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
  • C07K16/2896—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
• • 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
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
• • 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
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • 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

• the invention relates to the field of medicine.
• the invention further relates to disease-associated molecular markers and methods of obtaining and using these.
• the invention also relates to the diagnostic and/or medical use of binding molecules capable of recognizing and/or binding said disease-associated molecular markers.
• B-lymphocytes can produce antibodies in response to exposure to biological substances like bacteria, viruses and their toxic products. Antibodies are generally epitope specific and bind strongly to said biological substances carrying these epitopes.
• the hybridoma technique makes use of the ability of the B cells to produce monoclonal antibodies to specific antigens and to subsequently isolate and produce monoclonal antibodies by fusing B cells from mice exposed to the antigen of interest to immortalized murine plasma cells. This technology resulted in the realization that monoclonal antibodies produced by hybridoma' s could be used in research, diagnostics and therapies to treat different kinds of diseases like cancer and auto-immune related disorders.
• binding molecules Much interest is currently directed toward the use of binding molecules in the treatment of human disease.
• One application is the use of binding molecules to facilitate removal of undesired cells from a body.
• the binding molecule must comprise sufficient specificity for the cells to be removed in order not to result in undesired side effects.
• Such binding molecules comprise antibodies.
• An antibody is capable of binding to an epitope expressed by an undesired cell and thereby mark said cell for removal from the body. This can be done in several ways for instance mediated by the immune system or the complement system or a combination thereof. Removal can also be achieved in (combination with) other ways.
• Antibodies made in vivo can be capable of binding strongly to the invading micro-organisms or their products that elicited their production and aid in their elimination.
• the activity of the immune system of producing antibodies in response to an invading microorganism has been exploited in the production of monoclonal antibodies, a technology developed by K ⁇ hler and Milstein (1975) .
• monoclonal antibodies are all those immunoglobulin molecules that are produced by the progeny of a single B lymphocyte.
• monoclonal antibodies are obtained by immunizing a mouse with an antigen and fusing the spleen or lymphnode B lymphocytes with an immortalized murine plasma cell line.
• hybrid cell lines bear the characteristics of both parental cell types: they are immortal and produce a single species of monoclonal antibody specific for the antigen used to immunize the mouse.
• the advantage of monoclonal antibodies is that they represent a homogeneous population of immunoglobulin molecules with a pre-defined binding specificity. Over the years, monoclonal antibodies have proven invaluable tools in research and diagnostics.
• monoclonal antibodies were hypothesized, among others, to be capable of binding to viruses and bacteria and their toxic products facilitating their elimination.
• monoclonal antibodies were envisaged to specifically bind to tumor cells to promote their eradication or to bind to soluble molecules produced by cells of the immune system to neutralize their activity in harmful chronic inflammatory conditions and/or in autoimmune disease.
• monoclonal antibodies have been described as magic bullets that could be used in the treatment of a wide variety of human diseases (Bodey et al. 2000).
• the immunoglobulin variable regions of the murine monoclonal antibodies are genetically fused to human immunoglobulin constant regions (Fig.l).
• the resulting chimeric monoclonal antibody still contains >30% murine amino acid sequences.
• Clinical application in humans of chimeric monoclonal antibodies has shown that these proteins retain their immunogenicity in the majority of cases (Khazaeli et al. 1989; Elliot et al. 1994) .
• only immunoglobulin variable region sequences relevant for monoclonal antibody specificity are of murine origin; the constant regions of the immunoglobulin molecule as well as the framework regions of the variable region are of human origin (Fig.l).
• One method of obtaining human monoclonal antibodies employs transgenic mice harboring human immunoglobulin loci in combination with conventional hybridoma technology (Bruggeman and Neuberger 1996; Mendez et al. 1997) .
• human immunoglobulin heavy and light chain loci have been inserted in the mouse germ line while the endogenous murine immunoglobulin loci have been silenced by gene knockout.
• Immunization of these transgenic mice with an antigen results in the production of human antibodies specific for the antigen.
• Human monoclonal antibody-producing cell lines can be obtained from these mice by fusing the spleen cells of immunized mice with plasma cell lines in vitro to obtain immortalized monoclonal antibody-secreting hybridomas.
• phage display libraries Another method to obtain fully human monoclonal antibodies with desirable binding properties employs phage display libraries. This is an in vitro, recombinant DNA- based, approach that mimics key features of the humoral immune response (Burton et al. 1994).
• phage display libraries collections of human monoclonal antibody heavy and light chain variable region genes are expressed on the surface of bacteriophage particles, either in single chain Fv (scFv) or in Fab format.
• scFv single chain Fv
• Large libraries of antibody fragment-expressing phages typically contain > 10 9 antibody specificities and may be assembled from the immunoglobulin V regions expressed in the B lymphocytes of immunized or non-immunized individuals.
• phage display libraries may be constructed from immunoglobulin variable regions that have been partially assembled in vitro to introduce additional antibody diversity in the library (semi-synthetic libraries) .
• in vitro assembled variable regions contain stretches of synthetically produced, randomized or partially randomized DNA in those regions of the molecules that are important for antibody specificity.
• Recombinant phages expressing antibody fragments of desirable specificities may be selected from a library by one of several methods.
• Target antigens are immobilized on a solid phase and subsequently exposed to a phage library to allow binding of phages expressing antibody fragments specific for the solid phase-bound antigen.
• Non-bound phages are removed by washing and bound phages eluted from the solid phase for infection of Escherichia coli (E. coli ) bacteria and subsequent propagation. Multiple rounds of selection and propagation are usually required to sufficiently enrich for phages binding specifically to the target antigen. Phages may also be selected for binding to complex antigens such as complex mixtures of proteins or whole cells. Selection of antibodies on whole cells has the advantage that target antigens are presented in their native configuration, unperturbed by conformational changes that are introduced by immobilizing an antigen to a solid phase. The constraints imposed by the natural immune response and the influence of the immunogenicity of the target antigen do not permit the isolation of monoclonal antibodies against any antigen by conventional hybridoma technology.
• phage display libraries are used in combination with flow cytometry and cell sorting to isolate antibody fragments against molecules expressed on the plasma membrane of subpopulations of eukaryotic cells present in a heterogeneous mixture (US patent 6,265,150; De Kruif et al. 1995a). These published methods do not describe the processes of the present invention provided herein.
• a heterogeneous mixture of cells is incubated with the phage library allowing phages to bind to the different cell types.
• the cells are stained with fluorochrome-labeled monoclonal antibodies to permit identification of the subpopulation of target cells by immunofluorescence analysis and flow cytometry.
• Target cells and attached phages are collected by flow cytometry and the attached phages are eluted and propagated. This method is rapid, independent of the immunogenicity of the target antigen and yields antibody fragments against molecules in their native configuration. Specific antibodies against very small populations of cells in a heterogeneous mixture can be obtained (De Kruif et al. 1995a and 1996) .
• scFv For production of intact human monoclonal antibodies, scFv with desirable specificities can be inserted into mammalian expression vectors containing the genes encoding human immunoglobulin constant regions.
• Transfected cell lines harboring these constructs produce human monoclonal antibodies in vitro that are correctly assembled and glycosylated.
• a chimeric monoclonal antibody against the CD20 antigen was shown to give a response in 48% of patients with relapsed low grade or follicular lymphoma (McLaughlin et al. 1998).
• treatment of patients with the monoclonal antibody is sufficient for clinical effect.
• Binding of the monoclonal antibody to the target antigen results in the recruitment of components of the complement system and effector cells of the immune system with Fc receptor for antibody constant regions that act in concert to kill the tumor cell.
• binding of a monoclonal antibody to a target antigen on the membrane of a cell does not always result in tumor cell killing.
• the specificity of the monoclonal antibody employed is crucial.
• the monoclonal antibody should specifically bind to tumor cells with minimal cross-reactivity with normal tissues. This criterium is not always met as illustrated by the chimeric anti-CD20 monoclonal antibody that was approved for clinical use (see below) ' .
• the CD20 antigen is expressed by B cell tumors but also by non-malignant immature and mature B lymphocytes. Despite this cross-reactivity with non-malignant B cells, patients receiving treatment with the chimeric anti- CD20 monoclonal antibody causes few side effects and the clinical success is considerable. It is clear that although several antibodies have been approved for clinical use (such as the anti-CD20 antibody) that there is a strong need for monoclonal antibody based treatments in which the antibody only and specifically targets the diseased cells.
• the present invention discloses methods and means to treat Multiple Myeloma (MM) , which is a hitherto incurable neoplastic disease of the B-cell lineage, characterized by the presence of multifocal loci of monoclonal plasma cells in the bone marrow (BM) .
• MM Multiple Myeloma
• the disease can present itself with homogeneous serum immunoglubulin, osteolytic lesions, anemia, uremia, hypercalcemia, hyperviscosity, amyloidosis, secondary immunodeficiency and renal insufficiency. These clinical symptoms are dependend of the location, tumor load and the pathophysiology of malignant plasma cells.
• Allogeneic Bone Marrow Transplantation may proof beneficial for myeloma patients when the observed graft versus myeloma effect can be maximally exploited and the problems of occurring graft versus host disease are circumvented.
• the invention provides methods for selecting myeloma specific antigens and their specific interacting proteins. More in particular, the invention discloses binding molecules that selectively interact with tumor specific antigens, whereas the antigens are expressed on myeloma cells, other tumor cells and not on the normal CD46 positive cell types analyzed thus far.
• Antibody mediated therapies in myeloma have thus far been rather unsuccessful, partly because of absence of antigens with a plasma cell restricted expression pattern.
• vaccination experiments in a mouse model system using a tumor idiotype with comparable patho-biological features as human multiple myeloma suggest that the immune system can effectively be mobilized against myeloma tumor cells (King et al. 1998) .
• the invention further provides a novel Multiple Myeloma tumor marker namely human CD46, which is also known as Membrane Cofactor Protein (MCP) and it provides more in particular, binding molecules that specifically interact with differentially glycosylated forms of human CD46.
• CD46 is a one- or two-band profile type 1 membrane protein of approximately 60.000 Dalton in molecular weight and is expressed on all nucleated cells. A soluble form of said protein was also found (Hara et al. 1992). Said protein protects host cells from autologous complement attack and serves as a measles virus receptor, facilitating virus to cell and cell-to-cell attachment and fusion (Naniche et al. 1993; Dorig et al. 1993; Iwata et al. 1995) . The fysiological role of CD46 is to protect the host cell from complement- mediated cell damage (Oglesby et al. 1992; Seya et al.
• CD46 consists of four short consensus repeat domains, known as complement control protein (or CCP) repeats: a Ser/Thr-rich (ST) domain, a 13 amino acid unique sequence followed by a transmembrane (TM) portion and a cytoplasmic (CY) tail.
• CCP complement control protein
• ST Ser/Thr-rich domain
• TM transmembrane
• CY cytoplasmic tail
• CD46 glycosylation of CD46 was suggested to play a role in complement regulatory functions (Liszewski et al. 1998) . To date six isoforms of CD46 have been identified in human cell lines, testis and placental cDNA libraries (Post et al. 1991) .
• CD46 complement regulatory proteins
• DAF decay-accelerating factor
• CD55 protectin
• CD59 protectin
• human CD46 rather than CD55 is a key element in protection against complement activation (Van Dixhoorn et al. 2000).
• Overexpression of CD46 was observed in gastro-intestinal tumors, carcinomas of breast, cervix and endometrium, and hepatocellular carcinomas (Murray et al. 2000; Kinugasa et al . 1999; Schmitt et al . 1999; Thorsteinsson et al. 1998; Simpson et al. 1997).
• the difference in expression profiles of this membrane complement regulatory protein between normal and pathological tissues suggest resistance of tumor tissue to complement-mediated damage, thereby allowing tumor cells to escape from cytolysis and thus promoting tumor outgrowth.
• anti-CD46 monoclonal antibodies can be very useful to overcome the limitation of the potential of monoclonal antibodies mediated by complement resistance.
• the invention provides methods for determining differentially expressed, folded and/or post- translationally modified proteins on cells, in particular differentially glycosylated CD46 forms on tumor cells.
• the potential for structural diversity of glycans in metazoan cells is very large given the possible combinations of monosaccharides, linkages, branching, and variable lengths of glycan chains.
• glycans The structural variability of glycans is dictated, among others, by tissue specific regulation of glycosyltransferase genes, acceptor and carbohydrate availability in the Golgi, associationalization, and by competition between enzymes for acceptor intermediates during glycan elongation (reviewed in Dennis et al. 1999).
• tissue specific regulation of glycosyltransferase genes acceptor and carbohydrate availability in the Golgi, proximityalization
• enzymes for acceptor intermediates during glycan elongation Reviewed in Dennis et al. 1999.
• glycan structures At any particular glycosylation site of a mature glycoprotein, a range of biosynthetically related glycan structures may be present.
• the prevalence of particular glycan structures on specific glycoprotein molecules can affect their functions, including half-life, localisation and biological activity. Aberrant glycosylation has been associated with tumor cells of epithelial origin.
• epitopes expressed by the MUC- I antigen expressed on epithelial tumors are targets for various forms of immunotherapy.
• Previous experiments have suggested that deveating glycosylation of membrane and secreted proteins may be a characteristic of plasma cells and the malignant cells in multiple myeloma.
• An incompletely sialylated form of CD44 on a myeloma cell has been described and immunoglobulins produced by myeloma cells have a distinct oligosaccharide profile (Slupsky et al. 1993; Farooq et al. 1997) .
• Proteoglycans of B lymphocytes undergo structural changes during B cell ontogeny that may correspond to the specific requirements of the respective microenvironment of the maturing cell (Engelmann et al. 1995) . None in these studies suggests that certain post-translationally modified variants of proteins can or have been used to obtain specific binding molecules according to the present invention.
• Herceptin Trastuzumab
• HER2/neu is a proto-oncogen that due to amplification is found to be over-expressed in numerous malignant epithelial tumor cells.
• each of these antigens or disease associated molecular markers is characterized in that the marker is the result of the tissue specific expression or over-expression of an mRNA encoding the molecular marker, either through differential RNA transcriptional levels resulting in differential protein levels between healthy an diseased cells or between cells from different tissues, or through differentially RNA splicing patterns resulting in different splice variants from one particular gene.
• the CD55 (or Decay Accelerating Factor DAF) protein is a heavily glycosylated membrane protein for which different antibodies exist recognizing different subsets of the protein due to their glycosylation states.
• the inventors of the present invention realized that a wealth of disease associated molecular markers is waiting to be explored, which markers cannot be identified using conventional target- identification programs.
• the present invention fulfills in a need for methods and processes of obtaining this new type of molecular markers and developing medicines and therapies on the basis of these markers to diagnose, to prevent or to combat diseases with which the molecular markers are associated. Due to the present invention disease associated molecule markers can be identified and used for the development of medicines and therapeutic strategies against various diseases.
• the new type of disease associated molecular markers include, but are not limited to glycosylation variants, phosphorylation variants and conformational variants of otherwise known proteins and will for the purpose of this invention be referred to as disease associated molecular markers", or simply as "novel epitopes".
• Figure 2 Steps in selection of phages binding to subpopulations of cells using flow cytometry.
• a heterogeneous mixture of cells is incubated with the phage library. Non-binding phages are removed by washing.
• Cells with bound phages are stained with fluorochrome-labeled monoclonal antibodies and cells of interest are isolated using a cell sorter.
• Phages are eluted from the isolated cells and used to infect bacteria. Phage antibodies are isolated from single bacteria. IV. Phage antibodies are finally used in flow cytometric and immuno-histochemical analysis to assess the tissue and cellular distribution of the target antigen.
• CD46 Binding to glycosylation variants of CD46 by human monoclonal antibodies K19, K53 and the conventional murine anti-CD46 antibody J4.48, here depicted as CD46. Binding was determined in CHO cells, which were stably transfected with normal full length CD46 (BC1) , with NQ replacements of CCPl (NQ1)/ CCP2 (NQ2)/ CCP4 (NQ4) or with a CD46 deletion mutant in the serine/threonine/proline rich domain ( ⁇ STP) .
• BC1 normal full length CD46
• NQ1 NQ replacements of CCPl
• NQ2 CCP2
• NQ4 CCP4
• ⁇ STP serine/threonine/proline rich domain
• FIG. 12 A -(C) Analysis of bone marrow cells derived from non- multiple myeloma patients stained with fully human monoclonal antibody K53/IgGl (here depicted as L53) and negative control GBS III (here GBS3) . Abbreviations are explained in example 7. The gates were set for different markers (CD20, CD45 and CD19) as indicated.
• Figure 15 Staining of human colon tumor tissue using fully human monoclonal antibody K53/IgGl (left) and GBS III as a negative control (right) .
• Figure 16 Effect of tunicamycin on binding of fully human monoclonal antibody K53/IgGl (here indicated as K53, black bars), negative control antibody GBS III (open bars) and conventional murine anti-CD46 antibody J4.48 (here indicated as CD46, gray bars) to (A) LS174T colon tumor cells and on (B) T47D breast cancer cells.
• C Effect of swainsonine on binding of the antibodies mentioned in (A) and (B) to LS174T colon tumor cells.
• mice Mean tumor size in Balb/c (nu/nu) mice xenografted with the human colon carcinoma cell line LS174T and treated with fully human monoclonal K53/IgGl or control antibodies GBS III and UBS-54 antibodies on day 1, 3 and 6 (Group A) . All mice were included. When no tumor developed, the tumor size was adjusted to 0.
• mice xenografted with the human colon carcinoma cell line LS174T and treated with fully human monoclonal antibody K53/IgGl or control antibodies GBS III and UBS-54 on day 9, 12 and 15 (Group C) . All mice were included. When no tumor developed, the tumor size was adjusted to 0.
• Figure 21 Mean tumor size in Balb/c (nu/nu) mice xenografted with the human colon carcinoma cell line LS174T and treated with fully human monoclonal antibody K53/IgGl or control antibodies GBS III and UBS-54 on day 1, 3 and 6 (Group A) . Only tumor bearing mice were included.
• mice Mean tumor size in Balb/c (nu/nu) mice xenografted with the human colon carcinoma cell line LS174T and treated with fully human monoclonal antibody K53/IgGl or control antibodies GBS III and UBS-54 on day 6, 9 and 12 (Group B) . Only tumor bearing mice were included.
• mice Mean tumor size in Balb/c (nu/nu) mice xenografted with the human colon carcinoma cell line LS174T and treated with fully human monoclonal antibody K53/IgGl or control antibodies GBS III and UBS-54 on day 9, 12 and 15 (Group C) . Only tumor bearing mice were included.
• the present invention discloses processes for identifying post-translationally modified disease associated molecular markers (also named novel epitopes) , said post- translationally modified disease associated molecular markers being present on diseased cells in their post-translationally modified disease associated form.
• Said processes make use of phage display libraries displaying binding molecules, said binding molecules preferably being antibodies or antibody fragments.
• Said processes according to the invention make further use of cell sorting techniques and fluorescence based parameters .
• the invention also provides the identified post- translationally modified disease associated molecular markers as well as the binding molecules that bind to it.
• the present invention provides novel binding molecules such as scFv fragments or fully human IgG molecules that bind the human CD46 protein specifically present in a specific glycosylation state on diseased cells.
• said binding molecules are characterized, processed and recombinantly expressed in mammalian cells, preferably human cells.
• the invention also provides such cells, comprising a nucleic acid according to the invention encoding the binding molecule.
• Purified binding molecules according to the invention are used for the preparation of diagnostic tools, such as kits or medicaments for the treatment of diseases, such as neoplastic diseases as cancer (e.g.
• the present invention provides a process for identifying a disease associated molecular marker associated with a subset of cells comprising the steps of: a) incubating cells of a species with a library of binding molecules, combined with an incubation with diseased cells of said species; b) obtaining from said incubation, a collection of diseased cells essentially free from non-diseased cells, by sorting said collection of diseased cells from non-diseased cells according to parameters which distinguish between said collection of diseased cells and said non-diseased cells; c) obtaining binding molecules from said collection of diseased cells; d) selecting from said obtained binding molecules, an individual binding molecule capable of preferential binding to said diseased cells as compared to binding to said non-diseased cells; e) identifying a molecular marker which, in its disease associated form, binds to said individual binding molecule selected under step d) , said molecular marker being associated with said collection of diseased cells obtainable according to step b) ; and f)
• said process further comprises the step of establishing that said counterpart and said disease associated form differ in at least one post-translational modification. Genetic differences (i.e. where the modification is the result of a change in RNA or DNA encoding said marker) can typically also be found by other means.
• the present invention has clear advantages for identifying post-translationally modified disease associated markers.
• said post-translational modification comprises a glycosylation modification. This type of modification is relatively easy to identify and isolation binding molecules for.
• said process further comprises the steps of
• the invention provides for a process for identifying a binding molecule capable of binding a subset of diseased cells comprising the steps of: a) incubating cells of a species with a library of binding molecules, combined with an incubation with diseased cells of said species; b) obtaining from said incubation, a collection of diseased cells essentially free from non-diseased cells, by sorting said collection of diseased cells from non-diseased cells according to parameters which distinguish between said collection of diseased cells and said non-diseased cells; c) obtaining binding molecules from said collection of diseased cells; d) selecting from said obtained binding molecules, an individual binding molecule capable of preferential binding to said diseased cells as compared to binding to said non-diseased cells; e) recovering said individual binding molecule selected in step d) ; f) establishing that said individual binding molecule preferentially binds to a molecular marker in its disease associated form, said molecular marker in its disease associated form being associated with said diseased
• a post-translationally modified molecular marker does not need to comprise post-translational modifications in the disease associated form. What is needed is a difference in post-translational modification between the form in a normal cells and a disease associated form.
• a molecular marker of the invention preferably does not comprise amino-acid differences between it's diseased associated form and it's counterpart in non-diseased cells.
• post-translationally modified disease associated molecular markers or novel epitopes as used herein can be, but are not limited to, extra-cellular proteins or cell surface proteins, or parts thereof, that have undergone conformational or configurative changes due to differential N-glycosylation and/or O-glycosylation, phosphorylation, bridging (e.g. di-sulphide bridges), gamma-carboxylation and gamma-hydroxylation depending on the diseased state of the cells that they are associated with.
• Disease associated as used herein means that the marker is secreted by, bound by, attached to or targeted to a diseased cell; said cell being a diseased cell involved in disease.
• said library of binding molecules comprises a phage antibody display library.
• said library of binding molecules can also comprise but are not limited to, small molecules, peptides, polypeptides or other proteinaceous molecules.
• said phage antibody display library comprises at least lxlO 8 specificities. Said phage antibody display library preferably display scFv or Fab fragments on the surface of bacteriophage particles.
• said process of the invention is a process, wherein said sorting is conducted using a fluorescent activated cell sorter (FACS) and wherein said parameters are fluorescence based parameters.
• said diseased cells used in a process according to the invention, are present in a cell population derived from mammalian species suffering from diseases such as cancer (tumor cells, also referred to as neoplastic cells) , diabetes, Alzheimer, multiple sclerosis, rheumatoid arthritis, inflammatory disease or viral infections.
• said diseased cells are multiple myeloma cells, breast tumor cells or colon carcinoma cells.
• Said mammalian species can be, but is not limited to, human.
• the binding molecule and/or the post- translationally modified disease associated molecular marker are recovered from the diseased cells that are identified in said sorting by conventional methods known in the art and as described in the examples disclosed herein.
• said sorting comprises sorting using a fluorescent activated cell sorter.
• said binding molecule comprises an scFv antibody fragment.
• An scFv can be used for specificity studies, while still being present on the phage particle. It can also be used purified form. Association with phage allows characterization of its encoding DNA sequence.
• the DNA can be used to construct or to form a full sized fully human immunoglobulin molecule that can be cloned into mammalian expression vectors and expressed in eukaryotic cells.
• the produced immunoglobulin can be purified from the medium and subsequently used in experimentation (as explained in the examples) , and in the preparation of medicaments and/or diagnostic compounds.
• said eukaryotic cells are mammalian cells. Even more preferred are human cells for the expression of the fully human antibody comprising the binding moiety of the initially identified binding molecule.
• said post- translationally modified disease associated molecular marker comprises a glycosylation variant of a cell surface protein, such as the CD46 protein.
• said post-translationally modified disease associated molecular marker can also comprise phosphorylation or conformational variants of a (cell surface) protein, or a protein that is released from the cell but nevertheless associated with a diseased cell according to the invention.
• the present invention provides post-translationally modified disease associated molecular markers and binding molecules obtainable by a process according to the invention.
• said post-translationally modified disease associated molecular marker comprises a post- translationally modified CD46 protein.
• said binding molecule binds said post-translationally modified CD46 protein present on a subset of cells. Said binding molecule binding said post-translationally modified CD46 protein present on a subset of cells does not bind, or does bind to a significant reduced level to a post-translationally modified CD46 variant present on non-diseased cells.
• the present invention provides among others, novel disease associated molecular markers, methods for finding said novel epitopes and binding molecules capable of binding to said novel epitopes.
• said markers and/or said binding molecules are obtained by a method of the invention.
• a disease associated molecular marker may comprise of one proteinaceous molecule.
• said marker may also be part of a complex.
• the epitope on the disease associated marker of the invention can be present or provided by one proteinaceous molecule. However, it can also be formed in combination with one or more other molecules. However, preferably, said epitope is formed by or present on one proteinaceous molecule .
• the invention provides a binding molecule capable of specifically binding to an epitope present in a subset of CD46 proteins.
• Said binding molecule is capable of distinguishing between CD46 proteins belonging to the subset and CD46 proteins not belonging to the subset. Said binding molecule can thus be used to type samples containing CD46 protein, for example in diagnosing diseases. This property is also useful in, for instance, CD46 purification strategies.
• CD46 is a protein that is widely expressed on many different cell types and tissues.
• a binding molecule of the invention is capable of binding to a subset of CD46 expressing cells.
• said binding is specific for said subset of CD46 expressing cells.
• a cell belonging to said subset of cells comprises a detectable amount of CD46 protein comprising said novel epitope.
• This cell can also comprise CD46 protein not comprising said novel epitope.
• a CD46 positive cell that does not belong to the subset contains CD46 protein not comprising said novel epitope. Typically this cell does not comprise detectable levels of CD46 protein comprising said novel epitope, however, this may not always be true.
• CD46 positive cells With essentially specifically binding to a subset of CD46 positive cells is meant that the number of CD46 proteins comprising said novel epitope on a cell not belonging to said subset, is too low to be detected or to exert a biological effect upon binding of a binding molecule of the invention.
• a cell belonging to said subset of cells comprises a neoplastic cell.
• said neoplastic cell is derived from a hemopoietic cell, a cervix cell, a colon cell, a kidney cell or a liver cell.
• said neoplastic cell is derived from a B-cell. More preferably said neoplastic cell comprises a Multiple Myeloma (MM) cell.
• a novel epitope according to the invention can consist of any (combination of) substance (s) .
• a novel epitope is formed by an amino acid sequence, a sugar or lipid moiety, a (partly) post-translational modification or a combination of these elements.
• Post-translational modifications can be among other events the result of differential phosphorylation, differential glycosylation, conformational changes, such as di-sulphide bridging, multimerization in which two or more monomeric proteins form a novel epitope that can interact with a binding molecule of the invention, and the like.
• Said modifications can make up the epitope in a form that can interact with a binding molecule.
• An Example of a ycoprotein that is expressed on certain diseased cells and that has a different post- translational modification is MUC-1.
• one or more of the substances making up the epitope are not available for binding to a binding molecule.
• Said one more substances making up the epitope do not have to be absent from the molecule. All said substance (s) can still be present in or on the protein, however in this case, said one or more substances are present in a form that does not allow binding of a binding molecule of the invention. Typically, this is the case when the binding molecule is prevented from binding due to sterical hindrance and/or due to a conformational change in the protein leading to a different distribution of said one or more of the substance (s) in said protein.
• the invention provides different glycosylation forms of CD46.
• At least one variant glycosylation form of CD46 comprises a novel epitope that is expressed on e.g. Multiple Myeloma cells, whereas said novel epitope is not expressed by normal CD46-positive cell types thus far analyzed.
• a similar epitope is present in many other neoplastic cells (described below) .
• the CD46 epitope present on MM cells thus is a suitable marker for at least some types of tumor cells.
• the invention therefore provides the use of a binding molecule of the invention for the typing of a CD46 positive cell.
• said use comprises a diagnostic use.
• said use comprises a preventive and/or curative therapeutic use.
• CD46 proteins of various animal species have been isolated.
• a person skilled in the art is well capable of identifying CD46 protein in species from which the CD46 protein is not yet determined.
• a suitable strategy is to use the information from an identified CD46 in an evolutionary closely related species. This can be the nucleic acid and/or • amino acid sequence (for homology hybridization of nucleic acid libraries under more or less stringent conditions or nucleic acid amplification strategies using primers for conserved evolutionary conserved regions) .
• antibodies specific for conserved parts of a CD46 molecule of an evolutionary related species can be used to identify a CD46 protein in another species.
• said CD46 protein comprises a mammalian CD46 protein. More preferably, said CD46 protein comprises a human CD46 protein.
• a binding molecule of the invention can be any type of binding molecule known in the art.
• a binding molecule is capable of specifically binding a novel epitope, meaning that the binding molecule does not bind efficiently to proteins not comprising said novel epitope.
• Many different types of binding molecules are known in the art. Examples of binding molecules according to the invention are, but are not limited to, small molecules, lectins, peptides, polypeptides and proteins such as antibodies or immunoglobulins (Ig) or Ig- like molecules.
• a binding molecule of the invention comprises an antibody or a functional part or derivative thereof.
• Suitable parts and/or derivatives of antibodies are Fab fragments, single chain Fv fragments, CDR domains, single chain Fab fragments or variable regions of the antibody molecule.
• An antibody may be produced or first generated by a B-cell.
• An artificial antibody comprises a similar structure as a classical antibody. Such artificial antibodies can for instance be generated by in vitro assembly of amplified nucleic acid coding for various parts of an antibody.
• a functional part of an antibody comprises at least a part involved in epitope binding.
• a functional part of an antibody typically comprises the same epitope binding capacity in kind not necessarily in amount.
• a person skilled in the art is well capable of altering parts of the amino-acid sequence of an antibody without essentially affecting the binding capacity of said antibody. Alterations can comprise deletions, insertions, amino-acid substitutions or a combination of these alterations. Such derivatives of antibodies are also part of the invention.
• an antibody of the invention is a human antibody or a humanized antibody.
• Such antibodies closely resemble normal human antibodies and have similar pharmacodynamics upon administration to humans.
• a binding molecule of the invention comprises a tag.
• a tag can be used for detection purposes.
• a tag can comprise a toxic substance.
• a toxic substance can enhance removal of targeted cells from the body.
• said toxic substance comprises a toxin and/or a radioactive substance.
• the invention provides a method for the treatment of an individual suffering from or at risk of suffering from a disease, comprising administering to said individual a therapeutically acceptable amount of a binding molecule of the invention.
• Administration can be used to facilitate removal of, undesired cells that cause at least part of the disease from the body of said individual. Such cells may be present in said body upon administration or said individual may be at risk of comprising said undesired cells.
• said disease is a neoplastic disease.
• the invention also provides the use of a binding molecule of the invention for the preparation of a medicament for the treatment of certain diseases. Preferably, said medicament is used for the treatment of neoplastic disease.
• a binding molecule of the invention may be used in conjunction with other methods of treatments or medicaments.
• said other treatment or medicament comprises another binding molecule comprising a specificity for another epitope.
• said another epitope is present on a CD46 expressing cell. Since CD46 is involved in the complement pathway, a molecule of the invention can be used to modulate complement mediated effects of said antibody specific for said another epitope. Modulation can comprise stimulation or inhibition of complement activity toward cells capable of binding both, a binding molecule of the invention and said antibody specific for said another epitope.
• a binding molecule of the invention is used to type a cell. Now that different forms of CD46 can be detected it is possible to use this property in detection methods.
• a binding molecule of the invention is used to determine whether cells in a sample comprise neoplastic cells.
• said neoplastic cell comprises a multiple myeloma cell.
• the invention provides the use of an epitope expressed on a subset of CD46 expressing cells as a marker for tumor cells.
• the invention therefore further provides a kit comprising at least a binding molecule of the invention. Said kit preferably, further comprises a buffer suitable for allowing specific binding and/or means by which said binding can be detected, such as a fluorescence marker.
• the invention provides a nucleic acid encoding a binding molecule according to the invention, or a part involved in CD46 binding of such a molecule.
• nucleic acid can be obtained in various ways.
• One non- limiting example is amplification of nucleic acid encoding said binding molecule from a cell expressing said molecule.
• nucleic acid coding for said binding molecule is readily available.
• a binding molecule of the invention can be produced in variety of ways.
• a molecule of the invention is produced by a cell comprising a nucleic acid encoding said binding molecule.
• said cell is a primate-, a rodent-, a bird- or a plant cell.
• said cell is human cell.
• Human cells and the closely related primate cells have very similar post-translational modification machineries.
• a binding molecule of the invention can be provided with human-like and more preferably, human post-translational modifications such as glycosylation.
• Such human type modification is less immunogenic in humans than modifications introduced by cells of other species, thus leading to a better efficacy of the treatment.
• said cell further comprises a means for the conditional expression of a nucleic acid of interest.
• expression of a proteinaceous binding molecule of the invention can be induced at times when expression is desired. This is advantageous when expression of said proteinaceous binding molecule interferes with a function of said cell.
• a preferred system for the conditional expression of nucleic acid of interest comprises a tetracycline responsive expression system.
• the invention therefore also provides a cell comprising a nucleic acid encoding a binding molecule of the invention, said cell preferably further comprising a tetracycline responsive molecule capable of influencing expression of a promoter.
• said cell comprises nucleic acid encoding an early protein of adenovirus or a functional part, derivative and/or analogue thereof. Such a cell is capable of producing more functional binding molecule per time unit.
• said early protein comprises adenovirus El or a functional part, derivative and/or analogue thereof.
• Adenovirus El comprises transcriptional activation properties and generally has the effect of enhancing protein production in a cell.
• said adenovirus early protein comprises adenovirus E2A or a functional part, derivative and/or analogue thereof. E2A in a cell has a protein production enhancing effect. Derivatives of El or E2A can be generated in various ways.
• a functional part and/or derivative of adenovirus El or E2A comprises the same protein production enhancing qualities in kind not necessarily in amount. Many viruses use proteins with similar protein production enhancing qualities as adenovirus El or E2A. Such molecules form suitable analogues of El or E2A.
• the invention provides a plant or non- human animal comprising a cell capable of producing a binding molecule of the invention.
• a plant or non-human animal is transgenic for a nucleic acid encoding a binding molecule of the invention.
• said animal comprises a human immunoglobulin locus or a functional part thereof.
• the invention provides a gene delivery vehicle comprising a nucleic acid encoding a binding molecule according to the invention. Such a gene delivery vehicle can be used to target delivery of said nucleic acid contained in said gene delivery vehicle to diseased cells expressing a post-translationally modified protein belonging to a subset of proteins, such as the CD46 protein.
• the invention provides a method for selecting a binding molecule capable of binding specifically to a epitope on a protein wherein said epitope is present on a subset of cells expressing said protein, said method comprising providing a collection of cells comprising cells of one type, with a library of proteinaceous binding molecules and selecting from binding molecules bound to said collection of cells at least one binding molecule capable of binding to said novel epitope.
• the present invention demonstrates that molecular markers are present in disease associated, and preferably post-translationally modified, forms on the surface of diseased cells. These molecular markers can be identified and specifically targeted by binding molecules of the invention. More in particular, the invention demonstrates that tumor cells, for example but not limited to Multiple Myeloma cells, express certain types of glycosylated forms of CD46 proteins, which are not expressed on non-tumor cell types.
• the invention makes use of this feature and provides also a number of binding molecules and more in particular fully human monoclonal antibodies that specifically interact with the differentially glycosylated human CD46 proteins. In a particularly preferred embodiment of the invention these human monoclonal antibodies are used to diagnose, prevent and/or treat different kinds of human malignancies, in particular Multiple Myeloma.
• CDC Complement-dependent cytotoxicity
• ADCC antibody-dependent cellular cytotoxicity
• DCC complement plus antibody- dependent cellular cytotoxicity
• the numbers are expressed as percentage of cytotoxic LS174T colon cancer cells and are the mean of 8 different donors.
• GBS III served as a negative control, while J4.48 served as a positive, conventional an i-CD46 positive control (here depicted as Anti-CD46, right columns) .
• mice xenografted with LS174T cells.
• mice Mean tumor size in Balb/c (nu/nu) mice xenografted with the human colon carcinoma cell line LS174T and treated with the K53/IgGl or control antibodies GBS III and UBS-54. Only tumor bearing mice were included. On day 13 one GBS III treated mouse (bearing two tumors) from group B, and one K53/lgGl treated mouse (bearing one tumor) from group A were killed.
• HEK 293 cells Five separate harvests are depicted from cell line L53-7 that gave the highest yields .
• the antibodies were purified over protein-A and concentrations were measured by
• Example 1 Isolation of malignant plasma cell-specific phage antibodies K19, K29 and K53 for a semi-synthetic phage antibody display library.
• Phage antibody display in combination with flow cytometry was used to isolate human scFv antibody fragments that bind to malignant plasma cells. Details of this method have been described elsewhere (De Kruif et al. 1995a and 1995b) . The preparation of cell suspensions from blood, tonsil, spleen and bone marrow from healthy individuals and multiple myeloma patients is described in Van der Vuurst de Vries and Logtenberg (2000) .
• Mononuclear cells from these suspensions were isolated by Ficoll-Paque (Pharmacia) density centrifugation and subsequent washes in PBS plus 1% Bovine Serum Albumine (BSA) and used for FACS analysis as described (Van der Vuurst de Vries and Logtenberg 2000) .
• the heterogeneous mixture of mononuclear cells of a patient with Multiple Myeloma was mixed with a phage display library of human scFv fragments, made essentially as described by De Kruif et al. (1995a and 1995b) . Approximately 10 13 phage particles were blocked for 15 min in PBS/1% BSA containing 4% low-fat milk powder.
• Bone marrow cells from a healthy donor were added to the blocked phages and the mixture was slowly rotated for 16 h at 4°C. Non-binding phages were washed away with ice-cold PBS/1% BSA and cells were subsequently stained with PE-labeled anti- CD38 antibodies.
• Malignant plasma cells were isolated by sorting on a FACSstar plus (Becton Dickinson) based on high levels of CD38 expression and forward/side scatter profile (Fig.3) . It is known in the art that CD38 is highly expressed on these tumor cells and that they can be readily recognized by FACS analysis using staining procedures to detect CD38 proteins on their surface.
• Fig.3 shows what population is preferably selected for the expression levels of CD38 and can be used thereafter in the methods of the present invention.
• Phages bound to the sorted malignant plasma cells were eluted by incubation at room temperature with 1.5 volume of 76 mM citric acid pH 2.5, followed by neutralization with 1.5 volume of 1 M Tris.HCl pH 7.4.
• the eluted phages were rescued by infection of XLl-blue F' bacteria and propagated for a second round of selection.
• phages were prepared from individual bacterial colonies and used in immuno-fluorescent analysis as described (De Kruif et al. 1995a).
• Phage antibodies that bound to malignant plasma cells in the bone marrow of patients with Multiple Myeloma and that showed little staining (as performed by De Kruif et al . 1995a) of hematopoietic cells in other lymphoid organs were selected for further study.
• Three specifically and strongly interacting phage antibodies were identified and named K19, K29 and K53.
• Cell suspensions were prepared from blood, tonsil, bone marrow and spleen of healthy individuals and subsequently stained with the three identified phage antibodies K19, K29 and K53. For this, cells were stained with myc-tagged single chain Fv fragments followed by anti-myc antibody (9E10) and fluorochrome labeled goat anti-mouse antibodies (Southern Biotechnology Associates) staining. The details concerning the use of these antibodies were described in De Kruif et al. (1995a and 1995b) . Subsequently, binding capacity was analyzed by FACS.
• scFv's only bound to the malignant plasma cells in the bone marrow of patients with multiple myeloma and to a small population of CD38 br ⁇ ght cells in tonsil and normal bone marrow. No staining of hematopoietic cells in spleen or blood was observed.
• Fig.4 shows the binding of K53 to these cells as compared to a control phage antibody that is directed against thyroglobuline.
• the nucleotide sequence of the three scFv antibodies was determined and unveiled that K19, K29 and K53 were encoded by different immunoglobulin heavy and light chain variable genes (Fig.5) and thus represented three independent scFv antibody fragments.
• the amino acid FDY motif that seems to overlap between these antibodies is present in many (also non-CD46 binding) antibodies. The determination of crucial residues involved in the CD46 specific interaction is investigated and described in more detail in example 13.
• CD38 bright cells are DCis that exhi b it a very high C D38 expression (black dots in Fig.3). Cells of this kind are found in bone marrow and tonsil cells and have been previously shown to represent plasma cells and precursors of plasma cells, or so-called plasma blasts (Terstappen et al. 1990) .
• plasma blasts Teerstappen et al. 1990
• bone marrow cells from a MM patient were stained with scFv K53 and positive cells were isolated by cell sorting. For this, isolation, staining and cell sorting procedures were performed as described by De Kruif et al. (1995a) and Van der Vuurst de Vries and Logtenberg (2000).
• Cytospin preparations (80,000 cells were spun in 100 ⁇ l PBS at 500 rpm during 5 min) from the sorted cells were stained with May Gr ⁇ nwald Giemsa (Merck, 12% solution during 15 min in aquadest) , washed with water, covered with a coverslip and cells were visualized by light microscopy. As shown in Fig.6, sorted K53+ bone marrow cells displayed a distinct plasma cell morphology.
• the phage antibody K19 was used to screen expression libraries using baculovirus in Sf9 cells using two human placental cDNA libraries cloned in baculovirus .
• the construction of the human placenta cDNA libraries in the baculovirus transfer vector pBacPAK9 (Clontech) has been described in detail in Granziero et al. 1997). Two libraries were used, one containing cDNA inserts ranging in size from 1-3 Kb (1-3 Kb library) and the other containing cDNA inserts larger than 3Kb (> 3 Kb library) .
• the titer of the libraries was determined using the BackPAK rapid titer kit (Clontech).
• the titer of 1-3 kb library was l.lxlO 8 pfu/ml and the titer of the > 3kb library was 4xl0 7 pfu/ml.
• Sf9 insect cells ATCC were maintained at 27 °C in TC100 medium supplemented with 5% FCS, pen/strep and 0.1% pluronic-F68 (Gibco) .
• 2xl0 6 Sf9 insect cells were washed and exposed to 1 ml of the 1-3 Kb library (l.lxlO 8 pfu) or 1.5 ml of the > 3Kb library (6xl0 7 pfu) .
• Cells were left for 1 h at room temperature before 5 ml medium was added.
• the cells were left for 48 h at 27°C prior to staining with phage antibodies. By staining with phage antibodies, single positive cells were sorted.
• the virus which encodes the surface epitope was isolated by limiting dilution.
• Screening of the baculovirus expression library was performed 48 h after infection of Sf9 cells by incubating the cells with the scFv' s using M13-biotynilated antibodies followed by a Phycoerythrin (PE) -labeled goat anti-mouse Streptavidine antibody.
• PE Phycoerythrin
• Single positive cells were sorted by FACSstar, propagated and used for a next round of selection. For this, single positive cells were mixed with fresh insect cells to propagate the baculoviruses . Supernatants of insect cells were used to infect fresh Sf9 insect cells and the entire procedure was repeated twice.
• the viruses present in the supernatant of positive wells, were cloned by limiting dilution and the inserts were recovered by PCR and sequenced. Details of the cDNA library, the use of the library and virus identification by limiting dilutions are described by Granziero et al. (1997). The results of these experiments are shown in Fig.7. Nucleotide sequence analysis was subsequently performed by methods known to persons skilled in the art. The analysis of the cDNA inserts from clones obtained from two size- selected placental cDNA libraries, containing inserts smaller than 2 kb and inserts larger than 2 kb respectively, unveiled open reading frames that in both instances completely matched the reported nucleotide sequence of the human CD46 gene.
• CD46 is expressed on all nucleated cells including tumor cells; some tumor cells and cell lines express particularly high levels of CD46 (Kinugasa et al. 1999; Schmitt et al. 1999; Thorsteinsson et al. 1998; Simpson et al. 1997) .
• the CD46 protein is also expressed on various tissues and organs and soluble forms of CD46 are present in most bodily fluids (Hara et al. 1992) .
• the physiological role of CD46 seems to be to protect the host cell from complement-mediated cell damage (Oglesby et al. 1992; Seya et al.
• CD46 also known as Membrane Cofactor Protein
• This complement regulatory protein is a polypeptide of approximately 60 kDa and is composed of numerous repeating units (known as CCP's) containing several N-glycosylation sites, a serine-threonine-proline rich region (STP) containing several O-linked glycosylation sites, a transmembrane region and a cytoplasmic tail.
• CD46 transcripts results in different combinations of different parts of the protein (Liszewski et al. 1991).
• the N-terminus identical for all known isoforms, contains three potential N-glycosylation sites in CCP-1, CCP-2 and CCP-4.
• the STP domain is extensively O-glycosylated.
• the protein is found to be widely distributed among cell types, including fibroblasts, endothelial cells and epithelial cells in many organs.
• the murine anti-CD46 antibody (J4.48 Immunotech) stained CHO cells transfected with the wildtype CD46 cDNA as well as the transfectants in which each of the glycosylation sites in the CCP domains or the entire STP domain ( ⁇ STP) were deleted (Fig.8).
• different staining patterns were obtained with the scFv K19, K29 and K53 antibody fragments.
• the staining pattern of K19 and K29 were comparable. All three antibodies specifically stained the cell line transfected with the wildtype CD46 cDNA, as well as the CCP-1 and STP glycosylation mutants (Fig.8).
• K19 and K29 also bound to the CCP-2 glycosylation mutant but completely failed to bind to the CCP-4 glycosylation mutant.
• K53 lost binding to the CCP- 2 and CCP-4 glycosylation mutants.
• CD46 recognized by K19, K29 and K53 are all dependent on the N-glycosylation of CD46.
• the involvement of N-glycosylation in binding to CD46 by the human Monoclonal antibodies (huMabs) can also be demonstrated by using the glycosylation inhibitor tunicamycin (see also example 9) .
• Example 6 Generation of fully human IgGl monoclonal anti-CD46 antibodies from scFv's K19, K29 and K53.
• the engineering and production of the human IgGl monoclonal antibodies was described in detail by Boel et al . (2000) . Briefly, the VH and VL regions encoding the scFv CD46 antibodies were excised and recloned into vectors for expression of complete human IgGl/ ⁇ molecules in BHK cells transfected with the furine gene (to yield fur-BHK21 cells) in a two step cloning procedure. The scFv fragments were first cloned in pLEADER (Boel et al. 2000) to add the T cell receptor chain HAVT20 leader peptide sequence and a splice donor site.
• pLEADER Boel et al. 2000
• the scFv containing the leader sequence and donor splice site were subcloned in pNUT-C ⁇ (ECACC deposited) or pNUT-C ⁇ 2 (ECACC deposited) expression vectors. Both vectors were co-transfected in fur- BHK cells to generate stable cell lines expressing and secreting human monoclonal antibodies (Huls et al. 1999). For production, cells were cultured in serum free UltraCHO medium (Biowhittaker) . After 4 days, the medium was collected and the antibodies were purified using a protein A-sepharose column, using procedures known to persons skilled in the art. The resulting proteins were named K19/IgGl, K29/IgGl and K53/IgGl respectively.
• CD46 monoclonal antibodies Specific binding of CD46 monoclonal antibodies to myeloma and leukemic tumors .
• Bone marrow cells from healthy individuals and from Multiple Myeloma (MM) patients were screened for K53/IgGl human monoclonal anytibody (huMab) binding.
• K53/IgGl and control GBS III huMabs were labeled with PE or APC (IQ Systems, The Netherlands) .
• the bone marrow cells were incubated with the huMabs in the presence of 10% normal human serum (NHS) during 15 min at room temperature and were analyzed by FACS.
• the K53/IgGl monoclonal antibody co- stained with the myeloma markers CD38 and CD138 (Fig.9) .
• the tissues were co-stained with several (labeled) mouse monoclonal antibodies such as the ones directed against the myeloma markers CD38 and CD138.
• Co-staining experiments were performed using bone marrow cells as follows, with dilutions between brackets and huMab representing the different fully human IgGl's recognizing CD46:
• Fig.llA-E the dot plots of huMAb K53/IgGl (here also depicted as L53 on the left side of the specific dot- blots) and GBS III IgGl control human monoclonal antibodies in relation to CD38 staining are shown of all the patient material tested. All the multiple myeloma cells that were CD38 bright were highly positive for K53/IgGl staining. Specific K53/IgGl binding was observed in primary tumors, as well as in refractory/partly responsive tumors.
• Binding was not detected in bone marrow cells from healthy individuals and when the control human GBS III antibody was used. Also FACS experiments with whole blood of healthy persons was negative for all the human antibodies used. This negative result was also found with other types of leukemic tumors like Chronic Lymphatic Leukemia (CLL) . So, using normal leukocytes and cells from leukemic tumors, the K53/IgGl clearly binds to Multiple Myeloma cells, but not to non- neoplastic cells.
• CLL Chronic Lymphatic Leukemia
• Example 8 Binding of anti-human CD46 monoclonal antibodies to solid tumors, cells derived from solid tumors, myeloma cells and leukemic cells, in comparison to normal tissue.
• CD46 protein One of the functions of the CD46 protein is to protect the cell from autologous complement attack.
• One mechanism for tumor cells to deviate the complement attack is by over- expressing the CD46 protein. This over-expression is found in several neoplasia like breast-, cervix-, liver-, colorectal- and gastro-intestinal cancer.
• the experiments discussed in example 5. suggest that CD46 cannot only be over-expressed, but also differentially glycosylated.
• the MM-specific K19, K29 and K53 antibodies apparently distinguish between these different glycosylation forms (Fig.8).
• K19, K29 and K53 antibodies also bind to tumor cells that over-express CD46
• said antibodies were incubated with a set of tumor cell lines (MCF-7 (breast) , HeLa (cervix) , HepG2 (liver) and LS174T (colon, ATCC CL-188) and determined the staining with fluorescent labeled antibodies as described supra.
• Table I shows the increase in interaction between several solid tumor-derived cell lines with K19, K29 and K53 antibodies
• Table V shows an extensive number of myeloma cell lines, leukemic cell lines and solid tumor cell lines that were tested for binding to K53/IgGl (determined by FACS analysis) in comparison to a conventional murine anti-CD46 antibody J4.48 as a positive control and GBS III as a negative control.
• these data strongly suggest that these cells not only exhibit an over-expression of the CD46 protein but that the protein also has a different glycosylation pattern as compared to wild type, non-tumor cells. It is concluded that the antibodies interact with CD46 proteins on cells that were derived from solid tumors.
• the long tumor cell line GLC-1 and the human embryo kidney cell line (HEK293) were negative, whereas most of the colon cell lines (DLD-1, HT29, SW480, LS174T versus HCT116: 4 out of 5 tumor cell lines) were positive for staining with K53/IgGl.
• Most of the colon cell lines (DLD-1, HT29, SW480, LS174T versus HCT116: 4 out of 5 tumor cell lines) were positive for staining with K53/IgGl.
• T47D and MCF-7 were highly positive, whereas MDA-MB231 was less positive for K53/IgGl binding.
• a strong correlation between binding to the diseased form (the tumor-cell related post-translationally modified variant) of CD46 using K53/IgGl and murine J4.48 (depicted as CD46 in the right columns) was not found.
• the multiple myeloma associated variants are an example of a class of novel epitopes, herein referred to as post-translationally modified molecular markers, or as disease associated molecular markers, which are characterized by their aberrant post-translational modification as compared to their correctly post-translationally modified counterpart.
• the reason for the occurrence of a post-translational modification in a protein present on a diseased cell or associated with diseased cells (or a diseased state of a species) can be multiple.
• the over-expression identified on tumor cells for certain proteins can lead to incorrect post-translational modifications such as misfolding, multimerization and/or aberrant glycosylation and the like.
• tumor material was either obtained directly after surgery and pathology, or from commercial sources and stained with the different anti-human CD46 antibodies.
• tissue sections were derived from Novagen (Germany) and from the University Medical Center (Utrecht, The Netherlands) . Tissues were fixed in 4% paraformaldehyde, sectioned at 5-6 ⁇ m thickness and pre-treated with 10% normal human serum/PBS during 1 h at room temperature.
• the slides were incubated with PE-linked K53/IgGl (diluted 1:5) or PE- linked GBS III (diluted 1:5) in 1% BSA/5% NHS/PBS for 1 h at room temperature, washed three times for 10 min with PBS/0.1% Tween, followed by anti-rabbit PE (diluted 1:100) in 1% BSA/5% NHS/PBS (1 h, room temperature) , subsequently washed again three times for 10 min and incubated with anti-rabbit IgG-FITC (diluted 1:100) for 1 h at room temperature. After extensive washing, slides were covered with mounting medium and studied using fluorescent microscopy. The results of the im uno histochemistry are shown in Table VI for normal tissues and VII for tumor tissues.
• K53/IgGl positive tumor cell lines (LS174T cells and MCF7 cells) are treated with the N-glycosylation inhibitor tunicamycin during three days at concentrations of 0.3 and 1 ⁇ g/ml.
• the cells were stained for CD46 using PE-linked fully human monoclonal antibodies K53/IgGl and negative control antibody GBS III, and commercially available murine anti-CD46 antibody J4.48, which binds to all CD46 derivates (see also Fig.8).
• the antibodies were used in combination with a second PE-labeled goat-anti-mouse antibody.
• the binding capacity was detected and analyzed by Fluorescence-Activated Cell Sorting (FACS) using techniques well known to persons skilled in the art.
• FACS Fluorescence-Activated Cell Sorting
• swainsonine On K53/IgGl positive tumor cells was also tested.
• the target enzyme of swansonine is Mannosidase II and therefore this compound prevents the removal of mannose residues of the high mannose structure.
• Treatment with swainsonine generally results in an accumulation of high mannose type glycoproteins .
• LS174T colon tumor cells swainsonine treatment with 20 and 30 ⁇ g/ml during 3 days resulted in an significant increase of K53/IgGl binding, whereas the binding of J4.48 remained similar (Fig.16) • _ -_
• ADCC and CDCC assays in vitro killing assays.
• the tumor cell killing activity of the anti-CD46 antibody using human peripheral blood mononuclear cells was evaluated using Multiple Myeloma cells and tumor cell line LS174T derived from colon as target cells in Antibody and Complement Dependent Cellular Cytotoxicity (ADCC and CDCC) assays.
• the target cells were labeled with 30 ng/ml calcein
• Molecular Probes for 5 min at 37°C. After extensive washing, isolated human mononuclear cells were added to the target cells at an effector: target ratio of 40:1. Complement mediated lysis was performed with 50 ⁇ l serum in a final volume of 200 ⁇ l. Cells were incubated at 37°C in the presence of various concentrations of K53/IgGl, GBS III negative control antibodies or PBS. After 4 h, propidium iodide was added as a marker for cytotoxicity and cytolysis was determined by FACS analysis.
• V H and V genes encodig scFv K53, UBS-54 and GBS III were cloned into expression vectors for synthesis of complete IgGl molecules (Boel et al. 2000).
• the constructs were then stably expressed in BHK-21 cells transfected with the furine gene (Baby Hamster Kidney cell line fur-BHK21 (BHK-21, ATCC CCL- 10) .
• Cells were maintained at 37°C in a 5% C0 2 humidified incubator in Iscove's modified Dulbecco's medium containing 10% FCS, 2 mM glutamine, penicillin (100 IU/ml) and streptomycin (100 ⁇ g/ml) .
• K53/IgGl was produced in HEK 293 cells (human embryonal kidney cell line 293, German Collection of Microorganisms and cell Cultures, Dept. Human and Animal Cell Cultures ACC 305) .
• HEK 293 cells human embryonal kidney cell line 293, German Collection of Microorganisms and cell Cultures, Dept. Human and Animal Cell Cultures ACC 305) .
• the engineering and production of human K53/IgGl monoclonal antibodies in HEK 293 and PER.C6 cells is described in detail in example 14.
• the antibodies were purified using a protein-A column.
• the columns were washed with 50 ml PBS, and subsequenlty supernatant of the monoclonal antibody producing cells was applied to the column. After washing the column with 20 ml PBS, the proteins were eluted with 0.1 M citric acid pH 3.0. Fractions of 1 ml were collected and immediately neutralized with 200 ⁇ l 1 M Tris. The protein containing fractions, as determined by spectrophotometry at 280 nm, were pooled and dialyzed extensively against PBS at 4°C. The monoclonal antibodies were filtered (0.20 ⁇ m) and final concentrations were determined using the Biorad Protein Assay.
• LS174T human colon carcinoma cells day 0
• two groups of 6 animals each were treated: one group with 200 ⁇ g K53/IgGl and the other (control) group with 200 ⁇ g GBS III/IgGl human monoclonal antibodies.
• the treatment was repeated with 100 ⁇ g monoclonal antibodies.
• the treatment effects were evaluated by measuring the mean tumor size (maximal length x maximal width) during 3 weeks using methods known to people skilled in the art. Significantly, the tumor growth was markedly retarded by the CD46 human monoclonal antibody (Fig.10).
• mice seven week-old Balb/c (nu/nu) mice were injected subcutaneously into both flanks with 1x10 s LS174T cells (day 0).
• day 1 day 1
• day 6 Group B
• day 9 Group C
• three groups of five animals were treated with 300 ⁇ g antibody: one group with K53/IgGl, one with GBS III (negative control) and one with UBS-54 (positive control, Huls et al. 1999).
• 9 and 12 Group B
• 12 and 15 the treatment was repeated with 150 ⁇ g antibody.
• Table VIII The antibody treatment is summarized in Table VIII.
• K53/IgGl produced in BHK-21 cells was used for the antibody treatment on day 1 (group A) .
• a mixture of K53/IgGl produced in HEK 293 cells and K53/IgGl produced in BHK-21 cells (90% and 10% respectively) was used for the antibody treatment on day 9 (Group B and C) .
• K53/IgGl produced in HEK 293 cells was used.
• Treatment effects were evaluated by measuring the mean tumor size (maximal length x maximal height x maximal width) on day 9, 13, 15, 17 (Group A and B) or day 18 (Group C) .
• K53/IgGl The therapeutic potential of K53/IgGl was evaluated by measuring the mean tumor size using procedures well known to persons skilled in the art.
• the K53/lgGl antibody treatment started on day 1 (Group A)
• the tumor growth was significantly retarded when compared to the tumor growth in mice treated with the control antibody GBS III (Table IX, Fig.18).
• the effectivity of K53/IgGl was comparable to the UBS-54 treatment.
• mice After 17 days only 3 mice (3 out of 10 inoculation sites) developed a tumor.
• the mean size of the tumors that did develop was not significantly smaller than the tumors from the control animals (Table X, Fig.19).
• CD46-positive tumor cells MCF-7, LS174T and MM
• CD46- negative Peripheral Blood Lymphocytes PBL
• HRP-linked lectins These lectins discriminate between different sugar molecules present in the oligosaccharide backbones and are used for western blotting according to methods described by the manufacturers.
• the lectins can specifically recognize the following molecules: A.
• High mannose (con A from Canavalia ensiformis), B. Higher branched complex type oligosaccharides (WGA from Triticum vulgare and PHA-L from Phaseolus vulgaris) , C. Sialic acids (LFA from Limax flavus) and D. Fucose residue (UEX-I from Ulex europaeus) .
• the glycosylation status of the CD46 protein is determined on sets of tumor cells and compared to the status of the oligosaccharides present on CD46 which is expressed on the surface of normal cells or which is soluble and present in bodily fluids in healthy individuals and cancer patients.
• CD46 glycosylation is detected also within one tumor type and/or one tumor derived cell line.
• variable domains in the antibodies that are selected using the methods described supra are responsible for the specific interaction between the CD46 protein and the recombinant IgGl. Moreover, they specify the interaction with the different glycosylation patterns that are present on said protein expressed on the surface of Multiple Myeloma cells and other cells derived from solid tumors.
• the responsible amino acid residues are determined by randomly altering the amino acid order present in CDR3 and other variable regions. Subsequently, the mutagenized regions are incorporated into full IgGl molecules, expressed, purified and used in binding assays with MM and other tumor derived cells. Positive binding molecules are subsequently selected.
• K53/IgGl were cloned in several eukaryotic expression vectors that are described, amongst others, by Huls et al. (1999) and in WO 00/63403.
• the final expression vector (s) are subsequently transfected into human PER.C ⁇ cells (ECACC deposit 96022940) using transfection procedures well known to persons skilled in the art.
• cells that have stably integrated versions of both heavy and light chain are selected by double selection with G418 (GIBCO) and/or Hygromycin (GIBCO) .
• Another procedure involves subsequent selection in which first Hygromycin is added to the medium in which the cells are growing and outgrowing colonies are further selected with the addition of G418 to the medium or vice versa.
• both heavy and light chain are under the same selection pressure because they were cloned in one expression vector or they are cloned in similar expression vectors carrying the same selection marker.
• the method of using one expression vector carrying both the heavy and light chain in combination with the neomycin selection marker is described below in more detail.
• a Methotrexate-DHFR election method (Urlaub et al. 1983) is applied in which it is possible to amplify the integrated plasmids and thereby increasing the production levels of the recombinant antibodies.
• These methods have been applied by others by making use of for instance a hamster cell line, CHO that was deficient for its endogenous DHFR. Positive cell clones are picked and subcultured according to methods known to persons skilled in the art. Then specific production rates are determined and the best clones are selected for further outgrowth, banking, stability of expression, amplification, suspension growth and optimal growth versus production in large bioreactors.
• the recombinant fully human monoclonal antibodies directed against the differentially glycosylated CD4 ⁇ proteins are purified from the supernatant using methods well known to persons skilled in the art and used in specificity experiments, ADCC and CDCC assays and in vivo tumor-killing experiments.
• These fully human monoclonal antibodies produced in human cells are furthermore used in specificity- and pharmacokinetics studies and half-life experiments . Cloning of K53/IgGl mammalian expression vector pCD46- 3000/Neo.
• a backbone In order to construct a plasmid containing both the heavy and the light chain of the fully human anti-CD46 monoclonal antibody K53/IgGl, first a backbone had to be constructed. In contrast to the commercially available vectors this newly formed backbone contains: two CMV promoters, two Multiple Cloning Sites (MCS) and two Bovine Growth Hormone (BGH) poly-adenylation (poly (A)) sites. This was achieved by combining the described regions of two vectors. After establishment of the backbone, the light and heavy chains were cloned into the vector.
• a human IgGl consists of heavy and light chains which both contain variable and constant domains. The variable domain determines the specificity of the antibody, while the constant domain is preserved.
• pcDNA3.1/Hyg (-) (Invitrogen) was digested with Nrul and EcoRV, dephosphorylated at the 5' termini by temperature sensitive Shrimp Alkaline Phosphatase (tSAP, GIBCO Life Science Techn.) and the plasmid fragment lacking the immediate early enhancer and promoter from cytomegalovirus (CMV) was purified from agarose gel using a GeneClean kit (Bio 101, Quantum Biotechnologies Inc.).
• pAdApt (WO 99/60147 and WO 00/70071) containing the full length CMV enhancer/promoter (-735 to +95) next to overlapping Adenovirus serotype 5-derived sequences to produce recombinant Adenovirus, was digested with Avrll filled in with Klenow polymerase (GIBCO) and digested with Hpal.
• the fragment containing the CMV enhancer/promoter was isolated from agarose gel and ligated blunt/blunt into the NruI/EcoRV fragment of pcDNA3.1/Hyg (-) . This destroys the Hpal, EcoRV, Nrul and Avrll sites.
• the ligation product was transformed to competent DH5 ⁇ cells (GIBCO) and plated on LB/AMP plates. Thirty colonies were picked and cultured in LB/AMP medium for plasmid DNA isolation (according to the Qiagen miniprep procedure) .
• the plasmid DNA of the thirty clones was controlled by restriction enzyme analysis using the restriction enzyme Hindi. Eight clones turned out to contain the correct plasmid. One clone was used for further experiments.
• the resulting plasmid was designated pcDNA2000/Hyg(-) .
• pNUT-C ⁇ (ECACC deposited) comprises the constant domains, introns and hinge region of the human IgGl heavy chain (Huls et al. 1999).
• a synthetic intron and the variable domain of the gamma chain from the fully humanized monoclonal antibody UBS-54 was introduced upstream of the first constant domain in this plasmid resulting in pNUT-C ⁇ -UBS-54 essentially as described by Boel et al. (2000) .
• variable domain herein is preceded by the following leader peptide sequence: MACPGFLWALVISTCLEFSM (DNA sequence: 5'- ATG GCA TGC CCT GGC TTC CTG TGG GCA CTT GTG ATC TCC ACC TGT CTT GAA TTT TCC ATG -3')- pUBS-Heavy2000/Hyg (-) was generated in order to insert the Kozak sequence before the leader sequence of the heavy chain and place the sequence encoding the UBS-54 heavy chain under a CMV promoter.
• the entire gamma chain from UBS- 54 was amplified from pNUT-C ⁇ -UBS-54 by touchdown PCR using the upstream primer UBS-UP and the downstream primer CAMH- DOWN.
• UBS-UP The sequence of UBS-UP is as follows: 5 ' -GAT CAC GCG TGC TAG CCA CCA TGG CAT GCC CTG GCT TC-3' in which the introduced Mlul (ACGCGT) and Nhel (GCTAGC) sites are underlined and the perfect Kozak sequence is italicized.
• the sequence of CAMH-DOWN is as follows: 5' -GAT CGT TTA AAC TCA TTT ACC CGG AGA CAG-3' in which the Pmel recognition site is underlined.
• the resulting PCR product was digested with Nhel and Pmel restriction enzymes.
• the DNA fragment was purified over agarose gel using GeneClean and ligated to pcDNA2000/Hyg(-) digested with Nhel and Pmel, dephosphorylated with tSAP and purified over gel using
• the ligation product was transformed to competent DH5 ⁇ cells and plated on LB/AMP plates. Eight colonies were picked and cultured in LB/AMP medium for plasmid DNA isolation.
• the plasmid DNA of the clones was controlled by restriction enzyme analysis using Ncol. Six clones displayed the correct digestion pattern thereby confirming they contained the correct plasmid.
• One clone was stored as glycerol stock and used for further plasmid isolation.
• the resulting plasmid was named pUBS-Heavy2000/Hyg (-) . Instead of hygromycin, other selection markers were to be used in further constructs. For this, a plasmid was generated lacking a selection marker.
• pcDNA2000/Hyg (-) was digested with Pmll, and the 4.7 kb plasmid lacking the Hygromycin resistance marker gene was purified from agarose gel using GeneClean and subsequently religated.
• the ligation mixture was transformed to competent DH5c. cells and plated on LB/AMP plates. Four colonies were picked and cultured in LB/AMP medium for plasmid DNA isolation.
• the plasmid DNA of the clones was controlled by restriction enzyme analysis using the restriction enzyme Ddel. All clones turned out to contain the correct DNA plasmid. One clone was used for further plasmid isolation.
• pcDNA2000 The resulting plasmid was designated pcDNA2000 that was used to introduce the sequence of another selection marker: dehydrofolate reductase (DHFR) .
• DHFR dehydrofolate reductase
• pIG-GC9 Havenga et al. 1998), containing the wild type human DHFR cDNA was used to obtain the wild type DHFR- gene by polymerase chain reaction (PCR) with non-coding Pmll sites upstream and downstream of the cDNA.
• the primers were: DHFR up: 5' -GAT CCA CGT GAG ATC TCC ACC ATG GTT GGT TCG CTA AAC TG-3' and DHFR down: 5' -GAT CCA CGT GAG ATC TTT AAT CAT TCT TCT CAT ATA C-3' .
• the Pmll restriction sites are underlined.
• the PCR-product was digested with Pmll. The fragment was used for ligation into pcDNA2000 digested with Pmll, dephosphorylated by tSAP and purified from agarose gel using GeneClean.
• the ligation mixture was transformed to competent DH5 cells and 15 colonies were picked and cultured in LB/AMP medium for plasmid isolation.
• the plasmid DNA of the clones was controlled by restriction enzyme analysis using the restriction enzyme Ddel.
• One clone contained the correct plasmid.
• This plasmid was named pcDNA2000/DHFRwt .
• pcDNA2000/DHFRwt contains a MCS that has unique restriction sites. As this MCS does not contain the specific sites needed to sub-clone the full light chain of a human IgGl, another MCS was introduced.
• pIPspAdapt 6 (Galapagos Geno ics NV; WO 99/60147) was digested with Agel and BamHI.
• the resulting MCS fragment has the following sequence: 5' -ACC GGT GAA TTC GGC GCG CCG TCG ACG ATA TCG ATC GGA CCG ACG CGT TCG CGA GCG GCC GCA ATT CGC TAG CGT TAA CGG ATC C-3' .
• Agel and BamHI sites are underlined. This fragment contains several unique restriction enzyme recognition sites and was purified over agarose gel using GeneClean and subsequently ligated to an Agel/BamHI digested and agarose gel purified pcDNA2000/DHFRwt. This resulted in pcDNA2001/DHFRwt .
• pNUT-C ⁇ 2 (ECACC deposited) contains the variable and constant domain of the light chain of human IgGl kappa 2 (Huls et al. 1999).
• the light chain of UBS-54 and K53/IgGl were both of the kappa 2 type and therefore identical.
• the leader peptide sequence present is the same as the one in pNUT-C ⁇ described above.
• pUBS-Light2001/DHFRwt was created from pNUT-C ⁇ 2 and pcDNA2001/DHFRwt in order to obtain the light chain of UBS-54 preceded by the Kozak sequence and under control of a CMV promoter/enhancer.
• the entire (UBS-54 and K53) light chain of pNUT-C ⁇ 2 was amplified by touchdown PCR using the upstream primer UBS-UP and the downstream primer CAML-DOWN to modify the translation start site.
• the sequence of CAML-DOWN is as follows: 5' -GAT CGT TTA AAC CTA ACA CTC TCC CCT GTT G-3' .
• the Pmel restriction site is underlined. After purification the resulting PCR product was digested with Nhel and Pmel restriction enzymes and purified over agarose gel using GeneClean. The fragment was ligated to pcDNA2001/DHFRwt digested with Nhel and Pmel, treated with tSAP and purified over agarose gel using GeneClean.
• the ligation mixture was transformed to competent DH5 ⁇ cells and eight colonies were picked and cultured in LB/AMP medium for plasmid isolation.
• the DNA of the clones was controlled by restriction enzyme analysis using the restriction enzyme Ncol. Four clones displayed the correct restriction pattern, thereby confirming they contained the correct DNA. One clone was used to generate DNA for further experiments.
• the resulting plasmid was named pUBS-Light2001/DHFRwt .
• Neomycin selection marker was selected for the generation of stable cell lines. Therefore pUBS- Light2001/DHFRwt was used to generate a plasmid containing a Neomycin marker by the exchange of the selection marker sequences.
• pRc/CMV Invitrogen
• the 840 bp Neomycin resistance gene-containing fragment was purified from agarose gel using GeneClean.
• the fragment was ligated to pUBS-Light2001/DHFRwt digested with Xmal and Pmll restriction enzymes, followed by treatment with tSAP and purification over gel using GeneClean to remove the DHFR cDNA.
• the ligation of the Pmll end and the blunted BstBI site destroyed both restriction recognition patterns.
• the ligation mixture was transformed to competent DH5 ⁇ cells and fifteen colonies were picked and cultured in LB/AMP medium for plasmid isolation.
• the plasmid DNA of the clones was controlled using the restriction enzymes Nael and Sphl. The restriction enzyme analysis confirmed that all of the fifteen picked clones contained the correct DNA.
• One clone was used to generate DNA for further experiments.
• pcDNA3000/DHFRwt was created by the combination of pcDNA2000/DFHRwt and pcDNA2001/DHFRwt .
• the new vector would contain a double CMV promoter, a double MCS and a double BGH poly (A) .
• pcDNA2000/DHFRwt was partially digested with restriction enzyme PvuII. There are two PvuII sites present in this plasmid and cloning was performed into the site between the SV40 poly (A) and ColEl, not into the PvuII site downstream of the BGH poly (A) .
• a single site digested mixture of plasmid was dephosphorylated with tSAP and purified over agarose gel using GeneClean.
• pcDNA2001/DHFRwt was digested with Muni and PvuII restriction enzymes and filled in with Klenow to have both ends blunted.
• the resulting CMV promoter-linker-BGH poly (A) -containing fragment of 1269 bp was isolated over agarose gel using GeneClean and ligated into the partially digested and dephosphorylated pcDNA2000/DHFRwt. Due to the ligation the PvuII and Muni restriction recognition sites downstream of the SV40 poly (A) sites were destroyed.
• the ligation mixture was transformed to competent DH5 ⁇ cells and thirty colonies were picked and cultured in LB/AMP medium for plasmid isolation.
• the plasmid DNA of the clones was controlled by restriction enzyme analysis using Hindi. Six of the picked clones were containing the insert in the correct orientation. One positive clone was used to generate DNA for further experiments.
• the created plasmid was called pcDNA3000/DHFRwt .
• pcDNA3000/Neo was generated by the exchange of the selection marker sequences. It was generated because, as mentioned, the selection marker Neomycin was preferred.
• pRc/CMV was digested with BstBI, blunted with Klenow and subsequently digested with Xmal.
• the Neomycin resistant gene- containing fragment was isolated over agarose gel using GeneClean.
• the isolated fragment was ligated in pcDNA3000/DHFRwt digested with Xmal and Pmll, dephosphorylated with tSAP and gel purified using GeneClean. Due to the ligation both the restriction recognition sites of BstBI and Pmll were destroyed.
• the ligation mixture was transformed to competent DH5o( cells and 10 colonies were picked and cultured in LB/AMP medium for plasmid DNA isolation.
• the plasmid DNA was controlled by restriction enzyme analysis using Nael and Pstl/Bsml. Nine out of ten picked clones contained the correct DNA. A positive clone was used to generate DNA for further experiments.
• the generated vector was named pcDNA3000/Neo (-) .
• the heavy and light encoding sequences of the UBS-54 anti- EpCAM antibody were inserted.
• the next section describes how first the heavy chain was sub-cloned into the vector.
• the source used of the heavy chain was pUBS-Heavy2000/Hyg (-) since in this plasmid the heavy chain is preceded by the Kozak sequence.
• pUBS- Heavy2000/Hyg (-) was digested with first Pmel and subsequently with Nhel.
• the fragment containing the complete heavy chain including Kozak sequence and leader peptide was isolated from agarose gel using Geneclean.
• the fragment was ligated in pcDNA3000/Neo (-) digested with BstXI, blunted with T4 DNA polymerase and subsequently purified over agarose gel using GeneClean.
• the ligation mixture was transformed to competent DH5 ⁇ cells and thirty colonies were picked and cultured in LB/AMP medium for plasmid DNA isolation.
• the plasmid DNA was controlled by restriction enzyme analysis using Kpnl as well as Bglll/Pstl and Agel. Among the 30 colonies, 3 turned out to contain the correct plasmid. One of these was used to generate DNA for further experiments.
• the generated plasmid was named pUBS- Heavy3000/Neo(-) .
• pUBS-Heavy3000/Neo was used together with pUBS-Light2001/Neo (-) .
• the latter was used as source of the light chain since in this construct the sequence of the kappa chain was preceded by the Kozak sequence.
• pUBS-Light2001/Neo was digested with Pmel and Mlul . The fragment containing the complete light chain including Kozak sequence and leader peptide was isolated from agarose gel using GeneClean.
• the fragment was ligated in pUBS-Heavy3000/Neo (-) that was digested with Hpal and Mlul, gelpurified using GeneClean and dephosphorylated with tSAP. Due to the ligation the recognition sites of both Hpal and Pmel were destroyed.
• the ligation mixture was transformed to competent DH5 cells and 30 colonies were picked and cultured in LB/Amp medium for plasmid DNA was isolation.
• the plasmid DNA was controlled by restriction enzyme analysis with Kpnl as well as with Nael. With exception of one clone, all clones contained the correct plasmid. One positive clone was used to generate DNA for further experiments.
• the resulting plasmid was named pUBS3000/Neo(-) .
• Upstream of the first constant domain pNUT-C ⁇ received the variable domain of the gamma chain from the fully humanized monoclonal antibody K53/IgGl that is preceded by a leader peptide essentially according to procedures described by Boel et al. (2000).
• the leader peptide was identical to the one described above. This resulted in an insert of approximately 2 kb.
• the generated plasmid was named pNUT- C ⁇ K53. This plasmid contains a methallothionine promoter (MT- 4 promoter) .
• the heavy chain of K53/IgGl was subcloned into pcDNA3.1/Zeo (Invitrogen) .
• pNUT-C ⁇ K53 was digested with the restriction enzymes BamHI and EcoRI.
• the fragment containing the complete heavy chain including the proceeding leader sequence was purified and ligated in pcDNA3.1/Zeo digested with BamHI and EcoRI.
• the resulting plasmid was named pcDNA3. lK53/Zeo .
• the kappa 2 light chain was also ligated (Fig.24) into pcDNA3.1/Zeo (Invitrogen) to serve as expression vector in HEK 293 cells (see below) .
• pNUT-C ⁇ 2 was digested with BamHI and EcoRI restriction enzymes.
• the resulting 1.2 kb fragment was purified over agarose gel using QiaexII gel Extraction kit (Qiagen) and ligated into the 5.0 kb linearized pcDNA3.1/Zeo digested with BamHI and EcoRI restriction enzymes, purified over agarose gel.
• the ligation mixture was transformed to competent E . coli cells.
• plasmids of 4 generated clones were checked on correct inserts by restriction digestion with BamHI and EcoRI enzymes. One positive clone was used for further experiments.
• the correct plasmid was named pcDNA3. l ⁇ 2-K53/Zeo .
• the final construct (pCD46-3000/Neo) that would contain both the kappa 2 light chain and the heavy chain of K53/IgGl was generated by the exchange of the variable domain of the heavy chain in pUBS3000/Neo (-) with the variable domain of the heavy chain fragment of K53.
• the heavy chain of K53/IgGl did not contain the Kozak sequence in pcDNA3.
• lK53/Zeo so the 5' restriction site of the inserted variable domain had to be located within the leader sequence. Due to the fact that there was no unique restriction site present within the leader sequence, a three-point ligation was used to generate the construct pCD46-3000/Neo. For this pcDNA3.
• lK53/Zeo was digested with Sphl and Sfil.
• Sphl restriction site situated in the leader sequence and the Sfil in the intron following the CH2 domain a 1759 bp heavy chain fragment was isolated from agarose gel using GeneClean containing: part of the leader sequence + variable domain + intron + CHI + intron + Hinge + intron + CH2.
• pUBS-Heavy2000/Hyg (-) was digested with Muni and Sphl.
• a 922 bp fragment containing the sequence of the CMV promoter followed by the Kozak sequence and partly the leader sequence was obtained and isolated from agarose gel using GeneClean.
• pUBS3000/Neo (-) providing the backbone and the kappa 2 light chain was digested with the restriction enzymes Muni and Sfil.
• the backbone fragment of 7172 bp was isolated from agarose gel using GeneClean and subsequently de-phosphorylated with tSAP.
• a three-point ligation with the isolated fragments described above was performed.
• the ligation mixture was transformed to competent DH5 ⁇ cells and thirty colonies were picked and cultured on LB/AMP medium for plasmid DNA isolation.
• the plasmid DNA was controlled by restriction enzyme analysis with Bglll/Ndel and Bglll/Sacl. Of the 30 colonies 17 clones turned out to contain the correct DNA. One of these was used to generate DNA for further experiments.
• the resulting plasmid was named pCD46-3000/Neo and is depicted in Fig.25.
• the region from Muni to Sphl is derived from pUBS-Heavy2000Hyg (-) and contains one CMV promoter;
• the region from Sphl to Sfil is derived from plasmid pcDNA3.
• lK53/Zeo and contains the variable heavy chain and the first two heavy constant domains;
• the region Sfil to Muni is derived from pUBS3000/Neo (-) and contains the final heavy constant domain and poly (A) sites, resistance markers, plasmid replication sequences and the complete kappa 2 encoding region.
• PER.C6 cells (ECACC deposit 96022940) were transfected with pCD46-3000/Neo (construct described above, Fig.24 and 25) .
• This plasmid expresses both light and heavy chains of the K53/IgGl molecule, and also encodes a neomycin resistance marker (Neo) .
• Cells were grown in DMEM supplemented with serum in the presence of Geneticin (G418) to select PER.C6 clones that were stably transfected for this plasmid.
• PER.C6 cells (passage number # 41) were seeded in 10 cm tissue culture dishes in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% MgCl 2 at 3.5xl0 6 cells per dish. The cells were seeded one day before and cultured overnight at 37°C and 10% C0 2 . At day 1, transfections were performed in 49 dishes at 37°C with lipofectamine (Gibco) with 2 ⁇ g pCD46-3000/Neo per dish using procedures well known to persons skilled in the art. After 5 h, the medium of the cells was replaced by DMEM supplemented with 10 % FCS and 1% MgCl 2 . Replacement of the medium was performed regularly.
• the selection pressure was held at 500 ⁇ g G418 per liter. Of the colonies of cells that grew out, 571 were picked manually (on day 20, 21 and 22) to 96-well plates. After several weeks of growth in DMEM 4- serum, in the presence of G418, the culture supernatant from these clones was tested for the presence of monoclonal antibody by ELISA analysis using methodologies well known to persons skilled in the art (IgGl light chain/heavy chain capture: using anti-human IgGl kappa antibody M ⁇ H-Ig, Pharmingen cat.nr. 555789 for capture and biotin labeled anti-human M ⁇ H-Ig cat.nr. 555869 from
• cells were counted using procedures known to the skilled artisan and seeded in 96 well plates (4 plates per clone) at a density of 0.3 cells per well. The limiting dilution efficiency was between 2-15%. Of each clone about 5-14 sub-clones were passed from 96 wells to 24 wells (selected on growth) . An ELISA was performed, and most sub-clones were positive for antibody production. Two vials of all sub-clones were frozen. Based on criteria of expression levels, stability of expression and initial glyco- analysis results, five clones were subjected to a further round of limiting dilution to ensure that all were indeed single clones.
• CD46-007 -114, -124, -130 and -233.
• Sub-clones of the five clones were used for further culturing and analysis of antibody production levels.
• CD46-124 was a relatively bad growing cell line and left out of further experimentation.
• the other sub- clones were tested in ELISA and one clone of each was chosen for further experiments. The best performers reached production levels that ranged between 6.35 and 15.00 pg/cell/day.
• Culture supernatants from several PER.C6 clones producing recombinant K53/IgGl were purified using protein-A columns (Econo-Pac colums prepacked with 2 ml of Affi-Gel protein-A agarose) .
• the columns were rinsed with 50 ml PBS and subsequently the supernatant was applied on the column. After rinsing the column with 20 ml PBS, bound proteins were eluted with 0.1 M citric acid pH 3.0. Fractions of 1 ml were collected and immediately neutralized with 200 ⁇ l 1 M Tris. After elution the column was rinsed with at least 30 ml PBS before the next purification round was started.
• HEK 293 cells were grown at approximately 50-60% confluency in a six well plate in 2 ml/well DMEM supplemented with 10% FCS and Penicilline 10000 IU/ l and Streptomycin 10000 ⁇ g/ml (pen/strep) . The cells were seeded the day before and cultured overnight at 37°C and 5% C0 2 . Co-transfections (day 1) were performed in 10 cm culture-dish plate at 37 °C and 5% C0 2 .
• Cells were left at 4°C for 3.5 h, and thereafter transferred into the incubator at 37 °C and 5% C0 2 . Cells that did not integrate at least one of the two plasmids containing the Zeocin resistance gene, are considered to be killed in the selection procedure. Every 2 to 4 days, the cells were refreshed with medium containing the same concentration of zeocin. After 3 weeks, 13 distinct colonies were picked by scraping and pipetting the cells from the bottom of the dish. Cells were transferred into a 6-wells plate containing 2 ml medium with zeocin. Colonies were expanded for 2 weeks before they were tested for antibody production using an IgG-specific ELISA. The 5 highest producing colonies named L53-1, -2, -7, -8 and -10 were selected for further growth and frozen in liquid N 2 .
• clone L53-7 was selected as best candidate for antibody production.
• Clone L53-2 was not stable and turned out to have lost antibody production over time.
• clones L53-1 and -8 could not be subcultured.
• L53-7 was expanded in selection medium in triple flasks, and grown until a confluency of approximately 80% was reached.
• Ultra-CHO medium 100-120 ml per flask was added and cells were further cultured for 3 days.
• Supernatant containing the antibody was harvested, cells and debris were spun down and supernatant was filtered (0.22 ⁇ m) .
• the antibodies were purified using a protein A- sepharose column.
• the column was rinsed with 50 ml PBS, and subsequently supernatant was run over the column. After rinsing the column with at least 20 ml PBS, the proteins were eluted with 0.1 M citric acid pH 3.0. Fractions of 1 ml were collected and immediately neutralized with approximately 200 ⁇ l 1 M Tris. The purification was performed 5 times (Hl-5) . The protein containing fractions 3 and 4 (HI, 2, 3 and 4), as determined by spectrophotometry at 280 nm, were pooled and dialyzed extensively against PBS at 4°C. The monoclonal antibodies were filtered (0.22 ⁇ m) and final concentrations were determined using the Biorad Protein Assay (see Table XI) .
• a lxlO 6 CD46-overexpressing tumor cells (LS174T) are injected subcutaneously in immune deficient mice and treated one day later with 100 ⁇ g human anti-EpCAM (UBS-54) monoclonal antibody and/or 100 ⁇ g human K53/IgGl monoclonal antibody. On day 3 and 6, the treatment is repeated with 50 ⁇ g UBS-54 monoclonal antibody and/or 50 ⁇ g human K53/IgGl monoclonal antibody.
• UBS-54 human anti-EpCAM
• 50 ⁇ g human K53/IgGl monoclonal antibody As a control a Streptococcus specific GBS III antibody is used (see above) .
• MCP membrane cofactor protein

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