Phageantibodies:will new ‘coliclonaf’ antibodiesreplace monoclonalantibodies? David J. Chiswell and John McCafferty Antibodies can now be rapidly isolated from large and diverse recombinant libraries by displaying functional antibody fragments on the surface of bacteriophage particles and directly selecting with antigen. This method has been used to isolate antibodies, including human antibodies, with tind without immunization, and to improve the affinity and specificity of antigen binding.

Monoclonal antibodies (InAbs) wcrc arguably the most commcrcialIy succcssfi~l of the new biotcchnologjcs dwing the 138(!s, being used mainly in scnsitivc irt &O diagnostic tests. Although the potential for thcrapcutic and irr I-;VOdiagnostic USC for mAbs has still to bc fully rcalizcd, more products arc gaining rcgularorv approval. and antibodies could yet be the thcrapcuhc molecules ofthc 1990s aud beyond_ Mcs: mAhs are still isolated using csscntiaity the same tcchnoloby as originally dctcribcd by Kcihlcr and Milstcin’ : i.c. manipulation of the immune system :o incrcasc the number of B cells csprcssing antigenspecific antibodies, immortalization ofthcsc antibodyprcducing cells by cell fusion, and identification 0:’ stable cell Iinc~ sccrcting an antibody with the desired properties by cxtcnsivc scrcc-ninp. The co&wed USC oi thcsc tcchniqucs over 16 years of en~ml~~~ tcchnical change in the biosciences ampiy demonstrates the power and robustness of hybridoma technology, It now appcac;. howcvcr. that change is imminent.

‘Coliclonal’ antibodies Rcccnt advanrcs have combined

(1) the ability to prodw; functional antibody firgmrnn in bacteria, (2) the isolatiotx of antibody variable gcncs using polymcrasc chain reaction (PCR), and (3) powcrfid screening systems, to produce a radically mow cfflcicnt methodology that enables isolation of~antiboc’ics more rapidly and more easily than has hitherto been the cast with hybridoma-dcrivcd mAbs. WC suggestthat these ntw antibodies. isolated and manut%turcd in prokuryotic systems, bi- called ‘coliclonal’ antibodies. The new tcchnolo~~ has the potential to cstcnd rhc propcrtics ofcoliclonal antibodies so that they arc even mow widely applicablthan traditional mAbs have been.

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This article illustrates the application of thcsc new proccdurcs, emphasizing particularly the recent advances made at Cambridge in the devetopmcnt of a powxful, bactcriophagc-based screening system. For a recent comprehensive review deaIing in depth with the earlier advances made in the field and the uses made ofstandard scrcciling technology see Ref. 2.

Phage antibodies McCaff&ty rt ~rl.3 demonstrated the Gzasibility of displaying fragments of antibodies on the surface of a bactcnophagc and that thcsc fragments folded COTrcctly and bound antigen. Antibody variable-region genes were cloned into the gene encoding the minor coat protein (gene 3) ofthc filamentous bacteriophage fd. The fusion protein created, consisting of the antibody fragment at the N-terminus ofthc coat protein, is inccrporatcd into the phage particle (a ‘phagc antibody’; see Fig. 1). Each recombinant phagc gcnomc contains the DNA encoding the spccitic antibody displayed on its surface: allowing the phagc particle carrying antibody gene to be sclectcd directly using the binding properties of the csprcsscd protein. This discovery opcncd a novel route for antibody isolatic-1(Box 1). Re~~~&rs ofantibody gcncs’ arc amplified using PCR and cloned into phagc. thus creating a Iargc Iibrary of phagc each displaying a spccifc antibody. Wit%n thcsc large lib:&s ofphagc antibodies (potentially rcprcscnting betxvecn 1iY and IO” ditferent antigen-binding spcciticitics) each phagc cxptcsscs an individual heavy- and light-chain combination. To sclcct an antibody ofintercst, the library of phagc antibodies is passed over antigen bound to a solid surface such as a Scpharosc column or a coated tube. Phagc that displ: y antibody fragments specific for the antigen arc rctaincd on hc surface. Thcsc specific phagc antibodies can be &ted and used to infect E. mli to give stable clown. The phagc antibody rx be both analyscd and used directly from the culture wpematant as a rcagcnt in tcchniqucs such as ELBA.



This antigen-dircctcd selection is capable ofpicking out rare antigen-specific phage in huge populations; for example where only one in 107 phagc in a population is specific for the antigen3”. In addition, the technology has been used to display both sing&chain Fv (ScFv)” and Fab antibody fiagmen”9. Subsequently, other groups have used gene 3-display to isolate antibodies derived From libraries originally constructed in lambda phag&. It may also be possible to use antibody-gent 8 protein f&ion”. However, this latter approach involves the display of greater numbers of antibodies on each phage particle, and a variable number of antibodies on different phage particles produced by the same cell. Selection would be a fimction of avidity (i.e. a combination of affinity and valcncy) rather than affinity alone, limiting the usefuhiess of this approach, particularly when selecting high-affinity antibodies”.

Appkatioas of phage antibodies &placement of monocfonal anti:, Jdies Combining repertoire-cloning of antibody rarizble genes from hyperimmunized mouse spleens with phagc selection has been demonstrated to produce coliclonal antibodies with binding charactcristics equiv,&nt to those of traditiona: mAbs+. The phage system replaces all steps after immunization and isolation ofspleen cells with simple, rapid procedures utilizing DNA manipulation and bacteria. The time required from isolation of spleen cells to selected swble clones is reduced from several mcinths to several weeks. In the process, many millions, or billions, of potential binding molecult! may be surveyed using large phage libraries, compared with onlv severa1 thousand molecules screened using a traditional hybridoma fusion.

prove poGble, for example, to use these ;iairilsi libraries to isolate human antibodies directed ;ag.Gnst ‘self’ or other non-immunogenic antigen< aud to inlate research rcagrnts equir;lle:lt to mAbs \\-&our the need to use anin&

Hmnan antibodies and antibodies wihort immariiration Human mAts have proved difficult to isolate, especially if stable clones expressing high-afinity antibodies arc required. Traditional immortahzation techniques focus on human l3 cells and are technically ditl ficult and inefficient. Recombinant methods, in contrast, immortahze the genes rather than the ccl!?: and are exceptionally efficient and ‘qua& applicable to the isolation ofhdman and rodent antihody genes_ Another critical &tor has been the practical and ethical difficulties involved in the use of sp&& immunization regimes in humala. Marks cl nl.” have now demonstrated that the requirement for immunization can also he overcome by the use ofphage di_+ play. They have used 4 phage-antibody library derived Tom unimmunized donor-s to isolare human antibodies that bind specific antigens with at&it& in the region of l(H) n&q. ? hc Ibra&? were derived using I$ or IgM mRNA Tom peripheral blond Iymphocy’c~s ofdonon with no demonstrable serum ruponse to the antigens. This demonstration ofthe power ofphage-antibody technology has a number of implications. Ic should


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It-the immune system ii to be replaced alto+hcr i%r the isolation ofr-cr)-high-at&it?’ ~~ntibodies. ccchniqun need to be developed rhzi n&c the abilic?_of the immune system to imy:-ovc it< antibody rCypnse by rounds ofmutation aads&ction, i.e. afiG+ maturation. Ph’ha;s dispel)- appc;ln to hat-c J natural WI& ency to se!:ct the highc3t-.&nity rari.mts in any pop:~lation. making ijr &W at%+ matur3tion poscrhtr. For es~~~plc. using the phagc vstem, C&&on ~1a/.” hare shown that reFedted rounds ofantigcn-directed selection enrich for higher affinity variant\ in a population oiantibodics. directed qGart the -ame epitope. The combination ofphage ~&ction 112th mutagen

Phage antibodies: will new 'coliclonal' antibodies replace monoclonal antibodies?

Antibodies can now be rapidly isolated from large and diverse recombinant libraries by displaying functional antibody fragments on the surface of bact...
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