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WO2009019493A1 - Anticorps se liant a des cellules epitheliales de la prostate humaine - Google Patents

Anticorps se liant a des cellules epitheliales de la prostate humaine Download PDF

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Publication number
WO2009019493A1
WO2009019493A1 PCT/GB2008/002733 GB2008002733W WO2009019493A1 WO 2009019493 A1 WO2009019493 A1 WO 2009019493A1 GB 2008002733 W GB2008002733 W GB 2008002733W WO 2009019493 A1 WO2009019493 A1 WO 2009019493A1
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Prior art keywords
sequence
antibody
agent
cancer
kit
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PCT/GB2008/002733
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English (en)
Inventor
Tahereh Kamalati
Lakjaya Buluwela
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Imperial Innovations Limited
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Publication of WO2009019493A1 publication Critical patent/WO2009019493A1/fr

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    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the present invention relates to methods and reagents useful in relation to diagnosis and treatment of cancer, for example prostate cancer.
  • Cancer is a serious disease and a major killer. Although there have been advances in the diagnosis and treatment of certain cancers in recent years, there is still a need for improvements in diagnosis and treatment.
  • Carcinoma of the prostate has become a most significant disease in many countries and it is the most commonly diagnosed malignancy in men in the western world, its occurrence increasing significantly with age. By the year 2018 it is expected to be the biggest killer with 50% of the male population suffering from it (80% by age 80 years). Recent evidence suggests that prostate cancer is also increasing amongst younger men as well (Br J Cancer (1999) 79, 13-17). These increases and the recent deaths of many public figures from prostatic cancer have served to highlight the need to do something about this cancer. It has been suggested that the wider availability of screening may limit mortality from prostate cancer.
  • Prostate cancer screening currently includes a rectal examination and measurement of prostate specific antigen (PSA) levels. These methods lack specificity as digital rectal examination has considerable inter-examiner variability (Smith & Catalona (1995) Urology 45, 70-74).
  • PSA prostate specific antigen
  • Imaging of cancer cells is a potentially important tool in the diagnosis, management and understanding of cancer.
  • an object of the invention to provide tools and associated methods useful in imaging, diagnoses and prognoses of prostate cancer, and for aiding the clinician in the management of cancer, particularly prostate cancer.
  • an object of the invention is to provide tools and associated methods for detecting primary or metastatic prostate cancer.
  • Further objects of the invention include the provision of methods of treatment of cancer, in particular prostate cancer, and methods of identifying compounds which may be useful in treating, diagnosing or imaging cancer.
  • scFv Single Chain Antibody Fragment Phage Display
  • tumourogenicity can be assessed by ability to form tumours, for example in mice. It can be expressed as the % of introduced cells generating tumours, for example in mice. Tumourogenicity can also be assessed by the behaviour of cells in soft agar, as will be well known to those skilled in the art. Increasing tumourogenicity is considered to reflect increasing metastatic potential.
  • scFv's Single-chain antibody variable region fragments
  • scFv antibodies directed against growth factors and their receptors (3, 4), chemokines and their receptors (5), oncogenes (6) and transcription factors (7) have been effective in inhibiting tumour growth.
  • chemokines and their receptors (5), oncogenes (6) and transcription factors (7) have been effective in inhibiting tumour growth.
  • several bacteriophage-derived antibodies have been generated with clinical cancer applications (8).
  • scFv antibodies offer great flexibility in the development of targeting agents. Compared to whole IgG molecules and other antibody fragments, they represent affinity reagents which, by virtue of their small size, can penetrate into tumour masses more rapidly and, clear more quickly from normal tissues. This is important, as effective targeted therapy relies on efficient antibody retention after clearance from normal tissue.
  • a first aspect of the invention provides an isolated recombinant antibody that binds to a normal human prostate epithelial cell and/or cultured human prostate tumour cell to a greater extent than to a human prostate fibroblast or muscle cell, the recombinant antibody comprising at least one, two, three, four or five, or more preferably six, CDR sequence selected from a) GFTFSSYA (CDRl of IGHV3-30 like portion of 98.1); ISYDGSNK (CDR2 of IGHV3-30 like portion of 98.1); ARVYFRL WGQGTLVTV (CDR3 of IGHV3-30 like portion of 98.1); SSNIGSNY (CDRl of IGLV1-47 like portion of 98.1); RNN (CDR2 of IGLV1-47 like portion of 98.1); AAWDDSLPVFGGGTKLTVLGAAA (CDR3 of IGLV1-47 like portion of 98.1); with no or up to 1, 2, 3 or less preferably 4, 5, 6, 7, 8, 9 or 10 conservative or non-
  • GDSVSSNSAA CDRl of IGHV6-1 like portion of 95.2
  • TYYRSKWYN CDR2 of IGHV6-1 like portion of 95.2
  • ARLVD ASFD YWGQGTLVTV CDR3 of IGHV6-1 like portion of 95.2
  • QGISSW CDRl of IGKVl- 37 like portion of 95.2
  • AAS CDR2 of IGKVl- 37 like portion of 95.2
  • QQANSFRTFGQGTKVEIKRAAA CDR3 of IGKVl- 37 like portion of 95.2
  • the antibody is typically a scFv antibody.
  • the antibody typically has human framework regions and human CDR sequences but may alternatively have non- human framework sequences, for example mouse or rat framework sequences.
  • the framework and CDR sequences are typically human or may be modified in order to make them more antigenically similar to human sequences as well known to those skilled in the art.
  • the CDR sequences may have been arrived at using "affinity maturation" techniques based on the CDR sequences given above, for example in order further to improve the affinity or selectivity of the antibody binding. Suitable techniques will be well known to those skilled in the art, for example including techniques described in Pavoni et al (2006) BMC Cancer 6, 41.
  • the antibody may comprise at least one, two, three, four or five, or more preferably six, framework sequence selected from RGAVESGGGVVQPGRSLRLSCAAT (FRl from IGHV3-30 like portion of 98.1); MHWVRQAPGKGLEWVAV (FR2 from IGHV3-30 like portion of 98.1); YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (FR3 from IGHV3-3O like portion of 98.1); QSVLTQPPS ASGTPGQRVTISCSGS (FRl from IGLV1-47 like portion of 98.1); VYWYQQLPGTAPKLLIY (FR2 from IGLV1-47 like portion of 98.1);
  • QRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC (FR3 from IGLV 1-47 like portion of 98.1), with no or up to 1, 2, 3, 4, 5, or less preferably 6, 7, 8, 9 or 10 conservative or non- conservative amino acid substitutions in each framework sequence.
  • the antibody may comprise at least one, two, three, four or five, or more preferably six, framework sequence selected from QVQLQQSGPGLVKPSQTLSLTCAIS (FRl from IGHV6-1 like portion of 95.2); WNWIRQSPSRGLEWLGR (FR2 from IGHV6-1 like portion of 95.2); DYAVS VKSRITINPDTSKNQFSLQLNSVTPEDTAVYYC (FR3 from IGHV6-l like portion of 95.2); DIQLTQSPSSVSASVGDRVTITCRAS (FRI from IGKVl- 37 like portion of 95.2); LAWYQQKPGKAPKLLIY (FR2 from IGKVl- 37 like portion of 95.2); SLQSGVPSRPSGSGSGTDFTLTISSLQPEDF ATYYC (FR3 from IGKVl- 37 like portion of 95.2), with no or up to 1, 2, 3, 4, 5, or less preferably 6, 7, 8, 9 or 10 conservative or non- conservative
  • the antibody may comprise as the linker between the heavy-chain and light-chain like portions the sequence SSGGGGSGGGGSGGSAL with no or up to 1, 2, 3, or less preferably 4, 5, 6, 7, 8, 9 or 10 conservative or non-conservative amino acid substitutions.
  • the antibody may also comprise one or more tags to aid purification or identification, as well known to those skilled in the art, for example a His tag, for example HHHHHHGAA; or a Myc tag, for example EQKLISEEDLN.
  • the antibody may also comprise a leader sequence to aid secretion of the antibody, for example the sequence MKYLLPTAAAGLLLLAAQPAMA.
  • the antibody may have or comprise the sequence
  • antibody we include not only whole immunoglobulin molecules but also fragments thereof such as Fab, F(ab')2, Fv and other fragments thereof that retain the antigen-binding site.
  • antibody includes genetically engineered derivatives of antibodies such as single chain Fv molecules (scFv) and single domain antibodies (dAbs) .
  • scFv single chain Fv molecules
  • dAbs single domain antibodies
  • the term also includes antibody-like molecules which may be produced using phage-display techniques or other random selection techniques for molecules.
  • the term also includes all classes of antibodies, including: IgG, IgA, IgM, IgD and IgE.
  • variable heavy (V H ) and variable light (V L ) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies. Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
  • variable domains Antigenic specificity is conferred by variable domains and is independent of the constant domains, known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains.
  • variable domains include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the V H and V L partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad.
  • ScFv molecules we include molecules wherein the V H and V L partner domains are linked via a flexible linker oligopeptide.
  • the linker may have the sequence SSGGGGSGGGGSGGSAL.
  • antibody fragments rather than whole antibodies
  • the smaller size of the fragments may lead to improved pharmacological properties, such as better penetration to the target site. Effector functions of whole antibodies, such as complement binding, are removed.
  • Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining site.
  • the antibody may be a polyclonal antibody, it is preferred if it is a monoclonal antibody. In some circumstances, particularly if the antibody is going to be administered repeatedly to a human patient, it is preferred if the monoclonal antibody is a human monoclonal antibody or a humanised monoclonal antibody.
  • Non-human antibodies can be "humanised” in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.
  • the antibodies may be human antibodies in the sense that they have the amino acid sequence of human antibodies with specificity as defined herein but they may be prepared using methods known in the art that do not require immunisation of humans, for example as described herein.
  • a second aspect of the invention provides an isolated recombinant antibody that binds to a normal human prostate epithelial cell and/or cultured human prostate tumour cell to a greater extent than to a prostate fibroblast or muscle cell, and competes with the binding of an antibody of the first aspect of the invention, preferably an antibody having the sequence set out above, to a normal human prostate epithelial cell and/or cultured human prostate tumour cell.
  • Suitable antibodies can be identified by means of a screen.
  • a suitable method or screen for identifying antibodies, peptides or other molecules which selectively bind a target cell, protein or polypeptide may comprise contacting the target cell, protein or polypeptide with a test antibody, peptide or other molecule under conditions where binding can occur, and then determining if the test antibody, molecule or peptide has bound the target cell, protein or peptide. The ability of the antibody of the first aspect of the invention to block such binding can be assessed. Methods of detecting binding between two moieties are well known in the art of biochemistry. Preferably, the known technique of phage display is used to identify antibodies, peptides or other ligand molecules suitable for use as binding moieties. Examples of how suitable screens can be performed are provided in the Examples.
  • a further aspect of the invention provides a recombinant polynucleotide encoding an antibody of the invention.
  • a still further aspect of the invention provides a polynucleotide encoding at least one, two, three, four or five, or six CDR sequences selected from a) GFTFSSYA (CDRl of IGHV3-30 like portion of 98.1); ISYDGSNK (CDR2 of IGHV3-30 like portion of 98.1); ARVYFRLWGQGTLVTV (CDR3 of IGHV3-30 like portion of 98.1); SSNIGSNY (CDRl of IGLV1-47 like portion of 98.1); RNN (CDR2 of IGLVl- 47 like portion of 98.1); AAWDDSLPVFGGGTKLTVLGAAA (CDR3 of IGLVl -47 like portion of 98.1); with no or up to 1, 2, 3 or less preferably 4, 5, 6, 7, 8, 9 or 10 conservative or non-conservative amino acid substitutions in each CDR sequence; or
  • GDSVSSNSAA CDRl of IGHV6-1 like portion of 95.2
  • TYYRSKWYN CDR2 of IGHV6-1 like portion of 95.2
  • ARLVD ASFD YWGQGTLVTV CDR3 of IGHV6-1 like portion of 95.2
  • QGISSW CDRl of IGKVl- 37 like portion of 95.2
  • AAS CDR2 of IGKVl- 37 like portion of 95.2
  • QQANSFRTFGQGTKVEIKRAAA CDR3 of IGKVl- 37 like portion of 95.2
  • Such sequences encoding individual CDR sequences are considered to be useful in assembling a polynucleotide that encodes an antibody that has the desired binding characteristics (or a fragment thereof).
  • a further aspect of the invention provides an expression vector comprising a recombinant polynucleotide of the invention.
  • the genetic constructs of the invention can be prepared and manipulated using methods well known in the art.
  • the present invention also relates to a host cell transformed with a genetic (preferably DNA construct) construct of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RRl available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343).
  • Preferred eukaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line.
  • Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Preferred mammalian host cells includeBHKl, Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-I cells available from the ATCC as CRL 1650.
  • a further aspect of the invention provides an agent comprising an antibody of the invention.
  • the antibody may comprise or be bound covalently or non-covalently to a therapeutic or imaging or detection agent or may be provided in a kit alongside a therapeutic or imaging or detection agent.
  • the recombinant antibody of the invention allows, for example, the therapeutic or imaging agent to be targeted to prostate cancer cells, or the detection agent to bind to prostate cancer cells in a patient sample.
  • the Figures show that the difference in intensity of binding is sufficient to allow cancer cells to be distinguished from non-cancer cells.
  • the therapeutic or imaging or detection agent may be bound to the antibody (which may be tagged, as discussed further below) via one or more selective non- covalent interactions, for example selective antibody-antigen interaction or biotin- avidin/streptavidin interaction.
  • the recombinant antibody may comprise an antigen, biotin or avidin/strepatvidin tag, for example a His-6 tag or a Myc tag, as well known to those skilled in the art and as described in the Examples.
  • the imaging agent may be attached to a further entity, for example a further antibody. Such arrangements will be well known to those skilled in the art, are discussed further below and examples are also shown in the present Examples.
  • the antibody comprises or is bound covalently to a therapeutic or imaging or detection agent.
  • the antibody has human CDR and framework regions, as discussed above.
  • Agents indicated to be suitable as imaging agents may also be useful as detection agents. Additional detection agents will be well known to those skilled in the art and include Alexa 488, a fluorescent label for in vitro work, available from Invitrogen Ltd, 3 Fountain Drive, Inchinnan Business Park, Paisley. UK PA4 9RF. Other suitable fluorescent labels will be well known to those skilled in the art and include others available from Invitrogen (for example under the trademark Molecular ProbesTM). Isotope labels may be particularly suitable for in vivo use, as discussed further below.
  • the therapeutic agent may be or comprise a cytotoxic moiety; more preferably, the cytotoxic moiety is directly and/or indirectly cytotoxic.
  • directly cytotoxic we include the meaning that the moiety is one which on its own is cytotoxic.
  • directly cytotoxic we include the meaning that the moiety is one which, although is not itself cytotoxic, can induce cytotoxicity, for example by its action on a further molecule or by further action on it.
  • the cytotoxic moiety is a directly cytotoxic chemo therapeutic agent.
  • the cytotoxic moiety is a directly cytotoxic polypeptide.
  • Cytotoxic chemotherapeutic agents are well known in the art. Cytotoxic chemotherapeutic agents, such as anticancer agents, include: alkylating agents including nitrogen mustards such as mechlorethamine (HN 2 ), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimid
  • Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (rnithramycin) and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes.
  • VLB vinblastine
  • epipodophyllotoxins such as etoposide and teniposide
  • antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (rnithramycin) and mitomycin (mitomycin C)
  • enzymes such as L-a
  • Miscellaneous agents including platinum coordination complexes such as cisplatin (cw-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (o,/?'-DDD) and amino glutethimide; taxol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen.
  • agents of the invention comprising these agents may readily be made by the person skilled in the art.
  • carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159; incorporated herein by reference) may be used to conjugate a variety of agents, including doxorubicin, to antibodies or peptides.
  • Carbodiimide compounds attack carboxylic groups to change them into reactive sites for free amino groups.
  • the water soluble carbodiimide, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide is particularly useful for conjugating a functional moiety to a binding moiety and may be used to conjugate doxorubicin to tumour homing peptides.
  • the conjugation of doxorubicin and a binding moiety requires the presence of an amino group, which is provided by doxorubicin, and a carboxyl group, which is provided by the binding moiety such as an antibody or peptide.
  • EDC also can be used to prepare active esters such as N-hydroxysuccinimide (NHS) ester.
  • NHS N-hydroxysuccinimide
  • the NHS ester which binds only to amino groups, then can be used to induce the formation of an amide bond with the single amino group of the doxorubicin.
  • EDC and NHS in combination is commonly used for conjugation in order to increase yield of conjugate formation (Bauminger & Wilchek, supra, 1980).
  • cytotoxic moiety is a cytotoxic peptide or polypeptide moiety by which we include any moiety which leads to cell death.
  • Cytotoxic peptide and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor and the like. Methods for linking them to targeting moieties such as antibodies are also known in the art.
  • ricin as a cytotoxic agent is described in Burrows & Thorpe (1993) Proc. Natl. Acad. Sci. USA 90, 8996-9000, incorporated herein by reference, and the use of tissue factor, which leads to localised blood clotting and infarction of a tumour, has been described by Ran et al (1998) Cancer Res. 58, 4646-4653 and Huang et al (1997) Science 275, 547-550.
  • Tsai et al (1995) Dis. Colon Rectum 38, 1067-1074 describes the abrin A chain conjugated to a monoclonal antibody and is incorporated herein by reference.
  • Other ribosome inactivating proteins are described as cytotoxic agents in WO 96/06641.
  • Pseudomonas exotoxin may also be used as the cytotoxic polypeptide moiety (see, for example, Aiello et al (1995) Proc. Natl. Acad. Sci. USA 92, 10457-10461; incorporated herein by reference).
  • cytokines such as TNF ⁇ and IL-2, may also be useful as cytotoxic agents.
  • radioactive atoms may also be cytotoxic if delivered in sufficient doses.
  • the cytotoxic moiety may comprise a radioactive atom which, in use, delivers a sufficient quantity of radioactivity to the target site so as to be cytotoxic.
  • Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-I l l, rhenium- 186, rhenium- 188 or yttrium-90, or any other isotope which emits enough energy to destroy neighbouring cells, organelles or nucleic acid.
  • the isotopes and density of radioactive atoms in the agents of the invention are such that a dose of more than 4000 cGy (preferably at least 6000, 8000 or 10000 cGy) is delivered to the target site and, preferably, to the cells at the target site and their organelles, particularly the nucleus.
  • the radioactive atom may be attached to the binding moiety in known ways.
  • a chelating agent may be attached to the binding moiety and used to attach 111 In or 90 Y.
  • Tyrosine residues may be directly labelled with 125 I or 131 I.
  • the cytotoxic moiety may be a suitable indirectly-cytotoxic polypeptide.
  • the indirectly cytotoxic polypeptide is a polypeptide which has enzymatic activity and can convert a non-toxic and/or relatively non-toxic prodrug into a cytotoxic drug.
  • ADEPT Antibody-Directed Enzyme Prodrug Therapy
  • the system requires that the binding moiety locates the enzymatic portion to the desired site in the body of the patient and after allowing time for the enzyme to localise at the site, administering a prodrug which is a substrate for the enzyme, the end product of the catalysis being a cytotoxic compound.
  • the object of the approach is to maximise the concentration of drug at the desired site and to minimise the concentration of drug in normal tissues (see Senter, P.D. et al (1988) "Anti-tumour effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate" Proc. Natl. Acad. Sci. USA 85, 4842-4846; Bagshawe (1987) Br. J. Cancer 56, 531-2; and Bagshawe, K.D. et al (1988) "A cytotoxic agent can be generated selectively at cancer sites” Br. J. Cancer. 58, 700-703.)
  • the invention provides an agent wherein the cytotoxic moiety is capable of converting a non-cytotoxic prodrug into a cytotoxic drug.
  • the enzyme and prodrug of the system using a targeted enzyme as described herein may be any of those previously proposed.
  • the cytotoxic substance may be any existing anti-cancer drug such as an alkylating agent; an agent which intercalates in DNA; an agent which inhibits any key enzymes such as dihydro folate reductase, thymidine synthetase, ribonucleotide reductase, nucleoside kinases or topoisomerase; or an agent which effects cell death by interacting with any other cellular constituent.
  • Etoposide is an example of a topoisomerase inhibitor.
  • Reported prodrug systems include: a phenol mustard prodrug activated by an E.
  • coli ⁇ -glucuronidase Wang et al, 1992 and Ro filer et al, 1991
  • doxorubicin prodrug activated by a human ⁇ -glucuronidase Bosslet et al, 1994
  • further doxorubicin prodrugs activated by coffee bean ⁇ -galactosidase Azoulay et al, 1995
  • daunorubicin prodrugs, activated by coffee bean ⁇ -D-galactosidase (Gesson et al, 1994); a 5-fluorouridine prodrug activated by an E.
  • methotrexate prodrugs ⁇ e.g. methotrexate-alanine activated by carboxypeptidase A (Kuefher et al, 1990, Vitols et al, 1992 and Vitols et al, 1995). These and others are included in Table A, below.
  • Table A is adapted from Bagshawe (1995) Drug Dev. Res. 34, 220-230, from which full references for these various systems may be obtained; the taxol derivative is described in Rodrigues, M.L. et al (1995) Chemistry & Biology 2, 223).
  • Suitable enzymes for forming part of the enzymatic portion a agent of the invention include: exopeptidases, such as carboxypeptidases G, Gl and G2 (for glutamylated mustard prodrugs), carboxypeptidases A and B (for MTX-based prodrugs) and aminopeptidases (for 2- ⁇ -aminocyl MTC prodrugs); endopeptidases, such as e.g. thrombolysin (for thrombin prodrugs); hydrolases, such as phosphatases (e.g. alkaline phosphatase) or sulphatases (e.g.
  • aryl sulphatases (for phosphylated or sulphated prodrugs); amidases, such as penicillin amidases and arylacyl amidase; lactamases, such as ⁇ -lactamases; glycosidases, such as ⁇ -glucuronidase (for ⁇ -glucuronomide anthracyclines), ⁇ -galactosidase (for amygdalin) and ⁇ -galactosidase (for ⁇ -galactose anthracycline); deaminases, such as cytosine deaminase (for 5FC); kinases, such as urokinase and thymidine kinase (for gancyclovir); reductases, such as nitroreductase (for CB 1954 and analogues), azoreductase (for azobenzene mustards) and DT-diaphorase (for CB 1954
  • the prodrug is relatively non-toxic compared to the cytotoxic drug. Typically, it has less than 10% of the toxicity, preferably less than 1% of the toxicity as measured in a suitable in vitro cytotoxicity test.
  • each moiety of the agent of the invention is a polypeptide
  • the two portions may be linked together by any of the conventional ways of cross-linking polypeptides, such as those generally described in O'Sullivan et al (1979) Anal.
  • the binding moiety may be enriched with thiol groups and the further moiety reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP).
  • a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP).
  • the agent may be produced as a fusion compound by recombinant DNA techniques whereby a length of DNA comprises respective regions encoding the two moieties of the agent of the invention either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the agent.
  • the two portions of the agent may overlap wholly or partly.
  • the cytotoxic moiety may be a radiosensitizer.
  • Radiosensitizers include fluoropyrimidines, thymidine analogues, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3- aminobenzodiamide, etanixadole, pimonidazole and misonidazole (see, for example, McGinn et al (1996) J Natl. Cancer Inst. 88, 1193-11203; Shewach & Lawrence (1996) Invest. New Drugs 14, 257-263; Horsman (1995) Acta Oncol. 34, 571-587; Shenoy & Singh (1992) CHn.
  • genes into cells can radiosensitise them, for example delivery of the p53 gene or cyclin D (Lang et al (1998) J Neurosurg. 89, 125-132; Coco Martin et al (1999) Cancer Res. 59, 1134-1140).
  • the further moiety may be one which becomes cytotoxic, or releases a cytotoxic moiety, upon irradiation.
  • the boron- 10 isotope when appropriately irradiated, releases ⁇ particles which are cytotoxic (for example, see US 4, 348, 376 to Goldenberg; Primus et al (1996) Bioconjug. Chem. 7, 532-535).
  • the cytotoxic moiety may be one which is useful in photodynamic therapy such as photo frin (see, for example, Dougherty et al (1998) J Natl. Cancer Inst. 90, 889-905).
  • the further moiety may comprise a nucleic acid molecule which is directly or indirectly cytotoxic.
  • the nucleic acid molecule may be an antisense oligonucleotide which, upon localisation at the target site is able to enter cells and lead to their death.
  • the oligonucleotide therefore, may be one which prevents expression of an essential gene, or one which leads to a change in gene expression which causes apoptosis.
  • the cytotoxic moiety is a nucleic acid molecule encoding a directly and/or indirectly cytotoxic polypeptide.
  • Suitable oligonucleotides include those directed at bcl-2 (Ziegler et al (1997) J Natl. Cancer Inst. 89, 1027-1036), and DNA polymerase ⁇ and topoisomerase Il ⁇ (Lee et al ( 1996) A nticancer Res. 16, 1805-1811.
  • Peptide nucleic acids may be useful in place of conventional nucleic acids (see Knudsen & Nielsen (1997) Anticancer Drugs 8, 113-118).
  • the binding moiety may be comprised in a delivery vehicle for delivering nucleic acid to the target.
  • the delivery vehicle may be any suitable delivery vehicle. It may, for example, be a liposome containing nucleic acid, or it may be a virus or virus-like particle which is able to deliver nucleic acid.
  • the binding moiety is typically present on the surface of the delivery vehicle.
  • the binding moiety such as a suitable antibody fragment, may be present in the outer surface of a liposome and the nucleic acid to be delivered may be present in the interior of the liposome.
  • a viral vector such as a retroviral or adenoviral vector
  • a viral vector is engineered so that the binding moiety is attached to or located in the surface of the viral particle thus enabling the viral particle to be targeted to the desired site.
  • Targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the DNA is carried within the adenovirus, or adenovirus-like, particle.
  • Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell-selective moiety into a fibre protein.
  • Targeted retroviruses are also available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into pre- existing viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this and other targeted vectors for gene therapy).
  • Immunoliposomes may be used in which the binding moiety is an antibody.
  • MPB- PE N-[4-(p-maleimidophenyl)-butyryl]-phosphatidylethanolamine
  • MPB-PE is incorporated into the liposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the liposomal surface.
  • the liposome is conveniently loaded with the DNA or other genetic construct for delivery to the target cells, for example, by forming the said liposomes in a solution of the DNA or other genetic construct, followed by sequential extrusion through polycarbonate membrane filters with 0.6 ⁇ m and 0.2 ⁇ m pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA construct is separated from free DNA construct by ultracentrifugation at 80 000 x g for 45 min. Freshly prepared MPB-PE-liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4°C under constant end over end rotation overnight. The immunoliposomes are separated from unconjugated antibodies by ultracentrifugation at 80 000 x g for 45 min. Immunoliposomes may be injected intraperitoneally or directly into the tumour.
  • the nucleic acid delivered to the target site may be any suitable DNA which leads, directly or indirectly, to cytotoxicity.
  • the nucleic acid may encode a ribozyme which is cytotoxic to the cell, or it may encode an enzyme which is able to convert a substantially non-toxic prodrug into a cytotoxic drug (this latter system is sometime called GDEPT: Gene Directed Enzyme Prodrug Therapy).
  • Ribozymes which may be encoded in the nucleic acid to be delivered to the target are described in Cech and Herschlag "Site-specific cleavage of single stranded
  • Suitable targets for ribozymes include transcription factors such as c-fos and c-myc, and bcl-2.
  • Durai et al (1997) Anticancer Res. 17, 3307-3312 describes a hammerhead ribozyme against bcl-2.
  • EP 0 415 731 describes the GDEPT system. Similar considerations concerning the choice of enzyme and prodrug apply to the GDEPT system as to the ADEPT system described above.
  • the nucleic acid delivered to the target site may encode a directly cytotoxic polypeptide.
  • the further moiety may comprise a polypeptide or a polynucleotide encoding a polypeptide which is not either directly or indirectly cytotoxic but is of therapeutic benefit.
  • polypeptides include anti-proliferative or anti-inflammatory cytokines, and anti-proliferative, immunomodulatory or factors influencing blood clotting which may be of benefit in medicine, for example in the treatment of cancer.
  • the further moiety may usefully be an inhibitor of angiogenesis such as the peptides angiostatin or endostatin.
  • the further moiety may also usefully be an enzyme which converts a precursor polypeptide to angiostatin or endostatin.
  • Human matrix metallo -proteases such as macrophage elastase, gelatinase and stromolysin convert plasminogen to angiostatin (Cornelius et al (1998) J Immunol. 161, 6845-6852). Plasminogen is a precursor of angiostatin.
  • the invention provides an agent further comprising a readily detectable moiety. Such an agent may be useful as an imaging or diagnostic agent.
  • a "readily detectable moiety” we include the meaning that the moiety is one which, when located at the target site following administration of the agent of the invention into a patient, may be detected, typically non-invasively from outside the body and the site of the target located.
  • the agents of this embodiment of the invention are useful in imaging and diagnosis.
  • the readily detectable moiety is or comprises a radioactive atom which is useful in imaging.
  • Suitable radioactive atoms include 99m Tc and 123 I for scintigraphic studies.
  • Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRJ) such as 125 I, 123 I again, 131 I, 111 In, 19 F, 13 C, 15 N, 17 O, gadolinium, manganese or iron.
  • MRJ magnetic resonance imaging
  • the agent of the invention must have sufficient of the appropriate atomic isotopes in order for the molecule to be readily detectable.
  • the radio- or other labels may be incorporated in the agent of the invention in known ways.
  • the binding moiety is a polypeptide it may be biosynthesised or may be synthesised by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine- 19 in place of hydrogen.
  • Labels such as 99m Tc, 123 I, 186 Rh, 188 Rh and 1 11 In can, for example, be attached via cysteine residues in the binding moiety.
  • Yttrium-90 can be attached via a lysine residue.
  • the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Comm. 80, 49-57) can be used to incorporate 123 I. Reference (“Monoclonal Antibodies in Immunoscintigraphy", J-F Chatal, CRC Press, 1989) describes other methods in detail.
  • the readily detectable moiety comprises a radioactive atom, such as, for example technetium-99m or iodine- 123, Iodine- 125 or Iodine-131.
  • the readily detectable moiety may be selected from the group comprising: iodine- 125, iodine- 123; iodine-131 ; indium-I l l ; fluorine- 19; carbon- 13; nitrogen- 15; oxygen- 17; gadolinium; manganese; iron.
  • the invention provides an agent further comprising a moiety capable of selectively binding to a directly or indirectly cytotoxic moiety, or to a readily detectable moiety.
  • the further moiety is able to bind selectively to a directly or indirectly cytotoxic moiety or to a readily detectable moiety.
  • the further moiety may be any moiety which binds to a further compound or component which is cytotoxic or readily detectable.
  • the further moiety may, therefore be an antibody which selectively binds to the further compound or component, or it may be some other binding moiety such as streptavidin or biotin or the like.
  • the following examples illustrate the types of molecules that are included in the invention; other such molecules are readily apparent from the teachings herein.
  • a bispecif ⁇ c antibody wherein one binding site comprises the binding moiety (which selectively binds to a normal human prostate epithelial cell and/or cultured human prostate tumour cell to a greater extent than to a prostate fibroblast or muscle cell, as defined herein) and the second binding site comprises a moiety which binds to, for example, an enzyme which is able to convert a substantially non-toxic prodrug to a cytotoxic drug.
  • the agent may comprise an antibody of the invention, to which is bound biotin.
  • Avidin or streptavidin which has been labelled with a readily detectable label may be used in conjunction with the biotin labelled antibody in a two-phase imaging system wherein the biotin labelled antibody is first localised to the target site in the patient, and then the labelled avidin or streptavidin is administered to the patient.
  • Bispecific antibodies and biotin/streptavidin (avidin) systems are reviewed by Rosebrough (1996) QJNucl. Med. 40, 234-251.
  • the binding moiety and the further moiety are polypeptides which are fused.
  • a further aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody, agent or kit of parts of the invention and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is included that the formulation is sterile and pyrogen free.
  • Suitable pharmaceutical carriers are well known in the art of pharmacy.
  • the carrier(s) must be “acceptable” in the sense of being compatible with the agent of the invention and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free; however, other acceptable carriers may be used.
  • Methods of manufacturing a pharmaceutical composition or medicament using an active agent, such as the agent of the invention, for example an antibody are well known to persons skilled in the art of medicine and pharmacy.
  • a further aspect of the invention provides an antibody, agent, kit of parts or pharmaceutical composition of the invention for use in medicine.
  • a further aspect of the invention provides an antibody, agent, kit of parts or pharmaceutical composition of the invention for therapy, diagnosis or imaging of cancer, for example prostate cancer.
  • the recombinant antibody may have a therapeutic effect even if not associated with a further therapeutic agent.
  • the antibody can induce cell cycle arrest (block at Gl) - see Examples and Figure 2.
  • a further aspect of the invention provides a method of treating or imaging of cancer, for example prostate cancer, the method comprising the step of administering an effective amount of an appropriate antibody, kit of parts or pharmaceutical composition of the invention to a patient.
  • an effective amount we include an amount of the agent of the invention that is, as appropriate, sufficient to image or prevent and/or reduce a condition associated with the particular cancer to be treated, for example prostate cancer.
  • an effective amount may prevent and/or reduce the size and/or rate of growth and/or spread of a tumour.
  • An effective amount may prevent and/or reduce the cellular differentiation of a cancer cell, thereby preventing and/or reducing metastasis.
  • Appropriate tests for determining the therapeutic effect and most appropriate route of administration of an agent, composition or medicament of the invention will be known to those skilled in the relevant arts of medicine
  • a further aspect of the invention provides a method of diagnosing (or aiding in diagnosing) cancer, for example prostate cancer in a patient, the method comprising the step of detecting the level or presence or absence of binding of an antibody, agent or kit of parts of the invention to a sample from the patient.
  • the agent of the invention could be used to identify cells, such as cancer cells, potentially susceptible to treatment using an antibody, agent or kit of parts as defined herein and/or a medicament as defined herein and/or a pharmaceutical composition as defined herein.
  • the agent of the invention could be used (in accordance with the methods of the invention and those described in the accompanying Examples) as a diagnostic reagent to detect cancer cells, for example prostate cancer cells, in a test sample from a patient.
  • the identification of cells binding to an agent of the invention may indicate that the patient would be suitable for delivering a cytotoxic moiety using the agent of the invention and therefore susceptible to treatment using the agent and/or medicament and/or pharmaceutical composition of the invention. It will be appreciated that determining whether the cell displays a binding response associated with cancer or if imaging indicates the presence of cancer, may in itself be diagnostic of cancer or it may be used by the clinician as an aid in reaching a diagnosis.
  • the method may be useful in determining the susceptibility of a human patient to cancer, diagnosing cancer in a human patient or predicting the relative prospects of a particular outcome of a cancer in a human patient.
  • the method the invention may be useful in prognosis or aiding prognosis.
  • the method may be used as an adjunct to known prognostic methods such as histopathological examination of biopsy tissue, or measurement of plasma PSA levels (in relation to prostate cancer), external digital examination or other imaging techniques.
  • the imaging or diagnostic techniques of the invention will be useful to the clinician in determining how to manage the cancer in the patient.
  • the clinician may use the information concerning binding to the antibody or agent (either using imaging techniques or ex vivo binding tests), to facilitate decision making regarding treatment of the patient.
  • the response is indicative of a low metastatic potential of said prostate cancer, unnecessary radical surgery may be avoided.
  • radical surgery ie prostatectomy
  • Imaging may also indicate where surgery may be required or what drug treatment is appropriate.
  • any sample containing cells derived from the patient may be useful in the methods of the invention, it is preferred if the cells are derived from a sample of the tissue in which cancer is suspected or in which cancer may be or has been found.
  • the tissue in which cancer is suspected or in which cancer may be or has been found is prostate, it is preferred if the sample containing cells is derived from the prostate of the patient.
  • Samples of prostate may be obtained by surgical excision, laproscopy and biopsy, endoscopy and biopsy, and image-guided biopsy. The image may be generated by ultrasound or technetium-99-labelled antibodies or antibody fragments which bind or locate selectively at the prostate, such as antibodies of the present invention.
  • the sample may be directly derived from the patient, for example, by biopsy of the tissue, or it may be derived from the patient from a site remote from the tissue, for example because cells from the tissue have migrated from the tissue to other parts of the body.
  • the sample may be indirectly derived from the patient in the sense that, for example, the tissue or cells therefrom may be cultivated in vitro, or cultivated in a xenograft model.
  • the cells derived from the patient may have been physically within the patient, they may alternatively have been replicated from cells which were physically within the patient.
  • the tumour tissue may be taken from the primary tumour or from metastases.
  • the sample may be lymph nodes, lymph or blood and the spread of disease detected.
  • any sample containing cells derived from the patient is useful in the methods of the invention, it is preferred (in relation to prostate cancer) if the sample is selected from the group consisting of prostate tissue, blood, urine or semen.
  • Prostate tissue can be obtained from a patient using standard surgical techniques. Cells derived from the prostate are found in small numbers in the urine and in the blood.
  • the sample containing cells from the patient is, or is derived directly from, a cell of the patient, such as a prostate cell
  • a sample indirectly derived from a patient, such as a cell grown in culture is also included within the invention.
  • the tumour tissue may be taken from the primary tumour or from metastases, and particularly may be taken from the margins of the tumour.
  • the aforementioned methods may be used for presymptomatic screening of a patient who is in a risk group for cancer. For example, men older than about 60 years are at greater risk of prostate cancer than men below the age of 35. Similarly, the methods may be used for the pathological classification of tumours such as prostate tumours.
  • the sample is enriched for prostate-derived tissue or cells.
  • Enrichment for prostate cells may be achieved using, for example, cell sorting methods such as fluorescent activated cell sorting (FACS) using a prostate- selective antibody such as one directed to prostate-specific antigen (PSA) or prostate specific membrane antigen (PSMA).
  • FACS fluorescent activated cell sorting
  • PSA prostate-specific antigen
  • PSMA prostate specific membrane antigen
  • enrichment may be achieved using magnetic beads or other solid support, for example a column, coated with such a prostate-specific antibody, for example an anti-PSA antibody or an antibody of the present invention.
  • the source of the said sample also includes biopsy material and tumour samples, including fresh or frozen tissue.
  • Tissue preferably fresh tissue
  • sample containing cells derived from the patient is not a substantially pure sample of the tissue or cell type in question that the sample is enriched for the said tissue or cells.
  • Cells may be analysed individually, for example following single-cell immobilisation techniques. Methods by which single cells may be analysed include methods in which the technique of Laser Capture Microdissection (LCM) is used.
  • LCD Laser Capture Microdissection
  • This technique may be used to collect single cells or homogeneous cell populations for analysis and is described in, for example, Jin et al (1999) Lab Invest 79(4), 511-512; Simone et al (1998) Trends Genet 14(7), 272-276; Luo et al (1999) Nature Med 5(1), 117-122; Arcuturs Updates, for example June 1999 and February 1999; US 5,859,699 (all incorporated herein by reference).
  • the cells of interest are visualised, for example by immunohistochemical techniques, and transferred to a polymer film that is activated by laser pulses.
  • the technique may also be used for isolation of cells which are negative for a particular component.
  • Microscopes useful in performing LCM are manufactured by Arcturus Engineering, Inc., 1220 Terra Bella Avenue, Mountain View, CA 94042, USA.
  • LCM may be used with other isolation or enrichment methods.
  • LCM may be used following a method which enriches the sample for the target cell type.
  • FIG. 1 Reactivity of scFv clones 95.2 and 98.1 with human prostate cells as assessed by flow cytometry. FACS histograms demonstrating the binding of soluble scFv to prostate cancer cell lines (1), normal prostate cells (2) and primary tumour derived cell lines (3). Antibody binding was carried out at 4 °C using unfixed cells. Bound scFv 95.2 ( *) was detected through its His tag by using anti-6X His antibody followed by Alexa 488 ( +).
  • FIG. 1 Reactivity of scFv clones 95.2 and 98.1 with human prostate cells and tissue as assessed by Immunocytochemistry and immunohistochemistry. 1. Reactivity of scFv 95.2 and 98.1 with formaldehyde fixed but not permeablised prostate epithelial cells. Particulate deposits (arrow). 2. Reactivity of scFv antibodies with normal and pathogenic human prostate tissue sections, embedded in paraffin. Bound scFv antibody was detected through its His tag by using anti- 6X His antibody followed by Alexa 488 or HRP labeled antibody. 3. Biological effect of scFv 95.2 and 98.1 on the cell cycle distribution of LNCaP cells. Figure 3. Amino acid sequence of clone 95,2 Figure 4. Amino acid sequence of clone 98.1
  • scFv phage display we have successfully generated a panel of 15 human framework recombinant scFv antibodies specific to the cell surface of normal human prostate epithelial cells. Characterisation of this panel has demonstrated that two of these antibodies, scFv 95.2 and scFv 98.1, additionally exhibit a strong reactivity with prostate tumour cells that increases with the grade of tumour.
  • scFv 95.2 and scFv 98.1 also exhibit reactivity with the 957/hTERT (not tumourogenic in mice), RC-92a/hTERT (partially tumourogenic in mice) and RC-5T/hTERT (fully tumourogenic in mice) cell lines, each of which have been derived by telomerase immortalisation of cells from primary human prostate tumours (9).
  • scFv 95.2 and scFv 98.1 ' s reactivity with this panel of cells increases with increasing tumourogenicity.
  • the target epitope for scFv 98.1 appears to be concentrated at the polar tips of the cells ( Figure 2) while in prostate tumour cells it appears to be uniformly expressed on the entire surface of the cells ( Figure 2). Additionally, the absence of detectable particulate aggregates indicates that the target molecule for scFv 98.1 may be cell surface bound but not secreted. However, the possibility exists that the target molecule for scFv 98.1 is secreted but not laid down in aggregates as seen with that for scFv 95.2. To date, several prostate-specific proteins that are cell surface bound and/or secreted have been reported.
  • Table 1 Comparison of expression of the target epitope of scFv clones with a spectrum of prostate specific protein in established human prostate tumour cell lines.
  • scFv 92.5 and scFv 98.1 were used a human prostate cancer tissue microarray composed of 10 normal and 20 prostate cancer tissue samples (LF-PR-I, Cytomyx Ltd).
  • scFv 95.2 exhibited reactivity with 7 out of 10 normal and 17 out of 20 tumour samples present on this array.
  • scFv 98.1 exhibited reactivity with prostate epithelia in all the normal and tumour samples present on the array.
  • scFv 95.2 and scFv98.1 Towards characterisation of biological affects of scFv 95.2 and scFv98.1, we have found that the target epitope for scFv 95.2 is not trypsin sensitive while that of scFv98.1 does exhibit sensitivity to trypsin. Additionally, binding of scFv 95.2 and scFv98.1 to their target molecules does not elicit antibody induced cellular internalisation. This is a desirable feature in antibodies used for in vivo targeting. Further, addition of scFv 95.2 to growing cultures of LNCaP cells induces a significant Gl arrest of cell growth ( Figure 2). Similarly, addition of scFv 98.1 to growing cultures of LNCaP also elicits a significant Gl arrest of cell growth in these cells ( Figure 2).
  • ErbB2 the antigen targeted by the approved humanised mAb Herceptin is over-expressed in 20-30% of human breast and ovarian cancers. However, ErbB2 is also expressed at low levels in epithelial cells from a variety of organs (19). Similarly, expression of PMSA the antigen targeted by 7E11-C5.3, a murine monoclonal antibody approved for imaging prostate cancer, increases with tumour progression (20). However, PSMA is also expressed on a variety of normal epithelial cells in the body. Additionally, A33, a human antigen that is a promising molecular target for antibody therapy of colon cancer, is expressed in both normal and malignant colon epithelia (21).
  • a tumour crossreactive antibody that could be used for the treatment of a range of cancers of similar tissue type would be a unique and highly valuable tool.
  • the tissue miccroarrays intended for use in this proposal are purchased from Stretton Scientific, Cytomyx Ltd and TriStar Technology Group, LLC.
  • the clinical application of scFv 95.2 and scFv 98.1 in prostate cancer We are particularly keen to develop targeted antibodies for imaging and therapeutic purposes in prostate cancer. In this context, we have already established that scFv 95.2 and scFv 98.1 antibodies can both induce cell cycle arrest in prostate tumour cells in vitro and hence elicit an anti-growth effect.
  • the biophysical and pharmacokinetic properties of scFv 95.2 and scFv 98.1 are determined. Specifically, an optimised radio labelling procedure is established in order to ensure that the radio labelling procedure does not compromise optimal functionality of the antibodies. To do this, both antibodies are radio labelled with Iodine 125 using the IODO-GEN method (22) and their in vitro binding kinetics determined using saturation binding (Scatchard) assays. Finally, the biophysical and pharmacokinetic properties of scFv 95.2 and scFv 98.1 are determined with a view to the development of these antibodies for in vivo targeting i.e.; imaging or targeted therapy.
  • scFv radio labelled with ⁇ -emitting technetium-99m, 99m TC the preferred radionuclide for clinical imaging (23)
  • mice 8-10 wk old mice are inoculated, in the right/left flank, with 0.1 ml cell suspension containing 5X106 LNCaP cells (human prostate cancer cell line) suspended in Matrigel. After 14-18 days, tumours (100-300 mg) develop (approx. 5-8 mm). The tumour-bearing mice are used in biodistribution studies, radio labelled metabolites studies and imaging studies.
  • 5X106 LNCaP cells human prostate cancer cell line
  • tumour-bearing mice are injected via the tail vein with 0.1 ml saline containing radio labelled antibody.
  • mice are sacrificed by exsanguinations via cardiac puncture under general anaesthesia (isofluorane inhalation).
  • Blood, plasma, urine, faeces and tissues are obtained by dissection (tumour, liver, kidney, thyroid, spleen, lungs, heart, stomach, small intestine, large intestine, brain, muscle, skin and bone). Radioactivity in tissue and body fluids is determined. For each time point, 3 animals are studied. Hence a total of 18 animals is required for the biodistribution studies.
  • the rate of metabolism of the radiolabeled scFv is determined using HPLC.
  • HPLC High-Performance Liquid Chromatography
  • scFv 95.2 and scFv 98.1 are radiolabeled with Iodine 124 (positron emitting radionuclide) as established above, and their ability to selectively target human prostate tumours xenografted in nude mice evaluated using Small Animal Positron Emission Tomography.
  • the tail veins of tumour-bearing mice are cannulated after induction of anaesthesia (isofluorane/N 2 O/O 2 ) and the mouse placed within a thermostatically controlled jig.
  • anaesthesia isofluorane/N 2 O/O 2
  • dynamic emission scans are acquired on a small animal PET scanner or, images acquired using a ⁇ camera depending on the radiolabel utilised.
  • the mice are scanned at 0-60 min, 4 hrs and 24 hrs post antibody injection. This experiment requires a total of 5 mice.
  • the above uses may require modifications to improve antibody thermal stability or valency (generation of diabodies). This may constitute engineering of the antibody in order to obtain affinity maturation.

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Abstract

L'invention concerne un anticorps recombinant isolé se liant à une cellule épithéliale saine de la prostate humaine et/ou à une cellule tumorale de culture de la prostate humaine dans une plus grande mesure qu'à une cellule de muscle ou de fibroblaste de la prostate. Cet anticorps recombinant comprend au moins une, deux, trois, quatre, cinq ou six séquences CDR sélectionnées parmi a) GFTFSSYA (séquence CDRl); ISYDGSNK (séquence CDR2); ARVYFRL WGQGTLVTV (séquence CDR3); SSNIGSNY C (séquence CDRl); RNN (séquence CDR2); AAWDDSLPVFGGGTKLTVLGAAA (séquence CDR3); sans aucune ou jusqu'à 1, 2, 3, 4, 5, 6, 7, 8, 9 ou 10 substitutions d'acides aminés conservateurs ou non dans chaque séquence CDR; ou b) GDSVSSNSAA (séquence CDRl); TYYRSKWYN (séquence CDR2); ARLVDASFDYWGQGTLVTV (séquence CDR3); QGISSW (séquence CDRl); AAS (séquence CDR2); QQANSFRTFGQGTKVEIKRAAA (séquence CDR3); sans aucune ou jusqu'à 1, 2, 3, 4, 5, 6, 7, 8, 9 ou 10 substitutions d'acides aminés conservateurs ou non dans chaque séquence CDR. L'anticorps selon l'invention est utile dans l'imagerie, le diagnostic et le traitement du cancer, en particulier du cancer de la prostate.
PCT/GB2008/002733 2007-08-09 2008-08-11 Anticorps se liant a des cellules epitheliales de la prostate humaine WO2009019493A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005062977A2 (fr) * 2003-12-23 2005-07-14 The Regents Of The University Of California Anticorps humains d'internalisation specifiques du cancer de la prostate
EP1726650A1 (fr) * 2005-05-27 2006-11-29 Universitätsklinikum Freiburg Anticorps monoclonaux et fragments d'anticorps monocaténaires contre l'antigène spécifique de surface membranaire de la prostate

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005062977A2 (fr) * 2003-12-23 2005-07-14 The Regents Of The University Of California Anticorps humains d'internalisation specifiques du cancer de la prostate
EP1726650A1 (fr) * 2005-05-27 2006-11-29 Universitätsklinikum Freiburg Anticorps monoclonaux et fragments d'anticorps monocaténaires contre l'antigène spécifique de surface membranaire de la prostate

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHALLITA-EID PIA M ET AL: "Monoclonal antibodies to six-transmembrane epithelial antigen of the prostate-1 inhibit intercellular communication in vitro and growth of human tumor xenografts in vivo", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, BALTIMORE, USA, DE, vol. 67, no. 12, 15 June 2007 (2007-06-15), pages 5798 - 5805, XP002469628 *
POPKOV MIKHAIL ET AL: "Isolation of human prostate cancer cell reactive antibodies using phage display technology.", JOURNAL OF IMMUNOLOGICAL METHODS AUG 2004, vol. 291, no. 1-2, August 2004 (2004-08-01), pages 137 - 151, XP004549961, ISSN: 0022-1759 *

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