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WO1996012186A1 - Methode efficace de decouverte de mimotopes et appareillage correspondant - Google Patents

Methode efficace de decouverte de mimotopes et appareillage correspondant Download PDF

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Publication number
WO1996012186A1
WO1996012186A1 PCT/AU1995/000647 AU9500647W WO9612186A1 WO 1996012186 A1 WO1996012186 A1 WO 1996012186A1 AU 9500647 W AU9500647 W AU 9500647W WO 9612186 A1 WO9612186 A1 WO 9612186A1
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groupings
monomer
oligomers
binary
oligomer
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PCT/AU1995/000647
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English (en)
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Andrew M. Bray
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Chiron Mimotopes Pty. Ltd.
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Priority to EP95933253A priority Critical patent/EP0786085A4/fr
Priority to AU35998/95A priority patent/AU3599895A/en
Publication of WO1996012186A1 publication Critical patent/WO1996012186A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00504—Pins
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/0059—Sequential processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • This invention relates to an efficient method for mimotope discovery, and in particular to a method for elucidating which of a large number of possible oligomers is responsible for a given, known activity.
  • Mi otopes i.e. oligomers that give rise to essentially the same activity as the naturally occurring ligand, can then be identified and synthesized.
  • oligomers e.g. peptides
  • screening procedures are used, often involving the synthesis of many different oligomers of a given length, and conducting assays to determine which of the oligomers is likely to contribute to the observed activity.
  • the activity may be, for instance, binding activity with a particular receptor or enzyme.
  • Mimotope discovery refers to the identification of a novel oligomer (for example) that binds to a receptor.
  • the oligomer may or may not resemble the natural ligand.
  • Such an oligomer can be referred to as a mimotope (although if it were very similar to the native binding species it would be called an analog).
  • a peptide may be identified which binds strongly to a receptor which normally binds a totally unrelated compound.
  • Such a peptide would be a mimotope with respect to the peptide that normally binds to that receptor.
  • An example of this would be a peptide that binds to a receptor that recognizes adrenalin (epinephrine), which is an aromatic amine.
  • Such a peptide would be an adrenalin mimotope.
  • An example in nature is that of morphine (an alkaloid) and the enkephalins (a class of peptides) which both bind to the mu opioid receptor.
  • mimotope discovery involves the preparation of numerous oligomer mixtures, and each mixture is tested against the receptor preparation of interest. Through careful definition of the mixtures and correlation of results, oligomers that are likely to give rise to the observed activity may be elucidated. For the screening of oligomer mixtures, it is useful (1) to minimize the number of times a given oligomer is represented in the total set so that individual mixtures contain the minimum number of oligomers possible, and (2) to group the oligomers such that the smallest number of mixtures gives high activity results
  • the number of test mixtures must also be limited so that valuable receptors are used efficiently
  • the number of discrete peptide mixture to be synthesized could be reduced by grouping the monomer groupings into even larger groupings
  • the monomer groupings might be combined into larger grouping, such as by either (1) using small groupings (or individual monomers) together with complete mixtures, or (2) by using a smaller selection of larger groupings
  • the former was performed in the original approach described by H Mario Geysen in patent application PCT/AU85/00165, WO86/00991 on "Method of determining mimotopes", see also H M Geysen et al , A priori delineation of a peptide which mimics a discontinuous antigenic determinant, Molecular Immunology.
  • the method of the present invention involves first selecting a set of monomers, which will be used to construct a set of oligomers which will be tested for a known activity, such as binding with a receptor.
  • the oligomers may or may not bear resemblance to the native ligands, i.e. the compounds that naturally bind to the receptor. Accordingly, the monomers may or may not bear resemblance to chemical structure(s) present within the native ligand(s); indeed, these structures may not even be known.
  • the method of the invention can thus be carried out even if nothing is known about the native ligand, using oligomers that are not related in any way to the native ligand.
  • a set of such monomers is selected, and they are grouped, preferably according to similarity of chemical characteristics.
  • These monomer mixtures are used to build binary groupings of the sets of monomer mixtures; that is, the set of monomer groupings are divided into two (hence binary) mutually exclusive groupings, each including some of the monomer groupings and not others.
  • These binary groupings are used to form oligomers in numerous combinations on solid supports (for example, pins), with monomers in one of the two binary groupings present at each position in the oligomers.
  • oligomers of length 6 For instance, in an oligomer of length 6, if the first binary grouping is represented as 0 and the second is represented as 1, then all combinations of oligomers from 0-0-0-0-0-0 to 1-1-1-1-1 are formed, where for each 0 or 1 all the monomers in that group are represented in the final set of oligomers. This process is repeated for a second, different binary division of the monomer groupings, and again for a third. The result is three sublibraries of oligomers of varying combinations of the selected monomers, synthesized on solid supports. Additional monomer groupings and/or additional sublibraries can be prepared as required.
  • the monomer groupings can be applied to resins, and the elucidation of the mimotopes can be carried out by the "split resin” approach, wherein the resins that have been used in the preparation of the libraries are divided into discrete batches. These batches are reacted with individual monomers, and then remixed. Testing of the oligomers in this approach is done on the solution phase of the oligomers. This approach yields results as though the monomers were mixed, but avoids the problem of differential rates of coupling, hence variation in degree of monomer incorporation. This is a method that can be used with beaded resins and other beaded supports, but not with the pins used in the preferred embodiment of the invention described below.
  • Figure 1 represents the size of a library of peptide mixtures needed to isolate an active oligomer of size N (i.e. of length N in monomer units).
  • Figure 2 represents the reduction in library size as the number of components in each library mixture is increased.
  • Figure 3 represents the effective library size using sublibraries of mixtures according to the present invention.
  • Figure 4 represents an intersection of the sublibraries of Figure 3.
  • Figure 5 depicts an array of pins and wells used in the process for elucidating oligomers
  • Figure 6 illustrates the exposure of the pins of Figure 4 to a receptor preparation.
  • Figures 7-9 reflect test results from application of the method of the invention to a known serum identified herein as SI 479-1
  • Figures 10-12 are graphs representing the degree of binding of sublibraries of peptide mixtures prepared according to the invention with serum SI 479-1.
  • Figure 13 is a graph illustrating the degree to which particular points (residues) in high-binding peptides identified from Figures 6-8 are conserved
  • Figure 14 illustrates a replacement net study conducted by applicant on the epitope
  • a simple approach to mimotope discovery would be to synthesize each of the oligomers (e.g. peptides) suspected of activity, and to screen all of the synthesized oligomers As discussed above, this would require the synthesis of far too many oligomers to be practical
  • X n N
  • n the number of monomer groupings used in the library construction
  • N the length of the oligomer
  • Another approach to elucidating a mimotope such as an oligomer would be to increase the number of components (oligomers) in each mixture in the library, and decrease the number of mixtures This then decreases the total number of mixtures to be made and maintained in the library, as illustrated by the reduced-size libraries 20 and 30 in Figure 2, but decreases the potential resolution of the system, since if a given mixture is active it is not possible to tell which of the many components of the mixture is responsible for the activity, without a number of deconvolution steps using progressively smaller mixtures.
  • Figure 3 represents a library of mixtures according to the invention, and is constructed in a manner described below
  • monomers refers to a chemical entity which may be covalently linked to one or more other entities to form an oligomer
  • exemplary monomers include D- and L-amino acids, saccharides, nucleotides, peptoids, and the like
  • first and second ends e.g., C-terminal and N-terminal ends, or 5' and 3' ends
  • first and second ends e.g., C-terminal and N-terminal ends, or 5' and 3' ends
  • Terminal monomers need only one attachment site
  • Monomers may be formed "in situ", by the combination of two or more "submonomers” as described in WO94/06451, incorporated herein by reference
  • oligomer refers to a compound which is generated from two or more monomers in combination
  • oligomers will generally comprise about 2-50 monomers, preferably about 2-20, more preferably about 3-10 monomers
  • Oligomers may be linear, branched, or cyclic, and need not retain the original monomer structure apart from the diverse element Group theory basis for the method of the invention
  • monomer groupings are composed of appropriately derivatized (i.e. protected) monomers; they may, as noted above, alternatively be formed using the split resin approach. They are incorporated into oligomers as described below, and are thereby chemically altered, but may in context continue to be referred to as "monomer groupings" for the sake of the group analysis used for the described method of oligomer elucidation. (For example, amino acid mixtures are incorporated into peptides, and are then referred to as amino acid residues.)
  • Binary groupings are groups created, in the preferred embodiment, by mixing monomer groupings together in order to synthesize a sublibrary. For each binary grouping, there is another binary grouping which is its counterpart. Again, they may be formed alternatively by using the split resin approach.
  • Oligomer mixtures Each mixture generated in the library is called an oligomer mixture herein.
  • Each binary library, or "sublibrary" is composed of 2 N oligomer mixtures, which are used in the screening procedure. (4) Intersection mixtures.
  • intersection mixture is a mixture of oligomers common to a set of oligomer mixtures, where one oligomer mixture is drawn from each of the sublibraries; that is, an intersection mixture is the common intersection (in the sense of set intersection) of a number of oligomer mixtures Intersection mixtures are identified in the screening process by inter-sublibrary comparison
  • each sublibrary has a unique pair of binary groupings Discrete mixtures are synthesized using every possible permutation of the two groups of monomer groupings Consequently, each sublibrary contains 2 N discrete oligomer mixtures
  • the binary groupings are constructed by bisecting the total monomer set, as discussed in detail below Prior to bisection, the monomer groupings are organized by a group transformation (symmetry operation) designed to systematically swap monomer groupings between the two (binary) groups Each transformation is unique to each sublibrary Although the reorganization and bisection can be performed by two matrix operators, the total monomer grouping set can be divided into two equal, mutually exclusive groups in a variety of ways
  • Area W in Figure 4 represents all n N oligomer mixtures (defined by monomer groupings a-f) which are common to sublibraries A, B and C Active oligomer intersection mixture "m" is present in all sublibraries A, B and C as a component of the nonidentical discrete mixtures A', B' and C By comparing active mixtures A', B' and C, the common mixture (i.e.
  • intersection mixture "m" is elucidated, hence library resolution is restored by using the three dependent sublibraries
  • This example is very simplistic in that only a single mixture is assumed In practice, this would be demonstrated using a number of highest-activity oligomer mixtures drawn from each sublibrary Sublibrarv Requirements
  • NXm_pen > n ⁇ so Xmin > in n
  • the number of discrete mixtures synthesized is still relatively small compared to the number within library 10, namely; the number of discrete mixtures in library 10 is X n ., preparation-2 N .
  • N length of oligomer in monomer units
  • n number of monomer groupings used in oligomer synthesis
  • X n N (i.e. n raised to the power N), representing the maximum number of discrete mixtures that could be prepared, which is the same as the resolving power of the transformed group library
  • X mm '2 N represents the total number of discrete mixtures that need to be synthesized for the specified parameters.
  • n the number of potential intersections between the sublibraries represented by the (X naturally,i n -2 N ) discrete mixtures.
  • the 192 mixtures mentioned above have 262, 144 intersections, which encompass the 46,656 intersections representing the oligomer mixtures that would have to be made under the aforementioned direct approach.
  • 215,488 intersections are spurious, because for the present example only six, not eight, monomer groupings were used; i.e. only six of eight binary numbers, 000-1 1 1, were used to define monomer groupings.
  • each oligomer mixture within each sublibrary is determined.
  • the intersection mixtures that are identified using the high-activity oligomer mixtures encompass a set of compounds likely to contain high-activity oligomers. Potential constituent monomers and residue positions are identified in this process.
  • intersection 50 between these two sublibraries represents the set of mixtures that produced the expected activity, and thus should include the oligomer responsible for the activity.
  • Group Ao may be defined as including monomer groupings a, b and c, and group Ai as including monomer groupings d, e and f.
  • are used to construct sublibrary A; that is, sublibrary A is composed of the oligomers that are generated from these binary groupings.
  • An oligomer mixture represented by the concatenation can be synthesized, where the # symbol represents either a 0 or a 1 in each position.
  • the binary grouping (a,b,c) is present, and wherever Ai appears, the binary grouping (d,e,f) is present.
  • a concatenation of the form would refer to an oligomer mixture of the form (abc)-(def)-(def)-(def)-(abc)-(def).
  • Each of the monomer groupings stands for one or more monomers, e.g. "a” may stand for the amino acids (V, I and L), “b” may stand for (Y, F and homoPhe), and so on.
  • Table 5 below identifies the amino acids that were used for each of the monomer groupings (a-f) in experiments carried out by applicant. Since A * can stand for either one of two binary groupings, the set of all possible concatenations of 64 possible oligomer mixtures.
  • a ⁇ A ⁇ AKAHA ⁇ AA refers to a set of 64 different oligomer mixtures, and may be represented binarily as the series (000000, 000001, 000010, ..., 1 11 11 1), where a "0" refers to Ao and a " 1 " refers to A ( . Accordingly, the binary grouping (abc) is present wherever a "0" appears, and the binary grouping (def) is present wherever a " 1 " appears.
  • Oligomer mixtures are prepared in a manner to be described below.
  • the resulting mixtures are ⁇ mers (for this embodiment), with a binary grouping Ao or Ai represented at each position in the ⁇ mer. Since each binary grouping includes three monomer groups, and each monomer group in this embodiment includes up to three monomers, the number of ⁇ mers actually formed includes all possible variations on each monomer at each position.
  • the oligomer mixture 011 101 (for sublibrary A) represents a mixture including all possible variations on the ⁇ mer (abc)-(def)-(def)-(abc)-(def), where each of the monomer group designations (a-f) represents all of the up to three monomers represented thereby.
  • a single oligomer mixture such as AoAiAjAiAoAi represents many thousands of actually synthesized ⁇ mers.
  • the six monomer groups in the set (a,b,c,d,e,f) are arranged so that they can be divided into paired binary groupings (such as abc and def discussed above, or alternatively such as abd and cef, etc.) by a symmetry operation.
  • paired binary groupings such as abc and def discussed above, or alternatively such as abd and cef, etc.
  • the organization of the monomer groupings may become important in special cases.
  • the present embodiment uses only three of these possible pairings of binary groupings, which are used to generate sublibraries A, B and C, and which can be derived from one another by a simple set of transformations.
  • the transformation results in a pair of monomer groupings being swapped between groups 0 and 1.
  • Sublibrary A is a pair of binary groupings (abc) and (def) as discussed above, and sublibraries B and C are defined in Table 2.
  • binary grouping B includes the monomer groupings (a, b, f), binary grouping B) includes monomer groupings (c, d, e), and so on.
  • Each sublibrary is constructed using the two binary groupings specific to that sublibrary.
  • binary groupings Ao and Ai are used to construct sublibrary A
  • binary groupings B 0 and Bi are used to construct sublibrary B
  • binary groupings Co and Ci are used to construct sublibrary C (and so on, if there are additional sublibraries).
  • Each sublibrary thus has the same form as the others, and this form is expressible as a sequence of binary numbers, where each digit of each binary number represents the binary grouping incorporated into that position within a given oligomer mixture
  • Each binary number i.e. string of binary digits
  • composition of mixture 010100 in a given sublibrary will depend upon the particular monomer groupings used in its construction.
  • Each sublibrary is represented by the same set of binary numbers (000000 through 1 11 111), but a given binary number represents a different discrete oligomer mixture, depending on which sublibrary it is being used in reference to.
  • the first monomer grouping in the likely oligomer sequence is represented by the motif 001 (or Ao-B C ⁇ ), and the only monomer grouping common to those three mixtures is, in fact "c".
  • the second monomer grouping is in like manner identified as monomer grouping "b", and so on.
  • Each monomer grouping within the binary groups 0 and 1 can similarly be identified by a unique motif. Reorganization of Table 2 yields these motifs. A table of such motifs can be generated, with a unique binary number for each monomer grouping:
  • the motif "000" is indicative of "a" at the third oligomer position; that is, if each of the active peptide mixtures identified in sublibraries A, B' and C has its respective binary grouping represented at position 3, this means that position 3 for the A' set of mixtures included (a, b, c); for the B' set of mixtures it included (f, a, b); and for the C set of mixtures it included (e, f, a). Since the only common monomer grouping among these three sets is monomer grouping "a", it may be concluded that the active oligomer(s) contain, at position 3, one or more monomers from monomer grouping "a".
  • residues that are responsible for activity can be encompassed by an oligomer containing fewer than N residues, then residues at either or both of the N-and C-termini may play little role in the binding activity. In such cases, a "frame-shifting" of the residues may be possible. For instance, if a binding residue pattern of #001 1# is observed (assuming for the moment that the 001 1 sequence is responsible for the binding), the residue pattern 001 1## may bind equally or nearly as well, if the sequence of oligomers primarily responsible for the binding activity is the group 001 1. This example might yield good binding results for all of the following sequences
  • oligomers in general can be used with peptides, or indeed any amino acid or other monomer desired, such as groupings of amines, carboxylic acids, sugars, nucleic acids, and in general any chemical building blocks that may be linked in oligomers. See for example, WO91/19735, incorporated herein by reference, for a description of non-peptide oligomers ("peptoids") within the scope of this invention. Oligomers in the range of 3mer to >6mers may easily be studied by this approach, with the number of primary monomer groupings ranging from four and up (where n should be an even integer).
  • the method of the invention may be used in either support -bound or solution-phase assays, i.e. oligomers retained on solid supports or cleaved therefrom for testing.
  • the sublibraries are prepared, and are assayed by contacting the oligomers with a target (e.g., a receptor, enzyme, oligonucleotide, whole cell, organism, tissue culture, and the like) and detecting the presence or absence of a desired interaction between the target and the oligomer.
  • a target e.g., a receptor, enzyme, oligonucleotide, whole cell, organism, tissue culture, and the like
  • the desired activity may be inhibition or potentiation of an enzymatic activity (for example, inhibition of dihydrofolate reductase activity), growth or survival of a cell line, binding to a specific protein (for example, binding to antisera characteristic of a particular infection, used to diagnose presence of the infection in the serum donor), and the like
  • an enzymatic activity for example, inhibition of dihydrofolate reductase activity
  • a specific protein for example, binding to antisera characteristic of a particular infection, used to diagnose presence of the infection in the serum donor
  • the oligomers are presented bound to a solid phase and are contacted with the target The target is allowed to bind, and non-binding target is washed away
  • the presence of target bound to specific oligomers is detected, for example, by using an anti-target antibody labeled with a detectable label (e.g., radioactive atoms or enzymes capable of catalyzing a detectable color change)
  • a detectable label e.g.,
  • Alternative embodiments of the method include ( 1 ) application to oligomer mixtures where one or more positions are invariant, and (2) use in combination with complete mixtures, i.e. a hybrid between the defined-position strategy and the transformed- group strategy
  • a aliphatic amino acids
  • b aromatic amino acids
  • c acidic amino acids
  • d hydrophilic amino acids
  • e small amino acids
  • f basic amino acids
  • the pins were derivatized with Boc- hexamethylene-l,6-diamine, Boc deprotected by treatment with trifluoroacetic acid, treated with triethylamine in dimethylformamide, washed with dimethylformamide and methanol and air dried, and coupled with Fmoc-Gly- OH/diisopropylcarbodiimide/1- hydroxybenzotriazole (60 mM, 60 mM, 72 mM) in DMF to give a Gly loading of 1 micromole/detachable pin head (see R M Valerio, A M Bray, R A Campbell, A DiPasquale, C Margellis, S J Rodda, H M Geysen and N J Maeji (1993), Multipin peptide synthesis at the micromole scale using 2-hydroxyethyl methacrylate grafted polyethylene supports. International Journal of Peptide and Protein Research.
  • A(0) ⁇ or, alternatively notated, Ao' ⁇ a (4 ml) + b (4 ml) + a (4 ml)
  • the synthesis of peptide mixtures was then performed as follows: Fmoc deprotection.
  • the pins were treated with 20% piperidine/dimethylformamide (50 mL/96 pins) for 30 minutes.
  • the pins were then washed with dimethylformamide (50 mL/96 pins) for 5 minutes.
  • the pins were then fully immersed into methanol and then washed with two further lots of methanol (100 mL/96 pins) and then air dried. Coupling amino acid mixtures.
  • ⁇ B 0 ⁇ Bi', Co' and C were added to the wells of two 96 well polypropylene microtitre trays (60 microlitres/well ) in the dispensing patterns presented in Table 6 below.
  • a solution of BOP/hydroxybenzo- triazole/N-methylmorpholine ( 166 mM/166 mM/250 mM) in dimethylformamide was then added to each well (90 microlitres/well).
  • the pins were then immersed into the solution and allowed to react for at least 2 hours.
  • the pins were then removed from the wells and washed with dimethylformamide (50 mL/96 pins) and methanol (2 x 50 mL/96 pins), and air dried.
  • mixture 000000 for the sublibrary A set is prepared from six successive couplings of the Ao mixture to a given pin, which as noted above includes the monomer groupings (a, b, c) mixed together (and each of a, b and c includes the amino acids noted in Table 5 above)
  • a portion of At is placed in designated wells 40 of a reaction tray 50 such as that shown in Figure 5 (and as represented by the top left well ⁇ and indeed in all wells represented by "A0" — in Coupling 1, Tray 1 in Table 6)
  • the portion of Ao is coupled to a pin 80 attached to a carrier 90 by exposing the pin to the portion (see Figure 5), i.e. by placing the pins into the solutions in the wells In this way, A. is coupled (covalently bonded) to the pins (solid supports).
  • Ai, B 0 and Bi are coupled to other pins (see Table 6, Coupling #1, Tray 1).
  • Coupling #2 the second set of sublibrary mixtures is used in the wells (see Table 6, Coupling #2, Tray 1).
  • the oligomers are thereby synthesized on the pins. Note that, while an eight-by-eight array of pins and wells is represented in Figure 5, any convenient configuration may be used. For instance, in the procedure actually carried out using the wells represented in Table 6, and eight-by-twelve array was used.
  • oligomers This may be represented as 0-[support], where [support] represents the solid support, and 0 represents the addition of Ao.
  • An array of pins is used to build up the oligomers, in a conventional fashion. It will be appreciated that multiple peptide mixtures are built up simultaneously in this fashion, namely 64 at a time for each tray listed in Table 6. Thus, very rapidly thousands of oligomers are formed on the pins, and these oligomers are to be used in the testing.
  • synthesis media suitable for solid- phase synthesis could equally well be used, such as "tea bags", i.e. mesh packets of resin.
  • Alternative support structures suitable for use in the method of the invention include appropriately derivatized glass, functionalized cellulose paper, polymer-grafted plastics, and the like. If resin is used, then the split resin approach may be used, as mentioned above in the Summary of the Invention.
  • Each amino acid in the mixture such as A > will normally include a protecting group, as indicated above, which is removed in conventional fashion at this point.
  • Serum 1479- 1 is applicant's designation for an antihemagluttin monoclonal antibody (IgG) preparation available (as serum “17D09”) from the Scripps Clinic and Research Foundation Serum SI 479-1 is well understood (see Figure 14), and its antibody epitope is known to be DVPDYA.
  • IgG antihemagluttin monoclonal antibody
  • Figure 14 its antibody epitope is known to be DVPDYA.
  • the ELISA test was carried out according to the method described in the aforementioned Geysen article ("Strategies for epitope analysis using peptide synthesis").
  • the three sublibraries were formed on 192 pins (64 per sublibrary), and the pins were exposed to serum S I 479- 1, as illustrated in Figure 6, wherein the pins 80 mounted on holder 90 are submerged into a container 60 partially filled with a liquid 70 containing the serum.
  • the block After exposure to the serum preparation and rinsing to remove unbound antibody, the block is similarly submerged in a solution containing a horseradish peroxidase-labelled anti-IgG detecting antibody.
  • the detecting antibody binds to the S I 479-1 monoclonal antibody.
  • pins that contain peptides to which the monoclonal antibody binds also bind the detecting antibody.
  • the enzyme is detected by immersing the pins into microtitre plate wells which contain an enzyme substrate solution.
  • the target oligomers may be cleaved from the solid phase synthesis support by applying a cleavage reagent (usually a volatile acid), which is then removed, generally by evaporation.
  • a cleavage reagent usually a volatile acid
  • the dried peptides are then redissolved in a solution appropriate for subsequent testing.
  • the enzyme substrate solution into which the pins are immersed may be ABTS (Boehringer Mannheim cat. no 122661, 0.5 g/L) and hydrogen peroxide (120 vol, 0.3 mL/L) in pH 4 phosphate/citrate buffer. Color development was stopped simultaneously in all wells by removing the pins from the substrate solution. Absorbances were measured on a Titertek Multiskan MC plate reader at 405 nm against a reference wavelength of 492 nm.
  • FIG. 7 shows the results for sublibrary A; it will be noted that the second column includes an entry for each of the binary numbers listed in Table 7 above.
  • the first column of Figure 7 lists the ELISA absorbance results (in milliabsorbance units).
  • mixture Ao-Ao-Aj-Ao-Ao-Ai showed the highest binding with serum SI 479-1, with an ELISA absorbance value of 61 1.
  • the next highest binding occurred with Ao-Ao-Ao-Ao-Ao-Ao- A
  • Figure 8 shows the same type of results for the B sublibrary, with mixture 101101 showing the greatest activity
  • Figure 9 shows the results for the C sublibrary, where mixture 1 101 10 shows the greatest activity.
  • the numerical results of Figures 7-9 are graphically depicted in Figures 10-12.
  • Figure 13 is a graph showing the complement comparisons for each of the top- scoring mixtures of sublibraries A, B and C. The six points on each curve are taken from the six figures across the tops of Figures 7-9, respectively. Note that the higher the y- coordinate value, the greater the degree to which a given binary grouping (and hence one or more monomers within that binary grouping) is conserved at the respective position.
  • Table 8 below is a partial listing of the results shown in Figures 7-9: Table 8: Com lement Values for To -Scorin Mixtur
  • Residue 2 has a "preference” for b (reading the motif 001 in Table 9, with the "c” value taken from Table 3)
  • Residue 3 is not highly conserved, i.e. is relatively tolerant to change (note that even the highest complement values are rather low)
  • Residue 6 has a preference for "e” (motif 1 10), but is tolerant of "d” (motif 1 1 1, see the rightmost column at the top entry of library C, showing that a complement substitution leads to very little ELISA value change)
  • Figure 14 shows a single-point replacement study of the peptide DVPDYA, according to the technique described in the aforementioned Geysen article ("Strategies for epitope analysis using peptide synthesis") It will be seen that the results are quite similar to the above experimental results
  • the graphs of Figure 14 show how other amino acids in the positions noted affect the activity of the peptide
  • E is the amino acid that, when substituted at that point, compares best with the activity resulting from D In position 2, most residues could be acceptable substitutes for V, and in position 3, most residues could be acceptable substitutes for P In position 4, the native residue D is preferred, with C and H being acceptable replacements, all other substitutions would yield lower activity.
  • Position 5 is seen to be very highly conserved, inasmuch as the ability of the peptide to bind the monoclonal antibody is greatly diminished if Y is substituted by any of the other amino acids.
  • the native residue A is preferred, although G is an acceptable substitute.
  • a deconvolution procedure is used whereby the experimenter constructs a new library, including each of the possible combinations suggested by a predetermined number of top-scoring mixtures, and/or including each combination involving a mixture having an ELISA score above a certain threshold (such as about 200) — i e , only those mixtures that show activity significantly greater than the background result
  • position 6 can be modified or deleted without complete loss of binding activity.
  • the pattern cb@cb must be conserved for any binding to remain. Note that as these peptides are bound to a solid support at the C-terminal, i.e. the RHS (right- hand side), the linkage to the polymer may be acting as a poor substitute for the acceptable position 6 (e) in the non-frame-shifted species.
  • Table 1 1 presents rank-ordered ELISA results for the binding of 108 mixtures derived from the top intersection mixtures cbecbe, cbdcbe, and cbecbe to SI 479-1.
  • Five positions were defined, i.e. single amino acids coupled.
  • Position 3 which appears to be highly replaceable, was coupled as a mixture of all components of c, d and e, namely: A, G, P, D, E, S, N and Q (defined here as X).
  • HomoPhe is defined in Table 1 1 as #. These were compared with four copies each of DVPDYA (epitope, positive control) and ASQGGL (an unrelated peptide unlikely to bind to S I 479-1, negative control).
  • An even number of monomer groupings can be divided into two paired sets in a variety of ways.
  • the organization process can be described by two matrix operations. The first mixes the monomer groupings, and the second selects either group from the monomer grouping set.
  • the following example constructs binary grouping B(0) from the set of monomer groupings given above.

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Abstract

L'invention concerne une méthode permettant d'identifier quels oligomères, des peptides, par exemple, et ce, en dehors d'une pluralité d'oligomères de synthèse, sont susceptibles d'être responsables d'une activité connue. La méthode consiste d'abord à sélectionner un ensemble de monomères supposés se trouver dans les oligomères en question. Ces monomères sont groupés et mélangés, selon des propriétés similaires, chaque monomère n'étant représenté que dans un seul groupement de monomères. Les ensembles de groupements sont alors divisés pour former une première paire de groupements binaires tandis que des mélanges de monomères sont constitués à partir des groupements binaires, par synthèse, sur des supports, d'oligomères d'une longueur prédéterminée (par exemple de longueur 6, c'est-à-dire formant des hexamères). Chaque position dans chacun des hexamères est représentée par l'un ou l'autre des groupements binaires, le tout constituant une première banque secondaire d'oligomères synthétisés. Les ensembles de groupements de monomères sont alors divisés différemment pour former une deuxième paire de groupements binaires, tandis que d'autres mélanges d'oligomères sont confectionnées à partir de cette deuxième paire, instituant de la sorte une deuxième banque secondaire d'oligomères synthétisés. Une troisième banque est constituée de la même façon en faisant appel à une troisième paire de groupements binaires. Les trois paires de groupements binaires sont établies de manière à ce que deux groupements quelconques de monomères ne soient réunis que dans un seul groupement binaire d'une banque secondaire. On fait réagir les banques secondaires dans le cadre d'une activité connue et l'on détermine quels sont les contributeurs les plus efficaces dans le cadre de cette activité. Les mélanges d'oligomères qui manifestement s'avèrent être les plus grands contributeurs sont mis en corrélation croisée pour produire des oligomères susceptibles de témoigner d'une grande activité. Ces oligomères sont alors synthétisés et mis à l'épreuve, soit directement, soit en reprenant le procédé décrit ci-dessus, ce qui permet de déterminer des oligomèrs susceptibles de contribuer à l'activité susmentionnée, avec un nombre relativement peu élevé d'oligomères à synthétiser.
PCT/AU1995/000647 1994-10-14 1995-10-04 Methode efficace de decouverte de mimotopes et appareillage correspondant WO1996012186A1 (fr)

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AU35998/95A AU3599895A (en) 1994-10-14 1995-10-04 Method and apparatus for efficient mimotope discovery

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5647886A (en) * 1985-04-22 1986-10-30 Coselco Mimotopes Pty Ltd Improved method for determining mimotopes
AU3101989A (en) * 1988-03-04 1989-09-07 Board Of Trustees Of The University Of Alabama For And On Behalf Of The University Of Alabama In Huntsville, The Peptide equivalent of non-peptides and methods of design thereof
WO1992000091A1 (fr) * 1990-07-02 1992-01-09 Bioligand, Inc. Banque de bio-oligomeres aleatoires, son procede de synthese et son mode d'emploi
AU9052891A (en) * 1990-12-05 1992-07-08 Novo Nordisk A/S Proteins with changed epitopes and methods for the production thereof
US5384263A (en) * 1987-10-13 1995-01-24 Terrapin Technologies, Inc. Method to produce immunodiagnostic reagents
WO1995011998A1 (fr) * 1993-10-26 1995-05-04 United Biomedical, Inc. Bibliotheques structurees d'antigenes de synthese utilisables a des fins de diagnostic, de vaccin et de therapie
US5432018A (en) * 1990-06-20 1995-07-11 Affymax Technologies N.V. Peptide library and screening systems
AU1738395A (en) * 1994-01-31 1995-08-15 University Of North Carolina At Chapel Hill, The Reagents binding vinculin, dynein, and glutathione s-transferase from peptide libraries

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5647886A (en) * 1985-04-22 1986-10-30 Coselco Mimotopes Pty Ltd Improved method for determining mimotopes
US5384263A (en) * 1987-10-13 1995-01-24 Terrapin Technologies, Inc. Method to produce immunodiagnostic reagents
AU3101989A (en) * 1988-03-04 1989-09-07 Board Of Trustees Of The University Of Alabama For And On Behalf Of The University Of Alabama In Huntsville, The Peptide equivalent of non-peptides and methods of design thereof
US5432018A (en) * 1990-06-20 1995-07-11 Affymax Technologies N.V. Peptide library and screening systems
WO1992000091A1 (fr) * 1990-07-02 1992-01-09 Bioligand, Inc. Banque de bio-oligomeres aleatoires, son procede de synthese et son mode d'emploi
AU9052891A (en) * 1990-12-05 1992-07-08 Novo Nordisk A/S Proteins with changed epitopes and methods for the production thereof
WO1995011998A1 (fr) * 1993-10-26 1995-05-04 United Biomedical, Inc. Bibliotheques structurees d'antigenes de synthese utilisables a des fins de diagnostic, de vaccin et de therapie
AU1738395A (en) * 1994-01-31 1995-08-15 University Of North Carolina At Chapel Hill, The Reagents binding vinculin, dynein, and glutathione s-transferase from peptide libraries

Non-Patent Citations (10)

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Title
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, Vol. 3, No. 3, issued 1993, J.M. KERR et al., "Identification of Antibody Mimotopes Containing Non-Natural Amino Acids by Recombinant and Synthetic Peptide Library Affinity Selection Methods", pages 463-468. *
JOURNAL OF COMPUTER AIDED MOLECULAR DESIGN, Vol. 9, No. 3, issued 1995, FRENKEL D. et al., "PRO-LIGAND: An Approach to de Novo Molecular Design. 4. Application to the Design of Peptides", pages 213-225. *
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol. 117, No. 19, issued 17 May 1995, BENOIT DEPREZ et al., "Orthogonal Combinatorial Chemical Libraries", pages 5405-5406. *
MOLECULAR IMMUNOLOGY, Vol. 23, No. 7, issued 1986, H.M. GEYSEN et al., "A Priori Delineation of a Peptide Which Mimics a Discontinuous Antigenic Determinant", pages 709-715. *
NATURE, Vol. 251, issued 15 February 1991, S.P.A. FODOR et al., "Light-directed, Spatially Addressable Parallel Chemical Synthesis", pages 767-773. *
NATURE, Vol. 354, issued 7 November 1991, R.A. HOUGHTEN et al., "Generation and Use of Synthetic Peptide Combinatorial Libraries for Basic Research and Drug Discovery", pages 84-86. *
PEPTIDE RESEARCH, Vol. 5, No. 5, issued 1992, A. VAN AMERONGEN et al., "Peptides Reactive With a Transmission-Blocking Monoclonal Antibody Against Plasmodium Falciparum Pfs 25: 2000 - Fold Affinity Increase by PEPSCAN-Based Amino Acid Substitutions", pages 269-274. *
PEPTIDE RESEARCH, Vol. 7, No. 1, issued 1994, A. WALLACE et al., "A Multimeric Synthetic Peptide Combinatorial Library", pp. 27-31. *
SCIENCE, Vol. 249, issued 27 July 1990, J.K. SCOTT et al., "Searching for Peptide Ligands With an Epitope Library", pages 386-390. *
See also references of EP0786085A4 *

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