Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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HIGH-AFFINITY ANTIBODIES
Field of the Invention
This invention relates to antibodies and their
therapeutic use.
Backctround to the Invention
Antibodies have long been regarded as potentially
powerful tools in the treatment of cancer and other
diseases. However, although there have been some notable
exceptions, this potential has not generally yet been
10 realised.
This relative lack of success may be due, at. least in
part, to the u~~e of monoclonal antibodies derived from
rodents, which :seldom have affinities higher than 10-9 M.
Antibodies having this level of affinity are o:E limited
15 therapeutic utility, as it has proved difficult to deliver
enough antibody to the target to effect useful biological
activity. Antibody binding to an antigen is reversible,
and at the concentrations of antibody practical far in vivo
use, dissociation will be favoured over association. In
20 principle, it i:~ possible to counter the dissociation of
antigen by increasing the antibody concentration. However,
this may lead to unacceptable clinical side-effects and
would also increase the costs associated with the. therapy.
Summary of the Invention
25 The present invention is based on the realisation that
antibodies, or fragments thereof , can be produced which are
"acid-resistant" and that this property is associated with
high affinity binding of an antibody for its antigen.
According t:o the present invention, a high-affinity
30 antibody has affinity characterised by:
(i) incuba.ting first and second samples of the
antibody in antigen-coated microtitre plate wells at a
concentration chosen to be within the linear response part
of a standard curve at pH 7.2 for 1 hour at 37°C;
35 (ii) removing unbound antibody from both samples;
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(iii) incubating the first sample with PBS at pH 7.2
for 1 hour at 37°C, and reducing the pH of the second
sample to pH 3 or below and incubating for 1 hour at 37°C;
(iv} remov_~ng unbound antibody from both samples;
(v) incubating both samples with anti-antibody
alkaline-phosphatase conjugate for 1 hour at 37°C;
(vi) removing unbound conjugate from both samples; and
(vii) adding PNPP substrate to the samples, measuring
absorbance of the samples at 405nm, and determining the
amount of antibody bound to antigen, wherein the amount
bound in the second sample is >50% of that of 'the first
sample.
Preferably, the maximum pH in step (iii) is 2.5, more
preferably 2Ø
Antibodies or antibody fragments with the "acid
resistant" propE~rties are expected to favour association
rather than dissociation and they therefore have longer
localisation times at target sites, which results in a
higher concentration of antibodies localised at the target
sites.
In particular, this invention relates to the
production of a high affinity single-chain Fv antibody
fragment. This ScFv has particular advantages in that it
allows better targeting to a site in vivv.
Description of the Drawincx
Figure 1 illustrates the results achieved for acid-
resistance of sheep and mouse monoclonal antibodies and
single-chain Fvs with affinity to carcinoembryonic antigen
at various pH values.
Description of the Invention
The acid-resistant monoclonal antibodies according to
the present im~ention may be obtained using various
techniques. For' example, classical hybridoma technology
can be applied, comprising the fusion of B-lymphocytes from
immunised animals secreting high-affinity antibodies with
an appropriate fusion partner. An alternative method is to
purify the mRNA from selected lymphocytes and use the
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technique of PCR to amplify the antibody genes required.
Phage display technology and other techniques for the
display of antibody fragments may also be used to obtain
the antibody genets from naive or immunised libraries after
appropriate selection procedures.
The antibody gene can be co-expressed with or
otherwise chemically linked to toxins, radioisotopes or
enzymes or any other desirable molecules to provide a
fusion protein with strong binding characteristics. In a
further alternative, the antibodies may be produced by
transgenic anima7Ls as described in US-A-5770429.
The antibody may be a whole antibody, comprising heavy
and light chains, and constant and variable regions.
Alternatively, the antibody is an antibody fragment, e.g.
F(ab')2, Fab, Fv or single-chain Fv fragments, provided
that at least part of the variable region is present which
confers the propEarty of "acid resistance". The antibody
may also be an animal, chimeric or humanised antibody. A
suitable method for producing humanised antibodies is
disclosed in WO-A,-92/15699.
In a preferred embodiment of the invention, the
antibody is a single-chain Fv fragment. The single-chain
Fv fragment comprises both heavy chain and light chain
variable regions linked by a suitable peptide.
The antibodies of the present invention may be defined
by their acid-resistant properties, which can be
characterised by a~n acid-washed enzyme-linked immunosorbent
assay (EIA), as described above. Typically the A,os value
obtained by EIA will represent antibody binding of >50% for
a sample at pH 3 or below, compared to the value for the
sample at pH 7.2. Preferably, the A,oS value of a sample at
pH 2 will represent antibody binding of >60% more
preferably 70% of that obtained at pH 7.2.
The animal that is subjected to immunisation is not a
rodent, but is chosen to give higher affinity antibodies.
Any large mammal may be used and suitable animals include
rabbits, goats, cows and sheep.
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An antibody of the invention may be used in therapy
and may be formulated into any suitable composition with a
physi.ologically--acceptable excipient, diluent or carrier.
The following Examples illustrate the invention.
Example 1.
Sheep were immunised with carcinoembryonic antigen
(CEA) in complete Freund's adjuvant, then boosted three
times with antigen in incomplete Freund's adjuvant.
Animals were sacrificed after the final boost and lymph
nodes removed.
The lymph nade cells were then washed and fused with
sheep heteromye:loma fusion partner SFP3.2. Fused cells
were plated out at a total density of approximately 106 per
ml in medium containing HAT (Life Technologies). These
samples were them screened for hybridomas secreting high-
affinity antibodies to the specified antigen using both a
normal EIA and an acid-washed EIA.
Standard EIA screening assays were carried out as
follows:
Maxisorb assay plates (NUNC) were coated with CEA (0.4
~g/ml in phosphate-buffered saline at pH 7.2), 100~C1 per
well and left overnight at 4°C. The plates were then
washed three times using phosphate buffered saline at pH
7.2 with 0.01% Tween 20 detergent. Any remaining reactive
sites on the plates were blocked by the addition of 200,1
per well of 0.2% fat-free milk protein in PBS at pH 7.2 at
37°C for 2 haur. The plates were then washed in PBS as
described above amd 45,1 of the antibody samples were added
to the wells of the plates. The samples were incubated for
one hour at 37°C and then washed as described previously.
Bound antibody was detected using alkaline phosphatase-
conjugated donkey anti-sheep antibody (Sigma A5187 diluted
1/5000 in PBS at pH 7.2 with 1% BSA) . The plates were then
washed and 1001 per well of PNPP (Sigma N2770) solution
was added. Absorbance was measured using a
spectrophotometer at 405nm with phosphate buffered saline
as a control.
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Acid-wash EIA screening assays were carried out as
follows:
Coating and binding of antibody samples was as
described for 'the standard EIA above. However, after
5 incubation with the antibody samples, the plates were
washed and 200u:1 per well of HC1 (lOmM Stock solution) at
pH 2 was added for one hour at 37°C. After three washes
the antibody remaining bound to antigen was detected using
alkaline phosphatase-conjugated donkey anti-sheep antibody
and PNPP as described above. In order to ensure that a
proper comparison was being made between antibodies at
different concentrations, each sample was chosen to give an
AqpS value of app>roximately 1.0 in the normal EIA (i.e. in
the linear response part of the EIA curve).
Three hybridomas (1D2, 6611 and 6H9) secreted
antibodies which gave a greater than 50% retention of
binding in the acid washed EIA, in comparison to the
binding in the non-acid washed EIA.
Examgle 2
A single-clhain Fv fragment was produced from the
hybridoma 6H9 above, as follows:
mRNA was purified from the cultured hybridoma cells
using oligo-dZ' cellulose. Single-stranded DNA
complementary to the mRNA (cDNA) was synthesized by reverse
transcription. Universal primers, designed from the
constant regions. of sheep heavy and light chain antibody
genes, were u.ced in separate reverse transcription
reactions to synthesise the cDNA for the antibody variable
regions.
The cDNA wa.s then amplified by the polymerase chain
reaction to make double-stranded DNA using primers designed
from the heavy and light chain variable framework
sequences. Separate polymerase chain reactions were used
to amplify the heavy and light chain regions. The products
were then analysed by agarose gel electrophoresis and the
DNA bands equivalent to light and heavy chain genes were
cut from the gel and purified.
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Equimolar amounts of variable heavy and Light chain
DNA were mixed together with an oligonucleotide linker DNA.
The linker DNA coded for the amino acid sequence (Gly,Ser) 3
with additional nucleotides complementary to the 3' end of
the heavy chain 'variable region and the 5' end of the light
chain variable region. The three DNA molecules were
denatured, annealed and extended in the first stage
(without primers) of a two-stage PCR reaction so that the
fragments were joined, thereby assembling the single-chain
Fv.
The single-chain Fv DNA was amplified in the second
stage of the PCR using a pair of primers derived from the
heavy and light chain variable region termini with the
addition of the restriction enzyme recognition sites for
A1W44i and NotI., The single-chain Fv gene product was
analysed by agarose gel electrophoresis and purified. The
single-chain Fv was then digested with the restriction
enzymes AIW44i and Notl and cloned into an expression
vector. The vector was then used to transform E. coli HB
2151, and protein expression was allowed to occur. The
vector was designed so as to include a hexa-histidine tag
at the COOH terminus of the SFv. The single-chain Fv was
purified using nickel-chelate affinity chromatography and
analysed by SDS-PAGE. The amino acid sequence for the
heavy chain variable region and the light chain variable
region is disclosed in SEQ ID Nos. 2 and 4, respectively.
An acid-wash EI1~, was also carried out to determine the
acid-resistant properties of the single-chain Fv.
Acid-wash EI:A was carried out as follows:
Carcinoembryonic antigen (CEA)-coated microtitre
plates were prepa:red as described previously. Single-chain
Fv samples (6H9) were diluted to a range of concentrations
between lng/mI and 100ng/ml in PBS at pH 7.2 containing ix
bovine serum albumin (BSA). 1001 samples were added to
the mi.crotitre plate wells and incubated for 1 hour at
37°C. The platea were then washed, 200~C1 per well of
citrate added, and the plates incubated for 1 hour at 37°C.
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In this case, t:he acid preparations were made using a stock
solution of 100mM citrate diluted to pH values of. 4.0, 3.5,
3.0, 2.5 and 2.0 in the reaction mixture. PBS at pH 7.2
was used as a reference control. The plates were then
5 washed and 100u1 per well of mouse anti-tetra-histidine
antibody (Qiage:n) (100ng/ml diluted in PBS at pH 7.2 with
1% BSA) added and incubated for 1 hour at 37°C. After
plate washing t:he samples were incubated for 1 hour at 37°C
with 1001 per well of goat anti-mouse alkaline phosphatase
10 conjugate (Sigma A3688 diluted 1/1000 in PBS with 1% BSA at
pH 7 . 2 ) . The plates were then washed, treated with PNPP as
described previausly and the absorbance measured using a
spectrophotometer at 405nm.
As a control for acid resistance, sFv samples were
15 incubated with PBS at pH 7.2 to generate an EIA response
curve for the SFv samples. In the linear region, a
concentration of 10-20ng/ml of the SFv sample gave an
absorbance (A,oS,) of 1.0-1.5 and was therefore used to
determine the amount of antibody bound in the acid washed
20 samples as a percentage of the amount bound in the
reference sample.
The acid-resistant properties of the 6H9 whole
antibody and th,e 6H9 single-chain Fv were compared with
that for the mouse-derived anti-carcinoembryonic antigen
25 whole antibody, A5B7 and the single-chain Fv MFE. The
results are shown in Figure 1, with the antigen-binding of
the mouse-derived antibodies being substantially reduced at
pH 3.5 and less than 5% at pH 2.5. In contrast, the 6H9
antibodies retain >70% antigen at pH 3.5, >60% at pH 2.5
30 and >50% at pH 2Ø
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SEQUENCE LISTING
<110> KS Biomedix Ltd
<120> ANTIBODIES
<130> rep05827wo
<140>
<141>
<160> 4
<170> PatentIn Ver. 2.1
<210> 1
<211> 363
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Antibody
Fragment
<220>
<221> CDS
<222> (1)..(363)
<400> 1
cag gtg cag ctg cag gag tcg gga ccc agc ctg gtg aag ccc tca cag 48
Gln Val Gln Leu Gln Gl.u Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
acc ctc tcc ctc acc tgc a cg gtc tct gga ttc tca tta acc aag tat 96
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Lys Tyr
20 25 30
ggt gtt agt tgg gtc cgc cag get cca gga aag gcg ctt gag tgg cta 144
Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu
3.'i 40 45
ggt ggt gtg tcc agt ggt coca cta aca gcc tat aac aca gcc cta cag 192
Gly Gly Val. Ser Ser Gly Ala Leu Thr Ala Tyr Asn Thr Ala Leu Gln
50 55 60
tcc cga ctc agc gtc acc agg gac acc tcc aag agc caa ttc tcc ctg 240
Ser Arg Leu Ser Val Thr Arg Asp Thr Ser Lys Ser Gln Phe Ser Leu
65 70 75 80
1
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tca ctg agc agc gtg act act gag gac acg gcc att tac tac tgt gcg 288
Ser Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
aaa tct gtc aat ggt gac agt gtt cct tat ggt ttg gac tac tgg agc 336
Lys Ser Val Asn Gly Asp Ser Val Pro Tyr Gly Leu Asp Tyr Trp Ser
100 i05 110
cca gga ctc cta ctc acc gtc tcc tca 363
Pro Gly Leu Leu Leu Thr Val Ser Ser
115 120
<210> 2
<211> 121
<212> PRT
<213> Artificial Sequence
<223> Description of Artificial 5equence:Antibody
Fragment
<900> 2
Gln Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cy;s Thr Val Ser Gly Phe Ser Leu Thr Lys Tyr
20 25 30
Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Gly Gly Val Ser Ser Gly Ala Leu Tht Ala Tyr Asn Thr Ala Leu Gln
50 55 60
Ser Arg Leu Ser Val Thr Arg Asp Thr Ser Lys Ser Gln Phe Ser Leu
65 70 75 80
Ser Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys Ser Val Asn Gly Asp Ser Val Pro Tyr Gly Leu Asp Tyr Trp Ser
100 105 110
Pro Gly Leu Leu Leu Thi: Val Ser Ser
11.5 120
2
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WO 00/12556 PCT/GB99/02729
<210> 3
<211> 333
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Antibody
Fragment
<220>
<221> CDS
<222> (1)..(333)
<400> 3
cag gat gtg ctg act cag ccg tcc tcc gtg tct ggg tcc ctg ggc cag 48
Gln Asp Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln
1 5 10 15
agg gtc tcc atc acc tgc tct gga agc agc agc aac att gga ggt aat 96
Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn
20 25 30
get tat gtg ggc tgg t;ac caa cag gtc cca gga tca gcc ccc aga ctc 144
Ala Tyr Val Gly Trp T:yr Gln Gln Val Pro Gly Ser Ala Pro Arg Leu
35 40 45
ctc atc agt get aca acc gat cga gcc tcg ggg atc ccc gac cga ttc 192
Leu Ile Ser Ala Thr Thr Asp Arg Ala Ser Gly Ile Pro Asp Arg Phe
50 55 60
tcc ggc tcc agg tct ggg aac aca gcc acc ctg acc atc agc tcg ctc 240
Ser Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu
65 '10 75 80
cag get gag gac gag gc:c gat tat tac tgt gca tcg tat caa agt act 288
Gln Ala Glu Asp Glu Al.a Asp Tyr Tyr Cys Ala Ser Tyr Gln Ser Thr
85 90 95
tac agt ggt gtt ttc gc~c agc ggg acc agg ctg acc gtc ctg ggt 333
Tyr Ser Gly Val Phe Gl.y Ser Gly Thr Arg Leu Thr Val Leu Gly
100 105 110
<210> 4
<211> 111
<212> PRT
<213> Artificial Sequence
<223> Description of Artificial Sequence: Antibody
3
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WO 00/12556 PCT/GB99/02729
Fragment
<400> 9
Gln Asp Val Leu Thr Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln
1 5 10 15
Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn
20 25 30
Ala Tyr Val Gly Trp Tyr Gln Gln Val Pro Gly Ser Ala Pro Arg Leu
35 40 95
Leu Ile Ser Ala Thr T:hr Asp Arg Ala Ser Gly Ile Pro Asp Arg Phe
50 55 60
Ser Gly Ser Arg Ser G.ly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu
65 70 75 80
Gln Ala Glu Asp Glu A.La Asp Tyr Tyr Cys Ala Ser Tyr Gln Ser Thr
85 90 95
Tyr Ser Gly Val Phe G:Ly Ser Gly Thr Arg Leu Thr Val Leu Gly
100 105 110
9
