Note: Descriptions are shown in the official language in which they were submitted.
WO94/11403 2 1 4 ~ O l 3 PCT/SE93/00960
... `. '~:,~
1,
ARTIFICIAL ANTIBODIES, METHOD OF PRODUCING THE SAME AND
USE THEREOF
The present invention concerns artificial antibodies,
a method for producing the artificial antibodies, a method
for determination of an organic molecule in a fluid
sample, a method for separation or isolation of an organic
molecule and use of the latter methods in immunoassays as
well as a method of therapy or diagnostics.
Antibodies are used in several areas, such as thera-
py, immunoaffinity, purification and in particular in
immunoassays. As to the latter aspect the corresponding
antigens can either be small or large molecules.
Antibodies are normally produced by immunising ani-
15 mals with the corresponding antigen leading to polyclonal
antibodies, or by using fused cells (B cells) allowing the
obtained c~ll lines to produce monoclonal antibodies.
Recent efforts in obtaining other biologically deriv-
ed antibodies or at least antibody-like compounds involve
20 recombinant techniques applied to bacteria or plants.
Antibodies can be raised against most compounds; they
are versatile reagents employed in numerous applica-
- tionsl 5, ranging from basic research to clinical analy-
sis. However, being bio-macromolecules they re~uire care-
25 ful handling and their production is costly5.
A potentially useful alternative would be the produc-
tion of non-biologically derived antibody mimics or arti-
ficial antibodies, such as polymer structures that are
similar to biological antibodies in binding and recogni-
30 sing antigens. t
The inherent advantages of such systems would be that
the need for animal sources is obliviated, and that anti-
body mimics ran be obtained for cases where it is diffi-
cult or impossible to raise antibodies, as for immuno
35 suppressive agents, such as cyclosporin, certain structu-
res, such as macrolides or short peptides.
W O 94~11403 PC~r/SE93/00960 ~
21~9b 13
Furthermore, such non-biological systems could be
made more stable, allowing repeated use, higher temperatu-
res and easy sterilisation. ~
In addition the need for derivatisation of antigens
for immunisation purposes is made unnecessary, thereby
avoiding the often complicated chemistry and sometimes
decreased recognition for the original target molecule (=
antigen).
Since the development of the first radioimmunoassayl,
immunological techniques using labelled reactants have
gained an extraordinary prominence in the field of medical
research and in clinical diagnosis. In particular, the
discovery of monoclonal antibodies and their use in
immunoassays has offered novel advantages and more possi-
bilities. Despite the plethora of markers and differentprocedures3'4 that have been employed, all the immunologi-
cal techniques exploit the remarkable affinity and speci-
ficity of antibodies. However, antibodies are labile bio-
molecules which require careful handling and storage.
Their production is a time-consuming procedure5, including
several laborious steps like conjugation of the hapten to
a carrier protein, immunisation of animals and isolation
of immunoglobulins.
Thus, there was a need for an immunoassay-like tech-
nique in which stable and easily prepared highly selectivepol~mers, rather than antibodies are used.
The technique of molecular imprinting has attracted
much attention in the last few years . Recently, molecu-
lar imprinting has been developed to a stage of practical
application in enantiomeric separationsll 15, in particu-
lar in the resolution of racemic drugs such as . ~`-
~-blockersl ~ .
Furthermore, the technique has been applied to make ~ ~
synthetic enzymes9'10.
The technique of molecular imprinting and its special
form of non-covalent imprinting as developed by the inven-
tors makes it possible to achieve the above objects.
w o 94/11403 21 ~ 9 o 4 3 Pcr/sE93/00960
Briefly, the technique involves polymerisation of
functional monomers in the presence of a print molecule
(see Scheme 1). Subsequent removal of the print molecule
from the rigid polymer results in sites within the polymer
that are complementary to and have an affinity for the
original print molecule.
According to the invention there are provided artifi-
cial antibodies, which consist of polymers that carry
specific binding sites mimicking the properties of anti-
bodies.
There is also provided, according to another aspectof the invention, a method for producing artificial anti-
bodies, in which polymerisable monomers carrying functio-
nal groups and crosslinking monomers are polymerised in
the prese~ce of a print molecule and subsequently the
print molecule is removed leaving specific binding sites
complementary to the print molecule.
The invention also provides for a method for determi-
nation of an organic molecule in a fluid sample. According
to this method, a known amount of the organic molecule
provided with a label is added to the sample, the sample
is contacted with artificial antibodies having specific
binding sites for the organic molecule, whereby the
labelled and unlabelled organic molecules are competi-
tively bound to the binding sites, and the labelled orga-
nic molecule is determined either unbound in the superna-
tant or bound by the polymer.
There is also provi~ed a method for separation or
isolation of an organic molecule from a fluid sample, in
which the sample, labelled or not, is contacted with an
excess of artificial antibodies consisting of a polymer
ha~ing specific sites for the organic molecule, whereby
the organic molecule is bound to the binding sites, and
optionally the organic molecule is measured bound to the
artificial antibodies or eluted from the antibodies.
WO94/11403 PCT/SE93/00960 ~;
2 I g 9 0 9 3 r
4 `
The invention also provides fo a method of therapy or
diagnosis, in which artificial antibodies are administra-
ted to a mammal body, which artificial antibodies consist
of a biocompatible polymer carrying specific binding sites
S mimicking the properties of antibodies towards an organic
molecule.
In one embodiment of the invention, the polymers are
prepared by non-covalent polymerisation.
The polymers constituting the artificial antibodies
are preferably built up of polymerisable monomers carrying
functional groups and crosslinking monomers. Preferably
the polymerisable monomers carrying functional groups are
chosen among negatively charged monomers such as methacry-
lic acid, itaconic acid, basic monomers such as vinylpyri-
dine, vinylimidazole, hydrophobic monomers carrying alkylchains, monomers allowing ~ interactions, van der Waals
forces.
In one embodiment of the invention, ~olymers are
built up of methacrylic acid crosslinked by ethylene
glycol dimethacrylate.
If the artificial antibodies are to be used for
administration to a mammal body the polymers must be bio-
compatible. Preferably they must be of the size not more
than 5 ,um or the size of normal biological antibodies,
most preferred lO-lO0 nm.
In preparation of artificial antibodies according to
the invention, the polymer is ground to a particle size of
normally ~ 25 ~m $or use in so-called heterogenous assays.
The fines, that is particles with a size of lO-lO0 or
lO00 nm, resulting from the grinding, can be kept in solu-
tion or suspension and used for instance in so-called t:`
homogenous immunoassays. Such assays are extremely sensi-
tive and can be performed involving e.g. two different
antibodies.
Another advantage with the fine particles is that
they are more suitable for use in therapy or diagnostics.
W O 94/11403 2 14 ~ O 1 3 PC-r/SE93/00960
Preferably the binding sites are specific for a com-
pound chosen from the group consisting of drugs, rnetabo-
lites, nucleotides, nucleic acids, carbohydrates, pfo-
teins, hormones, toxins, steroids, prostaglandins and
leukotrienes.
In one embodiment the binding sites are specific for
theofylline or diazepam.
Suitable labels for use in the methods according to
the invention are radioligands, enzymes, biotin, steroids,
fluorochromes, gold.
The methods according to the invention are preferably
used in immunoassays, especially in radioimmunoassays.
The method of therapy or diagnosis according to the
invention comprises several different modes of action. For
example, it can be used to withdraw an undesired organic
molecule from a mammal body, such as a toxin. In another
embodiment the artificial antibodies assemble around a
cancer cell to indicate the presence of such a cell. In a
further embodiment the artificial antibodies are bringing
a drug to specific targets, for instance cancer cells.
In one embodiment of treating a mammal body an extra
corporal device containing the artificial antibodies is
coupled to the body via a shunt in the bloodstream, and
the bloodstream is passed through the device.
For the studies the inventors chose two chemically
unrelated drugs, theophylline and diazepam, as print
molecules. Theophylline, a commonly used drug in the
prevention and treatment of asthma r apnea and obstructive
lung diseases, has a narrow therapeutic index (56-112 ~mol
30 L serum) requiring careful monitoring of serum concent- j
rations17. Diazepam te.g. valium) is a member of the 3
benzodiazepine group of drugs widely used as hypnotics,
tranquilizers and muscle relaxants . Benzodiazepines are
one of the most commonly implicated substances in drug
35 overdose situations and their detection in body fluids is
very useful in clinical and forensic toxicology. Current
methods for measuring theophylline and benzodiazepines are
WO94/11403 PCT/SE93/00960
~14!~n i3
based on high-performance liquid chromatography
(HPLC) 9 and on immunologic~l techniques 6
The polymers were prepared using methacrylic acid
(MAA) as the functional monomer and ethylene glycol di-
methacrylate (EDMA) as the crosslinking monomer ~Scheme1). This is a well characterised polymer system that has
been used for the preparation of molecular imprints
against a number of compounds 12-14~16 Th
acid function of MAA has been shown to form ionic inter-
actions with amino groups12 and hydrogen bonds with polarfunctionalities of the print molecule . The inventors
assume that hydrogen bonding is the predominant type of
force operating during imprinting and subsequent recog-
nition in the present system. Dipole-dipole and hydro-
phobic interactions may also contribute.
The solvent compositions giving optimal binding andselectivity were determined for each polymer (see Example
2 and Fig. 1 below). As a general guidel4'27: i) in a more
apolar solvent the substrate binds more strongly to the
polymer than in polar solvents, and ii) small amounts of
acetic acid can be added to the solvent in order to
supress non-specific binding. The eqilibrium dissociation
constants (KD) for binding of the drugs to the correspon-
ding polymers were estimated by Scatchard plot analysis
using radio-labelled ligands. In both cases, the Scatchard
plots were nonlinear and fitted well with two KD values,
for high and low affinity binding sites. The inventors
believe that, as~in the case of polyclonal antibodies, the
polymers contain a heterogenous population of sites with
different affinities for the print molecule. The KD values
for the high and low affinity binding sites, calculated
with the LIGAND programme (Elsevier-Biosoft), were
3.46xlO 7 M and 6.55xlO M (associated with a population
of sites of 0.016 ~mol g and 1.28 ~mol g , respective-
ly) for theophylline and 3.76xlO 8 M and 7.36xlO 8 M(0.0071 ~mol g 1 and 0.51 ~mol g ) for diazepam.
W094/11403 2 1 4 ~ ~ 4 3 PCT~SE93/00960 ~ `
: . ' ' ' ' ,-
Polymers prepared against theophylline or diazepam
were used as antibody-substitut~s in the construction of
competitive binding for theophylline and diazepam de~ermi- ,
nation in human serum. The method, which we name Molecu- ,r
larly Imprinted Sorbent Assay (MIA), relies on the inhi-
bition of binding of radio-labelled ligand by the serum
analyte. The amount of radioligand bound to the polymer is
inversely related to the concentration of drugs present in
the sample. Dru~ free serum samples spiked with known
amounts of theophylline or diazepam were used for estab-
lishing the standard calibration curves. Prior to the
actual assay, the drug was extracted from the serum by
standard protocols used for HPLC analysis 9 21 (Fig. 1).
The MIA for theophylline was linear over the range
14-224 ~mol L 1 which is satisfactory for therapeutic
monitoring of the drug. The results for diazepam were
linear over the range which is normally used in standard
immunoassay techniques for benzodiazepines
(0.44-28 ,umol L 1),
The specificity of the method was tested by the
determination of cross-reactivity of major metabolites and
of drugs structurally related to theophylline or diazepam
~Table 1).
j .
WO 94/11403 PCr/SE93/00960
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C ~, C ~: ~ to~
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~_O ~ O ~ ~ Y ~ ~ ~ O N
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W094/11403 PCT/SE93~00960
- 2~ 0~3
The MIA method for theophylline (l,3-dimethylxanthine)
appears to be highly specific sjnce from all the compounds
tested only 3-methylxanthine showed some cross-reactivity.
In the case of the diazepam assay several other
benzodiazeplnes showed significant cross-reactivity~ This
was, however, expected because benzodiazepines are very
similar in structure, as seen below:
R R,
R ,~
Rl R2 R3 R4 R5
15 ~iazepam Cl Me O H H
Desmethyldiazepam Cl H O H H
Clonazepam NO2 H O H Cl
Lorazepam Cl H O OH Cl
I I .
Alprazolam Cl / ~ N H H
N/
and even antibodies have difficulty in distinguishing bet-
ween them25'26 (Table l)
The ability of the MIA method for accu~ate measure-
ment of theophylline was evaluated by analysing 32 patient
30 serum samples. The sample were also analysed with the ~`
Enzyme-Multiplied Immunoassay Technique (EMIT)28 and the
comparison of the results obtained showed excellent corre-
lation between the two methods (Fig. l). Furthermore, the
reliability of the assay was determined by measurement of
theophylline samples of known concentration (three clini-
cal significant concentrations; eleven repetitions;
coefficient of variation 5 6.5~).
SUE~STITUTE SHEET
WO 94/11403 : i ( P~/SE93/00960 ~r
2 1 4 9 0 1 3 .~
The results presented here demonstrate, for the first
time, the ability to use chemically prepared macromole-
cules with preselected specificity, instead of the tradi~
tional biomolecules, as receptors in competitive binding
assays. A grea~ advantage of molecularly imprinted poly-
mers is their simple and rapid (two to three days) prepa-
ration and their remarkable stability. They can be stored
in the dry state, even at elevated temperatures, for seve-
ral years without loss of recognition capabilities . In
addition, the potential to reuse the polymers may prove
valuable. Furthermore, by analogy to immunoaffinity chro-
matography, molecularly imprinted polymers could be useful
for the separation and isolation of different compounds.
Apart from the practical importance of the describecl pre-
parations, structural studies on the interactions of drugswith their artificial receptors could yield valuable in-
sight into the nature of molecular recognition pheno-
29-31
mena
Molecular imprints may be obtained against functiona-
lity complementary to the monomer14'27. There is a poten-
tial for molecularly imprinted artificial antibodies in
the analysis of many other drugs, metabolites, hormones,
toxins, etc.
It is also noteworthy that molecularly imprinted
polymers provide a potential alternative to the use of
laboratory animals for the production of antibodies.
Preliminary data from similar studies with an emphasis on
recognition in aqueous systems using other compounds such
as opiates and biologically active peptides, indicate that
this technique promises to become widely useful.
The invention is described more in detail with refe~
rence to the following examples and the accompanying
drawing.
Figure 1 shows a comparision of the competitive bind-
ing assays Enzyme-Multiplied Immunoassay Technique
(EMIT) and MIA for determination of serum concentration
av theophylline in patient samples (n=32).
WO~4~1~403 2 1 4 9 0 1 3 P~T/SE93/00960
11
Example 1
Preparation of molecularly imprinted polymers
The preparation follows the reaction of Scheme~
A) The functional monomer, methacrylic acid (MAA,l), is ,-
mixed with the print molecule, here theophylline (2), and
ethylene glycol dimethacrylate (EDMA), the crosslinking
monomer, in a suitable solvent. MAA is selected for its
ability to form hydrogen bonds with a variety of chemical
functionalities of the print molecule.
B) The polymerisation reaction is started with the addi-
tion of initiator (AIBN) and a rigid insoluble polymer is
formed. "Imprints", which are complementary in both shape
and chemical functionality to the print molecule, are now
present within the polymeric network.
C) The print molecule is removed by extraction.
The wavy lines in Scheme 1 represent an idealised
polymer structure but do not take into account the
acressibility of the substrate to the recognition site in
the macroporous polymer structure.
METHODS
Anti-theophylline polymer
To a glass bottle were added chloroform (250 ml),
theophylline (4.7 g), MAA (9 g), EDMA (93,5 g) and 2,2'-
-azobis(2-methylpropionitrile) (AIBN, initiator, 1.2 g).
The mixture was degassed under vacuum in a sonicating
waterbath and sparged with nitrogen for 5 min. The poly-
merisation reaction took place at 60C for 24 h. The bulk
polymer;was grounded in a mechanical mortar and wet sieved
(water) through a 25 ,um sieve. The fines were removediby
repeated settling in acetonitrile. The print molecule
(theophylline) was extracted by extensive washing of the ~:-
particles with methanol-acetic acid (9/1, v/v). Finally,
the polymer particles were dried under vacuum and stored
in a desiccator.
W O 94/11403 PC~r/SE93/00960
2143043 ~'"''~' ~''''`'"
Anti-diazepam polymer
Diazepam (1.27 g) was mlxed with MAA (2.26 g), EDMA
(26.1 g) and AIBN (0.5 g) in chloroform (39 ml). The poly- ~
merisation mixture was degassed under vacuum in a sonica- -
ting water-bath, sparged with nitrogen and then polymeri~
sed under W (366 nm) at 4C for 16 h. The resulting poly-
mer was then treated as described above.
Example 2
A comparison of the competitive binding assays
Enzyme-Multiplied Immunoassay Technique (EMIT) and MIA
for determination of serum concentration of theophylline
in patient samples (n=32) was performed. EMIT reagents
were supplied by the manufacturer (SYVA, Palo Alto, USA).
All enzyme immunoassays were preformed at the department
of Clinical Pharmacology, University Hospital, Lund,
Sweden, according to the method of the manufacturer. The
result is shown in Fig. 1:
Slope: 0.99, Intercept: 1.50 ~mol L , correlation
coefficient: 0.98.
METHCDS
The assay conditions were established by applying
similar protocols as is standard for the optimisation of
immunoassays using antibodies3 . 40 ~l of each sample was
mixed with 40 ~1 of HCt (0.2 M) and extracted with l ml of
dichloromethaneisopropanol (4/1, v/v). The organic layer
was evaporated at 40C under a stream of nitrogen. The
residue was redissolved in 100 ,ul of acetonitrile-acetic
acid (99/1, v/v) containing [ H]-theophylline (5 ng,
18.6 Ci mmol 1). Polymer imprinted against theophylline
3Q was ~hen added (12.5 mg of polymer in 0.9 ml of the same
solvent) and the mixture was incubated for 15 h at room
temperature. The binding equilibrium was reached after
8 h, 80 and 90~ of the binding occurred within 3 and 5 h.
After centrifugation, the unbound [3H]-theophylline in
200 ~l of the supernatant was measured by liquid scintil-
lation counting. The calibration graph was linear over the
range 14-224 ~mol L (correlation coefficient = 0.999)
WO94/11403 21 4 9 0 4 3 PCT/SE93/00960 ~-
--: Ç
13
and the detection limit of the assay was found to be
3.5 ~mol L l The diazepam assav, performed in a similar
i manner using 5 mg of polymer in toluene-heptane (4~
v/v)~ was linear from 0.44 to 28 ~mol L 1 (correlation
5 coefficient = 0,~gl) with a detection limit of
0.2 ~mol L
~ .
~, ..
WO94J11403 PCT/SE93/0~960 ~
21~90~3 I''-''';'': Ui~:
14
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WO 94/11403 PCI'/SEg3/00960
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