JP2001514188A - Crystals of SM3 antibodies (fragments) and recognition epitopes, their preparation, code data storage media containing their coordinates, and their use in diagnosis or medicine - Google Patents

Abstract

  (57) [Summary] Provided is a method for producing a crystal using cadmium. Using the structural factors and structural coordinates obtained from crystals of the SM3 antibody bound to the epitope, mimetics of the antibody or epitope can be designed. Such a mimic can be used for diagnosis and treatment of cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing a crystal, the use of the structural coordinates of a crystal containing an epitope-binding fragment of an antibody bound to a peptide, a mimic of an antibody and a peptide, recognizable by a mimic of an antibody or of a peptide. The present invention relates to products that recognize mimetics, molecularly engineered antibodies, and their use in diagnosis or therapy.

[0002] The monoclonal antibody SM3 (Accession No. 87010, Jan. 7, 1987)
The hybridoma HSM3 deposited with the ECACC at 701) secretes a tumor-associated epitope on the epithelial mucin MUC1 (International Patent Application WO-A-88 / 0).
5054, WO 90/05142).

[0003] MUC1 epithelial mucin is a transmembrane glycoprotein with an extracellular domain consisting of precise tandem repeats (tandem repeats) of mostly 20 amino acids, each of which is capable of 5 glycosylation (glycosylation). (Gend
lelet al. 1988, 1990, see FIG. 1). In breast and other cancers, MUC1 is overexpressed and abnormally glycosylated, thereby antigenically distinguishing it from normally processed mucin. In breast cancer, the added O-polysaccharide is short (Hanish et al., 1989; Hull et al., 19).
89; Lloyd et at. , 1996), resulting in the appearance of epitopes that are normally masked on the core protein (Burchell and
Taylor-Paradimitriou, 1993). Such an epitope is called a cryptic epitope. The SM3 antibody was raised against MUC1 which had been stripped of carbohydrates. SM3 recognizes such cryptic epitopes and shows high selectivity for specific reactions with cancer-associated mucins in more than 90% of breast cancers (Girling et al., 1989). Due to this high specificity, the SM3 antibody may be an effective tool for diagnosing and treating breast cancer (Gra
nowska et al. , 1996).

[0004] The sequence of the repetitive unit of the core protein of MUC1 contains a doublet of threonine and serine and binds to a highly immunogenic domain (Burcell et al.
al. , 1989; see Figure 1). Three-dimensional NM of three MUC1 peptide repeats
In the R structure, it is found to be a repetition of a knob-like structure, which corresponds to the immunogenic domains linked by an extended spacer (Fontenot et al., 1995). The epitope for SM3 has been mapped using overlapping peptides and contains five adjacent amino acids, Pro-Asp-Thr-Arg, within the knob-like domain and between the glycosylable sites of serine and threonine. -Pro (Bur
cell et a. 1989, hereinafter referred to as the MUC1 epitope). Recombinant Gal giving first sugar to Ser or Thr
In vitro studies using NAc transferase include Thr1, Se
r12 and Thr13 are glycosylated, suggesting that Thr6 (within the MUC1 epitope) and Ser2 are not glycosylated (Wanda
ll et al. , 1997).

[0005] The inventors crystallized the SM3 fragment bound to the peptide and performed X-ray diffraction on the crystal to measure the structural factor. The structural coordinates were elucidated from the structural factors. They can be used to create new diagnostic and therapeutic substances and for other research purposes. The present invention, as described below, provides structural factors and / or structural coordinates for identifying, characterizing, designing, and screening chemical entities (chemicals) for diagnostic and therapeutic applications. Including the use of

[0006] One embodiment of the present invention provides that a crystal comprising at least an epitope binding fragment of the SM3 antibody bound to a peptide recognized by the epitope binding site of SM3 is subjected to X-ray diffraction and, optionally, to subsequent structural coordinates. It provides the use of structure factors and / or structure coordinates obtained by analyzing the diffraction results for estimation.

The present invention specifically relates to moieties comprising at least an epitope binding fragment or substantially similar fragment of the SM3 antibody bound to a peptide, such as a crystalline peptide.
y) provides the use of structural coordinates. This portion may be the whole SM3 antibody or a Fab fragment obtained by digesting the whole SM3 antibody with papain. The portion may be a modified form of the entire SM3 or a modified form of the SM3 fragment. Such modifications include insertions and deletions of amino acids, substitutions with other amino acids, and the like. Other chemical modifications may be used.

[0008] The peptide may comprise the sequence Pro-Asp-Thr-Arg-Pro,
Alternatively, for example, the sequence Thr-Ser-Ala-Pro-Asp-Thr-Arg-
MUC1 tandem repeats, such as Pro-Ala-Pro-Gly-Ser-Thr, may be included.

The present invention provides the use of the crystal structure factor and / or structure coordinates of a Fab fragment of SM3 linked to a crystal peptide. Table 1 shows the structural factors of the crystals obtained in the examples, and Tables 2a and 2b show the structural coordinates. The structural coordinates shown in Tables 2a and 2b are derived from the structural factors. However, the coordinates shown in Table 2b have been calculated to a higher degree of refinement and include additional protein atoms. The present invention provides for the use of the structural coordinates shown in Table 2a and / or Table 2b.

[0010] The structural coordinates indicate the position of individual atoms in the crystal, and for each atom indicate the B-factor that gives some information about the mobility of that atom. Structure factors can be used to derive additional information about the mobility of individual atoms or groups of atoms in a crystal. This additional information, for example the anisotropic B factor, relates to the direction of possible movement for each atom. Thus, the structural factors and structural coordinates can indicate the space available for adjusting the position of individual atoms when designing a peptide or antibody mimic.

Mimics of SM3 The present invention provides the use of structural factors and / or structural coordinates to identify, characterize, design or screen mimics of SM3. By structural coordinates,
The epitope binding site bound to the peptide can be determined, for example, by using LIGPLOT shown in FIGS.
, A three-dimensional display of a physical model, or a display on a computer screen. Such indications can be used to design modifications of SM3. Such modifications include those that increase the binding activity of the epitope binding site for the binding peptide. This modification may preferentially increase the binding activity of the epitope binding site for aberrantly glycosylated MUC1.

[0012] Binding activity may be increased by increasing the amount and number of interactions favorable for peptide binding and / or by modifying the epitope binding site structure to reduce unfavorable interactions between the peptide and the antibody. Can be increased. The preferred interaction is
The structure of the epitope binding site may be increased by extending the space shown as being unoccupied or occupied by a water molecule in a two or three dimensional representation. Such water molecules include those shown in FIGS.

The representation of the structure may be used in other ways to modify the structure of SM3. It is believed that SM3 can bind to peptides by "inductive fit", which requires that a conformational change occurs in the structure of SM3 during the process of binding to the peptide. The SM3 binding site, or other portions of SM3, may be modified to make such changes more readily. Alternatively, a mimic of SM3 may include an SM3 epitope binding site that is constrained to the conformation employed when binding to the peptide. Using the representation of the epitope binding site, such forcing can be modeled by introducing putative covalent bonds between the SM3 atoms that approach each other when the SM3 binds to the peptide, or one or more between the SM3 atoms. Additional chemical linkers can be included to force the epitope binding site into the required conformation, and / or replace one or more amino acids of SM3 with analogs of the naturally occurring amino acids, and It can help to force home. Based on such modeling, mimetics of SM3 may be identified, characterized, designed or screened.

The reason that SM3 has low binding activity to abnormally glycosylated MUC1 is that, as shown in FIG. 1, the carboxyl moiety at positions 1, 12 and 13 of the peptide and the residue within or near the epitope binding site It is considered a steric barrier between the group. An indication of the epitope binding site bound to the peptide can also be used to predict which SM3 residues may be involved in this steric barrier. One such residue may be proline 56 of SM3. In order to increase binding activity, such residues can be modified, substituted, or deleted to reduce the steric barrier.

A simulated SM3 can be obtained, for example, by computer modeling technology. Using the structural coordinates, a three-dimensional representation of the epitope binding site surface bound to the peptide can be obtained from Catalyst / SHAPE, Catalyst / COMPARE,
Ca such as DBServer Hiphop Ludi, MCSS and Hook
It can be created using tallist Software. These softwares are available from Molecular Simulation Limited (240/250 The
Quorum, Barnwell Road, Cambridge, Engla
nd). A mimic of SM3 can be prepared by identifying a compound having a surface similar to the binding site of SM3 by computer calculation, or by designing a compound having a surface that easily binds to a peptide by computer calculation. In doing so, various methods can be used to create a three-dimensional surface that is the same or similar to the epitope binding surface. Based on this form, Catal
Using a package such as yst / SHAPE or Catalyst / COMPARE, a compound having the same three-dimensional shape as the epitope binding site can be selected from a database. Those with pharmacological action (Pharmacopharma, pharmacoph
To make ore), an analysis of the functional groups present may be performed to describe the epitope binding site surface in more detail. Based on pharmacological groups, DBServer1 and Hip
Using a package such as Hop, one can search the database for compounds with surfaces described by a similar pharmacist. Searchable databases include ACD, NCI, Maybridge, Derwent World In
dex and BioByte.

Packages such as Ludi and MCSS can be used to select from a database fragments or chemicals that can be positioned in various orientations or "docked" to the peptide surface. Once the appropriate chemicals or fragments that bind to sites on the peptide surface have been identified, the appropriate size and size must be used to support the chemicals and fragments in a suitable orientation and position and to form a mimic of SM3. Select a framework and bridging fragments with a geometric arrangement. Hook to choose a framework
You can use a package like

Once a candidate for a mimic of SM3 has been designed or selected in the manner described above, the effect of binding of the mimic to the peptide may be tested and optimized by computational or experimental evaluation. . Various parameters can be optimized depending on the desired result. The parameters are not limited, but include, for example, properties, binding activity, on / off ratios, and properties readily identifiable by those skilled in the art. Computer means for evaluating the selected SM3 mimic candidate include Catalyst / SHAPE, Catalyst / COMPARE, and DBSe.
Packages such as rver, HipHop, Ludi and MCSS can be employed. Experimental means include GST-MUC1 ELI as described below.
This includes the ELISA method using an SA plate and the technique described in Bynum et al.

As described above, in order to improve or modify the binding properties, some components of the SM3 mimic can be appropriately replaced, deleted, or inserted. Usually, the first substitution is conservative, that is, the substitution group is about the same in size, shape, hydrophobicity and charge as the original component. The mimetic modified in this way can also be evaluated by computer calculation or experiment, like the first SM3 mimic candidate, and can be further modified if necessary. This evaluation and modification process can be repeated any number of times.

Throughout this specification, the term “aberrant glycosylation MUC1” means a MUC1 that differs in level from the level of glycosylation normally found in MUC1 from a particular tissue. A different level of glycosylation may be a reduced level of glycosylation. Such reduced MUC1 glycosylation levels can be produced by tumor cells, such as, for example, cells from epithelial tumors of the large intestine, lung, ovary, pancreas, or adenocarcinoma, especially breast cancer cells.

The SM3 mimetic preferably has a high binding activity to abnormally glycosylated MUC1. Its binding activity may be higher than SM3. They may have higher selectivity for aberrantly glycosylated MUC1 than for normal glycosylated MUC1 compared to SM3.

The binding activity of the SM3 mimic that binds to aberrantly glycosylated MUC1 can be tested in an ELISA assay as described below.

GST-Muc-1 ELISA method GST-Muc-1 batch 5 Preparation of ELISA plate
ia). Transfer cells to L liquid medium (100 mg / ml ampicillin)
Growth overnight, then IPTG was added to a final concentration of 0.5 mM and incubated for a further 4 hours at 37 ° C. Pellet cells (20 min / 100
00 rpm), washed twice with PBSa, 25 mM TRIS pH8, 25 mM
Resuspend in 10 mM EDTA containing glucose, lysozyme (4 mg / ml)
It was left at room temperature for 30 minutes. Then 5 ml of lysis buffer was added and incubated on ice for 5 minutes. After centrifugation, the supernatant was applied to a glutathione Sepharose 4B column (Pharmacia) equilibrated with lysis buffer. The fusion protein product was eluted with PBSa / 15 mM glutathione and stored at 4 ° C. until needed.
SDS-PA and FPLC Superose6 (Pharmacia) size separation showed a single species mwt (molecular weight) of 43K.

Preparation of GST-Muc-1 Batch 5 ELISA Plate GST-Muc-1 was diluted to 50 mg / ml with PBSa and 50 ml
Added to each well of the well plate. Plates were incubated overnight at 4 ° C, blocked for 2 hours at room temperature with 200 ml of 2% BSA / PBSa, and stored at -70 ° C until needed.

Reagents / Buffers Alkaline phosphatase-goat anti-mouse (Fc-specific) Sigma No. A-2429 BSA-Albumin Bovine, fraction V powder, RIA grade, Sigma No. A-7888 ELISA plate-Nunc-Immuno plate No. 44240
4 ELISA substrate buffer-diethanolamine buffer pH9-8 400 ml diethanolamine (Sigma D-2286)
The pH was adjusted to 9.8 with 24.5 mg MgCl ₂ BHD (Anala®) HCl, and the volume was adjusted to 500 ml with distilled water. EDTA Ethylenediaminetetraacetic acid BDH Anala R Glutathione Reduced ICN 101814 IPTG Isopropylthio-bD-galactoside Promega Lysis buffer 150 mM NaCl, 16 mM Na ₂ HPO ₄ , 4 mM Na
H ₂ PO ₄ , 100 mM EDTA, 1% Triton X (BDH Anala®)
2 mM PMSF (phenylmethylsulfonyl fluoride) (Sigma) pNPP-p-nitrophenylphosphate Sigma No. N-9389 PBSAa-phosphate buffered saline (ICRF) TWEEN 20-polyoxyethylene (20) sorbitan monolaurate BDH 66368

GST-Muc-1 (a glutathione-S-transferase fusion protein (non-glycosylated) ELISA plate containing seven copies of a 20 amino acid tandem repeat) was pre-blocked with 2% BSA / PBSA at −70
Store at ° C. (PBSA-phosphate buffered saline BSA-bovine serum albumin, fraction V powder, RIA grade, Sigma No. A-7888)

1. Thaw plates at room temperature. 2. The test antibody sample is diluted with PBSA / 0.02% Tween20 (PBSA / T) to, for example, SM3 100 to 0.001 mg / ml. 3. Wash plate three times with PBSA / T. Samples are added in 50 ml volumes in triplicate and left at room temperature for 1 hour. 4. Carefully remove the antibody sample by aspiration and add 200 ml PBSA / T to each well. The removal by suction is repeated. Then, it is washed twice with PBSA / T. 5. 50 ml of 1/2000 (diluted in PBSA / T) goat anti-mouse (Fc-specific) alkaline phosphatase (Sigma No. A-24209) is added to each well and incubated for 1 hour at room temperature. 6. The plate is washed three times with PBSA / T, then diethanolamine buffer pH 9.8 (400 ml distilled water, 48 ml diethanolamine, 24.5 mg
PH 9.8 with MgCl ₂ , HCl, adjusted to 500 ml with distilled water)
Substrate 50 containing 1 p-dinitrophenyl phosphate (Sigma N-2765)
Add ml. 7. Incubate at room temperature for 15 minutes, and add Labsystems Multis
Read the OD at 405 nm on a can Plate reader.

[0027] The binding activity of the SM3 mimic can also be measured by the method described in Bynm et al.

[0028] These techniques also include aberrant glycosylated MUC1 and normal glycosylated MUC1.
It can be used to test the specificity of a SM3 mimic by testing the binding activity of the mimic to UC1.

Histological screening using SM3 can be performed using tumor tissue and normal tissue from breast cancer, for example, as described in Griling et al. (1989). This can be used to determine whether a mimetic is specific for aberrantly glycosylated MUC1 and is therefore suitable for breast cancer diagnostic methods. Mimetics can also be tested against unfixed liver tumor cells.

Cells with MUC1 with a reduced degree of glycosylation can be produced by using a metabolic inhibitor of O-linked chain elongation, such as O-benzylgalactosamine. Using such cells, low levels of glycosylated MUC1 can be converted to SM
The effect of the mimetic on the binding activity can be studied.

The mimic of SM3 can be used in a diagnostic test to detect the presence or absence of tumor cells in a tissue sample, for example, in histological screening. Mimetics used in this method may be labeled with a detectable label. Alternatively, the presence of a mimetic can be detected after the mimetic binds to aberrant glycosylation using a reagent capable of specifically binding to the mimetic.

[0032] SM3 mimetics may be used for the detection of tumor cells in vivo. They can be used for in vivo tumor imaging. Generally, such mimetics are labeled with a detectable label. Mimics of SM3 can also be used in therapies against cancer, especially adenocarcinomas such as ovarian, colon, pancreatic and epithelial breast cancer, especially breast cancer.

Mimetics that are antibodies or that are substantially similar to antibodies or antibody fragments bind to aberrantly glycosylated MUC1 on the surface of tumor cells and help kill tumor cells by enhancing the patient's immune system. Become. Mimics of SM3 may be chemically linked to cytotoxic agents such as toxins and radioisotopes. By binding such a toxin-linked mimic to a tumor cell, the tumor cell can be killed.

High levels of MUC1 or aberrant glycosylated forms of MUC1 are thought to have immunosuppressive effects. The tumor cells have a high level of MUC1 on their surface and / or are abnormally glycosylated. Therefore, in the treatment method of the present invention, the mimic of SM3 is i
It can be linked to MUC1 in vivo and used to prevent or reduce its immunosuppressive effects. This type of immunosuppressive action that can be prevented or suppressed is described below in connection with mimics of the MUC1 epitope. Such mimetics can be administered with an antitumor agent, such as an antitumor vaccine, and have an adjuvant-like effect (the mimic increases the immune response generated by the vaccine). The anti-tumor agent may be a mimic of the SM3 epitope. The SM3 mimetic can be administered at any time in relation to the administration of the anti-tumor agent, eg, before, simultaneously with, or after administration of the anti-tumor agent.

Thus, the present invention provides a method for treating humans or animals by surgery or therapy, such as anti-cancer therapy, or diagnostic methods used for humans or animals.
M3 mimics are provided. The present invention also provides a combination product of a mimic of SM3 and an antitumor agent for simultaneous, separate or sequential use in anticancer treatment.

The present invention provides a method for preparing a pharmaceutical composition for use in a method as described above.
Provides use of an M3 mimetic. The present invention provides a pharmaceutical composition comprising a mimic of SM3, a diluent or a carrier. The present invention provides a method for treating and diagnosing cancer by administering an effective and harmless amount of a mimic of SM3 to a human or animal as needed.

Hereinafter, the production of the mimic of SM3 and the method, procedure and dosage for using the same will be described.

The present invention enables the identification, characterization, and design of a MUC1 epitope peptide mimic using a structural factor and structural coordinates. Such mimetics can be used to generate a specific binding agent having the desired association with abnormally glycosylated MUC1. Peptide mimetics can be used to select specific binding agents from a library based on affinity for the mimetics. Such a library may be a microbial display library, such as a phage display library. Specific binding agents bind aberrantly glycosylated MUC1 and can therefore be used in a manner similar to SM3 mimetics. Mimetics of the peptide can be used in vaccines administered to humans or animals. Such a vaccine can stimulate the production of antibodies capable of binding aberrantly glycosylated MUC1. Accordingly, such vaccines can be used in cancer therapy, for example, in the prevention or treatment of cancer.

As described above, the present invention provides a MUC1 epitope mimic, a MUC1 epitope used in a method of treating humans or animals by surgery or therapy, such as anticancer therapy, or a diagnostic method practically used for humans or animals. It provides a simulated body. The invention also provides the use of such mimetics in the manufacture of a pharmaceutical composition for use in such a method of treatment. The invention provides a pharmaceutical composition comprising a MUC1 epitope mimic, diluent or carrier. The present invention provides a method for diagnosing and treating cancer by administering an effective and harmless amount of a MUC1 epitope mimic to a human or animal as needed.

Hereinafter, the production of the MUC1 epitope mimic and the method, procedure and dosage for using the same will be described. A MUC1 epitope mimetic is also administered to an animal, preferably a laboratory animal such as a rodent, eg, a mouse, to elicit an antibody response. The antibody-secreting cells can be recovered from the animal, and the hybridoma technology can then be used to generate hybridomas using the cells thus recovered. A hybridoma or other antibody expression system provides a source of a mimetic and, preferably, a monoclonal antibody capable of recognizing the MUC1 epitope, and can be used for diagnosis and therapy, similar to the SM3 mimic described above.

As mentioned above, MUC1 is also thought to cause an immunosuppressive effect. Thus, a mimic of the epitope can also cause an immunosuppressive effect and can be used to cause such an effect in humans or animals. The immunosuppressive effect is caused by mimics that reduce the activity of T cells such as CD4 and CD8 T cells. T cells are polyclonal.

Mimetics include (i) T cell proliferation, (ii) T cell cytokine secretion, (i)
ii) the interaction of T cells with other cells, such as the interaction mediated by ICAM-1 on the surface of T cells, and / or (iv) the cytotoxic activity of T cells. Mimetics can cause cross-linking of T cell surface molecules such as ICAM-1. Mimetics can make T cells anergic. The effect of the mimic can be reversed by the addition of IL-2 or anti-CD28 antibodies.

As described above, the mimetic is particularly effective when the immune reaction has a harmful effect on the body.
Can be used to reduce or prevent an immune response. Thus, the mimetics can be used to treat (ie, treat) transplant rejections such as diseases caused by autoimmune reactions (arthritis, multiple sclerosis, asthma, diabetes), allergies, inflammatory disorders, graft-versus-host disease. Or prevention).

Thus, the present invention provides MUC1 epitope mimetics for use in therapy to prevent or reduce an immune response and to treat or prevent a disease caused by the immune response. The invention also provides the use of such mimetics, ie, the use in the manufacture of a pharmaceutical composition for use in such a method of treatment. The present invention
Treating diseases caused by an immune response, such as autoimmune diseases, allergies, inflammatory disorders, transplant rejection, by administering to a human or animal an effective and harmless amount of a MUC1 epitope mimic as needed Provide a way.

Similar to the case of the SM3 mimetic, a mimetic can be designed using the two-dimensional and three-dimensional representations of the epitope binding site bound to the peptide described above. This mimic may be, for example, the sequence Pro-Asp-Th, which, when bound to a peptide, its derivative, SM3, is forced to adopt a conformation that is approximately the same as that assumed by the crystalline peptide.
r-Arg-Pro or the sequence Thr-Ser-Ala-Pro-Asp-Thr
-An analog of a peptide containing Arg-Pro-Gly-Ser.

The sequence can be forced to adopt a conformation that occurs when it binds to SM3 by introducing covalent bonds between atoms that approach each other. Alternatively, a chemical linker may be used to attach such atoms. The sequence may be forced by cyclizing the peptide in which the sequence resides. In addition, 1 of the peptide
Alternatively, more amino acids can be substituted with analogs of the naturally occurring amino acid, forcing the peptide analog to adopt the desired conformation when introduced.

Hydrogen bonds, such as intra-peptide hydrogen bonds, may be introduced into the mimic. This can force the mimetic to adopt a particular conformation, such as the one adopted when bound to SM3.

The epitope binding site bound to the crystal peptide can be indicated by the structural coordinates in a two-dimensional display such as LIGPLOT or a three-dimensional display on a computer screen, for example, in FIGS. Using such a display, for example, the “on rate” for the crystalline peptide is increased, and the “off rate” for the crystalline peptide is increased.
) ", It is possible to design a modification to the MUC1 epitope with increased SM3 binding activity. The binding activity of the MUC1 epitope mimic to the epitope binding site is determined by the method used to design the SM3 mimic. Mimetics can be favored with SM3 by, for example, extending the structure of the MUC1 epitope into a space that is indicated as being unoccupied or occupied by a water molecule. Such water molecules can be modified to increase the amount and number of interactions, such as those shown in Figures 8 to 11. The binding activity of the SM3 to the epitope binding site is MUC designed to increase
A mimic of one epitope has SM3 binding activity to aberrantly glycosylated MUC1.
Although such mimics do not appear to cause higher antibody production than that of E. coli, they induce increased antibody response stimulation due to their higher avidity for antibodies. A two-dimensional or three-dimensional representation of the epitope binding site bound to the crystalline peptide can be used to determine why SM3 binds to low avidity peptides, as described above. This information can then be used to select those SM3 mimetics with high avidity for aberrantly glycosylated MUC1 from the library,
A mimic of one epitope can be designed. Such mimetics can also stimulate the production of antibodies with higher avidity for aberrantly glycosylated MUC1 than SM3 when administered with a vaccine.

Such a mimic of the MUC1 epitope modifies the structure of the crystalline peptide that binds to SM3 in the region where unfavorable interaction between the crystalline peptide and the epitope binding site occurs, such as reducing the binding activity. Can be designed by For example, the structure of the MUC1 epitope may be extended in a region where steric hindrance occurs between the carbohydrate on the abnormally glycosylated MUC1 and the epitope binding site. By using such mimics, it is possible to select a specific binding agent (described above) from the library, reduce the degree of steric hindrance to carbohydrates, and generate antibodies having higher binding activity to abnormally glycosylated MUC1. You can stimulate.

Using computer modeling technology, mimics of the MUC1 epitope can be designed in a manner similar to the design of SM3 mimics using this technology. Again, Ca
Using a package such as tallyst / SHAPE or Catalyst / COMPARE, a compound having a three-dimensional shape similar to a peptide when bound to SM3 can be selected. Further, using a package such as DBServer or Hiphop, a compound having a pharmacological group similar to the peptide was found, and Ludi, M
Using packages such as CSS and Hook, mimetics predicted to bind well to the epitope binding site of SM3 can be designed.

Once a mimetic candidate has been designed or selected by the method described above, the effect of the mimetic binding to the epitope binding site of SM3 may be tested and optimized by computer or using experimental evaluations. Can be. As mentioned above,
lyst / SHAPE, Catalyst / COMPARE, DBServer
Mock-ups can be computer evaluated using packages such as, Ludi, and MCSS. Mimetics may also be evaluated in an ELISA competition assay described below. Various parameters can be optimized according to the desired result. Parameters include, but are not limited to, for example, specificity, binding activity, on / off ratio, and other properties that can be readily identified by those skilled in the art.

As described above, some components of the MUC1 epitope can be appropriately substituted or deleted in order to improve the binding characteristics. Usually, the first substitution is conservative, that is, the substitution group is about the same in size, shape, hydrophobicity and charge as the original component.

As in the case of the mimic of SM3, the steps of evaluation and modification can be repeated any number of times. A mimic of a MUC1 epitope may be glycosylated. The mimetic may have intra-peptide hydrogen bonds. This binding can be between the residues corresponding to Pro4 and Thr6 of the MUC1 epitope or between the residues corresponding to Asp5 and Arg7. The atom involved in the hydrogen bond is Pro4 (atom O)
, Thr6 (atomic N), Asp5 (atomic OD1) and / or Arg7 (atomic N)
May be an atom corresponding to

A mimic of the MUC1 epitope has higher, lower, or no binding activity to SM3 than aberrantly glycosylated MUC1. Mimetic binding activity can be tested in an ELISA competition assay. In this assay, MUC1 peptides or fragments thereof, such as abnormally glycosylated MUC1, deglycosylated MUC1 or completely naked core protein, are plated on MUC1
SM3 is added in the presence of the mimic of the epitope. By measuring the ability of the mimic to inhibit the binding of the antibody to the MUC1 peptide, the binding activity of the mimic to SM3 can be measured. Suitable mimetics include those that allow the selection of a specific binding agent from the library that binds aberrantly glycosylated MUC1 with higher binding activity and / or selectivity than SM3. Also preferred mimics of the MUC1 epitope are those that stimulate in vivo production of antibodies with higher affinity than SM3.

As described above, specific binding agents include those selected from libraries containing antibodies,
Alternatively, an antibody produced by a B cell that binds to the peptide mimic is included. Suitable specific binding agents are those that bind aberrantly glycosylated MUC1 with higher binding activity and / or higher selectivity than SM3.

Specific binding agents can be used in diagnostic tests to detect the presence or absence of tumor cells in a tissue sample, for example, in histological screening. The specific binding agent used in such a method may be labeled with a detectable label. Alternatively, the presence or absence of a binding agent can be detected after binding to the abnormally glycosylated specific binding agent using a moiety (moiety) that can specifically bind to the mimetic.

[0057] Specific binding agents can also be used to detect tumor cells in vivo. These can be used for in vivo tumor imaging. Usually, such specific binding agents are labeled with a detectable label. Specific binding agents can be used to prevent or reduce immunosuppression caused by MUC1.

[0058] The specific binding agent can be used in anti-cancer therapy, particularly in the treatment of breast cancer. A specific binding agent that is an antibody or that is substantially similar to an antibody or antibody fragment binds to abnormally glycosylated MUC1 at the tumor cell surface and helps the immune system kill the tumor cells. Specific binding agents may be chemically linked to cytotoxic agents, such as toxins and radioisotopes. By binding such a toxin link-specific binding agent to a tumor cell, the tumor cell can be killed.

Thus, the present invention provides a specific binding agent for use in a method of treating a human or animal by surgery or therapy, such as an anti-cancer therapy, or in a diagnostic method practiced on a human or animal. . The invention also provides the use of a specific binding agent, ie, in the manufacture of a pharmaceutical composition for use in such a method. The present invention provides a pharmaceutical composition comprising a specific binding agent and a diluent or carrier. The present invention also provides a method for treating and diagnosing cancer by administering an effective and harmless amount of a specific binding agent to a human or animal as needed.

The present invention further provides a product containing the specific binding agent and the antitumor agent as a combination for simultaneous, separate or sequential use in anticancer therapy. Hereinafter, the production of such specific binding agents and MUC1 epitope mimics, and methods, procedures and dosages for using the binding agents and mimics will be described.

[0061] When administered to animals or humans, the mimic of SM3 stimulates the production of antibodies that recognize the mimic. By combining B cells from animals or humans with hybridoma technology, such antibodies can be produced as monoclonal antibodies.
Such antibodies may have an epitope binding site similar in form to the crystalline peptide. When such an antibody is administered to an animal or a human, an antibody that recognizes abnormally glycosylated MUC1 is produced. Thus, antibodies that recognize SM3 can be used as vaccines or for producing anti-cancer vaccines.

An antibody or a fragment thereof that recognizes abnormally glycosylated MUC1 produced in response to an anti-SM3 antibody mimic can be used per se for anticancer treatment or diagnostic methods. Thus, the present invention also provides such antibodies or fragments thereof for use in methods of treating humans or animals by surgery or therapy, such as anti-cancer therapy, and diagnostic methods practiced on humans or animals. I do. The invention also provides the use of such an antibody or fragment thereof, ie, in the manufacture of a pharmaceutical composition for use in such a method of treatment. The present invention provides a pharmaceutical composition comprising such an antibody or fragment thereof and a diluent or carrier. The present invention also provides a method for treating and diagnosing cancer by administering an effective and harmless amount of such an antibody or a fragment thereof to a human or animal as needed.

The present invention provides the use of the H3 chain of SM3 as a therapeutic or diagnostic agent. The present invention also provides the use of the H1 chain of SM3 as a therapeutic or diagnostic agent. The present invention also provides H3 or H1 chains for use in methods of treating humans or animals by surgery or therapy, such as anti-cancer therapy, and diagnostic methods practiced on humans or animals. The invention also provides for the use of H3 or H1 chains, ie, in the manufacture of a pharmaceutical composition for use in such a method. The present invention provides a pharmaceutical composition comprising an H3 or H1 chain and a diluent or carrier. The present invention also provides a method for treating cancer by administering an effective and harmless amount of a H3 or H1 chain to a human or animal as needed.
Provide a method of diagnosing.

The present invention provides for the use of structure factors and / or structure coordinates to elucidate the structure coordinates of other crystals. Such crystals may be antibodies or fragments thereof. In particular, the structure coordinates can be used to elucidate the structure coordinates of crystals containing antibodies with non-proline cis binding. The cis peptide bond can be in the H3 chain of the antibody.

The present invention provides for the use of structural factors and / or structural coordinates to molecularly engineer, design, and modify antibodies. Molecular engineering can be performed for the purpose of applying the antibody to humans. The molecular engineering treatment may be a partial replacement of the antibody. Some of the antibodies can be replaced with parts from other proteins, such as human proteins, eg, human antibodies. The two-dimensional and three-dimensional representations described above are used in the molecular engineering of antibodies to ensure that the epitope binding site of the antibody is SM3
Can be the same or substantially the same as the antibody binding site. Molecular engineering of antibodies may be performed to increase the contribution of H3 and H1 in epitope binding.

The present invention provides the use of the above glycine in a protein to insert a non-proline cis peptide bond into the protein. The insertion of a glycine can cause a conformational change in the protein, for example, in connection with a specific binding reaction. The change is such that by taking a cis peptide bond adjacent to glycine, the affinity, avidity or selectivity of the specific binding reaction is modified. Glycine produced by molecular engineering is referred to as “Short Protocols in M”.
olecular Biology, 3 ^rd edition, published by John
Wiley and Sons, Inc. , USA ", using site-directed mutagenesis techniques. The protein may be an antibody or an antibody fragment. Glycine may be in the CDR loop of the antibody. Insertion of glycine into an antibody usually affects the binding of the antibody to the epitope.

The present invention also provides a method for producing a crystal including the use of cadmium. Generally, in this method, one of the portions to be crystallized is brought into contact with cadmium using a cadmium salt solution or the like. Cadmium ions may be present in the solution in which the crystals grow.

Where the mimic of SM3, the mimic of the MUC1 epitope or the specific binding agent comprises a peptide, administration of a nucleic acid encoding such a peptide may cause such an agent to occur in an animal or human. it can. Peptides are produced in vivo by transcription and translation or simply translation of nucleic acids. As those skilled in the art understand,
The nucleic acid may be in a suitable vector, particularly an expression vector such as a virus or organism to be administered, eg, a vaccine virus such as an attenuated vaccine virus, and may be “naked DNA” in the presence of a suitable diluent or carrier. May be administered. Aspects of the invention include nucleic acids encoding such peptides, their use in treating cancer, and compositions containing them.

Thus, the present invention provides nucleic acids for use in methods of treating humans or animals by surgery or therapy, such as anti-cancer therapy, and in diagnostic methods performed on humans or animals. The invention also provides the use of the nucleic acid, ie, in the manufacture of a pharmaceutical composition for use in such a method. The present invention provides a pharmaceutical composition comprising a nucleic acid and a diluent or carrier. The present invention also provides a method for treating and diagnosing cancer by administering an effective and harmless amount of a nucleic acid to a human or animal as needed.

The mimetic of SM3, the mimic of MUC1 epitope, the selective binding agent, the nucleic acid, the virus or the organism containing the nucleic acid (hereinafter, referred to as the substrate of the present invention) of the present invention comprises a pharmaceutically acceptable carrier or diluent. They can be mixed and formulated for clinical administration. For example, the substrate can be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, or transdermal administration. These can be mixed with vehicles such as diluents and carriers which are pharmaceutically acceptable and suitable for the desired method of administration. Pharmaceutical carriers for injection, as diluents, for example, water for injection (Water for Inj)
and sterile or isotonic solutions such as saline.

The dosage can be an effective and non-toxic amount of various parameters, particularly the nature and effect of the substrate used, the age, weight, and condition of the patient being treated, the mode of administration, the conditions of treatment, and the desired clinical regimen. Adjust according to. As a guide, in the case of the amount of a therapeutic substance such as a polypeptide to be administered by injection, usually between 10 and 10000 μg, for example 100
５００500 μg. Especially for the purpose of in vivo diagnostic imaging, the dosage depends on similar factors but needs to be considerably higher in order to obtain a suitable image.

The above administration methods and dosages are given merely as a guide, and those skilled in the art can easily determine the optimal administration method and dosage for a particular patient and condition.

The substances of the present invention can be produced using techniques well known to those skilled in the relevant art. Peptides can be synthesized de novo, or can be produced by transcribing or translating DNA or translating RNA in an appropriate expression system. Nucleic acids can be produced by novel synthesis, obtained from natural sources by conventional probing or cloning techniques, or can be generated from natural sources by modification by well known methods. Antibodies can be generated from appropriate immunization protocols or purified from immunized animals by conventional techniques. It can also be made from in vitro cultured cell lines that secrete hybridomas and similar antibodies, or obtained by expression from appropriate nucleic acids. Hybridomas and other antibody-secreting cells can be obtained by conventional methods, cultured, and used for antibody production. Chemical modification of substances such as peptides and antibodies can be performed by well-known methods. Antibody fragments can be produced by known chemical or enzymatic digestion, or by expression from appropriately modified nucleic acids. New chemicals, such as MUC1 epitope mimetics, can be generated by well known techniques of synthetic organic chemistry.

The substances according to the invention include the international patent applications WO-A-88 / 05054 and WO-A-
It does not include the peptides disclosed in 90/05142. The invention is illustrated by the following accompanying drawings.

Detailed Description of the Drawings Figure 1 shows the MUC1 tandem repeat sequence described from N-terminus to C-terminus using the internationally recognized three letter code (used throughout this specification). Is shown. Epitopes recognized by SM3 are in bold (Pro-Asp-Thr-Ar
g-Pro) with immunodominant regions indicated by double arrows. Putative glycosylation sites are shown in bold italics. Peptides used for crystallization studies (Thr-Ser-Ala-Pro-Asp-Thr-
Arg-Pro-Ala-Pro-Gly-Ser-Thr) is underlined (and referred to herein as "crystalline peptide").

FIGS. 2 and 3 show experimental electron density maps and refined atom models calculated using observed structure factors (30-1.95 °) and phases calculated after solvent correction.
This map is contoured at 1.0σ. FIG. 2 shows M showing immunodominant regions.
Shows the portion of the UC1 peptide antigen. The numbers are given according to FIG. FIG.
Figure 5 shows the cis-peptide binding of CDR H3 between ly96H and Gln97H. Two water molecules forming a hydrogen bond with the amide nitrogen are shown.

FIG. 4 shows the unbound peptide and the bound peptide. Unbound peptide is shown on the left. This structure represents a "knob-like" region and was determined in solution by NMR.
This includes parts of one tandem repeat and two flanking repeats (Fontenot et al., 1995). The conformation of the crystalline peptide bound to SM3 is shown on the right. Here, by SM3 coupling, "
It has been shown that the conformation of the “knob-like” region changes, leading to an extended peptide conformation. The main difference between the two is that Asp5P and A
It may be due to the φ angle with rg7P.

FIG. 5 shows a CPK model of the SM3 binding site with labeled individual CDR loops. The crystalline peptide has been shown to be in contact with all CDRs except H2. FIG. 6 shows an image of the electrostatic surface potential of the SM3 peptide complex. This surface is G
Calculated using RASP (Nicholls et al., 1991). The crystal model is shown as a rod model.

FIG. 7 shows the cis-peptide bond of H3. Residues Gly96H-Gln97
The interaction between and the crystalline peptide (solid line) is shown. Hydrogen bonds are indicated by dotted lines (MOLSCRIPT; drawn by Kraulis, 1991). FIGS. 8, 9, 10 and 11 are LIGPLOTs showing the major interactions that occur between the SM3 binding site, the crystalline peptide and the water molecule.

FIG. 12 shows an experimental electron density map calculated using the observed structure factors (30-1.95 A) and the phase calculated after solvent correction. This is 1.0σ (2Fo
−Fc) and 2.0σ (Fo−Fc). Residue numbering is as indicated herein. The model with this cis-peptide bond is colored by the type of atom. (A) trans Gly96H-Gl
The difference density of 2Fo-Fc calculated for the refined model with n97H peptide bond, (B) cis-Gly96H-
2Fo-Fc and Fo-F calculated for the refined model with Gln97H
c (negative electron density of residues; and positive electron density) map, (C) final cis-G
2Fo-Fc difference electron density map around the ly96H-Gln97H fragment, cis-
Shows hydrogen bonds that are thought to stabilize the peptide conformation. FIG. 13 shows a Ramachandran plot of the H3 fragment refined for cis and trans Gly96H-Gly97H peptide binding. Residues are labeled accordingly. PROCHECK, Laskowski et al. ,
Created by 1993.

FIG. 14 shows the backbone temperature factor of a fragment from H3 containing a non-proline cis-peptide bond between Gly96H and Gln97H. The refined value of the cis-conformation is indicated by a solid square (solid line), and the value of trans is indicated by a solid circle (dashed line). FIG. 15 shows a diagram of the MUC1 peptide at the antibody binding site. Peptide antigens are labeled and are shown in bold. SM3 F interacting with this peptide
The ab residues are labeled. Hydrogen bonds are shown as dotted lines with water molecules shown as black spheres. Plotted by MOLSCRIPT (Kraulis, 1991).

FIG. 16 shows cis peptide binding. Residues Gly96H-Gln97
The interaction between H and the portion of the peptide antigen (bold) is shown. Hydrogen bonds are indicated by dotted lines and water is indicated by black spheres (MOLSCRIPT, Kraulis, 1
991). Hydrogen bonds mediated by water molecules between Gly96H (N) and the peptide antigen are abolished in the Gly96H-Gln96H trans-peptide conformation.

The present invention will be further described by way of examples. Example 1 Evaluation of MUC1 Epitope Binding to SM3 The sequence of the MUC1 peptide in a MUC1 repeat situation is shown in FIG. In order to keep the peptide as soluble as possible and to maintain most of the tertiary structure of the knob region by the NMR structure of the mucin repeat, a region of the MUC1 repeat was selected for co-crystallization studies [Fontenot, J. et al. D. , Mariappan, S .; V
. S. , Catasti, P .; Domenech, N .; , Finn, O .; J.
and Gupta, G .; J. Biomol. Struct. and Dyna
m. 13, (1995) 245-260]. Binding of the MUC1 epitope to SM3 was assessed by inhibition in an ELISA assay. This assay uses the MU
Includes binding of SM3 to a recombinant fusion protein containing seven copies of the C1 tandem repeat, SM3 at a concentration of 1 μg / ml was incubated with increasing peptide concentration, and incubated at approximately 30 μg / ml (apparent Ka to 24 μM 50% inhibition was achieved at the peptide concentration of ()). In contrast, peptides of the same concentration but different sequences (Ala-Arg-Pro-Thr-Gly-Thr-Ser-Asp-Pr
o-Thr-Pro-Ala-Ser), no inhibition was observed at the same concentration.

Example 2 Preparation of SM3 Fab Fragment SM3 spiked at 4 mg / ml in 100 mM sodium acetate, pH 6.0, 3 mM EDTA, 50 mM cysteine was added to 160 μg / ml papain at 37 ° C. Incubated for hours. Fab in Superose in PBS
Gel filtration through 6, followed by dialysis to 20 mM Tris, Ph 8.0, followed by Mono Q ion exchange chromatography for purification. Fractions were analyzed by SDS PAGE and Fab fractions were analyzed for 10 mM Tris,
Concentrated to 4 mg / ml in pH 8.0. Total yield was 22%.

Example 3 Crystallization of SM3 Fab Fragment Bound to MUC1 Epitope The SM3 Fab fragment was mixed with a concentrated solution of the peptide at a molar ratio of 1: 5,
Incubated at 3 ° C. for 3 hours, then concentrated to 9.5 mg / ml. Crystallization was attempted using the hanging drop culture method. A commercially available screen, Hampton Res
search I and II [Jancarik, J. et al. and Kim, S.M. H.
J. Appl. Cryst. 24, (1991) 409-411 and Cudn.
ey, R .; Patel, S .; , Weisgraber K .; , Newhouse
e, Y. , And McPherson, A .; Acta Cryst. D50,
(1994) 414-423]. The first screen did not yield any crystals, but showed that polyethylene glycol (PEG) was the most promising precipitant. Subsequently, the test was carried out by changing the molecular weight of PEG, the pH range of the buffer solution (4-9) and the effect of the salt. Finally, PE as a precipitant
For the G4000 solution and acetate buffer at pH 5.0-7.0, a large amount of fine needles are seen in the equilibrated droplets and aggregate at higher pH 6.5-7.5. A columnar thing appeared. However, these first crystals were all too small for data collection. Since unsuccessful attempts at seeding, other methods for improving the morphology and size of the crystals were attempted, for example, metal ion screens. Addition of cadmium chloride often resulted in larger twinned or aggregated crystals.
However, single crystals could also be separated, one of which was used for data collection. In the final condition, 2.5 μl of the antibody-antigen complex was added to 0.2 M CdCl
₂ , 19% w / v PEG 4000, and 0.1 M acetate buffer, pH 6.0
Was mixed with 2.5 μl of a well solution containing

Example 4 Diffraction Measurement of SM3 Fab Fragment / MUC1 Epitope Crystal A single crystal having a size of 0.4 × 0.2 × 0.03 mm was obtained by DESY synchrotron
Humburg's X11 outstation
Used for diffraction measurement. As a result of analyzing the diffraction data, this crystal has a unit cell size of
a = 42.2, b = 83.9, c = 64.5 °, β = 93.4 ° monoclinic P2₁
It was shown to belong to the space group. Estimate one complex molecule per asymmetric unit
And the specific volume V of the protein_m= 2.50ų/ Da is equivalent to 51% solvent content
(Matthews, 1968). Because the number of available crystals was limited
Data collection was performed at 110K. The mother liquor with the glycerol concentration increased
Transfer 10% of the mother liquor water to glycerol) and then use standard techniques (T
eng, 1990) using Oxford Cryosystem.
It was flash frozen in a stream of nitrogen gas. Complete data set up to 1.95． resolution
Were collected using synchrotron radiation at a wavelength of 0.912 ° (Table 3). This
Data is 180 ° vibration range with 18cm MAR Research Image Plate
Over a 1.0 ° vibrating frame (oscillation frames),
Collected from single crystals. This frame is sent to DENZO software (Otwin
owski, 1993), the result is that 111, with I> 1.0σ (I), 111,
363 observations were obtained. Further scaling with CCP4 suite gives R_m
erge33,093 independent reflections with 6.9% were obtained.

Example 5 Structure Determination and Model Refinement All crystallographic calculations were performed using the CCP4 package [Collab, unless otherwise noted.
orativeComputational Project, No. 4Act
a Crystallogr. D50, (1994) 760-763]. The structure of the SM3-MUC1 peptide conjugate was determined as a search model for the Murine SE155-4 Fab fragment [Cygler attached to dodecasaccharide.
, M .; Rose, D .; R. and Bundle, D.C. R. Science
253, (1991) 442-445] and the program AmoRe [Na
vazza, J .; Acta Crystallogr. A50, (1994) 1
57-163]. Solutions consistent with rotation and translation functions were obtained only after removal of the bound antigen, CDR, N- and C-termini. All non-conserved residues are replaced with alanine and rigid body refinement by X-PLOR [Brunger, A. et al. T. (1992
) X-PLOR, ASystem for Crystalgraphy
and NMR (Yale Univ. Press, New Haven, CT
) Version 3.1] to give a result of 40% crystallographic R-factor and 49% R-free as calculated for a randomly selected set of reflections. [
Brunger, A .; T. ActaCrystallogr. D49, (199
3) 24-36]. At this stage, some omit maps were calculated to cover all molecules, and the models were programmed in program 0 [Jones, T .; A. an
d Kjeldgaard, M.D. (1994) 0-The Manual. Up
psala University, Sweden]. This model was further converted to a standard X-PLOR with a resolution of 3.0 ° cutoff.
Refinement by simulation annealing protocol followed by 0 and Tu
rbo-Frodo [A. Russel and C.I. Cambillau, M
arseille, France]. The R factor converges to 34% (43% R-free), close to the CDRs, residues 2-10 of the peptide
A substantially continuous positive electron density corresponding to was visualized. The peptide is incorporated into the electron density and REFMA provided in the CCP4 package and X-PLOR
Using C, the resolution was further increased to 1.95 °, while another round of refinement, DM solvent correction [Cowtan, K. et al. Protein Crystal
logy31, (1994) 34-38] and manual reconstruction. Inspection of the Fourier map of the difference between 2Fo-Fc and Fo-Fc showed a significant positive electron density corresponding to eight cadmium ions and a large number of solvent molecules. Several refinement cycles were repeated and the location of water and cadmium after each cycle was confirmed. All atoms were refined with individual isotropic B factors. Engh-Huber parameters as used in X-PLOR [Engh, R .; A. a
nd Huber, R .; (1991) Accurate bond andan
gle parameters for X-ray protein str
result refinement. Acta Crystallogr. A
47, 392-400] to change the Gly96H-Gln97H bond to cis
Made conformation. No electron density was seen for heavy chain residues 128-133 and some terminal residues (L2, L209-212, H214, P1 and P11-13). For the same reason, residues Asp41L, Glu123
L, Gln163L, Gln1H, Glu42H, Glu61H, Glu85H
, Ser134H, Ser160H, Ser172H, Asp173H, Ser
203H, Lys208H and Ser2P were modeled as alanine.
The final model contains 3227 atoms, including the peptide, 333 water molecules, 2 chlorines and 8 cadmium ions, and the final refinement statistics are shown in Table 4.
It was shown to. The full quality of this model is measured in the PROCHECK program [Laskow
ski, R .; A. , MacArthur, M .; W. , Moss, D.C. S. and
Thornton, J .; M. J. Appl. Crystallogr. 26,
(1993) 283-291]. The buried contact surface area is
The program PDBAREA [Lee et al. J. Mol. Biol (19
71), 55, 379-400]. During the refinement and manual reconstruction, it was found that some of the SM3 primary sequences were not accurate. S
The cDNA corresponding to the M3 Fab region was sequenced to confirm an error in the SM3 sequence.

This peptide was clearly incorporated into the electron density from the Fourier map of the differences. MU
Since the C1 peptide was almost symmetric, it was necessary to confirm the correct orientation of the peptide in electron density. This was done by backward modeling of the peptide, resulting in poor density fit and higher R-free and crystallographic R-factor after one round of refinement.

The accuracy of the cis-binding was confirmed by trying to refine it as a trans-binding. This resulted in a significant increase in the B-factor for Gly96H in the backbone, and thus poor quality electron density maps in this region (data not shown). Furthermore, when the peptide bond adopts a trans conformation,
Some possible hydrogen bonding between the peptide and the antibody becomes impossible. REF
Using MAC (CCP4, 1994), the results of such unrestricted refinement were always the cis-conformation of the Gly96H-Gln97H peptide bond and the electron density shown in FIG.

Example 6 SM3 Fab Fragment / MUC1 Epitope Structure The structure of the SM3-peptide antigen complex reveals some unexpected new insights into how antibodies recognize peptide antigens. .
In particular, CDR loop H3 contains a non-proline cis-peptide bond. SM3 f
The structure of the ab fragment / MUC1 epitope was confirmed to a resolution of 1.95 °.
The residues of this epitope all fall into the favored region of the Ramachandran plot, except for the two residues (Thr51L, Ser93L) of the CDRs (L2 and L2) involved in the interaction with the binding peptide ( Data not shown). Individual atoms B- factor is in the range of reasonable limits (mean 16.3Å ^2),
Higher values are only shown within residues located in mobile loops or fragments exposed to solvents (not shown). The quality of the density corresponding to the 9 residues of the MUC1 peptide antigen is shown in FIG. 2A. Peptide residues, the average atom 15.3Å ² B-
Factor, which indicates that the observed peptide is correctly located.

The SM3-peptide has eight cadmium sites, five of which are fully occupied and three with an occupancy of 0.75. They are mainly in contact with the carboxyl group of Glu or Asp and the histidine nitrogen atom between the symmetrically related molecules and / or chains (data not shown). All have distorted octahedral coordination completed by bound water and chloride ions. If crystallized without cadmium,
Cadmium ions are believed to stabilize contact between chains or molecules and contribute to crystal packing, as small needle-like crystals that were too small for diffraction experiments were generated. The SM3-cadmium structure is an example of an unusual crystal structure in which cadmium ions are introduced directly, rather than simply displacing other bound metal ions.

SM3-MUC1 Peptide Antigen Interaction The MUC1 peptide has two intra-peptide hydrogen bonds: Pro4 (atom O) and Th
Arg7 (atomic N) is formed with r6 (atomic N) and Asp5 (atomic OD1).
The MUC1 peptide is located in the extended groove of the SM3 antibody and contains all six CDRs.
Surrounded by loops. The peptide is immobilized by electrostatic interaction between Asp5P and Arg7P and binds to the SM3 antibody primarily through hydrophobic contact (Table 4). SM
Although all six CDRs of the three antibodies are involved in MUC1 peptide binding to some extent, H2
In the chain, only residue Arg52H contacts the peptide, due to water-mediated interactions. The major contribution to antigen binding is due to H1, and its three residues Asn
31H, Tyr32H, Trp33H are in contact with 7 residues of the peptide (
Table 4). These interactions account for 38% of the surface area of all CDRs buried in antibody-peptide bonds, and are primarily hydrophobic, with some hydrogen bonds and salt bridges. The cis-peptide bond between Gly96H and Gln97H plays an important role in antigen binding (Table 4). The nitrogen atom of Gly96H forms a hydrogen bond with both the carbonyl oxygen of Asp5P and the OD2 carboxyl group via a water molecule. The hydrophobic part of the Gln97H side chain consists of Trp33H (H1) and Tyr32L (L
Interacts with Pro4P located between 1). Pro4P is Trp91L (
L3), which is the most buried residue of all peptide antigens. The head group of the polarity of Gln97H is L1,
Direct hydrogen bond with L2 and Asp5P residues. The important antigenic residue Arg7P is
Interacts with Tyr32H (H1) and Asn31H (H1), while the C-terminal residues of the peptides are Pro56L (L2), Phe27H (H1) and Tyr10
It is located in a hydrophobic pocket surrounded by 2H (H3). In summary, there are several direct water-mediated hydrogen bonds between the peptide and the antibody (Table 4). Other SM3
-The peptide interaction is mainly hydrophobic.

On the SM3 antibody binding surface, the entire antigen-antibody contact region 185 °²Of which, 128Ų
Are occupied by hydrophobic interactions (Table 5), with hydrophobic contacts predominating. Pepti
Similarly, a high percentage was found for the deantigen, with 66% of the buried surface area (6% in total).
03Ų) Is represented by a non-polar residue. Such a high percentage indicates that hydrophobic interactions
It suggests that it is the main driving force in SM3-peptide complex formation. Average
Fabs provide space for binding about 10 residues of the peptide antigen, with the rest remaining
Very mobile and therefore often disturbed, not observed in electron density maps. SM3
There is apparent surface complementarity between the antibody and the peptide antigen. Of particular interest is H
3 around Pro4P likely to be facilitated by the presence of a cis-peptide bond within
Surface complementarity of the box (described below).

The Brookhaven database (Bernstein et al., 1)
977), no suitable H3 substitution could be found in the fragment search performed on the entire antibody structural library registered in 977). In the best solution of the search performed using the distance between the atoms of the base of H3, the Cα atom at the equivalent position of the crystal structure coordinate of H3 was shifted from the Cα atom of the fragment by a square root of 2.6 根 mean square displacement. Things. However, it is well known that it is difficult to classify and predict the conformation of CDR H3 (Reczko et al., 1995; Shirai).
et al. , 1996). In the SM3-peptide antigen structure, CDR H
Residues from 1 to a smaller range, H3, contribute primarily to peptide antigen binding (Table 4). This includes L3 and H3 (MacCallum et al., 199).
6) or H2 and H3 (Stanfield and Wilson, 1993)
) Is in contrast to other antibody-peptide complexes that primarily contribute to antigen binding. One obvious conclusion from this is that without the crystal structure of the complex, it is difficult to predict the contribution of each CDR to peptide antigen binding.

The presence of non-proline cis-peptide bonds in proteins is extremely rare, accounting for 0.05% of known protein structures containing such bonds (Step
art et al. Herzberg and Moult, 1;
991). To our knowledge, the SM3-peptide complex is the first such example in the hypervariable loop region of the antibody binding site. However, non-proline cis-peptide bonds have been found in the active site of many hydrolases (Perrakis et al., 1994). In these examples, ci
The s-peptide conformation is probably involved in promoting substrate contact and / or recognition (Perrakis et al., 1994). Interestingly, to date, all antibody-peptide structures contain a cis-proline residue within CDR L3, which is important for peptide-antibody binding. The reason for the conservation of Cis-Pro is not clear, but the two residues at each end of cis-Pro are at least 1
It is interesting to make one direct peptide contact and a second indirect contact via the other residue of the CDR. However, the SM3 structure does not contain an equivalent proline in L3, but has a non-proline but cis-peptide bond on the opposite side of the antibody binding surface of H3. From these observations, at least two conclusions can be drawn. (1) Antibody-
The cis-peptide bond within the antigen-binding site is an important and common feature of peptide-antigen binding. (2) SM3-peptide conjugates have a cis-peptide bond that is non-proline and a separate CDR loop. Is different from all other antibody-peptide conjugates. The role of the cis-peptide bond has been associated with specific interactions with the hydrophobic surface on the peptide antigen. Another reason that this binding is cis rather than trans is due to the small size of H3 due to extended peptide antigen epitopes running along its top surface. All trans in small size
CDRs have little freedom to accept such changes.

Comparison of Bound and Unbound MUC1 Peptide Antigen NMR Study of SM3-Bound MUC1 Peptide and Three MUC1 Repeats (Fo
ntenotet al. , 1995) is shown in FIG. The structure of the core region within each repeat in the solution structure is different from that of the antibody binding peptide antigen. The entire geometry of the “knob-like” projections, when bound to the SM3 antibody, is as seen in solution (Fontenot et al., 199).
5) Changed from two continuous II β-turns to an essentially extended geometry.
For the bound and unbound peptides, there is a difference in all of the twist angles φ and Ψ, the main difference being concentrated in the φ angles of Asp5P and Arg7P, both of which contribute a significant percentage (%) of total peptide contacts. (Table 4).

Even when rearranged, the charged headgroup in antibody binding remains partially solvated. This provides a dominantly hydrophobic surface at the SM3 antibody binding site. Therefore, if the NMR structure of the MUC1 repeat represents the most prominent conformation in vivo (Fontenot et al.
. , 1995), the SM3 antibody, upon binding, induces a change in the mucin molecular conformation, at least in the peptide epitope region. This suggests that recognition of the SM3-peptide is by an "induced fit" mechanism.

[0098]

Table 1 Structural factors of SM3 fragments / crystal peptide crystals described in the examples. This table is shown below. The data was arranged in four individual columns as described below. Each row of columns consisting of five numbers indicates the result of one reflection. From left to right, the first three numbers represent h, k, and l, respectively, and are the crystallographic reflection indices. The fourth number represents the structure factor F (hkl) defined as the sum of the Fourier series calculated for each reflection hkl. Since the same reflection was measured multiple times, the standard deviation for each structural factor is shown as the fifth number in the column.

[0100]

[Table 1]

Tables 2a and 2b are the structures (atomic coordinates) of the SM3 fragments / crystal peptide crystals described in the examples. These tables are shown below. The first column, CRYST1, defines the space group and the size of the vesicles. Scale 1, scale 2 and scale 3 define an orthogonalization matrix. The orthogonalization matrix, when applied to individual coordinates, converts those coordinates to each axis x, y
, Z are in a rectangular coordinate system of 90 degrees. Reading from left to right below these columns, the second column shows the chemical symbols of the atoms. OH2
Represents a water molecule. The next letter indicates the position of the atom in the amino acid residue expressed using conventional methods recognized by those skilled in the art. The letters refer to the corresponding Greek symbols (ie, A for alpha, B for beta, etc.). The third column is the residue where the atom is located. WAT represents a water molecule. The fifth column shows the number of the amino acid in the protein. The sixth, seventh, and eighth columns show the spatial positions of the atoms in xyz coordinates, respectively. The ninth column shows the occupancy of the atoms, 1.0 when the atom is present, 0 when the atom is not present (ie, not observed).
. 0. The tenth column represents factor B, which means mobility.

[0102]

[Table 2]

[Table 3] In the table, D is the resolution, and is defined as an inter-plane spacing d = λ / 2 sin θ.
This corresponds to the maximum 2θ value observed when λ = 1.54 °. The lower the resolution, the higher the number; the higher the resolution, the lower the number. Where I = intensity and σ (i) is
The standard deviation of the intensity for each batch of reflections. This indicates how well the data was measured. The higher the value, the stronger the data, and the lower the value, the weaker the data. This value decreases as the resolution increases, for example at 1.95 ° the diffraction angle is quite large. Table 4 shows a summary of the refined statistics for the structure of SM3-EP1.

[Table 4] Table 5 shows antigen-antibody contacts, with residues involved in cis-peptide binding underlined. SAS is the solvent-accepting surface of the peptide residue, residue no. 2 is Ser, but was modeled as Ala.

[Table 5] Table 6 shows the nucleotide and amino acid sequences of the variable light and heavy chains of SM3.
The numbering in this table is for convenience only and does not correspond to the conventional numbering of these sequences.

[Table 6] Table 7 shows the dihedral angles phi (φ) and psi (Ψ) for the bound MUC1 peptide antigen.

[Table 7] Table 8 shows SM3-MUC1 van der Waals contacts (vdW) and hydrogen bonding (Hd
B, direct and via water). Residues involved in cis peptide binding are underlined. Ser2P was modeled as Ala. The distance cutoffs used are 4.0 ° for vdW contact, 4.0 ° for direct HB (weak HBs above 3.5 ° are marked with an asterisk), and 3.5 ° for HB through water. MC and SC mean the main chain and side chain of the SM3 antibody, respectively.

[Table 8] Table 9 shows the buried MSA in complex formation of SM3-MUC1 peptide antigen. MSA was calculated for each SM3 CDR and peptide antigen alone. Residues involved in non-proline cis peptide binding are underlined.

[Table 9] Note that the MSA uses an MS program (Connoll) using a probe radius of 1.7 °.
y, 1983). Table 10 shows SAA buried in antibody-peptide complex formation, PDB
Calculated using AREA.

[Table 10]

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of MUC1 tandem repeat.

FIG. 2 shows an experimental electron density map and a refined atomic model of the epitope binding site of SM3 and the portion of the crystalline peptide.

FIG. 3 shows an experimental electron density map and a refined atomic model of the epitope binding site of SM3 and the portion of the crystalline peptide.

FIG. 4 shows a comparison of the conformation of unbound and bound peptides.

FIG. 5 shows the molecular surface properties of SM3-crystal peptide interactions.

FIG. 6 shows the molecular surface properties of SM3-crystal peptide interactions.

FIG. 7 shows a stereogram of the non-proline cis-peptide bond of CDR H3.

FIG. 8 is a LIGPLOT of a peptide binding site.

FIG. 9: LIGPLOT of the peptide binding site.

FIG. 10 is a LIGPLOT of a peptide binding site.

FIG. 11 is LIGPLOT of the peptide binding site.

FIG. 12 shows an experimental density map of the cis-peptide conformation of H3.

FIG. 13 shows a Ramachandran plot of H3 fragments.

FIG. 14 shows the backbone temperature factor of the fragment from H3.

FIG. 15 shows a stereogram of the MUC1 peptide at the antibody binding site.

FIG. 16 shows a stereogram of the non-proline cis-peptide bond of H3.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C07K 16/18 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 33/574 A 33/574 33/68 33/68 G06F 17/50 G06F 17/50 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA , BB, B , BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG , SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor David Sneary Kent B.R. B.L., Orpington, Crofton Lane 213 (72) Inventor Michael Joseph Ezra Stanberg Middlesex H.A.8.8.7 C.X, England, Edgware, Canons Drive 5 (7 2) Inventor Paul Alan Bates, UK, London E 18.2 P.Y., South Woodford, Derby Road 61 (72) Inventor Powell Docano, London ES, UK・ E ・ 6.4 ・ TS ・ Nelgard Road ・ 20 ・ B F Term (reference) 2G045 AA25 AA34 DA36 DA77 DA78 FA40 FB01 FB03 JA01 4C084 AA02 AA07 BA01 BA16 BA18 BA44 CA27 CA59 DC50 ZB092 ZB262 4C085 AA03 BB01 CC CC32 DD63 EE01 4H045 AA10 AA11 AA20 AA30 BA13 BA16 DA76 EA28 EA50 GA40 5B046 AA00

Claims (33)

[Claims] 1. A structural factor obtained by performing an X-ray diffraction measurement on a crystal containing at least a fragment bound to a peptide recognized by the epitope binding site of SM3, which is an epitope-binding fragment of the SM3 antibody. The use of chemicals to identify, screen, characterize, design or modify chemicals. 2. An X-ray diffraction measurement is performed on a crystal that is an epitope-binding fragment of the SM3 antibody and contains at least a fragment bound to a peptide recognized by the epitope-binding site of SM3. Use of structural coordinates obtained by estimation to identify, screen, characterize, design or modify chemicals. 3. The use according to claim 1, wherein the SM3 antibody fragment is a Fab fragment obtained by digesting SM3 with papain. 4. The use according to claim 1, wherein a substantially similar part to the SM3 antibody fragment is used in place of the SM3 antibody fragment. 5. The method according to claim 1, wherein the peptide comprises the sequence Pro-Asp-Thr-Arg-Pro. 6. The sequence having the sequence Thr-Ser-Ala-Pro-Asp-Thr-Arg-Pro-
6. Use according to claim 5, which is Gly-Ser-Thr. 7. A method for identifying, screening, characterizing, designing or modifying a chemical.
Use of the structural factors shown in Table 1. 8. A method for identifying, screening, characterizing, designing or modifying a chemical,
Use of the structural coordinates shown in Tables 2a, 2b. 9. Use of a structural factor or structural coordinates according to any one of claims 1 to 8 for identifying, screening, characterizing and designing mimetics of SM3. 10. The use according to claim 9, wherein the mimic of SM3 has a desired association with aberrantly glycosylated MUC1. 11. The use according to claim 10, wherein the desired association is a binding affinity for aberrantly glycosylated MUC1 that is higher than that of SM3 for aberrantly glycosylated MUC1. 12. The use according to any one of claims 9 to 11, wherein the mimic of SM3 is obtained by modifying the whole SM3 or a fragment of SM3. 13. Use according to claim 12, wherein the modification is a substitution of SM3 for proline 56. 14. Identifying a mimic of a peptide epitope of MUC1 recognized by SM3,
Use of a structural factor or structural coordinates according to any one of claims 1 to 13 for screening, characterizing and designing. 15. The use according to claim 14, wherein the mimic of the peptide is used to generate a moiety having the desired association with aberrantly glycosylated MUC1. 16. The use according to claim 15, wherein said moiety is an antibody. 17. The use of claim 16, wherein the antibody is produced by a method comprising vaccinating an animal or human with a mimic of the peptide. 18. The method according to claim 1, wherein the mimetic of the peptide is capable of preventing or reducing an immune reaction.
Use according to 4. 19. The use according to any one of claims 14 to 18, wherein the mimetic of the peptide is obtained by chemical modification of the peptide. 20. The mimetic of the peptide is a glycosylated peptide.
Use according to any one of the preceding claims. 21. A mimic of a peptide epitope of MUC1 recognized by SM3,
Chemicals not listed in any database as of the filing date of this application. 22. SM3 not listed in any database as of the filing date of this application
Mockup of. 23. An antibody having a conformation comprising a molecularly engineered non-proline cis peptide bond. 24. SM bound to a peptide recognized by the epitope binding site of SM3
3. A crystal comprising at least an epitope-binding fragment of an antibody. 25. The method for producing a crystal according to claim 24, comprising contacting the SM3 fragment or peptide with cadmium. 26. A machine readable data record comprising a data record material encoded with machine readable data capable of displaying a three-dimensional representation of the crystal of claim 24 when read by a suitable machine. Medium. 27. The method according to any one of claims 7 to 23, wherein the method is used in a method for treating a human or an animal by surgery or therapy, or for a diagnostic method used for a human or an animal.
A chemical substance, a mimic of SM3, a mimic of a peptide epitope of MUC1 recognized by SM3, a portion having a desired association with abnormally glycosylated MUC1, or an antibody manufactured by molecular engineering. 28. The chemical substance, a simulated form of SM3, SM3 according to any one of claims 7 to 23.
Mimetic of a peptide epitope of MUC1 recognized by MUC1, a portion having a desired association with aberrantly glycosylated MUC1 or an antibody engineered by molecular engineering, by anti-cancer treatment of human or animal by surgery or treatment, or human or Use in the manufacture of pharmaceuticals for cancer diagnosis in animals. 29. The chemical substance, a simulated form of SM3, SM3 according to any one of claims 7 to 23.
Mimic of a peptide epitope of MUC1 recognized by MUC1, a portion having the desired association with aberrantly glycosylated MUC1, or an effective and non-toxic amount of a molecularly engineered antibody can be administered to a human or non-human animal. And a method of diagnosing cancer or treating it as an anti-cancer treatment, which comprises administering as necessary. 30. A product comprising a mimetic or specific binding agent of SM3 and an antitumor agent as a combination for simultaneous, separate or sequential use in anticancer therapy. 31. A mimic of the MUC1 epitope recognized by SM3 for use in treating a disease caused by an immune response. 32. An immune response caused by an effective and non-toxic amount of a mimic of the MUC1 epitope recognized by SM3 to a human or non-human animal, as needed. How to treat the disease. 33. Use of a structure factor and / or structure coordinates according to any one of claims 1 to 8 for solving the structure coordinates of a crystal.