Antibody expression in bacteriophage systems: the future of monoclonal antibodies? Lisa I. Garrard and Eugene A. Zhukovsky Genentech Inc, South San Francisco, USA
Bacteriophage systems have been utilized to express and isolate antibodies. This promising technology has been evolving rapidly and has the potential to revolutionize the way in which monoclonal antibodies are generated. This review focuses on the many recent advances that have been made in obtaining monoclonal antibodies from bacteriophage systems.
Current Opinion in Biotechnology 1992, 3:474-480
Introduction
Antibody expression in bacteriophage 2~
Antibodies have played an essential role in the develo p m e n t of biotechnology as they have been utilized in research, diagnosis of disease, and are potential therapeutic agents. The many applications of monoclonal antibodies have b e e n made possible by the introduction of the hybridoma technology by Kohler and Milstein in 1975 [1]. This revolutionary technology permitred the production of monoclonal antibodies from mice. The recent objective, however, of obtaining human antibodies and the inability of the hybridoma or Epstein-Barr immortalization techniques [2] to adequately fulfill this need, has led to further advances in antibody technology. For example, a method was developed to 'humanize' antibodies b y grafting the complementarity-determining regions (CDRs) from a particular murine monoclonal antibody onto the framework of a human antibody [3]. Unfortunately, these types of molecules were often tedious to synthesize, required additional tailoring, and usually lost some degree of specificity and/or affinity [4-8]. Therefore, two additional methods have b e e n developed for the generation of human monoclonal antibodies. First, advances in the polymerase chain reaction (PCR) led to the possibility of cloning antibody repertoires [9-121. Second, as the expression of entire antibody molecules is not possible in Escherichia coli, methods for expression of antibody fragments in bacterial systems have b e e n found [13,14]. More recently, approaches employing the expression of antibodies in bacteriophage systems have provided an opportunity to obtain human antibodies directly [15-19]. This review describes recent developments in the rapidly evolving field of antibody expression in phage systems.
The cloning of antibody repertoires from immunized mice and the expression of these antibody fragments in a )v bacteriophage vector system was initially performed by Huse et al. [151. Plaques expressing antigenbinding antibody fragments were identified by obtaining plaque lifts that were then probed with labeled antigen. This type of antibody expression system has also been successfully applied to the isolation of murine antibodies specific for influenza virus hemagglutinin [16] and for h u m a n Fab fragments specific for tetanus toxoid [17,18]. Recently, Portolano et al. [20"] have obtained a high affinity (about 10-9M) human antibody to thyroid peroxidase (TPO) by screening a random library of combined heavy and light chain genes derived from the thyroid cDNA of patients with Graves' disease. The affinity of the selected Fab is comparable to the affinities of autoantibodies that patients afflicted with autoimmurfe thyroid disease produce in response to TPO. Sequences encoding the light and heavy chains produced from a particular cell, however, are not linked physically, and thus, will inevitably b e c o m e scrambled during the cloning process. Therefore, it is improbable that the particular heavy and light chain combination in the anti-TPO antibody reflects the original in vivo situation. This single anti-TPO Fab clone was isolated after screening 2 x 105 plaques. In a different application of a ~, phage expression system, it was shown that a human anti-(rhesus D) Fab fragment could be rescued from an Epstein-Barr-vims-transformed cell line that expressed an individual antibody clone [21].
Abbreviations CDR--complementarity-determiningregion; HBsAg--hepatitisB surface antigen; HIV--human immunodeficiencyvirus; PCR--polymerase chain reaction; phOx--2-phenyloxazol-5-one;se--single chain; TPO--thyroid peroxidase.
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Antibodyexpressionin bacteriophagesystemsGarrard Initially, the £ phage expression system was thought to hold great potential for isolating unique antibodies, as p r o b e d libraries were large in comparison to those that can be screened using conventional hybridoma technology. For example, a realistic u p p e r limit on the number of individual clones that can be sampled from a ~, phage system is about 106, whereas conventional hybridoma technology only allows access to about 103-104 individual clones at most. As alluded to previously, however, it is unlikely that the antibodies that result from the cloning of antibody repertoires will be composed of the original pairings of heavy and light chains. Therefore, it may be necessary to examine a million or more clones before an antibody with the desired property is obtained. Recently, a m e t h o d of displaying antibody fragments on the surface of filamentous bacteriophage has been developed, allowing extremely large numbers of individual antibody clones to b e screened in a convenient and timely manner. Therefore, this technology may actually supplant both £ phage expression and conventional hybridoma technologies in the future.
Antibody expression in filamentous phage systems Smith and coworkers [22,23] demonstrated that peptides could be displayed on the surface of filamentous phage in a multivalent fashion by inserting their sequences into gene III within the phage genome. The product of gene III (g3p) is a minor coat protein that is required for host infectivity, proper phage assembly, and is present as 3-5 copies at one end of the bacteriophage [24]. Peptides are selected based u p o n their specific binding to antibodies by affinity enrichment on columns or by panning on coated polystyrene wells. One important feature of this system is that the peptides are-physically linked to the DNA encoding them. Therefore, once individual clones are isolated, their sequence can easily be identified. Phage display technology has led to the display of other peptides and proteins, including antibody fragments, on the phage surface [19,25-29]. Further, it has the potential to advance the discovery of novel binding molecules and, in particular, to revolutionize antibody technology. Phage display was first applied to antib o d y fragments by McCafferty et al. [19]. This group demonstrated that a single chain antibody fragment ( s c F v - - t h e antibody light and heavy chain variable domains fused via a flexible linker to create a single chain) could be displayed on the filamentous phage surface b y fusing the scFv to the amino terminus of g3p. This scFv phage, which originated from the antilysozyme D1.3 antibody [30], could be enriched over non-cognate scFv phage via selection on the antigen lysozyme. Since this development, there have b e e n several other reports of antibody fragments displayed on filamentous phage. In addition, there have b e e n reports describing the display and selection of antibody fragments directly from libraries obtained from immunized and even unimmunized sources. These advance-
and Zhukovsky 475
ments will be reviewed in more detail in the sections that follow.
Display of specific antibody fragments Several recent articles have reported the successful display and enrichment of antigen-binding domains from previously identified antibodies [31"-36"]. In addition, a number of improvements in antibody phage display technology have been reported. Breitling and coworkers [31"] described a phagemid-based vector for low-copy scFv display. The major advantage of using a phagemid instead of inserting sequences directly into the phage genome, as had been done previously, is that antibody DNA can be manipulated in the plasmid form and its expression can be regulated. Kang et al. [32"] also employed a phagemid system to display heterodimeric Fab molecules. In this work, the major coat protein, e n c o d e d by gene VIII (g8p, typically present as approximately 2700 copies per virion), was used to display Fab-g8p fusion molecules on the surface of the filamentous phage particles. In this case, the carboxyl terminus of the Fd domain is fused to the amino terminus of g8p. Both the light chain and the Fd-g8p fusion protein are preceded b y the pelB signal sequence. This sequence directs the secretion of the light chain and the Fd-g8p fusion protein to the inner membrane of E. coli, where the phage particle is assembled. Multivalent display distributes the Fab fusion proteins along the entire length of the phage particles, as assessed by electron microscopy. The investigators utilized an Fab that was directed against the hapten pnitrophenyl phosphonamidate and demonstrated that it could bind specifically to its antigen. They also s h o w e d that it could be enriched 2700-fold over an unrelated Fab phage in one round of selection. Another example is the phage display of a specific Fab-g8p fusion protein that was directed against the p55 chain (Tac) of the human IL-2 receptor [33"]. In this case, the light chain was fused to g8p and two signal sequences, p h o A and stII, directed the heavy and light chains to the bacterial inner membrane where they were assembled on the surface of the phage. In addition, it was shown that enrichment of the anti-Tac Fab-bearing phage was accomplished by antigen selection on the p55-coupled Sepharose column. One potential disadvantage of the display of proteins as fusions to the major coat protein g8p is the avidity effect that can accompany the multivalent display of corresponding antibody fragments. Avidity or chelation effects have been attributed to the inability to discriminate between low and high affinity binders in both peptides and proteins [26,29]. This problem becomes a key consideration w h e n the objective is to select the strongest binding antibody and to discriminate between antibodies with different affinities. The development of monovalent display, which was first reported by Bass et al. [29] for human growth hormone, has the ability to avoid this avidity effect. Essentially monovalent display is produced by repression of the promoter that controls expression of the Fab-g3p fusion protein. As repression is not 100% efficient in most instances, low levels of the Fab fusion
476 Expressionsystems protein are produced relative to wild-type g3p and wild type is preferentially assembled into the phage particle. Enough of the Fab fusion protein is expressed, however, so that approximately 10% of the phage particles contain an Fab molecule. An additional advantage of monovalent display is that it allows the infectivi W of the p r o g e n y p h a g e m i d particles to be essentially identical to that of wild type, as most of the copies of g3p are derived from the wild-type gene III. Monovalent display of antibody fragments has n o w b e e n e m p l o y e d by two groups. Barbas et al. [34"] utilized anti-tetanus toxoid Fabs to s h o w that the m o n o v a lent display of a tight binder (10- 9 M) produced a 253fold enrichment over a w e a k binder (10-7 M). On the other hand, the multivalent display of the same Fabs generated only a fivefold enrichment. The limited enrichment of a tight binder over a w e a k one in the latter case is attributed to the avidity effect that can be prod u c e d by the multivalent display of Fab molecules. In a report by Garrard et al. [35"], monovalent Fab display was s h o w n to be effective in discriminating b e t w e e n Fab p h a g e possessing affinities in the picomolar to nanomolar range. Consequently, this type of display permitted preferential enrichment of the tightest binding phage. This group also demonstrated that the affinities for the free Fab and the Fab-phage were virtually identical and, therefore, antibodies selected from this type of display system could b e expected to possess affinities equal to the individual Fab monomer. Thus, the advantages of monovalent display in the selection of antibody fragments with different affinities has led most groups to use this technique. Another d e v e l o p m e n t which has further extended the monovalent antibody phage display technology was first reported by H o o g e n b o o m et al. [36"]. An a m b e r stop c o d o n (UAG) w a s inserted b e t w e e n the displayed Fab and g3p. When this type of construct is expressed in a strain of E. coli that suppresses the stop codon, read-through is accomplished and the fusion protein is p r o d u c e d for p h a g e assembly. Alternatively, if the same construct is placed in a host that does not suppress the codon, soluble Fab can be expressed, purified, and used for functional analysis.
Display and selection of antibody fragment libraries from immunized sources A major advantage of filamentous phage display technology is the ability to access large combinatorial libraries to achieve rapid and efficient selection. Therefore, this system s e e m s particularly well suited to selecting specific antibodies from combinatorial antibody fragment libraries originating from immunized sources. Clackson et al. [37"1 have shown that a range of w e a k to strong binders directed against the hapten 2-phenyloxazol-5-one (phOx) could be selected from scFv libraries of mice immunized with this hapten. It was also noted that a given heavy chain could pair with several light chains to produce antigen-binding scFvs and that a given light chain could also pair productively with a n u m b e r of heavy chains. This
p h e n o m e n o n , referred to as promiscuity, increases the n u m b e r of functional heavy and light chain combinations and forms the basis of hierarchical library construction. Hierarchical libraries w e r e generated b y taking a specific heavy or light chain that was found to bind to phOx, and then recombining each of these with heavy or light chains from the original library. Such a two-stage selection scheme, in which the initial selection of combinatorial libraries is followed b y hierarchical library screening, resulted in the identification of n e w pairings with significantly higher affinities than the original pairing (1.0 x 10- 8 M versus 1.8 x 10- 5 M). Burton et al. [38"] were able to identify several Fabs directed against the surface glycoprotein gp120 of type 1 h u m a n immunodeficiency virus (HIV) b y screening a r a n d o m combinatorial library expressed on the surface of filamentous phage. This library was generated from the b o n e marrow of an individual w h o had b e e n HIV-positive for six years. Inhibition enzyme-linked i m m u n o s o r b e n t assays using soluble gp120 w e r e employed to determine the affinities of the selected Fabs, which were found to be around 10-9 M. The authors note that a similar library constructed in ~, phage failed to yield any positives. Thus, this observation e m p h a sizes the potential of the filamentous phage display system for the identification of rare clones. Z e b e d e e et al. [39"] screened Fab libraries generated from h u m a n lymphocytes of individuals w h o had b e e n vaccinated with hepatitis B surface antigen (HBsAg) in order to isolate several antibodies that bind HBs a g specifically. Using phage display, Fabs w e r e obtained from individuals with b o t h high and low titers to HBsAg. Promiscuous pairings of the antibody chains w e r e also reported b y this group. For example, a particular heavy chain was capable of associating with several light chains to produce recognition of the same epitope, while another heavy chain could assemble with both ~ and ~, light chains and still maintain selective binding to the antigen.
Display and selection of antibody fragment libraries from unimmunized sources Although it is n o w possible to isolate antibodies from h u m a n s #immunized with specific antigens, it is not possible to immunize humans at will. In addition, a h u m a n immune system will not respond characteristically to self or h u m a n antigens except in conditions of autoimmune disease. Therefore, the ability to isolate functional antibodies b y circumventing immunization w o u l d b e a significant achievement in the utilization of antibody p h a g e display technology for the production of monoclonal antibodies. As antibodies obtained in this manner are the result of random combinations of h e a v y and light chains, it is h o p e d that this technology will permit the selection of h u m a n antibodies that bind to h u m a n antigens even though the h e a v y and light chains of antibody fragments arise from a hum a n source. Marks et al. [40"] have b e e n successful in obtaining antibody fragments against several antigens from a scFv p h a g e library generated from unim-
Antibody expression in bacteriophage systemsGarrard munized human peripheral blood lymphocytes. The human donors were not immunized with any of the antigens used in the experiment and were therefore considered to b e unimmunized with respect to these antigens. A library consisting of approximately 107-108 independent clones was screened against turkey lysozyme and p h O x antigens. Selection against these antigens yielded antibodies with moderate affinities of 10-7M and 5 x 10-7M. Although it depends on the ultimate use of the antibody, it is usually preferable to obtain antibodies with higher affinities. It seems possible that expanding the size of the original library to 1010-1012 independent clones will permit the selection of higher affinity antibodies. At the moment, however, it is feasible to generate libraries of only 108 members as a result primarily of limitations in E. coli transformation techniques. In order to increase the binding affinities of Fabs initially selected from random combinatorial libraries, several approaches could be pursued. First, hierarchical libraries could be constructed and screened for tighter binders. Second, higher affinities could be obtained by utilizing one of several methods of mutagenesis after the initial selection. For example, Gram et al. [41--] used a two-stage selection for isolating tight binding Fabs. Low affinity ( 1 0 - 5 - t 0 - 6 M ) Fabs, directed against progesterone and displayed multivalenfly as g8p fusions, were selected from combinatorial libraries that originated from unimmunized mice. Subsequent affinity maturation of the selected Fabs was performed by employing error-prone PCR to mutagenize only the variable domains from the selected Fabs. These mutagenesis domains were expressed monovalently as scFv-g3p fusions, and such a library was then subjected to further rounds of selection. Although direct affinity measurements were not determined, the results suggested that a 30-fold higher affinity could be achieved. Third, mutagenesis of a single CDR could be performed and, in fact, has recently bee used to alter the specificity of a particular antibody [42"]. In this study, Barbas et al. constructed a library by randomizing CDR3 of the heavy chain of an Fab directed against the hapten tetanus toxoid. Selection against a different hapten, fluorescein, yielded new Fabs, which specifically b o u n d this hapten and had affinities ranging from
Table 1.
and Zhukovsky
10-7 M to 10-8 M. Finally, other methods of mutagenesis such as passage of the antibody phage through a mutator strain (e.g.E. coli m u t D) may also yield improvements in affinity.
Considerations for the expression of antibodies in bacteriophage systems The expression of antibodies in phage systems is becoming an attractive and efficient approach for the identification of antibody fragments. As mentioned previously, however, both the )v and the filamentous phage systems present randomly combined heavy and light chains for selection and, therefore, make the possibility of creating original light and heavy chain pairings remote. This may be a consideration if the objective is to isolate antibodies from immunized sources and the desired antibody is rare. As observed by several groups, however, promiscuity of one or both chains may occur, thus increasing the potentially rare chance of generating a productive binder. If the goal is to obtain antibodies from unimmunized sources, both the size and complexity of the library b e c o m e even more important. Winter and Milstein [43] have p r o p o s e d that about 107-108 individual antibody seq u e n c e s exist at any given moment in vivo. As this degree of diversity is sufficient for ultimately generating antibodies in vivo, and as it is feasible to mimic this diversity with filamentous phage display, it should be possible to obtain at least low affinity binders. The reports by Marks et al. [40.-] and Barbas e t al. [42-] provide evidence to support this idea. Further investigation is required to determine whether libraries of this size will be sufficient to obtain antibodies against a large n u m b e r of antigens, and whether antibodies will be obtained against human antigens (particularly if the unimmunized source is human). So far, only low affinity antibodies have been generated using this approach. Therefore, in order to increase antibody affinity, multi-stage screening of the libraries could be performed. Such a strategy is analogous to the process of affinity maturation in the natural course of antibody development, and may be essential for the production of high affinity antibody fragments. It is also possible, however, that even moderate to high affinity binders
Summary of antibody fragment expression in phage.
Expression form
Source of antibody fragment DNA Immunized
Unimmunized
Individual clone
Filamentousphage g3p
Fab scFv
[38",39"] [37"]
[40",41 -']
[34",35",36",42"] [31 "]
g8p
Fab
-
[41 "]
[32",33"]
Fab
-
-
[20",21 ]
k phage
477
478
Expression systems can be selected in the beginning if methods that will significantly e x p a n d the size of the initial library are developed.
6.
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It is important to note that often the ultimate goal is to identify antibodies that elicit a particular biological response. Antibodies possessing the highest affinities will not necessarily possess the desired functional attribute. It might actually b e advantageous to obtain w e a k binders, and then determine if a specific function exists, for example in a cell-based assay. Multivalent display m a y b e preferred if the objective is to obtain an antibody with a specific function.
7.
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15.
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References and recommended reading
16.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest •. of outstanding interest
CATONAJ, KOPROWSK1H: Influenza Virus Hemagglutininspecific Antibodies Isolated f r o m a Combinatorial Exp r e s s i o n Library are Closely Related to t h e I m m u n e R e s p o n s e o f the Donor. Proc Natl Acad Sci USA 1990, 87:6450-6454.
17,
MULLINAXRL, GROSS EA, AMBERGJR, HAY BN, HOGREFEHI-I, KUBITZ MM, GREENERA, ALTING-MEESM, ARDOURELD, SHORT JM, ET AL,: Identification o f Hnra~tx Antibody Fragment Clones Specific for Tetanus Toxoid in a Bacteriophage I m m u n o e x p r e s s i o n Library. Proc Natl A c a d Sci USA 1990, 87:8095-8099.
18.
PERSSONMAA, CAOTHIEN RH, BURTON DR: G e n e r a t i o n o f Diverse High-aff3nity Hnm~n Monoclonal Antibodies b y R e p e r t o i r e Cloning. Proc Natl Acad Sci USA 1991, 88:2432-2436.
19.
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Conclusion The expression of antibody fragments in p h a g e systems is an exciting opportunity to produce monoclonal antibodies efficiently and without the need for immunization. Results discussed in this review highlight recent developments that contribute to this technological advance (Table 1). First, Fabs or scFvs can be expressed on the surface of the p h a g e and specific binders can b e selected. Second, a multi-stage approach, which initially employs selection of random combinatorial libraries followed b y selection of secondary or hierarchical libraries, is likely to result in the isolation of binders with enhanced affinities. Third, the resulting antibody fragments have affinities similar to those of antibodies obtained from conventional sources. In conclusion, it is possible to see h o w these advances in antibody technology will continue to c o m p l e m e n t conventional hybridoma technology and m a y replace it altogether in the near future.
Acknowledgements The authors thank Dr D Henner for advice and comments.
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31.
BREITLINGF, DUBEL S, SEEHAUS T, KLEWINGHAUS I, LITTLE M: A Surface Expression Vector for Antibody Screening. Gene 1991, 104:1147-1153. DNA coding for a scFab of anti-hen egg-white lysozyme was f u s e d to g3p of the filamentous p h a g e a n d expressed in E. coll. Expression resulted in a functionally specific antibody that w a s able to bind antigen. 32.
KANGAS, BARBAS CF, JANDA KD, BENKOVIC SJ: L | m k a g e o f Recognition and Replication Functions b y Assembling Combinatorial Antibody Fab Libraries along Phage S u r f a c e s . Proc Natl A c a d Sci USA 1991, 88:4363-4366. Multivalent display on the filamentous p h a g e of Fab, isolated from the original pmitrophenyl p h o s p h o n a m i d a t e combinatorial library, by fusing h e a v y chain to gBp is presented. It was s h o w n that o n e r o u n d of p a n n i n g enriched the phage displaying Fab by 2700-fold. CHANG CN, LANDOLFI NF, QUEEN C: Expression o f Antibody Fab D o m a i n s o n Bacteriophage Surface (Potential use for Antibody Selection). J I m m u n o l 1991, 147:3610-3614. Anti-Tac murine Fab was displayed in a multivalent fashion by fusing the light chain to the gBp of the filamentous phage. It was s h o w n that Fab expressed thus could be specifically selected o n an affinity matrix. 33.
BARBASCF III, KANG AS, LERNER RA, BENKOVIC SJ: Assembly o f C o m b i n a t o r i a l A n t i b o d y L i b r a r i e s o n Phage Surfaces: the Gene Ill Site. Proc Natl A c a d Sci USA 1991, 88:7978-7982. Previously characterized in bacteriophage ~, , a n anti-tetanus toxoid library was u s e d to construct a combinatorial Fab library displayed o n the surface of filamentous p h a g e via fusion of heavy chain to t h e carboxy-terminal domain of g3p. Selection by panning over tetanus toxoid resulted in 103- to 105-fold enrichment. 34.
35.
GARRARDLJ, YANG M, O'CONNELLMP, KELLEYRF, ITIENNERDJ: Fab Assembly and E n r i c h m e n t i n a Monovalent Phage D i s p l a y S y s t e m . Biotechnology 1991, 9:1373-1377. An Fab directed against the ne u receptor w a s displayed by fusing the Fd chain to the carboxy-terminal domain of g3p. Selection s c h e m e s that enriched for t h e tightest binder in a s u b n a n o m o l a r affinity range were performed. The affinities of soluble Fab a n d the p h a g e displayed Fab were s h o w n to be virtually identical. 36.
HOOGENBOOMHR, GRIFEITHSAD, JOHNSON KS, CHISWELLDJ, HUDSON P, WINTE~G: Multi-subunit Proteins o n the Surf a c e o f Filamentous Phage: Methodologies for D i s p l a y i n g Antibody (Fab) Heavy and L i g h t C h a i n . Nucl A c i d s Res 1991, 19:4133--4137. The heavy or the light chains o f Fab fragments from the m o u s e antip h O x antibody were fused to g3p of the filamentous phage a n d the c o m p l e m e n t chain was secreted into the periplasmic space of E, coB. T h u s formed, heterodimeric Fab showed specific binding to p h O x a n d bovine s e r u m albumin b u t not to bovine serum albumin alone. 37. ..
CLACKSONT, HOOGENBOOM HR, GRIFFITHS AD, WINTER G: M a k i n g A n t i b o d y Fragments Using Phage D i s p l a y Lib r a r i e s . Nature 1991, 352:6244528. In this paper a n approach employing a two stage selection is presented. A combinatorial scFv library displaying filamentous p h a g e via fusion with g3p was constructed by PCR amplification of mRNA obtained from mice i m m u n i z e d with hapten phOx. Following two r o u n d s of selection, two hierarchical libraries were made. By u s i n g soluble scFv o n e binder w a s f o u n d to have a dissociation constant o f 10 riM. 38.
BURTON DR, BARBAS CF 11I, PERSSON MAA, KOENIG S, CHANOCK RM, LERNERRA: A Large Array o f Hnm:~t Monoclonal A n t i b o d i e s to Type 1 H u m a n l m m u n o d e f i c i e n c y V i r u s f r o m C o m b i n a t o r i a l Libraries o f A s y m p t o m a t i c Seropositive I n d i v i d u a l s . Proc Natl A c a d Sct USA 1991, 88:10134-10137. RNA from the b o n e marrow lymphocytes of an asymptomatic HIV positive donor was used to produce a r a n d o m combinatorial library of Fab against gp120 (strain Inb) and w a s expressed o n the surface of the filamentous p h a g e by fusion of the heavy chain to g3p. After four rounds of panning, soluble Fabs were p r o d u c e d from selected phage. Affinity measurements were performed using inhibition enzyme-linked ilIkmunosorbent assays a n d resulted in dissociation constants of the order of 10 nM. ZEBEDEESL, BARBAS CF iii, HOM Y-L, CAOTHIEN RH, GRAFF R, DEGRAW J, PYATI J, LAPOLLA R, BURTON DR, LERNER RA, THORNTON GB: H u m a n C o m b i n a t o r i a l A n t i b o d y Lib r a r i e s t o H e p a t i t i s B S u r f a c e A n t i g e n . Proc Natl A c a d Sci U SA 1992, 89:3175-3179. Leukocyte RNA from h u m a n donors i m m u n i z e d with HBsAg w a s u s e d to construct a repertoire library in the filamentous phage system, in w h i c h Fab was fused to g3p. After p a n n i n g selection for three rounds, several functional Fabs were selected a n d affinity measurem e n t s were performed on a soluble form of Fab. Sequence analysis o f these clones showed h u m a n light-chain promiscuity and suggested that m u c h of the antigen recognition w a s d e p e n d e n t u p o n t h e h u m a n h e a v y chain. 39.
40. •.
MARKSJD, HOOGENBOOM HR, BONNERT YP, McCmFERTY J, GRtFFITHSA n , WINTER G: By-passing l m m u n | z a t i o n ( H u m a n Antibodies f r o m V-gene Libraries Displayed o n Phage). J Mol Biol 1991, 222:581-597. Peripheral blood lymphocytes of u n i m m u n i z e d h u m a n donors w e r e u s e d to construct a large (107-108 members) combinatorial library of scFv displayed on the surface o f the filamentous p h a g e via fusion to g3p. After four rounds of selection clones specifically binding turkey egg lysozyme, bovine serum a l b u m i n and p h O x were identified. T h e dissociation constant of the best scFv selected was 86 riM. 41. ,,
GRAMH, MARCONIL-A, BARBASCF II1, COLLETTA, LERNERRA, KANG AS: I n Vitro Selection and Afrmity Maturation o f A n t i b o d i e s from a N a i v e C o m b i n a t o r i a l l m m u n o g l o b ulin Library. Proc Natl A c a d Sci U SA 1992, 89:3576-3580. This paper describes a m e t h o d to access low affinity Fabs from a n immunoglobulin library of n o n - i m m u n i z e d mice with s u b s e q u e n t affinity maturation o f selected clones. Initially selected low
479
480
Expression systems affinity binders were subjected to affinity maturation by utilizing error-prone PGR. This scheme resulted in decrease of dissociation constant for one of the clones from 10 to 0.3 nM.
over hapten fluoroscein Oinked to bovine serum albumin) resulted in selection of clones with similar amino acid sequences and affinities of 10-100 nM.
BARBASGF III, BAIN JD, HOEKSTRA DM, LERNERRA: S e m i s y n thetic C o m b i n a t o r i a l A n t i b o d y Libraries: a C h e m i c a l S o l u t i o n to t h e D i v e r s i t y P r o b l e m . Proc Natl A c a d Sci USA 1992, 89:4457-4461. Heavy chain CDR3 of h u m a n tetanus toxoid-hinding Fab was randomized and the resulting library was displayed on the surface of the filamentous phage as an Fab-g3p fusion. Sorting of this library
43.
42.
WINTER G, MILSTEIN C: M a n - m a d e Antibodies. Na tu r e 1991, 349:293-299.
LJ Garrard and EA Zhukovsky, Genentech Inc, 460 Point San Bruno Boulevard, South San Francisco, CA 94080, USA.
Bacteriophage systems have been utilized to express and isolate antibodies. This promising technology has been evolving rapidly and has the potential to revolutionize the way in which monoclonal antibodies are generated. This review focuses on the many recent advances that have been made in obtaining monoclonal antibodies from bacteriophage systems.
Current Opinion in Biotechnology 1992, 3:474-480
Introduction
Antibody expression in bacteriophage 2~
Antibodies have played an essential role in the develo p m e n t of biotechnology as they have been utilized in research, diagnosis of disease, and are potential therapeutic agents. The many applications of monoclonal antibodies have b e e n made possible by the introduction of the hybridoma technology by Kohler and Milstein in 1975 [1]. This revolutionary technology permitred the production of monoclonal antibodies from mice. The recent objective, however, of obtaining human antibodies and the inability of the hybridoma or Epstein-Barr immortalization techniques [2] to adequately fulfill this need, has led to further advances in antibody technology. For example, a method was developed to 'humanize' antibodies b y grafting the complementarity-determining regions (CDRs) from a particular murine monoclonal antibody onto the framework of a human antibody [3]. Unfortunately, these types of molecules were often tedious to synthesize, required additional tailoring, and usually lost some degree of specificity and/or affinity [4-8]. Therefore, two additional methods have b e e n developed for the generation of human monoclonal antibodies. First, advances in the polymerase chain reaction (PCR) led to the possibility of cloning antibody repertoires [9-121. Second, as the expression of entire antibody molecules is not possible in Escherichia coli, methods for expression of antibody fragments in bacterial systems have b e e n found [13,14]. More recently, approaches employing the expression of antibodies in bacteriophage systems have provided an opportunity to obtain human antibodies directly [15-19]. This review describes recent developments in the rapidly evolving field of antibody expression in phage systems.
The cloning of antibody repertoires from immunized mice and the expression of these antibody fragments in a )v bacteriophage vector system was initially performed by Huse et al. [151. Plaques expressing antigenbinding antibody fragments were identified by obtaining plaque lifts that were then probed with labeled antigen. This type of antibody expression system has also been successfully applied to the isolation of murine antibodies specific for influenza virus hemagglutinin [16] and for h u m a n Fab fragments specific for tetanus toxoid [17,18]. Recently, Portolano et al. [20"] have obtained a high affinity (about 10-9M) human antibody to thyroid peroxidase (TPO) by screening a random library of combined heavy and light chain genes derived from the thyroid cDNA of patients with Graves' disease. The affinity of the selected Fab is comparable to the affinities of autoantibodies that patients afflicted with autoimmurfe thyroid disease produce in response to TPO. Sequences encoding the light and heavy chains produced from a particular cell, however, are not linked physically, and thus, will inevitably b e c o m e scrambled during the cloning process. Therefore, it is improbable that the particular heavy and light chain combination in the anti-TPO antibody reflects the original in vivo situation. This single anti-TPO Fab clone was isolated after screening 2 x 105 plaques. In a different application of a ~, phage expression system, it was shown that a human anti-(rhesus D) Fab fragment could be rescued from an Epstein-Barr-vims-transformed cell line that expressed an individual antibody clone [21].
Abbreviations CDR--complementarity-determiningregion; HBsAg--hepatitisB surface antigen; HIV--human immunodeficiencyvirus; PCR--polymerase chain reaction; phOx--2-phenyloxazol-5-one;se--single chain; TPO--thyroid peroxidase.
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©Current Biology Ltd ISSN 0958-1669
Antibodyexpressionin bacteriophagesystemsGarrard Initially, the £ phage expression system was thought to hold great potential for isolating unique antibodies, as p r o b e d libraries were large in comparison to those that can be screened using conventional hybridoma technology. For example, a realistic u p p e r limit on the number of individual clones that can be sampled from a ~, phage system is about 106, whereas conventional hybridoma technology only allows access to about 103-104 individual clones at most. As alluded to previously, however, it is unlikely that the antibodies that result from the cloning of antibody repertoires will be composed of the original pairings of heavy and light chains. Therefore, it may be necessary to examine a million or more clones before an antibody with the desired property is obtained. Recently, a m e t h o d of displaying antibody fragments on the surface of filamentous bacteriophage has been developed, allowing extremely large numbers of individual antibody clones to b e screened in a convenient and timely manner. Therefore, this technology may actually supplant both £ phage expression and conventional hybridoma technologies in the future.
Antibody expression in filamentous phage systems Smith and coworkers [22,23] demonstrated that peptides could be displayed on the surface of filamentous phage in a multivalent fashion by inserting their sequences into gene III within the phage genome. The product of gene III (g3p) is a minor coat protein that is required for host infectivity, proper phage assembly, and is present as 3-5 copies at one end of the bacteriophage [24]. Peptides are selected based u p o n their specific binding to antibodies by affinity enrichment on columns or by panning on coated polystyrene wells. One important feature of this system is that the peptides are-physically linked to the DNA encoding them. Therefore, once individual clones are isolated, their sequence can easily be identified. Phage display technology has led to the display of other peptides and proteins, including antibody fragments, on the phage surface [19,25-29]. Further, it has the potential to advance the discovery of novel binding molecules and, in particular, to revolutionize antibody technology. Phage display was first applied to antib o d y fragments by McCafferty et al. [19]. This group demonstrated that a single chain antibody fragment ( s c F v - - t h e antibody light and heavy chain variable domains fused via a flexible linker to create a single chain) could be displayed on the filamentous phage surface b y fusing the scFv to the amino terminus of g3p. This scFv phage, which originated from the antilysozyme D1.3 antibody [30], could be enriched over non-cognate scFv phage via selection on the antigen lysozyme. Since this development, there have b e e n several other reports of antibody fragments displayed on filamentous phage. In addition, there have b e e n reports describing the display and selection of antibody fragments directly from libraries obtained from immunized and even unimmunized sources. These advance-
and Zhukovsky 475
ments will be reviewed in more detail in the sections that follow.
Display of specific antibody fragments Several recent articles have reported the successful display and enrichment of antigen-binding domains from previously identified antibodies [31"-36"]. In addition, a number of improvements in antibody phage display technology have been reported. Breitling and coworkers [31"] described a phagemid-based vector for low-copy scFv display. The major advantage of using a phagemid instead of inserting sequences directly into the phage genome, as had been done previously, is that antibody DNA can be manipulated in the plasmid form and its expression can be regulated. Kang et al. [32"] also employed a phagemid system to display heterodimeric Fab molecules. In this work, the major coat protein, e n c o d e d by gene VIII (g8p, typically present as approximately 2700 copies per virion), was used to display Fab-g8p fusion molecules on the surface of the filamentous phage particles. In this case, the carboxyl terminus of the Fd domain is fused to the amino terminus of g8p. Both the light chain and the Fd-g8p fusion protein are preceded b y the pelB signal sequence. This sequence directs the secretion of the light chain and the Fd-g8p fusion protein to the inner membrane of E. coli, where the phage particle is assembled. Multivalent display distributes the Fab fusion proteins along the entire length of the phage particles, as assessed by electron microscopy. The investigators utilized an Fab that was directed against the hapten pnitrophenyl phosphonamidate and demonstrated that it could bind specifically to its antigen. They also s h o w e d that it could be enriched 2700-fold over an unrelated Fab phage in one round of selection. Another example is the phage display of a specific Fab-g8p fusion protein that was directed against the p55 chain (Tac) of the human IL-2 receptor [33"]. In this case, the light chain was fused to g8p and two signal sequences, p h o A and stII, directed the heavy and light chains to the bacterial inner membrane where they were assembled on the surface of the phage. In addition, it was shown that enrichment of the anti-Tac Fab-bearing phage was accomplished by antigen selection on the p55-coupled Sepharose column. One potential disadvantage of the display of proteins as fusions to the major coat protein g8p is the avidity effect that can accompany the multivalent display of corresponding antibody fragments. Avidity or chelation effects have been attributed to the inability to discriminate between low and high affinity binders in both peptides and proteins [26,29]. This problem becomes a key consideration w h e n the objective is to select the strongest binding antibody and to discriminate between antibodies with different affinities. The development of monovalent display, which was first reported by Bass et al. [29] for human growth hormone, has the ability to avoid this avidity effect. Essentially monovalent display is produced by repression of the promoter that controls expression of the Fab-g3p fusion protein. As repression is not 100% efficient in most instances, low levels of the Fab fusion
476 Expressionsystems protein are produced relative to wild-type g3p and wild type is preferentially assembled into the phage particle. Enough of the Fab fusion protein is expressed, however, so that approximately 10% of the phage particles contain an Fab molecule. An additional advantage of monovalent display is that it allows the infectivi W of the p r o g e n y p h a g e m i d particles to be essentially identical to that of wild type, as most of the copies of g3p are derived from the wild-type gene III. Monovalent display of antibody fragments has n o w b e e n e m p l o y e d by two groups. Barbas et al. [34"] utilized anti-tetanus toxoid Fabs to s h o w that the m o n o v a lent display of a tight binder (10- 9 M) produced a 253fold enrichment over a w e a k binder (10-7 M). On the other hand, the multivalent display of the same Fabs generated only a fivefold enrichment. The limited enrichment of a tight binder over a w e a k one in the latter case is attributed to the avidity effect that can be prod u c e d by the multivalent display of Fab molecules. In a report by Garrard et al. [35"], monovalent Fab display was s h o w n to be effective in discriminating b e t w e e n Fab p h a g e possessing affinities in the picomolar to nanomolar range. Consequently, this type of display permitted preferential enrichment of the tightest binding phage. This group also demonstrated that the affinities for the free Fab and the Fab-phage were virtually identical and, therefore, antibodies selected from this type of display system could b e expected to possess affinities equal to the individual Fab monomer. Thus, the advantages of monovalent display in the selection of antibody fragments with different affinities has led most groups to use this technique. Another d e v e l o p m e n t which has further extended the monovalent antibody phage display technology was first reported by H o o g e n b o o m et al. [36"]. An a m b e r stop c o d o n (UAG) w a s inserted b e t w e e n the displayed Fab and g3p. When this type of construct is expressed in a strain of E. coli that suppresses the stop codon, read-through is accomplished and the fusion protein is p r o d u c e d for p h a g e assembly. Alternatively, if the same construct is placed in a host that does not suppress the codon, soluble Fab can be expressed, purified, and used for functional analysis.
Display and selection of antibody fragment libraries from immunized sources A major advantage of filamentous phage display technology is the ability to access large combinatorial libraries to achieve rapid and efficient selection. Therefore, this system s e e m s particularly well suited to selecting specific antibodies from combinatorial antibody fragment libraries originating from immunized sources. Clackson et al. [37"1 have shown that a range of w e a k to strong binders directed against the hapten 2-phenyloxazol-5-one (phOx) could be selected from scFv libraries of mice immunized with this hapten. It was also noted that a given heavy chain could pair with several light chains to produce antigen-binding scFvs and that a given light chain could also pair productively with a n u m b e r of heavy chains. This
p h e n o m e n o n , referred to as promiscuity, increases the n u m b e r of functional heavy and light chain combinations and forms the basis of hierarchical library construction. Hierarchical libraries w e r e generated b y taking a specific heavy or light chain that was found to bind to phOx, and then recombining each of these with heavy or light chains from the original library. Such a two-stage selection scheme, in which the initial selection of combinatorial libraries is followed b y hierarchical library screening, resulted in the identification of n e w pairings with significantly higher affinities than the original pairing (1.0 x 10- 8 M versus 1.8 x 10- 5 M). Burton et al. [38"] were able to identify several Fabs directed against the surface glycoprotein gp120 of type 1 h u m a n immunodeficiency virus (HIV) b y screening a r a n d o m combinatorial library expressed on the surface of filamentous phage. This library was generated from the b o n e marrow of an individual w h o had b e e n HIV-positive for six years. Inhibition enzyme-linked i m m u n o s o r b e n t assays using soluble gp120 w e r e employed to determine the affinities of the selected Fabs, which were found to be around 10-9 M. The authors note that a similar library constructed in ~, phage failed to yield any positives. Thus, this observation e m p h a sizes the potential of the filamentous phage display system for the identification of rare clones. Z e b e d e e et al. [39"] screened Fab libraries generated from h u m a n lymphocytes of individuals w h o had b e e n vaccinated with hepatitis B surface antigen (HBsAg) in order to isolate several antibodies that bind HBs a g specifically. Using phage display, Fabs w e r e obtained from individuals with b o t h high and low titers to HBsAg. Promiscuous pairings of the antibody chains w e r e also reported b y this group. For example, a particular heavy chain was capable of associating with several light chains to produce recognition of the same epitope, while another heavy chain could assemble with both ~ and ~, light chains and still maintain selective binding to the antigen.
Display and selection of antibody fragment libraries from unimmunized sources Although it is n o w possible to isolate antibodies from h u m a n s #immunized with specific antigens, it is not possible to immunize humans at will. In addition, a h u m a n immune system will not respond characteristically to self or h u m a n antigens except in conditions of autoimmune disease. Therefore, the ability to isolate functional antibodies b y circumventing immunization w o u l d b e a significant achievement in the utilization of antibody p h a g e display technology for the production of monoclonal antibodies. As antibodies obtained in this manner are the result of random combinations of h e a v y and light chains, it is h o p e d that this technology will permit the selection of h u m a n antibodies that bind to h u m a n antigens even though the h e a v y and light chains of antibody fragments arise from a hum a n source. Marks et al. [40"] have b e e n successful in obtaining antibody fragments against several antigens from a scFv p h a g e library generated from unim-
Antibody expression in bacteriophage systemsGarrard munized human peripheral blood lymphocytes. The human donors were not immunized with any of the antigens used in the experiment and were therefore considered to b e unimmunized with respect to these antigens. A library consisting of approximately 107-108 independent clones was screened against turkey lysozyme and p h O x antigens. Selection against these antigens yielded antibodies with moderate affinities of 10-7M and 5 x 10-7M. Although it depends on the ultimate use of the antibody, it is usually preferable to obtain antibodies with higher affinities. It seems possible that expanding the size of the original library to 1010-1012 independent clones will permit the selection of higher affinity antibodies. At the moment, however, it is feasible to generate libraries of only 108 members as a result primarily of limitations in E. coli transformation techniques. In order to increase the binding affinities of Fabs initially selected from random combinatorial libraries, several approaches could be pursued. First, hierarchical libraries could be constructed and screened for tighter binders. Second, higher affinities could be obtained by utilizing one of several methods of mutagenesis after the initial selection. For example, Gram et al. [41--] used a two-stage selection for isolating tight binding Fabs. Low affinity ( 1 0 - 5 - t 0 - 6 M ) Fabs, directed against progesterone and displayed multivalenfly as g8p fusions, were selected from combinatorial libraries that originated from unimmunized mice. Subsequent affinity maturation of the selected Fabs was performed by employing error-prone PCR to mutagenize only the variable domains from the selected Fabs. These mutagenesis domains were expressed monovalently as scFv-g3p fusions, and such a library was then subjected to further rounds of selection. Although direct affinity measurements were not determined, the results suggested that a 30-fold higher affinity could be achieved. Third, mutagenesis of a single CDR could be performed and, in fact, has recently bee used to alter the specificity of a particular antibody [42"]. In this study, Barbas et al. constructed a library by randomizing CDR3 of the heavy chain of an Fab directed against the hapten tetanus toxoid. Selection against a different hapten, fluorescein, yielded new Fabs, which specifically b o u n d this hapten and had affinities ranging from
Table 1.
and Zhukovsky
10-7 M to 10-8 M. Finally, other methods of mutagenesis such as passage of the antibody phage through a mutator strain (e.g.E. coli m u t D) may also yield improvements in affinity.
Considerations for the expression of antibodies in bacteriophage systems The expression of antibodies in phage systems is becoming an attractive and efficient approach for the identification of antibody fragments. As mentioned previously, however, both the )v and the filamentous phage systems present randomly combined heavy and light chains for selection and, therefore, make the possibility of creating original light and heavy chain pairings remote. This may be a consideration if the objective is to isolate antibodies from immunized sources and the desired antibody is rare. As observed by several groups, however, promiscuity of one or both chains may occur, thus increasing the potentially rare chance of generating a productive binder. If the goal is to obtain antibodies from unimmunized sources, both the size and complexity of the library b e c o m e even more important. Winter and Milstein [43] have p r o p o s e d that about 107-108 individual antibody seq u e n c e s exist at any given moment in vivo. As this degree of diversity is sufficient for ultimately generating antibodies in vivo, and as it is feasible to mimic this diversity with filamentous phage display, it should be possible to obtain at least low affinity binders. The reports by Marks et al. [40.-] and Barbas e t al. [42-] provide evidence to support this idea. Further investigation is required to determine whether libraries of this size will be sufficient to obtain antibodies against a large n u m b e r of antigens, and whether antibodies will be obtained against human antigens (particularly if the unimmunized source is human). So far, only low affinity antibodies have been generated using this approach. Therefore, in order to increase antibody affinity, multi-stage screening of the libraries could be performed. Such a strategy is analogous to the process of affinity maturation in the natural course of antibody development, and may be essential for the production of high affinity antibody fragments. It is also possible, however, that even moderate to high affinity binders
Summary of antibody fragment expression in phage.
Expression form
Source of antibody fragment DNA Immunized
Unimmunized
Individual clone
Filamentousphage g3p
Fab scFv
[38",39"] [37"]
[40",41 -']
[34",35",36",42"] [31 "]
g8p
Fab
-
[41 "]
[32",33"]
Fab
-
-
[20",21 ]
k phage
477
478
Expression systems can be selected in the beginning if methods that will significantly e x p a n d the size of the initial library are developed.
6.
TEMPESTPR, BREMNERP, LAMBERTM, TAYLOR G, FURZEJM, CARR FJ, HARMS WJ: R e s h a p i n g a H u m a n M o n o d o n a l Antibody to Inhibit H u r n ~ Respiratory Syncytial Virus Infection I n Vivo. Biotechnology 1991, 9:266-271.
It is important to note that often the ultimate goal is to identify antibodies that elicit a particular biological response. Antibodies possessing the highest affinities will not necessarily possess the desired functional attribute. It might actually b e advantageous to obtain w e a k binders, and then determine if a specific function exists, for example in a cell-based assay. Multivalent display m a y b e preferred if the objective is to obtain an antibody with a specific function.
7.
KETTLEBOROUGHCA, SALDANHAJ, HEATH VJ, MORRISON CJ, BENDIG MM: Humanization o f a Mouse M o n o d o n a l Antibody b y CDR-grafting: t h e I m p o r t a n c e o f F r a m e w o r k Residues o n Loop C o n f o r m a t i o n . Protein Eng 1991, 4:773-783
8.
CARTERP, PRESTAL, GORMANCM, RIDGWAYJBB, HENNER D, WONG WLT, ROWLANDAM, KOTTS C, CARVERME, SHEPARD HM: Humanization o f a n Anti-p185 H~R2 A n t i b o d y for H u m a n Cancer Therapy. Proc Natl Acad Sct USA 1992, 89:42854289.
9.
ORLANDIR, GUSSOWDH, JONES PT, WINTERG: Cloning Imm u n o g l o b u l i n Variable Dom~/rts for E x p r e s s i o n b y the P o l y m e r a s e Chain Reaction. Proc Natl A c a d Sci USA 1989, 86:3833-3837.
10.
LARRICKJ'~?, DANIELSSON L, BRENNER CA, ABRAHAMSOMW, FRY KE, BORREBAECRCAK: Rapid Cloning o f Rearranged I m m u n o g l o b u l i n Genes f r o m H u m a n H y b r i d o m a Cells Using Mixed Primcrs a n d the Polymerase C h a i n Reaction. Biochem Biophys Res Commun 1989, 160:1250-1256.
11.
SASTRYL, ALTING-MEESM, I-IUSE WI), SHORTJM, SORGEJA, HAY BN, JANDA KD, BENKOVICSJ, LERNERRA: Cloning o f the Immunological Repertoire in E s c h e r i c h i a c o l i for Generation o f M o n o c l o n a l Catalytic Antibodies: Construction o f a Heavy Chain Variable Region-specific cDNA Library. Proc Natl Acad Sci USA 1989, 86:5728-5732.
12.
MULLISKB, FALOONAFA: Specific Synthesis o f DNA In Vitro via a Polymerase-cataly'zed Chain Reaction. Methods Enzymol 1987, 155:335-350.
13.
SKERRAA, PLUCKTHUNA: Assembly o f a Functional Imm u n o g l o b u l i n Fv Fragment i n E. coli. Science t988, 240:1038-1041.
14.
BETYERM, CHANG CP, ROBINSON RR, HORWITZ AH: Esc h e r i c h i a coli Secretion o f a n Active C h i m e r i c Antib o d y Fragment. Science 1988, 240:1041-1043.
15.
HUSE w n , LAKSHIMI S, IVERSON SA, KANG AS, ALTINGMEES M, BURTON DR, BENKOVIC SJ, LERNER RA: Generation o f a Large Combinatorial Ia'brary o f t h e Imm u n o g l o b u l i n Repertoire in Phage Lambda. Science 1989, 246:1275-1281.
References and recommended reading
16.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest •. of outstanding interest
CATONAJ, KOPROWSK1H: Influenza Virus Hemagglutininspecific Antibodies Isolated f r o m a Combinatorial Exp r e s s i o n Library are Closely Related to t h e I m m u n e R e s p o n s e o f the Donor. Proc Natl Acad Sci USA 1990, 87:6450-6454.
17,
MULLINAXRL, GROSS EA, AMBERGJR, HAY BN, HOGREFEHI-I, KUBITZ MM, GREENERA, ALTING-MEESM, ARDOURELD, SHORT JM, ET AL,: Identification o f Hnra~tx Antibody Fragment Clones Specific for Tetanus Toxoid in a Bacteriophage I m m u n o e x p r e s s i o n Library. Proc Natl A c a d Sci USA 1990, 87:8095-8099.
18.
PERSSONMAA, CAOTHIEN RH, BURTON DR: G e n e r a t i o n o f Diverse High-aff3nity Hnm~n Monoclonal Antibodies b y R e p e r t o i r e Cloning. Proc Natl Acad Sci USA 1991, 88:2432-2436.
19.
McCAFFERTYJ, GRIFFITHSAD, WINTERG, CHISWELLDJ: Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains, Nature 1990, 548:552-554.
20.
PORTOLANO S, SETO P, CHAZENBALK GD, NAGAYAMA Y, McLACHIaN SM, RAVOVORT B: A H u m a n Fab F r a g m e n t Specific for Thyroid P e r o x i d a s e Generated b y Cloning Thyroid Lymphocyte-derived I m m u n o g l o b u l i n Genes i n a Bacteriophage ~ Library. Btochem Btopbys Res C o m m u n 1991, 179:372-377.
Conclusion The expression of antibody fragments in p h a g e systems is an exciting opportunity to produce monoclonal antibodies efficiently and without the need for immunization. Results discussed in this review highlight recent developments that contribute to this technological advance (Table 1). First, Fabs or scFvs can be expressed on the surface of the p h a g e and specific binders can b e selected. Second, a multi-stage approach, which initially employs selection of random combinatorial libraries followed b y selection of secondary or hierarchical libraries, is likely to result in the isolation of binders with enhanced affinities. Third, the resulting antibody fragments have affinities similar to those of antibodies obtained from conventional sources. In conclusion, it is possible to see h o w these advances in antibody technology will continue to c o m p l e m e n t conventional hybridoma technology and m a y replace it altogether in the near future.
Acknowledgements The authors thank Dr D Henner for advice and comments.
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A n t i b o d y expression in bacteriophage systems G a r r a r d a n d Z h u k o v s k y A bacteriophage X expression library was u s e d to express a h u m a n Fab fragment specific for h u m a n TPO. Random combinations of heavy a n d light chain g e n e s produced a clone that b o u n d TPO with a dissociation constant of 1 nM. 21.
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BREITLINGF, DUBEL S, SEEHAUS T, KLEWINGHAUS I, LITTLE M: A Surface Expression Vector for Antibody Screening. Gene 1991, 104:1147-1153. DNA coding for a scFab of anti-hen egg-white lysozyme was f u s e d to g3p of the filamentous p h a g e a n d expressed in E. coll. Expression resulted in a functionally specific antibody that w a s able to bind antigen. 32.
KANGAS, BARBAS CF, JANDA KD, BENKOVIC SJ: L | m k a g e o f Recognition and Replication Functions b y Assembling Combinatorial Antibody Fab Libraries along Phage S u r f a c e s . Proc Natl A c a d Sci USA 1991, 88:4363-4366. Multivalent display on the filamentous p h a g e of Fab, isolated from the original pmitrophenyl p h o s p h o n a m i d a t e combinatorial library, by fusing h e a v y chain to gBp is presented. It was s h o w n that o n e r o u n d of p a n n i n g enriched the phage displaying Fab by 2700-fold. CHANG CN, LANDOLFI NF, QUEEN C: Expression o f Antibody Fab D o m a i n s o n Bacteriophage Surface (Potential use for Antibody Selection). J I m m u n o l 1991, 147:3610-3614. Anti-Tac murine Fab was displayed in a multivalent fashion by fusing the light chain to the gBp of the filamentous phage. It was s h o w n that Fab expressed thus could be specifically selected o n an affinity matrix. 33.
BARBASCF III, KANG AS, LERNER RA, BENKOVIC SJ: Assembly o f C o m b i n a t o r i a l A n t i b o d y L i b r a r i e s o n Phage Surfaces: the Gene Ill Site. Proc Natl A c a d Sci USA 1991, 88:7978-7982. Previously characterized in bacteriophage ~, , a n anti-tetanus toxoid library was u s e d to construct a combinatorial Fab library displayed o n the surface of filamentous p h a g e via fusion of heavy chain to t h e carboxy-terminal domain of g3p. Selection by panning over tetanus toxoid resulted in 103- to 105-fold enrichment. 34.
35.
GARRARDLJ, YANG M, O'CONNELLMP, KELLEYRF, ITIENNERDJ: Fab Assembly and E n r i c h m e n t i n a Monovalent Phage D i s p l a y S y s t e m . Biotechnology 1991, 9:1373-1377. An Fab directed against the ne u receptor w a s displayed by fusing the Fd chain to the carboxy-terminal domain of g3p. Selection s c h e m e s that enriched for t h e tightest binder in a s u b n a n o m o l a r affinity range were performed. The affinities of soluble Fab a n d the p h a g e displayed Fab were s h o w n to be virtually identical. 36.
HOOGENBOOMHR, GRIFEITHSAD, JOHNSON KS, CHISWELLDJ, HUDSON P, WINTE~G: Multi-subunit Proteins o n the Surf a c e o f Filamentous Phage: Methodologies for D i s p l a y i n g Antibody (Fab) Heavy and L i g h t C h a i n . Nucl A c i d s Res 1991, 19:4133--4137. The heavy or the light chains o f Fab fragments from the m o u s e antip h O x antibody were fused to g3p of the filamentous phage a n d the c o m p l e m e n t chain was secreted into the periplasmic space of E, coB. T h u s formed, heterodimeric Fab showed specific binding to p h O x a n d bovine s e r u m albumin b u t not to bovine serum albumin alone. 37. ..
CLACKSONT, HOOGENBOOM HR, GRIFFITHS AD, WINTER G: M a k i n g A n t i b o d y Fragments Using Phage D i s p l a y Lib r a r i e s . Nature 1991, 352:6244528. In this paper a n approach employing a two stage selection is presented. A combinatorial scFv library displaying filamentous p h a g e via fusion with g3p was constructed by PCR amplification of mRNA obtained from mice i m m u n i z e d with hapten phOx. Following two r o u n d s of selection, two hierarchical libraries were made. By u s i n g soluble scFv o n e binder w a s f o u n d to have a dissociation constant o f 10 riM. 38.
BURTON DR, BARBAS CF 11I, PERSSON MAA, KOENIG S, CHANOCK RM, LERNERRA: A Large Array o f Hnm:~t Monoclonal A n t i b o d i e s to Type 1 H u m a n l m m u n o d e f i c i e n c y V i r u s f r o m C o m b i n a t o r i a l Libraries o f A s y m p t o m a t i c Seropositive I n d i v i d u a l s . Proc Natl A c a d Sct USA 1991, 88:10134-10137. RNA from the b o n e marrow lymphocytes of an asymptomatic HIV positive donor was used to produce a r a n d o m combinatorial library of Fab against gp120 (strain Inb) and w a s expressed o n the surface of the filamentous p h a g e by fusion of the heavy chain to g3p. After four rounds of panning, soluble Fabs were p r o d u c e d from selected phage. Affinity measurements were performed using inhibition enzyme-linked ilIkmunosorbent assays a n d resulted in dissociation constants of the order of 10 nM. ZEBEDEESL, BARBAS CF iii, HOM Y-L, CAOTHIEN RH, GRAFF R, DEGRAW J, PYATI J, LAPOLLA R, BURTON DR, LERNER RA, THORNTON GB: H u m a n C o m b i n a t o r i a l A n t i b o d y Lib r a r i e s t o H e p a t i t i s B S u r f a c e A n t i g e n . Proc Natl A c a d Sci U SA 1992, 89:3175-3179. Leukocyte RNA from h u m a n donors i m m u n i z e d with HBsAg w a s u s e d to construct a repertoire library in the filamentous phage system, in w h i c h Fab was fused to g3p. After p a n n i n g selection for three rounds, several functional Fabs were selected a n d affinity measurem e n t s were performed on a soluble form of Fab. Sequence analysis o f these clones showed h u m a n light-chain promiscuity and suggested that m u c h of the antigen recognition w a s d e p e n d e n t u p o n t h e h u m a n h e a v y chain. 39.
40. •.
MARKSJD, HOOGENBOOM HR, BONNERT YP, McCmFERTY J, GRtFFITHSA n , WINTER G: By-passing l m m u n | z a t i o n ( H u m a n Antibodies f r o m V-gene Libraries Displayed o n Phage). J Mol Biol 1991, 222:581-597. Peripheral blood lymphocytes of u n i m m u n i z e d h u m a n donors w e r e u s e d to construct a large (107-108 members) combinatorial library of scFv displayed on the surface o f the filamentous p h a g e via fusion to g3p. After four rounds of selection clones specifically binding turkey egg lysozyme, bovine serum a l b u m i n and p h O x were identified. T h e dissociation constant of the best scFv selected was 86 riM. 41. ,,
GRAMH, MARCONIL-A, BARBASCF II1, COLLETTA, LERNERRA, KANG AS: I n Vitro Selection and Afrmity Maturation o f A n t i b o d i e s from a N a i v e C o m b i n a t o r i a l l m m u n o g l o b ulin Library. Proc Natl A c a d Sci U SA 1992, 89:3576-3580. This paper describes a m e t h o d to access low affinity Fabs from a n immunoglobulin library of n o n - i m m u n i z e d mice with s u b s e q u e n t affinity maturation o f selected clones. Initially selected low
479
480
Expression systems affinity binders were subjected to affinity maturation by utilizing error-prone PGR. This scheme resulted in decrease of dissociation constant for one of the clones from 10 to 0.3 nM.
over hapten fluoroscein Oinked to bovine serum albumin) resulted in selection of clones with similar amino acid sequences and affinities of 10-100 nM.
BARBASGF III, BAIN JD, HOEKSTRA DM, LERNERRA: S e m i s y n thetic C o m b i n a t o r i a l A n t i b o d y Libraries: a C h e m i c a l S o l u t i o n to t h e D i v e r s i t y P r o b l e m . Proc Natl A c a d Sci USA 1992, 89:4457-4461. Heavy chain CDR3 of h u m a n tetanus toxoid-hinding Fab was randomized and the resulting library was displayed on the surface of the filamentous phage as an Fab-g3p fusion. Sorting of this library
43.
42.
WINTER G, MILSTEIN C: M a n - m a d e Antibodies. Na tu r e 1991, 349:293-299.
LJ Garrard and EA Zhukovsky, Genentech Inc, 460 Point San Bruno Boulevard, South San Francisco, CA 94080, USA.
