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Structure factors or structural coordinates obtained from the crystal of SM3 antibody bound to an epitope can be used to design mimics of the antibody or epitope. Such mimics can be used in the diagnosis or therapy of cancer. The MUCl epithelial mucin is a transmembrane glycoprotein with the extracellular domain made up largely of exact tandem repeats of 20 amino acids, each of which contains five potential glycosylation sites Gendler et al. In breast and other carcinomas MUCl is over-expressed and is aberrantly glycosylated making it antigenically distinct from the normally processed mucin. In breast cancers, the 0- glycans which are added are shorter Hanish et al.
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Structure factors or structural coordinates obtained from the crystal of SM3 antibody bound to an epitope can be used to design mimics of the antibody or epitope. Such mimics can be used in the diagnosis or therapy of cancer. The MUCl epithelial mucin is a transmembrane glycoprotein with the extracellular domain made up largely of exact tandem repeats of 20 amino acids, each of which contains five potential glycosylation sites Gendler et al. In breast and other carcinomas MUCl is over-expressed and is aberrantly glycosylated making it antigenically distinct from the normally processed mucin.
In breast cancers, the 0- glycans which are added are shorter Hanish et al. Such epitopes are called cryptic epitopes. The high specificity of the SM3 antibody makes it a potentially useful tool in the diagnosis and treatment of breast cancer Granowska et al.
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. 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. 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 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 particularly provides a use of the structural coordinates of a moiety which comprises at least the epitope binding fragment of the SM3 antibody or a substantially similar fragment bound to a peptide such as the crystal peptide. The moiety may be whole SM3 antibody, or a Fab fragment derived from the digestion of whole SM3 antibody by papain.
The moiety may comprise a modified form of whole SM3 or a modified form of a fragment of SM3. Such modifications include the insertion or deletion of amino acids, or replacement of amino acids by other amino acids. Other chemical modifications may be made. 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 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 structure factors may be used to derive additional information about the mobility of any individual atom or group of atoms within the crystal.
This additional information, for instance, anisotropic B factors, may concern the direction of movement possible for each atom. The structure factors and structural coordinates thus give an indication of the available space for adjusting the position of individual atoms when designing mimics of the peptide or antibody.
The structural coordinates allow the epitope binding site bound to the peptide to be shown as a two dimensional representation, for example as in the LIGPLOTs of Figures 8 to 11 or a three dimensional representation by physical models or as displayed on a computer screen. Such representation can be used to design modifications of SM3. Such modifications includes modifications to increase the avidity of the epitope binding site for the bound peptide.
This modification may preferentially increase the avidity of the epitope binding site for abnormally glycosylated MUCl. 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 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. The SM3 binding site, or other parts of SM3 may be modified to allow such changes to occur more easily. Alternatively the mimic of SM3 may comprise the SM3 epitope binding site constrained in the conformation it adopts when it binds the peptide. On the basis of such modelling SM3 mimics may be identified, characterised, designed or screened.
It is believed that the reason why 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. Mimics of SM3 may for instance be obtained by computer modelling techniques. Mimics of SM3 can be produced either by computationally identifying compounds which have a similar surface to the binding site of SM3, or by computationally designing compounds with surfaces which are likely to bind the peptide.
Various methods can then be used to produce a three dimensional surface which is the same or similar to the epitope binding surface. The epitope binding site surface may be described in more detail by analysis of the functional groups present to produce a pharmacophore. Based on a pharmacophore, packages such as DBServerl and HipHop can be used to search databases for compounds whose surfaces are described by similar pharmacophores. Packages such as Ludi and MCSS can be used to select fragments or chemical entities from databases which can then be positioned in a variety of orientations, or “docked” with the surface of the peptide.
Once suitable chemical entities or fragments which bind sites on the surface of the peptide have been identified bridging fragments and framework structure are chosen with the correct size and geometry to support the che ical entities and fragments in the favourable orientation and location and to form the mimic of SM3.
Packages such as Hook can be used to select framework structures. Once a candidate mimic of SM3 has been designed or selected by the above methods, the efficiency with which that mimic may bind to the peptide may be tested and optimized using computational or experimental evaluation. Various parameters can be optimized depending on the desired result. Thus, one may optionally make substitutions, deletions, or insertions in some of the components of the SM3 mimics in order to improve or modify the binding properties.
Generally, initial substitutions are conservative, i. Such modified mimics can be computationally or experimentally evaluated in the same manner as the first candidate SM3 mimics, and if necessary further modifications can be made. This process of evaluating and modifying may be iterated any number of times. The term aberrantly glycosylated MUCl is used throughout this specification to refer to MUCl which has a different level of glycosylation than is normally found on MUCl from a particular tissue.
The different level of glycosylation may be a decrease in the level of glycosylation. MUCl with such decreased levels of glycosylation may be produced by a tumour cell, such as an adenocarcinoma cell for instance a cell from an epithelial tumour of colon, lung, ovary, pancreas or especially a breast tumour cell.
Such an avidity may be higher than that of SM3. Coli strain TGI. After centrifugation the supernatant was loaded onto a Glutathione Sepharose 4B column Pharmacia equilibrated in lysis buffer. Thaw out a plate at room temperature RT. SM3 from Add samples in a volume of 50 ml, in triplicate and leave at RT for 1 hour. Remove by aspiration and repeat. A to each well and incubate at R. Incubate at RT. The avidity of the mimic of SM3 can also be measured using methods described in Bynum et al.
These techniques may also be used to test the specificity of the mimic of SM3 by testing the avidity of the mimic for aberrantly glycosylated MUCl and for normally glycosylation MUCl. 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. 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. 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.
Therefore in the method of therapy of the invention 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.
Thus 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. Mimics of MUCl epitope peptides 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.
Such 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.
Such a library may be a microbial display library, such as a phage display library.
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