U.S. patent application number 12/041939 was filed with the patent office on 2008-06-26 for epitopes formed by non-covalent association of conjugates.
This patent application is currently assigned to Mozaic Discovery Limited. Invention is credited to Roger NEW, Istvan TOTH.
Application Number | 20080152703 12/041939 |
Document ID | / |
Family ID | 10856190 |
Filed Date | 2008-06-26 |
United States Patent
Application |
20080152703 |
Kind Code |
A1 |
NEW; Roger ; et al. |
June 26, 2008 |
EPITOPES FORMED BY NON-COVALENT ASSOCIATION OF CONJUGATES
Abstract
A method for producing and using in treatment a composition for
interacting with a ligand, which composition comprises a
non-covalent association of a plurality of distinct conjugates,
each conjugate comprising a head group and a tail group, wherein
the tail groups of the conjugates form a hydrophobic aggregation
and the conjugates are movable within the association so that, in
the presence of a ligand, at least two of the head groups are
appropriately positioned to form an epitope capable of interacting
with the ligand more strongly than each of the head groups
individually.
Inventors: |
NEW; Roger; (London, GB)
; TOTH; Istvan; (Moggill, AU) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W., SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Mozaic Discovery Limited
Tortola
VG
|
Family ID: |
10856190 |
Appl. No.: |
12/041939 |
Filed: |
March 4, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10019052 |
Apr 22, 2002 |
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PCT/GB00/02465 |
Jun 27, 2000 |
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12041939 |
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Current U.S.
Class: |
424/450 |
Current CPC
Class: |
C07K 5/1013 20130101;
G01N 33/5432 20130101; A61K 9/1271 20130101; C07K 5/0606 20130101;
G01N 33/5041 20130101; C07K 1/1077 20130101; G01N 2333/525
20130101; G01N 33/5008 20130101; C07K 7/06 20130101 |
Class at
Publication: |
424/450 |
International
Class: |
A61K 9/127 20060101
A61K009/127 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 28, 1999 |
GB |
99150740 |
Claims
1. A method for producing a composition for interacting with a
ligand, which method comprises: (a) providing a plurality of
distinct conjugates, each conjugate comprising a head group and a
tail group; and (b) forming from the plurality of conjugates a
non-covalent association thereof, in which the tail groups
aggregate hydrophobically and in which the conjugates are movable
so that, in the presence of a ligand, at least two of the head
groups are appropriately positioned to form an epitope capable of
interacting with the ligand more strongly than each of head groups
individually.
2. The method of claim 1, wherein the head group is selected from
the group consisting of an amino acid, a peptide, a peptide
analogue, a monosaccharide, a polysaccharide, a mononucleotide, a
polynucleotide, a sterol, a water-soluble vitamin, a porphyrin
nucleus, a haem nucleus, a metal ion chelate, a water-soluble drug,
a hormone, and an enzyme substrate.
3. The method of claim 2, wherein the head group comprises an amino
acid.
4. The method of claim 3, wherein the head group comprises a
peptide.
5. The method of claim 3 or claim 4, wherein the amino acid
comprises a terminal amino acid selected from the group consisting
of hydrophilic amino acids, hydroxylic amino acids, acidic amino
acids, amide amino acids, basic amino acids, and aromatic amino
acids.
6. The method of claim 1, wherein the tail group comprises a
lipophilic group selected from the group consisting of a straight
chain fatty acid, a branched-chain fatty acid, an alcohol having at
least 8 carbon atoms, an aldehyde having at least 8 carbon atoms, a
lipidic amino acid analogue, a prostaglandin, a leukotriene, a
monoglyceride, a diglyceride, a sterol, a sphingosine derivative, a
ceramide derivative, a silicon-substituted derivative thereof, and
a halogen-substituted derivative thereof.
7. The method of claim 6, wherein the lipophilic group comprises a
C.sub.10 to C.sub.14 fatty acid.
8. The method of claim 1, wherein the conjugate further comprises a
spacer group linking the head group to the tail group.
9. The method of claim 8, wherein the spacer group is
hydrophilic.
10. The method of claim 9, wherein the spacer group comprises an
amino acid, a hydroxy acid, a sugar or a polyethylene glycol.
11. The method of claim 1, wherein the non-covalent association
comprises a lamellar structure, a micelle or a liposome.
12. The method of claim 1, wherein the step of providing the
plurality of conjugates comprises: (a) selecting a set of
conjugates with an array of head groups; (b) forming a non-covalent
association therefrom, in which the tail groups aggregate
hydrophobically and in which the conjugates are movable; (c)
assaying for sufficient interaction between the non-covalent
association and the ligand; (d) optionally repeating steps (a) to
(c) using a set of conjugates with a modified array of head groups;
and (e) on finding sufficient interaction in step (c) selecting the
set of conjugates as the plurality of conjugates in step (a).
13. The method of claim 12, wherein the array of head groups
comprises (a) at least one terminal amino acid from each of the
following classes of amino acid: hydrophobic amino acids,
hydroxylic amino acids, acidic amino acids and amide amino acids;
and (b) at least two further terminal amino acids comprising at
least one basic amino acid and at least one aromatic amino acid, or
at least two basic amino acids or aromatic amino acids.
14. The method of claim 13, wherein the modified array of head
groups used in step (d) comprises the array of head groups used in
steps (a) to (c) in which the at least two further terminal amino
acids are different from those used in steps (a) to (c).
15. The method of claim 12, wherein the array of head groups
comprises at least one terminal amino acid from each of the
following classes of amino acid: hydrophobic amino acids,
hydroxylic amino acids, acidic amino acids, amide amino acids,
basic amino acids and aromatic amino acids.
16. The method of claim 15, wherein the modified array of head
groups used in step (d) comprises the array of head groups used in
steps (a) to (c) in which the at least one terminal amino acid from
one of the classes of amino acid is either absent or replaced by a
charged version thereof.
17. A method for producing a molecule for interacting with a
ligand, comprising: (a) producing a composition according to the
method of claim 1; (b) identifying the at least two head group
which form an epitope for the ligand in the composition; and (c)
producing a molecule incorporating the functional groups of the at
least two head groups optionally spaced apart by one or more linker
groups so that the molecule is capable of interacting with the
ligand more strongly than each of the head groups individually.
18. A method of treating a disease comprising administering to a
patient isolated micelles which comprise a plurality of conjugate
molecules non-covalently associating with one another to form the
micelles, each conjugate molecule comprising (1) a head group
molecule conjugated to a hydrophobic tail group molecule,
optionally via a spacer molecule, (2) a surface formed by the head
group molecules, which surface comprises a plurality of distinct
non-covalent associations of the head group molecules, and (3) a
hydrophobic core formed by the hydrophobic tail group molecules;
wherein the head group molecules in the non-covalent associations
change configuration through the movement of the head group
molecules on or along the surface, and the movement of the head
group molecules is facilitated by the movement of the conjugate
molecules in the micelle, and wherein a distinct non-covalent
association of the head group molecules forms an epitope which has
higher affinity to a ligand than each of the head groups of the
conjugates individually does.
19. The method of claim 18 wherein the head group of the micelles
is selected from the group consisting of an amino acid, a peptide,
a peptide analogue, a monosaccharide, a polysaccharide, a
mononucleotide, a polynucleotide, a sterol, a water-soluble
vitamin, a porphyrin nucleus, a haem nucleus, a metal ion chelate,
a water-soluble drug, a hormone, and an enzyme substrate.
20. The method of claim 19 wherein the head group comprises an
amino acid.
21. The method of claim 20, wherein the head group comprises a
peptide.
22. The method of claim 20 or of claim 21, wherein the amino acid
comprises a terminal amino acid selected from the group consisting
of hydrophilic amino acids, hydroxylic amino acids, acidic amino
acids, amide amino acids, basic amino acids, and aromatic amino
acids.
23. The method of claim 18, wherein the tail group comprises a
lipophilic group selected from the group consisting of a straight
chain fatty acid, a branched-chain fatty acid, an alcohol having at
least 8 carbon atoms, an aldehyde having at least 8 carbon atoms, a
lipidic amino acid analogue, a prostaglandin, a leukotriene, a
monoglyceride, a diglyceride, a sterol, a sphingosine derivative, a
ceramide derivative, a silicon-substituted derivative thereof, and
a halogen-substituted derivative thereof.
24. The method of claim 23, wherein the lipophilic group comprises
a C.sub.10 to C.sub.14 fatty acid.
25. The method according to claim 18, wherein the conjugate further
comprises a spacer group linking the head group to the tail
group.
26. The method according to claim 25, wherein the spacer group is
hydrophilic.
27. The method according to claim 26, wherein the spacer group
comprises an amino acid, a hydroxy acid, a sugar or a polyethylene
glycol.
Description
[0001] The present invention relates to a composition for
interacting with a ligand, a method for producing such a
composition and a method for producing a molecule based on the
composition.
BACKGROUND OF THE INVENTION
[0002] Protein receptors are known normally to bind to their target
ligands via epitopes, which constitute a small proportion of the
total protein molecule. For maximum binding or interaction, the
structure of the epitope needs to be maintained in a rigid
conformation in order to form a binding site containing all the
necessary components of the epitope in close proximity. Attempts to
produce an analogous peptide constructed solely of the amino acids
comprising the binding site often fail because these peptides do
not possess the same biological activity as the protein receptor.
This is attributed to the peptide having a different conformation
in free solution from that of the entire protein receptor. In
addition, where the binding site of a protein is constructed of
oligo-peptides from different, non-contiguous parts of a protein
chain, mixing isolated oligopeptides in free solution does not
result in reconstitution of the active binding site.
[0003] Being constrained to use such large proteins to present
binding-site epitopes gives rise to several problems in development
of new receptor-specific therapeutic strategies. One problem is
that such large proteins can readily evoke an immune response. A
second problem is that long peptide chains are susceptible to
attack by endopeptidases, such as those in the lumen of the gut.
Finally, these large proteins can be costly to manufacture, purify
and maintain in stable form.
SUMMARY OF THE INVENTION
[0004] The present invention aims to overcome the disadvantages of
the prior art.
[0005] In a first aspect, the invention provides a composition for
interacting with a ligand, which composition comprises a
non-covalent assembly of a plurality of distinct conjugates, each
conjugate comprising a head group and a tail group, wherein the
tail groups of the conjugates form a hydrophobic aggregation and
the conjugates have freedom of motion with respect to each other
within the assembly so that, in the presence of a ligand, at least
two of the head groups (which are the same or different) are
appropriately positioned to form an epitope capable of interacting
with the ligand more strongly than each of head groups
individually. The head groups are typically hydrophilic and the
tail groups typically hydrophobic, eg lipophilic, composed of
hydrocarbon chains, halophilic, constructed of fluorocarbon chains,
or silane based.
[0006] By constructing conjugates with a head group and a tail
group in accordance with the present invention, the tail groups can
associate to form a hydrophobic aggregation which is typically a
supramolecular assembly such as a micelle, a lamellar structure, a
liposome or other lipid structure, in which the conjugate are
oriented whereby the head groups are brought into close proximity
when in an aqueous phase. Because the conjugates are movable within
the assembly, the head groups are able to adopt a number of
different positions within the assembly. The head groups, which are
typically non-identical, are therefore free to move within the
assembly and, surprisingly, to interact cooperatively to induce
biological consequences which the head groups on their own are not
capable of eliciting. A further unexpected finding is that
assemblies composed of combinations of different headgroups are
capable of eliciting biological responses or participating in
binding with biological receptors while assemblies composed of
single headgroups are not capable of acting in this way.
[0007] As indicated above, these supra-molecular assemblies are
typically particulate or colloidal in nature, usually comprising
many hundreds of sub-units (the conjugates) all oriented with the
headgroups directed outwards from the centre of the particle as
shown in FIG. 1a. Each of the conjugates may change its location
within the assembly, being free to exchange places with adjacent
conjugates by a process of Brownian motion and, in so doing, may
migrate over the whole surface of the assembly. Other
manifestations of supra-molecular assemblies are cubic phases and
coated surfaces.
[0008] Each conjugate in the assembly may have a head group
selected from one chemical or biological class or a number of
different classes, such as an amino acid or peptide; a peptide
analogue; a mono-, di- or poly-saccharide; a mono-, di- or
poly-nucleotide; a sterol; an alkaloid; an isoprenoid; an inositol
derivative; a single or fused aromatic nucleus; a water-soluble
vitamin; a porphyrin or haem nucleus; a phthalocyanine; a metal ion
chelate; a water-soluble drug; a hormone; or an enzyme
substrate.
[0009] In one preferred embodiment, each head group comprises an
amino acid or oligo-peptide, which may be the terminal portion of a
peptide chain. It is desirable to keep the length of the peptide to
a minimum so as to avoid eliciting an immune response where the
composition is to be used in vivo. Accordingly, it is preferred
that the peptide is no more than six amino acids long.
[0010] The amino acids employed can be any of the natural amino
acids, substituted derivatives, analogues, and D-forms thereof.
[0011] The tail groups of the conjugates may be all the same or may
be a mixture of different tail groups, each of which preferably
comprises a hydrophobic group selected from a linear, branched,
cyclic, polycyclic, saturated or unsaturated construct, with or
without hetero-atoms included in the structure which can be
substituted or unsubstituted, for example, a lipidic amino acid
analogue; a prostaglandin; a leukotriene; a mono- or diglyceride; a
sterol; a sphingosine or ceramide derivative; and a silicon or
halogen-substituted derivative of such a hydrophobic group. The
tail group preferably has from 6 to 24 carbon atoms and more
preferably comprises from 10 to 14 carbon atoms. More than one tail
group may be present in each conjugate. For example, one or more
lipidic amino acids with hydrocarbon side chains may form part of
each conjugate, linked to one or more amino acids in the head
group.
[0012] Any chemical method may be used to link the head group to
the tail group. For example, each conjugate may further comprise a
spacer group linking the head group to the tail group so as to
facilitate presentation of the head group on the surface of the
non-covalent association. Such spacer groups are well known and
include, for example, amino acids, hydroxy acids, sugars and
polyethylene glycol.
[0013] In a further aspect, the present invention provides a
composition as defined above, for use as a medicament, a
prophylactic or a diagnostic.
[0014] An advantage of the invention is that strong specific
binding interactions can be achieved with conjugates in which the
head groups are small in comparison to conventional biological
receptors. If the head group is an oligo-peptide, for example, then
the length of the peptide chain would not normally exceed ten amino
acids and would preferably be six or less. Accordingly,
compositions according to the present invention can be made far
less immunogenic than their protein counterparts.
[0015] In accordance with this aspect of the invention, not only
can the composition of the present invention be formulated to
interact with a ligand in vitro but also the composition can be
used in vivo, optionally formulated with a suitable diluent,
excipient or carrier in accordance with a suitable delivery
route.
[0016] In a further aspect, the present invention provides use of a
conjugate comprising a head group and tail group for the
preparation of the composition as defined above.
[0017] There is further provided a method for producing a
composition for interacting with a ligand, which method comprises:
[0018] (a) providing a plurality of distinct conjugates, each
conjugate comprising a head group and a tail group; and (b) forming
from the plurality of conjugates, by noncovalent association
thereof, an assembly in which the tail groups aggregate
hydrophobically and in which the conjugates exhibit freedom of
motion relative to one another so that, in the presence of a
ligand, at least two of the head groups are appropriately
positioned to form an epitope capable of interacting with the
ligand more strongly than each of head groups individually. Each
conjugate is preferably as defined above.
[0019] The conjugates may be dispersed in aqueous phase by a
variety of known methodologies for the preparation of lipid
vesicles, including mechanical mixing, exposure to high shear
forces, sonication, solvent dispersion or codissolution with
detergents. Typically, the non-covalent supra-molecular assemblies
formed thereby will be composed of several different conjugates
mixed together. Additional lipidic materials may optionally be
added to alter surface properties, to aid in the dispersion of the
conjugates, to stabilise the non-covalently associated assembly of
conjugates, to aid in the presentation of head groups of the
conjugates, or to permit the construction of vehicles which can be
targeted by the epitopes formed upon random movement of the
conjugates and appropriate positioning of the head groups within
the assembly.
[0020] An important aspect of the method according to the present
invention involves the step of identifying the plurality of
conjugates which has the desired biological activity. In a
preferred aspect, this step comprises [0021] (i) selecting a set of
conjugates with an array of head groups; [0022] (ii) forming a
non-covalent association therefrom, in which the tail groups
aggregate hydrophobically and in which the conjugates exhibit
freedom of motion with respect to one another; [0023] (iii)
assaying for sufficient interaction between the non-covalent
association and the ligand; [0024] (iv) optionally repeating steps
(i) to (iii) using a set of conjugates with a modified array of
head groups; and [0025] (v) on finding sufficient interaction in
step (iii), selecting the set of conjugates as the plurality of
conjugates in step (a).
[0026] Examples of assays for "sufficient interaction" may include
binding assays such as those utilising the ELISA principle for
detection of association between antibody and antigen. Other
suitable in vitro assays include modification of fluorescence of
environmentally-sensitive membrane-bound fluorescent probes,
precipitation reactions, enhancement or inhibition of enzyme
activity etc. Assays relying on the ability of materials to alter
the behaviour of cells cultured in vitro may also be appropriate,
such as assays for cell death, cell proliferation, apoptosis,
inhibition or stimulation of cell-to-cell contact, secretion of
cytokines or other soluble products, synthesis of specific m-RNA,
intracellular vesicular transport, alteration of cell signalling
processes etc. In vivo assays in whole animals or humans may also
be carried out, for example incorporation of radiolabel into the
supramolecular assemblies, followed by investigation of its
subsequent distribution after administration by various routes.
[0027] According to this method a combinatorial approach is used in
which a range of different supra-molecular assemblies (or "probes")
is prepared, each containing a different combination of conjugates
selected from a pre-synthesised bank. Selection of the appropriate
conjugates may be based on known properties of the target ligand or
may simply involve the use of a very wide range of head groups to
increase the probability that two or more of the head groups will
form an epitope for the ligand. In this way, following the assay
for sufficient interaction between the probe and the ligand as
described above, the combination of conjugates found to be most
effective may be modified by adding further head groups, removing
some head groups, or both, and assaying the resultant probes once
again for sufficient interaction. Eventually, the most favourable
combination of head groups may be identified and selected for use
in the composition.
[0028] The present invention therefore has a very clear advantage
over traditional combinatorial chemistry. In combinatorial
chemistry, the identification of the most favourable sequence for
binding to a specific receptor must be carried out by synthesis of
hundreds of possible combinations of different groups such as amino
acids, in different orders, each one having to be tested for
efficacy. This process is time-consuming, expensive and is limited
by the nature of the chemistry which can be carried out in linking
the different components together. In contrast, the present
invention simply relies upon proximity of the head groups to
provide association-derived epitopes. Once a set of conjugates has
been synthesised, no further synthetic chemistry is required, only
simple mixing of the conjugates to form the different probes by
non-covalent association.
[0029] In a preferred simple embodiment, the present method uses
conjugates having a single terminal amino acid linked via a spacer
to a lipid tail group which can be combined simply by mixing in
aqueous medium to form micelles in which different amino acid side
chains would be presented together in a multiplicity of different
configurations. Accordingly, the need to present amino acids in a
specific order, or with a specific spacing or orientation, is
circumvented. On statistical grounds, a proportion of the
individual amino acid sub-units will always be associated in an
ideal configuration.
[0030] In one arrangement, each of the conjugates would have the
linear structure: X-spacer-spacer-lipid-lipid, where X represents a
single amino acid different for each of the distinct conjugates
employed.
[0031] When seeking to construct epitopes composed of natural amino
acids it is possible to simplify further the number of head groups
for selection. One can categorise the amino acid residues found in
natural proteinaceous materials into six fundamental classes
preferably using in any one class one amino acid rather than all
members of that class because of the increased spatial flexibility
of amino acids in the terminal position of the head group. This has
the effect of reducing considerably the total number of amino acids
required for constructing the pre-synthesised bank of conjugates
and thereby the total number of head groups used. The main classes
of amino acids are set out in Table 1 below.
TABLE-US-00001 TABLE 1 Class Representative Abbreviation
Hydrophobic Leucine L Hydroxylic Serine S Acidic Glutamate E Amide
Glutamine Q Basic Histidine H Aromatic Tyrosine Y
[0032] A number of strategies are available for identifying active
combinations of amino acid-containing conjugates.
[0033] In one embodiment, a restricted number of conjugates is
employed to form a range of distinct probes where each probe is an
aqueous suspension of supra-molecular assemblies, each assembly
consisting of selected conjugates mixed together, and each
differing from the other as a result of the inclusion of a
different additional conjugate as shown below where each of the
letters given represents a conjugate with a different terminal
amino acid:
TABLE-US-00002 Probe 1 A B C D Probe 2 A B C E Probe 3 A B C F
Probe 3 A B C G . . . . . . Probe x A B C Z
[0034] Each of the probes is tested separately in the biological
assays for sufficient binding as outlined above.
[0035] In a second simple embodiment, an initial probe can be
constructed which contains a large number of different conjugates
from the bank, and its efficacy compared with probes each lacking a
different conjugate in turn, to determine which headgroups in the
bank are essential, and which are redundant for the biological
interaction being investigated. This approach is illustrated
below:
TABLE-US-00003 Probe 1 A B C D E . . . Z Probe 2 A C D E . . . Z
Probe 3 A B D E . . . Z . . . Probe x A B C D E . . .
[0036] Combinations of the alternative approaches as outlined above
can be made.
[0037] A knowledge of the target ligand may assist in designing a
suitable starting array. For example, if the ligand is known to be
basic, it would make sense to impart an acidic character to the
conjugates by presenting them in the form where a free carboxyl
group of the terminal amino acid is exposed. Introducing additional
functionality by employing a particular amino acid as a spacer
group adjacent to the terminal amino acid may also confer increased
specificity. Where the involvement of, say, a short oligo-peptide
sequence of known structure has already been implicated in binding
to the target ligand, such a sequence may be incorporated into a
conjugate to be included in the set of conjugates making up the
composition.
[0038] In a final aspect, the present invention provides a method
for producing a molecule for interacting with a ligand. The method
comprises producing a composition according to one of the methods
defined above; identifying the at least two head groups which form
an epitope for the ligand in the composition; and producing a
molecule incorporating the functional groups of the at least two
head groups optionally spaced apart by one or more linker groups so
that the molecule is capable of interacting with the ligand more
strongly than each of the head groups individually.
[0039] Whilst the compositions of the present invention may
themselves be useful in in vitro or in vivo systems perhaps to
induce a biological response in a therapeutic, prophylactic or
diagnostic method, in some circumstances a molecule may be produced
based on the structure of the above compositions. By identifying
the functional groups of the at least two head groups which form
the epitope for the ligand a new molecule analogous to the
composition may be produced containing the same or a similar
epitope. The functional groups may, for example, be incorporated
into a single linear oligo-peptide possibly with one or more linker
groups to space the functional groups apart.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The invention will now be described in further detail, by
way of example only, with reference to the following Examples and
the attached drawings, in which:
[0041] FIG. 1 shows a schematic representation of the surface of a
supra-molecular assembly, and how such a composition according to
the present invention binds to a target ligand; and
[0042] FIG. 2 shows a schematic representation of the surface of a
supra-molecular assembly composed of two non-identical conjugates
whose headgroups consist of short-chain linear peptides.
DETAILED DESCRIPTION OF THE INVENTION
[0043] Referring to FIG. 1, a section 1 of a composition according
to the present invention is shown in the form of a micelle in which
the head groups 2 and tail groups 3 together form conjugates 4
(FIG. 1A). A target ligand 5 is presented to the composition 1.
Because the conjugates are movable, a rearrangement occurs (FIG.
1B) to allow positioning of the head groups 2 to bind the target
ligand 5. Referring to FIG. 2, a section of a composition according
to the present invention is shown in the form of a supramolecular
assembly, in which binding of a ligand to the surface of the
assembly is brought about by the creation of an epitope constructed
via the non-covalent association of two conjugates composed of
short-chain peptides (A), this epitope being able to interact with
the ligand more strongly than either of the individual conjugates
in isolation (B). The same principle applies for headgroups
containing structures other than amino acids.
EXAMPLES
[0044] In the examples given below, the standard convention for
representation of amino acids by single letters of the alphabet is
employed, except that in all cases the letter refers to conjugates
as described above in which that particular amino acid occupies the
terminal position in the peptide chain. In the examples described
here, the lipid comprises two amino acids linked via a peptide
bond, in which both of the amino acids are glycine analogues, where
in each case the alpha hydrogen has been replaced by a linear
hydrocarbon chain containing either 12 or 14 carbons. Linkages
between the headgroup and spacer and the spacer and lipid are all
via peptide bonds. The headgroup bears a free amino group and the
free end of the lipid bears a CONH.sub.2 group. The structure of
each conjugate is thus: NH.sub.2-headgroup-spacer-amino acid
(C.sub.14 side chain)-amino acid (C.sub.12 side
chain)-CONH.sub.2.
Example 1
Stimulation of TNF Secretion from Macrophages
[0045] 1. Individual conjugates E, Y, Q, S & H (linked to lipid
via a serine-glycine spacer) were prepared as solutions in
methanol/dichloromethane 1:1 at a concentration of 5 mg/ml. [0046]
2. Solutions of the conjugates were dispensed into 7 ml glass vials
in equal proportions, to give a final volume of 400 ul (2 mg of
solid) in all vials, as shown in the example overleaf. In cases
where the volume of organic solution available was insufficient,
adjustment was made at a later stage, when the quantity of water
added for reconstitution was reduced accordingly, as shown. [0047]
3. The contents of all vials were dried down under a stream of
nitrogen, then exposed to a vacuum of at least 1 mbar overnight in
a lyophiliser. [0048] 4. On the following day, distilled water was
added in volumes as indicated in the table overleaf, to give a
final concentration in all vials of 1 mg/ml. The vials were capped,
warmed to 37 deg C. and bath-sonicated until clarity was achieved.
[0049] 5. The samples were then applied to wells of 24-well cluster
plates into which cells of the J774A-1 macrophage cell line had
been plated (5.times.10.sup.4 cells/ml/well). Volumes of 100 ul and
10 ul of sample were added to individual wells, and the cells were
incubated overnight at 37 deg C. in an atmosphere of 5%
CO.sub.2/air. [0050] 6. The following day, duplicate volumes of 50
ul of supernate were taken from each well and measured for TNF
concentration in a capture ELISA assay. Results obtained are shown
in the table below.
TABLE-US-00004 [0050] Volume of Volume of conjugate dispensed water
E Y Q S H added E 260 ul 1.3 ml Y 400 ul 2.0 ml Q 310 ul 1.55 ml S
360 ul 1.8 ml H 400 2.0 ml EY 200 ul 200 ul 2.0 ml EQ 200 ul 200 ul
2.0 ml ES 200 ul 200 ul 2.0 ml EH 200 ul 200 ul 2.0 ml YQ 200 ul
200 ul 2.0 ml YS 200 ul 200 ul 2.0 ml YH 200 ul 200 ul 2.0 ml QS
200 ul 200 ul 2.0 ml QH 200 ul 200 ul 2.0 ml SH 200 ul 200 ul 2.0
ml QSH 133 ul 133 ul 133 ul 2.0 ml YSH 133 ul 133 ul 133 ul 2.0 ml
YQH 133 ul 133 ul 133 ul 2.0 ml YQS 133 ul 133 ul 133 ul 2.0 ml ESH
133 ul 133 ul 133 ul 2.0 ml EQH 133 ul 133 ul 133 ul 2.0 ml EYH 133
ul 133 ul 133 ul 2.0 ml EYS 133 ul 133 ul 133 ul 2.0 ml EYQ 133 ul
133 ul 133 ul 2.0 ml EQS 133 ul 133 ul 133 ul 2.0 ml EYQS 50 ul 50
ul 50 ul 50 ul 1.0 ml EYQH 50 ul 50 ul 50 ul 50 ul 1.0 ml EYSH 50
ul 50 ul 50 ul 50 ul 1.0 ml EQSH 50 ul 50 ul 50 ul 50 ul 1.0 ml
YQSH 50 ul 50 ul 50 ul 50 ul 1.0 ml EYQSH 40 ul 40 ul 40 ul 40 ul
40 ul 1.0 ml
TABLE-US-00005 OD.sub.450 in J774 supernates 100 ug 10 ug 0 ug E
0.628 0.098 0.013 Y 0.313 0.053 Q 0.083 0.015 S 0.348 0.143 H 0.632
0.206 EY 0.198 0.027 EQ 0.113 0.022 ES 0.211 0.225 EH 0.167 0.037
YQ 0.245 0.034 YS 0.786 0.363 YH 0.541 0.133 QS 0.212 0.025 QH
0.135 0.027 SH 0.515 0.177 QSH 0.253 0.032 YSH 0.712 0.229 YQH
0.290 0.020 YQS 0.519 0.119 ESH 0.380 0.246 EQH 0.107 0.026 EYH
0.254 0.042 EYS 1.289 0.355 EYQ 0.191 0.064 EQS 0.209 0.027 EYQS
0.777 0.206 EYQH 0.224 0.067 EYSH 0.262 0.146 EQSH 0.149 0.185 YQSH
0.319 0.045 EYQSH 0.375 0.073
[0051] It can be seen that some, but not all, of the combinations
of different headgroups elicit strong biological responses,
indicating that the response is specific to those particular
combinations. The example illustrates the way in which the
conjugates described can be employed in the combinatorial approach
to identify efficacious combinations for the purpose of eliciting a
desired biological response.
Example 2
TNF Secretion from Macrophages
[0052] Comparison of Supra-molecular Assemblies Containing a
Mixture of Conjugates, with a Mixture of Supra-molecular Assemblies
Each Containing a Single Conjugate
[0053] Samples were prepared as described in Example b 1, with or
without the inclusion of additional lipidic materials as described
below. The combination of conjugates Y, S and L was chosen since
this combination was a good performer in the experiment described
in Example 1.
[0054] Probes containing phosphatidyl choline were prepared at a
ratio of phospholipid to conjugate of 2:1 wt/wt.
[0055] Probes containing octyl glucoside were prepared at a ratio
of glycolipid to conjugate of 1:1 wt/wt.
[0056] Results shown in the table below are optical densities at
450 nm of TNF ELISAs conducted on 18 hour culture supernatants. The
concentration of conjugate in the wells was 10 .mu.ug/ml
TABLE-US-00006 OD.sub.450 of TNF ELISA EYS 0.390 E + Y + S 0.059
medium control 0.000 EYS:OG 0.559 (E + Y + S):OG 0.193 OG control
0.228 EYS:PC 0.320 (E + Y + S):PC 0.130 PC control 0.081
[0057] This example shows that combinations of the conjugates can
elicit biological responses either when presented alone, or when
presented in conjunction with other lipids, such as phospholipids
or lipid sugars. It also shows that for efficacy to be manifested,
it is important for all of the conjugates to be presented in
combination on the same supra-molecular assembly, and that activity
is not observed if the same conjugates are presented together at
the same time, but separated on different supra-molecular
assemblies. This suggests that it is important to present the
conjugates in close proximity to each other, in order to permit the
formation of epitopes formed by non-covalent association of the
conjugates, which can participate in specific binding with
cell-surface receptors.
Example 3
Enhancement of Oral Uptake
[0058] 1. Individual conjugates L, S, E & Q (conjugated to
lipid via a tyrosine-glycine spacer) were prepared as solutions in
benzyl alcohol at a concentration of 10 mg/ml. [0059] 2. 75 ul of
.sup.14C-cholesterol oleate (3.7 MBq/ml in toluene) was dispensed
into four 7 ml glass screw-capped vials and dried down under a
stream of nitrogen. [0060] 3. 400 ul of each of the solutions in
(1) was added to one of the vials in (2) and shaken overnight at
room temperature. [0061] 4. Solutions of the conjugates were
dispensed into 7 ml glass vials in equal proportions, to give a
final volume of 80 ul (0.8 mg of solid) in all vials, as shown in
the example below.
TABLE-US-00007 [0061] L S E Q L 80 ul -- -- -- S -- 80 ul -- -- E
-- -- 80 ul -- Q -- -- -- 80 ul LS 40 ul 40 ul -- -- LE 40 ul -- 40
ul -- LQ 40 ul -- -- 40 ul SE -- 40 ul 40 ul -- SQ -- 40 ul -- 40
ul EQ -- -- 40 ul 40 ul LSE 27 ul 27 ul 27 ul -- LSQ 27 ul 27 ul --
27 ul LEQ 27 ul -- 27 ul 27 ul SEQ -- 27 ul 27 ul 27 ul LSEQ 20 ul
20 ul 20 ul 20 ul
[0062] 5. 2 ml of distilled water was added to each of the vials
with vortexing. The vials were then capped and bath-sonicated for
20 minutes. [0063] 6. The samples were then frozen in liquid
nitrogen and lyophilised overnight. [0064] 7. The following day,
each vial was reconstituted with 2 ml of distilled water and
sonicated again until clear dispersions were achieved. [0065] 8.
The samples were administered by oral gavage to Balb/c female mice
(20-25 g weight--four mice per group) at a dose of 0.3 ml per
animal. [0066] 9. 75 ul heparinised blood samples were taken by
tail venupuncture at 45, 90 and 180 minutes after administration.
[0067] 10. Each sample was diluted in 0.5 ml of PBS, which was then
centrifuged, and 0.4 ml of the supernate was transferred to a
scintillation vial to which 2 ml of Optiphase Hisafe 3 (Wallac) was
added with mixing. [0068] 11. Activity in the samples was measured
in a scintillation counter.
[0069] Percentage uptake was estimated on the basis of a 2 ml blood
volume, of which 1 ml was assumed to be plasma.
[0070] Results are shown in the table below.
TABLE-US-00008 % uptake in bloodstream 45 mins 90 mins 180 mins L
0.90 1.39 0.61 S 1.12 1.14 0.81 E 0.85 1.55 0.79 Q 1.40 3.00 0.81
LS 2.87 2.38 0.66 LE 2.59 2.22 0.49 LQ 5.05 2.15 0.45 SE 4.21 1.66
0.70 SQ 4.67 1.45 0.67 EQ 3.72 2.65 0.59 LSE 1.91 1.20 0.97 LSQ
6.23 1.90 0.80 LEQ 2.77 1.73 0.98 SEQ 3.06 1.52 0.63 LSEQ 2.45 1.74
0.81
[0071] It can be seen that some, but not all, of the combinations
of different headgroups enhance uptake of label via the oral route,
indicating that the response is specific to those particular
combinations. The example illustrates the way in which the
conjugates described can be employed in the combinatorial approach
to identify efficacious combinations capable of acting as targeting
ligands.
Example 4
ELISA Fc Binding
[0072] 1. 100 ul of goat IgG (1 mg/ml) was added to 20 ml of PBS
and 100 ul was placed in each well of a flat-bottomed microtitre
plate. [0073] 2. The plate was incubated for several days at +4 deg
C. [0074] 3. 2 mg of each of the conjugates Y, F, W, L, S, E, Q
& R (each linked to lipid via a serine-glycine spacer) were
weighed into 1 ml glass vials and 200 ul of benzyl alcohol added to
give solutions of each conjugate at a concentration of 10 mg/ml.
[0075] 4. The solutions were dispensed in 7 ml glass screw-capped
vials as follows:
TABLE-US-00009 [0075] Vial No. Y F W L S E Q R 1 20 ul 20 ul 20 ul
-- 2 20 ul 20 ul -- 20 ul 3 20 ul -- 20 ul 20 ul 4 -- 20 ul 20 ul
20 ul 5 20 ul 20 ul 20 ul -- 6 20 ul 20 ul -- 20 ul 7 20 ul -- 20
ul 20 ul
[0076] 5. The contents of each vial were mixed well by vortexing,
then 1.5 ml of distilled water was added to each vial. [0077] 6.
The vials were capped and bath-sonicated for five minutes to give
crystal clear dispersions. [0078] 7. The plate from step (2) was
washed in PBS/0.02% Tween 20 and then blocked by incubating for one
hour with 1% BSA in PBS (300 ul/well). [0079] 8. The plate was then
washed as before, and 100 ul of sample from each of the vials in
step (6) was added to wells in column (1) of rows (1) to (7) Row
(8) was left as a blank control. [0080] 9. Doubling dilutions were
performed across the plate by transferring 100 ul from wells in
column (1) to the adjacent well on the same row in column (2) and
mixing, then transferring 100 ul to the next column as before, etc.
[0081] 10. The plate was then incubated overnight at +4 deg C.
[0082] 11. The following day, the plate was washed as before and 10
ul of commercial horseradish peroxidase-IgG conjugate (diluted
1/1000 in PBS) was added to each well and incubated at room
temperature for 40 minutes. [0083] 12. The plate was then washed
again, and 100 ul of OPD substrate for peroxidase was added to each
well and incubated at room temperature for 30 minutes. [0084] 13.
20 ul of 3M sulphuric acid was then added to each well to stop the
reaction. [0085] 14. The optical density of each of the wells was
measured at 450 nm on a plate reader, and the results obtained,
after adjustment for background, are recorded below.
TABLE-US-00010 [0085] Sample 1 in 4 1 in 8 1 in 16 1 in 32 1 in 64
1 YFW 0.001 0.039 0.048 0.053 0.083 2 YFL 1.504 1.484 1.325 0.723
0.051 3 YWL 0.803 0.192 0.022 0.023 0.060 4 FWL 1.034 0.778 0.208
0.031 0.034 5 SEQ 0.029 0.041 0.055 0.057 0.091 6 SER 0.013 0.030
0.044 0.062 0.075 7 SQR 0.000 0.045 0.031 0.054 0.065
[0086] It can be seen that maximal binding is achieved with samples
2, 3 and 4 (ie combinations YFL, YWL, and FWL).
[0087] It can be seen that some, but not all, of the combinations
of different headgroups enter into strong binding interactions,
indicating that the response is specific to those particular
combinations. The example illustrates the way in which the
conjugates described can be employed in the combinatorial approach
to identify efficacious combinations for the purpose of eliciting a
desired binding interaction.
* * * * *