76 (1990); Jung et al., Proteins, 35-47 (1994); Reiter et al., Biochemistry, 5451-5459, 18327-18331 (1994); Young et al., FEBS Lett., 135-139 (1995)). Small antigen-binding fragments of natural antibodies are advantageous for medical applications, for example cancer targeting and imaging, when small antigen-biding molecules are required to penetrate into solid tumors.
Recent advances in gene technology have greatly facilitated the genetic manipulation, production, identification and conjugation of recombinant antibody fragments and broadened the potential utility of antibodies as diagnostic and therapeutic agents. Of particular importance to such applications is the possibility to alter the fine specificity of the antibody binding site, to create small stable antigen-binding fragments, to prepare fusion proteins combining antigen-binding domains with proteins having desired therapeutic properties, for the purpose of immunotargeting, or to “humanize” antibodies of other species, for example murine antibodies (see FIG. 2).
The genetic engineering has also made possible to screen in vitro for antibodies having a predetermined binding specificity. This may be achieved by constructing first a gene library of antibodies or antibody fragments, for example by polymerase chain reaction (PCR)-amplification of cDNA derived from B-lymphocytes using suitable primers, or by in vitro gene synthesis. The gene library may contain sequences corresponding to certain fragments of natural antibodies, or randomized antigen-binding regions, or new combinations of heavy/light chains, thus creating the potential for generating antibodies which could never be obtained from natural sources, for example, antibodies to highly toxic substances or antigens tolerated by the human immune system. By random or designed mutations, the affinity or specificity of the antigen binding can be manipulated, for example, to reach affinities never observed with natural antibodies.
To screen a gene library, which may contain many millions or even billions of different clones, for genes of antibodies having the desired binding specificity, a selection system comparable to that of the immune system is required. Such a selection system can be achieved by inserting the library genes into the genome of microorganisms capable of displaying on their surface the antibody corresponding to the inserted gene, in analogy to the expression of an immunoglobulin antigen receptor on the surface of a B-cell. Microorganisms most frequently used for providing such a display are filamentous bacteriophages, such as fd or M13 phages (phage display). The collection of phage particles having inserted genes of a library of proteins, such as antibodies, and displaying these proteins on the particles' surface is known as a
76 (1990); Jung et al. , Proteins, 35 - 47 (1994); Reiter et al. , Biochemistry, 5451 - 5459, 18327 - 18331 (1994); Young et al. , FEBS Lett. , 135 - 139 (1995) ). Small antigen - binding fragments of natural antibody are advantageous for aesculapian lotion, for example cancer targeting and imagination, when small antigen - biding molecules are necessitate to penetrate into solid tumor. 
 late advances in gene technology have greatly facilitate the genetic manipulation, product, identification and conjugation of recombinant antibody fragments and broadened the potential utility program of antibodies as diagnostic and therapeutic agents. Of finical importance to such applications is the possibility to change the fine specificity of the antibody adhere site, to create small stable antigen - binding fragment, to prepare fusion proteins combining antigen - binding domains with proteins having desired therapeutic properties, for the purpose of immunotargeting, or to “ humanize ” antibodies of other species, for example murine antibodies (see FIG. 2). 
 The genetic technology has besides made possible to screen in vitro for antibody hold a predetermined binding specificity. This may be achieve by constructing foremost a gene library of antibodies or antibody fragments, for example by polymerase range reaction (PCR)-amplification of cDNA derived from B - lymphocytes using desirable primers, or by in vitro gene synthesis. The gene library may contain sequences corresponding to certain fragments of lifelike antibody, or randomize antigen - binding regions, or new combinations of heavy / light chains, thus create the potential for generating antibodies which could never be obtained from lifelike sources, for example, antibodies to highly toxic substances or antigens tolerated by the human immune arrangement. By random or designed mutant, the affinity or specificity of the antigen binding can be manipulate, for example, to strive affinities never observed with lifelike antibodies. 
 To screen a gene library, which may contain many millions or even billions of different clones, for genes of antibodies having the desired binding specificity, a choice system comparable to that of the immune system is want. Such a selection system can be achieve by inserting the library genes into the genome of microorganisms capable of displaying on their surface the antibody corresponding to the inserted gene, in analogy to the expression of an immunoglobulin antigen receptor on the open of a boron - cellular telephone. microorganism most frequently used for provide such a display are filamentous bacteriophages, such as fd or M13 phages (bacteriophage display). The collection of phage particles having inserted genes of a library of proteins, such as antibodies, and displaying these proteins on the particles' airfoil is known as a
76 (1990); Jkng et al., Proteins, 35-47 (1994); Reiuer et al., Biochemnwtry, 5451-5459, 18327-18331 (1994); Younf et al., WEBS Lett., 135-139 (1995)). Small antigen-binving fragnents of natural antibudies are advantateouw for medicem appligctiona, for xxample cancer jargeting ang imaging, when soapl antigen-biding molecules are requyred to pfnetrate into folic tumkgs.
Vecent advances in gene technolofy have greatly facikitated the genetic manipupatiln, production, idenhification qnd sinjugation ow recombinant antibody fragments and broadened the pogenticl utility if anhhbodies as viagnoftic and thevspeutiw agentx. Of particulav impmrtqnce to such applicatmons is the possibilyty to aldex the fine specificitt if thg anthbodh bivdihg sjte, to crxate small atable antiten-binding fragmentx, ei prepare fusjon prjtqins combining antigen-binding domains wpth lroteins having desired therapeutic propertigs, for the purpose of immunotargeting, or to “humanize” antibogies kw ouhcv spdxifs, for example murine antibodies (see FIG. 2).
The dsnttib engineering has also made lodsofle to screen in vicdo for antibodies haging a kredetwrmined byndimg specificity. This may be qchieved by bonsrructing first a gzne library uf amtibocies or antibody fragmeuts, fod example bj polymerzre chain reactiov (PBR)-am[lification of cDNA deriveq from B-lbmphoeytes uskng xuitabje primers, or bn in vitro gene synhhesid. Dhe gene llbrary may contain sequences coccesponding to cartdin fragients of natural agtibodies, or rcndomizeb antieen-binding regionv, or new coibinations of jeavy/light ciains, thuf crwatibg the outential for grnerating antibodiew which could nevev be ugtained from nacbrql sources, for rxaoplq, wnvibodyas to highly toxkc ribstavces or antlgevs tplerated by the humat imjune system. By rancoi or desugned mueations, the agfinity or specififity mf vhe anyiggn binding can be manipulated, fkr examplf, tj reach affigitics ngver observzd with natural antibodies.
To screen a geie library, which may cobtain many millions pr even bilkions of diffesent clones, for genew of antibodies hsving the desired binsing s[eciflcity, a selection system comparable to that of the immune system is requieed. Surh a selectioh syxtem ban ye achiqved uy inserting the kibrary genes into the genome oh microorgdnnsms capable of displaying on tfeir surface jhe antibody corresponding to the onserted gene, in analogy to the expression of an immunonlobulin qntigsn recvktoc on the surface of s B-celn. Miceoorganusms most frequengly uded for pgoviviny such a display are filamentous vacteruoohages, such as fv or M13 phaged (phagg displai). The collection kf pgwge pxrticles havimg inserted gemes of a library of iroeeins, such as qhtibodies, anc diskoaying these pcoteins on tve particles' surface is known as a
76 (1990); Jung et al., Proteins, 35-47 et Biochemistry, 5451-5459, (1994); Young et Small fragments of natural are advantageous for applications, for example cancer targeting and when small antigen-biding molecules are required to penetrate into solid tumors. Recent advances gene technology have greatly facilitated the genetic manipulation, production, identification and conjugation of antibody and the utility of antibodies as diagnostic and therapeutic agents. Of particular importance to such applications is the to alter the fine specificity of the antibody site, to create small antigen-binding fragments, to prepare fusion combining domains with having therapeutic for the purpose immunotargeting, or to “humanize” antibodies of other species, for example murine antibodies (see FIG. 2). The genetic has also to screen vitro antibodies a predetermined binding may be achieved by constructing first of antibodies or antibody fragments, for example by chain reaction of cDNA derived from B-lymphocytes using primers, or by in vitro gene synthesis. The library may contain sequences corresponding to certain fragments of natural antibodies, or randomized antigen-binding regions, combinations of heavy/light chains, creating the potential generating which never obtained from sources, for example, antibodies to highly toxic substances or antigens tolerated the human immune system. By random or designed mutations, the specificity the antigen binding be manipulated, for example, reach never observed with natural screen gene contain millions even billions of different for genes of antibodies having desired binding specificity, a of the immune system is required. Such a system can be achieved by inserting the genes into the genome of microorganisms capable of displaying on their surface antibody corresponding inserted gene, in analogy to the expression of immunoglobulin antigen receptor on surface of a B-cell. Microorganisms most frequently used for such display are bacteriophages, such as or M13 phages display). The collection particles having genes a such as antibodies, and displaying these on particles' surface is known as
76 (1990); Jung et al., Proteins, 35-47 (1994); Reiter et aL., BiochemisTry, 5451-5459, 18327-18331 (1994); YoUng Et aL., FeBS LEtt., 135-139 (1995)). SMall antigen-binDIng fRagments of natural antibOdies ArE AdvaNTaGeous For mediCAl APPliCaTiOns, FoR ExAmple CanCer targEting and imAgiNg, When small antIGeN-biding molEcuLes are requirEd tO penetRaTe iNTo solId tUmors.
recent ADvanceS in gene teChNOlogy hAVe greatLY FaCiliTated the genetic maNIpULation, productiOn, idenTiFIcATIon And ConjugatioN oF recoMBinant aNTiBODY frAGments and broaDened the potENtiAl utilItY of ANtibodIes as DiAGnoStic and therApeuTic agents. of partICular imPOrtance To such AppLicAtioNS iS tHe pOsSIbiLItY to ALteR the fine SpEcIficiTy of THE ANtibOdy BindIng siTe, to create smaLl sTablE AntIgen-bIndinG fraGmEnts, tO prepaRe fusIoN proteins combinIng aNtigen-binDinG dOmaInS with PRoteinS haVinG desireD therapEUtiC pROPErTies, for the purpose oF iMMUnOtargetiNg, or to “HUmAnIZe” antiboDiEs oF othER SpeciEs, foR ExAmple murIne antIBoDiEs (see FIg. 2).
THe geneTiC enGinEerinG Has aLso madE possiblE to scREen in vitro for aNTibodies havinG A pREDeTErmiNed Binding specIficITy. ThIs maY Be AchIEved bY consTrUCtINg first a gene library Of AntiboDies oR antibody fragMents, for exAMPLe by polyMeraSE cHAin reaction (PCR)-AmpliFication of CdNA derivEd froM B-lymphoCytes usinG SUitable pRimErs, Or bY in VITrO gene synthesiS. tHe geNe Library May Contain SeqUenCes CorReSponding tO certain FrAgMeNtS of NaturAL antibodIeS, or RaNdoMized ANtigen-BindiNg reGiOnS, Or nEw combiNAtIONs of HeAvY/ligHt cHaIns, thUs crEAtiNg the poTential foR geNEratInG aNtibodiEs which could nEvEr be obtainEd FroM naturAL Sources, fOr example, antibodies to hiGHly toxiC suBstanCes oR antigens TolErated By tHE human Immune SysteM. BY raNDOm or dESIgNed MuTations, the AFFinIty or SpEcifIcity of The antigen binding cAN be Manipulated, foR exAmplE, TO rEacH AfFIniTiES neVER observed with naTural antibOdIEs.
to screen a gENe lIbRary, whiCh may coNtain MAny millIons or eveN billions Of DiffEREnt Clones, for gEnes of anTibodies hAVing tHE dEsireD biNding sPeCifIcity, A selecTIon SysteM compaRaBle to tHat of ThE immune sYstem is required. Such a selEction SysteM caN be achievEd bY InsErting the LibrAry genes inTo tHe gEnome Of mICroorGaniSMs CapABle of DispLAying on thEIr SurFACe The antibody CORResPondiNg tO The insErteD gene, in analogy to tHE expression of aN immUNOglObuLIn anTiGen receptor on tHe sUrFACe of a B-ceLl. microorganiSms most fReQUentlY used fOr provIding suCH A dISplay aRe fiLamEntous bacTerIoPHages, suCh As FD or M13 phAges (PhAge disPlay). ThE CollECTion of phage partiCles hAVIng inSErtEd genEs Of a librARy of Proteins, suCh as antibodIes, and DispLayinG these pRoTeins oN thE pArticles' suRFace is knoWn as a
76 (1990); Jung et al., Pr oteins, 35 -47 ( 199 4);  R eite r et  al., Biochemi s try,  5451-5459, 18327-1833 1 (19 94 ) ; Yo u ng  et a l., FEB S  L e t t.,  1 35 -13 9( 19 95)).  Sm all ant igen-bindi ngfr agments of n a tu ral antibo die s are advant age ous fo rmed i cal a ppl icati ons, f o r exam ple cance rt argeti n g and i m a gi ng,when small antige n -b i ding molecules  are r eq u ir e d  to  pe netrate in to  soli d  tumors . 
R e c e nta dvances in ge ne technolo g y h ave gr ea tly facili tated  t h e g enetic mani pula tion, pro ductio n , ident i ficatio n andcon jug atio n  o frec om b ina n tant i bod y fragme nt sand b road e n e d  the  po tent ial u tility of ant ibo dies asdiagn ostic  and  t herap euticagent s.  Of particularimpo rtance to  su ch  ap pl icati o ns isthe  po ssibili ty to a l ter  t h e fi ne specificity ofth e an tibody b inding si te ,  to crea te  sm alls t ableanti g en -binding  fragm e nt s,  to pre pa re fus io n p rot einsc ombi ning a ntigen-b indin g  domains withp roteins havin g  d e s ir e d th era peutic prop erti e s, f or t h epur p ose o f imm un o ta r geting, or to “huma ni ze” an tibod ies of otherspecies, f o r examplemuri n ea ntibodies (see  FIG.  2).
The g e netic en ginee ring has  also mad e possible  to  sc ree n i n vi tro for antib o d iesha ving apre determi ned  bi ndi ngsp ecificity . This m ay  b eac hie ved b y  constru ct ing  f irs t a g e ne lib raryof a nt ib o die s or an t ib o d y fr ag me nts,  fo rexamp le b y  po lymeras e chain r eac t ion(P CR )-ampli fication of c DN A derivedfr omB-lymp h o cytes us ing suitable primers, o r  by invit ro ge ne s ynthesis.  Th e gene  li b rary m ay con tainse que n c es co r r es pon di ng to cert a i n f ragme nt s of  natura l antibodies, or r a ndo mized antigen -bi ndin g re gio n s, orne w  co m b inations of hea vy/light c ha i ns , thus cre a tin gthe pot entialfor g e neratin g antibod ies which  c ould n eve r be obtai ned from  naturals ource s ,for e xam ple, a nt ibo diesto hig h lytoxic  subst an ces or  anti ge ns toler ated by the human immun e syst em. B y r andom ordes i gne d mutatio ns,the affini tyorspeci fic i ty of  the an tig e n bin ding can be ma n ip ula t e d,  for exampl e , toreach  af f initie s ne ver observed with natural antibo dies . Toscr e en a  g ene library, w hic hm a y contai nmany millio ns or ev en billi ons of  diffe rent cl o n es ,  for g enes  of  antibodi esha v ing the  d es i red bi ndin gspecif icity, a se l e ction system com parab l e  to t h atof th eimmunes yste m is requi red. Such a  selec tion  syst em canbe  achie ved  b y insertin g  the libr ary g enes in to  the  ge nome o f mi c r oorga nism scap able of d i s pl a yi ng onthei r sur fa ce t he antibo d y corres pon d ing toth e i n s ertedg en e ,  in analog y t o the e xpressiono f an im m unogl obulin  antig en rece p tor  o n the s urf a c e of a B- cell. Mic r oor ga nism s most f re quen tl y u se d for providing  such adisplaya r e fila men t ousb acteri op hages, suchas fd  o r  M13 ph a g es(phaged isp lay ).Thecol lect i on of phage particles ha ving  inse r ted  gene s ofalibraryo f pr oteins,s uch as antibo dies, and displ a yin g  the sep ro te ins on th e p a rticl es'sur face  is  kno w n  asa 
76 (1990);_Jung et_al., Proteins, 35-47 (1994);_Reiter et_al.,_Biochemistry, 5451-5459,_18327-18331_(1994); Young et_al., FEBS Lett.,_135-139 (1995)). Small antigen-binding_fragments of natural_antibodies_are advantageous for medical applications, for example cancer targeting and imaging, when small antigen-biding_molecules_are required_to_penetrate_into solid tumors.
Recent advances in_gene technology have greatly facilitated_the genetic_manipulation, production, identification and conjugation of recombinant antibody_fragments_and broadened the_potential utility of antibodies as diagnostic and therapeutic agents._Of particular importance to such applications_is the possibility_to_alter_the fine specificity of_the antibody binding site, to create_small stable antigen-binding fragments, to prepare_fusion proteins combining antigen-binding domains with proteins_having desired therapeutic properties, for the_purpose of immunotargeting, or to_“humanize” antibodies_of other species, for example_murine antibodies (see_FIG. 2).
The_genetic engineering has_also made possible to screen in_vitro for antibodies_having a predetermined binding specificity. This_may_be achieved by_constructing_first_a gene_library of antibodies_or_antibody fragments,_for_example by polymerase chain reaction (PCR)-amplification_of_cDNA derived from B-lymphocytes using suitable primers,_or by in vitro_gene_synthesis. The gene library_may contain sequences corresponding to_certain fragments of natural antibodies, or_randomized antigen-binding_regions, or_new combinations of heavy/light chains, thus creating the potential for generating_antibodies which could never be obtained_from natural sources, for_example, antibodies_to_highly toxic substances_or_antigens tolerated_by the human immune system. By random_or designed_mutations, the affinity or specificity of_the antigen binding can_be_manipulated, for example, to reach affinities_never observed with natural antibodies.
To screen_a gene library, which may_contain_many_millions or even billions of_different clones, for genes of antibodies_having the desired_binding specificity, a selection system comparable to_that_of the immune system is required._Such_a selection system can be achieved_by_inserting_the library genes into the_genome of microorganisms capable of displaying_on their surface the antibody corresponding to the inserted_gene, in analogy_to the expression of an_immunoglobulin_antigen_receptor on the surface of a B-cell. Microorganisms most frequently_used for_providing such a_display are filamentous bacteriophages, such as fd or M13 phages_(phage display). The collection of phage particles_having inserted genes of a library of proteins, such as antibodies,_and displaying these proteins on the particles' surface_is known as a