Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
- 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
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~, . . ..
, . . .. :.,. ~ . ~, . .. , . .
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
SEQ I D NO 1 -
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
- 12 -
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
~:
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- ............................. ~
.;.: , . ~ , :: .
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.~
- 17 -
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~
18 -
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~
- 19 -
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
: ' , ' ' ' ' ,, ' ' ':
