CA2112193A1

CA2112193A1 - Process for preparing recombinant and synthetic peptides, and their use

Info

Publication number: CA2112193A1
Application number: CA002112193A
Authority: CA
Inventors: Ulrich Grundmann; Thomas Wissel; Gerd Zettlmeissl
Original assignee: Individual
Current assignee: Siemens Healthcare Diagnostics GmbH Germany
Priority date: 1992-12-23
Filing date: 1993-12-22
Publication date: 1994-06-24
Also published as: JP3386210B2; EP0603672A2; DE59309978D1; AU5255793A; JPH07303493A; AU669947B2; ES2144438T3; EP0603672A3; ATE190653T1; EP0603672B1; DE4243770A1

Abstract

Abstract Process for preparaing recombinant and synthetic peptides, and their use.

The invention relates to a process for preparing peptides, which bind to a predetermined, specific binding partner, which process includes the construction of a nucleotide bank, transformation of microorganisms, cultivation of the transformed microorganisms and selection of the microorganisms which express the sought-after peptides, as well as to the use of such peptides.

Description

- 21~2193 B~RINGW~RRB ARTIENG~S~LLSC~APT 92/B 033 - ~a 961 Process for preparing rocombinant and ~ynthotic poptidos, and their use.

The invention relates to a proceos for preparing peptides, which bind to a predetermined, specific binding partnor, which process includos tho construction of a nuclootide ban~, transformation of microorganisms, cultivation of tho transformed microorganisms and soloction of the mlcroorganisms which express tho sought-afeer p-ptides, as w-ll as to the use of such peptides.

A m~ltiplicity of important agonts react as binding partners in roceptor-ligand intoractions which aro of particular therapeutic and diagno~tic importanco.
Examplos which may b- montlonod aro inhibitors in protoolytic enzymo caecados, moduIators of tho immuno systom, ligand~ for hormone recoptors, proteins which bind nucleic acids, and binding partnors in antibody-antigon reactions.

It is froquontly of intorest to identify and to charàc-t-rizo binding sitos of r-coptor and ligand, and/or replaco ono of thè binding partnors by anothor compound, proforably a low molecular woight compound, which roacts ln a similar mannor with tho socond binding partnor.

In many casos, it is desirable to employ a synthetic poptido as the low lecular weight binding partner, which peptide can be synthesized simply and at low cost in large quantities and with a high degree of purity.
.
At prosent, structures whlch are constitutod by proteins or poptidos can, in somo cases, be replaced by synthetic peptidos. However, it is often very difficult, if not impossible, to identify the structure or amino acid sequence which contributes to the biological activity, or is responsible for it. Even if the amino acid sequence of , ".;

- 2 _ 2~21!~3 the binding side of a protein to a receptor can be successfully elucidated in some C8808, a~ was possible, for example, for some antigen-antibody bonds (Immunology ~ Today, 10, 266-272), it cannot then be pred$ctod whether a synthetic peptide comprising the amino acid seguence of the antigen in the region of the b$nding site will react in a similar way with the antibody, since it may some-times, in solution or on a solid phase, have a structure which is completely different from that of the antigen and therefore not react with the corresponding antibody.

~pirical methods are known which make it possible to identify and to characterize structures which are active in binding and which react with the eelected binding partner in a similar manner to that of the corresponding nativo ligands. Theso structuros can then be prepared as tho recombinant or synthetic peptide, and then employed.

Peptid- banks havo boon pr-parod using molecular blological methods, as described in Patent Applications WO 91/17271, WO 91/19818 and in the papers of Scott et al., Science 249, 386-390 (1990), Dovlin et al., Science 249, 404-406 (1990), Cwirla et al., Proc.Natl.Acad.Sci.
- USA 87, 6378-6382 (1990) and Felici ot al, ~. Mol. Biol.
(1991) 2~, 301-310, and using chemical methods as doscribed in Patent Application WO 92/00091 and by von Lam t al., Naturo 354, 82-84 (1991). They mako availablo extonsive pluralities of peptido oligomers as binding partnors, the plurality being achieved by permutations of the 20 ~ino acids at 5-15 positions in a peptide. The number of theoretically possible co~binatione with per~utations of 5-15 amino acids is between 3.2 x lOC and 3.3 x 101'. The peptide banks described in the literature comprise about 2.5 x iO~ to 3 x 10' different peptides.

Molecular biological processee for preparing p-ptide banks (i.e. Scott et al., see above), are based on the recombination of phage DNA with oligonucleotide~ which contain stochast1cally distributed or randomly varied ., : ,. .. ~ ~ . .. . .

3 21~21~3 nucleotide ~eguences in 15 to 60 positions. Ho~t bacterla are then transformed with the recombinant phage DNA. In the cases described, filamentous phage (e.g. M13, fd, fl) are employed. For this purpose, the oligonuclootides are inserted into phage genos which encode coat proteins and receptor proteins, such as pVIII (i.e. Felici et al., see above) and pIII ~i.e. Scott et al., see above), on the surface of the phage. The stochastically distributed nucleotide sequences are thus translated into stochastically distributed amino acid seguences and are presented on the surface of the phage.

- The employment of phage for preparing peptide banks has tho disadvantage, for example, that phage reguire bacteria as hosts and also that the number of copies of the recombinant protein on the surface of a phage is rolatively small.
' A ~uitablo scroonlng proc-ss for identifying the sought-aftor poptides must be ablo to idontify, from about 3 x 10~ to 4 x lO19 compounds (seo above), that which roacts spocifically with the receptor employed.
:
Consequently, the demands on screening methods are extraordinarily high and require a special approach.

Scrooning of the phage-poptide banks described in the state of the art, and consequently identificat~on of the binding protoins, is carriod out by a process which has boon described by Parmely et al. (Gene 73, 305-318 (1988)) as follows:
.

The recombinant phage are incubated with the receptor, which has been i~ ob~lized on potri dishos. Phage which aro not bound aro remo~od by washing. ~ound phago are roloa~od in a socond stop by treatmont with acidic buffers or wlth SH reagents and then amplified in nutrient médium. The purification step must be repeated ~3-4 times) until a sufficiently high enrichment of ~, . , ,::
~, . . ..

, . . .. :.,. ~ . ~, . .. , . .

specifically binding phage has been achievod The nueleotide sequence of the phage i8 then determined in the region of the inserted oligonucleotides and the amino acid seguence of the receptor-binding peptide is deduced The peptide can then be synthesized chemically Howevor, a di8advantage of this process is that the bound phage mu8t first be released from the earrier in order then to be amplified by way of infeetion in E eoli Depending on the ehoiee of relea8ing eondition8,~this entails the danger that either the relea8ed phage will be 80 seriously damaged that they can then no longer be amplified' or that phage with high affinity - and con~oguontly particularly important gualitatively - will pot be released and will therofore be lost , Th- ob~eet of th- pr-~ent invontion was, therefore, to mako availabl- a proeess whieh makes it possible to obtain the desirod peptides in high guantitative and gualitative yields ~hi8 ob~ect is aehi-ved by a proeess in whieh a multi-plicity of peptide8 are expr-ss-d direetly on'tho surface of suitablo microorganism~ in a manner which permit8 the p-ptid-s to bo directly aeeo~slble to receptors and to be id-ntified and isolatod using suitable screening proe-s~-s In this eontext, th- plurality of the peptide bank ~hould b- gr-at-r than 10 The invention therefore relates to a proeess for proparing peptidos whieh bind to a predetermined', ~peeifle binding partn-r, whieh proeess includes the following st~p~ ' a) isolation or synthosis of oligonucleotidos which eontain regions in which the nucleotide seguenees vary stochastically, b) r-eombination of the oligonucleotides with veetor .. . . . .... .. . . . . . . . .

^` _ 5 _ 23 ~2 1~ 3 -molecule~ in such a way that the oligonuclootides are inserted into a vector-encoded gene which expresses a protein of the outer coat of micro-organisms, an oligonucleotide being inserted into each vector molecule, thereby giving rise to a multiplicity o$ vector molecules which differ from each other in this region of their nucleotide sequence (nucleotide bank), .
c) transformation of the microorganisms- with the recombinant vector lecules, ~;

d) cultivation of the transformed microorganisms under conditions which permit the recombinant DNA
sequences to be expressed and the corresponding, recombinant proteins to be presented on the surface ~ ~:
of the microorgani~ms, o) incubation of tho microorganisms with tho pre-dotermined, specific binding partnor, which is bound to a solid phase, with those microorganisms being bound which have expressed the ought-after peptlde, f) romoval of the unbound microorganisms, g) replication of the bound microorganisms, h) cloning and isolation of bound microorganisms .

i) determination of the stochastically varied amino acid seguences by seguencing the corresponding DNA
seguences or by peptide seguencing, ~) obtaining the sought-after peptides by isolating the recombinant protelns from the microorgani~ms or by chemical synthesis of the corresponding peptides, with the microorganisms being selected from the group comprising bacteria and mammalian cells; E.coli is "

- 6 - 21~2~9~
advantageously used as tho microorganism.

It is known to the person skilled in the art that in the reeombination under step b) empty vectors or vectors containing a plurality of oligonucleotides can be obtained, it being sometimes advantageous deliberatoly to prepare vectors eontaining a plurality of oligo-nucleotides.

That process i8 preferred in which the organi~ isolated under stepo e-g) are brought into contact once again with the binding partner and tho selection and amplification steps are repeated.

In addition, that procèss is preferred in which the reeombinant protein~ on the surface of the organisms, and th~ s~leeted ree~ptor, are proeent in concentration~ sueh lS that only noval~nt binding roaetions are possiblo.

That process i8 partieularly advantageous in whieh the solld phase in stop o) is partieulate; a solid phase whieh is magnetieally attraetable is very partieularly advantageous.

That process 1~ preferr-d in which the vector is a pla~mid.

That proeess is also preferred in which the nucleotides eontain eodons whieh are represented by (NNK)X or (~NNS) X
where N represents the nueleotides A, C, G and T, ~
represents the nucleotides G and T, and Srepresents the nucleotides G and C, and X is a number from 4 to 25, preferably 6.

In addltlon to thls, that proc-ss 18 preferred ln which X ~ 8 and the nucleotldes encode only a fraction of the possible octapeptides.

The invention also relates to that process in which the .. . ... . . .

~ 7 ~ 2~2.l~3 synthetic oligonucleotides contain a sories of codons which encode random combinations of amino acids and, in the 5' and 3' regions of the codons, contain sequonc-s which are likewise not identical to coat protein seguences and which preferably encode the antibody regions Additionally, the invention relatss to that procoss in - which the synthesized oligonucleotides contain a series of codons which encode random combinations of amino acids and, in the 5' and 3' regions of the codons, contain seguences which encode a spacer, which is advantageously composed of proline residues -Tho invention also relates to that process in which the microorganisms ar- transform d by electroporation lS In addition, the invontion rolatos to that process in which th- nuclootide bank comprisos at least lOC, advantag-ously 10', constituent units Tho invention also rolateo to p-ptides which are prepared by at loast ono of tho procoss-s mentioned Tho invontion additionally rolates to antibodios which ar- producod using ouch poptidos, which poptides are, wh-ro appropriato, coupled to carrier proteins Th- invention furthormoro rolates to a proces~ whicb makes it possiblo to investigate simultaneously the ability of a large multiplicity of microorganisms to bind to a soloctod roceptor (scroening) Th- scro~ning proc~ss according to the invention is, intcr alia, slmpl-r, faster and more efficient than the known procos~o~ An approciable simplification and accoloration of the process is a~hieved by the fact that tho microorganisms no longer need to be released from the solid phase At the same time, this render~ it possible - 8 - 2 1~2.19 3 to identify and isolate peptides having high afflnity as well. It was possible to achieve further accsleration and simplification of the procedure by using a magnotically attractable, microparticulate, so1id phase.

Surprisingly, it was possible to demonstrate that a peptide ban~ of adequate complexity could be constructod dlrectly in E.coli, 80 that it was possible to dispense with the use of phage and the employment of an additional host.

l3y way of example, oligonucleotides were prepared which possssssd a region of stochastically distributsd nuclootidss. This region can comprise 12, 18, 24, 30, or rs, nucleotidss, a rsgion of 18-45 nucleotidss being prsfsrrsd. In ths present invsntion, a region of 18 nuclootidss was chosen, which rogion corrsspondingly oncodes 6 amino acids in random combinations.

Tho DNA prsparsd in this way was rscombinsd w1th the plasmid vector pHS164-E (EP O 335 737 A2), which sncodes ths outer membrane protoin A (O~pA). The variablo DNA was inserted into the OmpA gsne. Following transformation into E.coli, tho OmpA poss-s~ing ths stochastically pormutatod amino acids i~ prosont~d on the E.coli coat and is thus accossible to rocoptors.

Tho prosentation of ths stochastically varied amino aclds can bo sffsctod in a particular rsgion of ths OmpA
protsin such that it was possible, in combination with ths novel screening msthod introduced here, to identify usoful psptido sequences.

OmpA is composed of 325 amino acids and i~ a very frequontly occurring protoin in tho outer membr~no of E.coli. It serves as tho extornal receptor structuro for somo phagos and colicins during infection and lntoxication respectively. OmpA contains four regions possessing 10-13 predominantly hydrophilic amino acids.
.: -.

- 9 - 21~2193 These regions are separated by regions which are composed of 2 x 15 - 17 predominantly hydrophobic amino acids.
Although the three-dimensional structure of OmpA has not yet been determined, spectroscopic measuroments have supported a model which describes OmpA as a protein whose N-terminal half is composed of 8 transmembrane regions possessing a ~ structure. According to this model, the 4 hydrophilic rogions around amino acids 25, 70, 110 and 154 are xposed on the bacterial surface.

Pistor and Hobom (Rlin. Wochonschr 1988, 66, 110-116) were able to show that it is possible to insert foreign protein sequences into OmpA in the region around amino acid 110, which sequences are then themselves likewise expressed on the E.coli surface and do not appreciably alter the structure of the OmpA sequences.
, The presontation of hexapeptides as constituents of the outor membrano protein of OmpA provides a series of advantages, such a~, for example, simple culture of bacteria as compared with the hitherto customary phage and a significantly higher copy number of the outer membrane protein per cell.
The screening process employed here is based on the exprossion of poptides containing stochastically pormutated amino acids on the surface of bacteria in such a way that the poptides can bind to receptors. The roceptors are bound to a particulate, preferably magneti-cally attractable, ~olid phase. The plurality- of the bacteria is brought into contact with the solid phaso.
After the recoptor-ligand reaction has taken its course, the spocifically bound bacteria are separated from those which have not been bound. Surprisingly, it was possible further to replicate the specifically bound bacteria without relea~ing them from the solid phase. A further advantage lies in the abbreviated reaction time when using a microparticulate solid phase.

The application example described below describes a ~ ~ ! " I . , , ~
, :' , , ',', ,~,. . I '" ~ ' ' 21~219~
- 1 o preferred process for preparing and scroenlng peptide banks.

The advantages and possible applications of the procésses to which this invention relates are clarified below using the example of an antibody-antigen bond. This example is not-to be understood as being exclusive, since the term antibody-antigen bond can be u~ed synonymou81y, within the meaning of the invention, with the term receptor-ligand interaction.

The process is able to produce p}uralities of compounds in a complexity which makes it possible to isolate ligands for an arbitrary roceptor without having any information on the existence or the nature of the natural ligand.

lS In additlon to this, ligands can be isolated which pos~es~ differing affinities for each immobilizable roceptor, from which ligands those possessing the best properties can then be selectod.

An epitopo mapping can also be carried out significantly re fficlently than when using chemical methods. Thus, in addition to monoclonal antibodies, mixtures of anti-bodie~, such as polyclonal antibodies, can also be analyzed. If, for example, the primary structures of the antigen or the antigens are known, many epitopes, against which the polyclonal antibodles are directed, can bo identified in one operation.

~ ", ,,, .:

ll 2~21 es 1) Constructicn of a he~apeptide epitope ban~ in ~ch~richia coli (~ coli) Th~ pla~mid v~ctor p~8164-h (EP O 355 737 A2), which encode~ tho out-r membrane protein A (OmpA) of Gram-negativ- bacteria and po~e~se~ a polycloning ~ite, was --selected for presenting hexapeptide molecules on the surface of ~ coli Oligonucleotides, which wore intended to encode all th- hexapeptide combination~, w~r- inserted into the ClaI and XmaI restriction sites They were latorally flanked by five further hydrophilic amlno acidc, ~uch as proline and glycine, which, comparable to a ~tsm region, were intended to pre~ent tho hexapeptide region more effectively on the bacterial surfaco This gav- ri~o to the following nucleic acid sequence which was to be ~ynthe~ized 5' CG CCA GGA CCC CCG CCT NNR NNR NNR NNR NNR NNR CCT CCT
CCG CCA CC 3' In this context, ~N" ~ymbolize~ all four coding possibil~tie~ (A, C, G, T) and "R" only G and T Thi~

;` 23 ~2:~3 coding randomly gives all twenty naturally occurring amino acids. In order to be able to ligate a DNA double strand into the voctor opened with ClaI/XmaI, the singlo strand shown above was partially converted into a double strand at its end regions, which only encode proline and/or glycine and arginine, by means of two furthor, reverse complemontary, oligonucleotides. For this purpose, the oligonucleotides .
SEQ ID N0: 2 3' GGT CCT GGG GGC GGA 5' SEQ ID N0: 3 3' GGA GGC GGT GGG GCC 5' woro synthesized and hybridizod to tho presérved region of tho DNA ~inglo strand. As a rosult of this, tho oligonuclootido, which is doublo-strandod at its end regione, can be ligated into tho ClaI and XmaI
restriction sit-s of the opened vector in position 800, and then also transformed into ehe ho~t coll.

I ple--ntation:

To implomont tho DNA construction, the DNA of vector pHS164-L was oponsd at tho multiple polycloning site with the rostriction onzymos ClaI, which rocognizes the DNA
s-guenco 5AT'CGAT3, and XmaI, which rocognizos the DNA
soquonce 5C'CCGGG3, and th~n purified by gel electrophoresis. Tho oligonuclootido, which potentially oncodes 64 x 10' hexapeptidos as woll as tho ad~oining a~no acide proline and/or glycine, which contributo to tho stom region, was ligatod into those cloavago eitos.
The ligase roaction was carried out u~ing 10 ~g of oponed vector and 0.154 ~g of double-stranded, hybridized oligonucl-otid~ in a total ligase mixture of 10 ~1 at room tomp-rature for a period of 24 h. Prior to trans-formation by eloctroporation, the DNA was desalted using Centrikon ~30 microconcentrators (Amicon, Beverly, MA, USA). Transformation of this DNA into the cells of Escherichia coli strain 490 A, which had previously been made competent by standard methods, was carried out using .. . . . . . .
,,, . : .

:: .; . . . . .

- 13 _ 2~2~93 1 ~g of DNA from the ligase mixturs in 100 ~1 of competent cells by means of electroporation in a G-ne Pulser~ (BioRad, Richmond, CA, USA) at 2 4 kVolt, 400 Oh~ 25 ~FD in cuvsttes of 0 2 cm dimension Ten transformation mixtures, as described above, were collected and combined, and eventually yielded 3 x 10~
transformed cells It was possible to show, by restric-tion dige~tion of different cell clones, that 42% of the transformed cells had taken up the DNA encoding a hexapeptide Conseguently, the average transformation efficiency of the recombinant plasmid, exclusivoly - encoding the hexapeptides, was about 1 26 x 107/~g All theoretically possible hexapeptide combinations were thus represented two times The epitop- bank was aliguoted into 1 ml cultures and frozen at -80C together with 10%
glycerol 2) Coupling of ~450 ~agnetic particlos to antibodles Using tho example of thr-e monoclonal antibodies, which - are direct-d against different viral peptides, it can be d^m~nstrated below that the relevant antibodi-s select particular epitop-s, in a targ-ted manner, from the bacterial epitope bank in which more than 1 2 x 10' hexa-p-ptid-s are pr-s-nt-d ~ with r-gard to a MAb-144/158 dir-cted against the HBeAg of H-patitis B virus (Molecular Immunology 28, 1991, 719-726) with regard to b MAb-91-195/039 directed against a synth-tic peptide which contains ~mino acid~ 120-130 of the gag protein of human adult T-c-ll leukemia virus (Proc Natl Acad Sci USA 80, 1983, 3618-3622) `
with regard to c MAb-87-55/02/2 directed against an ~:
~,, - ............................. ~
.;.: , . ~ , :: .

i .. . . . . . . ..

- 14 - 2~?~.19 ~
epitope of the pp 150 protein of cytomegalo~irus (EPA 0 534 102 A1).

Implementation:

* with regard to 1: for screening the epitope bank with the monoclonal antibody MAb-144/158, the latter was coupled to M450 magnetic particles (Magnetobeads - -M450, Dynabeads~, Dynal A.S., Norway). For this, 250 ~1 of magnetic particles, and 75 ~g of antibody were taken in 1 ml of 40 mM borate buffer, pH=9.5.
This mixture was shaken at room temperature over-night' and then washed three times with storage buffer (50 mN 2-tN-cyclohexylamino] ethanesulfonic acid - CHES in brief - , 0.01% Na-azide) in the magnet~c separation unit. Finally, the antibodies coupled to the magnetic particles were stored at 4C
in 1 ml of storage buffer. Immediately prior to use (screening), they were washed three times with LB
medium.

* ' with regard to 2 and 3: for screening the epitope bank with the monoclonal antibodies MAb 91-195/039 and MAb-87-55/02/2, thes6 latter were coupled to Ml-070/40 magnetic particles (lot 383) (Estapor micro6pheros Rhône-Poulenc, France). 500 ~1 of magnetic particles were washed with 3 x 5 ml of coupling buffer (100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, -HEPES in brief -, p~ 4.7) in a magnetic separation unit, and then taken up in 8ml of coupling buffer. While shaking, 1.5 mg of antibody and 2 ml of a solution (2 mg/ml) of 1-othyl-3(3-dlmethylaminopropyl) carbodiimide HCl were addod, and tho ~olume of the mlxture wa~ made to 20 ml using coupling buffer. The suspension was incubated at 4C for 16 hours while shaking. The magnetic particles were ~ubsequently washed 3 times with ~torage buffer (50 mM 2-[N-cyclohexyl'amino]

.
.. . ,,: , , ~ 15 ~ 21~2~9~
ethanesulfonic acid - Ches in brief - 0.01%
Na-azide) in the magnetic soparatlon unlt. Tho antibodies coupled to the magnet$c particles were finally stored at 4C in 1 ml of storago buffer.
Immediately prior to use (screening), they were washed three times with LB medium.

3) Screening of the epitope ban~:

Impl~m~ntation:

l ml of a culture of the epitope bank which had been stored at -80C was thawed and incubated at 37C over-night in 200 ml of L broth medium/50 ~g/ml ampicillin. In the morning, 1 ml of this culture was added once again to 200 ml of L broth/ampicillin and incubated at 37C until the OD600 ~ = 0-5. The cells were induced in the presence of 1 mM IPTG (isopropyl ~-th~ogalactoside) and then incubated for a furth-r 2h undor the same condition~.

In ordor to screen the epitope bank with the monoclonal antibody, a further 900 ~1 of L broth/ampicill$n were then added,to 100 ~l of the cell culture and this mixture was then incubated in the presence of 1% Tween20 at 4C
for 1 h, while shaking gently, w$th 10 ~1 of the anti-bodie~ (75 ~g/ml) coupled to magnetic particles. Using the magnetic separation device, the magnetic particles, together with the MAbs and all the celIs bound -to them, wore separated off and washed a further four times with washing buffer (10 mM Tris/pH = 7.4, 0.9% NaCl, 1%
Tween20) and then taken up in 200 ~1 of broth/ampicillin. 10 ~1 of this mixture were plated out onto L broth agar containing ampicillln and incubated at 37C ovornight. On average, 500-1000 colonies grew on this plato.

For a further screening of these cells with the same MAbs bound to magnetic particles, the cells were washed with 1 ml of L broth and incubated once again in 300 ml of L
, ,' ' '' . ' ., ' ' ' ' '' ' ~'' , , ' "

-: ~' ' ' ' . ' -' ,-, ''' ' :~

-16 - 21~2~
broth/ampicillin at 37C until the OD~oo~ - 0.5. The cells were then once again induced with 1 mM IPTG; the second screening then took place under the same conditions as described above. The screening with an MAb was repeated four times in all.

4 ) Analy8i8 of po~itivo clones 4a) Screening with Mab 144/158 (HBeAG) - Three different methods were subsequently employed for analyzing positive clones which it had been possible to isolate by screening with the monoclonal antibody MAb 144/158. Inhibitlon experiments were u~od to test whother $t was possible to impede the binding of the positive clones to the magnetic particles coupled to MAb-144/158 by adding free MA:b-144/158. The bind$ng of -15the noclonal antibody 144/158 to the epitope region integrated into the QmpA protein was invostigatod with the aid of Westorn blot analysis, and the nucleic acid sequence of the corrosponding epitope rogion was finally determined by sequence analys$s. Tho~e experiments led to the predominant $dent$fication of one poptide, which had previously been determ$nod by Sallberg et al. 1991, Molecular Im~unology 28, 719-726, in a conventional manner by fine-mapping, to have the amino acid sequence T-P-P-A-Y-R.

T'Tl~tation: ~ -T~.h'Il~ition o~peri ~ ts Individual clones from the screening were incubated overnight and th- culture was then diluted the following morning and finally induced with IPTG. The cell culture was next diluted 1:100 with ~ broth/ampicillin (total sample vol~me 250 ~1) and then incubated at 4C for 1 h in the presence of 1% Tween20 and different concentrations of free MAb-144/158 (10-100 ~g/ml of sample). The magnetic particles w$th the bound MAb-;,, : :, : :
.. - .:

;:: , ~ ;

--` 21.~2.~

144/lS8 were then added and the mixture was incubated once again at 4C for 1 h. Subsoquently, tho cells were i~olated once again using the magnetic separation device and then washed three times with washing bu~fer. ~he cells were then taken up in 500 ~1 of L broth/ampicillin and in each case 20 ~1 of this suspension were plated out onto L broth agar/ampicillin. Parallel thereto, experimental series were carried out without the addition of free MAb-144/158 (positive control). In addition, parallel experiments were carried out in which cells were used which had been transformed with vector pHS164-L
which did not contain any insert (negative control). The oxporiments demonstrated that the binding of cells to the MAb-144/158 bound to the magnetic particles was reduced by a factor of 50 to 100 by the addition of free MAb-144!158 to the reaction ~amplo. By contrast, the binding of tho c-lls to tho magnetic particles was not impaired in tho po~itive control and, as expected, no cell binding took place in tho negative control.
~ ~.

Wostorn blot analy8i8 -Cell clones, which had been found by screoning with MAb-144/158 and whose binding was able to be inhibited by free NAb-144/158 in the inhlbition experiment, were sub~octed in the next step, to a Western blot analysis.
In this analy~is, all the clones exhibited a positiveIy r-acting protein band which corresponds to the size~of -tho membrane protein OmpA.

DNA seqnence ~n~lysis All the cell clone~ which roacted positiv-ly in the provious analysos were next sub~ected to a DNA seguence analysis. For this, the vector DNA was i~olated as double-stranded DNA by conventional processe~ and sequenced using vector-~pecific oligonucleotide primers by the method of Sanger (Saager et al., 1977, Proc.Natl.
Acad. Sci. USA, 74, 5463-5467). The analy~is demonstrated . : , . . -: . ~. : : .

-~ 21~2~

that, with one exeeption, all the positive clone~
eontained a DNA seguenee whieh eneode the original peptide sequenee whieh MAb-144/158 reeognizes - namely T-P-P-A-Y-R. The eorresponding DNA seguenee of a single cell clone diverged from this original sequence and had a nucleotide seguence whieh, instead, encodes the follo-wing amino acid seguence SEQ ID NO: 4.

SEQ ID NO:4 Leu Pro Pro Ala Phe Arg 4b) AnalysiJ of positive elones: sere-oinl with ~ab 91-195/039 ~TLV) : .
Three different methods were in turn employed for analyzing positive clones which it had been possible to isolate by oereoning with the monoelonal antibody MAb-91-195/039. Inhibition experimonts were usod to test whether it wa~ possible to impodo the binding of the po~ltlve elonos to the magnetle partieles eoupled to NAb-91-195/039 by adding free MAb-91-195/039. The binding of the noclonal antiboby 91-i95/039 to the epitope region ~ntegrated ~nto the QmpA protoin was investigated with the aid of Westorn blot analysis, and the nucleic acid soquence of the corrosponding epitope region was finally dot-rmined by sequence analysis. Poptides wers exelusively found whose soquence div rged from the P Y V E P Y A P Q V L region of the immunizing antigen ~Pro¢.Natl.Acad.Sci. USA 1983 80, 3618-3622). ~ ~

I pl _ ntation:

I~hl~ition e~cper~m^~ts Individual clones from th- screoning wero incubated overnight and tho culture was thon diluted the following morning and finally induced with IPTG. The cell culture was next diluted 1:100 w~th L broth/ampicillin (total sample volume i50 ~1) and then incubated at 4C for 1 h in the presence of 1% Tween20 and different 2112~

concentrationo of froe MAb-91-195/039 (10-100 ~g/ml of sample). The magnetic particles with the bound MAb-91-195/039 wore then added and the mixture was incubated once again at 4C for 1 h. Subsequently, the cells were isolated once again using the magnetic separation device and then washed three times with washing buffer. The cell~ were then taken up in 500 ~1 of L broth/ampicillin and in each case 20 ~1 of this suspension were plated out onto ~ broth agar/ampicillin.
Parallel thereto, experimental series were carried out without the addition of free MAb-91-195/039 (positive control). ln addition, parallel experiments were carried out in which cells were-used which had been transformed with vector pHS164-L which did not contain any insert (negative control). The experiments demonstrated that the binding of cells to the MAb-91-195/039 bound to the magnotic particles was reduced by a factor of 20 to 30 by the addition of fr-- MAb-91-195/039 to the reaction ~ampl-. By contrast, the blnding of the celle to the magnotic particlos was not impaired in the positive control and, as expected, no cell binding too~ place in the negative control.

Wostorn b~ot ~nalyd s Coll clonos,,which have boen found by screening with NAb-91-195/039 and whose blnding in the inhibition oxp-rimen was able to be clearly inhibited by free MAb-91-195/039, were subjected, in the next step, to a Wost-rn blot analysis. With one exception, all the clones demonstrated, in this analysis, a positively reacting protein band which corresponds to the size of the m~mbran- protein OmpA.

` DNa 8~quQnco analysis Cell clones, which ha~e been found by antibody screening, were next subjected to a DNA seguence analysis. For this, the yector DNA was isolated as double-stranded DNA by ,~

~ 20 - 2112~ ~
conventional processes and sequencod using vector-specific oligonucleotide primers by tho mothod of Sanger (Sanger et al., 1977, Proc.Natl.Acad.Sci. USA, 74, 5463-5467). The analysis de nstrated that, without exception, all the clone~ contained a DNA sequence which does not correspond to the wild-type sequence Pro-Tyr-Val-Glu-Pro-Thr-Ala-Pro-Gln-Val-Leu, which MAb-91-195/039 recognizes.
Rather, a total of 8 different peptide sequences were found among the positive clones, which sequences reacted positively either in the Western blot analysis or in the inhibition experiment, or in both procedures. The following peptide sequences were found:

SEQ ID NO: 5 Phe Leu Phe Pro Thr Ser Clone 5 SEQ ID NO: 6 Met Asn Phe Asn Ser Ser Clone 8 SEQ ID NO: 7 Ser Leu Ala Ala Thr Trp Clone 9 8EQ ID NO: 8 Val Asn Ile Asn Ser Gln Clono 12 g~Q ID NO: 9 Val Asn Tyr Asn Ser Sor Clono 13 SEQ ID NO: 10 Phe Ilo Ala Pro Mot Gly Clone 30 SEQ ID NO: 11 Tyr Ilo Leu Ala Thr Leu Clon- 31 SEQ ID NO: 12 Tyr ~eu Ser Pro Pho Gly Clone 33 4c) Analysis of positi~o clones: screening ~ith ~Db-87-55~02/2 (C~V) Threo different mothods were also employed for analyzing positiv- clones which it had boen possible to isolate by 25 screoning with the noclonal antibody NAb-87-55/02/2.
Inhibltion experiments wore used to test whether it was possible to impede the binding of the positive clonos to the magnetic particles coupled to MAb-87-55/02/2 by adding free MAb-87-55/02/2. The binding of tho monoclonal 30 antibody 87-55/02/2 to the epitope region intogratod into tho OmpA proteln was investigatod with tho aid of Wostern blot analysis, and tho nucleic acid sequenco of the corresponding epitope region was finally determined by sequence analysis. Peptides were found which are homologous to the CMV wild-type sequence of the pp 150 epitope, or come close to it. The epitope had previously , : , : : ., : , , : : ~ . , , ~ 21 - 2112~
been determined by Stuber et al. (EP-A-0 534 102), in a conventional manner by fine-mapping, to havo the amino acid sequence Asp-Met-Asn-Pro-Ala-Asn-Trp-Pro-Arg-Glu-Arg-Ala-Trp-Ala-~eu.

S rmpl~m~tation: -Inhibition ~xperiments Individual clones from the screening were incubated overnight and the culture was then diluted the following morning ant finally induced with IPTG. The cell culture was next diluted 1:100 with L broth/ampicillin (total sample volume 250 ~l) and then incubated at 4C for 1 h in the presence of 1% Tween20 and different concentrations of free MAb-87-55/02/2 (10-100 ~g/ml of sampl-). The magnetic particles with the bound MAb-87-55/02/2 wer- then added and the mixture was lncubated once again at 4C for 1 h. Subsequently, the cells were i~olated once again u~ing the magnetic separation device and then washed three times with washing buffer. The cells were then taken up in 500 ~1 of ~ broth/ampicillin and in each case 20 ~1 of this suspension were plated out onto L broth agar/ampicillin.
Parallel thereto, experimental series were carried out without the addition of free MAb-87-55/02/2 (positive control). In addition, parallel experiments were carried out in which cells were used which had been transformed with vector pHS164-L which did not contain any insert (negative control). The experiments demonstrated that the binding of cells to the MAb-87-55/02/2 bound to the magnetic particle~ wa~ reduced by a factor of 30 to 200 by the addition of free MAb-87-55/02/2 to the reaction sample. ~3y contrast, the binding of the cell~ to the magnetic parSicles wa~ not impalred in the positivs control and, as expected, no cell binding took place in She negative control.

- 22 - ~1~2~
Western blot analysis Cell clones, which had been found by scroening with MAb-87-55/02/2 and whose binding in the inhibition experiment was able to be clearly inhibited by free MAb-85-55/02/2, were subjected, in the next step, to a Western blot analysis. In this analysis, all the clones demonstrated~a positively reacting protein band which corresponds to the size of the m~mbrane protoin OmpA.

DNA sequence analysis 10 All the cell clones which reactod positively in the provious analyses were next subjected to a DNA seguence analysis. For this, ~the vector DNA was isolated as double-stranded DNA by conventional processes and 8eguenced using vector-~pocific oligonucleotido pri~ers by tho method of Sanger (Sanger et al., 1977, Proc.Natl.
Acad. Sci. USA, 74, 5463-5467). The analysis demonstrated that the positive clones contained peptide sequencos which come very closo to the original peptide sequence in the rogion Asp-Net-Asn-Pro-Ala-Asn-Trp-Pro-Arg. In all, five different hexapeptides were found which diverged from the wild-typo soguonco:

SEQ ID N0: 13 ~ou Vla Asn Pro Ala Asn Clono 4 SEQ ID N0: 14 Phe Asn Pro Ala Asn Pho Clono 7 SEQ ID N0: 15 Asp Arg Asn Pro Ala Asn Clone 8 SEQ ID N0: 16 Asp Tyr Asn Ala Ala Asn Clone 9 SEQ ID N0: 17 Phe Asn Pro Ala Asn Asn Clone 11 : ' , ' ' ' ' ,, ' ' ':

Claims

1. A process for preparing peptides, which bind to a predetermined, specific binding partner, which process includes the following steps:

a) isolation or synthesis of oligonucleotides which contain regions in which the nucleotide sequences vary stochastically, b) recombination of the oligonucleotides with vector molecules in such a way that the oligonucleotides are inserted into a vector-encoded gene which expresses a protein of the outer coat of micro-organisms, an oligonucleotide being inserted into each vector molecule, thereby giving rise to a multiplicity of vector molecules which differ from each other in this region of their nucleotide sequence (oligo-nucleotide bank), c) transformation of the microorganisms with the recombinant vector molecules, d) cultivation of the transformed microorganisms under conditions which permit the recombinant DNA
sequences to be expressed and the corresponding recombinant proteins to be presented on the surface of the microorganisms, e) incubation of the microorganisms with the pre-determined, specific binding partner, which is bound to a solid phase, with these microorganisms being bound which have expressed the sought-after peptide, f) removal of the unbound microorganisms, g) replication of the bound microorganisms, h) cloning and isolation of bound microorganisms i) determination of the stochastically varied amino acid sequences by sequencing the corresponding DNA sequences or by peptide sequencing, j) obtaining the sought-after peptides by isolating the recombinant proteins from the microorganisms or by chemical synthesis of the corresponding peptides wherein the microorganisms are selected from the group comprising bacteria and mammalian cells.

2. The process as claimed in claim 1, wherein the organisms isolated under steps e-g) are brought into contact once again with the binding partner and the selection and amplification steps are repeated.

3. The process as claimed in claim 1, wherein the recombinant proteins on the surface of the organisms and the selected receptor are present in concen-trations such that only monovalent binding reactions are possible.

4. The process as claimed in claim 1, wherein the microorganism is a bacterium.

5. The process as claimed in claim 1, wherein the microorganism is a mammalian cell in suspension.

6. The process as claimed in claim 4, wherein the microorganism is an E.coli

7. The process as claimed in claims 4 and 6, wherein the coat protein is OmpA.

8. The process as claimed in claim 1, wherein the solid phase in step e) is particulate.

9. The process as claimed in claim 8, wherein the solid phase is magnetically attractable.

10. The process as claimed in claim 1, wherein the vector is a plasmid.

11. The process as claimed in claim l, wherein the nucleotides contain codons which are represented by (NNK)?, wherein N represents the nucleotides A, C, G
and T and R represents the nucleotides G and T, and ? is a number from 4 to 25.

12. The process as claimed in claim 11, wherein ? = 6.

13. The process as claimed in claim 11, wherein ? = 8 and the nucleotides encode only a fraction of the possible octapeptides.

14. The process as claimed in claim 11, wherein ?= 10 and the nucleotides encode only a fraction of the possible decapeptides.

15. The process as claimed in claim 1, wherein the oligonuc-lotidos contain a series of codons which encode random combinations of amino acids and, in the 5' and 3' regions of the codon, contain sequences which likewise are not identical to coat protein sequences.

16. The process as claimed in claim 1, wherein the synthesized oligonucelotides contain a series of codons which encode random combinations of amino acids and, in the 5' and 3' regions of the codons, contain sequences which encode antibody regions.

17. The process as claimed in claim 1, wherein the synthesized oligonucleotides contain a series of codons which encode random combinations of amino acids and, in the 5' and 3' regions of the codons, contain sequences which encode the spacer.

18. The process as claimed in claim 17, wherein the spacer contains proline residues.

19. The process as claimed in claim 1, wherein the microorganisms are transformed by electroporation.

20. The process as claimed in claim 1, wherein the nucleotide bank comprises at least 106.

21. Peptides! which are prepared by at least one of the processes as claimed in claims 1-20.

22. Antibodies, which are produced using peptides as claimed in claim 21 which, where appropriate, are coupled to carrier proteins.

23. Peptides as claimed in claim 21, possessing the amino acid sequences of the peptides 4-17.

24. Antibodies, which are produced using peptides which, as claimed in claim 22, are, where appropriate, coupled to carrier proteins.

25. Antibodies, which are produced using peptides which, as claimed in claim 23, are, where appropriate, coupled to carrier proteins.