EP0442926A1 - Polypeptides - Google Patents

Polypeptides

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Publication number
EP0442926A1
EP0442926A1 EP89912475A EP89912475A EP0442926A1 EP 0442926 A1 EP0442926 A1 EP 0442926A1 EP 89912475 A EP89912475 A EP 89912475A EP 89912475 A EP89912475 A EP 89912475A EP 0442926 A1 EP0442926 A1 EP 0442926A1
Authority
EP
European Patent Office
Prior art keywords
pdtrp
epitope
antibody
human
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP89912475A
Other languages
German (de)
English (en)
Inventor
Joyce Taylor-Papadimitriou
Sandra Gendler
Joy Burchell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cancer Research Horizons Ltd
Original Assignee
Imperial Cancer Research Technology Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB888826362A external-priority patent/GB8826362D0/en
Priority claimed from GB898907660A external-priority patent/GB8907660D0/en
Application filed by Imperial Cancer Research Technology Ltd filed Critical Imperial Cancer Research Technology Ltd
Publication of EP0442926A1 publication Critical patent/EP0442926A1/fr
Withdrawn legal-status Critical Current

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Classifications

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

Definitions

  • the present invention relates to a polypeptide having a sequence corresponding to an antigenic epitope on human polymorphic epithelial ucin (PEM), to its production and use in diagnosis and therapeutic treatment of cancer.
  • PEM polymorphic epithelial ucin
  • WO88/05054 there is described the discovery of a tandem repeat sequence of a ino acid residues appearing in the human PEM protein expressed by normal or transformed epithelial cells. At least in the case of breast cancer, malignant cells express an under-glycosylated- form of PEM which is detected by the SM3 monoclonal antibody. That antibody does not react with fully processed PEM as produced by normal cells so that the antibody has uses in the diagnosis and therapy of breast cancer.
  • the epitope recognised by SM3 is a small, continuous amino acid sequence occurring within the tandem repeat sequence previously described. Identification of the epitope permits synthetic preparation of peptides mimicking the epitope which will be useful in generating antibodies with improved specificity for tumour as opposed to normal PEM.
  • the present invention therefore provides a
  • A,P,D,T and R are the internationally recognised one-letter symbols for the residues of the amino acids, alanine, proline, aspartic acid, threonine and arginine respectively;
  • PDTRP epitopes Poiypeptides as defined under (a), (b) and (c) above, subject to the same exclusions, will hereafter be referred to as "PDTRP epitopes".
  • the simplest poiypeptides according to the present invention are the pentapeptide PDTRP, immunological analogues thereof and glycosylated derivatives of the pentapeptide and its analogues.
  • Hexapeptides according to the invention include APDTRP, PDTRPA, immunological analogues thereof and glycosylated derivatives of the hexapeptides and analogues.
  • Heptapeptides according to the invention include APDTRPA and immunological analogues thereof and glycosylated derivatives of the heptapeptide and analogues.
  • poiypeptides include additional amino acid residues at the amino and/or carboxy terminal of the penta-, hexa- and hepta-peptides, analogues and derivatives.
  • Such poiypeptides may comprise, for instance, up to about 200, for instance up to about 50, particularly up to 30, preferably up to 20 and more preferably from 10 to 15 amino acid residues. Maximum and preferred values of x and z are determined accordingly.
  • the poiypeptides must include at least one five amino acid residue sequence PDTRP or an immunological analogue thereof and may include several repeats of this sequence, for instance 2 or 3 repeats and perhaps up to 100 or more such repeats.
  • Each such repeat independently may include one or both of the amino and/or carboxy terminal alanine residues, or immunological equivalents thereof and may be extended at either or both the N- and C-termini with other amino acids. Typically each repeat will comprise 19 or less amino acid residues.
  • any repeats of the PDTRP sequence or analogues thereof are independently glycosylated, for instance by attachment of a linkage sugar such as N-acetyl galactosa ine on the threonine residue of PDTRP and/or on threonine or serine residues outside the epitope PDTRP sequence or which are included within the epitope sequence of an immunological equivalent of PDTRP (for instance, wherein the threonine residue has been replaced by a serine residue).
  • a linkage sugar such as N-acetyl galactosa ine on the threonine residue of PDTRP and/or on threonine or serine residues outside the epitope PDTRP sequence or which are included within the epitope sequence of an immunological equivalent of PDTRP (for instance, wherein the threonine residue has been replaced by a serine residue).
  • Immunological analogues according to the present inventions are poiypeptides having a sequence corresponding to that of formula (I) except that the sequence (A) PDTRP(A) is modified at at least one amino acid residue by substitution and/or is modified by insertion of one or more additional amino acid residues provided that the modified sequence is capable of acting as an epitope for the generation of antibodies and provided that the antibodies so generated which recognise the modified epitope also recognise at least one of
  • Immunological analogues also known as mimotope ⁇
  • the poiypeptides of the present invention are synthetic.
  • PDTRP epitopes according to the invention may be produced de_ novo by synthetic methods or by expression of the appropriate DNA fragments described below by recombinant DNA techniques and expressed with or without glycosylation in human or non-human cells.
  • a peptide may be built up from single amino acids and/or preformed peptides of two or more amino acids in the order of the sequence of the desired peptide and optionally the resultant peptide may be converted into a pharmaceutically acceptable salt if desired.
  • Solid-phase or solution methods may be employed.
  • the amino acid sequence of the desired peptide is built up sequentially from the C-terminal amino acid which is bound to an insoluble resin. When the desired peptide has been produced, it is cleaved from the resin.
  • the desired peptide may again be built up from the C-terminal amino acid but the carboxy group of .this acid remains blocked throughout by a suitable protecting group, which is only removed at the end of the synthesis.
  • each amino acid added to the reaction system typically has a protected amino group and an activated carboxy group.
  • An amino group may be protected by the fluoren-9-ylmethoxycarbonyl (Fmoc) or t-butoxycarbonyl (Boc) group.
  • a carboxy group may be activated as a pentafluorophenyl or l-oxo-2-hydroxy- dihydrobenzotriazine ester.
  • Functional side-chain groups such as the side chain amino group of lysine and the side chain hydroxy group of threonine may be protected too.
  • Each condensation step may be effected in the presence of dicyclohexylcarbodiimide or 1-hydroxybe ⁇ zotriazole. After each step in the synthesis, the amir ⁇ o-protecting group is removed. Side-chain functional group protecting groups are generally removed at the end of the synthesis.
  • the resultant peptide may be treated with an organic or inorganic acid to form an acid addition salt.
  • Suitable acids include acetic, succinic and hydrochloric acid.
  • the peptide may be converted into a carboxylic acid salt such as the ammonium salt or an alkali metal salt such as the sodium or potassium salt by use of an appropriate base.
  • the PDTRP epitopes of the invention may also be prepared by recombinant DNA methodologies; an expression vector is prepared which incorporates a DNA sequence encoding the PDTRP epitope and which is capable of expressing the PDTRP epitope when provided in a suitable host.
  • the DNA sequence is located between translation start and stop signals in the vector.
  • Appropriate transcriptional control elements are also provided, in particular a promoter for the DNA sequence and a transcriptional termination site.
  • the DNA sequence is provided in the correct reading frame so as to enable expression of the peptide to occur in a host compatible with the vector.
  • the vector may be a plasmid or viral vector and the host may be any prokaryotic or eukaryotic cells such as bacterial or yeast cell or cells of a mammalian cell line.
  • the PDTRP epitopes may be obtained by stripping carbohydrate from native human mucin glycoprotein (which itself may be produced by isolation from samples of human tissue or body fluids or by expression and full processing in a human cell line) (Burchell e_t al.. , Cancer Research, 47, 5467-5482, (1987), Gendler e_t al.. , P.N.A.S. , 84 6060-6064, (1987)), and digesting the core protein.
  • native human mucin glycoprotein which itself may be produced by isolation from samples of human tissue or body fluids or by expression and full processing in a human cell line
  • PDTRP epitopes linked to carrier proteins such as keyhole limpet hae ocyanin, albumen or thyroglobulin are also within the invention. Linkage may be by known chemical methods or by expression of the PDTRP epitope and carrier protein as a fusion protein by genetically transformed cells as herein described.
  • the invention provides a nucleic acid fragment encoding a PDTRP epitope.
  • Fragments according to the invention may be RNA or DNA and DNA fragments may be single or double stranded.
  • Preferred such nucleic acid fragments are DNA fragments containing a continuous coding sequence of formula (II) which encodes a PDTRP epitope of formula (I) as hereinbefore defined,
  • X is any deoxyribonucleotide
  • XXX is a codon encoding an amino acid residue
  • Py is C or T
  • Pu is A or G; and A, C, G and T are the internationally recognised one-letter codes for the four deoxyribonucleotide bases.
  • Preferred nucleic acid fragments of the invention comprise the sequence
  • the invention further provides nucleic acid fragments encoding PDTRP epitopes which are immunologically analogous to peptides of formula (I) as hereinbefore defined.
  • fragments will be useful both for generating PDTRP epitopes as described below and as probes for detecting DNA encoding PDTRP epitopes.
  • detectable labels such as radioisotopes, fluorescent or enzyme labels
  • all of these may be achieved by conventional methods (Matthews e_t a_l. Anal. Biochem. 169, 1-25 (1988)).
  • the nucleic acid fragments of the invention may be produced d_e novo by conventional nucleic acid* synthesis techniques or obtained from human epithelial cells by conventional methods (Huynh e_t al, "DNA Cloning: A Practical Approach” ed Glover, D.M. ( RL, Oxford), 1985, Vol 1 pp49-48) .
  • the present invention provides cloning and expression vectors comprising nucleic acid fragments as hereinbefore defined (the expression vectors having the fragment in open reading frame relation to initiation and termination signals and being associated with appropriate regulatory and promoter sequences so as to form an expressible sequence) and host cells transformed with such cloning or expression vectors.
  • nucleic acid fragments of the present invention may also be used in active immunisation techniques.
  • the fragment, which codes for a PDTRP epitope may be extended at either or both the 5' and 3' ends with further coding or non-coding nucleic acid sequences including regulatory and promoter sequences, marker sequences and splicing or ligating sites.
  • coding sequences may code for other portions of the mucin protein chain or for other polypeptide chains.
  • the fragment according to the invention together with any necessary or desirable flanking sequences is inserted, in an appropriate open reading frame register, into a suitable vector such as a plasmid or a viral genome (for instance vaccina virus genome) and is then expressed as a polypeptide product by conventional techniques.
  • a suitable vector such as a plasmid or a viral genome (for instance vaccina virus genome) and is then expressed as a polypeptide product by conventional techniques.
  • the polypeptide product including the PDTRP epitope may be produced by culturing appropriate cells transformed with a vector, harvested and used as an immunogen to induce active immunity against the mucin core protein (Tartaglia e_t al. , Tibtech, 6, 43 (1988)).
  • the vector particularly in the form of a virus, may be directly inoculated into a human or animal to be immunised.
  • the vector then directs expression of the PDTRP epitope _ir ⁇ vivo and this in turn serves as an immunogen to induce active immunity against the mucin core protein.
  • the DNA sequence may, in one embodiment, be inserted into another gene and expressed as a fusion protein.
  • a nucleotide sequence encoding a PDTRP epitope flanked by other amino acid sequences is expressed in a genetically engineered poliovirus by inserting the oligonucleotide into the gene encoding capsid protein of the Sabin type 1 vaccine strain of poliovirus, as has been done for other protein epitopes (Evans et al., 1989, Nature 339, 385-388).
  • a genetically engineered poliovirus by inserting the oligonucleotide into the gene encoding capsid protein of the Sabin type 1 vaccine strain of poliovirus, as has been done for other protein epitopes (Evans et al., 1989, Nature 339, 385-388).
  • Such an engineered virus may readily be tested for its ability to induce tumour regression in humans or to delay relapse, and ultimately to prevent tumour development.
  • the invention therefore provides probes, vectors and transformed cells for use in the methods of treatment of the human or animal body by surgery or therapy and in diagnostic methods practised on the human or animal body and for use in the preparation of medicaments for use in such methods.
  • the invention also provides methods for treatment of the human or animal body by surgery or therapy and diagnostic methods practised j vivo as well as e_x vivo and ii vitro which comprise administering such fragments, probes, vectors or transformed cells in effective non-toxic amount to a human or other mammal in need thereof as well as diagnostic methods practised e_x vivo or _ir ⁇ vitro comprising contacting a body fluid or tissue sample with the fragments or probes.
  • the invention further provides a process for producing the PDTRP epitopes comprising culturing such host cells transformed with an expression vector and recovering expressed poiypeptides.
  • the invention provides ⁇ antibodies other than SM3 and HMFG-2 capable of recognising a PDTRP epitope as hereinbefore defined and preferably having reduced or no reaction with fully processed human PEM glycoprotein as produced by the normal lactating human mammary gland.
  • antibody is intended to include polyclonal and monoclonal antibodies and fragments of antibodies bearing antigen binding sites such as the F(ab') 2 fragments as well as such antibodies or fragments thereof which have been modified chemically or genetically in order to vary the amino acid residue sequence of one or more polypeptide chains, to change the species specific and/or isotype specific regions and/or to combine polypeptide chains from different sources.
  • Such antibodies may be obtained by conventional methods (Williams, Tibtech, 6_, 36, (1988)) and are useful in diagnostic and therapeutic applications, such as passive immunisation.
  • Antibodies according to the present invention react with PDTRP epitopes as hereinbefore defined; they also react with HPEM core protein, especially as expressed by colon, lung, ovary and particularly breast carcinomas, but have reduced or no reaction with corresponding fully processed HPEM.
  • the antibodies react with HPEM core protein but not with fully processed HPEM glycoprotein as produced by the normal lactating human mammary gland.
  • Antibodies according to the present invention preferably have no significant reaction with the mucin glycoproteins produced by mammary epithelial tissues lactating or pregnant individuals but react with the mucin proteins expressed by mammary epithelial adenocarcinoma cells. These antibodies show a much reduced reaction with benign breast tumours and are therefore useful in diagnosis and localisation of breast cancer as well as in therapeutic methods.
  • the antibodies may be used for other purposes including screening cell cultures for the polypeptide expression product of the human mammary epithelial mucin gene, or fragments thereof, particularly the nascent expression product.
  • the antibodies may conveniently be polyclonal or monoclonal antibodies.
  • the invention further provides antibodies linked by known techniques to therapeutically or diagnostically effective moieties or to linking moieties.
  • the linking moieties may be known linking agents for binding therapeutically or diagnostically effective moieties to the antibodies, for instance the linking moiety may be a specific binding partner for a specific binding agent, the therapeutically or diagnosically effective moiety being bound to the specific binding agent.
  • the linking moiety may be a specific binding partner for a specific binding agent, the therapeutically or diagnosically effective moiety being bound to the specific binding agent.
  • binding of the specific binding agent to the specific binding partner results in the therapeutically or diagnostically effective moiety being bound to the antibody.
  • a preferred example of a specific binding agent/ specific binding partner pair is biotin and avidin or streptavidin in which biotin may be the binding agent or the binding partner.
  • the therapeutically effective moieties are lethal agents to be delivered to cancerous breast or other tissue in order to incapacitate or kill transformed cells.
  • Lethal agents include toxins, radioisotopes and "direct killing agents" such as components of complement as well as cytotoxic or other drugs or enzymes which activate cytotoxic drugs at the tumour site.
  • the diagnostically effective moieties may be moieties such as solid supports and detectable labels such as enzyme labels, chromophores, fluorophores and radioisotopes and other directly or indirectly detectable labels.
  • detectable labels such as enzyme labels, chromophores, fluorophores and radioisotopes and other directly or indirectly detectable labels.
  • monoclonal antibodies are used in diagnosis.
  • the invention further provides a diagnostic test process or assay method comprising contacting a sample suspected to contain abnormal human mucin glycoproteins with an antibody as defined above.
  • diagnostic test process or assay method comprising contacting a sample suspected to contain abnormal human mucin glycoproteins with an antibody as defined above.
  • Such methods include tumour localisation involving administration to the patient of the antibody bearing detectable label or administration of an antibody and, separately, simultaneously .or sequentially in either order, administering a labelling entity capable of selectively binding the antibody or fragment thereof.
  • Particular uses of the antibodies include diagnostic assays for detecting and/or assessing the severity of breast, colon, ovary and/or lung cancers.
  • Diagnostic test kits are provided for use in diagnostic process and assays and comprise antibody and, optionally, suitable labels and other reagents such as buffers, reagents for labelling the antibody and/or for detecting labelled antibody and, especially for use in competitive assays, standard sera.
  • the invention further provides PDTRP epitopes and antibodies as hereinbefore defined for use in methods of surgery, therapy or diagnosis practised on the human or animal body or for use in the production of medicaments for use in such methods.
  • the invention also provides a method of treatment or diagnosis which comprises administering an effective non-toxic amount of a polypeptide or antibody as hereinbefore described to a human or animal in need thereof. Dosage rates will depend on the age, weight, size, sex and general health of the patient and will be sufficient to achieve appropriate levels of circulating antibodies without undesirable or intolerable side effects.
  • Administration may be by the oral or parenteral route, for instance by sub-cubtaneous, intravenous or intramuscular injection.
  • a PDTRP epitope is administered to a human adult in an amount up to 1 g per dose, preferably 1 to 200 mg per dose, by either the oral or the parenteral route.
  • the invention therefore also provides pharmaceutical compositions comprising a PDTRP epitope of the invention and a suitable carrier, diluent and/or adjuvant, which will, of course, be determined by the route of administration and selected for compatibility with the PDTRP epitope.
  • suitable carriers, diluents and adjuvants include Freunds incomplete adjuvant (IFA), aluminium hydroxide, saponin, DEAE-dextran, muramyl dipeptide, mineral oils, neutral oils such as miglyol, vegetable oils such as arachis oil, "Iscoms", liposo es, Pluronic (trade mark), polyols or the Ribi adjuvant system (GB-A-2189141) .
  • compositions may be in the form of aqueous sterile, non-pyrogenic solutions optionally containing buffers, antioxidants, biocides such as antibacterials and antifungals and agents to adjust the tonicity.
  • the compositions may be provided as dry powders, optionally containing excipients such as those mentioned above, for reconstitution by addition of water for injection.
  • Antibodies whether polyclonal or monoclonal, against PDTRP epitope may be used in therapeutic methods for treating humans and animals, for instance in the form of vaccines for passive immunisation. Preferably they are formulated as pharmaceutical compositions such as are described above.
  • the antibody may be appropriate to modify the antibody by coupling the Fab, or complementarity-determining region thereof, to the Fc, or whole framework region, of antibodies derived from the species to be treated (e.g. such that the Fab region of mouse monoclonal antibodies may be administered with a human Fc region to reduce immune response by a human patient) or in order to vary the isotype of the ' antibody (see EP-A-0 239 400).
  • the present invention also provides a process for producing polyclonal antibodies against a PDTRP epitope comprising inoculating a host animal with transfected cells capable of expressing the PDTRP epitope and recovering antibodies from the serum or other body fluid of the animal.
  • the present invention further provides a process for producing a cell capable of secreting antibodies against a PDTRP epitope comprising inoculating a host animal with PDTRP epitope or with transfected cells capable of expressing the PDTRP epitope and removing antibody-secreting cells from the animal.
  • the cells may be capable of secreting antibodies either iin vitro, for instance under appropriate cell culture conditions and/or in vivo, for instance by growth in ascites.
  • animal refers to any animal capable of producing antibodies for instance mammals such as laboratory rodents, and birds and includes humans.
  • Transfected cells are cells containing a DNA expression vector containing an expressible sequence encoding the PDTRP epitope.
  • the coding sequence may be natural or synthetic and the encoded polypeptide may be expressed directly or as a fusion protein.
  • DNA coding for the PDTRP epitope is identified and obtained from a natural source or produced by partial or total synthesis. Techniques for obtaining natural or artificial DNA encoding a particular polypeptide are well known in the field of genetic engineering and are described in Maniatis, T., Fritsch, E and Sambrook J. (1982) Molecular Cloning, a Laboratory Manual, published by Cold Spring Harbor Laboratory Press NY.
  • the vector is constructed by conventional methods to ensure expression in the cells to be transfected and may be provided with marker sequences enabling identification of the DNA and transfectant cells, (Maniatis loc. cit. ) .
  • Selective markers which are particularly suitable in the present invention include drug resistance markers such as the thymidine kinase (tK) gene for tK ⁇ cells. (Szybalska, E and Szybalski W. (1962) Proc. Natl. Acad Sci., USA, 4_2, 2026) .
  • promoter sequences may be inserted in the vector to increase expression of the PDTRP epitope in the transfectant cells (Subramani, S., Mullingan, R.C. and Berg, P. (1981) Mol. Cell. Biol. 1 , 854-864).
  • the cells to be transfected i.e. the cells into which the vector is to be inserted, (hereafter "donor cells") are obtained from donor animals, such as rodents, especially rats and mice, and primates, for instance monkeys.
  • the donor cells must be capable, once transfected, of expressing the PDTRP epitope, optionally with post translational processing of the PDTRP epitope.
  • the donor cells and the optionally modified DNA are chosen such that the PDTRP epitope appears extracellularly or at the cell surface although it is also possible to apply the present invention to poiypeptides which are only expressed intracellularly.
  • the donor cells are of a type not normally found in the peritoneum or circulating in the bloodstream of the host animals.
  • the cells to be transfected are derived from an animal (hereafter the "donor animal") syngeneic with the animal (hereafter the "host animal”) which is inoculated with the transfected cells.
  • the donor animal syngeneic with the animal (hereafter the "host animal") which is inoculated with the transfected cells.
  • the host and donor animals are substantially genetically identical and usually they will be from a well established inbred strain such as are well known in the art.
  • Transfection of donor cells is achieved by conventional methods such as using calcium phosphate (Austin, P., Trowsdale, J. , Rudd, C, Bodmer, W. , Feldmann, M. and Lamb, J. (1985) Nature, 313, 1-4) or by the electroporation technique (Neumann, E., Schaefer- Ridder, M., Wang, Y. and HofSchneider, P.H. (1982) EMBO J, 1, 841-845) or using retroviral vectors (Biotechniques, 6_, 608-614 (1988)).
  • Transfectant cells may be treated to enhance the expression of the gene product.
  • Various known enhancement methods can be used including treatment with sodium butyrate (Gorman, CM. and Howard, B.H. (1983) Nucleic Acids Research, 11, 7631-7648) or interferon (Balkwill, F.R., Stevens, M.H., Griffin, D.B., Thomas, J.A. and Bodmer, J.G. (1987) Eur. J. Cancer Clin. Oncol. , 23, 101-106).
  • the enhancement method will be selected as appropriate to the type of donor cells.
  • the host animal or animals are inoculated with PDTRP epitopes or transfected cells, preferably by intraperitoneal or intravenous injection of transfected cells in a suitable medium.
  • the inoculation is repeated at intervals and the animals' antibody titre is monitored to ascertain that the desired immune response occurs.
  • the route chosen for administration and the inoculation regime are preferably selected in order to favour production of the desired class of antibodies, eg production of cells secreting IgM antibodies is favoured by a short interval between inoculation and recovery of antibody-secreting cells, whereas production of cells secreting IgG antibodies is favoured by a long interval between repeated inoculations and eventual recovery of the antibody-secreting cells.
  • antibody -secreting cells eg, spleen cells
  • antibody -secreting cells are taken from the •inoculated host animal(s) and maintained in an appropriate medium. Conventional methods may be used for obtaining and maintaining the host cells.
  • Antibody producing cells may also be obtained from the body fluids or tissues of host animals having carcinoma, particularly lymphocytes from excised lymph nodes of human carcinoma, especially breast or ovarian cancer, patients. (J ⁇ Immunol. Methods, 105, 263-273 (1987)).
  • the invention provides a process for producing a cell capable of secreting antibodies against a PDTRP epitope comprising removing antibody-secreting cells from a host animal having carcinoma or inoculated with a PDTRP epitope or with transfected cells capable of expressing the PDTRP epitope.
  • the invention provides a cell capable of secreting antibodies against a PDTRP epitope which cell has been removed from a host animal having carcinoma or inoculated with a PDTRP epitope or with transfected cells capable of expressing the PDTRP epitope.
  • the cells are useful particularly for immortalisation in order to form an immortal cell line capable of secreting antibodies (hereafter monoclonal antibodies or MAb's) against the PDTRP epitope.
  • Immortalisation may be by any of the known methods but fusion with an immortal cell to form a hybridoma is perhaps the most convenient method.
  • the invention also provides a process for producing an immortal cell capable of secreting antibody against a PDTRP epitope comprising immortalising an antibody-secreting animal cell, the antibody-secreting animal cell having been taken from a host animal inoculated with PDTRP epitope or transfected cells capable of expressing the PDTRP epitope.
  • Immortal cells which cells are capable of repeated cell division under appropriate culture conditions
  • the immortal cell may be of the same inbred strain as the antibody-secreting cells and usually they will be of the same species as the antibody-secreting cells from the host animal but this is not essential.
  • Particularly convenient immortal cells are myeloma cells. Fusion is achieved by the method of Kohler and Milstein, Nature, 256, 495-497 (1975) and by variations thereon as described in the literature.
  • the present invention also provides an immortalised cell, preferably a hybridoma, capable of secreting antibodies against a PDTRP epitope, which cell comprises an immortalised, antibody-producing cell capable of secreting antibodies against the PDTRP epitope taken from a host animal inoculated with PDTRP epitope or transfected cells capable of expressing the PDTRP epitope, or is a descendant of such a cell.
  • an immortalised cell preferably a hybridoma, capable of secreting antibodies against a PDTRP epitope
  • which cell comprises an immortalised, antibody-producing cell capable of secreting antibodies against the PDTRP epitope taken from a host animal inoculated with PDTRP epitope or transfected cells capable of expressing the PDTRP epitope, or is a descendant of such a cell.
  • Immortal cells e.g. hybridoma cells
  • Immortal cells e.g. hybridoma cells
  • the present invention provides a process for producing monoclonal antibodies against a PDTRP epitope comprising culturing an immortalised cell as hereinbefore defined capable of secreting antibodies against a PDTRP epitope, or a descendant of such a cell, and recovering the monoclonal antibodies.
  • the invention further provides monoclonal antibodies against a PDTRP epitope which antibodies are obtained from growth in tissue culture or ascites of an immortal hybrid cell as hereinbefore defined.
  • Antibodies produced by the immortalised cells as described above may be screened by conventional techniques to identify those cells producing MAb's against a PDTRP epitope. Such cells may be multiplied and cultured to produce MAb's according to the invention in large quantities. Suitable culturing techniques and ascites growth conditions are well known and are chosen as appropriate to the particular immortalised cell line involved and the MAb's to be produced.
  • the present invention further provides a fusion. protein comprising a PDTRP epitope as hereinbefore defined and a carrier polypeptide or protein bound to the C-terminal or the N-terminal of the PDTRP epitope.
  • the carrier polypeptide or protein may be any suitable polypeptide or protein.
  • Convenient carrier proteins are thyroglobulin and bovine serum albumin.
  • the fusion proteins of the invention may be produced by conventional techniques of polypeptide synthesis and protein chemistry from individual amino acid precursors or by coupling smaller poiypeptides.
  • the fusion proteins may be produced by recombinant DNA techniques involving expression of a gene encoding the fusion protein. These methods are all well known to those skilled in the art.
  • the invention further provides fusion proteins as hereinbefore defined for use in a method of treatment of the human or animal body by surgery or therapy or a diagnostic method practised on the human or animal body; the use of fusion proteins in the manufacture of a medicament for use in the therapeutic, surgical or diagnostic treatment of the human or animal body; a pharmaceutical composition comprising the fusion protein and a pharmaceutically acceptable diluent or carrier therefor and, optionally, conventional accessory ingredients, and a method for treating the human or animal body comprising administering an effective, non-toxic amount of a fusion protein to a human or animal in need thereof.
  • the fusion proteins are particularly useful in the treatment or prophylaxis of cancers such as breast cancer by increasing the patient's immunity to the tumour cells and in the production of polyclonal and monoclonal antibodies for use in diagnosis and therapeutic treatment of cancer.
  • Fig.l is a graph of the O.D. at 450 n of a series of octapeptide/SM3 reactions in accordance with Example 1.
  • Fig. 2 shows the design of overlapping peptide octamers.
  • the starting sequence consisted of a twenty amino acid tandem repeat with four amino acids of adjacent repeats on the amino and carboxyl ends. Overlapping octamers are indicated [ ] and [A] refers to the alanine used as the spacer on the pins. Note, octamers 1 and 21 are identical. The one letter code for the amino acids is used.
  • Fig. 3 is a graph of the binding of the monoclonal antibody SM-3 to octamer peptides.
  • Monoclonal antibody 50 g/ml, 100 ⁇ l per pin
  • octamers 1-21 defined in Fig. 2
  • the boxed sequences in the insert represent the epitope recognized by SM-3.
  • Fig. 4 is a series of graphs showing the binding of monoclonal antibodies HMFG-2, LICR-LON-M8 and onc-Ml5 to octamer peptides.
  • Monoclonal antibody 50 / /g/ml, 100 ⁇ l per pin for HMFG-2 and onc-Ml5; 1:500 dilution of LICR-LON-M8 ascites fluid
  • octamers 1-21 defined in Fig. 2
  • the sequence in brackets indicates the epitope.
  • Fig. 5 is a diagrammatic representation of epitopes recognised by monoclonal antibodies reactive with the PEM mucin core protein. Potential glycosylation sites are marked with arrows.
  • Fig. 6 is a series of graphs showing the binding- of HMFG-2 to lymphoblastoid and breast carcinoma " cell lines detected by fluorescence activated cell sorter.
  • Cell ' s in suspension were incubated with HMFG-2 (-) or TAL-14.
  • TAL-14 an anti HLA-DR monoclonal antibody, and the binding detected using a fluorescence labelled rabbit anti-mouse.
  • A lymphoblastoid cell line PGF which expresses HLA-DR2
  • the ⁇ chain contains the sequence Asp-Thr-Arg-Pro (DTRP)
  • B lymphoblastoid cell line Mann which expresses HLA-DR7
  • the ⁇ chain contains the sequence Asp-Thr-Gln-Pro (DTQP)
  • C breast carcinoma cell line T47D which expresses the PEM mucin.
  • Fig. 7 is a graph of the binding of anti-peptide antiserum to octamer peptides.
  • the anti-serum diluted 1:200 was reacted with the peptides as described in Fig. 2.
  • the boxed sequence in the insert represents the dominant epitope recognized by this anti-serum.
  • Fig. 8 is a pair micrographs showing staining of breast cancer cell lines with antipeptide antiserum.
  • SM-3 monoclonal antibody recognises an epitope within the 20 amino acid tandem repeat found in the PEM core protein (Gendler et al., J. Biol. Chem. , 263, 12820-12823 (1988)).
  • epitope scanning kit purchased from Cambridge Research Biochemical (Cambridge, England) we have further defined the epitope recognised by SM-3 and have shown that the minimum epitope consists of Proline - Aspartic Acid - Threonine - Arginine - Proline
  • the method consists of synthesizing small, overlapping peptides on the end of the polypropylene pins, 96 pins being held in a plastic support in the format and spacing of a microtitre plate.
  • the tips of the pins have been chemically derivatized to produce functional groups t which amino acids may be coupled and the peptides are synthesised on to the tips of the pins by the stepwise elongation of the peptide chain with the addition of one amino acid per day.
  • the pins can be assayed for their reactivity with antibody in a standard ELISA type assay, dispensing the reagents into 96 well microtitre plates in which the pins are then placed.
  • Octamers 1 and 6 to 21, each of which contains only a part or none of the PDTRP sequence and which have little or no reaction with SM-3 are not PDTRP epitopes.
  • the results obtained with such a set of octamers are shown in Fig. 1. indicating that the epitope for SM-3 is PDTRP.
  • PEM polymorphic epithelial mucin
  • the PEM component is of particular interest because, although it is produced by several normal epithelial tissues, and abundantly by the lactating mammary gland, it is also expressed by breast and other carcinomas (Taylor-Papadimitriou et al., 1986; Girling et al., 1989). Moreover, the mucin appears to be glycosylated differently by cancer cells, vith the result that novel carbohydrate epitopes appear (Kjeldsen et al., 1988), and core protein epitopes vhich are masked in the normal mucin are exposed on the cancer-associated component.
  • SM-3 One such core protein epitope is detected by the antibody SM-3, which was developed using the chemically deglycosylated normal PEM mucin (Burchell et al., 1987). This antibody reacts vith more than 90£ of breast cancers and vith the deglycosylated mucin, but shovs little or no reaction vith the normal breast or the native PEM mucin purified from milk (Burchell et al., 1987; Girling et al., 1989). The specificity of the SM-3 antibody makes it a potentially useful tool in the diagnosis and treatment of breast cancer, and it was therefore of great interest to identify the SM-3 epitope.
  • Peptides The peptides were synthesized by solid phase techniques on a 430A peptide synthesizer (Applied Biosystems), using commercially available t-boc protected amino acids and resins as previously described (Townsend et al., 1986).
  • Antibodies The development and characterization of the monoclonal antibodies HMFG-1, HMFG-2 and SM-3 (Taylor-Papadimitriou et al., 1981; Burchell et al., 1983, 1987; Girling et al. r 1989), LICR-L0N-M8 (Foster et al., 1982), onc-M15, onc-M23, onc-M27 (Linsley et al. , 1988) has been described elsewhere.
  • the monoclonal antibody TAL 14.1 is directed to HLA-DR.
  • Anti-peptide mouse antiserum was produced by immunizing intraperitoneally vith 25 ⁇ g of PDTR peptide 1-24 (see Table 2)
  • ELISA Monoclonal antibodies (50 ug/ml or 1:500 dilution of ascites fluid, 100 ⁇ l per pin) and polyclonal serum (1:200) were tested for their reactivity with the octamers in an ELISA assay as previously described (Burchell et al., 1983), using peroxidase conjugated rabbit antimouse antibody (DAK0, Denmark).
  • the octamers were synthesized onto pins conferring to the format of a 96 well microtitre plate and so for the assay pins were inserted into microtitre dishes containing the antibodies or reagents.
  • HMFG-2 Binding of HMFG-2 to cell lines: Cell pellets containing 10 cells were washed with RPMI containing 10% foetal calf serum and suspended in 1 ml of hybridoma tissue culture supernatant containing 0.02% sodium azide and incubated at 4°C for 1 hour. After two washes with RPMI containing 10% FCS, 0.02% azide (wash buffer) the cell pellets were suspended in 1 ml of fluorescence conjugated rabbit anti mouse (DAK0, Denmark) vhich had been diluted 1:20 in wash buffer and filtered prior to use.
  • DAK0 fluorescence conjugated rabbit anti mouse
  • the sequence of the tandem repeat unit of the PEM mucin is shown in Figure 2. Arbitrarily the sequence has been written as starting vith PDTR since this was the beginning of the first tandem repeat in one of the cDNA clones (Gendler et al. , 1987).
  • the antibody SM-3 has been shown previously to react with the PDTR peptide (1-24) which corresponds to one tandem repeat and four amino acids of the next repeat (see Table 1).
  • a series of overlapping peptide octamers, covering a single repeat and eight amino acids of adjacent repeats were synthesized onto the tips of polypropylene pins (see Materials and Methods).
  • Figure 3 shows the results obtained when the .peptides were reacted with SM-3 in an ELISA. It can be seen that all the peptides reactive with the SM-3 antibody contain the sequence Pro-Asp-Thr-Arg-Pro which appears therefore to represent the minimum epitope.
  • Monoclonal antibodies reactive with the PEM mucin have been developed using a variety of immunogens, including human milk fat globule extracts, vhich contain the mucin, (Taylor-Papadimitriou et al., 1981; Hilkens et al., 1984; Foster et al., 1982; Ceriani et al., 1983) and carcinoma cells (Bramwell et al. , 1983; Price et al. , 1985). Some were obtained from mice injected with both the mucin and cultured Table 1 Binding of antibodies to peptides based on the sequence of the PEM mucin of tandem reoeat
  • PDTR peptide (1-24) PDTRPAPGSTAPPAHGVTSAPDTR 2 PDTR peptide (1-14) PDTRPAPGSTAPPAC* J VTSA peptide (17-20, 1-10) VTSAPDTRPAPGST
  • Antibodies HMFG-2 and LICR-L0N-M8 show a remarkably similar pattern of reaction with the octamers and in both cases, the minimum epitope appears to be Asp-Thr-Arg (DTR), while that recognised by onc-15 is Thr-Arg-Pro-Ala (TRPA).
  • DTR Asp-Thr-Arg
  • TRPA Thr-Arg-Pro-Ala
  • the ⁇ chain of HLA DR2 contains the sequence Asp-Thr-Arg-Pro while the ⁇ chain of HLA DR7 contains the sequence Asp-Thr-Gln-Pro (Young et al., 1987), however the adjacent amino acids show no homology with the PEM mucin.
  • the binding of HMFG-2 to lymphoblastoid cell lines expressing HLA DR2 (PGF cells) and HLA DR7 (MANN cells) was analyzed and compared with the binding of HMFG-2 to a breast carcinoma cell line expressing the PEM mucin (T47D).
  • Figure 6 shows that as expected HMFG-2 did not bind to MANN cells expressing DR7, nor did it bind to cells expressing DR2 vhich contains the sequence Asp-Thr-Arg. Hovever, HMFG-2 did show strong binding to the breast carcinoma cell line T47D.
  • the minimum epitope for HMFG-2 consists of only three amino acids in the context of a conformationally free peptide while in the native protein, which has conformational restraints, the adjacent amino acids have an important role in determining the antigenic site.
  • the core protein of the polymorphic epithelial mucin is made up of a number of tandem repeats, each of which contains five potential 0-linked glycosylation sites (Gendler et al., 1988). These tandem 31
  • repeats contain the epitopes recognized by many of the monoclonal antibodies directed to this mucin. Using overlapping octamers the epitope recognized by SM-3, has been defined and has been shown to include the single threonine at position 3. Furthermore, epitopes reactive with other monoclonal antibodies have also been shown to map to this region. It therefore appears that, at least in mice, this sequence of the tandem repeat is highly immunogenic.
  • HMFG-2 Although the epitopes recognized by HMFG-2, LICR-L0N-M8, one 15 and SM-3 are overlapping, the reactivity of these antibodies with the mucin is quite different. All react with the mucin expressed by breast carcinoma cells but the HMFG-2, one 15 and M8 determinants are also expressed on the normal mucin (Burchell et al., 1983; Linsley et al., 1988; ' Foster et al., 1982), whereas the SM-3 determinant is not (Burchell et al., 1987; Girling et al., 1989).
  • the SM-3 monoclonal antibody was raised to the chemically deglycosylated normal milk mucin suggest that carbohydrate may play a role in masking this epitope on the PEM produced by normal cells.
  • the combined epitopes recognized by the three antibodies which react with the normal mucin cover the sequence, DTRPA, which is centrally placed between the flanking glycosylation sites (T 18' , S19' , S9, S10 in figure2. , see also figure 5 .
  • the SM-3 epitope PDTRP contains a proline at the amino end which is only one amino acid away from a glycosylation site (serine 19 ). This suggests that in the normal mucin the proline on the amino end of the SM-3 epitope is masked by carbohydrate while in tumours, because of premature termination of the sugar chains
  • BT20 oligosaccharide side chains of the mucin produced by the breast carcinoma cell line, BT20 consists of only 3 or 4 sugars (Hull et al., 1988).
  • HMFG-2 epitope Another feature of the HMFG-2 epitope is that the affinity for the antibody appears to vary, depending on the micro environment. Upon comparing the binding of the antibody to the normal mucin and to a cancer associated mucin, it is found that the antibody shows a higher affinity for the normal mucin, suggesting that the normal oligosaccharide side chains are required for maximum affinity. That oligosaccharide side chains may be important is also shown by the fact that both HMFG-2 and SM-3 bind more avidly to the partially deglycosylated mucin than to the mucin stripped of all carbohydrate (Burchell et al., 1987).
  • oligosaccharide side chains may play a role in determining the conformation of the core protein epitopes recognized by antibodies HMFG-2 and SM-3, it is quite clear that there are antigenic determinants in the core protein of the PEM mucin vhich are immunogenic and are expressed by breast cancers.
  • Using a synthetic peptide corresponding to the tandem repeat sequence coupled to a protein carrier we have been able to induce antibodies in the mouse which show reactivity with breast carcinomas. Epitope mapping with the antiserum indicate that dominant epitopes are found in the
  • CARLSTEDT I., LINDGREN, H. , SHEEHAN, J., ULMSTEN, U. , and WINGERUP, L. , Isolation and characterization of human cervical-mucus glycoproteins. Biochem. J. 211, 13-22 (1983).
  • GENDLER S.J., BURCHELL, J.M., DUHIG, T., LAMPORT, D. , WHITE, R. , PARKER, M., and TAYLOR-PAPADIMITRIOU, J., Cloning of partial cDNA encoding differentiation and tumor-associated mucin glycoproteins expressed by human mammary epithelium. Proc. natl. Acad. Sci. USA., 84, 6060-6064 (1987).
  • GENDLER S., TAYLOR-PAPADIMITRIOU, J., DUHIG, T., R0THBARD, J., and BURCHELL,- J.
  • a highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J. Biol. Chem., 263, 12820-12823 (1988).
  • GIRLING A., BARTK0VA, J., BURCHELL, J., GENDLER, S., GILLETT, C. , and TAYLOR-PAPADIMITRIOU, J., A core protein epitope of the PEM mucin detected by the monoclonal antibody SM-3 is selectively exposed in a range of primary carcinomas. Int. J. Cancer (1989) (in press). HANISCH, F.-G., UHLENBRUCK, G. , PETER-KATALINIC, J. , EGGE, H., DABROWSKI, J. and DABROWSKI, U.
  • HILKENS J., BUIJS, F., HILGERS, J., HAGEMAN, P.H., CALAFAT, J., SONNENBERG, A., and VAN DER VALK, M.
  • SEKINE H., OHNO, T., and KUFE, D. , Purification and characterization of a high molecular weight glycoprotein detectable in human milk and breast carcinomas. J. Immunol. 135, 3610-3615 (1985).
  • the human tumour-associated epithelial mucins are coded by an expressed hypervariable gene locus PUM. Nature, 328, 82-84 (1987).
  • TOWNSEND A.R.M. , ROTHBARD, J. , GOTCH, F.M., BAHADUR, G., WRAITH, D. and MCMICHAEL, A.J.
  • the epitopes of influenza nucleoprotein recognised by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell, 44, 959-968 (1986).
  • Clone 7 which was isolated by Gendler et al. (PNAS 84, 6060-6064, 1987) and is made up of approximately 10 tandem repeat units, was cut out of the Bluescript vector SK with EcoRl and inserted into the expression vector pRX-1 (Rimm and Pollard, Gene 75, 323-327, 1989). Selection for expression of the mucin fragment in E. coli DH-1 strain was done by probing lifts of the transformants with antibody. DNA from plasmid expressing the mucin fragment was prepared and E. coli strain CAG-456 (see Rimm and Pollard) was transformed for higher expression of the mucin fragment. The fragment containing 18 a.a.
  • Clone 7 was excised with EcoRl from the Bluescript vector (Stratagene, California) and inserted into the EcoRl site of a vector based on the pJ3 expression vector so that the correct reading frame of the tandem repeat was maintained. Clones vith plasmids containing inserts in the correct orientation were selected using oligonucleotides which overlapped the vector insert sequence. DNA was prepared and purified according to standard methods and transfected into Cos cells. After 48 hours cells were dissolved in sample buffer for acrylamide gel electrophoresis and gels were blotted on to nitrocellulose paper. Probing of the blot with antibody using an ELISA to detect bound antibody showed a band around 30 Kd in the transfected cells.
  • Example 3 & 4 The procedures of Example 3 & 4 may be used to express and test PDTRP epitope poiypeptides or fusion proteins according to the inventon.

Abstract

Des polypeptides comprenant la séquence PDTRP et des analogues immunologiques correspondent à un épitope important de la mucine épithéliale polymorphe humaine anormalement exprimée dans des cancers du sein, et sont utiles dans l'immunisation et pour la production d'anticorps destinés au diagnostic et à la thérapie. Des fragments d'acide nucléique codant PDTRP ou un analogue immunologique sont utiles pour les sondages à des fins de diagnostic et pour produire les polypeptides.
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