Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
  • A61K49/0058—Antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/14—Peptides, e.g. proteins
  • A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • 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
  • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • 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
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/77—Internalization into the cell

Definitions

• compositions and methods of use described herein are in the area of materials and methods for enhancing cellular internalization.
• anti-mitotic drugs such as adriamycin, vincristine, cisplatin, doxorubicin, daunomycin and methotrexate, toxins such as diphtheria toxin, pseudomonas toxin and ricin, and anti-tumor drugs such as cyclophosphamide and isophosphamide in cancer chemotherapy has acute undesirable side effects on the normal cells of the patient, thus severely limiting the doses that can safely be administered.
• anti-mitotic drugs such as adriamycin, vincristine, cisplatin, doxorubicin, daunomycin and methotrexate
• toxins such as diphtheria toxin, pseudomonas toxin and ricin
• anti-tumor drugs such as cyclophosphamide and isophosphamide in cancer chemotherapy has acute undesirable side effects on the normal cells of the patient, thus severely limiting the doses that can safely be administered.
• drug targeting One approach has been to attempt to specifically direct anticancer drugs to malignant cells, so that their effect on normal cells would be minimal.
• drug targeting is generally referred to as "drug targeting.”
• the drug is conjugated to a biodegradable polyamino acid macromolecular base or to such a polyamino acid base that is also linked to a carrier agent.
• a carrier agent e.g., a biodegradable polyamino acid macromolecular base
• degradation of the polyamino acid base in the target cells releases the cytotoxic drug.
• the use of a polyamino acid base may reduce the ability of the conjugate to penetrate many tumors efficiently.
• carrier agents there is a dearth of appropriate carrier agents.
• several compounds have been considered as carrier agents, they suffer from disadvantages including unpredictability of their internalization into cells.
• HerceptinTM A humanized monoclonal antibody, HerceptinTM, has been used to successfully treat some women with HER"2 /neu overexpressing breast and ovarian cancers, demonstrating that pl85 HER 2 can be used to selectively target human cancers (10, 1 1). While outcomes have improved for patients treated with HerceptinTM, more effective classes of antibody-based therapies need to be developed to capitalize on cancer specific antigen expression. Moreover, because relatively little is known about cellular responses after binding of a vector construct there is a need to assess the efficiency of post-binding receptor-mediated endocytosis, which will help to improve the efficacy of novel therapies.
• the present invention relates to compositions and methods for enhancing intracellular delivery of bioactive and/or diagnostic agents and to provide less invasive methods for delivering high molecular weight and labile drugs, such as proteins and nucleic acid molecules, and diagnostic agents.
• the invention relates to a carrier compound, such as a monoclonal antibody, to deliver other molecules or gene therapy vectors specifically to cells that can be identified by a specific surface marker protein and which, once bound, would be internalized by a normal cellular process called receptor-mediated endocytosis.
• a carrier compound such as a monoclonal antibody
• compositions and methods for improving cellular internalization of one or more compounds are disclosed.
• the invention provides a drug conjugate composition that can be delivered to a target cell, which comprises a carrier compound that has a binding specificity for a receptor molecule and is conjugated to a therapeutic or diagnostic moiety.
• a target cell which comprises a carrier compound that has a binding specificity for a receptor molecule and is conjugated to a therapeutic or diagnostic moiety.
• the carrier compound binds to the receptor and is internalized by the target cell.
• the invention provides methods for treating disease states and inhibiting tumors using the compositions of the invention. Also described are methods of using the compositions and compounds of the invention to detect disease states and tumors, and to screen for putative compositions of the invention.
• the invention also provides four novel monoclonal antibodies that recognize the extracellular domain of pl85 " , a membrane receptor protein, for use in internalization-dependent therapies. All four antibodies bind to pl85 HER 2 and two of them recognize accessible epitopes of pl85 HER"2 on viable cells. Because the successful gene therapy vector will require not only cell targeting, but internalization of the vector and expression of a therapeutic gene, it is important to evaluate as many of these steps as possible prior to construction of the vector. To this end, the invention discloses characterization of the internalization potential of the four anti- pi 85 HER ⁇ 2 monoclonal antibodies using a rapid, quantifiable radioimmunoassay.
• HFR 7 results of the invention show that two pi 85 " monoclonal antibodies, 8H1 1 and 10H8, will bind to viable, intact target cells, and will be internalized and trafficked through an endosomal pathway and, thus, establish that the antibodies will be useful in targeting treatment vectors that require internalization for therapeutic effect.
• FIG. 1 Purification of recombinant m ⁇ 2 /neu extracellular domain (ECD) protein with poly-His tag.
• ECD extracellular domain
• the cDNA of HER"2 / «ew was cut between the Ncol and Sphl sites and subcloned into an expression vector for production of a 70 kDa epitope-tagged protein.
• TM transmembrane domain.
• Purified protein was resolved by SDS-PAGE, transferred to nitrocellulose and detected by an antibody to the poly- His tag.
• FIG. 1 Western Blots of the total protein lysates from human and mouse cell lines.
• Monoclonal antibodies 5A7, 11F11, 8H11, and 10H8 were tested for binding to p 185 " in total protein lysates from normal human mammary epithelial cells (HMEC), human breast carcinoma cells overexpressing HER ⁇ 2 / «e « (SKBR-3), human epidermoid carcinoma cells overexpressing EGF receptor (A431), NIH3T3 cells which lack HER 2 /new expression, and NIH/189 cells engineered to overexpress " Ineu.
• HMEC normal human mammary epithelial cells
• SKBR-3 human breast carcinoma cells overexpressing HER ⁇ 2 / «e «
• human epidermoid carcinoma cells overexpressing EGF receptor (A431) human epidermoid carcinoma cells overexpressing EGF receptor (A431)
• NIH3T3 cells which lack HER 2 /new expression
• FIG. 1 Immunohistochemical localization of pi 85 HER 2 .
• Tissue sections from a single archival paraffin-embedded breast tumor were analyzed for monoclonal antibody localization to membranes of carcinoma cells.
• Membrane localization of anti-pl85 HER"2 monoclonal antibodies were detected by the peroxidase- anti-peroxidase method.
• FIG. 5 Flow cytometry using novel monoclonal antibodies to detect pl85 HER"2 on SKBR-3 cells.
• SKBR-3 cells overexpressing pi 85 HER"2 were incubated with indicated monoclonal antibody and then tagged with secondary FITC-labeled antibodies.
• 8H11 and 10H8 monoclonal antibodies detected pl85 HER"2 on the surface of viable SKBR-3 cells.
• IgG isotype control, 5A7 and 1 1F11 show no fluorescence.
• Figure 6 Receptor-mediated endocytosis assays showing internalization and catabolism of monoclonal antibodies 8H11 and 10H8 by NIH/189 cells.
• Figure 7 shows the sequences of the variable regions of monoclonal antibody 8H11 both for the light chain (upper) and for the heavy chain (lower).
• Figure 8 shows the sequences of the variable regions of monoclonal antibody 10H8 both for the light chain (upper) and for the heavy chain (lower).
• antibody or antibody molecule in the various grammatical forms is used herein as a collective noun that refers to a population of immunoglobulin molecules and or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope.
• an "antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
• monoclonal antibody in its various grammatical forms refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with a particular epitope.
• a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g., a bispecific monoclonal antibody.
• nucleotide sequence refers to a heteropolymer of nucleotides or the sequence of nucleotides.
• nucleic acid and polynucleotide are also used interchangeably herein to refer to a heteropolymer of nucleotides.
• nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.
• oligonucleotide fragment or a “polynucleotide fragment,” “portion,” or “segment” refer to a stretch of nucleotide residues which is long enough to use in polymerase chain reaction (PCR) or various hybridization procedures to identify or amplify identical or related parts of mRNA or DNA molecules.
• PCR polymerase chain reaction
• Oligonucleotides comprise portions of such a polynucleotide sequence having at least about 15 nucleotides and usually at least about 20 nucleotides.
• Nucleic acid probes comprise portions of such a polynucleotide sequence having fewer nucleotides than about 6 kb, usually fewer than
• probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue.
• probes includes naturally occurring or recombinant or chemically synthesized single- or double-stranded nucleic acids. They may be labeled by nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al, 1989. Molecular Cloning: A Laboratory Manual, Cold Spring
• recombinant when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, mammalian, or insect-based) expression systems.
• Microbial refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.
• recombinant microbial defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.
• recombinant expression vehicle or vector refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.
• An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.
• Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
• recombinant protein when expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
• recombinant expression system means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.
• Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.
• This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.
• Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.
• the cells can be prokaryotic or eukaryotic.
• active refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.
• naturally occurring polypeptide refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
• derivative refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.
• recombinant variant refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using recombinant DNA techniques.
• Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, such as cellular trafficking, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology.
• amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.
• Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
• nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
• “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
• insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides.
• Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.
• such alterations may change polypeptide characteristics such as ligand- binding affinities, interchain affinities, or degradation/turnover rate.
• such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.
• cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.
• substantially equivalent can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.
• a substantially equivalent sequence varies from one of those listed herein by no more than about 2% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.02 or less).
• Such a sequence is said to have 98% sequence identity to the listed sequence.
• a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 2% (98% sequence identity); in a variation of this embodiment, by no more than 0.5% (99.5% sequence identity); and in a further variation of this embodiment, by no more than 0.1 % (99.9% sequence identity).
• Substantially equivalent, e.g., mutant, amino acid sequences according to the invention generally have at least 98% sequence identity with a listed amino acid sequence, whereas substantially equivalent nucleotide sequence of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code. For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded.
• an expression vector may be designed to contain a "signal or leader sequence" which will direct the polypeptide through the membrane of a cell.
• a signal or leader sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.
• a polypeptide "fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, often at least about 7 amino acids, typically at least about 9 to 13 amino acids, and, in various embodiments, at least about 17 or more amino acids. To be active, any polypeptide must have sufficient length to display biologic and or immunologic activity.
• recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic code.
• Various codon substitutions such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.
• Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.
• activated cells are those which are engaged in extracellular or intracellular membrane trafficking, including the export of neurosecretory or enzymatic molecules as part of a normal or disease process.
• purified denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.
• the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99.8% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).
• isolated refers to a nucleic acid or polypeptide separated from at least one other component (e.g. , nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.
• the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.
• isolated and purified do not encompass nucleic acids or polypeptides present in their natural source.
• salt it is meant those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
• Pharmaceutically acceptable salts are well known in the art. For example, S. M.
• Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphersulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pe
• alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
• ester refers to esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof.
• Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.
• Examples of particular esters includes formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.
• prodrugs refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.
• prodrug refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A. C. S. Symposium Series, and in Edward B.
• ORF open reading frame
• EMF expression modulating fragment
• infection refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.
• transformation means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.
• transfection refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.
• RME receptor-mediated endocytosis
• the second prong of a successful drug delivery regimen requires the use of well-characterized reagents that specifically recognize tumor cells in the host background and preferentially bind to such cells rather than to normal cells.
• Recent identification of differences in the genotype and molecular stucture of cancer cells has provided an opportunity to develop such targetable reagents for the specific therapy of selected human cancers (1-4).
• amplification of the 2 /neu oncogene in human breast cancers, endometrial cancers, ovarian cancers, gastric cancers, and salivary gland carcinomas provides a potential molecular target for these cancers (5- 9).
• HER"2 /neu gene amplification is associated with overexpression of the pl85 HER"2 protein.
• Both monoclonal antibodies have been characterized extensively for binding to the protein product of the HER"2 /neu oncogene. They have also been shown to be internalized specifically in " /neu overexpressing cells and show evidence of endosomal location followed by degradation and extrusion into extracellular spaces as protein fragments found floating outside the cells.
• the genes encoding each VH and VL region of monoclonal antibodies 8H11 and 10H8 have been isolated, cloned, and sequenced. Predicted amino acids were cross-referenced to a database of amino acid sequences of monoclonal antibodies and confirmed as genuine monoclonal antibody variable regions. Amino acid sequence analysis of a partial tryptic digest of one of the expressed genes confirmed that the predicted amino acid sequence was correct.
• monoclonal antibodies of the invention may be exploited for the ability to trigger internalization of "piggybacked" gene therapy vectors, therapeutic toxins, or imaging molecules.
• Toxin proteins, chemicals, or isotopic labels may also be conjugated to the monoclonal antibodies to deliver these effector molecules into a cell in order to enhance targeted therapies or create imaging agents which rely on endosome localization.
• Gene therapy and immunotoxin therapy may be effectively enhanced when the effector vector or protein can be predictably delivered into an endosome of a targeted cell.
• radiographic imaging may be established by such targeted moieties.
• the carrier component of the conjugate has a cell- specific binding component (or site) and a moiety-binding component (or site).
• the cell-specific binding component specifically binds a cellular surface structure which mediates its internalization by, for example, the process of endocytosis.
• the surface structure can be a protein, polypeptide, carbohydrate, lipid or combination thereof. It is typically a surface receptor which mediates endocytosis of a ligand.
• the binding component can be a natural or synthetic ligand which binds the receptor.
• the ligand can be an antibody, a protein, polypeptide, carbohydrate, lipid or a combination thereof which has functional groups that are exposed sufficiently to be recognized by the cell surface structure. It can also be a component of a biological organism such as a virus, cells (e.g., mammalian, bacterial, protozoan) or artificial carriers such as liposomes.
• Ligands useful in forming the carrier will vary according to the particular cell to be targeted.
• glycoproteins having exposed terminal carbohydrate groups such as asialoglycoprotein (galactose-terminal) can be used, although other ligands such as polypeptide hormones may also be employed.
• asialoglycoproteins include asialoorosomucoid, asialofetuin and desialylated vesicular stomatitis virus.
• Such ligands can be formed by chemical or enzymatic desialylation of glycoproteins that possess terminal sialic acid and penultimate galactose residues.
• asialoglycoprotein ligands can be formed by coupling galactose terminal carbohydrates such as lactose or arabinogalactan to non-galactose bearing proteins by reductive lactosamination.
• the cell-specific binding agent can be a receptor or receptor-like molecule, such as an antibody which binds a ligand (e.g., antigen) on the cell surface.
• a ligand e.g., antigen
• the moiety conjugated to the carrier for internalization is an oligonucleotide, an antibody, monoclonal or polyclonal, an antibody fragment, a therapeutic toxin, or an imaging molecule, such as an isotope label.
• the invention is a conjugate composition for targeted delivery to a target cell that comprises a carrier compound that has a binding specificity for a receptor molecule and a moiety conjugated to the carrier compound. This composition is internalized by the target cells when the carrier compound binds to the receptor molecule.
• the carrier compound is an antibody, either monoclonal or polyclonal, or a fragment of such antibodies.
• the invention provides for monoclonal antibodies Mab 8H11, 10H8, 5A7 and 11F11 as carrier compounds.
• the conjugates of the invention are useful for delivering different moieties to various kinds of cells including, but not limited to, cancer cells.
• the receptor molecule in certain embodiments, is a polypeptide overexpressed by cancer cells, for example, the pi 85 HER- " protein.
• the conjugate of the invention targets delivery of a moiety to cancer cells that overexpress the HER"2 /neu oncogene.
• This composition comprises a monoclonal antibody that is anti-pl85 HER 2 protein and a moiety conjugated to the antibody.
• the antibody has a binding specificity for the pl85 HER"2 protein and it is internalized when it binds to the protein.
• the monoclonal antibody in certain embodiments of the invention, is one of Mab 8H11, Mab 10H8, Mab 5A7 and Mab 1 1F1 1.
• the moiety conjugated to the antibody is a therapeutic moiety or an imaging compound.
• the moiety-binding component conjugates the moiety to be delivered. Conjugation with the moiety must be sufficiently stable in vivo to prevent significant uncoupling of the moiety extracellularly prior to internalization by the target cell. However, the conjugate is cleavable under appropriate conditions within the cell so that the moiety is released in functional form.
• the conjugate can be labile in the acidic and enzyme rich environment of lysosomes. A non-covalent bond based on electrostatic attraction between the binding component and the moiety provides extracellular stability and is releasable under intracellular conditions.
• the moiety and carrier are mixed and incubated under conditions conducive to complexation. For example, the moiety and carrier can be mixed at the appropriate ratio in 2 M NaCl and the solution can be diluted to 0.15 M and filtered to provide an administrable composition.
• the carrier compounds of the present invention are antibodies, monoclonal or polyclonal, or fragments thereof.
• Example monoclonal antibodies are Mab 8H11, 10H8, 5A7 and 11F11.
• moieties of the conjugate, therapeutic or imaging can also be antibodies.
• Any animal which is known to produce antibodies can be immunized with a peptide, e.g., the receptor molecule, pi 85 HER"2 protein.
• Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the peptide. One skilled in the art will recognize that the amount of the peptide used for immunization will vary based on the animal which is immunized, the antigenicity of the peptide and the site of injection.
• the peptide that is used as an immunogen may be modified or administered with an adjuvant to increase the peptide's antigenicity.
• Methods of increasing the antigenicity of a peptide are well known in the art and include, but are not limited to, coupling the antigen with a heterologous protein (such as globulin or ⁇ -galactosidase) or through the inclusion of an adjuvant during immunization.
• spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Agl4 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
• myeloma cells such as SP2/0-Agl4 myeloma cells
• Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al, Exp. Cell Research, 175: 109-124 (1988)).
• Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and
• HFR 2 peptides e.g., pl85 " protein.
• antibody containing antiserum is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.
• the present invention further provides the above-described antibodies in detectably labeled form.
• Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labeling are well-known in the art, for example, see (Sternberger, L.A. et al, J. Histochem. Cytochem. 18:315 (1970); Bayer, E.A. et al, Meth. Enzym. 62:308 (1979); Engval, E. et al, Immunol. 109:129 (1972); Goding, J.W. J. Immunol. Meth.
• the antibodies can also be used to make anti-idiotype atibodies which in turn can be humanized as is known in the art to prevent immunological responses.
• Humanized monoclonal antibodies offer particular advantages over murine monoclonal antibodies, particularly insofar as they can be used therapeutically in humans. Specifically, human antibodies are not cleared from the circulation as rapidly as “foreign” antigens, and do not activate the immune system in the same manner as foreign antigens and foreign antibodies.
• the antibody of the invention can also be a fully human antibody such as those generated, for example, by selection from an antibody phage display library displaying human single chain or double chain antibodies such as those described in de Haard, H.J. et al. (1999) J. Biol. Chem. 274:18218-30 and in Winter, G. et al. ( ⁇ 99A) Annu. Rev. Immunol. 12:433-55.
• the labeled antibodies of the present mvention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues in which a fragment of the receptor molecule of interest is expressed.
• the antibodies also may be used directly in therapies or other diagnostics.
• the present invention further provides the above- described antibodies immobilized on a solid support.
• solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir, D.M. et al, Handbook of Experimental Immunology 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W.D.
• the immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as for im uno- affinity purification of receptors for which the antibodies have a binding affinity.
• the antibodies can also be used in research to further elucidate the functioning of the signaling pathways in activation of cells by growth factors, hormones, cytokines or the like.
• Antisera titer may be established through several means known in the art, such as by dot blot and density analysis, and also by precipitation of radiolabeled peptide-antibody complexes using a protein, secondary antisera, cold ethanol or charcoal-dextran followed by activity measurement with a gamma counter. If desired, the highest titer antisera may be purified on affinity columns. For example, a peptide such as the receptor molecules pl85 HER"2 may be coupled to a commercially available resin and used to form an affinity column. Antiserum samples may then be passed through the column so that antibodies to the peptide bind (via the peptide) to the column. These bound antibodies are subsequently eluted, collected and evaluated for determination of titer and specificity.
• An additional way to determine whether a monoclonal antibody has the specificity of a monoclonal antibody of the invention is to determine the amino acid residue sequence of the CDR regions of the antibodies in question.
• Antibody molecules having identical, or functionally equivalent, amino acid residue sequences in their CDR regions have the same binding specificity. Methods for sequencing polypeptides are well known in the art. This does not suggest that antibodies with distinct CDR regions cannot bind to the same epitope.
• Exemplary antibodies for use in the present invention include intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v), and also referred to as antibody fragments.
• the Fab fragment, lacking Fc receptor, is soluble, and affords therapeutic advantages in serum half life, and diagnostic advantages in modes of using the soluble Fab fragment.
• the preparation of a soluble Fab fragment is generally known in the immunological arts and can be accomplished by a variety of methods.
• Fab and F(ab') 2 portions (fragments) of antibodies are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibodies by methods that are well known. See for example, U.S. Pat. No. 4,342,566 to Theofilopolous and Dixon.
• Fab' antibody portions also are well known and are produced from F(ab') .sub.2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
• Nucleotide sequences of the invention are reported below.
• the present invention also provides genes corresponding to the cDNA sequences disclosed herein.
• the corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
• compositions of the present invention include,but are not limited to, isolated polynucleotides, including recombinant DNA molecules, and cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants.
• SEQ ID NO: 1 encodes the variable light region of Mab 8H11
• SEQ ID NO:2 encodes the variable heavy region of Mab 8H11
• SEQ ID NO:3 encodes the variable light region of Mab 10H8
• SEQ ID NO:4 encodes the variable heavy region of Mab 10H8.
• the isolated polynucleotides of the invention include, but are not limited to, polynucleotides comprising the nucleotide sequence of SEQ ID NO: 1 , or polynucleotides comprising the nucleotide sequence of SEQ ID NO: 2, or polynucleotides comprising the nucleotide sequence of SEQ ID NO: 3, or polynucleotides comprising the nucleotide sequence of SEQ ID NO: 4.
• the polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.
• the invention also provides for polynucleotides as moieties to be used in the conjugate compositions of the invention.
• the invention provides for targeted delivery of gene therapy vectors.
• the polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.
• the invention also provides the complement of the polynucleotides including a nucleotide sequence that has at least about 95%, more typically at least about 99%), and even more typically at least about 99.5%, sequence identity to a polynucleotide encoding a polypeptide recited above.
• the polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions which can routinely isolate polynucleotides of the desired sequence identities.
• a polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).
• Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.
• the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.
• Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
• a host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism. The sequences falling within the scope of the present invention are not limited to the specific sequences herein described, but also include allelic variations thereof.
• allelic variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1, a representative fragment thereof, or a nucleotide sequence at least 98 % identical to SEQ ID NO: 1 or nucleotides 226-2836 of SEQ ID NO: 1, with a sequence from another human isolate.
• An allelic variation is more typically at least 99%o identical to SEQ ID NO: 1 and even more typically 99.8% identical to SEQ ID NO: 1.
• the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein. In other words, in the coding region of an
• ORF substitution of one codon for another which encodes the same amino acid is expressly contemplated. Any specific sequence disclosed herein can be readily screened for errors by resequencing a particular fragment, such as an ORF, in both directions (i.e., sequence both strands).
• the present invention further provides recombinant constructs comprising a nucleic acid having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or a fragment thereof.
• the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having the sequence of one of the above-noted sequences or a fragment thereof is inserted, in a forward or reverse orientation.
• the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.
• Bacterial Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
• nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids. These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide. There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.
• the amino acid sequence variants of the nucleic acids are preferably constructed by mutating the polynucleotide to give an amino acid sequence that does not occur in nature. These amino acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions). Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g. , hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site. Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to
• Amino acid insertions include amino- and/or carboxy-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues. Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells.
• polynucleotides encoding the novel nucleic acids are changed via site-directed mutagenesis.
• This method uses oligonucleotide sequences that encode the polynucleotide sequence of the desired amino acid variant, as well as a sufficient adjacent nucleotide on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed.
• site-directed mutagenesis is well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al, DNA 2:183 (1983).
• a versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res.
• PCR may also be used to create amino acid sequence variants of the novel nucleic acids.
• primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.
• PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer. The product DNA fragments replace the corresponding region in the plasmid and this gives the desired amino acid variant.
• a further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al, Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al, supra, and Current Protocols in Molecular Biology. Ausubel et al, supra Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.
• the conjugates of the invention may be used in combination with other compositions and procedures for the treatment of diseases.
• a tumor may be treated conventionally with surgery, radiation or chemotherapy combined with a conjugate of the present invention.
• the conjugates of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained- release matrices, such as biodegradable polymers, to form therapeutic compositions.
• a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids.
• a sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid) polyanhydrides, poly(ortho) esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxcylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
• a preferred biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and glycolic acid).
• a therapeutically effective amount of one of the conjugates of the present invention may be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form.
• terapéuticaally effective amount of the compound of the invention is meant a sufficient amount of the compound to treat an angiogenic disease, (for example, to limit tumor growth or to slow or block tumor metastasis) at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the conjugate of the present invention will be decided by the attending physician within the scope of sound medical judgment.
• the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the conjugate at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
• the conjugate compositions of the present invention can be used in the form of salts derived from inorganic or organic acids.
• These salts include but are not limited to the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethansulfonate (isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, pcrate, pivalate, propionate, succinate, tartrate, thiocyan
• acids which may be employed to form pharmaceutically acceptable addition salts include such inorganic acids as hydrochloric acid, sulphuric acid and phosphoric acid and such organic acids as maleic acid, succinic acid and citric acid.
• Other salts include salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium or magnesium or with organic basis.
• Preferred salts of the conjugate compositions of the invention include phosphate, tris and acetate.
• the total daily dose of the conjugate compositions of this invention administered to a human or lower animal may range from about 0.001 to about 1 mg/kg of patients body mass/day. If desired, the effective daily dose may be divided into multiple doses for purposes of administration; consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
• a conjugate of the present invention may be administered as pharmaceutical compositions containing the conjugate of interest in combination with one or more pharmaceutically acceptable excipients.
• a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• the compositions may be administered parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), rectally, or bucally.
• parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
• compositions for parenteral inj ection comprise pharmaceutically-acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
• suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols
• glycerol such as glycerol, propylene glycol, polyethylene glycol, and the like
• carboxymethylcellulose and suitable mixtures thereof vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
• Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
• Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
• the injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
• Topical administration includes administration to the skin or mucosa, including surfaces of the lung and eye.
• Compositions for topical administration, including those for inhalation, may be prepared as a dry powder which may be pressurized or non-pressurized.
• the active ingredient in finely divided form may be used in admixture with a larger-sized pharmaceutically-acceptable inert carrier comprising particles having a size, for example, of up to 100 micrometers in diameter.
• Suitable inert carriers include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.
• the composition may be pressurized and contain a compressed gas, such as nitrogen or a liquified gas propellant.
• a compressed gas such as nitrogen or a liquified gas propellant.
• the liquified propellant medium and indeed the total composition is preferably such that the active ingredient does not dissolve therein to any substantial extent.
• the pressurized composition may also contain a surface active agent, such as a liquid or solid non-ionic surface active agent or may be a solid anionic surface active agent It is preferred to use the solid anionic surface active agent in the form of a sodium salt.
• a further form of topical administration is to the eye.
• a conjugate of the invention is delivered in a pharmaceutically acceptable ophthalmic vehicle, such that the conjugate is maintained in contact with the ocular surface for a sufficient time period to allow the conjugate to penetrate the corneal and internal regions of the eye, as for example the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid retina and sclera.
• the pharmaceutically-acceptable ophthalmic vehicle may, for example, be an ointment, vegetable oil or an encapsulating material.
• the conjugates of the invention may be injected directly into the vitreous and aqueous humour.
• compositions for rectal or vaginal administration are preferably suppositories which may be prepared by mixing the conjugates of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active conjugate.
• suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active conjugate.
• Conjugates of the present invention may also be administered in the form of liposomes.
• liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi- lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any nontoxic, physiologically-acceptable and metabolizable lipid capable of forming liposomes can be used.
• the present compositions in liposome form can contain, in addition to a conjugate of the present invention, stabilizers, preservatives, excipients, and the like.
• the preferred lipids are the phospholipids and the phosphatidyl cholines
• liposomes both natural and synthetic.
• Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.
• conjugates of the invention can be administered as the sole active pharmaceutical agent, they may also be used in combination with one or more agents which are conventionally administered to patients for treating angiogenic diseases.
• the conjugates of the invention are effective over the short term to make tumors more sensitive to traditional cytotoxic therapies such as chemicals and radiation.
• the conjugates of the invention also enhance the effectiveness of existing cytotoxic adjuvant anti-cancer therapies.
• the conjugates of the invention may also be combined with other antiangiogenic agents to enhance their effectiveness, or combined with other antiangiogenic agents and administered together with other cytotoxic agents.
• conjugates of the invention when used in the treatment of solid tumors, conjugates of the invention may be administered with IL-12, retinoids, interferons, angiostatin, endostatin, thalidomide, thrombospondin-1, thrombospondin-
• captopryl anti-neoplastic agents such as alpha inteferon, COMP (cyclophosphamide, vincristine, methotrexate and prednisone), etoposide, mBACOD (methortrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone), PRO-MACE/MOPP (prednisone, methotrexate (w/leucovin rescue), doxorubicin, cyclophosphamide, taxol, etoposide/mechlorethamine, vincristine, prednisone and procarbazine), vincristine, vinblastine, angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG and the like as well as with rediation.
• Total daily dose of the compositions of the invention to be administered to a human or other mammal host in single or divided doses may be in amounts, for example, from 0.0001 to 300 mg/kg body weight daily and more usually 1 to 300 mg/kg body weight.
• agents which can be combined with the conjugate of the present invention for the inhibition, treatment or prophylaxis of angiogenic diseases are not limited to those listed above, but include in principle any agents useful for the treatment or prophylaxis of angiogenic diseases.
• the invention provides for methods for treating various disease states.
• the methods comprise administering to a subject the conjugates of the invention.
• the conjugates are administered in a therapeutically effective amount and with a pharmaceutically acceptable carrier as discussed above.
• the disease state to be treated is, inter alia, cancer, psoriasis, macular degeneration, a neurological disease or restenosis in a tissue.
• the cancers to be treated include, but are not limited to, colorectal cancer, breast cancer, gastric cancer, esophageal cancer, small cell lung carcinoma, and different types of lymphoma such as Burkitt's lymphoma and B follicular cell lymphoma.
• the conjugates of the invention are administered to inhibit tumor growth or metastasis in a tissue.
• the tumor may be a melanoma, carcinoma, sarcoma, fibrosarcoma, glioma or astrocytoma.
• conjugates of the invention can also be used to inhibit angiogenesis, which plays an important role in a variety of disease processes.
• angiogenesis inhibitors conjugates of the invention are useful in the treatment of both primary and metastatic solid tumors, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract, (including cervix, uterus, and ovaries as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as
• conjugates may also be useful in treating solid tumors arising from hematopoietic malignancies such as leukemias (i.e. chloromas, plasmacytomas and the plaques and tumors of mycosis fungoides and cutaneous T-cell lymphoma/leukemia) as well as in the treatment of lymphomas (both Hodgkin's and non-Hodgkin's lymphomas).
• leukemias i.e. chloromas, plasmacytomas and the plaques and tumors of mycosis fungoides and cutaneous T-cell lymphoma/leukemia
• lymphomas both Hodgkin's and non-Hodgkin's lymphomas.
• these conjugates or genes which encode their expression may be useful in the prevention of metastases from the tumors described above either when used alone or in combination with radiotherapy and/or other chemotherapeutic agents.
• the patient treated in the present invention in its many embodiments is desirably a human patient, although it is to be understood that the principles of the invention indicate that the invention is effective with respect to all mammals, which are intended to be included in the term "patient".
• a patient can be, for example, a pig, a cow, a horse, a goat, a sheep, a mule, a donkey, a dog, a cat, a rabbit, a mouse and a rat.
• a tissue to be treated is a tumor tissue of a patient with a solid tumor, a metastases, a skin cancer, a breast cancer, a hemangioma or angiofibroma and the like cancer.
• Typical solid tumor tissues treatable by the present methods include lung, pancreas, breast, uterus, salivary gland, stomach, colon, laryngeal, ovarian, Kaposi's Sarcoma and the like tissues.
• the invention contemplates the practice of the method in conjunction with other therapies such as conventional chemotherapy or radiation therapy directed against solid tumors and for control of establishment of metastases.
• therapies such as conventional chemotherapy or radiation therapy directed against solid tumors and for control of establishment of metastases.
• the administration of the invention's conjugates is typically conducted during or after chemotherapy.
• the dosage ranges for the administration of a conjugate of the invention depend upon the form of the conjugate, and its potency, and are amounts large enough to produce the desired effect in which the disease symptoms are ameliorated.
• the dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like.
• the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage also can be adjusted by the individual physician in the event of any complication.
• conjugates of the invention can be administered parenterally by injection or by gradual infusion over time.
• tissue to be treated can typically be accessed in the body by systemic administration and therefore most often treated by intravenous administration of therapeutic compositions, other tissues and delivery means are contemplated where there is a likelihood that the tissue targeted contains the target molecule.
• conjugates of the invention including monoclonal antibodies, polypeptides, and derivatives thereof can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, topically, intraocularly, orally, intranasally and can be delivered by peristaltic means.
• compositions of this invention are conventionally administered intravenously, as by injection of a unit dose, for example.
• unit dose when used in reference to a composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
• the compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
• the quantity to be administered and timing depends on the patient to be treated, capacity of the patient's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual.
• suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration also are variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
• the conjugates of the invention also are suitable for detection of disease in tissues.
• the tissue is ex vivo.
• the conjugate is an antibody
• it can be used in immunohistochemical techniques to stain tissues ex vivo. Immunological techniques such as immunostaining and ELISA are described in, for example, Receptor Binding Techniques. Methods in Molecular Biology. 106. ed. M. Keen. Humana Press, 1999; Brooks et al. (1998) Cell 92:391-400; Brooks et al. (1996) Cell 85:683-693; and Brooks et al. (1993) J. Cell.
• conjugates of the invention once bound to the target tissue can be detected either directly or indirectly.
• Direct detection can be performed on conjugates that comprise a moiety, which is detectable label such as a fluorochrome, a radioactive tag, paramagnetic heavy metal or diagnostic dye.
• a detectably labeled conjugate For in vivo detection, it is preferable to use a detectably labeled conjugate.
• the labeled conjugate is administered to a patient intravenously, intramuscularly, transdermally, intrasynovially, intratumorally, intraocularly, intranasally, intrathecally, topically or orally.
• Labels suitable for detection within a patient are particularly preferred.
• paramagnetically labeled conjugates can be detected by magnetic resonance imaging. Radioactively tagged conjugates also can be detected.
• the invention also provides for a method for screening for the carrier compounds of the invention.
• such methods comprise providing a putative carrier, measuring the (first) affinity of the putative carrier compound to bind to pl85 HER"2 , and measuring the (second) affinity of an antibody for binding with pl85 HER"2 .
• the putative carrier is selected as a carrier of the invention, if the second affinity is less than the first affinity.
• the second affinity is measured for antibodies that include Mab 8H11, Mab 10H8, Mab 5A7 and
• the invention provides for determining the internalization potential of putative carrier compounds.
• the fraction of Mab 8H11 that is internalized by NIH/189 cells is compared to the fraction of the putative carrier that is internalized by NIH/189 cells.
• the internalization potential of the putative carrier is high, if the fraction of the putative carrier internalized by NIH/189 cells is higher than the fraction of Mab 8H11 internalized by NIH/189 cells.
• Mab 10H8 is used to determine the internalization potential of the putative carrier.
• the carrier compounds of the present invention may be chemically coupled to isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules, chemiluminescent, bioluminescent and other compounds for a variety of applications.
• a carrier compound may be labeled to facilitate testing of its ability to bind antisera or to detect cell types which possess a relevant receptor.
• the coupling technique is generally chosen on the basis of the functional groups available on the amino acids of the peptide including, but not limited to amino, sulfhydral, carboxyl, amide, phenol, and imidazole groups.
• reagents used to effect such couplings include among others, glutaraldehyde, dizodized benzidine, carbodiimide, and p-benzoquinone.
• the efficiency of the coupling reaction is determined using different techniques appropriate for the specific reaction. For example, radiolabeling of the carrier compound with I 125 may be accomplished using chloramine T and Nal 125 of high specific activity. The reaction is terminated with sodium metabisulfite and the mixture is desalted on disposable columns. The labeled peptide is eluted from the column and fractions are collected. Aliquots are removed from each fraction and radioactivity measured in a gamma counter. In this manner, a labeled carrier compound may be obtained which is free from unreacted Nal 125 .
• Kits for measurement of receptors that facilitate cellular internalization are also contemplated as part of the present invention.
• Antisera that possess the highest titer and specificity and can detect receptor molecules in extracts of plasma, tissues, and in cell culture media may be used to establish assay kits for rapid, reliable, sensitive, and specific measurement and detection of receptors.
• kits may employ (but are not limited to) the following techniques: competitive and non-competitive assays, radioimmunoassay (RIA), bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry.
• RIA radioimmunoassay
• bioluminescence and chemiluminescence assays fluorometric assays
• sandwich assays sandwich assays
• immunoradiometric assays sandwich assays
• dot blots enzyme linked assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry.
• enzyme linked assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry
• kits may be used to visualize or localize receptor moleculse in tissues and cells. Immunohistochemistry techniques and kits, for example, which employ such techniques are well known to those of ordinary skill in the art. Such a kit provides antisera to the receptor, and possibly blocking serum and secondary antiserum linked to a fluorescent molecule such as fluorescein isothiocyanate, or to some other reagent used to visualize the primary antiserum. Using this methodology, biopsied tumors may be examined for the presence of the peptide receptor.
• fluorescent molecule such as fluorescein isothiocyanate
• Monoclonal antibodies mimic the effects of ligands in experimental systems.
• Monoclonal antibodies directed against the rat pi 85 receptor trigger receptor dimerization, phosphorylation, and downregulation of rat pl85,
• a truncated pl85 HER"2 protein was synthesized and injected into mice as the initial immunogen for generating anti-pl85 HER 2 monoclonal antibodies.
• the cDNA of the HER"2 /neu gene was cut between the Ncol and Sphl sites, yielding a portion of the open reading frame which includes the initiation codon extending to the transmembrane domain (20).
• the 2-kilobase DNA insert was ligated into the multiple cloning site immediately downstream of an inducible trp-lac promoter of a pTrcHisA expression vector (Invitrogen, San Diego, CA).
• pi 85 " overexpressing intact cells were used to immunize BALB/c mice.
• NIH/189 and NIH 3T3 cells were used as sources of intact or solubilized pl85 HER"2 protein for immunologic assays.
• the NIH/189 cell line which has been described pprreevviiously as a pl85 HER"2 overexpressor, was a generous gift from C. Richter King
• NIH 3T3, NIH/189, and A431 human epidermoid carcinoma cell lines were grown in DMEM (Life Technologies, Grand Island, NY) supplemented with
• HMEC human mammary epithelial cell
• HFR ⁇ HFR ⁇
• Sp2/0-Agl4 mouse myeloma cells were grown in RPMI-1640 media supplemented with sodium pyruvate, L-glutamine, penicillin/streptomycin, and 15%) FBS prior to cell fusion. Immuization
• ECD HER /neu protein.
• the first immunization of ECD " was an intraperitoneal injection of protein emulsified in Freund's complete adjuvant (Sigma, St. Louis, MO). At weeks 3 and 5, mice were given two immunizations of 50 mg of protein mixed with Freund's incomplete adjuvant (Sigma). The mouse that yielded two monoclonal antibodies (5A7 and 11F1 1) in this work was given three further boosts of 50 mg ECD HER"2 .
• mice were also inoculated with live pl85 HER"2 -overexpressing cells in order to ensure the widest range of monoclonal antibodies against all potential
• ECD HER"2 epitopes including glycosylated regions.
• Initial immunizations were intraperitoneal injections of 2-5 xl06 SKBR-3 human breast cancer cells in serum- free RPMI-1640 media over a 7 month period.
• the mouse that yielded monoclonal antibodies 8H11 and 10H8 had another boost of 5x106 NIH/189 cells in Freund's incomplete adjuvant injected intraperitoneal ly and 1x106 cells injected subcutaneously.
• the final immunizations before fusion were an intraperitoneal boost of 10x106 NIH 189 cells in PBS and an intravenous injection of 50 mg of ECD HER"2 protein in a buffer containing 0.1M NaH2P04 and 10 mM TrisHCl, pH 8.0.
• Hybridoma production and monoclonal antibody screening Spleen cells from BALB/c mice immunized with either protein or cells were fused to Sp2/0-Agl4 mouse myeloma cells to generate hybridoma cells (22). Cells were fused with polyethylene glycol in a method modified from Kohler and Milstein (23). Hybridoma cells were selected for anti-pl85 HER 2 antibody production by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Protein and cell ELISA
• ECD HER"2 250 ng/well protein was coated onto 96-well plates in PBS. After blocking in 3% bovine serum albumin (BSA), 50 mL of supernatant were incubated from 1-2 hours at room temperature. After washing 3 times with PBS, a 1 :2000 dilution (in 3% BSA) of horseradish peroxidase- conjugated goat anti-mouse secondary antibody (Bio-Rad, Hercules, CA) was incubated for 30 minutes at room temperature. Positive wells were visualized by o- phenylene diamine (OPD) substrate (Sigma) and read at O.D. 490 nm on an ELISA plate reader (Bio-Tek Instruments, Inc., Winooski, VT).
• BSA bovine serum albumin
• cell ELISA using NIH3T3 and NIH/189 cells was employed to detect such clones.
• Cells (lxl04/well) were fixed using 100% methanol onto 96- well Linbro Titertek (ICN, Irvine, CA) plates after overnight attachment onto 1.5% gelatin (Difco, Detroit, MI).
• the protocol for protein ELISA was used for cell ELISA. limn ui lohistochem islry
• Frozen tissue section immunohistochemistry was used as a secondary screening method to detect positive hybridomas that secreted antibodies to intact pl85 HER"2 from human tissue.
• pl85 HER"2 in tissue sections were detected by the peroxidase-anti-peroxidase technique as previously described (24).
• Monoclonal antibodies were used to probe for full-length pl85 HER"2 in total protein lysates of both mouse and human cell lines loaded equally in SDS- PAGE. The method used has been previously described (26).
• Immunoprecipitations demonstrated the ability of monoclonal antibodies to bind to full length HER"2 /neu in soluble detergent lysates of mouse and human cell lines.
• the inimunoprecipitation method used has been previously described (27).
• a rabbit polyclonal antibody (R60) previously reported for use in p 185 HER"2 Western blots was used (9).
• a prerequisite for the determination of cellular trafficking of monoclonal antibody-antigen complexes is the binding of monoclonal antibody to its
• FACS Fluorescence-activated cell sorting
• HFR-2 described to characte ⁇ ze anti-pl85 " monoclonal antibodies with this potential (29, 30).
• Monoclonal antibodies 8H11 and 10H8 were labeled with 1251 (Amersham Pharmacia) using the Iodo-Gen method (Pierce).
• NIH 3T3 and NIH/189 cells were pulsed with labeled monoclonal antibodies and chased with 1%> BSA/RPMI-1640. Supernatant, acid-releasable surface-bound, and intracellular label were collected and counted on a Cobra Automated Gamma Counter (Packard, Meriden, CT).
• BALB/c mice were also immunized with viable cells overexpressing pl85 HER"2 as well as the recombinant ECD HER"2 protein in order to isolate hybridoma clones 8H1 1 and 10H8. Both protein and cell ELISA were used as the primary screening methods for detection of antibodies to pl85 HER"2 from hybridoma supematants.
• Supematants positive in either of these screens were further tested for the presence of antibodies which could detect full-length endogenous pl85 HER"2 in frozen breast cancer tissue from a patient with HER"2 /neu gene overexpression.
• the monoclonal antibodies from hybridomas which bound pl85 HER"2 in these screens were 5A7, 1 IFl 1, 8H11, and 10H8.
• the hybridoma cells were isolated as monoclonally derived cell lines after cloning twice by limiting dilution. 5A7, 1 IFl 1, and 8H11 were of the IgGl/k class and isotype. 10H8 was of the IgG2a/k class and isotype.
• both 8H11 and 10H8 were found exclusively as intact proteins (data not shown). Thus, both 8H11 and 10H8 neither bound to proteins on the surface of NIH 3T3 cells nor were internalized by endocytosis, trafficked to lysosomes, and exocytosed.
• IHC immunohistochemistry

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