U.S. patent number 4,833,092 [Application Number 07/010,088] was granted by the patent office on 1989-05-23 for method for determining mimotopes.
This patent grant is currently assigned to Commonwealth Serum Laboratories Commission. Invention is credited to Hendrik M. Geysen.
United States Patent |
4,833,092 |
Geysen |
May 23, 1989 |
Method for determining mimotopes
Abstract
A method of detecting or determining the sequence of monomers
which is a topographical equivalent of the ligand which is
complementary to a particular receptor of interest, the method
comprising the steps of: 1. synthesizing a plurality of catamers,
each said catamer being of the general formula: wherein D.sub.1
represents a designated monomer selected from a first set of
monomers, and D.sub.2 represents a designated monomer selected from
a second set of monomers which may be the same as or different to
said first set of monomers; said plurality of catamers comprising
catamers in which each designated monomer is systematically varied
to contain members from the respective set of monomers; 2.
contacting each catamer with the receptor of interest, and, 3.
detecting or determining the presence or absence of binding between
each catamer and said receptor.
Inventors: |
Geysen; Hendrik M. (Knoxfield,
AU) |
Assignee: |
Commonwealth Serum Laboratories
Commission (Victoria, AU)
|
Family
ID: |
3771065 |
Appl.
No.: |
07/010,088 |
Filed: |
December 22, 1986 |
PCT
Filed: |
April 22, 1986 |
PCT No.: |
PCT/AU86/00110 |
371
Date: |
December 22, 1986 |
102(e)
Date: |
December 22, 1986 |
PCT
Pub. No.: |
WO86/06487 |
PCT
Pub. Date: |
November 06, 1986 |
Foreign Application Priority Data
Current U.S.
Class: |
436/501; 436/543;
436/518; 436/548 |
Current CPC
Class: |
G01N
33/53 (20130101); G01N 33/6878 (20130101); G01N
33/6803 (20130101) |
Current International
Class: |
G01N
33/53 (20060101); G01N 33/68 (20060101); G01N
033/53 (); G01N 033/543 (); G01N 033/577 () |
Field of
Search: |
;436/518,543,548,501 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
|
|
|
|
|
25428 |
|
Sep 1984 |
|
AU |
|
36563 |
|
Jun 1985 |
|
AU |
|
36562 |
|
Jun 1985 |
|
AU |
|
36560 |
|
Jun 1985 |
|
AU |
|
45339 |
|
Jan 1986 |
|
AU |
|
WO84/02983 |
|
Aug 1984 |
|
WO |
|
WO84/03564 |
|
Sep 1984 |
|
WO |
|
WO85/02121 |
|
May 1985 |
|
WO |
|
Other References
Hopp, Thomas P. et al., Proc. Natl. Acad. Sci., U.S.A., 78(6),
3824-3828 (Jun. 1981). .
Geysen, H. Mario et al., Molecular Immunology, 23(7), 709-715
(1986). .
Scientific American, Feb. 1983, pp. 48-56, (p. 50, lines 33-39), R.
A. Lerner, "Synthetic Vaccines"..
|
Primary Examiner: Marantz; Sidney
Attorney, Agent or Firm: Sughrue, Mion, Zinn, Macpeak &
Seas
Claims
I claim:
1. A method of detecting or determining the sequence of monomers
which is a topographical equivalent of the ligand which is
complementary to a particular receptor of interest, the method
comprising the steps of:
1. synthesizing a plurality of catamers, each said catamer being of
the general formula:
wherein D.sub.1 represents a designated monomer selected from a
first set of monomers, and D.sub.2 represents a designated monomer
selected from a second set of monomers which may be the same as or
different from said first set of monomers; said plurality of
catamers comprising catamers in which each designated monomer is
systematically varied to contain members from the respective set of
monomers;
2. contacting each catamer with the receptor of interest, and,
3. detecting or determining the presence or absence of binding
between each catamer and said receptor.
2. A method according to claim 1, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
3. A method according to claim 1, wherein each of said plurality of
catamers is synthesized on a solid support, and has the general
formula:
wherein D.sub.1 represents a designated monomer selected from a
first set of monomers, and D.sub.2 represents a designated monomer
selected from a second set of monomers which may be the same as or
different from said first set of monomers, Lk represents a linker
molecule, and Y is a end group of the catamer.
4. A method according to claim 1, comprising the further step of
systematically replacing the monomers in any of the catamers with
their optical isomers, either individually or in combinations of
monomers, and again performing the following steps: contacting each
catamer with the receptor of interest, and detecting or determining
the presence or absence of binding between each catamer and said
receptor.
5. A method according to claim 4, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
6. A method according to claim 1 comprising the further steps
of:
A. synthesizing a plurality of additional catamers, each said
additional catamer being of the general formula:
wherein D.sub.1 represents a designated monomer selected from a
first set of monomers, and D.sub.2 represents a designated monomer
selected from a second set of monomers which may be the same as or
different from said first set of monomers, and Sp is a spacer
molecule which can modify the relative orientation of the monomers
D.sub.1 and D.sub.2 ;
B. contacting each said additional catamer with the receptor of
interest, and,
C. detecting or determining the presence or absence of binding
between each said additional catamer and said receptor.
7. A method according to claim 6, wherein each of said plurality of
additional catamers is synthesized on a solid support, and has the
general formula:
wherein D.sub.1 represents a designated monomer selected from a
first set of monomers, and D.sub.2 represents a designated monomer
selected from a second set of monomers which may be the same as or
different from said first set of monomers, Sp is a spacer molecule
which can modify the relative orientation of the monomers D.sub.1
and D.sub.2, Lk represents a linker molecule and Y is an end group
of the catamer.
8. A method according to claim 6, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
9. A method according to claim 6, comprising the further step of
systematically replacing the monomers in any of the catamers with
their optical isomers, either individually or in combinations of
monomers, and again performing the following steps: contacting each
catamer with the receptor of interest, and detecting or determining
the presence or absence of binding between each catamer and said
receptor.
10. A method according to claim 9, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
11. A method according to claim 1, comprising the further steps
of:
a. synthesizing a further plurality of catamers, each said catamer
being of the general formula:
wherein D.sub.1 represents a designated monomer selected from a
first set of monomers, and D.sub.2 represents a designated monomer
selected from a second set of monomers which may be the same as or
different from said first set of monomers, and D.sub.3 represents a
designated monomer selected from a third set of monomers which may
be the same as or different from either the first or the second set
of monomers, and
b. contacting each said catamer with the receptor of interest,
and,
c. detecting or determining the presence or absence of binding
between each said catamer and said receptor.
12. A method according to claim 11, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
13. A method accordinag to claim 11, comprising the further step of
systematically replacing the monomers in any of the catamers with
their optical isomers, either individually or in combinations of
monomers, and again performing the following steps: contacting each
catamer with the receptor of interest, and detecting or determining
the presence or absence of binding between each catamer and said
receptor.
14. A method according to claim 13, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
15. A method according to claim 11, wherein steps a., b. and c. are
repeated with the systematic introduction of a spacer molecule, Sp,
which can modify the relative orientation of the monomers D.sub.1
and D.sub.2, into all possible positions of the further pluralities
of the catamers.
16. A method according to claim 15, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
17. A method according to claim 15, comprising the further step of
systematically replacing the monomers in any of the catamers with
their optical isomers, either individually or in combinations of
monomers, and again performing the following steps: contacting each
catamer with the receptor of interest, and detecting or determining
the presence or absence of binding between each catamer and said
receptor.
18. A method according to claim 17, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
19. A method according to claim 11 comprising the further steps
of:
A. synthesizing a plurality of additional catamers, each said
additional catamer being of the general formula:
wherein D.sub.1 represents a designated monomer selected from a
first set of monomers, and D.sub.2 represents a designated monomer
selected from a second set of monomers which may be the same as or
different from said first set of monomers, and Sp is a spacer
molecule which can modify the relative orientation of the monomers
D.sub.1 and D.sub.2 ;
B. contacting each said additional catamer with the receptor of
interest, and,
C. detecting or determining the presence or absence of binding
between each said additional catamer and said receptor.
20. A method according to claim 19, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
21. A method according to claim 19, wherein each of said plurality
of additional catamers is synthesized on a solid support, and has
the general formula:
wherein D.sub.1 represents a designated monomer selected from a
first set of monomers, and D.sub.2 represents a designated monomer
selected from a second set of monomers which may be the same as or
different from said first set of monomers, Sp is a spacer molecule
which can modify the relative orientation of the monomers D.sub.1
and D.sub.2, Lk represents a linker molecule and Y is an end group
of the catamer.
22. A method according to claim 19, comprising the further step of
systematically replacing the monomers in any of the catamers with
their optical isomers, either individually or in combinations of
monomers, and again performing the following steps: contacting each
catamer with the receptor of interest, and detecting or determining
the presence or absence of binding between each catamer and said
receptor.
23. A method according to claim 22, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
24. A method according to claim 11, wherein steps a., b. and c. are
repeated with the systematic addition of further monomers to the
catamers.
25. A method according to claim 24, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
26. A method according to claim 24, comprising the further step of
systematically replacing the monomers in any of the catamers with
their optical isomers, either individually or in combinations of
monomers, and again performing the following steps: contacting each
catamer with the receptor of interest, and detecting or determining
the presence or absence of binding between each catamer and said
receptor.
27. A method according to claim 26, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
28. A method according to claim 24, wherein steps a., b. and c. are
repeated with the systematic introduction of a spacer molecule, Sp,
which can modify the relative orientation of the monomers D.sub.1
and D.sub.2, into all possible positions of the further pluralities
of the catamers.
29. A method according to claim 28, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
30. A method according to claim 28, comprising the further step of
systematically replacing the monomers in any of the catamers with
their optical isomers, either individually or in combinations of
monomers, and again performing the following steps: contacting each
catamer with the receptor of interest, and detecting or determining
the presence or absence of binding between each catamer and said
receptor.
31. A method according to claim 30, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
32. A method according to claim 24 comprising the further steps
of:
A. synthesizing a plurality of additional catamers, each said
additional catamer being of the general formula:
wherein D.sub.1 represents a designated monomer selected from a
first set of monomers, and D.sub.2 represents a designated monomer
selected from a second set of monomers which may be the same as or
different from said first set of monomers, and Sp is a spacer
molecule which can modify the relative orientation of the monomers
D.sub.1 and D.sub.2 ;
B. contacting each said additional catamer with the receptor of
interest, and,
C. detecting or determining the presence or absence of binding
between each said additional catamer and said receptor.
33. A method according to claim 32, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind with the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
each catamer and said receptor.
34. A method according to claim 32, wherein each of said plurality
of additional catamers is synthesized on a solid support, and has
the general formula:
wherein D.sub.1 represents a designated monomer selected from a
first set of monomers, and D.sub.2 represents a designated monomer
selected from a second set of monomers which may be the same as or
different from said first set of monomers, Sp is a spacer molecule
which can modify the relative orientation of the monomers D.sub.1
and D.sub.2, Lk represents a linker molecule and Y is an end group
of the catamer.
35. A method according to claim 32, comprising the further step of
systematically replacing the monomers in any of the catamers with
their optical isomers, either individually or in combinations of
monomers, and again performing the following steps: contacting each
catamer with the receptor of interest, and detecting or determining
the presence or absence of binding between each catamer and said
receptor.
36. A method according to claim 35, comprising the further step of
systematically replacing the monomers in any of the catamers which
bind the receptor of interest, either individually or in
combinations of monomers, with other monomers selected from the
respective set(s) of monomers, and again performing the following
steps: contacting each catamer with the receptor of interest, and
detecting or determining the presence or absence of binding between
eact catamer and said receptor.
Description
This invention relates to a method of detecting or determining a
sequence of monomer molecules which corresponds to the ligand
molecule for a particular receptor. The sequence of monomer
molecules so determined is the mimotope (defined below) of the
particular ligand. The mimotope which is determined by this method
may not have any obvious or direct relationship to the natural
ligand molecule, but will share with it the ability to react with
the receptor, and indeed, the mimotope so determined may be
modified to incorporate specific or additional properties in the
reaction with the receptor. Such a mimotope could then be used to
replace the natural ligand in the treatment or prevention of
particular disease or it may be used to mediate a particular
biological effect.
As used throughout this specification, the terms listed below have
the following meanings:
receptor: a molecule or molecular complex which will combine
specifically with its particular ligand molecule. It is those
receptors which on binding with their particular ligand(s) mediate
a biological function that are of most interest. Examples of
receptors include, but are not restricted to, the common class of
receptors associated with the surface membrane of cells and
include, for instance, the immunologically important receptors of
B-cells, T-cells, macrophages and the like. Another example is
receptors for acetyl choline on nerve cells which cause a nerve
pulse to be transmitted down the length of the neuron when the
receptor molecule reacts with its ligand, acetyl choline.
epitope: the specific surface of an antigen molecule which is
delineated by the area of interaction with the sub-class of
receptors known as antibodies.
catamer: a polymer molecule which is a precisely defined linear
sequence formed by the condensation of small molecules. Note that
this term includes molecules in which different types of
condensation reactions are used to join the small molecules. A
number prefixed to the word "catamer" implies that the catamer is
formed by the condensation of the indicated number of small
molecules, for example, 8-catamer means that the catamer is made up
from eight small molecules. Examples of catamers include any given
peptide and any given oligo-saccharide.
monomer: a member of the set of small molecules which can be
condensed together to form a catamer. The set of monomers includes
but is not restricted to, for example, the set of common L-amino
acids, the set of D-amino acids, the set of synthetic amino acids,
the set of nucleotides, and the set of pentoses and hexoses.
peptide: a catamer in which the small molecules are alpha-amino
acids and which are joined together through a peptide bond. In the
context of this specification it should be appreciated that the
amino acids may be the L-optical isomer or the D-optical
isomer.
mimotope: a catamer which in at least one of its conformations has
a surface region with the equivalent molecular topology to the
binding surface of the ligand molecule of which it is the mimic. In
the context of immunological receptors, the mimotope mimics the
epitope of the antigen.
complementary: refers to the matching together of the reacting
surfaces of an ligand molecule and its receptor. Thus, the receptor
and its ligand can be described as complementary, and furthermore,
the contact surface characteristics are complementary to each
other.
paratope: the combining site of an antibody which is complementary
to a particular epitope.
ligand molecule: is the molecule which binds to a particular
receptor and when bound to it mediates the biological function
associated with that particular receptor.
Examples of receptors which can be investigated by this method
include, but are not restricted to:
hormone receptors: for instance the receptors for insulin and
Growth Hormone; determination of the mimotopes of the ligands
binding to these receptors may lead to the development of an oral
replacement of the daily injections which diabetics must take to
relieve the symptoms of diabetes, and in the other case, a
replacement for the scarce human Growth Hormone which can only be
obtained from cadavers or by recombinant DNA technology. Other
examples are the vasoconstrictive hormone receptors; determination
of the mimotope of the ligand binding to these receptors may lead
to the development of drugs to control blood pressure.
opiate receptors: determination of mimotopes of the ligands binding
to the opiate-receptors in the brain may lead to the development of
less-addictive replacements for morphine and related drugs.
microorganism receptors: determination of mimotopes of the ligands
binding to a receptor such as specific transport proteins or
enzymes essential to survival to microorganisms, may lead to a new
class of antibiotics. Of particular value would be antibiotics
against protozoa and those bacteria resistant to the antibiotics in
current use.
enzymes: for instance, the enzymes responsible for cleaving neural
transmitters; determination of mimotopes able to modulate the
action of the enzymes which cleave the different neural
transmitters may lead to the development of drugs which can be used
in the treatment of disorders of neural transmission; and,
antibodies: for instance the ligand-binding site on the antibody
molecule which combines with the epitope of an antigen of interest;
determination of a mimotope for the epitope may lead to the
development of vaccines of which the immunogen is based on one or
more mimotopes or diagnostic agents or compounds useful in the
therapeutic treatment of autoimmune diseases.
Examples of ligands which can be investigated by this method
include, but are not restricted to:
toxins and venoms: for instance, the combining site of the toxin
molecule which reacts with a particular receptor in the body to
give the particular symptom(s) of intoxication; determination of
mimotopes to the combining site of the ligand may lead to the
development of drugs which can be used to treat envenomation by
snakes and other poisonous animals without the side effects of
heterologous antivenenes.
virus and other microorganism capsid molecules: for instance, the
combining site on the virus coat molecule which reacts with a
particular receptor on the cell membrane in the body and which
allows the virus to invade and thus infect the particular cell;
determination of mimotopes to this combining site may lead to the
development of drugs which specifically prevent intracellular
invasion by the virus and thus prevent their replication.
It is a primary object of this invention to detect or determine one
or more short sequences of monomers (catamers) which selectively
combine with a particular receptor so as to mediate its biological
function. These catamers are the mimotopes of the ligand. This
information is invaluable for the design of very specific
diagnostic and therapeutic agents.
The most usual group of small molecules which may be condensed
together to form a catamer is the group of alpha-amino acids.
However, other molecules which are consistent with a different
chosen chemistry may also be used; for example, catamers formed
from the specified sequential condensation of nucleotides or
saccharides. Another group of small molecules would be the
non-genetically coded amino acids such as beta-amino acids, which
may be used to advantage to add an additional bond at specified
positions within the catamer.
The method of the present invention is based on the realisation
that a given receptor will specifically react with a catamer which
is the mimotope for the ligand to which the receptor is directed.
It further relies on modern techniques of immunology to detect
reaction between a receptor and its ligand when both are
present.
In Australian Patent Specification No. 45339/85 it is proposed to
delineate mimotopes based on an overall length of about 8 monomers.
It is now clear that the preferred method for the delineation of
mimotopes is from shorter catamers (2 or 3 monomers long) made up
from all combinations of monomers from either:
(i) two sets of monomers, which may be identical; or
(ii) three sets of monomers, of which the centre monomer of the
catamer is selected from a set of special chosen monomers which
confer known spatial relationships on the other two monomers, the
remaining monomers of the catamer coming from two sets of monomers
which may be identical.
Furthermore, it has now been shown that the sensitivity of
detection of binding to receptors is reduced in some instances
where the monomers are synthesized in catamer preparations as
disclosed in Patent Specification No. 45339/85.
We have now demonstrated that reaction is readily detected between
a receptor and short mimotopes. These short mimotopes when
condensed together then bind to the receptor with either a greater
affinity or with greater specificity for that receptor. This
reaction can be detected even when the short mimotope presented to
the receptor is as small as two monomer molecules long. By
determining the optimum short mimotope at each stage and then
testing further variants, the final structure of a strongly-binding
mimotope can be determined.
According to the present invention there is provided a method of
detecting or determining the sequence of monomers which is a
topographical equivalent of the ligand which is complementary to
the particular receptor of interest and prior knowledge is
irrelevant about the identity, structure and sequence of the
receptor or its ligand. The method comprises the steps of:
1. synthesizing a plurality of catamers, each said catamer being of
the general formula:
wherein D.sub.1 represents a designated monomer selected from a
first set of monomers, and D.sub.2 represents a designated monomer
selected from a second set of monomers which may be the same as or
different to said first set of monomers; said plurality of catamers
comprising catamers in which each designated monomer is
systematically varied to contain members from the respective set of
monomers;
2. contacting each catamer with the receptor of interest, and,
3. detecting or determining the presence or absence of binding
between each catamer and said receptor.
The method may also comprise the further steps of:
a. synthesizing a further plurality of catamers, each said catamer
being of the general formula:
wherein D.sub.1 and D.sub.2 are as defined above, and preferably
represent a combination of monomers corresponding to a catamer
which binds to said receptor, and D.sub.3 represents a designated
monomer selected from a third set of monomers which may be the same
as or different to either the first or the second set of monomers,
and
b. performing steps 2 and 3 as described with the further plurality
of catamers.
The procedure of steps a. and b. above may be repeated to further
"extend" the catamers by systematically adding further monomers to
the catamers, and testing in the same manner as in step b,
above.
In another important aspect, the method of this invention may
comprise the steps of:
A. synthesizing a plurality of additional catamers, each said
additional catamer being of the general formula:
wherein D.sub.1 and D.sub.2 are as defined above and Sp is a spacer
molecule which can modify the relative orientation of the monomers
D.sub.1 and D.sub.2 ; and
B. performing steps 2 and 3 as described above with the plurality
of additional catamers.
The spacer molecule "Sp" as described above may also be
systematically introduced into all possible positions of the
"extended" catamers referred to above, and tested in the same
manner as in Step B. above.
In yet another aspect, the method of the invention may further
comprise the steps of systematically replacing the monomers in any
of the catamers referred to above with their optical isomers,
either individually or in combinations of monomers, and again
performing steps 2 and 3 as described above.
In a further aspect, the method may further comprise the steps of
systematically replacing the monomers in any of the catamers
referred to above which bind with the receptor of interest, either
individually or in combinations of monomers, with other monomers
selected from the respective set(s) of monomers, and again
performing steps 2 and 3 as described above.
It will be apparent that the method of this invention requires no
previous information about the nature of the ligand and in
particular it requires no foreknowledge of the sequence of monomers
which make up the ligand. In fact, it is not necessary for the
application of this invention to know the source or identity of the
ligand to which the receptor is directed. Furthermore, this
invention makes no assumptions about the nature of the ligand of
the particular receptor. This method will identify mimotopes of
discontinuous as well as continuous ligands. Because of the very
nature of the method of the invention it will be appreciated that
mimotopes may or may not consist of members of the same set of
monomers which make up the ligand of which it is the mimic.
The plurality of catamers required to be synthesized in the
application of this invention may be prepared by any of the known
method of catamer synthesis. The preferred methods when the
monomers are amino acids is to use the solid phase technique
described in Australian Patent Specification No. 25429/84, whereby
each catamer is synthesized on a polyethylene support.
The following is a detailed description of one embodiment of the
present invention when applied to the determination of a mimotope
for a ligand able to bind to a receptor when that receptor is an
antibody. In this context, the ligand is usually referred to as the
epitope for the antibody. Preferably the method of the present
invention is carried out by screening a plurality of synthesized
catamers against the antibody of interest. Ideally the antibody
will be a monoclonal antibody which can be prepared by any of the
usual methods. Polyclonal antiserum from humans and animals may be
used if a source of monoclonal antibody with the desired
characteristics is not available, however, analysis of the
resulting data may be complicated because the reaction observed
could be from more than one monoclonal antibody. When using
polyclonal antiserum it may be necessary to reduce the antibody
diversity by using techniques well known to those skilled in the
art, for example iso-electric focusing, HPLC-based chromatography
or affinity chromatography.
Current indications suggest that an epitope mimicked by a catamer
which is usually about six monomers in length when the monomers
come from the set of alpha-amino acids. It is to be understood,
however, that the present invention is not retricted to sequences
formed from six monomers. The ability of the 6-catamer to be the
mimotope of the epitope is not critically dependent on every
position having a designated monomer. It has been found that
certain positions in most mimotopes are not restricted to a single
designated monomer for binding with the receptor.
A. Synthesis of a plurality of catamers.
As noted above, the preferred method of applying this invention is
to synthesize the catamers on a solid support. In this embodiment,
the plurality of catamers will all have the general formula:
where "Lk" represents a linker molecule which provides a suitable
end group for condensing the monomers to the solid support.
"D.sub.1 " and D.sub.2 " represent designated positions occupied by
monomers which are selected from known sets of monomers; but which
are altered systematically between catamers. It should be noted
that the set of monomers used for the D.sub.1 designated position
need not be the same set of monomers used for the D.sub.2
designated position. "Y" in the general formula is an end group of
the catamer and may be, but is not restricted to, for example a
hydrogen atom or an acetyl group. "Y" may also be another molecule
which is coupled to the catamer to preserve particular
characteristics of the molecular environment of a peptide bond at
the amino terminal designated position.
If i is the number of members in the set of monomers to be coupled
in the D.sub.1 position and j is the number of members in the set
of monomers to be coupled in the D.sub.2 position then a total of
i. j different catamers will be synthesized.
In the present embodiment, the support rods are prepared so that
the monomers can be coupled to them by coupling an appropriate
linker molecule.
For the coupling at the D.sub.1 position, each rod will be treated
with a reaction mixture which contains only a single monomer such
as a protected amino acid or the like. In this position each of the
i monomers are coupled to j rods. For the coupling at the D.sub.2
position each rod is treated with a reaction mixture which contains
a single monomer such as a protected amino acid or the like. Each
of the j rods which has a particular monomer in the D.sub.1
position will have a different monomer coupled at the D.sub.2
position. In this way every combination of the members of the
set(s) of monomers will be found in the i. j rods.
The desired end group, "Y", is then coupled using the appropriate
chemistry.
After synthesis of the plurality of catamers any side-chain
protective groups are removed from the catamers using the
appropriate techniques and the rod-coupled catamers are washed.
It has been found to be a preferred embodiment of the invention to
synthesize more than one set of plurality of catamers to aid in the
analysis of data. Thus, as well as synthesizing catamers with the
general formula
as described above, additional sets of catamers may be prepared
with the general formula
where "Sp" is a spacer molecule which may restrict the relative
orientation of the monomers at the designated positions to a
particular geometrical configuration(s). The spacer molecule may
also be deliberately chosen to allow a greater flexibility to the
relative geometric configuration between monomers in the designated
positions, D.sub.1 and D.sub.2. It should be noted that members of
the set of spacer molecules may be made up from the condensation of
more than one monomer. Examples of spacer molecules include, but
are not restricted to glycine (approximately linear extension),
beta-alanine (increased flexibility), proline (forced bend),
glycyl-proline (extended bend, otherwise known as reverse bend in
protein structure terminology) and o-aminobenzoic acid (a planar
bend).
By analyzing the results from these sets of catamers the preferred
spatial relationships between monomers in the mimotopes can be
deduced as will be shown in the appropriate examples given
below.
B. Testing of the plurality of catamers.
The plurality of catamers prepared as in A. above are then
contacted with the particular antibody of interest. The reaction
between antibody and each catamer can then be detected by any of
the usual methods, for example, radioimmunoassay (RIA). However,
the preferred method of detection is to use the well known
enzyme-linked immunosorbent assay (ELISA).
At the end of each assay antibodies can be removed from the
catamers by, for example, washing with a solution of 8M urea, 0.1%
2-mercaptoethanol and 0.1% sodium dodecylsulphate followed by
several washes in phosphate buffered saline. In this way the
plurality of catamers may be used for testing with many other
antibodies.
C. Analyses of the data
In the testing of a set of catamers with antibody it has been found
that certain catamers will show detectable binding with the
antibody. These reacting catamers identify useful combinations of
the members of the set(s) of monomers. These combinations of
monomers are short mimotopes which, when extended, may bind to the
antibody with a greater affinity or alters specificity. Analysis of
the data is greatly facilitated by including a number of control
peptides in the synthesis which aid the determination of
significant responses. Further analysis of the data can be carried
out in a number of ways. These include:
1. permutating each of these reacting combinations of monomers to
create a list of catamers which will include mimotopes which bind
to the paratope; each catamer in this list can be regarded as a
possible mimotope;
2. selecting candidates from the list of reacting combinations of
monomers and further synthesizing sets of catamers in which the
known reacting combination of monomers is held constant and further
monomers are added systematically at either end; thus in the
example above, a suitable set of such catamers would include
catamers with the formulae
where A.sub.1 and A.sub.2 constitute the reacting combination of
monomers from the previous results and D.sub.3 is the new
designated position where each of the members of the set of
monomers is systematically varied; and
3. combining the results from different sets of a plurality of
catamers and analyzing the results to deduce a single sequence of
monomers, or a small number of such sequences which would bind to
the antibody of interest. Analysis of this data is possible as the
reacting combinations of monomers when they are adjacent to each
other are now known, as well as the reacting combinations of
monomers when they have particular geometrical configurations
between them. In this way, these data can then be interpreted to
predict the structure of the mimotope when bound to the antibody;
this approach will be of greatest benefit when the antibody used to
test the plurality of catamers is a monoclonal antibody.
The procedure given in 2. above can be repeated until no further
enhancement of binding is achieved by additional extending of the
mimotope. Ideally, the sequence of the mimotope should be checked
by regularly synthesizing and testing replacement nets of the
mimotope to obtain the optimally-binding mimotope for further
extension as described in Australian Patent Specification No.
25428/84.
D. Synthesis of selected catamers
The selected catamers can be synthesized using similar methods to
those used in A. After the selected catamers have been synthesized
they are reacted with the antibody of interest. It is then simple
to select the catamer which binds most strongly with the
antibody.
The binding of the mimotope with the antibody can be further
enhanced by synthesizing a further plurality of catamers based on
the sequence of the most strongly binding mimotope. This plurality
of catamers consists of adding spacer molecules (--Sp--)
systematically at all positions of the mimotope; and where feasible
systematically replacing each monomer of the catamer with its
optical isomer. Testing of this set of catamers with the antibody
will give invaluable information about the relative orientation of
the monomers and their stereochemistry as required for binding with
the antibody.
In Examples 1, 2, 3 and 4 given below, the mimotopes are being
determined for the antigen against which different monoclonal
antibodies were raised. The defined set of monomers is the set of
the common L-alpha acids unless otherwise specified. The linker
molecule, --Lk--, was 3 amino-N.sup.1 (6-aminohexyl)-propanamide
and the end group, Y--, was the acetyl moiety. In Example 5 below,
the mimotope is determined for an antigenic determinant of human
chorionic gonadotrophin which induced a monoclonal antibody. The
linker molecule, --Lk--, is the same as that used in earlier
Examples. The end group, Y--, is either
.beta.-alanyl-.beta.-alanine or .beta.-alanine. The defined set of
monomers was extended to include the D-optical isomers of the
common alpha amino acids and several unusual amino acids. In
Example 6 below, the mimotope is determined for a receptor site on
viruses. The linker molecule, --Lk--, is the same as for earlier
Examples. The end group, Y--, is either
.beta.-alanyl-.beta.-alanine or .beta.-alanine. The defined set of
monomers was extended as described for Example 5.
EXAMPLE 1
A monoclonal antibody was raised against sperm whale myoglobin
using the usual techniques. This monoclonal antibody was tested
against a set of catamers with the general formula:
Three pairs of reacting monomers bound to the catamers with
approximately equal response and were significantly higher than
other pairs of monomers. The sequences were E-F, E-L and E-H.
A further set of catamers was synthesized which comprised all
4-catamers which could be made from the set of monomers; Glutamic
acid (E), Phenylalanine (F), Histidine (H) and Leucine (L). The
following catamers were found to bind significantly higher than the
remaining catamers:
This small group of short mimotopes can now become the candidates
for further extensions.
EXAMPLE 2
A monoclonal antibody was raised against sperm whale myoglobin
using the usual techniques. This monoclonal antibody was tested
against a set of catamers with the general formula:
Three pairs of reacting monomers bound to the catamers with
approximately equal response and were significantly higher than
other pairs of monomers. The sequences were E-F, E-L and E-H.
Six further sets of catamers were synthesized using the pairs E-F,
E-L and E-H as starting points. Using the E-F pair as an example,
the catamers in each set had the general formula:
where Sp is a spacer molecule from the set of beta-alanine, glycine
and L-proline. Furthermore the D-optical isomer of both the
Glutamic acid and Phenylalanine were systematically substituted for
the L-optical isomer.
The catamers which gave the best response from each set of catamers
were D-Glutamic acid--L-Proline--L-Phenylalanine L-Glutamic
acid--L-Leucine, and D-Glutamic acid--L-Proline--D-Histidine. These
results show that the monomers F and H were better positioned
non-adjacent to E; furthermore, E and L are best positioned
adjacent. The optical isomers in the catamers which gave the best
binding suggest a structure for a strongly binding mimotope as set
out below. It is to be noted that the sperm whale myoglobin
molecule does have a region which is similar to the predicted
mimotope. Obviously, this predicted mimotope becomes a candidate
for further extension.
The structure below is a two dimensional representation of the
spatial relationship between the amino acids, F, E, L, and H when
bound to a monoclonal antibody against sperm whale myoglobin (see
Example 2). It must be noted that this is an illustration only and
must not be interpreted to mean that the catamer illustrated is
planar. Furthermore, the bonds joining F to E and L to H are meant
to represent a distance greater than that of a peptide bond.
##STR1##
When this structure is compared with the X-ray crystallography
structure of myoglobin, it is of considerable interest to note that
the sequence --F--L--E--L-- appears at positions 135 to 138 and
that there are two histidine residues (at positions 81 and 82) in
close proximity to the glutamic acid at position 136. This gives
considerable credence to the postulated structure of the epitope of
the monoclonal antibody.
EXAMPLE 3
A mimotope to a monoclonal antiserum raised against Foot and Mouth
Disease Virus was delineated to the sequence W--Q--M--G--H--S. A
series of catamers were synthesized in which a beta-alanine residue
was introduced systematically between monomers. The sequence
W--Q--M--.beta.--G--H--S gave a response which was significantly
larger than the base sequence, where .beta. represents
beta-alanine. It was further found that excellent binding to the
antibody was achieved with the sequences:
which suggests that the glycine in the starting mimotope was a
spacer between two reacting elements, W--Q--M and H--S. Thus it can
be clearly seen that the mimotope is made up of two parts;
furthermore, the joining together of these parts is not critical
for the mimotope to be able to react strongly with the
antibody.
A further set of catamers were synthesized in which the D-optical
isomer systematically replaced the L-optical isomer monomer in the
starting mimotope. The results of testing with the antibody showed
that for strongest binding, the monomers in each element should
have the same optical isomer and that there was a preference for
the monomers in the W--Q--M element to be the D-optical isomers
whereas the monomers in element H--S should be L-optical isomers.
These results can be interpreted to mean that the epitope to the
antibody is made up of two adjacent anti-parallel chains in which
the chain direction is M--Q--W and the second chain is H--S. This
prediction leads to the suggestion that another mimotope to the
monoclonal antibody would be:
This was synthesized and found to react with comparable binding to
the antibody as the strongly binding catamer:
Thus, the way in which the two elements W--Q--M and H--S are joined
together is irrelevant to the ability to combine with the antibody
so long as their relative positions remain the same. The best
mimotope as a potential immunogen will be one in which these
elements are joined together from both sides in order to minimize
conformational mobility.
EXAMPLE 4
A monoclonal antibody (SO93-7) raised against Foot and Mouth
Disease virus was tested with catamers with the general
formula:
It was found that the dipeptide Q-F reacted significantly more
strongly with the monoclonal antibody than with any other
dipeptide.
Further sets of catamers were synthesized with the general
formulae:
When these sets of catamers were reacted with the antiserum it was
found that the following catamers reacted significantly better with
the monoclonal antibody than any of the others:
This small group of short mimotopes can now become the candidates
for further extensions.
EXAMPLE 5
A monoclonal antibody (S218-4) was raised against human chorionic
gonadotrophin using the usual techniques. This was tested with
catamers with the general formula:
where Y.sub.1 -- is .beta.-alanyl-.beta.-alanine. It was found that
the dipeptide in the designated positions which bound most strongly
to the monoclonal antibody was F-A.
Further sets of catamers were synthesized with the general
formulae:
where Sp is a spacer which is an element of the set [null,
.beta.-alanine], and Y.sub.2 -- is the end group, .beta.-alanyl.
The set of monomers used in the designated position D.sub.3 was
extended to include both the L-- and D-- optical isomers of the
common alpha amino acids, and the unusual amino acids,
.alpha.-amino butyric acid, .gamma.-amino butyric acid,
L-norcleucine, sarcosine, ornithine, L-norvaline,
L-homophenylalanine, .beta.-alanine. When these sets of catamers
were reacted with the monoclonal antibody it was found that the
catamers which reacted strongly were:
where P.sub.D and A.sub.D represent the D-optical isomers of
proline and alanine, respectively.
Further sets of catamers were synthesized with the general
formulae:
where the extended set of monomers was used in the designated
position D.sub.4. When these sets of catamers were reacted with the
monoclonal antibody it was found that the catamer which reacted
most strongly was:
where D.sub.D represents the D-optical isomer of aspartic acid.
Further sets of catamers were synthesized with the general
formulae:
where the extended set of monomers was used in the designated
position D.sub.5. When these sets of catamers were reacted with the
monoclonal antibody it was found that the catamer which reacted
most strongly with the antibody was:
where .beta. represents .beta.-alanine and had been included in the
synthesis of the catamer as a spacer.
Pretreatment of the monoclonal antibody with human chorionic
gonadotrophin completely removed the ability of the antibody to
react with the catamer .beta.--R.sub.D --.beta.--P.sub.D
--F--A--D.sub.D thus illustrating the specificity of the
mimotope.
EXAMPLE 6
This Example illustrates the application of the invention to the
detection of a mimotope to a receptor which is not an antigen to
which an antibody has been raised. This Example detects mimotopes
of a receptor to which a virion binds. In this case the test system
for detecting binding to catamers has to be modified. In this
Example, the synthesized catamers are allowed to react with
influenza virus particles (strain A-Shearwater/1/72). After
reaction the catamers are washed to remove unbound virus particles.
The presence of bound virus particles was detected by reacting with
a polyclonal antibody (S227-1) which had been raised against the
haemaglutinin of influenza virus A/Shearwater/1/72. Antibodies
which had reacted with the bound virus were detected in the usual
way by ELISA.
A set of catamers was synthesized with the general formula:
where the end group, Y.sub.1 --, is .beta.-alanyl-.beta.-alanine.
The set of monomers which was used in the designated positions
D.sub.1 and D.sub.2 consisted of the D- and L- optical isomers of
the common alpha amino acids. When the catamers were reacted with a
suspension of influenza virions strain A/Shearwater/1/72, particles
bound to dipeptides at the designated positions:
where the suffix "D" indicates the D-optical isomer of the
indicated amino acid. After removal of the bound virus from the
catamers, the catamers were reacted with the polyclonal antibody
S227-1 at the same concentration as that used to detect bound virus
to ensure that the peaks found were due to binding of virus rather
than binding of the polyclonal antibody.
Further sets of catamers were synthesized with the general
formulae:
where Y.sub.1 -- represents the .beta.-alanine end group and Sp is
an element of the set of spacers, [null, .beta.-alanine]. The set
of monomers used at the designated position D.sub.3 was the
extended set as described in Example 5. When these sets of catamers
were reacted with influenza virus strain A/Shearwater/1/72, virions
bound most strongly to the catamer:
This Example demonstrates that the invention can be applied to the
determination of mimotopes of receptor molecules and ligands in
general. Implementation of the method is limited only by the
ability to detect the presence of binding to the set of
catamers.
* * * * *