JPH04501719A - polypeptide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 20. Methods of treating the human or animal body by surgery or therapy; or of drugs for use in diagnostic methods or carried out on the body of animals. In preparation, the polypeptide according to any one of the above items 1 to 4, the above items 5 to 7 The nucleic acid fragment according to any one of the above items, the construct according to the above item 8, the above item 9 to 11 The vector according to any of the above, the cell according to the above item 12, the protein according to the above item 13 the antibody according to item 14 or 15 above, the pharmaceutical formulation according to item 16 above Alternatively, use of the cells described in item 17 or 18 above.

21. A non-toxic amount effective for humans or animals in need of treatment or diagnosis. The polypeptide according to any one of Items 1 to 4, the polypeptide according to any one of Items 5 to 7 above. A nucleic acid fragment, a construct according to item 8 above, a vector according to any one of items 9 to 11 above. a cell according to item 12 above, a probe according to item 13 above, a cell according to item 14 above, or a cell according to item 12 above. is the antibody according to item 15, the pharmaceutical formulation according to item 16 above, or the pharmaceutical composition according to item 17 above. or a method of treatment or diagnosis comprising administering the cell according to item 18.

22. Samples suspected of containing abnormal human mucin glycoproteins are A diagnostic test method comprising contacting with the antibody according to item 4 or 15.

23. A diagnostic test kit comprising the antibody according to item 14 or 15 above.

Specification polypeptide The present invention provides a sequence corresponding to an antigenic epitope on human pleomorphic epithelial mucin (PEM). Polypeptides having sequences thereof, their production and their use in the diagnosis and treatment of cancer Regarding.

Expressed by normal and transformed epithelial cells in WO38105054 The sequence of tandem repeats of amino acid residues that appear in the human PEM protein Are listed. In the case of breast cancer, malignant cells are expresses a glycosylated form of PEM that is detected by a null antibody. the Antibodies are produced by normal cells and do not react with fully processed PEM. Therefore, it is used in the diagnosis and treatment of breast cancer.

Further studies surprisingly revealed that the epitopes recognized by 5M-3 A loop is a small, contiguous sequence of amino acids that occurs within a previously described tandem repeat sequence. It was revealed that it was a column. Identification of this epitope suggests that, contrary to normal PEM, Epitopes that are useful in generating antibodies with improved specificity for tumors allows the synthetic preparation of peptides that mimic

Therefore, the present invention provides (a) formula (I) (Xl, ((A), %PDTRP (A), +7(Xl □ (I) in the formula, X and 2 are the same or different, and each is 0 or 1 or is an integer greater than or equal to y is an integer of 1 or more, Each X, if present, is independently selected from amino acid residues and m and n are the same or different, and each is O or 1, and A, P, DST and R are amino acid residues, alanine, proline, a synthetic polypeptide of aspartic acid, threonine and arginine, (b) (A),,PDTRP (A), and (c) one or more polypeptides containing a sequence immunologically equivalent to A polypeptide as defined in (a) and (b) above having a saccharide of , and a pharmaceutically acceptable salt thereof, However, polypeptides and bases specifically disclosed in WO38105054 Excludes targlucuronidase.

The polypeptides defined in (a), (b) and (C) above have the same limitations. hereinafter referred to as the [PDTRP epitope].

The simplest polypeptide according to the invention is the pentapeptide PDTRP.

Immunological analogues thereof and glycosylated derivatives of pentapeptides and analogues thereof It is the body. The hexapeptides according to the present invention include APDTRP, PDTRPA, Immunological analogues and glycosylated derivatives of pentapeptides and their analogues. include. The heptapeptide according to the invention is APDTRPA, its immunological analogue and glycosylated derivatives of pentapeptides and analogs thereof. Book Larger polypeptides according to the invention include penta-, hexa- and hepta-pepeptides. Additional compounds at the amino and/or carboxy terminus of compounds, analogs and derivatives Contains amino acid residues. Such polypeptides may include, for example, up to about 200, e.g. up to about 50, especially up to 30, preferably up to 20, more preferably from 10 to It can contain 15 amino acid residues. The maximum preferred values of X and 2 are It will be decided according to the Polypeptides are small (with one five-amino acid residue sequence PDT RP or an immunological analogue thereof, and this sequence ( A few repetitions, e.g. 2 or 3 repetitions, and perhaps 100 or more repetitions. can include revenge. Each such repeat independently has an amino terminus and/or may include one or both carboxy termini, or immunological equivalents thereof. and extended with other amino acids at either or both the N- and C-termini. It can be long. Typically each repeat has 19 or fewer amino acid residues. It will consist of If the polypeptide is glycosylated, the PDTRP sequence The repeats of the string or analogs thereof are independently glycosylated, e.g., the sugars of the chain, For example, N-acetylgalactosamine is placed on the threonine residue of PDTRP and / or installed on threonine or serine residues outside the epitope PDTRP sequence. or within the epitope sequence of the immunological equivalent of PDTRP. (e.g., where the threonine residue is (substituted by a serine residue).

Immunological analogues according to the invention are polypeptides having a sequence corresponding to the sequence of formula (I). however, sequence (A), PDTRP (A), has at least one Modified by substitution and/or one or more amino acid residues modified by the insertion of additional amino acid residues, but the modified sequence is The modified epitope can act as an epitope for the generation of antibodies. Antibodies so generated that recognize the pitope are (a) polypeptide of formula (1), Cb) from a serial repeating array as defined in WO88105054 a polypeptide, and (c) human PEM core protein, In each case, a normal lactating human A fully processed human PE glycoprotein produced by the human mammary gland. reaction is reduced or does not react with it, or preferably Does not respond qualitatively.

Immunological analogs are also known as mimotopes. For example, “Advances in Peptide Protein and Gene Technology” Protein and Gene Techn.

1ogy Advances) J, Publication No. 2, Cambridge Research Library. Iochemical (Cambridge Re5earch Bjochemica) l) (copy attached) and “mimicking the assembled epitope” The delineation of Pepti de able t, o mimiccassembled Epitopes) J, H, M, Geysen et al., “Synthetic peptides (Sy) as antigens. nthetic Peptides as Antigens) J [Ciba Fa Ciba Foundation Symposium ium 19) J, R, Porter and J, Uniran (Whelan) (ed.), Pitman, London 1986, pp. 130-1. It can be designed as described in 49.

Preferably, the polypeptides of the invention are synthetic.

PDTRP epitopes according to the invention can be obtained by synthetic methods or as described below. By expressing a suitable NA fragment, it can be synthesized de novo by recombinant DNA technology and glycosylated or non-glycosylated in human or non-human cells It can be expressed in

Peptides can be made from a single amino acid and/or from two or more amino acids. can be constructed from preformed peptides in the desired peptide sequence order; If necessary, it can be converted into a pharmaceutically acceptable salt.

Solid phase or solution methods can be used. In solid phase synthesis, the desired peptide The amino acid sequence of Tide is sequentially composed of C-terminal amino acids bound to an insoluble resin. be done. When the desired peptide is produced, it is cleaved from the resin. solution phase When using a peptide, the desired peptide is again composed of the C-terminal amino acids, but The carboxy group of this acid remains blocked with a suitable protecting group, and this protecting group is removed only at the end of the synthesis. When using either technique, the reaction Each amino acid added to the system typically has a protected amino group and an activated It has a carboxy group. The amino group is fluoren-9-ylmethylcarbonyl (F moc) or t-butoxycarbonyl (Boc) groups. Ru. Carboxy group is pentafluorophenyl or 1-oxy-2-hydro It can be activated as a xydihydrobenzotriazine ester. functional side chain groups, such as the side chain amino group of lysine and the side chain carboxy group of threonine. Groups can also be protected. Each condensation step consists of dicyclohexyl carposiimide The reaction can be carried out in the presence of hydrogen or 1-hydroxybenzotriazole.

After each step in the synthesis, the amino protecting group is removed. The protecting group for the side chain functional group is Generally removed at the end of synthesis.

To convert the resulting peptide into a pharmaceutically acceptable salt, it is treated with an organic acid or or immunoglobulinic acid to form acid addition salts. A suitable acid is , acetic acid, succinic acid and hydrochloric acid. Alternatively, the peptide can be a carboxylate salt, For example, ammonium salts or alkali metal salts, such as sodium salts or carbon Conversion to lithium salts can be achieved by using a suitable base.

PDTRP epitopes of the invention can also be prepared by recombinant DNA methods. can be used: an expression vector is used that expresses the PDTRP epitope. When incorporated into a DNA sequence encoding a PD and placed in a suitable host TRP epitopes can be expressed.

The DNA sequence is placed between the translation start and stop signals in the vector.

Also suitable transcriptional control elements, such as promoters of DNA sequences and Prepare a transcription stop site. Expression of the peptide occurs in a host compatible with the vector. Prepare the DNA sequence in the correct reading frame so that it can be read.

Any suitable host-vector system can be used. For example, vectors are plasmids. can be a prokaryotic or viral vector, and the host can be any prokaryotic or viral vector. or eukaryotic cells, such as bacterial cells or yeast cells or mammalian cells. It can be a cell of an animal cell line.

Alternatively, the PDTRP epitope may be derived from the natural human mucin glycoprotein (which itself expression in human cell lines or by isolation from human tissue samples Stripping carbohydrates from (which can be produced through complete processing) [Burche et al., Cancer Research (Canc. er　Re5earch), 47.5467-5482　(1987)], and can be obtained by digesting core proteins.

carrier proteins, e.g. keyhole limpet hemocyanin, albumin PDTRP epitopes linked to thyroglobulin or thyroglobulin are also within the scope of the present invention. It is within the range. The linkage may be by known chemical methods or as described herein. PDTRP epitope and carrier tan by genetically transformed cells It can be formed by expressing protein as a fusion protein.

In another aspect, the invention provides nucleic acid fragments encoding PDTRP epitopes. provide

A fragment according to the invention can be RNA or DNA, and a DNA fragment is It can be single-stranded or double-stranded. Preferred such nucleic acid fragments are A sequence of formula (II) encoding a defined PDTRP epitope of formula (I) (XXX), ((GCX), CC X GAPV ACX [CGX] [or] CCX (GCX), 1, (XXXI, (I I) [AGP ul where mSn, xSy and 2 are all as defined with respect to formula (1), X is a deoxyribonucleotide and XXX encodes an amino acid residue a codon, py is C or T, Pu is A or G, and A, C, G and T are internationally recognized for the four deoxyribonucleotide bases. This is a one-character code.

Preferred nucleic acid fragments of the invention have the sequence (GCC), CCG GACACCAGG CCG (GCC).

Consisting of

Another sequence encoding a PDTRP epitope is β-glucof E. coli. Coding sequence present in lonidase and also for PDTRP Contains CCG GAT ACCCGT CCG. contains this sequence and requires If necessary, it can be further related to the β-glucuronidase sequence. Flanking sequences that cannot be related ・National Academy of Sciences (Proc, Natl, Aca d, Sc i,) USA, 8447-8451. Fragments consisting of [other than β-glucuronidase gene and fragments] of the present invention Form the top surface.

The present invention further provides PDs that are immunologically similar to the peptides of formula (I) defined above. Nucleic acid fragments encoding TRP epitopes are provided.

These fragments are used to generate PDTRP epitopes as described below and Both as probes to detect DNA encoding PDTRP epitopes. Useful. For such purposes, detectable labels (e.g. radioactive isotopes) fluorescent, fluorescent or enzymatic labels) to the fragments or to a solid support. It may be convenient to combine. All of this can be achieved using conventional methods. [Matthews et al., Analytical ・Biochemistry (Analyt, Biochem,), 169. ], − 25 (1988)].

Can the nucleic acid fragments of the present invention be produced de novo by common synthetic techniques? or can be obtained from human epithelial cells by conventional methods [Huynh ) et al., rDNA cloning: a practical approach (DNA Cloning: A Practical Approach) J., Grover, D. M. (ed.) (IRL, Oxford), 1985, Vol, 1, pp, 49-48].

In another aspect, the invention provides cloning and Expression vectors (expression vectors are open read sources for start and stop signals) have the fragment in frame and make appropriate adjustments and adjustments to form the expressed sequence. and associated promoter sequences) and such cloning or expression vectors. provides a host cell transformed by the vector.

Nucleic acid fragments of the invention can also be used in active immunization techniques.

The genetic information of the PDTRP epitope is designated for use in such methods. The defined fragment is either the 5' or 3' end, or both, and further analysis is required. Readable or unreadable nucleic acid sequences include regulatory or promoter sequences, marker sequences and Includes splicing or binding sites. Encodes PDTRP epitope In addition to The genetic information of the chain can be specified. Fragments according to the invention may be any necessary or a suitable oven reading frame along with the desired flanking sequence. alignment of a suitable vector, e.g. a plasmid or viral genome (e.g. for example, into the vaccinia virus genome), and then the polypeptide product expressed by ordinary techniques. In one aspect, a PDTRP epitope The polypeptide product containing activity against mucin core proteins, harvested, and used as an immunogen. [Tartaglia et al., T. ibtech, 6.43 (1,988)E. In other aspects, the vector is Particularly in the form of viruses, which can be directly inoculated into the human or animal to be immunized. Wear.

The vector then directs the expression of the PDTRP epitope, which subsequently It acts as an immunogen and induces immunity against mucin core proteins.

The DNA sequence, in one embodiment, is inserted into and fused into another gene. Can be expressed as a protein. For example, other amino acid sequences (kore lah, anc can be the same as or different from the tandem repeat sequence) The nucleotides encoding the PDTRP epitope flanked by Sequencing is performed using oligonucleotides as has been done for other protein epitopes. capsidan of the poliovirus Sabin type 1 vaccine strain. Expressed by insertion into a gene encoding a protein [Evan Evans et al., 1989, Nature, 339.385-388]. child Engineered viruses such as can be easily tested for its ability to delay and ultimately prevent tumor development. can be done.

Accordingly, the present invention relates to methods of surgical or therapeutic treatment of humans or animals. for use in diagnostic methods carried out on the human or animal body. and for use in the preparation of drugs for use in such methods. Probes, vectors, and transformed cells are provided. The present invention also , such lobes, vectors and transformed cells can be used in humans in effective non-toxic amounts. surgical or therapeutic administration to humans or other mammals. methods of animal treatment and diagnostic methods carried out in vivo and in vitro; An in vitro procedure that consists of contacting a sample of body fluid or tissue with a fragment or probe or in vivo.

The present invention further provides for culturing host cells transformed with the expression vector and The process consists of recovering the expressed polypeptide without producing a PDTRP epitope. provide a method for

In another aspect, the invention provides recognition of a PDTRP epitope as defined above. and preferably produced by normal milk-producing human mammary glands. whether the reactivity with human PEM glycoproteins processed by provides an antibody other than 5M-3 and HMFG-2 that does not react with .

As used herein, the term "antibody" refers to a polyclonal antibody that has an antigen-binding site. Antibodies and monoclonal antibodies and fragments of antibodies, such as F(ah')2 fragments , and one or more polypeptides, chemically or genetically modified. Species-specific and/or isotype-specific This method combines polypeptide chains of varying regions and/or from different sources. is intended to include antibodies or fragments such as. Such antibodies can be prepared using conventional methods. [Williams, Tfbtech, 6.36 ( (1988)] and can be obtained for diagnostic and therapeutic applications, e.g. Useful in dynamic immunization.

Antibodies according to the invention react with the PDTRP epitopes defined above; they are Also, HPEM core protein, especially colon, lung, ovarian and especially breast cancer, reacts with the expressed core protein but with the corresponding fully processed H Reduced reactivity with PEM or no reaction with it. on a particular surface The antibody reacts with HPEM core protein, but normal milk-producing human mammary glands does not react with fully processed HPEM glycoproteins produced by stomach. The antibodies according to the invention are preferably used in mammals of lactating or pregnant individuals. does not significantly react with mucin glycoproteins produced by the epithelial tissue of mammals, but Reacts with mucin proteins expressed by animal epithelial adenocarcinoma cells.

These antibodies show very reduced reactivity with benign breast tumors and therefore breast cancer. useful in the diagnosis and localization of cancer and in therapeutic methods.

The antibody is a polypeptide expression product of the human mammary epithelial mucin gene, or a fragment thereof. , particularly involving screening of cell cultures for nascent expression products. can be used for. In this case, the antibody is conveniently polyclonal. It can be an antibody or a monoclonal antibody.

The present invention further lies in the therapeutically or diagnostically effective portion of known techniques. or provide an antibody linked to a linkage moiety. The linking moiety is a linkage moiety that gives the antibody a therapeutic or can be a known linkage factor for binding the diagnostically effective moieties, e.g. , the linkage moiety can be a specific binding partner of a specific binding factor and has therapeutic or The diagnostically effective moiety is conjugated to a specific binding agent. Antibody: linking part (specific adducts of (binding partners) and therapeutically or diagnostically active moieties: specific binding agents Separate administration of the adducts, binding of the specific binding agent to the specific binding partner, binds to the antibody. resulting in a combined therapeutically or diagnostically effective moiety. Specific binding factor: specific Preferred examples of binding partners are biotin and avidin or biotin is a binding agent or or streptavidin, which can be the binding partner.

For therapeutic use of antibodies, the therapeutically effective moiety is cancerous breast or other tissue. It is a lethal factor that is released into the cells and disables or kills the transformed cells. lethal Agents include toxins, radioactive isotopes and "direct killing agents" such as complement components. minutes and cytotoxic or other drugs or cytotoxic drugs at the tumor site. Includes activating enzymes.

For diagnostic use, the diagnostically effective moiety is a solid support and a detectable label. such as enzyme labels, chromogenic factors, fluorescent factors and radioisotopes and other The moiety can be a direct or indirect detectable label. Preferably , monoclonal antibodies are used in diagnosis.

゛゛　The present invention further provides for a diagnostic test method comprising: causing a sample to contain an antibody as defined above; and assay methods. Such methods involve adding antibodies to the patient and selected antibodies or fragments thereof, in any order, separately, simultaneously or sequentially. localization of the tumor, including administering a labeled entity capable of binding to Includes existence.

Certain uses of antibodies may be used to detect the extent of breast, colon, ovarian and/or lung cancer. and/or diagnostic assays that evaluate

Diagnostic test kits are provided for use in diagnostic methods and assays; This kit contains antibodies and, if necessary, appropriate labels and other reagents, e.g. For example, buffers, labeled antibodies and/or reagents for the detection of labeled antibodies. and antiserum, especially for use in competition assays.

The invention further relates to surgical, therapeutic or diagnostic procedures performed on the human or animal body. or of drugs used in such a method. PDTRP epitopes and antibodies as defined above for use in the preparation of provide. The present invention also provides the aforementioned polypeptides or peptides of effective non-toxic substances. Provides a method of treatment or diagnosis comprising administering to a human or animal Ru. The rate of administration depends on the patient's age, weight, size, sex and general health; and at appropriate levels without undesirable or intolerable side effects. The rate would be sufficient to circulate Bell's antibodies.

Administration can be by oral or parenteral route, e.g. by subcutaneous, intravenous or intramuscular injection. This can be done by radiation. Typically, PDTRP epitopes are present in human adults. Orally or parenterally in amounts up to Ig/dose, preferably 1 to 200 mg/dose. Administered via targeted route.

Therefore, the invention also provides a PDTRP epitope of the invention and a suitable carrier. , a diluent and/or an adjuvant; The body, diluent and/or adjuvant will, of course, be determined by the route of administration. and selected for compatibility with the PDTRP epitope. suitable carrier, Diluents and adjuvants are as follows: Freund's incomplete adjuvant (IFA), aluminum hydroxide, saponin, DEAE-dextran, Murami mineral oils, neutral oils, e.g. miglyol, vegetable parts, e.g. Nut oil, Iscoms J, Liposome, Pluronitac Pluronjc™, polyol or Rtbi adjuvant [British Patent Application (GB-A) No. 2189141]. For injection, the composition An aqueous, sterile, non-biogenic solution containing buffers, antioxidants and disinfectants as required. Contains microbial agents, such as antibacterial and antifungal agents and tonicity regulators do. Alternatively, the composition is reconstituted by adding water for injection. can be provided as a dry powder, and optionally with excipients, e.g. For example, it can contain those mentioned above.

Antibodies against PDTRP epitopes can be polyclonal or monoclonal. In therapeutic methods of treating humans and animals, e.g. It can be used in the form of a vaccine for passive immunization. Preferably it are formulated into a pharmaceutical composition, eg, as described above.

Particularly in therapeutic applications, the Fab or its complementarity-determining region should be treated. Coupling to the Fc or entire framework region of a species-derived antibody ( For example, the Fab region of a mouse monoclonal antibody can be administered together with the human Fc region. modification of the antibody (which can reduce the immune response by human patients) Alternatively, it may be appropriate to alter the antibody profile. [see European Patent Application (EP-A) No. 239 400].

The present invention also provides a host animal capable of expressing a PDTRP epitope. Inoculate the transfected cells and inject the antibody with the animal's serum or other body fluid. A polyclonal antibody against a PDTRP epitope recovered from Provides a method for producing.

The present invention further provides a host animal with a PDTRP epitope or a PDTRP epitope. inoculate transfected cells capable of expressing the antibody for the PDTRP epitope, consisting of removing cells from the animal that secrete A method for producing cells capable of secreting antibodies is provided. cells in vitro , e.g. under appropriate cell culture conditions and/or in vivo, e.g. in ascites. Growth allows antibodies to be produced.

As used herein, the term "animal" refers to any animal capable of producing antibodies. , for example, refer to mammalian animals, such as laboratory serrated animals, and birds, and humans. includes.

The transfected cells contain a protein that encodes a PDTRP epitope. A cell containing a DNA expression vector containing an expressible sequence. The decoding sequence is can be natural or synthetic, and the encoded polypeptide is directly or can be expressed as a fusion protein. For example, PDTRP epitope DNA specifying the genetic information of the group can be identified and obtained from natural sources, or Produced partially or completely synthetically. encodes a specific polypeptide Natural or artificial DNA is commonly known in the field of genetic engineering. Maniatis, T, Fritoshi ), E.

and Sambrook, J. (1982), Molecular Crow. Molecular Cloning: Laboratory Manual (Molecular Cloning: A La laboratory Manual), :+-Rudeau Spring Harbor Laboratory Laboratory Press (Cold Spring Harbor Laborato ry Press), New York. Vectors become common practice to ensure expression in the cells to be transfected, and the DN A marker sequence was established to allow identification of A and transfected cells. [Maniatis et al., foe, c. it, ]o In the present invention, suitable selective markers for and are drug resistance markers. , including, for example, the thymidine kinase (tK) gene for tK-cells. Szybalski (5ka), E, and Szybalski (Szy balski), W. (1962), Proceedings Eve National. ・Academy Eve Sciences (Proc, Natl, Acad, Sci) ) USA, 42.2062].

Suitably, the promoter sequence is inserted into the vector and transfection is carried out. can increase the expression of PDTRP epitopes in cells [subrama Subramani, Sl, Mulingan, R , C., and Berg, P. (1,981), Molecular & ・Cellular Biology (Mo1.Ce11, Biol, L 1.854-' 864].

Cells to be transfected, i.e., cells into which the vector should be inserted (hereinafter referred to as "Donor cells") are donor animals, such as rodents, especially rats and mice. , and from primates, such as monkeys. Once the donor cells are in trans Once transfected, the PDTRP epitope is post-translational processing of epitopes that cannot be expressed stomach. Preferably, the donor cells and optionally modified DNA are PDTR Although the P epitope is selected to appear extracellularly or on the cell surface, the present invention also Brightness can be applied to polypeptides that are expressed only intracellularly.

Especially for intracellularly expressed polypeptides, donor cells normally have intraperitoneal Preferably, it is absent or of a type that does not circulate in the bloodstream of the host animal. Delicious.

Preferably, the animal inoculated with the transfected cells (hereinafter referred to as [host animal] j) and the cells to be syngenetically transfected are animals (hereinafter referred to as I). - a donor animal). I don't want to be bound by a particular theory, but Shinjie Use cells from a naked donor animal! − by transfection cells that can be detected and acted upon by the host animal's immune system. It is believed that the differences between the host animal's own cells will be minimal. Thus, the immune response is produced by the transfected cells by expression of the DNA sequence. are likely to be directed against foreign polypeptides.

host animal and donor animal to maximize the directed effect on the host animal's immune system. are virtually genetically identical, and usually they are well-represented in this field. Preferably a well-established inbreeding strain, such as a known strain.

- irrespective of whether the oocyte donor animal and the host animal form an inbred lineage; These uses are conceivable, including cells from the host animal itself or even cells from the host animal itself.

Transfection of donor cells is carried out by conventional methods, e.g. [Austin, P., ) Rowsdale (Tr. owsdale), jl, Rudd, C,, podo? -(Bodme r), W, , Feldmann, M, and Lamp (L amb), J. (1985), Nature 1.313. 1-41 or by electroporation technique [= , , -man (Neu man), E., Schaefer-Rfdder ), M, , Wa ng, Y, and Hofs (1982), EMBO Journal (J,), 1 ．． 841-845] or using retroviral vectors [Biology Biotechniques, 6.608-614 (1988)].

After transfection, screen cells or clones or their subclones. lean and select those capable of expressing PDTRP epitopes. Sometimes things are convenient. For gene products expressed on the cell surface, fluorescent Antibody and fluorescence-activated cell sorters are particularly useful [Austin stin), loc, cit,] to screen and select other techniques. It can be used for.

Transfected cells are selected and processed as necessary to produce the gene product. can increase the expression of Various known enhancement methods can be used, e.g. Treatments can be used: Sodium butyrate [Gorman ), C, M, and Howard, B, H, (1983) Nucleic acids Nucleie Ac1ds Research, 11.7631-7 6481 or interferon [Ba Ikwf I 1] , F., R., Stevens, M. H., Glitzin ( Griffin), D.B.

, Thoma, s., J.A., and Bodmer. ), J, G, (1987L Eur, J, Cancer C11n, 0nco ＋、　、23.101-1061゜Enhancement method is appropriate for donor/cell type. selected as follows.

PDTRP epitope or transfection into one or more host animals inoculate the transfected cells, preferably in a suitable medium. Inoculate by intraperitoneal or intravenous injection of the cyst. Preferably, inoculations are spaced repeat and monitor the animal's antibody titer to ensure the desired immune response occurs. confirm. The route chosen for the method of administration and inoculation is preferably as desired. selected to be preferentially produced in the class of antibodies, e.g., IgM antibodies. The production of antibody-secreting cells shortens the interval between inoculation and collection of antibody-secreting cells. Therefore, production of IgG antibodies occurs favorably, but the production of IgG antibodies requires repeated inoculations and antibody-secreting cells. This preferably occurs by increasing the interval between ultimate withdrawals.

Once inoculation is complete, antibody-secreting cells, such as lung cells, are removed from the inoculated animal. and maintained in a suitable medium. Obtain host cells using conventional methods and can be maintained. Cells that produce antibodies can also be used in hosts with cancer. from body fluids or tissues of animals, especially human cancer patients, especially breast or ovarian cancer patients. can be obtained from lymphocytes from removed lymph nodes [Journal Eve Im Nological Methods (J.

Immunologtcal Methods), 105.263-273 ( 1987)].

In another aspect, the invention provides a method for treating patients with cancer or with PDTRP epitopes or is a transfected cell capable of expressing the PDTRP epitope. PD, which consists of removing antibody-producing cells from a host animal inoculated with Provides a method for producing cells capable of secreting antibodies against TRP epitopes. provide

In a further aspect, the present invention provides a method for treating patients with cancer or with PDTRP epitopes. transfectants capable of expressing PDTRP epitopes or PDTRP epitopes. against the PDTRP epitope, which was removed from a host animal inoculated with cells Cells capable of secreting antibodies are provided.

At this stage, a population of antibody-secreting cells obtained from the host animal is screened. to select those that secrete antibodies against PDTRP epitopes. can be useful.

The cells were incubated with antibodies against the PDTRP epitope (hereinafter monoclonal antibodies or M upon immunization to form permanently proliferative cell lines capable of secreting Ab) and (Ab). It is useful for Imparting permanent proliferation is carried out by any known method. can form hybridomas by fusion with permanently proliferating cells. is the most convenient method.

Therefore, the present invention also provides antibody-producing animal cells, PDTRP epitope antibody-secreting cells taken from a host animal inoculated with PDTRP epitope or PDTRP epitope. confers permanent division potential on transfected cells capable of expressing A permanent protein that is capable of producing antibodies against PDTRP epitopes. A method of producing proliferative cells is provided.

Permanently proliferating cells suitable for fusion (these cells can be replicated under appropriate culture conditions) ) is well known. Permanently proliferating cells secrete antibodies can be of the same lineage as cells from the host animal and usually secrete antibodies from the host animal may be of the same species as the cells that contain the cells, but this is not required. and (useful for permanent proliferation) The cells are myeloma cells. Fusion is Kohler and Milstein (Milstein), Nature, 25th, 495-49 7 (1975) and its modifications as described in the literature. be done.

After fusion, the resulting population of hybrid cells is screened to identify the PDTRP epitope. It may be convenient to select cells that secrete antibodies against the tope.

The present invention also relates to the use of PDTR epitopes obtained from host animals inoculated with PDTRP epitopes. Antibody-producing cells or PDTRs capable of secreting antibodies against P epitopes Consisting of transfected cells capable of expressing the P epitope or the progeny of such cells, antibodies against PDTRP epitopes. Permanently endowed cells, preferably hybrids, capable of secreting provide a market.

Permanently proliferating cells (e.g. hybridoma cells) according to the invention are It can be used to generate MAbs against pitopes.

Therefore, the present invention provides secreted antibodies against the PDTRP epitopes defined above. cells endowed with the ability to permanently divide, or the progeny of such cells PDTRP epitope, consisting of culturing and collecting monoclonal antibodies. Provided are methods for producing monoclonal antibodies directed against antibodies.

The present invention further provides a tissue culture or a permanently proliferating hybrid cell as defined above. is a monoclonal anti-PDTRP epitope obtained from growth in ascites. Provide your body.

As mentioned above, antibodies produced by cells endowed with the ability to permanently divide can be produced using ordinary techniques. to generate MAbs against PDTRP epitopes. Cells can be identified. Such cells can be grown and cultured to achieve the desired results according to the present invention. MAbs can be produced in large quantities.

Appropriate culture techniques and growth conditions for antibody-secreting cells are well known and available. cell lines endowed with the specific permanently dividing capacity involved and appropriate for the MAb to be produced. selected as appropriate.

The present invention further provides PDTRP epitopes and PDTRP epitopes as defined above. a carrier polypeptide or protein attached to the C-terminus or N-terminus of the The present invention provides a fusion protein consisting of protein.

The carrier polypeptide or protein can be any suitable polypeptide or protein. It can be a protein. A useful carrier protein is thyroglobulin and bovine serum albumin.

The fusion proteins of the invention can be synthesized using conventional techniques of polypeptide synthesis and protein chemistry. from individual amino acid precursors or from smaller polypeptide couplings. It can be produced by Alternatively, the fusion protein is produced by recombinant DNA technology involving the expression of genes encoding be able to. All these methods are well known in the art.

The invention further provides a method or method of surgically or therapeutically treating the human or animal body. or for use in diagnostic methods carried out on the human or animal body; Fusion proteins as defined above; therapeutic, surgical or diagnostic applications of the human or animal body; Use of fusion proteins in the manufacture of drugs for use in the treatment of cancer; protein and its pharmaceutically acceptable diluent or carrier, and if necessary A pharmaceutical composition consisting of common auxiliary ingredients, and an effective non-toxic amount of the fusion agent. treatment of the human or animal body, consisting of administering a protein to the human or animal provide a method to do so.

The fusion protein can be used in the treatment or prevention of cancer, such as breast cancer, to by increasing immunity against tumor cells, and in the diagnosis and treatment of cancer. Production of polyclonal and monoclonal antibodies for use in medical procedures It is useful in

The invention will now be described by reference to the accompanying drawings.

Figure 1 shows a series of octapeptide/SM3 reactions at 450 nm according to Example 1. It is a graph of optical density (0, D, ) at .

Figure 2 shows the design of overlapping peptide octamers. Departure arrangement The string consists of tandem repeats of 20 amino acids, with the 4 amino acids of adjacent repeats being the amino terminal present on the end and carboxy terminus. Overlapping octamers [− ] and [A] indicates the alanine used as a spacer on the bottle. . Note, octamers 1 and 21 are identical. One letter code for amino acids use.

Figure 3 is a graph of the binding of monoclonal antibody 5M-3 to the octamer peptide. It is. Monoclonal antibody (50 μg/ml, 100 μm/bottle) was added to the octama 1-21 and repeated in Figure 2) and binding was determined by ELISA. Visualized using assay. The boxed array in the insert is 5M-3. represents a more recognized epitope.

Figure 4 shows monoclonal antibodies HMFG-2, LICR-LON-M8 and Figure 2 is a series of graphs showing the binding of the c5 octamer to peptides. monochrome null antibodies (50 μg/ml, 100 μm/bottle, HMFG-2 and onc- For M15, LICR-LON-M8 ascites not: 500 dilution) (defined in Figure 2) and the bond is ELI Visualized using SA assay. Sequences in parentheses indicate epitopes.

Figure 5 shows the detection using monoclonal antibodies reactive with PEM mucin core protein. 1 is a diagrammatic representation of identified epitopes. Potential glycosylation sites are indicated by arrows. It is.

Figure 6. Lymphoblastoid and breast cancer detected by fluorescence-activated cell sorter. 1 is a series of graphs showing HMFG-2 binding to cell lines. cells in suspension HMFG-2 (-) or TAL-4 (---) anti-HLA-DR monoclonal Rabbit anti-method in which the binding was incubated with the antibody and fluorescently labeled. Detected using. A, lymphoblastoid cell line PGF expressing HLA-DR2. , the β chain contains the sequence Asp-Thr-Arg-Pro (DTRP). B.S. The lymphoblastoid cell line Mann expressing HLA-DR7, the β chain has the sequence Asp-T. Contains hr-GIn-Pro (DTQP). Expresses CSPEM mucin Breast cancer cell line T47D0 Figure 7 is a graph of the binding of anti-peptide antiserum to octamer peptides. Ru. 1: React the antiserum diluted to 200 with the peptide as shown in Figure 2. I let it happen. The boxed sequence in the insert represents the dominant antigen recognized by this antiserum. Represents pitope.

Figure 8: Paired photomicrographs showing staining of breast cancer cell lines using anti-peptide antiserum. It is.

The invention is further illustrated by the following examples. These examples illustrate how the present invention It is not intended to be limiting in any way.

As we have shown previously, the monoclonal antibody MaellI is a PEM core protein. recognizes epitopes within tandem repeats of 2o amino acids found in proteins. [Gendler et al., Journal Eve Biological Research] Mistry (J, Biol, Chem,),! Dan, 12820-12823 ( 1988)]. Cambridge Research Biochemicals 　Re5search　Bi.

Epitope scanning kit purchased from C11emicC11e (Cambridge, UK). Using this method, we further revealed the epitope recognized by 5M-3. and the smallest epitope is proline-aspartate-threonine-arginate. It was shown that it consists of nin-proline.

This method involves forming small, overlapping pellets at the end of a polypropylene bottle. It consists of synthesizing peptides and storing 96 bottles in a microtiter plate format. - held in plastic supports with mats and spacing. chemical pin tip can be derivatized to produce functional groups to which amino acids can be coupled. , and the peptide is added stepwise to the peptide chain on the tip of the bottle with the addition of 1 amino acid/day. Synthesize by elongation.

Once the desired length of peptide has been synthesized, the pin can be attached to a standard ELISA-type assay. can be assayed for their reactivity with antibodies in a dispense the reagents into 96-well microtiter plates and then Place a pin inside.

Choose to synthesize overlapping octamers, along serial repeats. move one amino acid with each octamer synthesized, and all The first and last peptides are identical, covering the possible octamers. so that Thus, to design an octamer, one series of iterations and the next 8 amino acids of repeats, i.e. VTSAPDRTPAPGSTAPPAHGV TSAPDTR was used. Thus, the first octamer VTSAPDTR, and the second TSAPDTRP, and the third 5APDTRPA. Therefore , octamers 2-5 are rPDTRPJ epitopes as defined herein. . Octamers 1 and 6-21 are not PDTRP epitopes (each of them contains only a portion of the PDTRP sequence or does not contain it, and There is little or no reaction with 5M-3. The results obtained using the 5M-3 octamer are shown in Figure 1. The pitope is PDTRP, indicating epithelial specificity and recognizing tumor-associated antigens. Many of the monoclonal antibodies used are antibodies found on large molecular weight mucin molecules. Reacts with Pitope. Until recently, the biological characterization of mucins was limited to e.g. , carried out using large mucins produced in the lungs and neck [Haun et al. Cell (Houns e I I) and Fage (Fe i zz i), 1 982; Maushall and Alien, 197 8; Slayter et al., 1984; Carl stedt et al., 1983]. However, it includes the mammary glands, sweat glands, and salivary glands. Other glands produce mucin, which we term pleomorphic epithelial mucin (PEM). One name is the large number of monoclonal antibodies that recognize epithelial or cancer-related determinants. [Burche II and Tay for Papad imi t r 1ou), 1989].

PEM components are produced by several normal epithelial tissues and are present in the milk-producing mammary glands. It is also of interest to breast cancer because it is produced in greater abundance. and other cancers [Taylor-Papadimitriou Papadimitriou et al., 1986; Giring et al. 1989], moreover, mucins are differentially glycosylated by cancer cells. As a result, novel carbohydrate epitopes appear [Kuzierdsen ( Kjeldsen et al., 1988] and masked in normal mucin. Epitopes of core proteins that are present are exposed in cancer-associated components. one thing Epitopes of the core protein such as -3 was generated using chemically deglycosylated normal PEM mucin [ Burche et al., 1987]. This antibody is responsible for 90% of breast cancers Reacts with higher amounts (but with normal breast mucin or PEM mucin purified from milk) There is little or no reaction with [Burch heI1) et al., 1987 Giring et al., 1989] , the specificity of the 5M-3 antibody makes it potentially useful in the diagnosis and treatment of breast cancer. The identification of the 5M-3 epitope is therefore of great importance.

Recently, the amino acid sequence of the immunogenic domain of PEM mucin [Gendler [1er) et al., 1988] developed a cDNA clone specifying the genetic information of this region. deduced from the nucleotide sequence of [Gendler et al., 19 87]. This domain is composed of repeating units 20 amino acids long, and The number of repeats varies among individuals and accounts for the polymorphisms seen at the DNA and protein levels. [Swallow et al., 1987]. Reedy of serial iterations The coding frame consists of three core proteins for synthetic peptides with corresponding sequences. To demonstrate the binding of quality-reactive antibodies (HMFG-1, HMFG-2 and 5M-3) clearly established by [Gendler et al., 1988] .

This sequence matches what can be expected for mucin, and proline serine and threonine separated by a rich extension (possibly O-glycosylated) ), suggesting a linear structure. Contains a sequence of tandem repeats of 20 amino acids Using overlapping octamers to Amino acid sequence, Pro-Asp-Thr-Arg-Pro to 5M-3 epitope was able to be identified as the core of Interestingly, both normal and cancer-related Other core proteins found on human mucin and recognized by other antibodies Epitopes of the 5M-3 protein are also mapped to this region, but both Does not contain proline at the amino terminus of the epitope. This data indicates that cancer-related mucin Exposure of the 5M-3 epitope on an aberrant process resulting in a shorter carbohydrate side chain This is consistent with the idea that it can be attributed to sing. In a preliminary study, Arrangements of tandem repeats were used to generate antibodies against PEM mucin that are reactive with breast cancer. It has been shown that peptides corresponding to part of a column can be used.

material and method Peptides: Peptides are produced using solid-phase technology using a 430A peptide synthesizer [Appla Applied Biosystems, a commercial using the locally available t,-boc protected amino acids and restrictions. Synthesized as described in [Townsed et al., 1986] .

Synthesis of overlapping peptide octamers 2 Overlapping peptides The octamer was originally published by Geysen et al. (1984) Method-based epitope scanning kit [Cambridge Research Base Iochemicals (Cambridge Re5each Biochemica) ls), Cambridge, UK] onto the ends of the polypropylene pins. accomplished. The pin has an alanine attached to the end that acts as a spacer. The amino acids at the carboxy terminus of the tuS-synthesized peptide are It is attached via a peptide bond to ranin.

Antibody: Monoclonal antibody HMFG-1,! (MFG-2 and 5M-3 Taylor-Papadimi Tortou et al., 1981; Burchel 1 Ia11983; 19 87; Girling et al., 1989], LICR-LON- M8 [Foster et al., 1982], one-M2S, one -M23, one-M27 [Linsley et al., 1988] Occurrence and characterization were described here. Monoclonal antibody TAL14.1 is directed towards HL A - D R.

Anti-peptide mouse antiserum contained 25 μg of P coupled to troglobulin. Immunization intraperitoneally with DTR peptide 1-24 (see Table 2) followed by bovine serum by three injections of 25 μg PDTRI-14 conjugated to albumin. was produced.

ELISA: Monoclonal antibody (504g/ml or 1:500 dilution ascites, 100 μI/pin) and polyclonal serum (1:200) using an oxidase-conjugated rabbit anti-mouse antibody (DAKO, Denmark). [Burchell et al., 1983] ELI They were tested for their reactivity with octamers in the SA assay. Octa The markers are placed on pins giving a 96-well microtiter plate format. Microtameter containing antibodies or reagents to synthesize and therefore assay pins I inserted it into the itadish.

Binding of HMFG-2 to cell lines: 10% cell pellet containing 106 cells of fetal calf serum and 0.02% sodium azide. Suspend in 1 ml of hybridoma tissue culture supernatant containing H. and incubated for 1 hour at 4°C. 10% Fe2, 0.02% Wash twice with RPMI (wash buffer) containing sodium azide, then wash Fluorescently conjugated rabbit anti-mass diluted 1=20 in buffer and filtered before use. Us (DAKO.

The cell pellets were suspended in 1 ml of (Denmark). After 30 minutes at 4°C, The pellet was washed once with wash buffer (20 ml) and PBSA (20 ml). ) and then suspended in 1 ml of PBSA. Next, fluorescent bonding Spectra-Phys i cs) Analyzed in a fluorescent cell sorter.

result Identification of the epitope recognized by antibody 5M-3 Direct repeat unit of PEM mucin Figure 2 shows the arrangement of the parts. Optionally, sequences were written starting with PDTR. Na This may be through the first tandem repeat in one of the cDNA clones. Antibody 5 [Gendler et al., 1987] M-3 is a PDTR sequence corresponding to one tandem repeat and 4 amino acids of the next repeat. It has previously been shown to react with peptide (1-24) (see Table 1). 5M-3 anti Single repeats and A series of overlapping peptides encompassing 8 amino acids of adjacent repeats. A catamer (Figure 2) was synthesized on the tip of a polypropylene pin (see Materials and Materials). method). Figure 3 shows that the peptide was reacted with 5M-3 in ELISA. The results obtained are shown below. As you can understand, all of the 5M-3 and reactivity All peptides contained the sequence Pro-Asp-Thr-Arg-Pro, but This seems to represent the minimal epitope.

one-M2S MCF-7, HMFG + + Linsley et al. one- M23 MCF-7, HMFG + + onc-M27 W5-6. HMF G + + + PDTR peptide (1-24) PDTRPAPGSTAPP ATIGVTSAPDTR2PDTR peptide (1-14) PDTRPAr' GSTAPPAC*3VTSA peptide (17-20,1,-10) VTSA PDTRPAPGST’ F! MFG human milk fat globules Cystine was included to enable Kyo coupling.

5M-3 epitope-containing domain reactive with immunogenic PEM mucins Clonal antibodies can be produced using various immunogens containing mucin, such as human milk fat globule extract. Things [Taylor-Papadj, m1tori ou) et al., 1981; Hilkens et al., 1984; Foster Foster et al., 1.982: Certani et al., 1983 ] and cancer cells [Plumwell et al., 1983 Nibla [Price et al., 1985]. Some things are mucin [Tayfor-Babazi Mitriou] -Papadimitortou et al., 1981; l) et al., i, 983; see Table 1]. We are directly testing some of these antibodies. tested for their reactivity with peptides encoding sequence repeats, and Many showed a strong positive reaction with the PDTR peptide (amino acids, 1--24, Table 1). It was discovered that These are the antibodies listed in Table 1 as well as the antibody NCRC II [Price et al., 1985] and DF3 [Sekin e) et al., 1985]. domains reactive with some of these antibodies. Their reactivity with smaller peptides was examined for more detailed identification. . As shown in Table 1, all of the antibodies tested reacted with two smaller peptides. However, both of them contained the first 10 amino acids of the PDTR peptide. this territory The region also contains the 5M-3 epitope, and this part of the tandem repeats is present in mice. This suggests that the protein contains an epitope that is immunodominant. of the antibodies shown in Table 1. Three of them, namely HMFG-2, LICR-LON-M8 and one- The specific epitopes reactive with M2S are overlapping as illustrated in Figure 2. The octamer was used for more detailed identification and the results are shown in FIG. Antibody HM FG-2 and LICR-LON-M8 have strikingly similar patterns to octamers and in both cases the smallest epitope is Asp-Thr-Ar g (DTR), but what is recognized by one-Mi5 is T h r -A r g-P r o-A 1 a (TRPA).

Detailed epitope mapping results show single threonine and potential glycosylation The flanking amino acids between the zylation sites are highly immunogenic in tandem repeats of PEM. Indicates that a domain is configured. The overlap between different epitopes is 5 is shown.

Role of adjacent amino acids Search PIR's protein database for Ebitobu shown in Figure 5. When testing, numerous tags in the region from the bacterial protein to the HLA-DRβ chain are analyzed. The protein was shown to contain the sequence Asp-Thr-Arg. But long have shown that antibodies HMFG-2 and M8 show limited reactions with pleomorphic epithelial mucin. was reported. As one of these antibodies has different flanking amino acids To study whether it binds to proteins containing p-Thr-Arg. , we are targeting natural proteins known to contain the smallest epitope. The binding of HMFG-2 was studied. The β chain of HLA-DR2 has the sequence Asp-Thr- contains Arg-Pro, but the β-strand of HLA-DR7 has the sequence Asp-Thr-G in-Pro [Young et al., 1987], however , adjacent amino acids show no homology to PEM mucins.

Expressing HLA-DR2 (PGF cells) and HLA-DR7 (MANN cells) analyzed the binding of HMFG-2 to lymphoblastoid-like cell lines and PEM whip. The binding of HMFG-2 to a breast cancer cell line (T47D) expressing HMFG-2 was compared.

As Figure 6 shows, as expected, HMFG-2 Expressing DR2 that does not bind to N cells and also contains the sequence Asp-Tbr-Arg. It did not bind to the cells. However, HMFG-2 is actually associated with breast cancer cell line T47. It showed strong binding to D. Thus, the minimal epitope of HMFG-2 is conformational. Compositionally, the free peptide consists of only three amino acids; In natural proteins with limited conformation, adjacent amino acids are It has an important role in determining sexual sites.

In the expression of peptide 5M-3 as an immunogen for the generation of breast cancer-reactive antibodies, The monoclonal antibodies described above are isolated from milk or associated with glycosylated mucin cells. and in these cases the carbohydrate side chains are linked to the core protein. protein epitopes and play a major role in determining which ones are exposed or not. will perform.

We here present the PDTR-peptide (1-24) and the carrier protein. PDTR peptides (1-14) coupled to protein (see Materials and Methods ) was compared with the response of mice injected with antiserum and overlapping octamer. It was tested by tracking. As shown in Figure 7, the overall connection is monochrome. Although higher than the local antibody, there are binding peaks at peptides 4 and 5. It seemed like that. This is an epitope that recognizes the sequence P-D-T-R-P-A-P. It corresponds to the antibody present in serum that recognizes Antisera are primary breast cancer and cell lines (see Figure 8), some of the antibodies present are cancer-related mucins. Indicates that it reacted with the epitope exposed above. This first experiment suggests Peptides based on tandem repeat sequences can be used as immunogens to target cancer-associated PEMs. Antibodies reactive with mucin can be generated.

essay The core protein of pleomorphic epithelial mucin is composed of a number of tandem repeats; each contains five potential 〇1-linked glycosylation sites [Gendler (Ge ndler) et al., 1988]. These serial iterations are directed against this mucin. contains an epitope recognized by a large number of monoclonal antibodies. Oh Recognized by 5M-3 by using bar wrapping octamer The epitope has been revealed and contains a single threonine at position 3. Shown. Furthermore, epitopes reactive with other monoclonal antibodies may also It was shown that it was mapped to the area of Therefore, at least in mice This tandem repeat sequence appears to be highly immunogenic.

By HMFG-2, LICR-LON-M8, onc-M2S and 5M-3 Although the epitopes recognized overlap, mucins and these antibodies The reactivity of is very different. All react with mucins expressed by breast cancer cells However, HMFG-2, onc-M2S and M8 determinants are also present on normal mucin. [Burche Il et al., 1983; Linsley et al., 1988; Foster et al., 1982 ], but the 5M-3 determinant does not react [Burchel et al., 1 987; Girling et al., 1989]. 5M-3 monochrome Null antibodies were raised against chemically deglycosylated normal pore mucin. The fact that carbohydrates absorb this epitope on PEM produced by normal cells Suggests that scales play a role when masking. Reacts with normal mucin The combined epitope recognized by the three antibodies encompasses the sequence DTRPA. This sequence is centrally located between the flanking glycosylation sites (the second In the figure, T18', 819'', see S-8IO1, also Fig. 5). However, However, the 5M-3 epitope PDTRP contains proline at the amino terminus; is the only amino acid distant from the glycosylation site (serine 19'). This is As suggested, in normal mucin, a protein on the amino terminus of the 5M-3 epitope Lorin is masked by carbohydrates, but in tumors serine 1111 and and for premature termination of the glycan on threonine 18' be done. Recent results support this idea, showing that normal mucin isolated from human milk is Containing long and/or branched poly-N-acetyl-lactosamine side chains It was shown [Hanisch et al., 1989] that breast cancer cell line B The oligosaccharide side chains of mucins produced by T20 contain only 3 or 4 It consists of sugars [HuI1 et al., 1988].

All four of the mapped epitopes contain threonine in their core Therefore, this amino acid in the reactive epitope can be glycosylated. do not have. However, fewer HMFG-2-specific epitopes on cancer-associated mucins It should be noted that the protein is present on the normal mucin found in milk. Burche et al., 1983]. As this means, Some threonines in the DTR epitope are in fact normal mucins and glycosyl be converted into In other words, due to the heterogeneity in the length of carbohydrate side chains, Some DTR epitopes are associated with long or branched polylactosamine chains. are masked, but others are not. Other features of the HMFG-2 epitope The symptoms appear to be that the affinity for antibodies changes depending on the microenvironment. Is Rukoto. Comparing antibody binding to normal mucin and cancer aromatic mucin, Antibodies were discovered to exhibit higher affinity for normal mucins and normal oligosaccharides. We suggest that the tucaride side chain is required for maximum affinity. Oligosatsuka The lido side chain may also be important, as both HMFG-2 and 5M-3 , partially deglycosylated to mucin stripped of all carbohydrates [Burtier] cell) et al., 1987.

The oligosaccharide side chain is the core recognized by antibodies HMFG-2 and 5M-3. play a role in determining the conformation of a protein epitope of the core protein of PEM mucin, which is immunogenic and expressed by breast cancer. It is quite clear that there are antigenic determinants within. to protein carrier Using synthetic peptides corresponding to coupled tandem repeats, we was able to induce antibodies in mice that were reactive with breast cancer. antiserum In mapping the epitopes to be used, the dominant epitope is sequence 1) DTRP Thr”’, Set”’ and 5re9 between the glycosylation sites in AP, Thr10 (Figure 2), again confirming the immunogenicity of this domain. was shown. As these results suggest, this time, 5M-3 that reacts with breast cancer It is possible to take a direct approach to the production of monoclonal antibodies with similar specificity. Yes, and such research is currently underway.

Furthermore, the identification of epitopes selectively exposed in cancer cells focused research on the aberrant processing of mucins, and peptides or polypeptides for use as synthetic vaccines for certain human cancers. This suggests that it should be made into a Noh play.

References for Example 2 Bramwe 11. M, E, , Bhavanandan%V, P,, Wj Seman, G., and Harr i slH, C. Structure and Function of Antigens, B. r, J. Cancer, 48.177-183 (1983).

Burchell, J., Burbjn, H., and Taylor-Papa. dimi tr 1ouSJ, normal and malignant human breast epithelial cells. Antigen recognized by monoclonal antibodies HMFG-1 and HMFG-2 Complexity of determinant expression, J. Immunol, 131.508-513 (19 83).

Burchel I, J, Gndler SS, Taylor-Pap adimitriou, J, , Gtl ingSA, , Lewis.

A., Millis, R., and Lamport, D., Co., Ltd. of human milk mucin. Development and characterization of breast cancer-reactive monoclonal antibodies directed against protein Sex determination, Cancer Res, 4U, 5476-5482 (1987).

Burehel ISJ, and Talylor-Papadfmitriou , J., Antibodies against human milk fat globule molecules, Cancer Invest, (1 989) Printing in progress.

Carlstedt, 1. ,LindgrenXH,,5heehan,,J , , Ulmsten, U., and Wingerup, L. , Human 11.13 -22 (1,983).

Ceriani, R.L., Peterson, J.A., Lee.

J, Y, , MoncadaSR, and B], ankSE, W, , human milk. Cell culture of human breast epithelial cells using monoclonal antibodies prepared against lipid globules. Characterization of cell surface antigens, Som, Cel 1. Genet, 9.415-427 (1983).

Foster, C.S., EdwardsSP., A.W., Dinsda. Ie, E., A., and Neville, A. M., Model for human mammary gland. Noclonal antibody, Virchoes Arch, (Path, Anat,) 3 94.279-293 (1982).

Gendler, S. J., Burchel 1. J., M., Duhig. T., Lamport, D., Whyte, R., Parker.

M., and Talylor-Papadimitriou, J., Encouraging differentiation. Cloning of a partial cDNA encoding and expressed by human mammary epithelium Tumor-associated mucin glycoprotein, Proc, Natl, Acad, Sci, USA , 84.6060-6064 (1987).

Gendler, S. J., Talylor-Papadimi trio , J., , Duhig, T., , Rothbard, J., and Burche. 11. J., highly immunogenic regions of human pleomorphic epithelial mucin expressed by cancer. The area is constructed by serial repetitions of J, Bi.

1, Chem, 263.12820-12823 (1988).

Geysen, H.M., Meloen, R., and Barteling, S. , Probing viral antigens for epitopes into single amino acid splits Use of peptide synthesis for, Proc, Nat 1. Acad, Sci, USA Sdan↓, 3998-4002 (1984).

Gir ing, A., Bartkova, J., Burchel 1. J.

, Gendler SS, , G11lett, C., and Ta1ylor-Pa. pad imi tr touSJ, by monoclonal antibody 5M-3 The detected epitopes of the PEM mucin core protein are associated with a range of primary cancers. Int, J, Cancer (1989) (in press) .

Hanisch, F.-G., Uhlenbruck, G., Peter -Katal 1nic SJ, EGGE, H, Dabrowski, J , and Dabrowski SU, Neutral 〇1 chain on human skim milk mucin. Structure of polylactosaminoglycan. Glaβ(1-4)G1cNAcβ(1- 6) Linearly extended poly-N-acetyl-lactosamine with repeating units A novel type of main chain, J, Btol, Chem, (in press).

Hf1kensS.J., Buijs, F., Hilges, J., Hag. eman, P.H., ,Ca1afatXJ, ,Sonnengerg, ,A. and Van DERValk, M., Detecting differentiation antigens of the mammary gland and its tumors. Monoclonal antibody against human milk fat globule members, Int, J, Cancer , 34.197-206 (1984).

I (ownsell, E. F., and Felzi, E., Gastrointestinal mucin, Med , Btol, , Dans, 227-236 (1982).

Hul ISS, R., Bright, A., Carraway K.

L, , Abe, M, and Kufe SD, , BT-20 human breast cancer cell line. +7) Oligosaccharide of DF3 antigen, J, Ce1l, Biochem, 5u ppl, 12E, 130, Abstract (1988).

Kjeldsen, T., C1ausen, H., HirohashilS. , Ogawa, T, , I t j ima, , H, and Hakomori. .

G9, tumor-related 〇 monochain sialosyl-2-6α-N-acetylgalactosaminyl Preparation of monoclonal antibodies directed against the (Sialosyl-Tn) epitope and Characterization, Cancer Res, 48.2214-2220 (1 988).

Lin5 ley, P, Brown, J, Magnan i, J , and Horn, J., Mucin glycoproteins found in serum from breast cancer patients. Monoclonal antibody reactive with protein, Cancer Res, April 213 8-2148 (1988).

PriceSM, R., Edwards, S., Owafnat i.

A, , Bul 1ock1J, E, , Ferry, B, , Robinsl R, A, and Baldwin, R, W, , polypeptides on human breast cancer-related antigens. Number of epitopes, Int, J, Cancer, 36.567-574 (198 5).

5ekine, H, 0hno, T, and K u f e s D -, human Purification and characterization of high molecular weight glycoproteins detectable in breast and breast cancer , J. Immn, ol., 1985, 3610-3615 (1985).

5Layter, H., Lamble in G., Letreut, A.

, Galabert SC, , Houdret, N., , Degand, P.

, Complex structure of human bronchial mucosal glycoproteins, Eur, J., Bjochem, . 1↓S, 209-218 (1984).

Swallw, D.M., Gendler, S., Griffithi B. , ,Corney, G. ,Tarylor-Papadjmi triou , J., and Bramwe [1], M.E., Human tumor-associated epithelial mucins are Nature, 3 encoded by the revealed hypervariable locus PUM 28.82-84 (1987).

Taylor-Papadimi Triou, J., Peterson, J., A., Arklie, J., Burchell, J., Cerian. i, R.L., and Bodmer, W.F., Epithelial-specific formation of milk globule members. Monoclonal antibodies against minutes: production and culture of Talylor-Pap adimi triou, J, , Mi l l l l l .

R., Burchell, J., Na5h, R., Pang, L., and G1. 1bert, benign breast tissue and breast cancer and monoclonal antibodies HMFG-1 and Pattern of reactivity with J, Exptl, Pathol, 2.247-2 60 (1986).

Townsend, A.R.M., Rothbard, J., Gotch. F, M, , Bahadur, G, , Wra i th, D, and Mcmi. Chael, A.J., Influenza recognized by cytotoxic T lymphocytes. Ce nucleoprotein epitopes can be defined with short synthetic peptides, Ce 11.44.959-968 (1986).

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W., and Trowsdale, J., HLA-DH7 and DRW53 related. Sequence and evolution of side chain genes, Proc, Nat 1. Acad.

Sc, USA, 84.4929-4933 (1987).

Example 3 Genetic information of several tandem repeats as a fusion protein in E. colt Expression of specified partial cDNAs Gendler et al. (PNAS) 84.6060-6064.1987) and approximately 10 series counters. Clone 7, which is composed of the recovery unit, is K was excised with EcoRI and inserted into the expression vector pRX-1 [ Rim (Ri mm) and bollard (Pol 1 ard), Gene.

Mutual, 323-327.1989], E, col i. Whip in DH-1 strain Selection for expression of protein fragments is performed by blobbing lift of transformation using antibodies. It was carried out by Prepare DNA from plasmids expressing mucin fragments and and E. coli strain CAG-456 [see Rimm and Bollard ( Pollard)] was transformed for higher expression of mucin fragments. 18a The fragment containing the amino acid trpE protein was found to be approximately 3. It could be detected with the antibody as a fragment with a molecular weight of 0 kD. Gel separated bacterium Coomassie blue staining of the rear lysate can visualize this band. , which was increased in cells induced with indolyl acrylic acid and 10 It appeared to be composed of ~20% bacterial protein.

Example 4 Partial cDNA specifying genetic information of several tandem repeats in mammalian cells Expression of A Clone 7 was cloned into Bluescript vector [Stratagene ne), cut with EcoRI from California, and the positive of serial repeats. vectors based on the pJ3 expression vector to ensure that the correct reading frame is maintained. into the EcoR1 site of the vector.

Align the clone containing the insert in the correct orientation with the vector insert sequence. was selected using a bar-wrapping oligonucleotide.

DNA was prepared, purified, and transformed into Cos cells according to standard methods. I had an ection. After 48 hours, cells were sampled for agarose gel electrophoresis. The gel was dissolved in buffer solution and the gel was blotted onto nitrocellulose paper. P Probing the lot with antibodies using ELISA results in transfection. A band around 30 kD was shown in the cells analyzed.

Using the procedures of Examples 3 and 4, polypeptides of PDTRP epitopes according to the invention were prepared. Peptides or fusion proteins can be expressed and tested.

Pevenado 5M3 Pep ÷ Do ONI Knee M15 (TRPAI Gu7゛yudo Copy and translation of amended sound) Submission (Article 184-8 of the Patent Law) May 1991 IO date wise

Claims (1)

[Claims] 1. Formula (I) {X}x{(A)mPDTRP(A)n}y{X}2(I) where, x and z are the same or different and each is 0 or 1 or is an integer greater than or equal to y is an integer of 1 or more, Each X, if present, is independently selected from amino acid residues and m and n are the same or different, and each is 0 or 1, and A, P, D, T and R are each an amino acid residue, alanine, proline, Synthetic polypeptides of aspartic acid, threonine and arginine, or or array (A)mPDTRP(A)n During the ceremony, Each symbol is as defined above, said synthetic polypeptide containing a sequence immunologically equivalent to is said synthetic polypeptide containing two or more saccharide moieties, or its pharmaceutical formulation. -acceptable salts, excluding beta-glucuronidase. 2. (a) PDTRP APDTRP PDTRPA APDTRPA (b) a polypeptide (a) having one or more saccharide moieties, and (c) a pharmaceutically acceptable salt of the above polypeptides (a) and (b); A polypeptide selected from. 3. The polypeptide according to item 1 or 2 above linked to a carrier protein . 4. Carrier protein is keyhole limpet hemocyanin, albumin and thyroglobulin. 5. Nucleic acid fragment encoding the polypeptide according to any one of items 1 to 4 above. . 6. Formula (II) {XXX}x{(GCX)mCCXGAPyACXRRRCCX(GCX)n} y{XXX}2(II) in the formula, RRR is CGX or AGPu {X}x{(A)mPDTRP(A)n}y{X}2, x and z are the same or different and each is 0 or 1 or is an integer greater than or equal to y is an integer of 1 or more, Each X, if present, is independently selected from amino acid residues and m and n are the same or different, and each is 0 or 1, and A, P, D, T and R are each an amino acid residue, alanine, proline, aspartic acid, threonine and arginine, and X is deoxyribonucleate. octide, XXX is a codon encoding an amino acid residue, and Py is C or T, Pu is A or G, and A, C, G and T are internationally recognized for the four deoxyribonucleotide bases. Item 5 above, containing a sequential decoding sequence of, which is a one-letter code determined by Fragment described. 7. Deciphered sequence (GCC)mCCGGACACCAGGCCG(GCC)n, the sixth Fragment described in section. 8. Consists of an engineered gene that encodes a fusion protein and is produced in an expressible form. A DNA construct containing the fragment according to any one of items 5 to 7 above. 9. A clone containing the nucleic acid fragment or construct according to any of items 5 to 8 above. ning or expression vector. 10. Encoding the nucleic acid fragment or construct according to any of items 5 to 8 above. consisting of the poliovirus genomic DNA containing the coding sequence in an expressible form. expression vector. 11. The fragment is contained together with the poliovirus capsid protein gene. The vector according to item 10 above. 12. In the cloning or expression vector according to any of the above items 9 to 11. Transformed host cells. 13. A nucleus comprising the fragment according to any of items 5 to 7 above and a detectable label. Acid probe. 14. Can recognize the polypeptide according to any one of items 1 to 4 above. , SM-3 and HMFG-2. 15. The antibody according to paragraph 14 above, linked to a therapeutically or diagnostically effective moiety. body. 16. Carrier or diluent or adjuvant and as described in item 14 or 15 above. A pharmaceutical formulation consisting of an antibody. 17. Secretes antibodies against the polypeptide according to any one of items 1 to 4 above. Cells other than HSM-3 that can be used. 18. Permanently proliferating cell according to item 17 above. 19. Methods of treating the human or animal body by surgery or therapy; or for use in a diagnostic method carried out on or on the body of an animal; The polypeptide according to any one of paragraphs 1 to 4, the polypeptide according to any one of paragraphs 5 to 7 above. a nucleic acid fragment, a construct according to item 8 above, a vector according to any one of items 9 to 11 above. vector, the cell described in item 12 above, the probe described in item 13 above, the cell described in item 14 above. or the antibody according to item 15, the pharmaceutical formulation according to item 16 above, or the antibody according to item 17 above. or the cell described in item 18. 20. Methods of treating the human or animal body by surgery or therapy; or of drugs for use in diagnostic methods or carried out on the body of animals. In preparation, the polypeptide according to any one of the above items 1 to 4, the above items 5 to 7 The nucleic acid fragment according to any one of the above items, the construct according to the above item 8, the above item 9 to 11 The vector according to any of the above, the cell according to the above item 12, the protein according to the above item 13 the antibody according to item 14 or 15 above, the pharmaceutical formulation according to item 16 above Alternatively, use of the cells described in item 17 or 18 above. 21. A non-toxic amount effective for humans or animals in need of treatment or diagnosis. The polypeptide according to any one of Items 1 to 4, the polypeptide according to any one of Items 5 to 7 above. A nucleic acid fragment, a construct according to item 8 above, a vector according to any one of items 9 to 11 above. a cell according to item 12 above, a probe according to item 13 above, a cell according to item 14 above, or a cell according to item 12 above. is the antibody according to item 15, the pharmaceutical formulation according to item 16 above, or the pharmaceutical composition according to item 17 above. or a method of treatment or diagnosis comprising administering the cell according to item 18. 22. Samples suspected of containing abnormal human mucin glycoproteins are A diagnostic test method comprising contacting with the antibody according to item 4 or 15. 23. A diagnostic test kit comprising the antibody according to item 14 or 15 above.