WO2006022690A1 - Methode et compositions destinees au traitement de maladies ciblant cd71 - Google Patents

Methode et compositions destinees au traitement de maladies ciblant cd71 Download PDF

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WO2006022690A1
WO2006022690A1 PCT/US2004/025271 US2004025271W WO2006022690A1 WO 2006022690 A1 WO2006022690 A1 WO 2006022690A1 US 2004025271 W US2004025271 W US 2004025271W WO 2006022690 A1 WO2006022690 A1 WO 2006022690A1
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protein
cell
antibody
cells
expression
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PCT/US2004/025271
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English (en)
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Bruno Domon
Ian Mccaffery
Vaihhav Narayan
Scott Patterson
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Applera Corporation
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Publication of WO2006022690A1 publication Critical patent/WO2006022690A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70582—CD71
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention relates to the fields of molecular biology and oncology. Specifically, the invention provides a molecular marker and a therapeutic agent for use in the diagnosis and treatment of cancers.
  • Cancer currently constitutes the second most common cause of death in the United States. Carcinomas of the pancreas are the eighth most prevalent form of cancer and fourth among the most common causes of cancer deaths in this country.
  • pancreatic carcinoma The prognosis for pancreatic carcinoma is, at present, very poor, it displays the lowest five-year survival rate among all cancers. Such prognosis results primarily from delayed diagnosis, due in part to the fact that the early symptoms are shared with other more common abdominal ailments.
  • diagnostic imaging methods like ultrasonography (US), endoscopic ultrasonography (EUS), dualphase spiral computer tomography (CT), magnetic resonance imaging (MRT), endoscopic retrograde cholangiopancreatography (ERCP) and transcutaneous or EUS-guided fine-needle aspiration (FNA), distinguishing pancreatic carcinoma from benign pancreatic diseases, especially chronic pancreatitis, is difficult because of the similarities in radiological and imaging features and the lack of specific clinical symptoms for pancreatic carcinoma.
  • targets are either cell surface proteins or cytosolic proteins.
  • Antibodies or other biomolecules or small molecules which will specifically recognize and bind to the targets in the cancerous cells potentially provide powerful tools for the diagnosis and treatment of the particular malignancy.
  • CD 71 also known as transferrin receptor and OKT9, functions in the import of iron into cells by binding iron-carrying transferrin in the serum.
  • the expression of CD71 is posttranscriptionally regulated by intracellular iron, where an iron-responsive binding protein activated by low iron levels stabilizes the CD71 mRNA.
  • Stimulation of lymphocytes with mitogen results in the elevation of CD 71 expression in a population of the stimulated cells (Rittenhouse-Diakun et al., 1995, Photochem. Photobiol., 61 : 523-528).
  • CD71 is necessary for erythropoiesis.
  • CD71 haploinsufficiency results in erythroid microcytosis, hypochromia and impairment of iron homeostasis (Levy et al., 1999, Nat. Genet. 21: 396-399).
  • a diseased, e.g. malignant, cell often differs from a normal cell by a differential expression of one or more proteins.
  • differentially expressed proteins, and suitable fragments thereof, are useful as markers for the diagnosis and treatment of the disease.
  • the present inventors discovered that CD71 is differentially expressed in pancreatic tumor cells in comparison to normal pancreatic cells. Accordingly, the present invention provides methods and compositions for treating pancreatic diseases, especially malignant pancreatic tumors, using CD71 as a target.
  • the differentially expressed CD71 proteins (SEQ ID NOS: 1 encoded by SEQ ID NO: 3 or 4; and SEQ ID NO: 2 encoded by SEQ ID NO: 5) and suitable fragments thereof, and nucleic acids (SEQ ID NOS: 3, 4 and 5) encoding said protein, and suitable fragments thereof, are respectfully referred to herein as CD71 proteins, CD71 peptides or CD71 nucleic acids, and collectively as CD71.
  • the CD71 proteins of the present invention may serve as a target for one or more classes of therapeutic agents, including antibody therapeutics.
  • CD71 proteins of the present invention are useful in providing a target for diagnosing a pancreatic cancer or tumor, or predisposition to a pancreatic cancer or tumor mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, a CD71 protein of the present invention in a biological sample such as tissues, cells and biological fluids isolated from a subject.
  • the diagnosis method may detect CD71 nucleic acids, proteins, peptides and fragments thereof that are differentially expressed in pancreatic diseases in a test sample, preferably in a biological sample.
  • the further embodiment includes but is not limited to, monitoring the disease prognosis (recurrence), diagnosing disease stage, preventing the disease and treating the disease.
  • the present invention provides a method for diagnosing or detecting a pancreatic cancer or tumor in a subject comprising: determining the level of CD71 in a test sample from said subject, wherein a differential level of said CD71 in said sample relative to the level in a control sample from a healthy subject, or the level established for a healthy subject, is indicative of the pancreatic cancer or tumor.
  • the test sample includes but is not limited to a biological sample such as tissue, blood, serum or biological fluid.
  • the diagnostic method of the present invention may be suitable for monitoring the disease progression or the treatment progress.
  • the present invention provides additionally a pharmaceutical composition comprising an antagonist to CD71 of the present invention, and a pharmaceutically acceptable excipient, for treating a pancreatic tumor or cancer.
  • the present invention further provides a method for screening for agents ; that modulate CD71 protein activity, comprising the steps of (i) contacting a candidate agent with a CD71 protein, and (ii) assaying for CD71 protein activity, wherein a change in said activity in the presence of said agent relative to CD71 protein activity in the absence of said agent indicates said agent modulates said CD71 protein activity.
  • Candidate agents include but are not limited to protein, peptide, antibody, nucleic acid such as antisense RNA, RNAi fragments, small molecules.
  • the screening method may also determine a candidate agent's ability to modulate the expression level of a CD71 protein or nucleic acid.
  • the method comprises (i) contacting a candidate agent with a system that is capable of expressing a CD71 protein or CD71 mRNA, (ii) assaying for the level of a CD71 protein or a CD71 mRNA, wherein a specific change in said level in the presence of said agent relative to a level in the absence of said agent indicates said agent modulates said CD71 level.
  • the present invention further provides a method to screen for agents that bind to the CD71 protein, comprising the steps of (i) contacting a test agent with a CD71 protein and (ii) measuring the level of binding of agent to said CD71 protein.
  • the fusion protein does not affect the activity of the peptide or protein per se.
  • the fusion protein can include, but is not limited to, fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions.
  • fusion proteins for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions.
  • Such fusion proteins, particularly poly- His fusions can facilitate the purification of recombinant CD71 proteins or peptides.
  • expression and/or secretion of a protein can be increased by using a heterologous signal sequence.
  • a chimeric or fusion CD71 protein or peptide can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al, Current Protocols in Molecular Biology, 1992).
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • CD71 peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the CD71 peptide as well as being encoded by the same genetic locus as the CD71 peptide provided herein. Genetic locus can readily be determined based on the genomic information. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a CD71 peptide encoding nucleic acid molecule under stringent conditions as more fully described below.
  • Non-naturally occurring variants of the CD71 peptides of the present invention can readily be generated using recombinant techniques.
  • Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the CD71 peptide.
  • one class of substitutions is conserved amino acid substitution.
  • Such substitutions are those that substitute a given amino acid in a CD71 peptide by another amino acid of like characteristics.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu, and He; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and GIu; substitution between the amide residues Asn and GIn; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr.
  • Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).
  • Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. MoI. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).
  • the present invention further provides fragments of CD71, in addition to proteins and peptides that comprise and consist of such fragments.
  • a fragment comprises at least 8, 10, 12, 14, 16, 18, 20 or more contiguous amino acid residues from CD71.
  • Such fragments can be chosen based on the ability to retain one or more of the biological activities of CD71 or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen.
  • Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length.
  • Such fragments will typically comprise a domain or motif of CD71, e.g., active site, a transmembrane domain or a substrate-binding domain.
  • the CD71 of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature CD71 is fused with another compound, such as a compound to increase the half-life of CD71 (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature CD71, such as a leader or secretory sequence or a sequence for purification of the mature CD71 or a pro-protein sequence.
  • a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included
  • the mature CD71 is fused with another compound, such as a compound to increase the half-life of CD71 (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature CD71, such as a leader or secretory sequence or a sequence for purification of the mature CD71 or a pro-protein sequence.
  • antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical, heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.
  • VH variable domain
  • VL variable domain at one end
  • the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. Chothia et al., J. MoI. Biol. 186, 651-63 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82 4592-4596 (1985).
  • An "isolated" antibody is one which has been identified and separated and/or recovered from a component of the environment in which it is produced. Contaminant components of its production environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • an "antigenic region” or “antigenic determinant” or an “epitope” includes any protein determinant capable of specific binding to an antibody. This is the site on an antigen to which each distinct antibody molecule binds. Epitopic determinants usually consist of active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as charge characteristics.
  • Antibody specificity is an antibody, which has a stronger binding affinity for an antigen from a first subject species than it has for a homologue of that antigen from a second subject species.
  • the antibody bind specifically to a human antigen (i.e., has a binding affinity (Kd) value of no more than about 1 x 10 "7 M, preferably no more than about 1 x10 " M and most preferably no more than about 1 xlO " M) but has a binding affinity for a homologue of the antigen from a second subject species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen.
  • Kd binding affinity
  • the antibody can be of any of the various types of antibodies as defined above, but preferably is a humanized or human antibody (Queen et al., US Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370).
  • the present invention provides an "antibody variant,” which refers to an amino acid sequence variant of an antibody wherein one or more of the amino acid residues have been modified. Such variant necessarily have less than 100% sequence identity or similarity with the amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the antibody, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. Since the method of the invention applies equally to both polypeptides, antibodies and fragments thereof, these terms are sometimes employed interchangeably.
  • An "Fv" fragment is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V H -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define ani antigen-binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the present invention further provides monoclonal antibody, polyclonal antibody as well as humanized antibody.
  • an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse.
  • the full-length protein, an antigenic peptide fragment or a fusion protein of the CD71 protein can be used. Particularly important fragments are those covering functional domains.
  • Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In additional to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal" antibody indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), or may be made by recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567.
  • the monoclonal antibodies for use with the present invention may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624-628 (1991), as well as in Marks et al., J. MoI. Biol. 222: 581-597 (1991). For detailed procedures for making a monoclonal antibody, see the Example below.
  • humanized antibody may comprise residues, which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Polyclonal antibodies may be prepared by any known method or modifications of these methods including obtaining antibodies from patients. For example, a complex of an immunogen such as CD71 protein, peptides or fragments thereof and a carrier protein is prepared and an animal is immunized by the complex according to the same manner as that described with respect to the above monoclonal antibody preparation and the description in the Example. A serum or plasma containing the antibody against the protein is recovered from the immunized animal and the antibody is separated and purified. The gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE SEPHADEX, or other techniques known to those skilled in the art.
  • the antibody titer in the antiserum can be measured according to the same manner as that described above with respect to the supernatant of the hybridoma culture. Separation and purification of the antibody can be carried out according to the same separation and purification method of antibody as that described with respect to the above monoclonal antibody and in the Example.
  • antibodies are preferably prepared from regions or discrete fragments of the CD71 protein.
  • Antibodies can be prepared from any region of the peptide as described herein. In particular, they are selected from a group consisting of SEQ ID NOS: 1-3 and fragments thereof.
  • An antigenic fragment will typically comprise at least 8 contiguous amino acid residues.
  • the antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues.
  • Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness.
  • Antibodies may also be produced by inducing production in the lymphocyte population or by screening antibody libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (1989; Proc Natl Acad Sci 86:3833-3837) or Winter et al. (1991; Nature 349:293-299).
  • a protein may be used in screening assays of phagemid or B- lymphocyte immunoglobulin libraries to identify antibodies having a desired specificity. Numerous protocols for competitive binding or immunoassays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Smith G. P., 1991, Curr. Opin. Biotechnol. 2: 668-673.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen- binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and M 13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.
  • Antibody can be also made recombinantly.
  • the antibody variant can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody variant is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli.
  • cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 minutes.
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore PELLICON ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibodies or antigen binding fragments may also be produced by genetic engineering.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human .delta.1, .delta.2 or .delta.4 heavy chains . (Lindmark et al., J. Immunol Meth. 62: 1-13 (1983)).
  • Protein G is recommended for all mouse isotypes and for human .delta.3 (Guss et al., EMBO J. 5: 1567-1575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a CH3 domain
  • the BAKERBOND ABXTM resin J.T. Baker, Phillipsburg, NJ.
  • Isolated CD71 nucleic acid molecules of the present invention consist of, consist essentially of, or comprise a nucleotide sequence that encodes CD71 peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.
  • an "isolated" nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • flanking nucleotide sequences for example up to about 5KB, 4KB, 3KB, 2KB, or 1KB or less, particularly contiguous peptide encoding sequences and peptide encoding seqt-chces within the s. «m ⁇ ; genfi. but sep-'fated by introns in the genomic sequence, flit important point is that the U nucleic acid Ic isolated fiom remote and unimportant flanking sequences such that it can ⁇ be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.
  • an "isolated" nucleic acid molecule such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when, chemically synthesized.
  • the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
  • isolated DNA molecules contained in a vector are considered isolated.
  • isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • the isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance).
  • the isolated nucleic acid molecules include, but are not limited to, the sequence encoding CD71 peptide alone, the sequence encoding the mature peptide and ⁇ additional' coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or; pro-protein sequence), the sequence encoding the mature peptide, with or ⁇ . ⁇ without , the additional coding sequences, plus additional non-coding sequences, for . •% example introns and non-coding 5' and 3' sequences such as transcribed but non- translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA.
  • the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.
  • a fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide
  • Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other.
  • the conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
  • stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 0 C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65C.
  • SSC sodium chloride/sodium citrate
  • Examples of moderate to low stringency hybridization conditions are well known in the art.
  • the invention also provides vectors containing the nucleic acid molecules described herein.
  • the term "vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules.
  • the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid.
  • the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.
  • a vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules.
  • the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.
  • the invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules.
  • the vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).
  • Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell.
  • the nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription.
  • the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector.
  • a trans-acting factor may be supplied by the host cell.
  • a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.
  • the regulatory sequences to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long- terminal repeats. • :.•- . • i
  • expression vectors ⁇ may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
  • expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation.
  • Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals.
  • the person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001).
  • a variety of expression vectors can be used to express a nucleic acid molecule.
  • Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.
  • Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g.
  • the regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand.
  • host cells i.e. tissue specific
  • inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand.
  • a variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.
  • the nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology.
  • the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.
  • the vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques.
  • Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium.
  • Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.
  • Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S- transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione S- transferase
  • suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 1 Id (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).
  • the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors.
  • mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).
  • the expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein.
  • the invention also relates to recombinant host cells containing the vectors described herein.
  • Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.
  • bacteriophage and viral vectors these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction.
  • Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects. -:c ⁇ '
  • the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.
  • secretion of the peptide is desired, which may be difficult to achieve with a multi-transmembrane domain containing protein such as CD71, appropriate secretion signals are incorporated into the vector.
  • the signal sequence can be endogenous to the peptides or heterologous to these peptides.
  • the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria.
  • the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.
  • the recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing CD71 protein or peptide that can be further purified to produce desired amounts of CD71 protein or fragments. Thus, host ⁇ cells containing expression vectors are useful for peptide production.
  • Host cells are also useful for conducting cell-based assays involving the CD71 protein or CD71 protein fragments, such as those described above as well as other formats known in the art.
  • a recombinant host cell expressing a native CD71 protein is useful for assaying compounds that stimulate or inhibit CD71 protein function.
  • a biological sample can be collected from tissues, blood, sera, cell lines or biological fluids such as, plasma, interstitial fluid, urine, cerebrospinal fluid, and the like, containing cells.
  • a biological sample comprises cells or tissues suspected of having diseases (e.g., cells obtained from a biopsy).
  • a “differential level” is defined as the level of CD71 protein or nucleic acids in a test sample either above or below the level in control samples, wherein the level of control samples is obtained either from a control cell line, a normal tissue or body fluids, or combination thereof, from a healthy subject.
  • the expression of CD71 is preferably greater than about 20%, or prefereably greater than about 30%, and most preferably greater than about 50% or more of pancreatic disease sample, at a level that is at least two fold, and preferably at least five fold, greater than the level of expression in control samples, as determined using a representative assay provided herein. While the protein is under expressed, the expression of CD71 is preferably less than about 20%, or preferably less than 30%, and most preferably less than about 50% or more of the pancreatic disease sample, at a level that is at least 0.5 fold, and preferably at least 0.2 fold less than the level of the expression in control samples, as determined using a representative assay provided herein. ... ⁇ . ,
  • a "subject" can be a mammalian subject or non mammalian subject, preferably, a mammalian subject.
  • a mammalian subject can be human or non- human, preferably human.
  • a healthy subject is defined as a subject without detectable pancreatic diseases or pancreatic associated diseases by using conventional diagnostic methods.
  • the "disease(s)" include pancreatic diseases and pancreatic associated disease.
  • the disease is a pancreatic cancer.
  • This invention further pertains to novel agents identified by the screening assays described below. It is also within the scope of this invention to use an agent identified for treatment purposes.
  • an agent identified as described herein e.g., a CD71 -modulating agent, an antisense CD71 nucleic acid molecule, a CD71-RNAi fragment, a CD71 -specific antibody, or a CD71 -binding partner
  • an agent identified as described herein can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • Modulators of CD71 protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by CD71, e.g. by treating cells or tissues that express CD71 at a differential level.
  • Methods of treatment include the steps of administering a modulator of CD71 activity in a pharmaceutical composition to a subject in need of such treatment.
  • Treating includes: (1) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms, or (2) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.
  • prophylaxis is used to distinguish from “treatment,” and to encompass both “preventing” and “suppressing,” it is not always possible to distinguish between “preventing” and “suppressing,” as the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, the term “protection,” as used herein, is meant to include “prophylaxis.”
  • a “therapeutically effective amount” means the amount of an agent that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the agent, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • a "pancreatic disease” includes pancreatic cancer, pancreatic tumor (exocrine or endocrine), pancreatic cysts, acute pancreatitis, chronic pancreatitis, diabetes (type I and II) as well as pancreatic trauma.
  • the method of the present invention is preferably used for treating a pancreatic cancer.
  • an inhibitor, antagonist, antibody and the like or a pharmaceutical agent containing one or more of these molecules may be delivered. Such delivery may be effected by methods well known in the art and may include delivery by an antibody specifically targeted to the protein.
  • the formulations of the present invention may incorporate a stabilizer.
  • Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids, which may be used either on their own or as admixtures. These stabilizers are preferably incorporated in an amount of 0.11-10,000 parts by weight per part by weight of immunogen. If two or more stabilizers are to be used, their total amount is preferably within the range specified above. These stabilizers are used in aqueous solutions at the appropriate concentration and pH.
  • the specific ⁇ osmotic pressure of such aqueous solutions is generally in the range of 0.1-3.0 osmoles, ' preferably in the range of 0.8-1.2.
  • the pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8.
  • anti-adsorption agent may be used.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • the nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.
  • nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.
  • the invention thus provides a method for identifying a compound that can be used to treat a pancreatic tumor or cancer associated with differential expression of the CD71 gene.
  • the method typically includes assaying the ability of the compound to modulate the expression of CD71 nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired CD71 nucleic acid expression.
  • the assays can be performed in cell-based and cell-free systems.
  • Cell-based assays include cells naturally expressing CD71 nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.
  • the assay for CD71 nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the CD71 protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as ' luciferase.
  • RNAi also operates on a post-transcriptional level and is sequence specific, but suppresses gene expression far more efficiently than antisense RNA.
  • RNAi fragments particularly double-stranded (ds) RNAi, can be also used to generate loss-of- function phenotypes.
  • the present invention provides methods for diagnosing or detecting the differential presence of CD71 protein. Where CD71 is overexpressed in diseased cells, CD71 protein is detected directly.
  • the present invention provides a method for monitoring pancreatic diseases treatment in a subject comprising: determining the level of CD71 protein or any fragment(s) or peptide(s) thereof in a test sample from said subject, wherein a level of said CD71 protein similar to the level of said protein in a test sample from a healthy subject, or the level established for a healthy subject, is indicative of successful treatment.
  • the present invention provides a method for diagnosing recurrence of pancreatic diseases following successful treatment in a subject comprising: determining the level of CD71 protein or any fragment(s) or peptide(s)thereof in a test sample from said subject; wherein a changed level of said CD71 protein relative to the level of said protein in a test sample from a healthy subject, or the level established for a healthy subject, is indicative of recurrence of pancreatic diseases.
  • Immunological methods for detecting and measuring complex formation as a measure of protein expression using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), fluorescence-activated cell sorting (FACS) and antibody arrays. Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody. These assays and their quantitation against purified, labeled standards are well known in the art (Ausubel, supra, unit 10.1-10.6).
  • the antibody or its variant typically will be labeled with a detectable moiety.
  • a detectable moiety Numerous labels are available which can be generally grouped into the following categories:
  • Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are available.
  • the fluorescent labels can be conjugated to the antibody variant using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorometer.
  • the enzyme generally catalyzes a chemical alteration of the chromogenic substrate which can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above.
  • the chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor.
  • the label is indirectly conjugated with the antibody.
  • the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody is conjugated with a small hapten (e.g. digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g. anti-digoxin antibody).
  • a small hapten e.g. digoxin
  • an anti-hapten antibody e.g. anti-digoxin antibody
  • the biological samples can then be tested directly for the presence of CD71 by assays (e.g., ELISA or radioimmunoassay) and format (e.g., microwells, dipstick, etc., as described in International Patent Publication WO 93/03367).
  • assays e.g., ELISA or radioimmunoassay
  • format e.g., microwells, dipstick, etc., as described in International Patent Publication WO 93/03367
  • proteins in the sample can be size separated (e.g., by polyacrylamide gel electrophoresis (PAGE)), in the presence or absence of sodium dodecyl sulfate (SDS), and the presence of GD71 detected by immunoblotting (e.g., Western blotting).
  • Immunoblotting techniques are generally more effective with antibodies generated against a peptide corresponding to an epitope of a protein, and hence, are particularly suited to the present invention.
  • Antibody binding may be detected also by "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • the immunogenic peptide should be provided free of the carrier molecule used in any immunization protocol. For example, if the peptide is conjugated, to KLH, it may be conjugated to BSA, or used directly, in a screening assay. In some embodiments, an automated detection assay is utilized.
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, or the protein to be detected.
  • the test sample to be analyzed is bound by a first antibody, which is immobilized on a solid support, and thereafter a second antibody binds to the test sample, thus forming an insoluble three-part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an antiimmunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • the antibodies may also be used for in vivo diagnostic assays.
  • the antibody is labeled with a radionucleotide (such as 111 In, 99 Tc, 14 C, 131 1, 3 H, 32 P or 35 S) so that the tumor can be localized using immunoscintiography.
  • a radionucleotide such as 111 In, 99 Tc, 14 C, 131 1, 3 H, 32 P or 35 S
  • antibodies or fragaments thereof bind to the extracellular domains of two or more CD71 targets and the affinity value(Kd) is less than 1 x 10 8 M.
  • Antibodies for diagnostic use may be labeled with probes suitable for detection by various imaging methods.
  • Methods for detection of probes include, but are not limited to, fluorescence, light, confocal and electron microscopy; magnetic resonance imaging and spectroscopy; fluoroscopy, computed tomography and positron emission tomography.
  • Suitable probes include, but are not limited to, fluorescein, rhodamine, eosin and other fluorophores, radioisotopes, gold, gadolinium and other lanthanides, paramagnetic iron, fluorine- 18 and other positron-emitting radionuclides. Additionally, probes may be bi- or multi-functional and be detectable by more than one of the methods listed.
  • Attachment of probes to the antibodies includes covalent attachment of the probe, incorporation of the probe into the antibody, and the covalent attachment of a chelating compound for binding of probe, amongst others well recognized in the art.
  • the disease tissue sample may be fresh or frozen or may be embedded in paraffin and fixed with a preservative such as formalin (see Example).
  • the fixed or embedded section contains the sample are contacted with a labeled primary antibody and secondary antibody, wherein the antibody is used to detect CD71 protein expression in situ.
  • the detailed procedure is shown in the Example.
  • Antibodies against CD71 protein or peptides are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development.
  • antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full-length protein can be used to identify turnover.
  • kits for using antibodies to detect the presence of a protein in a biological sample can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use.
  • a kit can be supplied to detect a single protein or epitope, or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nucleic acid arrays and similar methods have been developed for antibody arrays.
  • the antibody of the present invention can be used for therapeutic reasons. It is contemplated that the antibody of the present invention may be used to treat a mammal, preferably a human with a pancreatic disease.
  • the antibodies are also useful for inhibiting protein function, for example, blocking the binding of CD71 protein or peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function.
  • An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity.
  • Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated within a cell or cell membrane. The functional blocking assays are provided in detail in the Examples.
  • the antibodies of present invention can also be used as means of enhancing the immune response.
  • the antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at a range of about 1 mg to about 100 mg per patient.
  • an antibody subclass for therapy will depend upon the nature of the antigen to be acted upon. For example, an IgM may be preferred in situations where the antigen is highly specific for the diseased target and rarely occurs on normal cells. However, where the disease-associated antigen is also expressed in normal tissues, although at much lower levels, the IgG subclass may be preferred, since the binding of at least two IgG molecules in close proximity is required to activate complement, less complement mediated damage may occur in the normal tissues which express smaller amounts of the antigen and, therefore, bind fewer IgG antibody molecules. Furthermore, IgG molecules by being smaller may be more able than IgM molecules to localize to the diseased tissue.
  • the therapeutically effective amount of the antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat a disease or disorder.
  • the antibody may optionally be formulated with one or more agents currently used to prevent or treat the disorder in question.
  • Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof.
  • Preferred radionuclides include 90 Y, 123 I, 125 I, 131 I, 186 Re, 188 Re 211 At, and 212 Bi.
  • Preferred drugs include methotrexate, and pyrimidine arid purine analogs.
  • Preferred differentiation inducers include phorbol esters and butyric acid.
  • Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein
  • a therapeutic agent may be coupled (e.g., covalently bonded) to a suitable antibody either directly or indirectly (e.g., via a linker group).
  • a direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other.
  • a nucleophilic group such as an amino or sulfhydryl group
  • on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.
  • a linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities.
  • a linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.
  • CD71 protein or their peptides may be administered as a vaccine in a pharmaceutically acceptable carrier.
  • Ranges of the protein that may be administered are about 0.001 to about 100 mg per patient, preferred doses are about 0.01 to about 100 mg per patient. Immunization may be repeated as necessary, until a sufficient titer of anti-immunogen antibody or immune cells has been obtained. ⁇ ⁇ ; ⁇
  • mammals preferably human, at high risk for pancreatic diseases, particularly cancer, are prophylactically treated with the vaccines of this invention.
  • examples include, but are not limited to, humans with a family history of pancreatic diseases, humans with a history of pancreatic diseases, particular cancer, or humans afflicted with pancreatic cancer previously resected and therefore at risk for reoccurrence.
  • the vaccine is provided to enhance the patient's own immune response to the diseased antigen present on the pancreatic diseases or advanced stage of pancreatic diseases.
  • the vaccine which acts as an immunogen, may be a cell, cell lysate from cells transfected with a recombinant expression vector, or a culture supernatant containing the expressed protein.
  • Parameters that may be assessed to determine the efficacy of these sensitized T- lymphocytes include, but are not limited to, production of immune cells in the mammal being treated or tumor regression. Conventional methods are used to assess these parameters. Such treatment can be given in conjunction with cytokines or gene modified cells (Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3: 75-90; Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3: 57-73).
  • the proteins of the present invention can be used to screen a compound or an agent for the ability to stimulate or inhibit interaction between CD71 protein and a molecule that normally interacts with CD71 protein, e.g. a substrate or an extracellular binding ligand or a component of the signal pathway that CD71 protein normally interacts (for example, a cytosolic signal protein).
  • a molecule that normally interacts with CD71 protein e.g. a substrate or an extracellular binding ligand or a component of the signal pathway that CD71 protein normally interacts (for example, a cytosolic signal protein).
  • Hs766T correlates well with normal tissue. For this reason, this cell line is reported in the literature as being a good surrogate for normal tissue in analyses of differential expression between pancreatic adenocarcinoma (and derived tumor lines) and normal tissue (or surrogate, Hs766T).
  • the model system employed here involves the use of Hs766T as a "normal" reference to which cell surface expression in tumor derived cell lines is compared.
  • a library of scFvs is constructed from the RNA of human PBLs as described in PCT publication WO 92/01047.
  • To rescue phage displaying antibody fragments approximately 10 9 E. coli harboring the phagemid are used to inoculate 50 ml of 2x TY containing 1% glucose and 100 ⁇ g/ml of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking.
  • Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phages are then used to infect 10 ml of mid-log E. coli TGl by incubating eluted phage with bacteria for 30 minutes at 37 0 C. The E. coli are then plated on TYE plates containing 1% glucose and 100 ⁇ g/ml ampiciJlin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.
  • CMNS Complete Media No Sera
  • BD Hybridoma medium CM - HAT ⁇ Cell Mab
  • FBS or HS
  • Origen HCF hybridoma cloning factor
  • Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza (1 X) stored in the refrigerator at 4 0 C.
  • mice are immunized with 5-50 ug of antigen either intra-peritoneal (i.p.) or by intravenous injection in the tail vein (i.v.).
  • the antigen used is a recombinant protein that is generated as described above.
  • the primary immunization takes place 2 months prior to the harvesting of splenocytes from the mouse and the immunization is typically boosted by i.v. injection of 5-50 ug of antigen every two weeks. At least one week prior to expected fusion date, a fresh vial of myeloma cells is thawed and cultured.
  • the optimum density is determined to be 3-6 xl ⁇ 5 cells/ml.
  • a final immunization is administered of ⁇ 5ug of antigen in PBS i.p. or i.v.
  • Myeloma cells are washed with 30 ml serum free media by centrifugation at 500 x g at 4 0 C for 5 minutes. Viable cell density is determined in resuspended cells using hemocytometry and vital stains. Cells resuspended in complete growth medium are stored at 37°C during the preparation of splenocytes. Meanwhile, to test aminopterin sensitivity, 1x10 6 myeloma cells are transferred to a 15ml conical tube and centrifuged at 50Og at 4°C for 5 minutes. The resulting pellet is resuspended in 15 ml of HAT media and cells plated at 2 drops/well on a 96 well plate.
  • RPMI-CMNS is added to the PEG cells to slowly dilute out the PEG.
  • Cells are centrifuged and diluted in 5 ml of Complete media and 95 ml of Clonacell Medium D (HAT) media (with 5 ml of HCF). The cells are plated out at 10 ml per small petri plate.
  • HAT Clonacell Medium D
  • RNA is isolated from cancer model cell lines using the RNEASY kit (Qiagen) per manufacturer's instructions and included DNase treatment.
  • Normal human tissue RNAs are acquired from commercial vendors (Ambion, Austin, TX; Stratagene, La Jolla, CA, BioChain Institute, Newington, NH) as are RNAs from matched disease/normal tissues.
  • Target transcript sequences are identified for the differentially expressed peptides by searching the BlastP database.
  • TAQMAN assays PCR primer/probe set
  • CDS CELERA DISCOVERY SYSTEM
  • TAQMAN primers and probe sequences are designed by Applied Biosystems (AB) as part of the ASSAYS ON DEMAND product line or by custom design through the AB ASSAYS BY DESIGN service.
  • RT-PCR is accomplished using AMPLITAQGOLD and MULTISCRIBE reverse transcriptase in the ONE STEP RT-PCR Master Mix reagent kit (AB) according to the manufacturer's instructions. Probe and primer concentrations are 250 nM and 900 nM, respectively, in a 15 ⁇ l reaction. For each experiment, a master mix of the above components is made and aliquoted into each optical reaction well. Eight nanograms of total RNA is used as the template. Each sample is assayed in triplicate.
  • Quantitative RT- PCR is performed using the ABI PRISM 7900HT SEQUENCE DETECTION SYSTEM (SDS). Cycling parameters follow: 48°C for 30 min. for one cycle; 95 °C for 10 min for one cycle; 95 °C for 15 sec, 60 °C for 1 min. for 40 cycles.
  • the SDS software calculates the threshold cycle (Cx) for each reaction, and C T values are used to quantitate the relative amount of starting template in the reaction. The C T values for each set of three reactions are averaged for all subsequent calculations.
  • the WESTERN BREEZE kit from Invitrogen is used for western blot analysis.
  • Primary antibodies are either purchased from commercially available sources or prepared using one of the methods described in Section 3. For this application, antibodies are typically diluted 1 :500 to 1 : 10,000 in a diluent buffer. Blots are developed using Pierce NBT.
  • Post tissue processing cells are sorted by flow cytometry known in the art to enrich for epithelial cells.
  • cells isolated from pancreatic tissue are stained directly with EpCAM (for epithelial cells) and the specific antibody to CD71.
  • Cell numbers and viability are determined by PI exclusion (GUAVA) for cells isolated from both normal and tumor pancreatic tissue. A minimum of 0.5 x 10 6 cells are used for each analysis. Cells are washed once with Flow Staining Buffer (0.5% BSA, 0.05% NaN3 in D-PBS).
  • ICAT Immunotope Coded Affinity Tag
  • the proteins from cells are prepared by methods known in the art.
  • the LC/MS spectra are collected for the labeled samples and processed using the following steps:
  • Paraffin embedded, fixed tissue sections are obtained from a panel of normal tissues (Adrenal, Bladder, Lymphocytes, Bone Marrow, Breast, Cerebellum, Cerebral cortex, Colon, Endothelium, Eye, Fallopian tube, Small Intestine, Heart, Kidney (glomerulus, tubule), Liver, Lung, Testes and Thyroid) as well as 30 tumor samples with matched normal adjacent tissues from pancreas, lung, colon, prostate, ovarian and breast.
  • other tissues are selected for testing such as bladder renal, hepatocellular, pharyngeal and gastric tumor tissues.
  • Esophageal replicate sections are also obtained from numerous tumor types (Bladder Cancer, Lung Cancer, Breast Cancer, Melanoma, Colon Cancer, Non-Hodgkins Lymphoma, Endometrial Cancer, Ovarian Cancer, Head and Neck Cancer, Prostate Cancer, Leukemia [ALL and CML] and Rectal Cancer). Sections are stained with hemotoxylin and eosin and histologically examined to ensure adequate representation of cell types in each tissue section.
  • a positive and negative control sample is generated using data from the ICAT analysis of the pancreatic cancer cell lines.
  • Cell lines are selected that are known to express low levels of a particular target as determined from the ICAT data. This cell line is the reference normal control "Hs766T.”
  • a pancreatic tumor line known to overexpress the target is selected as positive control.
  • Sections are deparaffinized and rehydrated by washing 3 times for 5 minutes in xylene; two times for 5 minutes in 100% ethanol; two times for 5 minutes in 95% ethanol; and once for 5 minutes in 80% ethanol. Sections are then placed in endogenous blocking solution (methanol + 2% hydrogen peroxide) and incubated for 20 minutes at room temperature. Sections are rinsed twice for 5 minutes each in deionized water and twice for 5 minutes in phosphate buffered saline (PBS), pH 7.4.
  • PBS phosphate buffered saline
  • sections are deparrafinized by High Energy Antigen Retrieval as follows: sections are washed three times for 5 minutes in xylene; two times for 5 minutes in 100% ethanol; two times for 5 minutes in 95% ethanol; and once for 5 minutes in 80% ethanol. Sections are placed in a Coplin jar with dilute antigen retrieval solution (10 mM citrate acid, pH 6). The Coplin jar containing slides is placed in a vessel filled with water and microwaved on high for 2 - 3 minutes (700 watt oven). Following cooling for 2 - 3 minutes, steps 3 and 4 are repeated four times (depending on the tissue), followed by cooling for 20 minutes at room temperature. Sections are then rinsed in deionized water, two times for 5 minutes, placed in modified endogenous oxidation blocking solution (PBS + 2% hydrogen peroxide) and rinsed for 5 minutes in PBS.
  • PBS + 2% hydrogen peroxide modified endogenous oxidation blocking solution
  • Sections are blocked with PBS/1% bovine serum albumin (PBA) for 1 hour at room temperature followed by incubation in normal serum diluted in PBA (2%) for 30 minutes at room temperature to reduce non-specific binding of antibody. Incubations are performed in a sealed humidity chamber to prevent air-drying of the tissue sections.
  • PBA bovine serum albumin
  • the choice of blocking serum is the same as the species of the biotinylated secondary antibody.
  • Excess antibody is gently removed by shaking and sections covered with primary antibody diluted in PBA and incubated either at room temperature for 1 hour or overnight at 4°C. (Care is taken that the sections do not touch during incubation). Sections are rinsed twice for 5 minutes in PBS, shaking gently. Excess PBS is removed by gently shaking.
  • the sections are covered with diluted biotinylated secondary antibody in PBA and incubated for 30 minutes to 1 hour at room temperature in the humidity chamber. If using a monoclonal primary antibody, addition of 2% rat serum is used to decrease the background on rat tissue sections. Following incubation, sections are rinsed twice for 5 minutes in PBS, shaking gently. Excess PBS is removed and sections incubated for 1 hour at room temperature in VECTASTAIN ABC reagent (Vector Laboratories, Burlingame, CA) according to kit instructions. The lid of the humidity chamber is secured during all incubations to ensure a moist environment. Sections are rinsed twice for 5 minutes in PBS, shaking gently.
  • Sections are incubated for 2 minutes in peroxidase substrate solution that is made up immediately prior to use as follows: lOmg diaminobenzidine (DAB) dissolved in 10ml 50 mM sodium phosphate buffer, pH 7:4; 12.5 microliters 3% CoCl 2 ZNiCl 2 in deionized water; 1.25 microliters hydrogen peroxide.
  • DAB diaminobenzidine
  • Fresh tissues are embedded carefully in OCT in a plastic mold, without trapping air bubbles surrounding the tissue. Tissues are frozen by setting the mold on top of liquid nitrogen until 70-80% of the block turns white at which point the mold is placed on dry ice. The frozen blocks are stored at - 80°C. Blocks are sectioned with a cryostat with care taken to avoid warming to greater than -10 0 C. Initially, the block is equilibrated in the cryostat for about 5 minutes and 6- 10mm sections are cut sequentially. Sections are allowed to dry for at least 30 minutes at room temperature. Following drying, tissues are stored at 4 0 C for short term and -80°C for long term storage.
  • Sections are fixed by immersing in acetone jar for 1-2 minutes at room temperature, followed by drying at room temperature.
  • Primary antibody is added (diluted in 0.05 M Tris-saline [0.05 M Tris, 0.15 M NaCl, pH 7.4], 2.5% serum) directly to the sections by covering the section dropwise to cover the tissue entirely. Binding is carried out by incubation a chamber for 1 hour at room temperature. Without letting the sections dry out, the secondary antibody (diluted in Tris-saline/2.5% serum) is added in a similar manner to the primary and incubated as before (at least 45 minutes).
  • Chromium release assays to assess complement-mediated cytotoxicity are performed for each patient at various time points (Dickler, et ah, 1999, Clin. Cancer Res. 5, 2773-2779). Cultured tumor cells are washed in FCS-free media two times, resuspended in 500 ⁇ l of media, and incubated with 100 ⁇ Ci 51 Cr per 10 million cells for* 2 h at 37°C. The cells are then shaken every 15 min for 2 h, washed 3 times in media to . achieve a concentration of approximately 20,000 cells/well, and then plated in round- bottom plates.
  • the plates contain either 50 ⁇ l cells plus 50 ⁇ l monoclonal antibody, 50 ⁇ l cells plus serum (pre- and post-therapy), or 50 ⁇ l cells plus mouse serum as a control.
  • the plates are incubated in a cold room on a shaker for 45 min.
  • Human complement of a 1:5 dilution (resuspended in 1 ml of ice-cold water and diluted with 3% human serum albumin) is added to each well at a volume of 100 ⁇ l.
  • Control wells include those for maximum release of isotope in 10% Triton X-100 (Sigma) and for spontaneous release in the absence of complement with medium alone.
  • % cytotoxicity [(experimental release-spontaneous release)/(maximum release-spontaneous release)] x 100. A doubling of the CDC to >20% is considered significant.
  • LIPOFECTAMINE is purchased from Invitrogen (Carlsbad, CA) and GENESILENCER from Gene Therapy Systems (San Diego, CA).
  • Synthetic siRNA oligonucleotides are from Dharmacon (Lafayette, CO), Qiagen (Valencia, CA) or Ambion (Austin, TX)
  • RNEASY 96 Kit is purchased from Qiagen (Valencia, CA).
  • APOP-ONE homogeneous caspase-3/7 kit and CELLTITER 96 Aqueous One Solution Cell Proliferation Assay are both purchased from Promega (Madison, WI).
  • Cell invasion assay kits from purchased from Chemicon (Temecula, CA).
  • RIBOGREEN RNA Quantitation Kit is purchased from Molecular probes (Eugene, OR).
  • RNAi is performed by using SMARTPOOLS (Dharmacon), 4-FOR SILENCING siRNA duplexes (Qiagen) or scrambled negative control siRNA (Ambion). Transient transfections are carried out in triplicate by using either LIPOFECTAMINE 2000 from Invitrogen (Carlsbad, CA) or by using GENESILENCER from Gene Therapy Systems (San Diego, CA) in methods described below. One to four days after transfections, total RNA is isolated using the RNEASY 96 Kit (Qiagen) according to manufacturer's instructions and expression of mRNA is quantitated using the TAQMAN ' ' technology. Protein expression levels are examined by flow cytometry. Apoptosis and « proliferation assays are performed daily using APOP-ONE homogeneous caspase-3/7 kit and CELLTITER 96 Aqueous One Solution Cell Proliferation Assay.
  • Transient transfections are carried out on sub-confluent pancreatic cancer cell lines as previously described. Elbashir, S.M. et al. (2001) Nature 411: 494-498; Caplen, NJ. et al. (2001) Proc Natl Acad Sci USA 98: 9742-9747; Sharp, P.A. (2001) Genes and Development 15: 485-490. Synthetic siRNA to gene of interest or scrambled negative control siRNA is transfected using LIPOFECTAMINE according to manufacturer's instructions. Cells are plated in 96 well plates in antibiotic-free medium.
  • the transfection reagent and siRNA are prepared for transfection as follows: Each 0.1- IuI of LIPOFECTAMINE 2000 and 10-15OmM siRNA are resuspended 25ul serum-free media and incubated at room temperature for 5 minutes. After incubation, the diluted siRNA and the LIPOFECTAMINE 2000 are combined and incubated for 20 minutes at room temperature. The cells are then washed and the combined siRNA- LIPOFECTAMINE 2000 reagent added. After further 4 hours incubation, 50 ⁇ l serum containing medium is added to each well. One and four days after transfection, expression of mRNA is quantitated by RT- PCR using the TAQMAN technology and protein expression levels are examined by flow cytometry. Apoptosis and proliferation assays are performed daily using APOP-ONE homogeneous caspase-3/7 kit and CELLTITER 96 Aqueous One Solution Cell Proliferation Assay.
  • Transient transfections are carried out on sub-confluent pancreatic cancer cell lines as previously described (Elbashir, et al., 2001, Nature 411: 494-498; Caplen, et ah, 2001, Proc. Natl. Acad. Sci. USA 98: 9742-9747; Sharp, 2001, Genes and Development 15: 485-490).
  • Synthetic siRNA to gene of interest or scrambled negative control siRNA is transfected using GENESILENCER according to manufacturer's instructions. Cells are plated in 96 well plates in antibiotics free medium.
  • the transfection reagent and the synthetic siRNA are prepared for transfections as follows: predetermined ⁇ amount of GENE SILENCER is diluted in serum-free media to a final volume of 20 ⁇ l*u per well. After resuspending 10-150 mM siRNA in 20 ⁇ l serum-free media, the reagents are combined and incubated at room temperature for 5-20 minutes. After incubation, the siRNA-GENE SILENCER reagent is added to each well and incubated in a 37 0 C incubator for 4 hours before an equal volume of serum containing media is added back to the cultured cells. The cells are then incubated for 1 to 4 days before mRNA, protein expression and effects on apoptosis and proliferation are examined.
  • Sub-confluent pancreatic cancer cell lines are serum-starved overnight. The next day, serum-containing media is added back to the cells in the presence of 5-50ng/ml of function blocking antibodies. After 2 or 5 days incubation at 37 0 C, 5% CO 2 , antibody binding is examined by flow cytometry and apoptosis and proliferation are examined by using protocols described below.
  • Apoptosis assay is performed using the APOP-ONE homogeneous caspase- 3/7 kit from Promega according to the manufacturer's instructions.
  • Cell proliferation assay is performed using the CELLTITER 96 Aqueous One Solution Cell Proliferation Assay kit from Promega. 20 ⁇ l of CELLTITER 96 Aqueous One Solution is added to 100 ⁇ l of culture medium. The plates are then incubated for 1-4 hours at 37 0 C in a humidified 5% CO 2 incubator. After incubation, the change in absorbance is read at 490 nm.
  • Cell invasion assay is performed using the 96-well cell invasion assay kit : available from Chemicon. After the cell invasion chamber plates are adjusted to room temperature, 100 ⁇ l serum-free media is added to the interior of the inserts. 1-2 hours later, cell suspensions of 1 x 10 6 cells/ml are prepared. Media is then carefully removed from the inserts and 100 ⁇ l of prepared cells are added into the insert along with 0 to r 50 rig of function-blocking antibodies, the cells are pre-incubated for 15 minutes' at 37 0 C before 150 ⁇ l of media containing 10% FBS is added to the lower chamber. The cells are then incubated for 48 hours at 37°C.
  • the invasion chamber plates are then placed on a new 96-well feeder tray containing 150 ⁇ l of pre-warmed cell detachment solution in the wells.
  • the plates are incubated for 30 minutes at 37°C and are periodically shaken.
  • Lysis buffer/ dye solution (4ul CYQUANT Dye/ 300 ⁇ l 4X lysis buffer) is prepared and added to each well of dissociation buffer/ cells on feeder tray.
  • the plates are incubated for 15 minutes at room temperature before 150 ⁇ l is transferred to a new 96-well plate. Fluorescence of invading cells is then read at 480 nm excitation and 520 nm emission.
  • ELISA assays are performed essentially as described by Daunt et ah, 1997, MoI. Pharmacol. 51, 711-720.
  • the cell lines are plated at 6x10 5 cells per in a 24-well tissue culture dishes that have previously been coated with 0.1mg/ml poly-L-lysine. The next day, the cells are washed once with PBS and incubated in DMEM at 37°C for several minutes. The agonist to the cell surface target of interest is then added at a pre-determined concentration in prewarmed DMEM to the wells.
  • the cells are then incubated for various times at 37°C and reactions are stopped by removing the media and fixing the cells in 3.7% formaldehyde/TBS for 5 min at room temperature.
  • the cells are then washed three times with TBS and nonspecific binding blocked with TBS containing 1% BSA for 45 min at room temperature.
  • the first antibody is added at a pre-determined dilution in TBS/BSA for 1 h at room temperature.
  • Incubation with goat anti-mouse conjugated alkaline phosphatase (Bio-Rad) diluted 1:1000 in TBS/BSA is carried out for Ih at room temperature.
  • the cells are washed three times with TBS and a colorimetric alkaline phosphatase substrate is added. When the adequate color change is reached, 100- ⁇ l samples are taken for colorimetric readings.
  • RNA is isolated from cancer model cell lines using the RNEASY 96 kit (Qiagen) per manufacturer's instructions and included DNase treatment.
  • Target transcript sequences are identified for the differentially expressed peptides by searching the BlastP database.
  • TAQMAN assays PCR primer/probe set
  • CDS CELERA DISCOVERY SYSTEM
  • the TAQMAN primers and probe sequences are as designed by Applied Biosystems (AB) as part of the ASSAYS ON DEMAND product line or by custom design through the AB ASSAYS BY DESIGN service.
  • RT-PCR is accomplished using AMPLITAQGOLD and MULTISCRIBE reverse transcriptase in the ONE STEP RT-PCR Master Mix reagent kit (AB) according to the manufacturers instructions.
  • Probe and primer concentrations are 900 nM and 250 nM, respectively, in a 25 ⁇ l reaction. For each experiment, a master mix of the above components is made and aliquoted into each optical reaction well. 5ul of total RNA is the template. Each sample is assayed in triplicate.
  • Quantitative RT-PCR is performed using the ABI PRISM 7900HT Sequence Detection System (SDS). Cycling parameters follow: 48 ° for 30 min. for one cycle; 95 °C for 10 min for one cycle; 95 °C for 15 sec, 60 °C for 1 min. for 40 cycles. [00304] The SDS software calculates the threshold cycle (C T ) for each reaction, and C T values are used to quantitate the relative amount of starting template in the reaction. The C ⁇ values for each set of three reactions are averaged for all subsequent calculations.
  • SDS ABI PRISM 7900HT Sequence Detection System
  • Total RNA is quantitated using the RIBOGREEN RNA Quantitation Kit according to manufacturer's instructions and the percent mRNA expression is calculated using total RNA for normalization. Percent knockdown is then calculated relative to the no- addition control.
  • Anti-CD71 antibodies inhibit cell proliferation, as measured using MTS (3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethanoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium) cell proliferation assays (see e.g. Emerman and Eaves, 1994, Bone Marrow Transplantation 13:285) ( Figure 2).
  • pancreatic cancer cells Treatment of pancreatic cancer cells with monoclonal antibodies.
  • Pancreatic cancer cells are seeded at a density of 4 X 10 4 cells per well in 96- well microtiter plates and allowed to adhere for 2 hours. The cells are then treated with different concentrations of anti-CD71 monoclonal antibody (Mab) or irrelevant isotype matched (anti-rHuIFN- ⁇ Mab) at 0.05, 0.5 or 5.0 mug/ml. After a 72 hour incubation, the cell monolayers are stained with crystal violet dye for determination of relative percent viability (RPV) compared to control (untreated) cells. Each treatment group consists of replicates. Cell growth inhibition is monitored.
  • NIH 3T3 cells transfected with either a CD71 expression plasmid or the neo- DHFR vector are injected into nu/nu (athymic) mice subcutaneously at a dose of 10 6 cells in 0.1 ml of phosphate-buffered saline.
  • 100 ⁇ g (0.1 ml in PBS) of either an irrelevant or anti-CD71 monoclonal antibody of the IG2A subclass is injected intraperitoneally. Tumor occurrence and size are monitored for 1 month.

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Abstract

L'invention concerne des méthodes et des compositions destinées à diagnostiquer, détecter et traiter une maladie pancréatique associée à l'expression différentielle de CD71 par comparaison avec des cellules saines. L'invention concerne également des antagonistes ou des agonistes de CD7 1, et des méthodes destinées à cribler des agents modulant le niveau ou l'activité de CD71 in vivo ou in vitro. Invading cells
PCT/US2004/025271 2004-08-06 2004-08-06 Methode et compositions destinees au traitement de maladies ciblant cd71 WO2006022690A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114848665A (zh) * 2022-06-22 2022-08-05 中南大学湘雅三医院 CD71-CD44-GEMs在制备治疗膀胱癌药物中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002044329A2 (fr) * 2000-11-30 2002-06-06 Uab Research Foundation Absorption de peptides se liant au recepteur de la transferrine humaine par ledit recepteur
WO2002059368A1 (fr) * 2000-12-08 2002-08-01 The Board Of Regents Of The University Of Nebraska Expression specifique de la mucine utilisee comme marqueur du cancer pancreatique
EP1416279A1 (fr) * 2002-10-31 2004-05-06 F. Hoffmann-La Roche Ag Methodes et compositions pour le traitement et le diagnostic du cancer du pancreas

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002044329A2 (fr) * 2000-11-30 2002-06-06 Uab Research Foundation Absorption de peptides se liant au recepteur de la transferrine humaine par ledit recepteur
WO2002059368A1 (fr) * 2000-12-08 2002-08-01 The Board Of Regents Of The University Of Nebraska Expression specifique de la mucine utilisee comme marqueur du cancer pancreatique
EP1416279A1 (fr) * 2002-10-31 2004-05-06 F. Hoffmann-La Roche Ag Methodes et compositions pour le traitement et le diagnostic du cancer du pancreas

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
RYSCHICH E ET AL: "Transferrin receptor is a marker of malignant phenotype in human pancreatic cancer and in neuroendocrine carcinoma of the pancreas", EUROPEAN JOURNAL OF CANCER, vol. 40, no. 9, June 2004 (2004-06-01), pages 1418 - 1422, XP002323379, ISSN: 0959-8049 *
YANG BO ET AL: "Role of detection of PCNA and transferrin in human pancreatic cancer", ZHONGGUO ZHONGLIU LINCHUANG, vol. 25, no. 3, March 1998 (1998-03-01), pages 207 - 209, XP008045227, ISSN: 1000-8179 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114848665A (zh) * 2022-06-22 2022-08-05 中南大学湘雅三医院 CD71-CD44-GEMs在制备治疗膀胱癌药物中的应用
CN114848665B (zh) * 2022-06-22 2023-09-12 中南大学湘雅三医院 CD71-CD44-GEMs在制备治疗膀胱癌药物中的应用

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