WO1999010379A1 - Cristal de l'anticorps sm3 (fragment) et de l'epitope de reconnaissance, sa preparation, support de donnees codees contenant ses coordonnees et son utilisation diagnostique ou medicale - Google Patents

Cristal de l'anticorps sm3 (fragment) et de l'epitope de reconnaissance, sa preparation, support de donnees codees contenant ses coordonnees et son utilisation diagnostique ou medicale Download PDF

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Publication number
WO1999010379A1
WO1999010379A1 PCT/GB1998/002542 GB9802542W WO9910379A1 WO 1999010379 A1 WO1999010379 A1 WO 1999010379A1 GB 9802542 W GB9802542 W GB 9802542W WO 9910379 A1 WO9910379 A1 WO 9910379A1
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WIPO (PCT)
Prior art keywords
peptide
mimic
mucl
antibody
epitope
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PCT/GB1998/002542
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English (en)
Inventor
Paul Simon Freemont
David Snary
Michael Joseph Ezra Sternberg
Paul Alan Bates
Pawel Dokurno
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Imperial Cancer Research Technology Limited
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Publication date
Application filed by Imperial Cancer Research Technology Limited filed Critical Imperial Cancer Research Technology Limited
Priority to EP98940371A priority Critical patent/EP0996634A1/fr
Priority to CA002300370A priority patent/CA2300370A1/fr
Priority to IL13454798A priority patent/IL134547A0/xx
Priority to AU88711/98A priority patent/AU751386B2/en
Priority to JP2000507705A priority patent/JP2001514188A/ja
Publication of WO1999010379A1 publication Critical patent/WO1999010379A1/fr
Priority to NO20000845A priority patent/NO20000845L/no

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4727—Mucins, e.g. human intestinal mucin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • SM3 antibody was raised against MUCl stripped of its carbohydrate (Burchell et al . , 1987). SM3 recognises such a cryptic epitope, and shows high selectivity in reacting specifically with the carcinoma associated mucin in more than 90% of breast carcinomas (Girling et al . , 1989) .
  • the high specificity of the SM3 antibody makes it a potentially useful tool in the diagnosis and treatment of breast cancer (Granowska et al . , 1996).
  • the sequence of the repeating unit of the core protein of MUCl contains doublets of threonine and serine, bounding a highly immunogenic domain (Burchell et al . , 1989; see Figure 1).
  • a three-dimensional NMR structure for three multiple MUCl peptide repeats reveals repeating "knob-like" structures, corresponding to the immunogenic domains which are connected by extended spacers (Fontenot et al . , 1995).
  • the epitope for SM3 has been mapped using overlapping peptides and found to correspond to just five contiguous amino acids, Pro-Asp- Thr-Arg-Pro (Burchell et al .
  • the MUCl epitope lying within the "knob-like" domain and between the potential serine and threonine glycosylation sites.
  • the MUCl epitope lies within the "knob-like" domain and between the potential serine and threonine glycosylation sites.
  • the inventors have crystallised a fragment of SM3 bound to a peptide, subjected the crystal to X-ray diffraction studies and measured the structure factors. From the structure factors they have solved the structural coordinates. These may be used to generate new diagnostic and therapeutic materials and for other investigative purposes .
  • the invention includes the use of the structure factors and/or structural coordinates to identify, characterise, design or screen chemical entities, which have uses in diagnosis and therapy, as will be discussed below.
  • the present invention provides the use of the structure factors and/or structural coordinates obtainable by subjecting a crystal comprising at least the epitope binding fragment of the SM3 antibody, bound to a peptide recognised by the epitope binding site of SM3 to X-ray diffraction measurements and optionally thereafter analysing the diffraction measurements to deduce structural coordinates.
  • the invention provides the use of the structure factors and/or structural coordinates of a crystal of the Fab fragment of SM3 bound to the crystal peptide.
  • the structure factors of such a crystal obtained as in the Examples are shown in Table 1 and the structural coordinates are shown in Tables 2a and 2b.
  • the structural coordinates shown in each of Tables 2a and 2b are derived from the structure factors . However the coordinates shown in Table 2b have been calculated to a higher refinement and include additional protein atoms.
  • the invention provides the use of the structural coordinates shown in Table 2a and/or Table 2b.
  • the structural coordinates indicate the positions of individual atoms within the crystal and indicate the B factor for each atom which gives some information about the mobility of the atoms.
  • the avidity may be increased by modifying the epitope binding site structure to increase the amount and number of interactions favourable to peptide binding and/or diminish unfavourable interactions between the peptide and antibody.
  • Favourable interactions may be increased by extending the structure of the epitope binding site into spaces which are shown in the two dimensional or three dimensional representations to be unoccupied or filled with water molecules . Such water molecules may include those which are shown in Figures 8 to 11.
  • the representations of the structures may be used in other ways to modify the structure of SM3. It is believed that SM3 may bind the peptide by an "induced fit" method which requires that conformation changes occur in the structure of SM3 during the process of binding the peptide.
  • SM3 has a low avidity for aberrantly glycosylated MUCl is steric hindrance between residues close to or in the epitope binding site and carbohydrate moieties attached to positions 1, 12 and 13 of the peptide as shown in Figure 1.
  • the representation of the epitope binding site bound to the peptide may be used to predict which residues of SM3 are likely to be involved in the steric hindrance.
  • One such residue may be Proline 56 of SM3.
  • Such residues may be modified, replaced or deleted to decrease the steric hindrance in order to increase avidity.
  • the avidity of the mimic of SM3 to bind aberrantly glycosylated MUCl can be tested in an ELISA assay as detailed below: GST-Muc-1 ELISA Method
  • the cells were grown in L broth (lOOmg/ml ampicillin) overnight and then IPTG was added to a final concentration of 0.5 mM followed by a further incubation of 4 hr at 37 C
  • the cells were pelleted (20min/10000rpm) , washed x2 with PBSa and resuspended in 25mM TRIS pH 8, 25mM Glucose, 10 mM EDTA containing lyzozyme (4mg/ml) and left for 30 min at RT . 5ml of lysis buffer was then added and incubated on ice for 5 min.
  • Lysis Buffer 150 100 mM NaCl, 16 mM Na2HP04, 4mM NaH2P04, lOOmM EDTA, 1% Triton X (BDH Anala R) 2 mM PMSF (Phenylmethylsulfonyl fluoride)
  • Test antibody samples are diluted in PBSA/ 0.02% Tween 20 (PBSA/T). e.g. SM3 from 100-0.001 mg/ml.
  • a histological screen using the mimic of SM3 can be performed using tumour tissue from a breast tumour and normal tissue, for example, as described in Girling et al . , 1989. This can be used to determine if the mimic is specific for the aberrantly glycosylated MUCl and therefore suitable for use in a method of diagnosing breast cancer.
  • the mimic can also be tested against live tumour cells which are not fixed. Cells which have MUCl with reduced levels of glycosylation can be produced by the use of metabolic inhibitors of O-linked chain extension, such as 0- benzylgalactosamine . Such cells can be used to study the effects of low levels of glycosylation of MUCl on the avidity of the mimics of SM3.
  • Mimics of SM3 may be used in a diagnostic test to detect the presence of tumour cells in a tissue sample, for example in a histological screening. Mimics used in this manner may be labelled with a detectable label. Alternatively agents able to specifically bind such mimics may be used to detect the presence of the mimics once the mimics have bound the aberrantly glycosylated.
  • the mimics of SM3 may be used in vivo for the detection of tumour cells. They may be used in tumour imaging in vivo . Generally, such mimics would be labelled with a detectable label.
  • the mimics of SM3 can be used in a method of therapy against cancer, particularly adenocarcinomas such as ovary, colon, lung, pancreas and breast epithelial cancers, especially breast cancers.
  • adenocarcinomas such as ovary, colon, lung, pancreas and breast epithelial cancers, especially breast cancers.
  • Mimics which are antibodies or substantially similar to antibodies or fragments of antibodies may bind to aberrantly glycosylated MUCl on the surface of tumour cells and aid the killing of the tumour cells by recruiting the patients immune system.
  • Mimics of SM3 may be chemically linked to a cytotoxic agents such as a toxin or a radioisotope . Binding of such toxin linked mimics to the tumour cells would lead to the killing of the tumour cell. It is believed that high levels of MUCl or aberrantly glycosylated forms of MUCl may have an immunosuppressive effect. Tumour cells may have high levels of MUCl on their surface and/or aberrantly glycosylated forms.
  • a mimic of SM3 could be used to bind to MUCl in vivo and prevent or decrease its immunosuppressive effects.
  • the types of immunosuppressive effects that may be prevented or decreased are discussed below in relation to mimics of the MUCl epitope.
  • Such a mimic could be administered in conjunction with an anti- tumour agent, such as an anti-tumour vaccine, and may have an adjuvant-like effect (the mimic may increase the immune response generated by the vaccine) .
  • the anti- tumour agent could be a mimic of the SM3 epitope.
  • the mimic of SM3 could be administered at any time in relation to the administration of the anti-tumour agent, for example before, with or after the administration of the anti-tumour agent.
  • the invention provides a mimic of SM3 for use in a method for treatment of the human or animal body by surgery or therapy or in a diagnostic method practised on the human or animal body, such as in anti-cancer therapy.
  • the invention also provides a product comprising a mimic of SM3 and an anti-tumour agent as a combined preparation for simultaneous, separate or sequential use in anti- cancer therapy.
  • the invention provides a use of a mimic of SM3 in the production of a pharmaceutical composition for use in the methods discussed above.
  • the invention provides a pharmaceutical composition containing a mimic of SM3 and a diluent or carrier.
  • the invention provides a method of treating or diagnosing cancer by administering to a human or non-human animal in need thereof an effective non- toxic amount of a mimic of SM3.
  • the production of mimics of SM3 and the methods, routes and dosages for use of the mimics of SM3 are discussed below.
  • the invention allows the use of the structure factors and the structural coordinates to identify, characterise or design a mimic of the MUCl epitope peptides.
  • a mimic may be used to produce a specific binding agent which has a desired association with aberrantly glycosylated MUCl.
  • the mimic of the peptide can be used to select a specific binding agent from a library on the basis of its affinity to the mimic.
  • a library may be a microbial display library, such as a phage display library.
  • the specific binding agent may bind aberrantly glycosylated MUCl, and therefore be used in a similar manner to the mimics of SM3.
  • the mimic of the peptide may be used in a vaccine administered to a human or animal. Such a vaccine would stimulate the production of antibodies able to bind aberrantly glycosylated MUCl. Such a vaccine could therefore be used as a therapy against cancer, for example to prevent or treat cancer.
  • the invention provides a mimic of the MUCl epitope, a mimic of the MUCl epitope for use a method for treatment of the human or animal body by surgery or therapy or in a diagnostic method practised on the human or animal body, such as in anti-cancer therapy.
  • the invention also provides a use of such a mimic in the production of a pharmaceutical composition for use in such a method.
  • the invention provides a pharmaceutical composition containing a mimic of the MUCl epitope and a diluent or carrier therefor.
  • the invention provides a method of diagnosing or treating cancer by administrating an effective non-toxic amount of a mimic of the MUCl epitope to a human or non-human animal in need thereof.
  • the production of mimics of the MUCl epitope and methods, routes and dosages for use of the mimics of the MUCl epitope are discussed below.
  • the MUCl epitope mimics may also be administered to an animal preferably a laboratory mammal, such as a rodent, for instance a mouse, in order to induce an antibody response.
  • Antibody secreting cells may be recovered from the animal and hybridoma technology may then be used to produce a hybridoma using such recovered cells.
  • the hybridoma or any other antibody expression system would be a source of monoclonal antibodies capable of recognising the mimic and preferably the MUCl epitope and thus have diagnostic and therapeutic utilities as for the SM3 mimics described above.
  • the mimic may cause a decrease in (i)the proliferation of the T cells, (ii) their secretion of cytokines, (iii) their interaction with other cells, such as interaction mediated by ICAM-1 on the T cell surface, and/or (iv) their cytotoxic activity.
  • the mimic may cause the cross-linking of a surface molecule on the T cells, such as ICAM-1.
  • the mimic may cause T cells to become anergic.
  • the effects of the mimic may be reversible by the addition of IL-2 or anti-CD28 antibody.
  • the mimic may be used to decrease or prevent an immune response, particularly when the response has a deleterious effect on the body.
  • the mimic may be used in the therapy (i.e. to treat or prevent) of diseases caused by autoimmune responses (such as arthritis, multiple sclerosis, asthma or diabetes), allergies, inflammatory disorders or transplant rejections, such as graft versus host disease.
  • the invention provides a mimic of the MUCl epitope for use in therapy which prevents or decreases an immune response, and so can treat or prevent a disease caused by an immune response.
  • the invention also provides a use of such a mimic in the production of a pharmaceutical composition for use in such a method.
  • the invention provides a method of treating a disease caused by an immune response, such as autoimmune disease, allergy, inflammatory disorder or transplant rejection; by administrating an effective non-toxic amount of a mimic of the MUCl epitope to a human or non-human animal in need thereof.
  • the two dimensional and three dimensional representations of the epitope binding site bound to the peptide mentioned previously can be used to design a mimic which is, for instance, a peptide or a derivative thereof or an analogue of a peptide comprising the sequence Pro-Asp-Thr-Arg-Pro or the sequence Thr-Ser-Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-Ser constrained in the conformation approximately that which the crystal peptide adopts when bound to SM3.
  • the sequence can be constrained by the introduction of covalent bonds between atoms which come close together in the conformation adopted when bound to SM3. Alternatively a chemical linker may be used to join such atoms together.
  • the mimic can be modified to increase the amount and number of favourable interactions with SM3, for example by extending the structure of the MUCl epitope into spaces which are shown to be unoccupied or filled with water molecules.
  • water molecules may include those shown in Figures 8 to 11. It is appreciated that such mimics of the MUCl epitope which are designed to have an increased avidity with the epitope binding site of SM3 may not cause the production of antibodies with a higher avidity to aberrantly glycosylated MUCl than SM3 has, however such mimics may lead to the stimulation of an increased antibody response due to their higher avidity to the antibody.
  • the two dimensional and three dimensional representation of the epitope binding site bound to the crystal peptide can as mentioned previously be used to determine why SM3 binds the peptide with a low avidity.
  • This information can then be used to design mimics of the MUCl epitope which can select from a library those mimics of SM3 which have a high avidity to aberrantly glycosylated MUCl.
  • Such mimics when administered in a vaccine may also stimulate the production of antibodies with a higher avidity to aberrantly glycosylated MUCl than SM3.
  • Such mimics of the MUCl epitope may for example be designed by modifying the structure of the crystal peptide which binds SM3 in those areas where unfavourable interactions occur between the crystal peptide and epitope binding site which cause a lowering of avidity.
  • the structure of the MUCl epitope may be extended in those areas where steric hinderance occurs between the carbohydrates on the aberrantly glycosylated MUCl and the epitope binding site.
  • Use of such a mimic may lead to the selection of specific binding agents (as described above) from a library or the stimulation of antibodies which have a reduced level of steric hinderance with the carbohydrate and therefore a higher avidity for aberrantly glycosylated MUCl.
  • Mimics of the MUCl epitope may be designed by computer modelling techniques in a similar manner to the designing of mimics of SM3 using these techniques. Again, packages such as Catalyst/SHAPE and
  • Catalyst/COMPARE can be used to select compounds with a similar three dimensional shape to the peptide when it is bound to SM3.
  • Packages such as DBServer and HipHop can be used to find compounds with similar pharmacophores to the peptide and packages such as Ludi, MCSS and Hook can be used to design a mimic which is predicted to bind well to the epitope binding site of SM3.
  • the efficiency with which that mimic may bind to the SM3 epitope binding site may be tested and optimized using computational or experimental evaluation.
  • computational or experimental evaluation As described previously packages such as Catalyst/SHAPE, Catalyst/COMPARE, DBServer, Ludi and MCSS may be used to computationally evaluate the mimic.
  • the mimic may be experimentally evaluated in the competition ELISA assay described below.
  • Various parameters can be optimized depending on the desired result. These include, but are not limited to, specificity, avidity, on/off rates, and other characteristics readily identifiable by the skilled artisan .
  • substitutions, deletions, or insertions in some of the components of the MUCl epitope in order to improve or modify the binding properties.
  • initial substitutions are conservative, i.e. the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original component.
  • the mimic of the MUCl epitope may be glycosylated.
  • the mimic may have an intra-peptide hydrogen bond. This may be between the residues equivalent to Pro 4 and Thr 6 of the MUCl epitope or between the residues equivalent to Asp 5 and Arg 7.
  • the atoms involved in the hydrogen bond may be the equivalent of Pro 4 (atom 0) , Thr 6 (atom N) , Asp 5 (atom OD1) and/or Arg 7 (atom N) .
  • the mimic of the MUCl epitope may have higher avidity to SM3 than aberrantly glycosylated MUCl, or a low avidity or may not bind at all.
  • the avidity of the mimic can be tested in a competition ELISA assay in which a MUCl peptide such as aberrantly glycosylated MUCl, deglycosylated MUCl or the fully stripped core protein or a fragment thereof is attached to the plate and SM3 is added in the presence of the mimic of the MUCl epitope.
  • a MUCl peptide such as aberrantly glycosylated MUCl, deglycosylated MUCl or the fully stripped core protein or a fragment thereof is attached to the plate and SM3 is added in the presence of the mimic of the MUCl epitope.
  • Preferred mimics include those which lead to the selection of specific binding agents from a library which bind aberrantly glycosylated MUCl with a higher avidity and/or higher selectivity than SM3.
  • Preferred mimics of the MUCl epitope are also those which stimulate the production of antibodies in vivo which have a higher affinity than SM3.
  • specific binding agents include those which have been selected from a library, which may include antibodies, or those antibodies produced by B cells which bind the mimic of the peptide.
  • Preferred specific binding agents are those which bind aberrantly glycosylated MUCl with an avidity and/or specificity higher than SM3.
  • the specific binding agents may be used in a diagnostic test to detect the presence of tumour cells in a tissue sample, for example in a histological screening. Specific binding agents used in this manner may be labelled with a detectable label. Alternatively moieties able to specifically bind such mimics may be used to detect the presence of the specific binding agents once the specific binding agents have bound the aberrantly glycosylated.
  • the specific binding agents may be used in vivo for the detection of tumour cells. They may be used in tumour imaging in vivo . Generally, such specific binding agents would be labelled with a detectable label. The specific binding agents may be used to prevent or decrease immunosuppression caused by MUCl.
  • the specific binding agents can be used in a method of therapy against cancer, particularly breast cancer.
  • Specific binding agents which are antibodies or substantially similar to antibodies or fragments of antibodies may bind to aberrantly glycosylated MUCl on the surface of tumour cells and aid the killing of the tumour cells by the immune system.
  • Specific binding agents may be chemically linked to a cytotoxic agents such as a toxin or a radioisotope .
  • Binding of such toxin linked specific binding agents to the tumour cells would lead the killing of the tumour cell .
  • the invention provides a specific binding agent for use a method for treatment of the human or animal body by surgery or therapy or in a diagnostic method practised on the human or animal body, such as in anti-cancer therapy.
  • the invention also provides a use of a specific binding agent in the production of a pharmaceutical composition for use in such a method.
  • the invention provides a pharmaceutical composition containing a specific binding agent and a diluent or carrier therefor.
  • the invention also provides a method of diagnosing or treating cancer by administering an effective non-toxic amount of a specific binding agent to a human or non-human animal in need thereof.
  • the invention also provides a product comprising a specific binding agent and an anti-tumour agent as a combined preparation for simultaneous, separate or sequential use in anti-cancer therapy.
  • the mimic of SM3 may lead to the production of antibodies which recognise the mimic.
  • B cells from the animal or human may be used in conjunction with hybridoma technology to produce such antibodies as monoclonal antibodies.
  • Such antibodies may have a epitope binding site which is similar in shape to the crystal peptide.
  • the administration of such antibodies to an animal or human may lead to the production of antibodies which recognise aberrantly glycosylated MUCl. Therefore the antibodies which recognise SM3 may be used as a vaccine or may be used to produce a vaccine against cancer.
  • the antibodies or fragments thereof which recognise aberrantly glycosylated MUCl produced in response to the anti-mimic of SM3 antibody may themselves be used in an anti-cancer treatment or in a diagnostic method.
  • the invention also provides such an antibody or fragment thereof for use in a method for treatment of the human or animal body by surgery or therapy or in a diagnostic method practised on the human or animal body, such as in anti-cancer therapy.
  • the invention also provides use of such an antibody or fragment thereof in the production of a pharmaceutical composition for use in such a method.
  • the invention provides a pharmaceutical composition containing such an antibody or fragment thereof and a diluent or carrier therefor.
  • the invention also provides a method of diagnosing or treating cancer by administering an effective non-toxic amount of such an antibody or fragment thereof to a human or non-human animal in need thereof.
  • the invention provides the use of H3 chain of SM3 as a therapeutic or diagnostic agent.
  • the invention also provides the use of HI chain of SM3 as a therapeutic or diagnostic agent.
  • the invention also provides such an H3 or HI chain for use in a method for treatment of the human or animal body by surgery or therapy or in a diagnostic method practised on the human or animal body, such as in anti-cancer therapy.
  • the invention also provides use of such an Hi or H3 chain in the production of a pharmaceutical composition for use in such a method.
  • the invention provides a pharmaceutical composition containing such an HI or H3 chain and a diluent or carrier therefor.
  • the invention also provides a method of diagnosing or treating cancer by administering an effective non-toxic amount of such an HI or H3 chain to a human or non-human animal in need thereof.
  • the invention provides the use of the structure factors and/or structural coordinates to solve structural coordinates of other crystals.
  • a crystal may comprise an antibody or antibody fragment.
  • the structural coordinates can be used to solve the structural coordinates of a crystal comprising an antibody which has a non-proline cis peptide bond.
  • the cis peptide bond may be in the H3 chain of the antibody.
  • the invention provides the use of the structure factors and/or structural coordinates to engineer, design or modify an antibody.
  • the engineering may be for the purpose of humanising the antibody.
  • the engineering may comprise the replacement of portions of the antibody.
  • the portions of the antibody may be replaced with portions from another protein, such as a human protein, for instance a human antibody.
  • Two dimensional and three dimensional representations may be used during the engineering of the antibody to ensure that the epitope binding site of the antibody is the same or substantially similar to the antibody binding site of SM3.
  • the engineering of the antibody may be for the purpose of increasing the contribution made by the H3 and HI chains in the binding of the epitope.
  • the invention provides the use of an engineered glycine in a protein to insert a non-proline cis peptide bond into the protein.
  • the insertion of the glycine may enable the protein to undergo conformational change, for instance in connection with a specific binding reaction where the affinity, avidity or selectivity of the specific binding reaction is modified by adoption of a cis peptide bond adjacent to the engineered glycine.
  • the engineered glycine can be inserted using site directed mutation techniques, such as those described in Short Protocols in Molecular Biology, 3rd edition, published by John Wiley and Sons, Inc., USA.
  • the protein may be an antibody or fragment of a antibody.
  • the engineered glycine may be in a CDR loop of the antibody. The insertion of the engineered glycine in an antibody will generally affect the binding of the antibody to an epitope .
  • the invention also provides a method of producing a crystal which comprises the use of cadmium.
  • the method comprises contacting any one of the moieties to be crystallised with cadmium such as by use of a solution of a cadmium salt.
  • the cadmium ions may be present in the solution in which the crystal is grown.
  • the mimic of SM3 the mimic of the MUCl epitope or the specific binding agent comprise a peptide then such agents may be delivered to animal or human by the admimistration of a nucleic acid which encodes such a peptide. Transcription and translation or merely translation of the nucleic acid would lead to the production of the peptide in vivo.
  • the nucleic acid may be within a suitable vector, especially an expression vector, for instance a virus or an organism which is administered, such as a Vaccinia virus, for instance an attenuated Vaccinia virus or the nucleic acid may be administered in the presence of a suitable diluent or carrier as in the use of "naked DNA" .
  • Nucleic acids encoding such peptides form a further aspect of the present invention as do their uses in cancer therapy and pharmaceutical compositions containing them.
  • the invention also provides such nucleic acids for use in a method for treatment of the human or animal body by surgery or therapy or in a diagnostic method practised on the human or animal body, such as in anti- cancer therapy.
  • the invention also provides use of such nucleic acids in the production of a pharmaceutical composition for use in such a method.
  • the invention provides a pharmaceutical composition containing such nucleic acids and a diluent or carrier therefor.
  • the invention also provides a method of diagnosing or treating cancer by administering an effective non-toxic amount of such nucleic acids to a human or non-human animal in need thereof.
  • the mimic of SM3 mimics of the MUCl epitope, selective binding agents, nucleic acids, viruses or organisms containing nucleic acids of the invention
  • substances of the invention may be formulated for clinical administration by mixing them with a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent for example the substances can be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, or transdermal administration. They may be mixed with any vehicle, e.g. a diluent or carrier which is pharmaceutically acceptable and appropriate for the desired route of administration.
  • the pharmaceutical carrier or diluent for injection may be, for example, a sterile or isotonic solution such as Water for Injection or physiological saline.
  • Chemical modification of such materials as peptides and antibodies may be achieved by well known methods. Fragments of antibodies may be produced by known chemical or enzymatic digestions or by expression from suitably modified nucleic acids. New chemical entities such as mimics of the MUCl epitope may be produced by the well known techniques of synthetic organic chemistry.
  • the substance of the invention does not include those peptides disclosed in WO-A-88/05054 or in WO-A- 90/05142.
  • Figure 1 shows the amino acid sequence of the MUCl tandem repeat .
  • Figures 2 and 3 show experimental electron density maps and the refined atomic models of the epitope binding site of SM3 and part of the crystal peptide.
  • Figure 4 shows a comparison of the conformations of the unbound and bound peptide .
  • Figures 5 and 6 show molecular surface features of the SM3-crystal peptide interaction.
  • Figure 7 is a stereo view of the non-proline cis- peptide bond in CDR H3.
  • Figures 8, 9, 10 and 11 are LIGPLOTs of the peptide binding site.
  • Figure 12 shows experimental density maps of the cis- peptide conformation in H3.
  • Figure 13 shows a Ramachandran plot for the H3 fragment .
  • Figure 14 shows the main chain temperature factors of the fragment from H3.
  • Figure 15 shows a stereo view of the MUCl peptide in the antibody combining site.
  • Figure 16 shows a stereo view of the non-proline cis- peptide bond in H3.
  • Figure 1 shows MUCl tandem repeat sequence written from N- to C-terminal using the Internationally recognised 3-letter code (which is used throughout this specification) .
  • the epitope recognised by SM3 is shown in bold (Pro-Asp-Thr-Arg-Pro) with the immunodominant region indicated by the double headed arrow.
  • the probable glycosylation sites are shown in bold italics.
  • the peptide used for the crystallisation studies (Thr-Ser- Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-Ser-Thr) is underlined (and is herein referred to as "the crystal peptide" ) .
  • Figures 2 and 3 show experimental electron density maps and refined atomic models calculated using the o observed structure factors (30 to 1.95 A) and calculated phases after solvent modification. The maps are contoured at 1.0 ⁇ .
  • Figure 2 shows part of the MUCl peptide antigen showing the immunodominant region. The numbering refers to Figure 1.
  • Figure 3 shows the cis- peptide bond in CDR H3 between residues Gly96H and
  • Figure 4 shows the unbound and bound peptide.
  • the unbound peptide is shown on the left.
  • the structure represents the "knob-like" region and was determined in solution by NMR; it includes the whole of one tandem repeat and parts of the two flanking repeats (Fontenot et al . , 1995).
  • the conformation of the crystal peptide as bound by SM3 is shown on the right. There appears to be a conformational transition of the "knob-like" region upon SM3 binding, which leads to a more extended peptide conformation.
  • the main differences can be attributed to the ⁇ angles of Asp5P and Arg7P.
  • Figure 5 shows a CPK model of the SM3 combining site with the individual CDR loops labelled. The crystal peptide is shown contacting all of the CDR's except H2.
  • Figures 8, 9, 10 and 11 are LIGPLOTS showing the main interactions which occur between the SM3 binding site, the crystal peptide and water molecules.
  • Figure 12 shows experimental electron density maps calculated using the observed structure factors (30 to 1.95 A and calculated phases after solvent modification, contoured at 1.0 ⁇ level (2Fo-Fc) and 2.0 ⁇ (Fo-Fc). Residue numbering as in the text.
  • the model with this cis - peptide bond is coloured according to atom type.
  • Example 1 Assessment of the Binding of the MUCl epitope to SM3.
  • the sequence of the MUCl peptide in the context of the MUCl repeat is shown in Figure 1.
  • the region of the MUCl repeat for the co-crystallisation studies was chosen to keep the peptide as soluble as possible, while maintaining most of the tertiary structure of the knob region as based upon the NMR structure of the mucin repeats [Fontenot, J.D., Mariappan, S.V.S., Catasti, P., Domenech, N., Finn, O.J. and Gupta, G.J. Biomol . Struct , and Dynam . 13, (1995) 245-260] .
  • the binding of the MUCl epitope to SM3 was assessed by inhibition in an ELISA assay.
  • the assay involved SM3 binding to a recombinant fusion protein containing seven copies of the MUCl tandem repeat, a l ⁇ g/ml concentration of SM3 was incubated with increasing concentrations of peptide, a 50% inhibition was achieved at a peptide concentration of approximately 30 ⁇ g/ml (Apparent Ka ⁇ 24 ⁇ M) .
  • a peptide of the same composition but different sequence (Ala-Arg-Pro-Thr- Gly-Thr-Ser-Asp-Pro-Thr-Pro-Ala-Ser) gave no detectable inhibition at the same concentration.
  • SM3 at 4mg/ml in 100 mM sodium acetate pH 6.0, 3mM EDTA, 50mM cysteine was incubated with 160 ⁇ g/ml of papain for 4 hr at 37°C.
  • Fabs were purified by gel filtration on Superose 6 in PBS, followed by dialysis into 20mM Tris pH 8.0 and then Mono Q ion exchange chromatography. Fractions were analyzed by SDS PAGE and Fab fractions concentrated to 4 mg/ml in lOmM Tris pH 8.0. The overall yield was 22%.
  • Example 3 Crystallisation of the SM3 Fab fragment bound to the MUCl epitope.
  • the SM3 Fab fragments were mixed with concentrated solution of the peptide at a molar ratio 1:5, incubated for 3 hours at 37°C, and then concentrated to 9.5 mg/ml.
  • the crystallisation trials were conducted using the hanging drop method.
  • a wide range of conditions were tested using commercial screens, namely Hampton Research I and II [Jancarik, J. and Kim, S.H. J. Appl . Cryst . 24, (1991) 409-411 and Cudney, R., Patel, S., Weisgraber K., Newhouse, Y., and McPherson, A. Acta Crys t .
  • Example 4 Diffraction measurements of the SM3 Fab fragment/MUCl epitope crystal.
  • a monocrystal of dimensions 0.4 x 0.2 x 0.03 mm was used for the diffraction measurements at the Xll outstation of the DESY synchrotron in Hamburg.
  • the o specific volume V ra 2.50
  • a 3 /Da of protein corresponds to a solvent content of 51% (Matthews, 1968).
  • the CCP4 package [Collaborative Computational Project, No. 4 Acta Crystallogr . D50, (1994) 760-763]. was used for all crystallographic calculations unless otherwise stated.
  • the structure of the SM3-MUC1 peptide complex was solved by the Molecular Replacement method as implemented in the program AMoRe [Navazza, J. Acta Crystallogr . A50, (1994) 157-163], using the Murine SE155-4 Fab fragment complexed with the dodecasaccharide [Cygler, M., Rose, D.R. and Bundle, D.R. Science 253, (1991) 442-445] as a search model.
  • the peptide was unambiguously built into the electron density from difference Fourier maps. Since the MUCl peptide is almost symmetrical, it was necessary to confirm the correct orientation for the peptide in the electron density. This was done by modelling the peptide backwards which resulted in a poorer fit into the density, and a higher R-Free and crystallographic R- factor after one round of refinement.
  • a cis-peptide bond within the antibody-antigen combining site is an important and common feature of peptide- antigen binding;
  • the SM3-peptide complex differs from all other antibody-peptide complexes in that the cis- peptide bond is non-proline and is located in another CDR loop.
  • the role of the cis-peptide bond it may relate to specific interactions with the hydrophobic surface on the peptide antigen.
  • An additional reason for this bond to be cis rather than trans may be due to the small size of H3 resulting from the extended peptide antigen epitope running across its upper surface.
  • a small all-trans CDR would have less degrees of freedom to accommodate such a change.
  • the protein data bank A computer based archival file for macromolecular structures. /. Mol. Biol.
  • PROCHECK a program to check the stereochemical quality of protein structures./ Appl. Crystallogr. 26, 283-291.
  • AMORE An automated package for molecular replacement.
  • the top row defines the spacegroup and crystal cell dimensions.
  • Scale 1, Scale 2 and Scale 3 define an orthogonalisation matrix which when applied to the individual coordinates puts them into an orthogonal axis system i.e. 90 degrees between each of the axes x, y and z .
  • Below these rows reading left to right the second row gives the chemical symbol of the atom.
  • OH2 represents a water molecule.
  • the letter following indicates the position of the atom in the amino acid residue using a convention recognised by a skilled partisan.
  • the letter refers to the equivalent Greek symbol (i.e. A for alpha, B for beta etc.) .
  • the third column gives the identity of the residue in which the atom is present.

Abstract

L'invention concerne un procédé de préparation d'un cristal comprenant l'utilisation de cadmium. Des facteurs de structure ou des coordonnées structurelles obtenues à partir du cristal de l'anticorps SM3 lié à un épitope peuvent être utilisés pour construire des analogues de cet anticorps ou de cet épitope. De tels analogues peuvent être utilisés dans le diagnostic ou le traitement du cancer.
PCT/GB1998/002542 1997-08-22 1998-08-24 Cristal de l'anticorps sm3 (fragment) et de l'epitope de reconnaissance, sa preparation, support de donnees codees contenant ses coordonnees et son utilisation diagnostique ou medicale WO1999010379A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP98940371A EP0996634A1 (fr) 1997-08-22 1998-08-24 Cristal de l'anticorps sm3 (fragment) et de l'epitope de reconnaissance, sa preparation, support de donnees codees contenant ses coordonnees et son utilisation diagnostique ou medicale
CA002300370A CA2300370A1 (fr) 1997-08-22 1998-08-24 Cristal de l'anticorps sm3 (fragment) et de l'epitope de reconnaissance, sa preparation, support de donnees codees contenant ses coordonnees et son utilisation diagnostique ou medicale
IL13454798A IL134547A0 (en) 1997-08-22 1998-08-24 Crystal of sm3 antibody (fragment) and recognizing epitope, its preparation, encoded data storage medium containing its coordinates and its diagnostical or medical use
AU88711/98A AU751386B2 (en) 1997-08-22 1998-08-24 Crystal of SM3 antibody (fragment) and recognizing epitope, its preparation, encoded data storage medium containing its coordinates and its diagnostical or medical use
JP2000507705A JP2001514188A (ja) 1997-08-22 1998-08-24 Ｓｍ３抗体（断片）および認識エピトープの結晶、その調製、その座標を含むコードデータ記憶媒体、およびその診断又は医療への使用
NO20000845A NO20000845L (no) 1997-08-22 2000-02-21 Krystall av SM3 antistoff (fragment) og gjenkjennede epitop, dets fremstilling, kodet datalagringsmedium inneholdende dets koordinater samt diagnostisk eller medisinsk anvendelse derav

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1994144A1 (fr) * 2006-03-06 2008-11-26 Agency for Science, Technology and Research Procédés d'identification de cellules souches embryonnaires humaines et expression de la protéine de type podocalyxine (podxl)
US9469684B2 (en) 2004-12-07 2016-10-18 University of Pittsburgh—of the Commonwealth System of Higher Education Therapeutic and diagnostic cloned MHC-unrestricted receptor specific for the MUC1 tumor associated antigen

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007087266A2 (fr) * 2006-01-23 2007-08-02 Errico Joseph P Procedes et compositions de developpement d’un medicament cible

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988005054A1 (fr) * 1987-01-07 1988-07-14 Imperial Cancer Research Technology Limited Sonde
WO1990005142A1 (fr) * 1988-11-10 1990-05-17 Imperial Cancer Research Technology Ltd. Polypeptides
US5353236A (en) * 1992-04-23 1994-10-04 The Board Of Trustees Of The Leland Stanford University High-resolution crystallographic modelling of a macromolecule
WO1994025860A1 (fr) * 1993-04-28 1994-11-10 Immunex Corporation Procede et systeme de modelisation de proteines

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988005054A1 (fr) * 1987-01-07 1988-07-14 Imperial Cancer Research Technology Limited Sonde
WO1990005142A1 (fr) * 1988-11-10 1990-05-17 Imperial Cancer Research Technology Ltd. Polypeptides
US5353236A (en) * 1992-04-23 1994-10-04 The Board Of Trustees Of The Leland Stanford University High-resolution crystallographic modelling of a macromolecule
WO1994025860A1 (fr) * 1993-04-28 1994-11-10 Immunex Corporation Procede et systeme de modelisation de proteines

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BATES E.A.: "Conformational analysis of the first observed non-proline cis peptide bond ocurring within the CDR of an antibody", J.MOL.BIOL., vol. 284, no. 3, 4 December 1998 (1998-12-04), pages 549-555, XP002091260 *
BURCHELL E.A.: "A short sequence within the amino acidtandem repeat of a cancer associated mucin, contains immunodominant epitopes", J.MOL.BIOL., vol. 44, no. 4, 15 October 1989 (1989-10-15), pages 691 - 696, XP002091256 *
DOKURNO E.A.: "Crystallization of an antitumour antibody SM3 complexed with a peptide epitope", ACTA CRYSTALLOGRAPHICA SECTION D BIOLOGICAL CRYSTALLOGRAPHY, vol. 53, no. 6, 1 November 1997 (1997-11-01), pages 780 - 781, XP002091258 *
GRANOWSKA E.A.: "Breast cancer (99m)TcSM3 radioimmunoscintigraphy", ACTA ONCOLOGICA, vol. 35, no. 3, 1996, pages 319 - 321, XP002091257 *
STEWART E.A. OCURRENCE OF ROLE OF CIS PEPTIDE BONDS IN PROTEIN STRUCTURES, J.MOL.BIOL., vol. 214, no. 1, 1990, pages 253 - 260, XP002091259 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9469684B2 (en) 2004-12-07 2016-10-18 University of Pittsburgh—of the Commonwealth System of Higher Education Therapeutic and diagnostic cloned MHC-unrestricted receptor specific for the MUC1 tumor associated antigen
US11325962B2 (en) 2004-12-07 2022-05-10 University of Pittsburgh—of the Commonwealth System of Higher Education Therapeutic and diagnostic cloned MHC-unrestricted receptor specific for the MUC1 tumor associated antigen
EP1994144A1 (fr) * 2006-03-06 2008-11-26 Agency for Science, Technology and Research Procédés d'identification de cellules souches embryonnaires humaines et expression de la protéine de type podocalyxine (podxl)
EP1994144A4 (fr) * 2006-03-06 2009-09-09 Agency Science Tech & Res Procedes d'identification de cellules souches embryonnaires humaines et expression de la proteine de type podocalyxine (podxl)
US8173374B2 (en) 2006-03-06 2012-05-08 Agency For Science, Technology And Research Human embryonic stem cell methods and PODXL expression
US8710190B2 (en) 2006-03-06 2014-04-29 Agency For Science, Technology And Research Human embryonic stem cell methods and PODXL expression
EP3275901A1 (fr) * 2006-03-06 2018-01-31 Agency for Science, Technology and Research Procédés de cellules souches embryonnaires humaines et expression podxl

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GB9717946D0 (en) 1997-10-29
CA2300370A1 (fr) 1999-03-04
AU8871198A (en) 1999-03-16
IL134547A0 (en) 2001-04-30
AU751386B2 (en) 2002-08-15

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