EP1601695A1 - Inhibiteurs de mcam - Google Patents

Inhibiteurs de mcam

Info

Publication number
EP1601695A1
EP1601695A1 EP03763867A EP03763867A EP1601695A1 EP 1601695 A1 EP1601695 A1 EP 1601695A1 EP 03763867 A EP03763867 A EP 03763867A EP 03763867 A EP03763867 A EP 03763867A EP 1601695 A1 EP1601695 A1 EP 1601695A1
Authority
EP
European Patent Office
Prior art keywords
seq
mcam
antibody
cells
polypeptide
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Withdrawn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
EP03763867A
Other languages
German (de)
English (en)
Inventor
Christine Margarete Unger
Carolin Zehetmeier
Claudia Torella
Jens NIEWÖHNER
Bianca Ahrens
Gerald Beste
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.)
Xerion Pharmaceuticals AG
Original Assignee
Xerion Pharmaceuticals AG
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
Application filed by Xerion Pharmaceuticals AG filed Critical Xerion Pharmaceuticals AG
Priority to EP03763867A priority Critical patent/EP1601695A1/fr
Publication of EP1601695A1 publication Critical patent/EP1601695A1/fr
Withdrawn legal-status Critical Current

Links

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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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)

Definitions

  • Metastasis is a multi-step process in which tumor cells detach from the primary tumor, invade the cellular matrix, penetrate through blood vessels, thus enter the circulatory system (intravasation), arrest at a distant site, exit the blood stream (extravasation), and grow.
  • MCAM cell adhesion molecules
  • MCAM cell adhesion molecules
  • MUC18 MUC18
  • Mel-CAM CD 146
  • MCAM is an integral membrane glycoprotein with an apparent molecular weight of M 113,000 Da. It contains five immunoglobulin-like domains, and its cytoplasmic domain contains several protein kinase recognition motifs, which suggests the involvement of MCAM in cell signaling (See, C. Sers et al. (1993) Proc. Nat. Sci. USA 90, 8514-8518).
  • MCAM-interacting proteins varies from tissue to tissue (Shih et al. (1997) Cancer Res. 57(17), 3835-3840) and for this reason the effects of MCAM overexpression must be seen in context. This is demonstrated by results obtained with another tumor type, i.e. breast cancer. Shih et al. (1997) Am. J. Pathol. 151(3), 745- 751 revealed that in this context invasiveness was inhibited at high expression levels of MCAM. In this context MCAM even acted as a tumor suppressor. Therefore, the physiological role of MCAM in invasiveness and metastasis is still unclear.
  • metastasis formation depends on the invasiveness of tumor cells. It would, therefore, be useful to identify molecules and subsequently develop drugs, which inhibit invasiveness and therewith prevent metastasis of primary tumors.
  • the present invention relates to said polypeptides, which can specifically bind to the extracellular domain of MCAM and inhibit MCAM function. In preferred embodiments these polypeptides are antibody fragments or antibodies.
  • the polypeptides of the invention can be labeled with detectable groups, if desired, or can be part of a bioconjugate.
  • the invention further relates to compositions comprising a polypeptide of the invention and optionally pharmaceutical acceptable additives and diluents.
  • the invention relates to nucleic acid molecules encoding a polypeptide of the invention, as well as to vectors comprising such a nucleic acid and to host cells comprising such a vector.
  • the invention relates to the use of molecules inhibiting MCAM function for the manufacture of a medicament for the treatment or prevention of invasion and/or metastasis of naturally occurring cancer cells, wherein invasiveness and/or metastatic potential of said cancer cells depends on MCAM function.
  • molecules bind specifically to already expressed MCAM and inhibit MCAM function in invasion and/or metastasis.
  • Such molecules are small chemical compounds or certain polypeptides binding to the extracellular region of MCAM, particularly a polypeptide or a bioconjugate of this invention.
  • the invention relates to a method of treating or preventing invasion and/or metastasis in a patient, wherein the invasiveness and/or metastatic potential of said cancer cells depends on MCAM function.
  • the invention relates to a method to determine the dependency of the invasiveness of a naturally occurring cancer cell on the functionality of MCAM.
  • the invention relates to a method for the identification of antibody fragments useful for inhibiting the invasiveness of sarcoma cells, particularly the identification of such antibody fragments that bind to the extracellular domain of MCAM.
  • a "polypeptide” as used herein is a molecule comprising more than 10, preferably more than 20, most preferably more than 30, and less than 10000, more preferably less than 2500, most preferably less than 1000 amino acids. Also polypeptides with substantial amino acid sequence identity and polypeptides, which contain a low percentage of modified or non-natural amino acids are encompassed.
  • the terms " antibody” and “immunoglobulin”, as used herein refer to any inimunological binding agent, including polyclonal and monoclonal antibodies.
  • antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as IgGl, IgG2, IgG3, IgG4, and the like.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are termed alpha, delta, epsilon, gamma and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Antibodies may be also selected from modified immunoglobulins, for example chemically or recombinantly produced antibodies, CDR grafted antibodies or humanized antibodies, site directed mutagenized antibodies that exhibit substantial amino acid sequence identity in their CDR regions, particularly in their CDR3 region, to the corresponding antibody fragments of the invention and retain substantially the same affinity for MCAM binding as the corresponding antibody fragments.
  • the CDRs (complementary determining region) of an antibody are the parts of these molecules that determine their specificity and make contact with specific ligands.
  • the CDRs are the most variable parts of the molecule and contribute to the diversity of these molecules.
  • CDR- Ll amino acids 24 to 41
  • CDR-L2 50 to 57
  • CDR-L3 amino acids 26 to 38
  • CDR-Hl amino acids 26 to 38
  • CDR-H3 amino acids 26 to 38
  • CDR-H3 amino acids 26 to 38
  • CDR-H3 amino acids 26 to 38
  • CDR-H3 amino acids 26 to 38
  • CDR-H3 amino acids 26 to 38
  • CDR-H3 amino acids 26 to 38
  • CDR-H3 amino acids 26 to 38
  • CDR-H3 51 to 70
  • CDR-H3 100 to 125
  • the CDR regions of an antibody fragment can easily be determined by somebody skilled in the art by aligning the antibody fragment with said human IgG, e.g. using a program of the NCBI that allows to "Blast”, and thereby align, two sequences with one another, and identifying the amino acids of the antibody fragment corresponding to the CDRs of a human IgG.
  • Substantial amino acid sequence identity means that at least 70%, preferably at least 85%, more preferably all but 5, still more preferably all but 3 and even more preferably all but 1 of the amino acids of two aligned amino acid sequences, particularly of aligned CDRs, are identical.
  • antibody fragment is used to refer to any fragment of an antibody-like molecule that has an antigen binding region, and this term includes antibody fragments such as scFv, dsFv, Fab', Fab, F(ab') 2 , Fv, single domain antibodies (DABs), diabodies, and the like.
  • scFv antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • a “Fv” fragment is the smallest antibody fragment that retains an intact antigen binding site.
  • a “dsFv” is a disulfide stabilized Fv.
  • a “Fab” fragment is an antigen binding fragment, containing complete light chains paired with the VH and CHI domains of the heavy chain.
  • a “Fab”' fragment is a reduced F(ab') 2 fragment.
  • a “F(ab') 2 " fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • a “single domain antibody (DAB)” is an antibody with only one (instead of two) protein chain derived from only one of the domains of the antibody structure. Dabs exploit the finding that, for some antibodies, half of the antibody molecule binds to its target antigen almost as well as the whole molecule (Davies et al. (1996) Protein Eng. 9: 531-537.
  • Diabodies are bivalent, or bispecif ⁇ c antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448).
  • label refers to a detectable marker or the incorporation of such, respectively, e.g., by incorporation of a fluorophore-, chromophore- or radio-labeled amino acid or attachment of a fluorophore-, chromophore- or radiolabel to a polypeptide or attachment of moieties that can be detected by a labeled second molecule containing a fluorescent marker or enzymatic activity that can be detected by an optical or a colorimetric method.
  • An example for such a two-step detection system is the well-known biotin-avidin system.
  • An "epitope” includes any protein determinant capable of specific binding to an immuno globulin or an antibody fragment. Epitopic determinants usually consist of chemically active surface groupings of molecules such as exposed amino acids, aminosugars, or other carbohydrate side chains and usually have specific three- dimensional structural characteristics, as well as specific charge characteristics.
  • a “naturally occurring cancer cell” as used herein is a cell that has not been transfected, transduced or otherwise genetically engineered in the laboratory. Such a cell does not comprise artificial DNA sequences, e.g. of vectors, or DNA sequences being found only in other species, but not usually in the species from which the naturally occurring cancer cell was derived. However, a naturally occurring cancer cell may comprise sequences that are not usually found in the species from which it was derived, if those sequences have arisen due to the processes of viral infection or mutation and selection that took place within the individuum from which the naturally occurring cancer cell was derived, and/or during continued culture of the naturally occurring cancer cell.
  • Selected cancer cell-types as preferably used and/or treated in the context of this invention comprise of melanoma, breast carcinoma, prostate carcinoma, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural ectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinomas, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolarcarcmoma, epithelial adenocarcinoma, and liver metastases thereof, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovioma
  • Treating metastatic tumors means that the metastasis of the tumor is prevented, delayed, or inhibited.
  • Metalstatic tumors as used herein include both tumors at the primary site capable of metastasizing and metastasized tumors at a secondary site. Such metastatic tumors can be of a tissue origin of the lung, liver, kidney, stomach, small intestine, bone, spleen, brain, peripheral nervous system, thyroid, pancreatic, endometrial, ovarian, cervical, skin, colon or lymphoid tissue.
  • “Invasiveness” as used herein is the ability of a cell to migrate through a layer of other cells or to migrate through the extracellular matrix. Invasiveness can be assessed by the Matrigel assay described in Example 5.
  • Invasion is measured as cells that reach the lower surface of the filter during a certain incubation period.
  • the naturally occurring cancer cell is defined as invasive.
  • the control cells instead form only 5% colonies in the same time frame and are defined as non-invasive.
  • Adhesiveness is the ability of a cell to reattach after they have been removed from the matrix on which it had been grown, resuspended as a solution of single cells (not in direct contact with other cells of the solution), and replated on a matrix to which adhesion is possible.
  • a cell is defined as adhesive if in an assay as described in Example 7, more than 40%) of the cells adhere within a time of 30-120 min. Instead, only 5% of the control cells adhere within the same time frame.
  • Proliferation as used herein is the ability of a eukaryotic, and thus mammalian cell, to proliferate and divide itself into daughter cells. Proliferation leads, e.g. in a cell culture, to an increase in overall cell number over time.
  • Metastatic potential as used herein is the ability of a tumor cell to form a new tumor at a site distant from the primary tumor of which the tumor cell was derived (a metastase). Metastatic potential can be measured by injecting, e.g. lxl 0 6 , cells into the lateral tail vein of athymic nude mice and determining the number of tumor nodules in the lung, e.g. 2 months post injection, e.g. as described in the section "Tumor cell injections" on page 2346 of Huang et al (1996) Oncogene 13: 2339-2347, or the sections "Animals and production of tumors" and " Histochemical analysis for calcified matrix” on page 1882 of Radinsky et al.
  • a cell line to produce more than 3, preferably more than 8, more preferably more than 20 tumor nodules in the lung in this assay is considered metastatic.
  • Therapeutically effective amounts are amounts which eliminate or reduce the patient's tumor burden, or which prevent or reduce metastasis.
  • the dosage will depend on many parameters, including the nature of the tumor, patient history, patient condition, the possible co-use of cytotoxic agents, and methods of administration. Methods of administration include injection (e.g., parenteral, subcutaneous, intravenous, intraperitoreal, etc), for which the molecule inhibiting MCAM function is provided in a nontoxic pharmaceutically acceptable carrier.
  • suitable carriers and diluents are selected so as not to significantly impair biological activity of the binding agent (e.g., binding specificity, affinity or stability), such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oils, ethyloleate, or liposomes.).
  • Acceptable carriers can include biocompatible, inert or bio-absorbable salts, buffering agents, oligo- or polysaccharides, polymers, viscoelastic compound such as hyaluronic acid, viscosity- improving agents, preservatives, and the like.
  • composition or formulation may also include other carriers, adjuvants, or nontoxic, non-therapeutic, non-immunogenic stabilizers and the like.
  • Typical dosages may range from about 0.01 to about 20 mg/kg, or more particularly from about 1 to about 10 mg/kg.
  • Therapeutic methods employing molecules inhibiting MCAM function may be combined with chemotherapy, surgery, and radiation therapy, depending on type of the tumor, patient condition, other health issues, and a variety of factors.
  • a "molecule inhibiting MCAM function” is a molecule resulting in inhibition of the biological activity of MCAM.
  • This inhibition of the biological activity of MCAM can be assessed by measuring one or more indicators of MCAM's biological activity, such as MCAM dependent invasiveness or MCAM dependent adhesion. These indicators of MCAM's biological activity can be assessed by one or more of several in vitro or in vivo assays (see, Examples 5, 7 and 6.2).
  • the ability of a molecule to inhibit MCAM activity is assessed by inhibition of MCAM-induced invasiveness, adhesion or proliferation of invasive human sarcoma cells, particularly the cells used in Examples 5, 7 and 6.2.
  • a "molecule inhibiting MCAM function" of the invention is not a molecule which is a general inhibitor of protein function, like a protease, like a denaturing agent, e.g. urea or guanidinium hydrochloride, like heavy metal atoms or like small molecules (e.g. aldehyds or isocyanates) reacting covalently and non-specifically with biomolecules (lipids, proteins, sugars).
  • a molecule inhibiting MCAM function is characterized by its ability to inhibit MCAM function at a concentration at which it does not inhibit the function of the insulin receptor (e.g. as determined in an anti-Phosphotyrosine Western Blot Assay, see e.g. B. Cariou et al.
  • a molecule inhibiting MCAM function as used in this patent. Inhibition is understood to be at least a 30%, preferably a 40%, more preferably a 50%, even more preferably a 60% decrease in function, as defined by MCAM function in an invasion assay as mentioned above, when compared to a negative control with the same experimental conditions, but without the molecule of the invention.
  • a molecule is defined as not significantly affecting the function of the other three receptors if the decrease in function affected by the molecule of the invention is less than 20%, more preferably less than 15%>, even more preferably less than 10%.
  • such a molecule of the invention decreases MCAM expression by more than 50%, preferably by more than 80%, still more preferably by more than 90%), most preferably by more than 95% when measured in a quantitative western blot normalized to the level of beta tubulin present, when present in an experiment at a concentration of 10 nM to 100 ⁇ M, preferably at around 1 ⁇ M, in which the amount of MCAM is compared between two otherwise identical samples, wherein in one sample the molecule of the invention was allowed to inhibit MCAM expression.
  • the molecule of the invention does not decrease the amount of the beta tubulin present per cell by more than 20%, and said molecule does not decrease the relative level of the insulin receptor and the B-CAM cell surface glycoprotein by more than 20%.
  • the polypeptide of the invention is considered to inhibit the biological function of MCAM if it reduces the invasiveness of naturally invasive cancer cells in an experiment as in Example 5 by more than 30%), preferably more than 60%, when said antibody fragment is present at a concentration of 1 nM to 50 ⁇ M, preferably around 20 ⁇ M.
  • a small chemical compound of the invention is considered to inhibit the biological function of MCAM if it reduces the invasiveness of naturally invasive cancer cells in an experiment as in Example 5 or 7 by more than 30%, preferably by more than 60%), when present at a concentration of 10 nM to 100 ⁇ M, preferably at around 1 ⁇ M, while not affecting cell morphology, cell cycle progression (determined by analyzing the DNA content of a cell population by propidium iodide staining and FACS analysis), and not increasing the percentage of the cells of the culture that show signs of apoptosis (determined by measuring the percentage of cells showing DNA fragmentation, e.g. by a so called tunnel-assay).
  • a small chemical compound as used in this invention is a molecule with a molecular weight between 50 Da and 10000 Da, preferably between 100 Da and 4000 Da, more preferably between 150 Da and 2000 Da, or a physiologically acceptable salt thereof.
  • a molecule "binding specifically to MCAM” or “specific for MCAM” as mentioned herein is a molecule which binds to MCAM expressing HT 1080 cells, but not to Hs-27 cells or MCAM-negative SBcl-2 cells (Shih et al. (1997) Cancer Res. 57(17), 3835-3840) under the conditions given in Examples 1 and 2.
  • binding to HT 1080 cells is at least 2 fold higher, preferably 5 fold higher and most preferably 20 fold higher than binding to Hs-27 cells or SBcl-2 cells when said molecule is tested at a concentration of 0,1 nM to 10 ⁇ M, preferably 1 nM to 1 ⁇ M, still more preferably 10 nM to 500 nM and most preferably around 100 nM.
  • Such a molecule may additionally have a binding constant for MCAM lower than 10 ⁇ M, preferably lower than 1 ⁇ M, more preferably lower than 100 nM, most preferably from 0,1 nM to 20 nM. Binding constants can be determined, e.g., by standard methods like the BIACORE system according to the instructions of the manufacturer's manual.
  • the term "at least one” as used here means “one and more than one", particularly one, two, three, four and five.
  • the present invention demonstrates for the first time that for specific, naturally occurring tumor cells the expression and function of MCAM is essentially involved in the process of adhesion, invasion and/or metastasis.
  • the present invention provides an unbiased screen to identify molecules inhibiting MCAM function.
  • the present invention relates to molecules or polypeptides, which can specifically bind to the extracellular region of MCAM and inhibit MCAM function in invasion, proliferation, adhesion and/or metastasis.
  • the polypeptides according to the invention comprise at least on sequence selected from the group consisting of ' - SEQ ID NO. 39 to SEQ ID NO. 57, which represent CDR's specifically involved in binding the tumor specific cell surface molecules like e.g. MCAM; or
  • polypeptide of the invention is an antibody fragment, in particular a scFv, dsFv, Fab', Fab, F(ab')2, Fv, single domain antibody or diabody, more particularly a scFv, dsFv, Fv, single domain antibody or diabody, still more particularly a scFv, single domain antibody or diabody and even more preferably a scFv.
  • polypeptide of the invention is an antibody, in one preferred embodiment an antibody derived from a scFv antibody fragment, in another preferred embodiment a polyclonal or monoclonal antibody, particularly a human monoclonal antibody.
  • the CRD3 region of the antibody or the antibody fragment is identical to one of the CDR3 regions, which are shown in Table 1 by underlining the relevant nucleotides in the sequence of the scFv's.
  • Anti-human MCAM binding antibodies may be selected from modified immunoglobulins, for example chemically or recombinantly produced antibodies or humanized antibodies, site directed mutagenized antibodies, that exhibit substantial amino acid sequence identity in their CDR regions, particularly in their CDR3 region, to the corresponding antibody fragments of the invention and retain substantially the same affinity for MCAM binding as the corresponding antibody fragments.
  • the polypeptide of the invention is a human antibody selected from the group consisting of IgA, IgD, IgE, IgG, and IgM, in particular IgG and IgM, more particularly IgGl, IgG2a, IgG2b, IgG3, IgG4.
  • a polypeptide of the invention is labeled with a detectable label.
  • detectable labels are radioisotopes, chromophores, fluorophores, enzymes or radioisotopes.
  • the detectable label can, for example, be selected from this group.
  • the polypeptide of the invention can be covalently or non- covalently conjugated and/or coupled to or with, respectively, another protein, a solid matrix (e.g. like a bead), with itself to form multimers, a cytotoxic agent further enhancing the toxicity to targeted cells, a cytostatic agent, a prodrug, or an effector molecule, which is able to modify the cell expressing MCAM or to recruit immune cells. All these conjugates are "bioconjugates" of the invention.
  • cytotoxic agents include, but is not limited to, daunorubicin, taxol, adriamycin, methotrexate, 5 FU, vinblastin, actinomycin D, etoposide, cisplatin, doxombicin, genistein, andribosome inhibitors (e.g., trichosantin), or various bacterialtoxins (e.g., Pseudomonas exotoxin; Staphylococcus aureus protein A).
  • Bioconjugates comprising the polypeptides of the invention, particularly the antibody fragment or antibody of the invention, together with said cytotoxic moieties are made using a variety of bifunctional protein coupling agents.
  • Some examples of such reagents are N- succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), bifunctional derivatives of imidoesters such a dimethyl adipimidate HC1, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bisazido compounds such as his (R- azidobenzoyl) hexanediamine, bisdiazonium derivatives such as bis-(R- diazoniumbenzoyl)ethylenediamine, diisocyanates such as tolylene 2,6-diisocyanate, and bis-activated fluorine compounds such as l,5-difluoro-2,4-dinitrobenzene.
  • SPDP
  • a metastasis-associated MCAM antigen can be detected by using a bioconjugate or a polypeptide of the invention, particularly an antibody or an antibody fragment of the invention.
  • a sample is taken from the subject, e.g., a biopsy specimen taken from tissue suspected of having a metastatic tumor. Generally, the sample is treated before an assay is performed. Assays, which can be employed including ELISA, RIA, EIA, Western Blot analysis, immunohistological staining and the like.
  • the antigens or the antibodies can be labeled by an enzyme, a fluorophore or a radioisotope. (See, e.g., Coligan et al. (1994) Current Protocols in Immunology, John Wiley & Sons Inc., New York, New York; and Frye et al. (1987) Oncogene 4: 1153-1157.)
  • one embodiment of the invention relates to the use of at least one polypeptide of the invention and/or at least one labeled polypeptide of the invention and/or at least one bioconjugate of the invention for the detection of MCAM.
  • one polypeptide of the invention or one labeled polypeptide of the invention or one bioconjugate of the invention can be used for the detection of MCAM, or one labeled polypeptide together with one bioconjugate or two or three polypeptides or two labeled polypeptides can be used.
  • the polypeptide will bind to the extracellular region of MCAM.
  • the extracellular region of MCAM is defined as that part of the MCAM protein outside of the cellular membrane, particularly the extracellular amino acid loops between amino acid 24 and amino acid 553.
  • the present invention encompasses a diagnostic kit.
  • a diagnostic kit comprises at least one bioconjugate and/or at least one labeled polypeptide of the invention and/or at least one polypeptide of the invention, particularly an antibody fragment or an antibody of the invention, or a labeled version of these, and consists additionally of the reagents and materials necessary to carry out a standard competition or sandwich assay.
  • Said diagnostic kit may be used for the determination of the invasive potential of biological samples, in particular of certain cancer cell types.
  • a kit will further typically comprise a container.
  • anti-idiotypic antibodies which can be used to screen antibodies to identify whether the antibody has the same binding specificity as a human monoclonal antibody of the invention and can also be used for active immunization (Herlyn et al. (1986) Science, 232:100).
  • anti-idiotypic antibodies can be produced using well-known hybridoma techniques (Kohler et al. (1975) Nature, 256:495).
  • An anti-idiotypic antibody is an antibody, which recognizes unique determinants present on the antibody of interest. These determinants are located in the hypervariable region of the antibody.
  • An anti-idiotypic antibody can be prepared by immunizing an animal with the polypeptide, particularly the antibody fragment or antibody, of interest. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an antibody to these idiotypic determinants. By using anti-idiotypic antibodies, it is possible to identify other hybridomas expressing monoclonal antibodies having the same epitopic specificity.
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region, which is the "image" of the epitope bound by the first antibody.
  • the anti-idiotypic monoclonal antibody can be used for immunization, since the anti-idiotype monoclonal antibody binding domain effectively acts as an antigen.
  • the polypeptide of the invention particularly the antibody fragment or antibody of the invention reduces the invasiveness of invasive tumor cells by 30-60%, or preferably by 30-55%, 40-50%) or even at least 60%, when tested in an invasion assay (see example 5). While preferably the invasiveness is reduced via a specific binding of the polypeptide to an extracellular domain of the MCAM, according to a further embodiment the invasiveness is also reduced by polypeptides binding to other surface molecules such as integrins or ephrins.
  • polypeptide of the invention particularly the antibody or antibody fragment reduces the adhesiveness of invasive tumor cells by 30-60%, or preferably by 30-55%, 40-50% or even at least 60%>, when tested in an adhesion assay (see example 7).
  • the polypeptide of the invention particularly the antibody or antibody fragment reduces the proliferation of invasive tumor cells by 30-60%, or preferably by 30-55%>, 40-50%) or even at least 60%, when tested in a proliferation assay (see example 6.2).
  • the antibody fragment of the invention specifically recognizes one or more epitopes of MCAM, or epitopes of conserved variants of MCAM, or peptide fragments of the MCAM.
  • the invention relates to the use of a molecule of the invention, particularly selected from the group consisting of a small chemical compound of the invention, a molecule inhibiting gene expression of MCAM, a bioconjugate or a polypeptide of the invention, more particularly an antibody fragment or an antibody of the invention as a medicament.
  • the present invention relates to a composition
  • a composition comprising effective amounts of at least one, particularly one, molecule of the invention, particularly at least one of any of the polypeptides or nucleotide sequences of the invention, or at least one of a molecule inhibiting gene expression of MCAM and a pharmaceutically acceptable carrier and/or a diluent.
  • the pharmaceutical composition can be used for the treatment of conditions related to the over-expression or ectopic expression of human MCAM, especially the treatment of metastatic tumors, especially of metastatic tumors derived from the group selected of cancer cell-types as described above.
  • compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one molecule inhibiting MCAM function, particularly a molecule inhibiting MCAM function by binding to the extracellular region of MCAM, more particularly wherein the molecule is a small chemical compound, a nucleic acid sequence, a polypeptide or the bioconjugate of the invention, still more particularly wherein the molecule is an antibody fragment of the invention, even more preferably wherein the molecule is a scFv of the invention or an antibody derived from such a scFv of the invention.
  • the invention relates to administering a molecule inhibiting MCAM function in a pharmaceutical acceptable composition, particularly wherein the molecule inhibits gene expression of MCAM via a receptor mediated pathway.
  • the molecule inhibiting MCAM function can particularly be a molecule which can bind to the extracellular region of MCAM, more particularly wherein the molecule is a small chemical compound or an antibody or an antibody fragment or a polypeptide of the invention or a bioconjugate of the invention, still more particularly wherein the molecule is an antibody fragment of the invention, even more preferably wherein the molecule is a scFv of the invention or an antibody derived from such a scFv of the invention.
  • the present invention further relates to a method to produce the polypeptide of the invention by recombinant techniques. These techniques are well known in the art (Skerra et al. (1993), Curr. Opin. Immunol. 5, 256-62; Chadd et al. (2001), Curr. Opin. Biotechnol. 12, 188-94).
  • nucleic acid sequences encoding a polypeptide of the invention can be isolated and cloned into one or more polynucleotide expression vectors, and the vector can be transformed into a suitable host cell line for expression of a recombinant polypeptide of the invention.
  • Expression of the gene encoding the polypeptide of the invention provides for increased yield of the polypeptide, and also allows for routine modification of the polypeptide by introducing amino acid substitutions, deletions, additions and other modifications, for example humanizing modifications (Rapley (1995) Mol. Biotechnol.
  • the present invention therefore relates to an above mentioned isolated nucleic acid molecule encoding any one of the polypeptides of the invention, particularly an antibody fragment of the invention, more particularly a scFv, dsFv, Fv, single domain antibody or diabody of the invention, still more particularly a scFv, single domain antibody, diabody of the invention or an antibody derived from such a scFv of the invention, and even more preferably a scFv of the invention or an antibody derived from such a scFv of the invention.
  • SEQ ID NO. 39 to SEQ ID NO. 57 which represent CDR's specifically involved in binding the tumor specific cell surface molecules like e.g. MCAM; or
  • SEQ ID NO. 1 to SEQ ID NO. 9 SEQ ID NO. 19 to SEQ ID NO. 23 and SEQ ID NO. 29 to SEQ ID NO. 33, which represent scFv's specifically involved in binding the tumor specific cell surface molecules like e.g. MCAM.
  • the isolated nucleic acid sequence of the invention comprises any one of the nucleic acid sequences selected from the group consisting of SEQ ID NO. 10 to SEQ ID NO. 18, SEQ ID NO. 24 to SEQ ID NO. 28 and SEQ ID NO. 34 to SEQ ID NO. 38.
  • the present invention further relates to a nucleic acid sequence, which hybridizes under stringent conditions to any of the sequences of SEQ ID NO. 10 to SEQ ID NO. 18, SEQ ID NO. 24 to SEQ ID NO. 28 and SEQ ID NO. 34 to SEQ ID NO. 38.
  • the term under stringent conditions as used herein refers to hybridization conditions and temperatures, which allow the aggregation of nucleic acid sequences with high homology (preferably above 70%) only. Typical hybridization conditions are well-known to the skilled practitioner. In brief, for applications requiring high selectivity, one will typically desire to employ relatively low salt conditions and/or high temperature conditions, such as provided by 0.02M - 0.15M NaCI at temperatures of 50°C to 70°C.
  • the present invention further relates to a vector comprising a nucleic acid of the invention.
  • the vector is a plasmid, a phagemid, or a cosmid.
  • the nucleic acid molecule of the invention can be cloned in a suitable fashion into procaryotic or eucaryotic expression vectors (Sambrook et al., "Molecular cloning: a laboratory manual” Second edition, Cold Spring Harbor Laboratory Press (1989)).
  • Such expression vectors comprise at least one promotor, at least one signal for translation initiation, at least one nucleic acid sequence of the invention and - in the case of procaryotic expression vectors - a signal for translation termination, while in the case of eucaryotic expression vectors preferably additional signals for transcriptional termination and for polyadenylation.
  • prokaryotic expression vectors are, for expression in Escherichia coli, e.g. expression vectors based on promotor' s recognized by T7 RNA polymerase, as described in US 4,952,496, for eucaryotic expression vectors for expression in Saccharomuces serevisiae , e.g., the vectors p426Met25 or 526GAL1 (Mummberg et al. (1994) Nucl. Acids Res., 22, 5767-5768), for the expression in insect cells, e.g., Baculovirus-vectors as e.g.
  • EP-B1-0 127 839 or EP-B1-0 549 721 and for the expression in mammalian cells, e.g., the vectors Rc/CMV and Rc/RSV or SV40-vectors, which are commonly known and commercially available.
  • the present invention further relates to a host cell comprising a nucleic acid of the invention and/or a vector of the invention, particularly wherein the host cell is a microorganism like yeast or other fungi, like Escherichia coli, Bacillus subtilis or other bacteria.
  • the host cell can also be a cell of higher eucaryotic origin, like an insect cell, preferably a virus infected insect cell, more preferably a baculovirus infected insect cell, or like a mammalian cell like COS, MDCK 293-EBNA1, NS0 or a hybridoma cell.
  • the present invention relates further to a method for the production of a polypeptide of the invention, particularly an antibody fragment of the invention, comprising culturing a microorganism transformed with a recombinant vector comprising DNA encoding a polypeptide of the invention, particularly an antibody fragment of the invention, and recovering said a polypeptide of the invention, particularly an antibody fragment of the invention or a fusion protein containing it, from the medium.
  • the present invention shows that blocking MCAM function inhibits invasiveness of certain cancer cells derived from group selected of cancer cell-types as listed earlier in the specification, and particularly inhibits invasiveness of selected cancer cells, e.g., derived from human sarcoma cells, epithelial tumors, mesenchymal tumors, reticuloendothelial tumors, nervous system tumors, teratomas, even more preferably human sarcoma cells.
  • One embodiment of the invention is therefore the use of at least one, particularly one, molecule inliibiting MCAM function in the manufacture of a medicament for the treatment or prevention of invasion and/or metastasis of naturally occurring cancer cells, wherein proliferation, adhesiveness, invasiveness and/or metastatic potential of said cancer cells depends on MCAM function.
  • the molecule inhibiting MCAM inhibits the function of expressed MCAM.
  • Expressed MCAM is to be understood in this context as MCAM protein already present on naturally occurring cancer cells before any kind of treatment is initiated. These molecules are particularly molecules, which bind to the extracellular region of receptors and inhibit MCAM expression via an internal pathway.
  • Such molecule is selected from the group consisting of a small chemical compound, an antibody against MCAM, an antibody fragment against MCAM, a polypeptide of the invention, an anti-idiotypic antibody of the invention and/or a bioconjugate of the invention, especially wherein the molecule is a polypeptide and/or a bioconjugate of the invention.
  • the naturally occurring cancer cells which depend on MCAM function for proliferation, adhesion, invasiveness and/or the metastatic potential can be any cancer cells selected from the group consisting of the tissues or cancer cell- types as mentioned before, particularly they can be of a tissue origin selected from the group consisting of the lung, liver, kidney, stomach, small intestine, bone, spleen, brain, peripheral nervous system, thyroid, pancreatic, endometrial, ovarian, cervical, skin, colon or lymphoid tissue, more particularly the cancer cells are sarcoma cells.
  • the invention further pertains to the MCAM antigen as a draggable target.
  • Another aspect of the present invention pertains to antibody fragments that bind to human MCAM with high neutralizing capacity.
  • At least one polypeptide of the invention and/or a bioconjugate of the invention are used for identifying additional molecules that specifically bind human MCAM, particularly in screening assays.
  • These methods entail contacting a reference anti-MCAM antibody fragment with a target species comprising the MCAM domain in the presence of a putative competitor test-binding agent. This step of contacting is conducted under conditions suitable for complex formation between the reference antibody fragment and the target species in the absence of the test-binding agent. Complex formation between the reference antibody fragment and the target species in the presence of the test-binding agent is detected as an indicator of specific binding activity of the test- binding agent to MCAM.
  • This screening method is useful for high throughput screening of, e.g., other antibody libraries or antibody fragment libraries, antisense oligonucleotide libraries or peptide and small molecule libraries to identify and characterize additional "molecules binding specifically to MCAM".
  • Competition is determined by an assay in which the antibody fragment, or other binding agent under test substantially inhibits specific binding of the reference antibody fragment to the target species containing the MCAM domain. This can be determined for example by measuring binding of the reference antibody fragment to a target species comprising MCAM domain in the presence and absence of a putative competitor, i.e. a "molecule binding specifically to MCAM " under conditions suitable for complex formation.
  • a putative competitor i.e. a "molecule binding specifically to MCAM "under conditions suitable for complex formation.
  • Numerous types of competitive binding assays are known and routinely practicable within the invention, as described for example in U.S. Pat.
  • such assays involve the use of a target species containing the MCAM domain (e.g., purified MCAM or a cell line expressing the MCAM antigen), an unlabeled "molecule binding specifically to MCAM ", and a labeled reference antibody fragment or other binding agent.
  • a target species containing the MCAM domain e.g., purified MCAM or a cell line expressing the MCAM antigen
  • an unlabeled "molecule binding specifically to MCAM " e.g., purified MCAM or a cell line expressing the MCAM antigen
  • a labeled reference antibody fragment or other binding agent e.g., antigen binding to MCAM
  • Competitive inhibition is measured by determining the amount of label bound to the target species in the presence of the "molecule binding specifically to MCAM ".
  • the "molecule binding specifically to MCAM " is present in excess.
  • “Molecules binding specifically to MCAM" identified by these competition assays” include antibodies, antibody fragments, peptides, antisense oligonucleotides, small molecules and other binding agents that bind to an epitope or binding site bound by the reference antibody fragment, as well as a "molecule binding specifically to MCAM " that bind to an epitope or binding site sufficiently proximal to an epitope bound by the reference antibody fragment.
  • competitive binding agents of the invention will, when present in excess, inhibit specific binding of a reference antibody fragment to a selected target species by at least 10%>, preferably by at least 25%, more preferably by at least 50%), and even more preferably by at least 75%-90% or greater.
  • a polypeptide of the invention particularly an antibody fragment or an antibody of the invention
  • natural or artificial ligands, peptides, anti-sense, or other small molecules capable of specifically targeting human MCAM may be employed.
  • Drugs can be designed to bind or otherwise interact and inhibit human MCAM based upon the present invention.
  • rational drug design techniques such as X-ray crystallography, computer-aided (or assisted) molecular modeling (CAMM), quantitative or qualitative structure-activity relationship (QSAR), and similar technologies can be utilized to focus drug discovery efforts.
  • Rational design allows prediction of molecules, which can interact with proteins or specific parts thereof. Such molecule structures can be synthesized chemically and/or expressed in biological systems.
  • Small molecules may be produced by synthesizing organic compounds according to methods that are well known in the art.
  • a plurality of peptides, semi-peptidic compounds or non-peptidic, and organic compounds may be synthesized and then screened in order to find compounds, which bind to MCAM with high neutralizing capacity.
  • Particularly compounds that inhibit MCAM related invasion See generally Scott and Smith, "Searching for Peptide Ligands with an Epitope Library", Science (1990), 249, pp. 386-90 and Devlin et al., "Random Peptide Libraries: A Source of Specific Protein Binding Molecules", Science, (1990), 249, pp. 40407.
  • the present invention also provides methods of using the antibody or antibody fragments to inhibit human MCAM activity or to detect human MCAM in sarcoma cells, either in vitro or in vivo.
  • treating cells expressing the antigen with one or more antibody fragments causes or leads to a reduction of proliferation or adhesion and/or inhibition of the invasive ability of human sarcoma cells.
  • the migration of tumor cells into tissue is an important step in metastasis.
  • the processes of adhesion and invasion can be studied in the transendothelial model (See, Woodward et al. (2002) Invest Ophthalmol Vis Sci 43, 1708-14 and Vachula et al. (1992) Invasion Metastasis 12, 66-81).
  • the transendothelial model provides a useful in vitro system for the investigations of cellular interactions during the invasion process.
  • the present invention therefore further provides an in vitro method to determine the dependency of the invasiveness of a naturally occurring invasive cancer cell on the functionality of MCAM. This method comprises the steps of:
  • gel-like matrix as used herein is understood to be a semi-solid substance with a water content of at least 90%, which allows cultivation of cancer cells in contact with the matrix and allows migration of invasive cancer cells through a slab of said "gel- like matrix" of 0,1 mm to 1 mm, preferably 0,3 mm thickness, but not migration of non- invasive cells.
  • examples for such a “gel-like matrix” are substances resembling the extracellular matrix in protein and carbohydrate composition, particularly the commercially available "Matrigel”.
  • the "gel-like matrix” comprises one of the proteins selected from the group consisting of the proteins collagen type IV, fibronectin and laminin. More particularly the gel-like matrix comprises the proteins collagen type IV, fibronectin and laminin. More preferable the gel-like matrix comprises the proteins collagen type IV, laminin, entactin, nidogen and heparan sulfate proteoglycans.
  • the interfering molecule of step a) is a polypeptide that specifically binds to an extracellular epitope of MCAM, particularly a polypeptide of the invention, more particularly an antibody or an antibody fragment of the invention, still more particularly an antibody fragment, even more particularly a scFv, dsFv, Fv, single domain antibody or diabody, especially a scFv, single domain antibody or diabody and even more preferably a scFv.
  • the present invention further provides a method of identifying an antibody or antibody fragment binding specifically to the extracellular region of MCAM, by screening a na ⁇ ve antibody fragment phage display library, wherein these antibody fragments or the antibody (see WO 91/17271 for phage display of an antibody) is capable of inhibiting and, in particular, inhibits the invasiveness of sarcoma cells.
  • Said method comprises the steps of:
  • the identity of phages representing the antibody or the antibody fragment obtained with step d) can be determined by, e.g., sequencing the DNA encoding the antibody or the antibody fragment, or, in the case of a commercial library with gridded or numbered phages, by determining the grid position or the number of the phage. The grid position or the number then can reveal the identity of the antibody or the antibody fragment represented by the phage.
  • the pool of phages is enriched in phages binding to MCAM.
  • Those phages binding to MCAM can finally be identified by numerous methods known in the art. Phages can be separated to form individual clones and the clones of the phages can be probed with labeled MCAM protein, or a labeled part of the MCAM protein, e.g. an at least seven amino acid long peptide of the extracellular region of MCAM. Clones binding to such a probe are identified as MCAM-binders. Phages can also be affinity purified on purified MCAM protein or on recombinant MCAM
  • the open reading frame encoding the antibody or antibody fragment can be recloned from the whole enriched pool into an expression vector, the antibody or the antibody fragment can then be expressed in clones of another host cell, and the clone of the host cell carrying the expression vector comprising a nucleic acid encoding for the antibody or the antibody fragment binding specifically to MCAM can be identified, e.g. by the method described above for the identification of relevant phage clones, by the method of Examples 2 or 9, or by affinity purification on recombinant MCAM.
  • a particular advantage of this method is that an antibody or an antibody fragment specific for the accessible part of the extracellular region of MCAM is obtained, since the initial selection step is performed on intact cells, which present the accessible part of the extracellular region of MCAM for binding of the phages.
  • the above method comprises instead of step e) the further steps of:
  • the antibody fragments expressed on the phages comprise an antibody or an antibody fragment selected from the group consisting of scFv, dsFv, Fab', Fab, F(ab') 2 , Fv, single domain antibodies (DABs) and diabodies, more particularly selected from the group consisting of scFv, dsFv, Fv, single domain antibody or diabody, still more particularly selected from the group consisting of scFv, single domain antibody or diabody and even more preferably a scFv.
  • the "detergent” used in steps c) and f) is a detergent solution, preferably buffered, and can be Tween in a concentration of 0.001- 0.5%, particularly 0.01-0.1 %.
  • "High salt” in step f) is a high salt solution, preferably buffered, and has an ionic strength of lOmM-lM, particularly 20-500mM, more particularly 50-350mM, even more preferably 80-250mM.
  • Typical useful anions are, for example, chloride, citrate, phosphate, hydrogen phosphate or borate.
  • Typical useful cations are, for example, sodium, potassium, lithium, calcium or magnesium.
  • the buffered solution in the above paragraph typically has a pH of 7-8.
  • DMEM or PBS particularly with 1-20%, more particularly 5-15%o, even more preferably about 10% FCS, can be used as buffers.
  • Isolation of cells with phages bound to them is effected by gentle centrifugation at g values from 200 to 300 for 3 to 20 minutes, particularly 5 to 10 minutes.
  • Elution of bound phages, both to cells and to immobilized MCAM is effected by a wash with 2-lOOmM, particularly 4-50mM, more particularly 5-20mM, even more preferably around lOmM Glycine at a pH of from 0 to 2.5, particularly from 1 to 2.5, more particularly from 1.5 to 2.5.
  • the MCAM inhibitory activity of these antibody fragments, particularly of these scFvs may be assayed as described above, in vivo or in a cell culture experiment.
  • Cell culture assays include assays that determine the inhibitory effect of the antibody fragments of this invention in an invasion or adhesion assay as described in Examples 5 and 7. For example, results of the invasion assay are provided in Fig 2.
  • Figure 2 shows - presented as a table - results of the Invasion assay performed with HT1080 (human fibrosarcoma) cells.
  • the scFv tested are identified by their number as shown in Fig. 6.
  • the symbol "*" indicates that the respective scFv - as identified by its number as shown in Fig. 6 - was cloned into an IgG4 format before performing the Invasion assay.
  • the invasion of cells through the matrix is measured and shown in %> of inhibition of invasion.
  • Figure 3 shows - presented as a table - results of FACS analysis performed on various cell types, such as HT1080 (human fibrosarcoma), KHOS-NP (human osteosarcoma), MCF-7 (human breast adenocarcinoma), BT-474 (human breast cancer, mammary gland), PC-3 (human adenocarcinoma, prostate), Jurkat (human T cell leukemia), HL-60 (human acute myeloid leukemia), HeLa (human cervix carcinoma), SW480 (human colon carcinoma), LS174T (human colon carcinoma), HT-29 (human colon carcinoma) in comparison to a control cell (Hs27 (human skin fibroblast)).
  • HT1080 human fibrosarcoma
  • KHOS-NP human osteosarcoma
  • MCF-7 human breast adenocarcinoma
  • BT-474 human breast cancer, mammary gland
  • PC-3 human adenocarcinoma, prostate
  • Jurkat human T cell leukemia
  • Table 3 a depicts scFv identified by its number as shown in Fig. 6.
  • Table 3b depicts FACS results of IgG.
  • the symbol * means that the respective scFv -as identified by its number as shown in Fig. 6 was cloned into an IgG4 format before performing the FACS experiment.
  • FIG. 4 shows results of the immunoprecipitation experiments.
  • the immuno-complexes were separated by SDS-PAGE and silver stained.
  • Figure 5 show the vector map of pXP14 (SEQ ID No. 39) as well as sequence of the scFv expression vector.
  • Figure 6 shows in Table 1 the peptide sequences of the identified single chains: scFvl to scFcl9.
  • the CDR3 region is underlined in the depicted peptide sequence. Relevant SEQ ID No. ' s are indicated aside.
  • Figure 7 shows in Table 2 the nucleotide sequences encoding for the polypeptides scFvl to scFcl9. Relevant SEQ ID No.'s are indicated.
  • Figure 8 shows - presented as a table - results of the Adhesion assay performed on various matrixes, such as CI (collagen S type I), CIV (collagen IV), FN (fibronectin) and LN (laminin), with various cell types, such as HT1080 (human fibrosarcoma), PC-3 (human adenocarcinoma, prostate), HeLa (human cervix carcinoma) and HT-29 (human colon carcinoma).
  • the scFv tested are identified by their number as shown in Fig. 6.
  • the symbol "*" indicates that the respective scFv -as identified by its number as shown in Fig. 6- was cloned into an IgG4 format before performing the Adhesion assay.
  • the adhesion is measured and shown in % of inhibition of adhesion.
  • inhibition values “+” represents 1-10% inhibition, "++” represents an inhibition value of > 10-40% and "+++” represents an inhibition value of >40-100%.
  • “n.d.” represents not determined.
  • Figure 9 shows the vector map of scFv expression vector pXPIO (SEQ ID No. 40) and sequence of the pXPIO.
  • Figure 10 shows the results of the immunohistochemistry using the MCAM specific antibodies (scFv7* and scFv9*) (scFv7 and scFv9 were cloned into IgGl format).
  • Figure 11 shows a MALDI-MS spectrum (a) of the peptide mixture obtained from the band with an approximate size of 110 kDa. Two trypsin auto digestion peaks, indicated as T, were used for internal calibration.
  • Figure 12 shows the results of the Proliferation assay as the reduction of SW-480 and PC- 3 proliferation by IgG's.
  • scFv 7, scFv9 and scFvl ⁇ were cloned into an IgG4 format before performing the proliferation assay.
  • Proliferation of SW-480 colon cancer cells (Fig. 12b) and of PC-3 prostate cancer cells (Fig. 12a) was measured with an MTS cell viability assay at the indicated time points after first antibody addition. Data are shown as means of % medium control, pooled from 3 independent experiments. Error bars: Tukey HSD 95% confidence intervals.
  • Figure 13 shows — presented as a table - the anti-tumor effect of compounds in a subcutaneously grown human tumor xenograft in athymic mice.
  • a lung adenocarcinoma (LXFA) was used as a xenograft.
  • the table shows the tumor size at day 0 and day 7 after compound injection.
  • scFv7* scFv7 cloned into IgGl format
  • Docetaxel A result of the combination therapy of scFv* with Docetaxel is also shown.
  • the following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting upon the present invention.
  • Example 1 Selection and screening of scFv (Selection on cells in suspension)
  • Single chain Fv were selected from a large non-immune phage displayed repertoire of human origin containing 1011 independent clones, provided by Cambridge Antibody Technology Ltd., Cambridge, UK.
  • HT1080 cells human fibrosarcoma cell line; ATCC, CCL-121
  • ATCC human fibrosarcoma cell line
  • FCS DMEM + 10% FCS.
  • 1012 cfu of phage library/107 cells were pre-blocked for 1 hour at 25°C with DMEM + 10%) FCS and subsequently incubated with end-over-end rotation for 1.5 hour at 25°C with the cells in Eppendorf tubes pre-blocked with DMEM + 10% FCS.
  • the diversity of the enriched repertoire is decreased compared to the original repertoire.
  • the eluate containing the enriched repertoire was amplified by infecting exponentially growing E. coli TGI. Phagemid containing E. coli were selected and propagated by overnight growth at 30°C on LB agar plates supplemented with lOO ⁇ g/ml ampicillin and 1% glucose.
  • the enriched repertoire can either be amplified as a polyclonal pool and used for further rounds of selection in an iterative manner even until convergence to desired properties is achieved or be spatially separated and screened for a desired function on a single clone level.
  • Phage particles for the next round of selection were produced by super- infecting exponentially growing cultures of the previous round of selection with helper phage VCS-M13 (Stratagene, La Jolla, CA) and growing the cultures overnight at 20°C in 2xTY supplemented with 100 ⁇ g/ml ampicillin and 50 ⁇ g/ml kanamycin. Selection ready phage were precipitated with 0.5 M NaCl/4% PEG-6000 from the cleared bacterial supernatant and re-suspended in PBS. In this example two rounds of selection were performed followed by screening on a single clone level.
  • helper phage VCS-M13 (Stratagene, La Jolla, CA)
  • the genes encoding the selected scFv, contained in the phage display vector were re-cloned to the expression vector pXP14.
  • This vector directs the expression of a scFv in fusion with a Streptag and E-tag and does not contain a filamentous phage gene-3.
  • Expression vector containing E. coli TGI from single colonies were grown in individual wells of a microtiter plate so that each well contains only one scFv clone.
  • the bacteria were grown at 30°C in 2xTY supplemented with 100 ⁇ g/ml ampicillin and 0.1 %> glucose in 96-well microtiter plates (#9297, TPP) until an OD600 of 0.7.
  • Expression was induced with IPTG at a final concentration of 0.5 mM and continued at 25°C overnight.
  • Single chain Fv containing cleared lysates were prepared by addition of hen-egg lysozyme (#L- 6876, Sigma) to a final concentration of 50 ⁇ g/ml for 1 hour at 25°C and centrifugation for 15 minutes at 3000xg. Prior to the screening ELISA, the cleared lysates were blocked by the addition of an equal volume of DMEM + 10% FCS for 1 hour. For the screening ELISA, HT1080 cells were seeded in a 96-well microtiter plate (#9296, TTP) at a density of 3x104 cells/well in DMEM + 10% FCS overnight at 37°C.
  • Sequencing of scFv genes was performed by Sequiserve GmbH, Vaterstetten, Germany using the primer pXP2 Seq2 (5'-CCCCACGCGGTTCCAGC-3'; SEQ ID No. 41) and pXP2 Seql (5 ACCTATTGCCTACGGC-3'; SEQ ID No. 42). Amino acid sequences are shown in Table 1 and nucleotide sequences are shown in Table 2. Unique clones identified by sequencing were streaked out from glycerol stocks onto LB/Amp(100 ⁇ g/ml)/l% Glucose Agar plates and incubated o/n at 30°C.
  • the lysed bacteria were centrifuged at 20000 x g for 15 minutes at 4°C and the supematants (lysate) transferred to a 15 ml plastic tube.
  • the lysates were loaded with 1 ml/min onto 1ml StrepTactin (# 2-1505-010, IBA) columns equilibrated with 10 column volumes (CV) PBS-0.5 M Na buffer via a parallel protein purification system (self-made). After a 10 CV wash with PBS the elution was done with 5 CV PBS/5 mM Desthiobiotin (#D-1411, Sigma) and 1 ml fractions collected.
  • fractions were measured at UV280, protein containing fractions were pooled and concentrated with Amicon Ultra Centrifugal Filter Devices 10.000 MWCO (#UFC801024, Millipore) at 4700 x g.
  • the concentrated scFv were checked on 12% Bis-Tris SDS-PAGE gels stained with Coomassie Blue for purity and frozen in aliquots with 20 % glycerol at -80°C.
  • the scFvs consist of the sequence of a variable light and heavy chain linked by a linker sequence.
  • the variable light chain and the variable heavy chain were amplified by PCR separately with the usage of primer, which contain restriction sites. Those restriction sites are also present in the vectors, which contain the appropriate constant domains for the heavy and light chain.
  • the amplified variable domains were cut with the restriction enzymes and cloned into the cut vectors. The correct sequence was confirmed via sequencing
  • the vectors contained an Epstein Barr virus origin of replication (oriP sequence) which enhances the level of transcription in 293- EBNA-HEK cells, because the EBNA protein leads to the replication of the episomal vector.
  • oriP sequence Epstein Barr virus origin of replication
  • a co-transfection was carried out with the vector for the heavy chain and the vector for the light chain leading to the expression of both chains in the cell and the assembly of the IgG in the Endoplasmic Reticulum.
  • the assembled IgG was then secreted to the medium.
  • Calcium-phosphate transfection was used, where a precipitate of Calcium-phosphate and the DNA is formed and incorporated into the cell. After the transfection the medium was changed to serum-free medium. Three harvests per IgG were done every 3 days. The supernatant (media) were sterile-filtrated and stored at 4°C.
  • the supematants were purified via Protein A Sepharose either by gravity flow or by HPLC depending on the volume.
  • FACS fluorescence-activated cell sorter
  • Example 5 Invasion assay The ChemoTx® system (Neuro Probe Inc.#106-8, Gaithersburg) was used as a disposable chemotaxis/cell migration chamber in a 96 well format with an 8 ⁇ m filter Track etched Polycarbonate pore size, 5.7 mm diameter/site.
  • Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm- Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins. Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycan, entactin and nidogen.
  • TGF- fibroblast growth factor also contains TGF- fibroblast growth factor, tissue plasminogen activator, and other growth factors which occur naturally in the EHS tumor
  • Becton Dickenson, BD #356234 diluted in Dulbeccos PBS (Gibco #14040-091) was applied on the membrane filter of the 96-well plate on row B-H and on row A 1,2 ⁇ g/site of collagen S type I (Roche #10982929) diluted in 0,05 M HCl (Sigma #945-50) and incubated over night at 20°C in a desiccator for gelation.
  • HT1080 cells were grown to 70-80% confluence in DMEM supplemented with Glutamaxl (862mg/l (Gibco #31966-021) with 10% FCS (Gibco #10270106). The cells were then labeled in situ with Bisbenzimide H 33342 (Sigma #B-2261) diluted 1 :100 in DMEM/GlutamaxI/0.1 % BSA (Sigma #A-7030) for 15 min at 37°C, 7,5% CO 2 . Cells were washed 2x with DMEM/GlutamaxI/0.1 % BSA and loaded with DMEM/GlutamaxI/0.1 % BSA for 15 min at 37°C, 7,5% CO 2 for recovering.
  • Bisbenzimide H 33342 Sigma #B-2261
  • DMEM/GlutamaxI/0.1 % BSA Sigma #A-7030
  • the cells were detached with 0.5mM EDTA (Sigma #E8008), collected with Dulbeccos PBS/0.1% BSA/lOmM Hepes (Gibco #15630-056), washed 2x with Dulbeccos PBS/0.1% BSA lOmM Hepes, suspended in Dulbeccos PBS/0.1% BSA/lOmM Hepes and diluted to 6,7 x 10 6 cells/ml with Dulbeccos PBS/0.1% BSA/lOmM Hepes.
  • DMEM/GlutamaxI with 5% FCS was used as a chemo attractant in the lower chamber.
  • a standard curve from lxlO 4 to 4x10 cells/site is performed on collagen S type I coated row A of the chemotaxis chamber.
  • DMEM/GlutamaxI/0.1%> BSA was used in the lower chamber (cells are not migrating).
  • fluorescence of cells, which had migrated through the membrane was measured on the Fluostar Galaxy (bMG) microplate reader using excitation/emission wavelengths of 370/460nm.
  • bMG Fluostar Galaxy
  • Viable cells were detected by measuring the conversion of the tetrazolium dye MTS (MTS, Celltiter Aqueous one, Promega #G4000) to formazan.
  • MTS tetrazolium dye
  • HT1080 cells and HT1080 cell/scFv dilutions obtained from the dilutions prepared in the Invasion assay were pipetted in triplicate at a density of 3,4 x 10 4 cells/well were plated in a 96-well plate (black, ultra thin clear flat bottom, special optics, Costar #3615) 10 ⁇ l MTS was added to each well and incubated for 1 hour at 37°C, 7,5%) CO .
  • Absorbance was measured at 492 nm with the Fluostar Galaxy (bMG) microplate reader. For all tested scFvs, no effect on viability of cells was seen.
  • SW-480 and PC-3 cells were cultured in RPMI containing L-Glutamine and 10%) FCS (Invitrogen #21875-034, Carlsbad, California).
  • SW-480 and PC-3 cells were seeded in culture medium in a volume of lOO ⁇ l/well into a 96well plate (Corning Costar #3904, Acton, Massachusetts). Cells were incubated 24h at 37°C, 5% CO 2 . Medium was aspirated and 15 ⁇ g/ml antibody or 50 mM NaN 3 was added diluted in culture medium. Control cells were incubated in medium alone. 48h after the first addition, antibody was added a second time at the same concentration. MTS absorbance was measured before, 24h, 48h and 72h after the first treatment.
  • TPP #9296 cell culture treated
  • collagen S type I l ⁇ g/well (Roche #10982929)
  • Dulbeccos PBS (Gibco #14040-091) at 4°C over night.
  • the Adhesion assay was also performed using several matrix proteins, such as CIV (collagen IV), FN (fibronectin) and LN (laminin), and with various cell types, such as HT1080 (human fibrosarcoma), PC-3 (human adenocarcinoma, prostate), HeLa (human cervix carcinoma) and HT-29 (human colon carcinoma) cells. Additionally to the scFv also IgG cloned from the single chains according to Example 3.2 were tested for their capacity to inhibit adhesion of tumor cells. Results are shown in Figure 8.
  • CIV collagen IV
  • FN fibronectin
  • LN laminin
  • HT1080 human fibrosarcoma
  • PC-3 human adenocarcinoma
  • HeLa human cervix carcinoma
  • HT-29 human colon carcinoma
  • single cell suspensions of HT1080 were harvested with 0,5mM EDTA/PBS. Approximately 1 x 10 6 cells were incubated in CellWash (BD, #349524) with lO ⁇ g/ml scFv for one hour at 4°C. After washing with Cell Wash lO ⁇ g/ml FITC labeled scFv was added and incubated for 20 min at 4°C. Signals of bound FITC labeled scFvs with and without pre incubation of other binders were analyzed on a Becton Dickinson FACSscan.
  • Example 9 Immunoprecipitation HT1080 and Hs-27 cells (10 8 ) were lysed in 3ml 50 mM Tris-HCl, pH 8.0, 150 mM NaCI, 1% Triton X-100 (v/v) containing protease inhibitor cocktail (1 pill in 50ml buffer) (Boehringer Mannheim, Cat.-No. 1697498) and 100 ⁇ M Pefablock (Roth, Cat. -No. A154.1). Lysates were pre-incubated for 2h at 4°C with Streptactin Sepharose (IBA, # 2- 1201-010) and the supematants used for the immunoprecipitation reactions.
  • IBA Streptactin Sepharose
  • HT1080 specific single chain Fv 50 ⁇ g/1 mg cell extract
  • the immuno-complexes were separated by SDS-PAGE and silver stained for MS analysis.
  • scFv 2 pulled down a protein, detected as a band on SDS-PAGE by silver staining at a molecular weight of approximately 120 kDa. This band was absent in the control samples with Hs-27 cells.
  • Immunoprecipitations were performed using cell extract from HT1080 cells and Hs-27 cells as control cells. It was observed that this particular band was also present in the control samples, but in all cases to a much lower extent indicating that this protein is likely to be over-expressed in the HT1080 cell line. In the case of the scFv3 this band was only detected when HT1080 cell extract was used and not with the Hs-27 cells.
  • Single chain Fv 4, 11 and 14 pulled down 2 proteins, detected as 2 bands on SDS-PAGE by silver staining at a molecular weight of approximately 150 kDa and approximately 130 kDa, respectively.
  • the upper band was also present in the control samples, but a much lower extent, whereas the lower band was absent or extremely faint in the controls, indicating that the 2 proteins are likely to be overexpressed in the HT1080 cell line.
  • Example 10 Protein identification via mass spectrometry The gel bands obtained from immunoprecipitations followed by SDS PAGE were subjected to a tryptic in-gel digest over night at 37 °C. Peptides were extracted using 5% formic acid and the resulting peptide mixture was desalted using ZipTip ⁇ C18 (Millipore) and eluted first with 2 ⁇ l of 30% ACN/0.1% TFA, then with 2 ⁇ l of 70% ACN/0.1% TFA.
  • the two fractions were pooled and one microliter of the obtained peptide mixtures was mixed in a 1 :1 ratio with a solution of -cyano-4-hydroxy cinnamic acid (3 mg/ml), co- crystallized on a Teflon-coated stainless steel target and analyzed on a MALDI-TOF instrument yielding peptide mass fingerprints (PMF) in a mass range of m/z 800-3000.
  • the obtained PMF were used to search all entries for the species Homo sapiens in the NCBI and SwissProt databases. In all cases, only peptides matching a given protein with a mass deviation of less than 13 ppm were considered for identification.
  • the band with an approximate size of 110 kDa yielded 6 to 15 peptides in different MALDI-TOF-MS experiments, which matched cell surface glycoprotein MUC18 precursor, with a maximum protein coverage of 23% (151/646 residues). Multiple entries of the same protein are available in the database, which contain slight deviations in the amino acid sequences.
  • a MALDI spectrum that led to the identification of MCAM is shown in Figure 11a.
  • the peptide coverage is shown in Figure lib.
  • the molecular weight of any of the melanoma adhesion proteins is around 72 kDa, but since the protein is expected to be glycosylated it is not surprising to find the band on the gel around 110 kDa.
  • the molecular weight of this protein is around 119 kD, but it is expected to be glycosylated, thus explaining the higher observed molecular weight observed on SDS- PAGE.
  • the band with an approximate size of 150 kDa which is the higher one of the two bands obtained by using scFvl l-scFvl4, yielded 17 to 21 peptides, which matched integrin alpha-2, with a maximum protein coverage of 19% (225/1181 residues).
  • the molecular weight of integrin alpha-2 is around 129 kDa
  • the molecular weight of integrin beta-1 is around 88 kDa.
  • both proteins are expected to be highly glycosylated, which might explain the higher apparent molecular weight observed on SDS- PAGE.
  • Example 11 Methods for epitope mapping
  • fragments of the cDNA for the antigen of interest are expressed as recombinant (fusion)proteins and probed in various assays such as Westernblot or ELISA.
  • Example 11.2 Phage display technology The technique of epitope mapping using random peptide phage display libraries was developed to clone small random fragments of the cDNA for the antigen of interest into the phage protein pill of filamentous phages and display them on the surface of the phage (Fack et al., (1997) J. Immunol. Methods 7, 43-52). Epitope-displaying phages can be captured with antibodies in a procedure called "bio-panning”. Sequencing of the inserts of the corresponding phages gives some information on the epitopes. This procedure is in principle capable to identify conformational epitopes.
  • oligo-peptide chains are stepwise synthesized from C- to the N-terminus. Oligo-peptides containing natural as well as modified amino acids can be synthesized up to a length of 20 amino acids. Following synthesis the membranes are equilibrated and unspecific binding sites are blocked.
  • Example 13 Anti-tumor effect of compounds in subcutaneously growing human tumor xenografts
  • a lung adenocarcinoma (bronchial adenocarcinoma) was used as a xenograph.
  • Nude mice of NMRI background approximately 7 weeks old at the start of treatment with an average weight of 35g were used.
  • Animal health was examined before study commencement to ensure that only animals of good health entered testing procedures. Identification of mice was achieved by individual ear tag number, each cage by labeling with a record card, indicating the number of the experiment, date of randomization, mouse strain, gender, individual mouse number, test compound, dosage, schedule, and route of administration.
  • the animals were housed in Macrolon M type III cages with filter hoods in air-conditioned rooms at 24 ⁇ 1°C and relative humidity at 60 ⁇ 10%o and fed Altromin Extrudat 1439 Rat/Mouse diet, demineralized sterilized water containing 0.9 g/1 potassium sorbate with the pH adjusted to 2 with IN HCl. Water consumption was monitored visually daily, food and water provided ad libitum.
  • the animal bedding S AWI produced by Altromin GmbH was analyzed and certified by Jelu Werk and autoclaved and distributed by Charles River and renewed twice a week. Antibodies were administered at a single high dose level (50 mg/kg twice a week for 4 weeks).
  • the group size is 6 mice and in order to obtain 24 mice with similar-sized tumors, 32 mice were implanted with a total of 64 tumor fragments (bilateral implantation).
  • Tumor fragments for implantation were obtained from xenografts in serial passage in nude mice. After removal of the tumor from donor mice, the tumor was cut into fragments (2-3 mm diameter) and placed in RPMI 1640 culture medium (maximally for 30-45 minutes) until subcutaneous implantation in the mice.
  • the mice were anaesthetized by inhalation of vaporized isoflurane and two small incisions were made in the skin of the back (left and right), and 2 tumor fragments were transplanted per mouse. Treatment started if tumors had grown to mean diameters of between 6 and 8 mm.
  • animals were divided into 3 categories according to tumor diameter: large: >8 mm; medium: between 6 and 8 mm; small: 5 mm. At randomization, tumor categories were equally distributed among the different groups.
  • Tumor size and mouse body weight were measured twice a week and mortalities and clinical signs recorded daily.
  • Relative volume of individual tumors was calculated as the ratio between the tumor volume on Day x and the tumor volume on Day 0 (Tx/TO, Day 0 is the day of randomization and the first day of treatment).
  • Relative body weights of individual mice were calculated correspondingly. Plots of the relative tumor volume and the relative body weight over time were generated.
  • T/C% value the ratio of the median relative tumor volumes for the antibody-treated and the vehicle-treated groups on a particular day was calculated (T/C% value).
  • a minimum requirement for rating an antibody as active was a minimum T/C% value of ⁇ 50%>.
  • Tumor-bearing mice were treated twice a week for 4 weeks.
  • Tumors were collected at study termination for further analysis. A comparison of the tumor size after different treatments at Day 7 is shown in Figure 13.
  • blood samples of 300 ⁇ l were retrieved by sublingual bleeding of mice in the antibody-treated group at 6 different time points during the in vivo therapy study. Trough levels were determined on Day 4 just before administration of the second dose and on Day 22 before admmistration of the 7 th dose.
  • Blood samples (three blood samples per time point) were obtained on Day 26, 27, 28 and 29. Samples on Days 4, 26 and 28 were obtained from mice 1-3, samples on Days 22, 27 and 29 from mice 4-6. The blood sample size was sufficient to prepare at least 100 ⁇ l plasma. Plasma samples were prepared using EDTA, stored at -80°C until analysis.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Wood Science & Technology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne des polypeptides inhibant la fonction de la molécule MCAM dans des cellules tumorales d'origine naturelle ainsi que leur utilisation. Cette invention concerne également l'utilisation d'autres molécules inhibant la fonction de la MCAM dans le traitement de cancers, notamment afin de réduire le pouvoir envahissant, la prolifération, l'adhésion et/ou le potentiel métastatique de cellules cancéreuses spécifiques. Ladite invention concerne en outre une méthode permettant de déterminer si le pouvoir envahissant, l'adhésivité, la prolifération et/ou le potentiel métastatique d'une cellule tumorale d'origine naturelle dépendent de MCAM fonctionnelles ainsi qu'une méthode permettant d'identifier un anticorps ou un fragment d'anticorps capable d'inhiber le pouvoir envahissant, la prolifération ou l'adhésivité de cellules tumorales.
EP03763867A 2002-07-15 2003-07-15 Inhibiteurs de mcam Withdrawn EP1601695A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP03763867A EP1601695A1 (fr) 2002-07-15 2003-07-15 Inhibiteurs de mcam

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP02015591 2002-07-15
EP02015591A EP1382615A1 (fr) 2002-07-15 2002-07-15 Inhibiteurs de MCAM
US41033102P 2002-09-11 2002-09-11
US410331P 2002-09-11
PCT/EP2003/007661 WO2004007550A1 (fr) 2002-07-15 2003-07-15 Inhibiteurs de mcam
EP03763867A EP1601695A1 (fr) 2002-07-15 2003-07-15 Inhibiteurs de mcam

Publications (1)

Publication Number Publication Date
EP1601695A1 true EP1601695A1 (fr) 2005-12-07

Family

ID=29762615

Family Applications (3)

Application Number Title Priority Date Filing Date
EP02015591A Withdrawn EP1382615A1 (fr) 2002-07-15 2002-07-15 Inhibiteurs de MCAM
EP03763867A Withdrawn EP1601695A1 (fr) 2002-07-15 2003-07-15 Inhibiteurs de mcam
EP03763866A Withdrawn EP1592792A1 (fr) 2002-07-15 2003-07-15 Criblage proteomique pour identifier des molecules biologiques associees a des maladies et leurs inhibiteurs

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP02015591A Withdrawn EP1382615A1 (fr) 2002-07-15 2002-07-15 Inhibiteurs de MCAM

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP03763866A Withdrawn EP1592792A1 (fr) 2002-07-15 2003-07-15 Criblage proteomique pour identifier des molecules biologiques associees a des maladies et leurs inhibiteurs

Country Status (6)

Country Link
EP (3) EP1382615A1 (fr)
JP (1) JP2006516085A (fr)
CN (1) CN1675244A (fr)
AU (2) AU2003250956A1 (fr)
CA (1) CA2492559A1 (fr)
WO (2) WO2004007550A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2650311A2 (fr) 2007-11-27 2013-10-16 Ablynx N.V. Séquences d'acides aminés dirigées contre des cytokines hétérodimériques et/ou leurs récepteurs et polypeptides les comprenant

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7601351B1 (en) 2002-06-26 2009-10-13 Human Genome Sciences, Inc. Antibodies against protective antigen
EP1539235A2 (fr) 2002-07-01 2005-06-15 Human Genome Sciences, Inc. Anticorps qui se lient specifiquement a reg iv
GB0404936D0 (en) * 2004-03-04 2004-04-07 Univ London Screen
CN101389756A (zh) * 2006-02-23 2009-03-18 田边三菱制药株式会社 单克隆抗体、编码该抗体的基因、杂交瘤、药物组合物和诊断试剂
USD564883S1 (en) * 2006-03-21 2008-03-25 Saverglass, Inc. Bottle
US8450278B2 (en) 2008-10-15 2013-05-28 Board Of Regents, The University Of Texas System MUC18 targeting peptides
EP2216399A1 (fr) * 2009-01-30 2010-08-11 Université de la Méditerranée CD146 humaine soluble, preparation et utilisations
US20130096070A1 (en) * 2009-04-07 2013-04-18 University Of Kansas Peptide targeting of inner ear cells
US8293468B2 (en) 2009-06-10 2012-10-23 Centre Hospitalier De L'université De Montréal MCAM modulation and uses thereof
EP2491401B1 (fr) * 2009-10-21 2018-06-06 Mycartis N.V. Mcam en tant que biomarqueur pour l'homéostasie des fluides
US9220733B2 (en) 2012-03-19 2015-12-29 The Regents Of The University Of California Solubilization of antigen components for removal from tissues
TW201922795A (zh) * 2012-09-10 2019-06-16 愛爾蘭商尼歐托普生物科學公司 抗ｍｃａｍ抗體及相關使用方法
UY35457A (es) 2013-03-14 2014-10-31 Bayer Healthcare Llc ANTICUERPOS MONOCLONALES CONTRA ANTITROMBINA ß
TW201623331A (zh) * 2014-03-12 2016-07-01 普羅帝納生物科學公司 抗黑色素瘤細胞黏著分子（ｍｃａｍ）抗體類及使用彼等之相關方法
EP3116911B8 (fr) * 2014-03-12 2019-10-23 Prothena Biosciences Limited Anticorps anti-mcam et procédés d'utilisation associés
US10059761B2 (en) 2014-03-12 2018-08-28 Prothena Biosciences Limited Anti-Laminin4 antibodies specific for LG4-5
CN104725483A (zh) * 2015-04-06 2015-06-24 苏州普罗达生物科技有限公司 一种黑色素瘤细胞粘附分子拮抗剂多肽及其应用
CN104725489A (zh) * 2015-04-06 2015-06-24 苏州普罗达生物科技有限公司 关于黑色素瘤细胞粘附分子拮抗剂多肽及其应用
CN104710513A (zh) * 2015-04-06 2015-06-17 苏州普罗达生物科技有限公司 黑色素瘤细胞粘附分子拮抗剂多肽及其应用
CA3019588A1 (fr) * 2016-04-20 2017-10-26 Merck Sharp & Dohme Corp. Proteines de liaison a un antigene de neutralisation des cmv
US11899020B2 (en) 2017-03-31 2024-02-13 The Regents Of The University Of California Shotgun proteomic antigen identification
WO2018220467A1 (fr) 2017-05-30 2018-12-06 Foroogh Nejatollahi Immunotoxine humaine anti-muc18 et ses applications

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000058501A2 (fr) * 1999-03-26 2000-10-05 The Wistar Institute Nouveaux elements suppresseurs genetiques et methodes de fabrication
WO2001077153A2 (fr) * 2000-04-10 2001-10-18 The Curators Of The University Of Missouri Selection par expression phagique de peptides antifongiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004007550A1 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2650311A2 (fr) 2007-11-27 2013-10-16 Ablynx N.V. Séquences d'acides aminés dirigées contre des cytokines hétérodimériques et/ou leurs récepteurs et polypeptides les comprenant
US8975382B2 (en) 2007-11-27 2015-03-10 Ablynx N.V. Amino acid sequences directed against HER2 and polypeptides comprising the same for the treatment of cancers and/or tumors
US9969805B2 (en) 2007-11-27 2018-05-15 Ablynx N.V. Amino acid sequences directed against HER2 and polypeptides comprising the same for the treatment of cancers and/or tumors

Also Published As

Publication number Publication date
EP1382615A1 (fr) 2004-01-21
JP2006516085A (ja) 2006-06-22
CA2492559A1 (fr) 2004-01-22
EP1592792A1 (fr) 2005-11-09
CN1675244A (zh) 2005-09-28
AU2003250065A1 (en) 2004-02-02
WO2004007717A1 (fr) 2004-01-22
WO2004007550A1 (fr) 2004-01-22
AU2003250956A1 (en) 2004-02-02
WO2004007717A8 (fr) 2005-05-12

Similar Documents

Publication Publication Date Title
WO2004007550A1 (fr) Inhibiteurs de mcam
US20120058117A1 (en) Neuropilin-1 inhibitors
US20090215175A1 (en) Modulation of the poliovirus receptor function
EP3878863A1 (fr) Anticorps anti-claudin18.2 et son utilisation
EP1623997B1 (fr) Anticorps de recombinaison et fragments reconnaissant le ganglioside n-glycolyl gm3 et leur utilisation dans le diagnostic et le traitement des tumeurs
CN109369808B (zh) 用于治疗ror1癌症并抑制转移的抗体和疫苗
MXPA05006828A (es) Anticuerpos contra pd-1, y sus usos.
EP3851455B1 (fr) Anticorps monoclonal se liant de manière spécifique à l'antigène cd38 humain et de singe, son procédé de préparation et son utilisation
CN110121509A (zh) 中和抗tl1a单克隆抗体
HUE026846T2 (en) Antibodies to Recognize Carbohydrate-Containing Epitope on CD43 and CEA Expressed on Cancer Cells and Procedures for their Use
MXPA04006980A (es) Anticuerpos novedosos anti-receptor del factor de crecimiento i tipo insulina, y usos de los mismos.
EP3741777A1 (fr) Anticorps pd-l1, fragment de liaison à l'antigène de celui-ci et utilisation pharmaceutique associée
KR102611444B1 (ko) 플렉틴-1 결합 항체 및 그의 용도
CA2463943A1 (fr) Anticorps humains presentant une activite de liaison a mn et de neutralisation de l'adhesion cellulaire
CN101370826A (zh) 抗分泌型卷曲相关蛋白-4(sfrp-4)的抗体
AU2004294893A1 (en) Antibodies specific for cancer associated antigen SM5-1 and uses thereof
EP4424708A1 (fr) Anticorps dérivé d'un survivant de cancer et son utilisation
KR102589409B1 (ko) Gpr87 결합 항체
WO2023124857A1 (fr) Anticorps et son utilisation
RU2735956C1 (ru) Анти-dr5-антитело и его применение

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050215

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20061013