2 http://www.microbialcellfactories.com/content/2/1/1 chromatography using C-terminal polypeptide tails, like Production of antibody fragments by lower eukaryotes poly-His or FLAG [88,89]. Recently, production of solu- An attractive possibility for the cost-effective large-scale 6 ble and functional scFv by E. coli could be increased by im- production of antibody fragments and antibody fusion proving disulfide bond formation activity in the proteins are yeast or fungal fermentations. Large-scale fer- cytoplasm, using mutants and overexpression of di- mentation of these organisms is an established technolo- sulfide-bond isomerase [90]. Finally, E. coli is unable to gy already used for bulk production of several other carry out eukaryotic post-translational modifications and recombinant proteins and extensive knowledge is availa- is therefore not suitable when glycosylation of antibody ble on downstream processes. Besides that, yeasts and fil- fragments or more importantly the fusion proteins is amentous fungi are accessible for genetic modifications required. and the protein of interest may be secreted into the culture medium. In addition, some of their products have the so- Alternative prokaryotic expression systems called GRAS (Generally Regarded As Safe) status and they E. coli is not the only available prokaryotic expression sys- do not harbour pyrogens, toxins or viral inclusions. tem, although it is rather dominant in the field. Alterna- tive prokaryotic expression systems are available for Methylotrophic and other yeasts antibody fragment production (Table 1). However, these The methylotrophic and other yeasts like Candida boidinii, will encounter similar limitations as E. coli, even though Hansenula polymorpha, Pichia methanolica and Pichia pas- most organisms described in Table 1 secrete the investigat- toris are well known systems for the production of heter- ed antibody fragment into the culture medium. A field ologous proteins (reviewed in [98]). High levels of where production of antibody fragments in prokaryotic heterologous proteins (milligram-to-gram quantities) can cells could still be interesting, is in food grade organisms be obtained and scaling up to fermentation for industrial used for delivery passive immunisation in humans, by applications is possible [99–101]. means of functional foods. In a recent article, Kruger et al. [91] reported the production of scFv antibody fragments Especially the P. pastoris system is used in several industri- against Streptococcus mutans by the Gram positive food al-scale production processes [102]. Ridder et al. [103] grade bacteria Lactobacillus zeae. In experimental animals were the first to report the expression of a scFv fragment a decrease of S. mutans and reduced development of caries by P. pastoris. From then on several papers reported about was observed. the use of P. pastoris for the production of recombinant antibodies and fragments thereof [104,105]. In shake- Eukaryotic expression systems flask cultures a level of 250 mg/L scFv was obtained [106] Also several eukaryotic systems can be envisaged for large- and Freyre et al. [107] were able to obtain even an expres- scale production of antibody fragments and antibody fu- sion level of 1.2 g/L scFv fragment under fermentation sion proteins (see also [34]), like mammalian cells, insect conditions. However, Cupit et al. [108] also showed that cells, plants, transgenic animals and lower eukaryotes (see the production of antibody fragments by P. pastoris is not Table 2). always a success story. The production of therapeutical whole antibodies is well Based on the described results the commercial recom- established in mammalian cells. However, large-scale pro- binant antibody production by P. pastoris is promising. duction is expensive and time-consuming. However, products currently obtained from P. pastoris are not regarded as GRAS, which may limit its use. 'Plantibodies' can be produced in several plant target or- gans (reviewed in [92]). Roots, storage organs (seeds and Wood et al. [109] were the first to report the production of tubers) and fruiting bodies can be suitable for mass oral mouse IgM by the baker's yeast S. cerevisiae, although only (edible) applications (see [93] and references therein). Ex- unassembled chains were detected in the culture medium. pression of scFv in transgenic plants has been proposed as However, the production of Fab fragments was possible as a way to produce and store pharmaceutical antibodies was first shown by Horwitz et al. [110]. Although the ob- [94,95] and as means to block physiological processes in tained levels were low, functional Fab fragments were se- the plant itself [96] or establish plant pathogen resistance creted in the culture medium. Davis et al. [111] expressed [97]. Plants show several advantages as large-scale anti- scFv antibody fragments in Schizosaccharomyces pombe. body production systems, like the ease and low costs of Studies on the scFv production in the non conventional growing plants, even in large quantities. However, the yeasts Yarrowia lipolytica and Kluyveromyces lactis resulted generation of transgenic plants that express antibodies is in 10–20 mg/L functional and soluble anti-Ras scFv [112]. a time consuming process and the downstream processing to isolate the expressed antibodies from the plant parts is relatively expensive and laborious. Page 8 of 15 (page number not for citation purposes) Microbial Cell Factories 2003, 2 http://www.microbialcellfactories.com/content/2/1/1 Filamentous fungi: Trichoderma reesei and Aspergillus spp an important role in the accumulation of scFv in the cell Filamentous fungi, in particular species from the genera [128]. Shusta et al. [129] reported the increase of scFv pro- Trichoderma and Aspergillus have the capacity to secrete duction up to 20 mg/L in S. cerevisiae by optimising the ex- large amounts of proteins, metabolites and organic acids pression system by overexpression of two ER resident into their culture medium. This property has been widely chaperones and reduction of growth temperature. Kauff- exploited by the food and beverage industries where com- man et al. [130] showed that overexpression of scFv in S. pounds secreted by these filamentous fungal species have cerevisiae resulted in cellular stress, displayed by decreased been used for decades. This has led to the GRAS status for growth rates and induction of the Unfolded Protein Re- some of their products. Filamentous fungi like A. awamori, sponse (UPR). It was hypothesised that a functional UPR A. niger and A. oryzae are therefore suitable organisms for was required to decrease the malfolded scFv in the ER, the production of commercially interesting homologous leading to a recovery from cell stress. and heterologous proteins [113–115]. Strategies to im- prove protein secretion by filamentous fungi are exten- As further improved levels were desired also a fungal ex- sively reviewed in [116–119]. pression system was considered [4]. In shake-flask cul- tures a production level of 10 mg/L was achieved by using Production strains of Trichoderma reesei (Hypocrea jecorina) A. awamori as production host. As secretion of a heterolo- have an exceptional secretion capacity up to 35 g protein/ gous protein can be greatly enhanced by fusing it to a "car- L, where half of the secreted protein consists of the cellu- rier" protein such as glucoamylase (GLA; [117,118]), also lase cellobiohydrolase I (CBH1; [120]). Therefore, Tri- this fusion-approach was employed. Analysis of the cul- choderma is considered as an excellent host for the ture medium of transformants carrying the fusion con- production of heterologous proteins (reviewed in struct revealed a production of approximately 50 mg/L [121,122]). Nyyssönen et al. [123] reported a production scFv in the culture medium [4]. Several commercially in- of 1 mg/L in shake-flasks of Fab antibody fragments by T. teresting scFv fragments were investigated for their ability Reesei Rut-C30. More strikingly, when the Fab antibody to be produced by A. awamori using the GLA-fusion strat- fragment chain was fused to the core-linker region of egy. The results showed that the production levels differed CBH1, a production level of 40 mg/L in shake-flasks and significantly between the different scFv transformants. In- 150 mg/L in bioreactor cultivations was obtained terestingly, in some cases increased levels of scFv detected [123,124]. in the culture medium corresponded to an increase of transcription level of the ER chaperone BiPA [131], indi- The use of S. cerevisiae and A. awamori for the cating that the antibody fragments, like in S. cerevisiae, large-scale production of antibody fragments may have problems with correct folding and aggregate in and fusion proteins the fungal cell. In our own laboratory at TNO Nutrition and Food Re- search in Zeist (The Netherlands) and in collaboration To increase production levels, successful 10 L and 1,5 × with Unilever Research Vlaardingen (The Netherlands) re- 104 L scale fermentations were carried out resulting in 200 search on antibody fragment production in S. cerevisiae mg/L scFv under optimal conditions. However, variable and A. awamori has been carried out [125,126]. The aim amounts of scFv dimers and other multimers were ob- of this project was a detailed comparison of both served. Recent fermentation experiments performed by expression systems, in relation to their possible large-scale Sotiriadis et al. [132] showed that the highest scFv level production process of antibody fragments and fusion pro- was observed when induction was started in the late expo- teins. In the framework of this collaboration also a new A. nential phase. An increase of the carbon and nitrogen awamori expression system, based on xylose induction source concentrations and a decreased of the concentra- was developed [127]. tion of the inducer, resulted in increased product yields. The use of S. cerevisiae and A. awamori for the large- Production of Llama V antibody fragments by S. cerevi- HH scale production of scFv siae and A. awamori To investigate the feasibility of a large-scale cost-effective Although the production of scFv fragments by S. cerevisiae process for the extracellular production of (functional- and A. awamori was successful, levels up to several g/L ised) scFv fragments initially S. cerevisiae was used. How- were not achieved. Possibly the hydrophobic regions of ever, it was shown that S. cerevisiae was a poor host for the the scFv, responsible for keeping the variable regions of production of scFv, since the secretion of scFv was ham- the heavy and light chains together, could also interact pered by improper folding of the fragments, because large with other molecules in the cell. Aggregation of scFv in S. aggregates were formed in the ER and vacuolar-like or- cerevisiae may result in accumulation and subsequent deg- ganelles. It was hypothesised that the exposure of the hy- radation (of a part) of the antibody fragment molecules drophobic surfaces on the V and V chains of scFv plays [128] as frequently observed when expressing heterolo- L H Page 9 of 15 (page number not for citation purposes) Microbial Cell Factories 2003, 2 http://www.microbialcellfactories.com/content/2/1/1 gous proteins that exhibit hydrophobic surfaces [133]. In- the production of Magic bullets by filamentous fungi or terestingly, antibody fragments devoid of these yeasts is of interest. An enzyme coupled to an antibody hydrophobic surfaces could be obtained from camels, fragment recognising persistent stains from e.g. azo-dyes dromedaries and llamas (V s, see Llama Heavy-chain [134] results in a more directed bleaching process, result- HH antibody fragments (V s) and [13]), providing an op- ing in lower amounts of required detergent, reduction of HH tion to improve production levels in relevant microorgan- harmful effects of the enzyme to the textile and lower en- isms [126]. vironmental burden (see Figure 3). V s could be produced in E. coli up to levels of 6 mg/L, Currently we are investigating the feasibility of produc- HH were found to be extremely stable, highly soluble and re- tion of V -enzyme fusions by A. awamori. One of the HH acted specifically and with high affinity with antigens V s used is a model llama V , recognising the azo-dye HH HH [52]. V s were produced in S. cerevisiae at levels over 100 Reactive Red 6 (RR6 [134]). As a bleaching enzyme, the HH mg/L in shake-flask cultures [134], although considerable Arthromyces ramosus peroxidase (ARP) [137,138] was ge- amounts of V s were detected intracellularly. From a 1,5 netically linked to the V fragment. This peroxidase uti- HH HH × 104 L fed-batch fermentation, 1.3 kg of V s was ob- lises hydrogen peroxide to catalyse the oxidation of a wide HH tained, which clearly showed that these fragments could range of organic and inorganic compounds, which makes be produced in this host more efficiently than scFv frag- the enzyme suitable for use in bleaching processes [139]. ments [135]. For a cost-effective large-scale process for the ARP alone could be produced in high amounts by A. production of V s in S. cerevisiae further improvement is awamori (800 mg/L; Lokman et al. submitted). Prelimi- HH required. Van der Linden et al. [54] showed that produc- nary results showed the feasibility of fusion protein pro- tion of V s by S. cerevisiae could be improved by DNA duction by A. awamori, yielding high levels of ARP-V HH HH shuffling techniques, in which three homologous V fusion protein in controlled fermentation experiments HH genes were randomly fragmentated and reassembled (Joosten et al. manuscript in preparation). The fusion pro- subsequently. tein showed both ARP activity and azo-dye binding activity. Based on the fact that A. awamori performed superior for scFv also the possibility of V production by A. awamori In future experiments V s fragments can be replaced by HH HH was investigated. As a model V s against the hapten RR6 other more relevant antibody fragments, for example HH were chosen [134]. Gene fragments coding for anti-RR6 those binding tomato or blood spots. Also the peroxidase V s were cloned in an expression vector containing the part of the fusion can be further optimised. HH highly inducible endoxylanase promoter. Recent experi- ments (Joosten et al. submitted) showed that functional Conclusions and future prospects V s could be produced in the culture medium in shake- Recent developments in the fields of antibody engineering HH flask cultures, albeit at relatively low levels. For further op- and expression systems have enabled the engineering and timisation a carrier strategy and controlled fermentations production of antibodies and antibody fragments for a will be carried out. wide variety of applications. A lot of examples are already mentioned, but presumably more applications can be en- Production of 'Magic bullets' by A. awamori visaged. The development of the 'Magic bullet' approach A major research interest is the production of fusion pro- will even increase the interest in antibodies and their re- teins or 'Magic bullets', consisting of an antibody frag- lated products, also for applications in human medicine. ment (scFv or V fragment) fused to an enzyme of A recently envisioned application that is of much interest, HH interest. In our laboratory research has been carried out is the use of antibody fragments in micro-arrays. Antibody with a few examples of scFv fragments coupled to glucose arrays can be used for proteomic analysis by comparing oxidase (GOX). GOX is already for many years an interest- the differences in presence of proteins in healthy and dis- ing enzyme for coupling to antibodies for killing cells eased cells. For this purpose antibody fragments derived [136]. A scFv, which recognises for example oral Strepto- from large phage-antibody libraries can be used as probes mycetes, when fused to GOX, which is an antimicrobial en- to capture proteins on chips in a high-throughput system zyme, may kill bacteria by generation of the bactericidal (reviewed in [140,141]). In this respect, V s fragments HH hydrogen peroxide. In activity assays it was shown that the are of great interest, due to their simple and stable fusion protein produced by A. awamori was functional, structure. both in binding to the antigen and GOX activity [4]. In this review we evaluated whether the yeast S. cerevisiae In the detergent industry enzymatic bleaching may be a and the filamentous fungus A. awamori are suitable ex- good alternative to the current chemical bleaching used. pression systems for the large-scale production of anti- To make these laundry-cleaning products more effective, body fragments and antibody fusion proteins. Although Page 10 of 15 (page number not for citation purposes)