Antibody engineering

Antibody engineering Bjiirn

Nilsson

Pharmacia AB, Stockholm,

Antibody years.

engineering

(ii) new structural

of essentially

antibodies

discussed

in this

research area for many

review

include:

in the field of selection of antigen-specific

on bacteriophages; cloning

has been an extremely intensive

Recent achievements

improvements

Sweden

work, in particular

the complete set of human

(i) significant

antibody fragments using NMR;

(iii) the

VH genes; (iv) the use of

to catalyze complicated chemical reactions; and (v) novel antibody

fusion proteins to potentiate immune therapy. An interesting is the replacement of antibodies

future biotechnological

Current Opinion

new development

with more stable protein scaffolds for many

in Structural

applications.

Biology

1995,

5:450-456

Introduction

Antibody

This review article describes recent trends and accomplishments in the field of antibody engineering, a field which is undoubtedly one of the most active in the very active area of protein engineering, for several reasons. First, antibody engineering provides a molecular link to understanding details of the complex humoral immune response. For decades scientists have been striving to elucidate the molecular mechanism by which the antibody maturation system in viva in mammals is able to generate a set of molecules that can bind to any foreign antigen exposed to the system. Detailed structural-function analysis of antibodies could lead to enhanced detailed understanding of the different steps involved in the maturation of specific antibodies. Second, the Ig fold is rather common in biology [l], featuring in many cell-bound receptors. Many of these receptors, such as the T-cell receptor, are directly involved in the triggering of the immune system, but this super-family includes many other receptors, including many cytokine receptors. Thus, knowledge of antibody molecules can to some degree be extended to other molecular systems. Third, antibodies are used in many biotechnological applications, including diagnostics and therapy.

The basic antibody three-dimensional structure, which comprises two heavy chains and two light chains as repeated Greek-key domains, has been known since as early as 1973 [13], when the first structure of a Fab fragment was solved (Fig. 1). In spite of this, much of the structural work of importance has been performed in recent years, and much remains to be done in the field. It was not until 1992 that the first three-dimensional structure of a complete antibody was solved [14]. The structure revealed a rather asymmetrical structure.

This review focuses mainly on recent trends in the protein engineering of antibodies or antibody tiagments. Many recent and excellent reviews in the antibody field provide more extensive background reading on specific antibody topics: structure/function [2], antibody engineering [3,4], antibody-antigen recognition [5], recombinant production methods [3,6,7], phage display and library selection [8,9’,1 O**] and humanization [11,121.

structure,

stability

and function

Other recent break-throughs include analysis of structures of single-chain Fv (scFv) molecules. The scFv concept was originally adopted to simplify expression of antigen-binding antibody fragments [ 151. Although the approach has been extremely useful, in particular when selecting specific antibodies on phage (see below), the structural analysis has been rather unsuccessful in spite of many attempts. It was not until 1993 that an un-complexed scFv molecule was crystallized and analyzed structurally [16]. NMR is predicted to aid in the understanding of structures and dynamics of protein-protein interactions; so far, however, direct studies of Fv fragments have been hampered by poor solubility and short 1H relaxation times. Despite these problems, multi-dimensional NMR techniques have successfully been utilized to compare Fv and scFv structures [17]. An isolated VL domain has been completely assigned [18], but NMR analysis of VH has until now, not been as revealing. Recently, a VH domain was engineered in the surface normally packed against VL, to mimic the corresponding surface of VH domains from a class of heavy chains

Abbreviations CDR-complementarity-determining 450

region; scfv-single-chain 0 Current

Fv, where Fv is the variable portion of the immunoglobulin.

Biology Ltd ISSN 0959-440X

Antibody engineering Nilsson

heavy chain is much more variable structure predictions very uncertain.

in length,

making

Some of the existing predictive models for antigen binding have been used to improve antigen binding [24] and to design an antibody de novo [25**]. Although this design was based on other known similar systems for which structural data were available, remarkable similarities were found between the model and the structure determined by X-ray crystallography Complete de novo design of antibodies that are specific for an antigen with a known three-dimensional structure still remains to be demonstrated. The computational tools that may be used in the design process are continuously being improved, and now there is even a specialized commercial software product for this application (Oxford Molecular Ltd, UK). Another interesting trend in the de novo design area is the design of small-molecule mimics of antibodies, based on antibody-antigen structures. The first successful design of a cyclic peptide, based on CDR3 from the heavy chain, that mimics rather well the antibody in its antigen interaction was recently reported [26].

Fig. 1. Structure of the Fab fragment of the McPC603 antibody 1751. The heavy chain (V,, Ct+l) is on the left-hand side, the light chain (V,, CL) on the right-hand side. The antigen-binding loops, which harbour the CDRs, are positioned at the top of the molecule. The figure was generated from the 1 mcp coordinates in the Brookhaven protein data bank using the software RasMol.

from camel antibodies which lack light chains. This ‘camelized’ VH shows significantly improved NMR properties [ 193. The heavy-chain portion generally seems to be much more important than the light chain in determining the recognition process, possibly because of the large variation in the structure of the complementarity-determining region 3 (CDR3) (see below) [20]. The production of engineered VH domains will most probably lead to increased efforts in utilizing NMR to understand the antigen-recognition process. Antigen binding is mainly found performed and determined by six hyper variable loops which overlap with the CDRs; three on the heavy chain and three on the light chain. Over the years much effort has been devoted to understanding the mechanism of antigen recognition. CDRl and CDR2 of the heavy and light chains are encoded by the V genes, and essentially all of the human V-genes have been cloned and sequenced [21]. Most interestingly, all of the peptide sequences encoded by these 50 genes can be grouped into a surprisingly small number of structural classes [22]. Further, comparison of the human and murine immunoglobulin genes suggests that the V regions are at least as conserved as the core gamework sequences [23]. This will encourage those endeavouring to design de novo antibodies with a predicted specificity. In contrast, the CDR3 loop on the

The low thermodynamic conformational stability of antibodies makes them unsuitable for many biotechnological applications. Therefore, a trend has emerged towards enhancing the stability of antibodies, or fragments of antibodies, by engineering [27,28]. Of course, the original design of scFv [15] can be considered just such a stabilizing design. Indeed, the single-chain concept is constantly being improved in order to maximize stability and minimize interference with antigen binding [29]. If the single-chain is constructed ‘short’, the VH and VL domains are forced to pair with the complementary domains of another chain to form a dimer with two antigen-binding sites, termed a ‘diabody’ [30]. These small molecules, with two antigen-binding sites, may find many interesting applications. Further, attempts to thermally stabilize Fv fragments have revealed that the CDR regions also have a significant impact on thermal stability [3 11.

Antibody

production

The possibility of producing antibody Fab fragments in Errherichia coli [32] has been a major break-through in the field of antibody engineering. A fast and reliable method of producing a purified target protein is a prerequisite for efficient protein engineering. However, the production of antibody fragments in E. coli shows a large variability of success and may not be as straightforward as initially predicted. These problems have motivated many laboratories to develop production systems in alternative microorganisms, including Saaharomyces cerevisiae [33,34], Pichia pastoris [35], Trihodema reesei [36] and insect cells [37]. Yet

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Engineering and design

although production levels in reactors exceeding several hundred milligrams per liter have been reported using some of these alternative host systems, E. coli is still the system of choice in most laboratories, possibly because of the overall simplicity of cloning work using this host. Instead, attempts are now being made to improve E. coli production systems by strains improvements, for example by co-expressing chaperone proteins [38], or by production as fusion proteins to facilitate recovery [39,40]. Interesting data that explain the wide range of production levels in E. coli of different antibodies or antibody fragments are starting to emerge. The approach has been, where possible, to utilize protein engineering to convert an antibody that shows a low production level into a variant with high-level production while mutating only regions of the molecule outside the CDRs. Comparison of production levels of antibody fragments directed against 2-phenyloxazolone [41] has shown that the CH~ domain is important for differences in secretion efficiencies. Further, it was recently shown that replacement of only three amino acid residues in the Fv fragment of antibody McPC603 increased 60-fold, the yield of secreted soluble material in E. coli [42*]. These data clearly demonstrate that the production yields of different antibodies in E. coli differ significantly and that low yields can, in some cases, be improved by protein engineering.

Selection of antibody

fragments using phage

display The technology to display antibody fragments [43] on the surface of filamentous phages using the Gene III protein is an extension of the principle of peptide display [44]. The method opened up the possibility of mimicking the B-cell selection system in vitro by specifically enriching phage particles that display antibodies with a given desired specificity [45]. The basic method has been iteratively improved, and many of these improvements are described in detail in two excellent recent reviews [8,9*,10**]. One current trend in the field has been to apply antibody-fragment combinatorial libraries to new applications, for example to raise specific antibodies against human self-antigens using human antibody fragments [46], and to generate larger and larger libraries in order to _ select for ‘nanomolar binders’ from a single pot. Only a few years ago, it was thought that this goal would be hard to reach because of the size limitations of the libraries. A practical limit of 109 independent phage particles is often mentioned. In nature, the maturation of B-cells into plasma cells/memory cells involves somatic mutations of selected antibodies to improve further affinity from the

primary response. This mechanism has been mimicked to some degree in the ‘chain-shuffling technology’ (reviewed in [lo**]). The method utilizes a heavy or a light chain of a selected antibody, which pairs with a library of heavy or light chains. From this second selection step significantly higher afftnities can be isolated by comparison with the primary response [lo”]. Many variants of a second library step based on a primary selected antibody exist. One such alternative to emerge recently involves adding one specific light chain to a phage library of heavy chains in vitro by renaturation [47]. This method enabled a hapten antibody to evolve from a 108 M-1 binder to a 109 M-1 binder. Yet another variation on this theme, designated ‘CDR walking’, was demonstrated to yield an antigp120 antibody with both higher affinity and broader specificity [48]. It can be concluded that two-step methods, which form second hierarchical libraries, will continue to be important in the field. Currently, the approach is gathering an increasing number of applications, including novel and faster methods of humanizing murine antibodies [49]. An alternative to the chain-shuflling approach has been to simply make larger and larger libraries and use these for selection. The hope has been of finding in a single phage library high-affinity antibodies against any antigen. It was demonstrated recently that specific binders to a set of as many as 18 different antigens can be obtained from a single phage-displayed scFv library [50*]. Although the affinities recorded may not be high enough for many applications, the report does show the feasibility of the method. A recent and very interesting method is to recombine light and heavy chains in vivo in E. coli using the cre/lox recombination system [51]. The system has been used to generate an antibody fragment library with 6.5 x 1010 entries [52**]. Antibodies specific against a number of antigens of different types were selected from the library; the results are comparable with those of antibodies from a secondary immune response in mice (affinity constants of up to nM) [51]. Thus, the goal to select for tight binders in a single step was reached and the me/lox system must be considered a breakthrough in antibody engineering. Therefore, as predicted [53], the approach of phage display will soon replace many of the applications of traditional hybridoma techniques. Another trend in the field is to work on simplified selection methods. For a number of years, a major technical consideration has been the procedure to immobilize the target molecules to plates, columns, tubes or magnetic beads. All of these methods have advantages and drawbacks. Recently, Dueiias and Borrebaeck [54*] have presented a new approach to the selection problem. Instead of selection in vitro, an in vivo method was outlined. The method utilizes phage particles that are devoid of Gene III function. The selection is made by including a Gene III-target protein fusion protein

Antibody engineering Nilsson and E. coli F+ hosts cells. The fusion protein makes only phages that are displaying target protein binders competent for infection; this forms the basis of an efficient selection strategy. This system could also be used to mimic the clonal selection mechanism in the humoral immune response in mammals. Although the value of the strategy remains to be confirmed with more extensive characterization of any antibodies obtained and with the emergence of new examples, the method is elegant and might become a popular method in years to come.

Applications

of antibody

partners. The fusion approach is likely to expand to the use of antibodies as targeting molecules in new clinical applications. Although the fusion concepts remain to be fully evaluated in the clinic, the approach will continue to evolve as new molecular discoveries in the field of immune cell activation are divulged.

engineering

Antibody engineering has many applications. An early application was humanization, in which CDRs are grafied from a murine antibody into the framework of a human antibody [55]. The method has proven extremely use&l in the generation of more biologically active antibodies for therapeutic situations. In addition to the CDRs, substitutions of several framework residues may be needed to restore the affinity of the original murine antibody [56]. Most recently, the cumbersome methods of humanization have been significantly simplified using the chain-shuflling approach [49]. Even though the chain-shuffling method will simplify a number of applications in the hture, the use of human antibody fragments on phages (see above) should eventually decrease the overall need for humanization. Another interesting application of antibody engineering has been the generation of catalytic antibodies. Several recent trends in this field have emerged. One such trend is to increase the number of types of reactions that antibodies can catalyze, including the complicated ryn elimination to a cis olefin [57] and stereo-selective oxygenation reactions [58]. Another trend is to use protein engineering to dissect the mechanism of a catalytic antibody in more detail, as with the antibody 43C9, which catalyzes amide hydrolysis [59]. Further, phage display techniques (see above) are being used to select directly for catalytic functions out of a library [60]. Yet another trend in the area is to develop catalytic antibodies for specific biotechnological applications, such as peptide synthesis [61,62] and prodrug activation [63,64]. Antibody engineering in therapy is an interesting area, in which of course humanization plays an important role (see above) in avoiding the production of human anti-mouse antibodies. Antibodies are also engineered by making fusions to effector molecules in order to potentiate their effects in immunotherapy situations in vivo. Such fusion partners recently reported include interleukin 2 [65], staphylococcal enterotoxin A [66-l and Pseudomonas exotoxin [67]. Antibody, or antibody fiapent, fusion molecules retain antigen-binding activity and incorporate the new functionality of the fusion

Fig. 2. The protein A domain molecule. IgG binding is mediated by a surface comprising side chains of helices 1 and 2 (shown in cyan). The 13 side chains that have been subjected to random substitutions to form a combinatorial library are shown [69*]. The figure was generated using the modeled structure described in 1761 using the software Molscript [77].

Future aspects of antibody

engineering

Why do research groups continue to work on antibody engineering? One reason is, of course, to learn more about the immune system Gem a molecular standpoint. The question, however, becomes relevant when considering many of the biotechnological applications where the purpose is ‘only’ to find specific binders to a target molecule. Antibodies are rather unstable molecules and sometimes hard to produce in recombinant systems [68]. Therefore, a simpler molecular system involving the production of a small, stable and easily folded domain could be favourable in many applications. Recently, such a system based on one domain of staphylococcal protein

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Engineering and design

A was described [69*]. In this system, 13 amino acid residues on the surfaces of two of the CL helices of the 58-amino-acid domain were replaced by random amino acid residues (Fig. 2). Whether this system, or other similar systems, will provi& an alternative to antibodies in specific applications remains to be confirmed. Although the surface area of the protein A domain is similar to that of the antigen-binding sites of antibodies, the domains differ with regard to many other properties. It has been suggested that the dynamic properties are extremely important for the function of a protein, as was recently demonstrated by comparing the catalytic properties of a set of mutants of T4 lysozyme

[701. Another trend in the future will be to expand the antibody systems into other Ig-folded molecules such as T-cell receptors. These molecules are generally difficult to express in E. coli. The crystal structure of the p chain of the T-cell receptor was solved recently [71] and promising production systems are emerging [72]. With these tools in hand, the time is right to study the diversity and antigen recognition of T-cell receptor molecules with the approaches and systems described in this review for antibody engineering. Another Ig-fold protein that is being studied extensively using protein engineering and structural biological techniques, is the extracellular portion of the human growth hormone receptor. The receptor is dimerized by the hormone, and the individual amino acid residues that are responsible for this interaction have been mapped in detail [73]. Most surprisingly, 90% of the binding energy is found in only a subset of the interaction surface defined by X-ray crystallography [74].

Concluding

References and recommended Papers of particular interest, published review, have been highlighted as: . of special interest .. of outstanding interest

Acknowledgement Lan AbrahmsCn is acknowledged for fruitful critical comments on the manuscript.

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remarks

Antibody engineering has been a fast moving field during the past few years; in structural work, in making large libraries for selection, and in new applications. The years to come will continue to teach us more about how antibodies recognize antigens. In addition, protein engineering will continue to be utilized in the applied field to introduce novel effector functions to the antibody molecule. For many biotechnological applications, however, antibodies will perhaps be replaced by other protein families that are more stable.

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29.

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