WO2010065711A1 - Épitope abcb5 et anticorps connexes pour le traitement du cancer - Google Patents

Épitope abcb5 et anticorps connexes pour le traitement du cancer Download PDF

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Publication number
WO2010065711A1
WO2010065711A1 PCT/US2009/066539 US2009066539W WO2010065711A1 WO 2010065711 A1 WO2010065711 A1 WO 2010065711A1 US 2009066539 W US2009066539 W US 2009066539W WO 2010065711 A1 WO2010065711 A1 WO 2010065711A1
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antibody
cell
abcb5
antibodies
group
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PCT/US2009/066539
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English (en)
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Maximiliano Vasquez
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Adimab, Inc.
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Publication of WO2010065711A1 publication Critical patent/WO2010065711A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • ABCB5 ATP-Binding Cassette Subfamily B (MDR/TAP), member 5) is an ATP-binding cassette (ABC) transporter and P-glycoprotein family member known to play a role in progenitor cell fusion (Frank et al., J. Biol.
  • the present invention relates to the discovery of a previously unrecognized extracellular loop in ABCB5.
  • This loop provides a novel target for therapeutic and diagnostic antibodies binding ABCB5.
  • ABCB5 contains an additional extracellular loop located N- terminal to the two previously identified extracellular loops.
  • polypeptides comprising, consisting of, or consisting essentially of amino acids about 273 to about 288, about 275 to about 284, about 265 to about 288, or about 273 to about 283 of the C-terminal portion of ABCB5 (SEQ ID NO: 1 ).
  • these polypeptides may be fusion polypeptides and/or cyclic polypeptides.
  • antibodies that bind to one or more of the polypeptides of the invention, or a polypeptide having at least about 80% identity thereto.
  • these antibodies are full-length, heterodimeric antibodies and/or antibody fragments.
  • the antibodies are human.
  • the antibodies may have a biological activity selected from the group consisting of antibody-dependent cell-mediated cytotoxicity, complement- dependent cytotoxicity, cell lysis, cell death, reduction in tumor size and/or inhibition of ABCB5-mediated efflux of an ABCB5 substrate. Some antibodies will have more than one of these biological properties.
  • the antibodies are conjugated to therapeutic or cytotoxic agents, such as antimetabolites, cytokines, anti-angiogenic agents, anti-mitotic agents, toxins, or apoptotic agents.
  • the antibodies are conjugated to detectable moieties, such as radiolabels, enzymes, fluorescent labels, luminescent labels, or bioluminescent labels.
  • the antibodies of the invention may bind a cell with tumor-initiating phenotype and/or a stem cell phenotype.
  • inventions relate to polynucleotides encoding the polypeptides of the invention, antibodies of the invention, or portions thereof.
  • Vectors comprising these polynucleotides and host cells comprising these vectors are also within the scope of the invention.
  • Any host cell suitable for producing a polypeptide can be used, such as mammalian cells, bacteria, or yeast.
  • these host cells are yeast.
  • the yeast are S. cerevisiae or P. pastoris.
  • the invention relates to methods of treating a cell with an antibody that binds ABCB5, wherein the cell is contacted with an anti-ABCB5 antibody.
  • the contacting is performed in vitro or in vivo.
  • Certain methods of the invention relate to methods of inhibiting ABCB5-mediated efflux of an ABCB5 substrate and/or inducing ADCC in a cell in a patient in need thereof, by administering an effective amount of an anti-ABCB5 antibody.
  • the methods of the invention relate to the treatment and/or diagnosis of cancer. These methods can be used for the treatment of any cancer expressing ABCB5.
  • the cancer is a melanoma.
  • the anti-ABCB5 antibody may be co-administered (at the same time, before or after) with a second agent.
  • the second agent is a substrate of ABCB5.
  • the invention relates to methods of detecting ABCB5 expressing cells by contacting cells with the anti-ABCB5 antibody under conditions to permit the formation of a complex between the cell and the antibody and detecting the complex.
  • compositions and kits comprising one or more antibodies, polypeptides and/or polynucleotides of the invention.
  • Figure 1 shows the results obtained when analyzing ABCB5 using the HMMTOP algorithm. Extracellular regions are designated as “o” in the "pred” row.
  • Figure 2 shows the results obtained when analyzing ABCB5 using the TOPPRED algorithm. Extracellular regions are underlined and bolded in the "Input Sequence”. Six candidate membrane-spanning regions are shown.
  • Figure 3 shows the results obtained when analyzing ABCB5 with the SAM- T2K algorithm, using Salmonella MsbA to generate the homology model.
  • the novel extracellular loop is highlighted in dark gray.
  • Figure 4 shows a schematic representation of the data from Table 1 , focusing on residues 200 to 600 of ABCB5.
  • Figure 5 shows the results obtained when analyzing the full-length ABCB5 ⁇ -helical (12 TM) "core" built with the Swissmod program, using the murine ABCB1 structure as template to generate a homology model.
  • Figure 6 shows the application of the worm-like chain model to select the optimal linker length.
  • Figure 7 shows the chemical structure of LCBiot-(mPEG2).
  • antibody is used herein in the broadest sense and encompasses at least monoclonal antibodies, polyclonal antibodies, multi-specific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and antibody fragments.
  • An antibody is a protein comprising one or more polypeptides encoded or partially encoded by immunoglobulin genes, fragments of immunoglobulin genes, or polynucleotides with "substantial identity” to such genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • a “full-length, heterodimeric” antibody is an antibody comprised of two full- length heavy chains and two full-length light chains.
  • isolated refers to a substance (e.g., an antibody, a polypeptide, a polynucleotide) that has been separated from one or more components of the environment in which it was produced. Contaminant components of its production environment (e.g., a biological, recombinant, or synthetic production environment) that interfere with research, diagnostic, or therapeutic uses for the substance (e.g., enzymes, hormones, proteinaceous or non-proteinaceous solutes) are present at a lesser concentration than in the environment in which the substance was produced.
  • an isolated substance may be purified to at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99% by weight, as determined, for example, by chromatography, gel electrophoresis, and/or other methods known in the art.
  • an "isolated antibody” may also refer to an antibody which is substantially free of other antibodies having different antigenic specificities. However, this is not intended to preclude the mixing, use, and/or administration of an antibody of the invention with another antibody (e.g., in combination therapies), or the fact that an antibody may be cross-reactive with closely-related antigens.
  • human antibody is intended to include antibodies having variable and constant regions with “substantial identity” to human germline immunoglobulin sequences.
  • Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro mutagenesis or by in vivo somatic mutation).
  • antibody fragment is a portion of an antibody that has the ability to bind an antigen, for example, one or more portions of the antigen-binding region of an antibody.
  • antibody fragments include Fab, Fab', F(ab')2, Fd, and Fv fragments, diabodies, linear antibodies, single domain antibodies, single-chain antibodies, and multi-specific antibodies formed from intact antibodies and antibody fragments.
  • alternative scaffold refers to a molecule in which one or more regions may be diversified to produce molecules with specificities and affinities that are similar to those of antibodies.
  • exemplary alternative scaffolds include those derived from fibronectin (e.g., AdNectin), the ⁇ -sandwich (e.g., iMab), lipocalin (e.g., Anticalin), EETI-II/AGRP, BPTI/LACI-D1/ITI-D2 (e.g., Kunitz domain), thioredoxin (e.g., peptide aptamer), protein A (e.g., Affibody), ankyrin repeats (e.g., DARPin), gB-crystallin/ubiquitin (e.g., Affilin), CTLD 3 (e.g., Tetranectin), and (LDLR- A module)3 (e.g., Avimers).
  • fibronectin e.g., AdN
  • the term "specific binding” refers to antibody binding to a predetermined antigen.
  • the antibody binds to the predetermined antigen with an affinity corresponding to a "K d " of at least about 10 ⁇ 6 , 10 ⁇ 7 , 10 “8 M or less, and with an affinity that is at least about 10-fold greater, and preferably at least about 100-fold greater (i.e., a Kd that is at least about 10-fold less or about 100-fold less) than its affinity for a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
  • a non-specific antigen e.g., BSA, casein
  • the antibody can bind to the predetermined antigen with an affinity corresponding to a "K 3 " of at least about 10 6 , 10 7 , 10 8 M “1 or greater, and with an affinity that is at least about 10-fold greater, and preferably at least about 100-fold greater (i.e., a K a that is at least about 10- fold greater or about 100-fold greater) than its affinity for a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
  • a non-specific antigen e.g., BSA, casein
  • Kd (M), as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction.
  • K 3 (M "1 ), as used herein, is intended to refer to the association equilibrium constant of a particular antibody-antigen interaction.
  • k d (sec "1 ), as used herein, is intended to refer to the dissociation rate constant of a particular antibody-antigen interaction. This value is also referred to as the k O ff value.
  • k a (M "1 x sec “1 ), as used herein, is intended to refer to the association rate constant of a particular antibody-antigen interaction. This value is also referred to as the k on value.
  • the term “high affinity” for an IgG antibody refers to a K 3 of at least about 10 7 M “1 , preferably at least about 10 8 M “1 , more preferably at least about 10 9 M “1 , 10 10 M “1 , 10 11 M “1 or greater, e.g., up to 10 15 M “1 or greater.
  • “high affinity” binding can vary for other antibody isotypes.
  • “high affinity” binding for an IgM isotype refers to a binding affinity of at least about 1 x 10 7 M "1 .
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Cancer cells include, but are not limited to, carcinomas, such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma
  • CDC complement-dependent cytotoxicity
  • fusion polypeptide refers to a chimeric protein containing a protein of interest ⁇ e.g., ABCB5 or a portion thereof, an antibody) joined to a different protein (e.g., an affinity tag, an epitope tag, an adjuvant).
  • a “host cell” may be any cell containing a nucleic acid or vector of the invention.
  • Host cells include mammalian, fungal, insect, plant, and bacterial cells.
  • the fungal cells may be yeast.
  • Exemplary non-limiting yeast cells include, for example, S. cerevisiae and P. pastoris.
  • Exemplary non-limiting mammalian cells include Chinese hamster ovary cells, HEK-293 cells, and NSO cells.
  • subject includes a human or non-human animal, including all vertebrates (e.g., mammals and non-mammals).
  • patient refers to a human.
  • the term "effective amount,” as used herein, refers to the amount of an antibody of the invention necessary to achieve the desired effect.
  • Non-limiting examples of the use of this nomenclature include, for example, identifying a polypeptide as being “substantially identical to ABCB5", “at least about 60% identical to ABCB5", or “at least about 75% identical to residues 265 to 288 of ABCB5". Percent identity (i.e., ⁇ number of identical positions / total number of positions ⁇ * 100) can be calculated after optimal alignment using, for example, the Basic Local Alignment Search Tool (BLAST) suite (see ncbi.nlm.nih.gov). The default parameters of the program may be used, though this is not a limitation.
  • BLAST Basic Local Alignment Search Tool
  • Exemplary default parameters for BLASTN include an Expect Threshold of 10; a Word Size of 1 1 ; Match/Mismatch Scores of 2,-3; and Gap Costs of Existence: 5, Extension: 2.
  • Exemplary default parameters for BLASTP include an Expect Threshold of 10; a Word Size of 3; use of the BLOSUM62 matrix; and Gap Costs of Existence: 1 1 , Extension: 1 .
  • Exemplary default parameters for BLASTN comparing two sequences include a Reward for a Match of 1 ; a Penalty for a Mismatch of -2; an Open Gap of 5 and Extension Gap 2 penalties; a Gap_X_Dropoff of 50; an Expect of 10; and a Word Size of 11 .
  • Exemplary default parameters for BLASTP comparing two sequences include use of the BLOSUM62 matrix, an Open Gap of 1 1 and Extension Gap 1 penalties; a Gap_X_Dropoff of 50; an Expect of 10; and a Word Size of 3. These parameters and BLAST algorithms are provided for exemplary purposes only, and are non- limiting.
  • wash buffers may comprise about 0.1 % to about 1 % SDS.
  • the duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
  • tumor-initiating phenotype refers to a cellular phenotype which includes the capabilities of self-renewal and differentiation.
  • Cells with a tumor- initiating phenotype may be distinguished, for example, by their increased tumorigenicity in comparison to cells without a tumor-initiating phenotype.
  • these tumor-initiating cells are distinguished, at least in part, by the expression of ABCB5 (Schatton et ai, Nature, 451 : 345-349, incorporated by reference in its entirety).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • plasmid a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • viral vector Another type of vector.
  • Vectors may be episomal or non- episomal. Vectors that are capable of directing the expression of genes to which they are linked are called "expression vectors”.
  • immunoconjugate includes antibodies that are conjugated to a second molecule, for example a drug, toxin, and/or detectable moiety.
  • Immunoconjugates may be used in both diagnostic and therapeutic applications. Immunoconjugates that include one or more cytotoxins are referred to as “immunotoxins”. Immunoconjugates that include one or more radioactive moieties are called “radioimmunoconjugates". These radioimmunoconjugates may be used solely for imaging (e.g., using radioactive moieties with limited or no cytotoxicity), and/or for therapeutic purposes ⁇ e.g., with cytotoxic radioactive moieties).
  • the term "pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity, and absorption delaying agents, and the like, that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, oral, transdermal, anal, vaginal, topical, intraperitonealy, and/or buccal administration. These routes of administration are provided only for the purposes of exemplification and are non- limiting. Other routes of administration known in the art and suitable for administration of the antibodies of the invention may also be utilized.
  • a “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge et al., J. Pharm. ScL, 66: 1 -19, 1977, incorporated by reference in its entirety). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous acids and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous acids and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N- methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • polypeptides comprising, consisting essentially of, or consisting of the polypeptide sequences from about: (1 ) 273-288; (2) 275-284; (3) 265-288; and/or (4) 273-283 of the C-terminal portion of ABCB5 (SEQ ID NO: 1 ), or peptides substantially identical thereto.
  • the polypeptides comprise, consist essentially of, or consist of 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, or 2 contiguous amino acids from said polypeptide sequences.
  • the polypeptides comprise, consist essentially of, or consist of amino acids 265 to 270, 265 to 275, 265 to 280, 270 to 275, 270 to 280, 270 to 285, 270 to 288, 275 to 280, 275 to 285, 275 to 288, 280 to 285, 280 to 288 and/or 283 to 288 of ABCB5.
  • the polypeptides may be fusion polypeptides, comprising all or a portion of any of said polypeptide sequences.
  • a polypeptide of the invention may also be a fragment derived from any of said peptide sequences.
  • a fusion polypeptide or a peptide fragment may comprise about 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, or 2 contiguous amino acids from any of said peptide sequences.
  • Polynucleotides and vectors encoding each of the polypeptides of the present invention are also within the scope of the invention. Vectors containing these polynucleotides and host cells containing the vectors also fall within the scope of the invention.
  • Certain embodiments of the invention relate to polynucleotides encoding polypeptides or antibodies of the invention, or portions thereof (e.g., a CDR, a variable region), and polynucleotides substantially identical thereto.
  • the polynucleotides may be present in whole cells, in a cell lysate, or in a partially purified or isolated form.
  • a polynucleotide of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences.
  • Substantially identical polynucleotides also include those which hybridize, under selective hybridization conditions, to the complement of a selected polynucleotide.
  • the invention also relates to antibodies that bind to one or more residues of amino acids of any of said peptides, or peptides substantially identical thereto.
  • the invention further relates to antibodies that recognize homologs, paralogs, and orthologs of ABCB5.
  • the antibodies of the invention may recognize a polypeptide sequence comprising at least about 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, or 2 contiguous amino acids from amino acids 265 to 288 of ABCB5.
  • Polynucleotides encoding each of the antibodies of the present invention, or subunits thereof, are also within the scope of the invention.
  • Vectors containing these polynucleotides are also within the scope of the invention.
  • Host cells containing these vectors, and host cells producing the antibodies described herein are also within the scope of the invention.
  • the antibodies of the invention may be whole antibodies (i.e., full-length, heterodimeric) or antibody fragments.
  • the antibodies of the invention may be of several isotypes, including IgA (1 -2), IgD, IgG (1 -4), IgE, and IgM.
  • the antibodies of the invention may be human, non-human, humanized, or chimeric. They may be derived from animals, via recombinant procedures that include amplification of DNA or RNA from animal cells, or via synthetic procedures that rely upon chemical synthesis of oligonucleotides. Alternative scaffold-based binding proteins that recognize the specified extracellular domain of ABCB5 are also within the scope of the invention.
  • an antibody may have a Kd of about 10 ⁇ 4 , 10 ⁇ 5 , 10 ⁇ 6 , 10 ⁇ 7 , 10 ⁇ 8 , 10 ⁇ 9 , 10 ⁇ 10 , 10 ⁇ 1 1 , 10 ⁇ 12 , 10 ⁇ 13 , 10 ⁇ 14 , 10 "15 M, or less.
  • Certain aspects of the invention relate to an antibody with a biological activity that is elicited upon binding to its target.
  • This biological activity can include, for example, ADCC, CDC, cell lysis, cell death, reduction in tumor size, and/or inhibition of ABCB5-mediated efflux.
  • immunoconjugates for example antibodies carrying a therapeutic and/or detectable moiety.
  • Information on producing immunoconjugates for therapeutic and/or diagnostic purposes is contained in, for example, US Patent Nos. 4,444,744; 4,490,473; 4,478,815;
  • Therapeutic moieties include, for example, cytotoxins, drugs, and radiotoxins.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vin
  • An example of a calicheamicin antibody conjugate is commercially available (MylotargTM; Wyeth-Ayerst).
  • Immunoconjugates of the invention can be produced using linker technology available in the art.
  • linker types that have been used to conjugate a moiety to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers.
  • a linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue, for example cathepsins (e.g., cathepsins B, C, D).
  • detectable moieties include, for example, radioactive moieties, enzymes, fluorescent moieties, luminescent moieties, and bioluminescent moieties.
  • radioactive isotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine 131 , indium 111 , yttrium 90 , technicium 99m , and lutetium 77 . Methods for preparing radioimmunconjugates are established in the art.
  • radioimmunoconjugates examples include ZevalinTM (RIT Oncology, LLC) and BexxarTM (GlaxoSmithKline), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention.
  • the immunoconjugates of the invention can be used to modify a given biological response, and the moiety conjugated to the antibody is not to be construed as limited to classical chemical therapeutic agents.
  • the moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon ⁇ ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (IL-1 ), interleukin-2 (IL- 2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM- CSF), granulocyte colony stimulating factor (G-CSF), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM- CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • the antibodies of the invention may also be combined with one or more pharmaceutically acceptable carriers to produce a pharmaceutical composition for systemic or local administration.
  • exemplary pharmaceutically acceptable carriers and pharmaceutical compositions may be found in, for example, US Patent No. 6,803,039, incorporated by reference in its entirety.
  • the invention also relates to kits containing the antibodies of the invention.
  • kits may have utility for diagnostic assays used to quantify the presence, fraction, and/or number of ABCB5+ cells in a cell or tissue sample.
  • these cells have a stem cell ⁇ e.g., CD133+) or tumor initiating cell phenotype.
  • the antibodies of the invention may be produced in a host cell using, for example, a combination of recombinant DNA techniques and gene transfection methods that are well known in the art (Morrison, Science, 229: 1202-1207, 1985 incorporated by reference in its entirety).
  • Antibodies can be produced using, for example, a bacterial cell, a mammalian cell, a fungal cell, an insect cell, or a plant cell.
  • the antibodies are produced in yeast.
  • the yeast is S. cerevisiae or P. pastoris.
  • DNAs encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., DNA synthesis, PCR amplification, site directed mutagenesis) and can be inserted into expression vectors such that the genes are operatively linked to transcriptional control sequences.
  • operatively linked is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate expression vector or into the same expression vector.
  • the antibody genes are inserted into the expression vector by standard methods ⁇ e.g., recombination or ligation).
  • the light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment within the vector and the VL segment is operatively linked to the CL segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell.
  • the term "regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • Such regulatory sequences are described, for example, in Goeddel, Methods in Enzymology, 185: 3-7, 1990, incorporated by reference in its entirety. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed and the level of expression of protein desired.
  • Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells ⁇ e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al., incorporated by reference in their entirety).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques.
  • the various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • the antibodies of the invention can also be produced by a variety of other techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of K ⁇ hler and Milstein (Nature, 256: 495-497, 1975, incorporated by reference in its entirety). Although somatic cell hybridization procedures may be used, many other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes.
  • One animal system for preparing hybridomas is the murine system.
  • Hybridoma production in the mouse is a very well-established procedure.
  • Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.
  • Antibodies may also be obtained by any other method known in the art, including immunization of animals, isolation from synthetic antibody libraries, and/or isolation from template-derived antibody libraries.
  • display technologies may be utilized to isolate cells or phage expressing an antibody that binds an antigen.
  • Ribosomal display methods may also be used to isolate antibodies of the invention. Polyclonal antibodies are also within the scope of the invention.
  • references relevant to synthetic antibody libraries include, for example, co- owned USSN 12/210,072; Knappik et al. US Patent Nos. 6,300,064 and 6,696,248; Enzelberger et al. WO2008053275; and Ladner et al. US20060257937, each incorporated by reference in its entirety.
  • References relevant to template-derived antibody libraries include, for example, Wigler et al. US Patent No. 6,636,424 and Griffiths et al. US Patent No. 6,593,081 , each incorporated by reference in its entirety.
  • references relevant to antibody display technologies include, for example, Wittrup et al. US Patent No. 6,696,251 ; Frenken et al. US Patent No. 6,1 14,147; Ladner et al. US Patent Nos. 5,837,500 and 7,208,293; and Fandl et al. US Patent No. 6,919,183, each incorporated by reference in its entirety.
  • the antibodies of the invention may also contain certain modifications in the constant region. Such modifications may include, for example, amino acid substitutions and/or changes in glycosylation characteristics.
  • Relevant references include US Patent Nos. 5,624,821 , 5,648,260, and 7,029,872; WO200042072; WO199429351 ; and Shields et al., J. Biol. Chem., 276: 6591 -6604, 2001 , each of which is incorporated by reference in its entirety.
  • the invention also relates to methods of contacting a cell with an antibody of the invention.
  • a cell is contacted with an antibody that binds to one or more residues comprising the polypeptides from about (1 ) 273-288; (2) 275-284; (3) 265-288; and/or (4) 273-283 of the C-terminal portion of ABCB5 (SEQ ID NO: 1 ).
  • This contacting may be performed in vitro, for example for drug screening or the identification of ABCB5+ cells in a sample, or in vivo, for example for the diagnosis and/or treatment of cancer.
  • the antibody may also induce a biological effect, for example ADCC, CDC, cell lysis, cell death, reduction in tumor size and/or inhibition of ABCB5-mediated efflux. Dosage regimens are adjusted to provide the optimum desired response
  • a therapeutic response ⁇ e.g., a therapeutic response
  • a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • Actual dosage levels of the antibodies of the present invention may be varied so as to obtain an amount which is effective to achieve the desired therapeutic response for a particular subject without being toxic to the subject.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular antibody, the route of administration, the time of administration, the rate of excretion of the antibody, the duration of the treatment, other drugs, compounds and/or materials used in combination with the antibody, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the antibody of the invention at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of an antibody of the invention will be that amount of the antibody which is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target.
  • the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • an antibody of the present invention may be administered alone, it is preferable to administer the antibody as a pharmaceutical formulation (composition).
  • the dosage can be determined or adjusted by measuring the amount of circulating anti-ABCB5 antibody at different time points following administration in a biological sample by making use of anti-id iotypic antibodies targeting the anti-ABCB5 antibody or by using other specific methods to detect the anti- ABCB5 antibody, for instance, by an ELISA assay using ABCB5 as coating.
  • the antibodies of the invention may be administered by infusion in a dosage of, for example, about 0.15 to about 10 mg/kg, for example about 0.15, 0.5, 1 , 1 .5, 2, 3, 4, 8 or 10 mg/kg, on day 0 followed by 2 to 8 administrations once a week, such as 4 administrations once a week starting at day 28.
  • the administration may be performed by continuous infusion over a period of 24 hours or over a period of more than 24 hours, in order to reduce toxic side effects.
  • the antibodies are administered by maintenance therapy, such as, for example, once a week, once every second week or once a month for a period of 6 months or more.
  • the efficient dosages and the dosage regimens for the antibodies of the invention depend on the disease or condition to be treated and can be determined by persons skilled in the art.
  • the anti-ABCB5 antibody may be co-administered with a second agent, for example, a chemotherapeutic agent that is a substrate for ABCB5.
  • chemotherapeutic agents that are substrates for ABCB5 include: camptothecin, mitoxantrone, pyrazofuhn, menogaril, amsacrine, etoposide, anthrapyrazole, teniposide, daunorubicin, doxorubicin, oxanthrazole, zorubicin, uracil mustard, piperazinedione, hepsulfam, melphalan, bisantrene, triethylenemelamine, spiromustine, yoshi-864, chlorambucil, piperazine mustard, hydroxyurea, porfiromycin, mechlorethamine, fluorodopan, mitomycin, cytarabine, dianhydrogalacti
  • Administration of the second agent may occur before, during, or after administration of the anti-ABCB5 antibody.
  • Inhibition of ABCB5-mediated efflux promotes the intracellular accumulation of these agents, leading to enhanced cytotoxicity. This may enhance the potency of the agents, enabling the administration of a lower dose that induces fewer systemic side-effects.
  • Methods for determining whether a particular anti-ABCB5 antibody inhibits ABCB5-mediated efflux are described herein and are known to those skilled in the art.
  • the invention provides methods of evaluating the susceptibility of a cell, cancer cell, tissue, or organ to treatment with an antibody of the invention by ex vivo or in vivo quantification of ABCB5.
  • Ex vivo quantification may be performed, for example, by contacting a sample ⁇ e.g., a tissue sample, a body fluid sample or a cell sample) to be tested, optionally along with a control sample (e.g., not containing ABCB5), with an antibody of the invention under conditions that allow for formation of a complex between the antibody and ABCB5. Complex formation can then be detected.
  • complex When using a control sample along with the test sample, complex can be quantified in both samples and any statistically significant difference in the formation of complexes between the samples can be used to quantify the amount of ABCB5 in the test sample.
  • the amount of ABCB5 can be used to determine whether the cell, cancer cell, tissue, or organ may be susceptible to treatment with an antibody of the invention.
  • the expression of ABCB5 in a cell of a patient may be evaluated via biopsy prior to the administration of an anti-ABCB5 antibody. This may enable the identification of patients with one or more diseases that is likely to be responsive to treatment with an antibody of the invention.
  • the antibodies may be used for the isolation of ABCB5+ cells. These cells may have utility, for example, in modulating an immune response (Frank, US Pub. No. 2008/0003206, incorporated by reference in its entirety).
  • This example presents the identification of a novel extracellular loop of ABCB5.
  • the loop was identified by analyzing a part of the C-terminal portion of the ABCB5 primary sequence (residues 232-554 of SEQ ID NO: 1 were analyzed) with the HMMTOP and TOPPRED algorithms, followed by three-dimensional homology modeling using SAM-T2K and Swissmod (Arnold et al., Bioinformatics, 22: 195-201 , 2006; Kiefer et al., Nuc. Acids Res., 37: D387-D392, 2009; Peitsch, Bio/Technology, 13: 658-660, 1995, each incorporated by reference in its entirety).
  • Figure 1 shows the results obtained when analyzing residues 232-554 of SEQ ID NO: 1 using the HMMTOP algorithm.
  • the algorithm predicts three extracellular loops, designated as "o" in the "pred" row. These loops are predicted to span from about position 273 to about position 288, from about position 365 to about position 380, and from about position 493 to about position 508 of SEQ ID NO: 1 .
  • Figure 2 shows the results obtained when analyzing residues 232-554 of
  • SEQ ID NO: 1 using the TOPPRED algorithm.
  • the algorithm predicts three extracellular loops (bolded and underlined), spanning from about position 265 to about position 288, from about position 366 to about position 389, and from about position 493 to about position 505 of SEQ ID NO: 1 .
  • FIG. 3 shows the results of this analysis, with the novel extracellular loop highlighted in dark gray.
  • the three-dimensional model predicts three extracellular domains that are in agreement with the predictions of the HMMTOP and TOPPRED algorithms. These domains reside from about position 275 to about position 284, from about position 386 to about position 392, and from about position 493 to about position 508 of SEQ ID NO: 1.
  • Table 1 summarizes the regions of residues 232-554 of SEQ ID NO: 1 that are predicted to form extracellular loops, according to THMM1.0, HMMTOP, TOPPRED, and SAM-T2K algorithms.
  • Figure 4 shows a schematic representation of the same data, focusing on residues 200 to 600 of ABCB5.
  • HMMTOP, TOPPRED, and SAM-T2K are all in agreement on the presence and location of the novel extracellular loop (from about position 265 to about position 288).
  • Example 2 Isolation and Characterization of Antibodies Binding to the Novel Extracellular Loop of ABCB5
  • Antibodies recognizing a sequence contained within loop 3 (i.e., the third loop from the N-terminus) of ABCB5 have previously been shown to recognize the native ABCB5 protein (e.g., mAb 3C2-1 D12; Frank et al., J. Biol. Chem., 278: 47156-47165, 2003, incorporated by reference in its entirety).
  • Antibodies to loop 3 can thus serve as a positive control for binding to ABCB5.
  • Tables 2 and 3 show eight exemplary peptides (SEQ ID NOS: 2-9) that are synthesized to serve as antigens for the isolation of antibodies binding loops 1 and 3 of ABCB5, respectively.
  • Each of the peptides corresponds to an amino acid sequence located within the respective loop, flanked by 0, 1 , or 2 glycine residues.
  • the glycine residues are designed to provide flexibility in the loop, and therefore allow it to adopt a structure that is not overly constrained and that may be similar to its native structure.
  • these constructs are flanked by cysteine residues which, through the formation of intramolecular disulfide bonds, can be used to cyclize the peptide. Without being bound by theory, this may more closely mimic the topology of the extracellular loop.
  • the constructs are synthesized in the absence of cysteine residues, though a terminal cysteine residue (or any other reactive moiety) may be added during synthesis or after, for conjugation to a carrier, adjuvant, or label (e.g. a fluorescent label).
  • Validation of antigen binding and measurement of antibody affinity is carried out using surface plasmon resonance, bio-layer interferometry, equilibrium dialysis, or other methods known in the art to quantify antigen binding and affinity. Affinity is also measured by flow cytometry, by contacting ABCB5+ cells with various concentrations of fluorescently-labeled antibody and measuring the corresponding mean fluorescence intensity.
  • Binding of isolated antibodies to ABCB5 expressed on the surface of cells is validated by staining cells expressing ABCB5 (e.g., HEM and G3361 melanoma cells) with the isolated antibodies, after conjugation of a fluorescent label. This method is also used to isolate antibodies binding ABCB5 (e.g., using ABCB5+ cells as an antigen). A cell line not expressing ABCB5 (e.g., MCF-7) is used as a negative control. Binding of the labeled antibody to the cells is quantified by flow cytometry or fluorescence microscopy. Competitive binding studies are used to define and confirm the region of ABCB5 to which each isolated antibody binds. The peptides provided in Tables 2-5 are used in these competitive binding studies.
  • Inhibition of ABCB5-mediated efflux is evaluated in MCF-7 cells (ABCB5-) transfected with ABCB5 DNA or with control DNA (e.g., encoding lacZ). Cells are incubated with rhodamine-123 for 60 min at 37 0 C. Cells are then washed and fluorescence measurements are acquired by flow cytometry. A decrease in the intracellular fluorescence of rhodamine-123, at a rate that is greater than the decrease observed in the control, is indicative of ABCB5-mediated efflux. Inhibition of ABCB5-mediated efflux is evaluated by introducing each of the isolated antibodies to be evaluated at various times in the experiments, for example, prior to incubation with rhodamine-123 and after washing.
  • the dose and time of introduction of the antibody used in the assay will depend on the binding rate constants (i.e., k on , k Of ⁇ ) and the equilibrium binding constant (K d ). These are determined by surface plasmon resonance or bio-layer interferometry, prior to in vitro evaluation.
  • rhodamine-123 Although the lack of toxicity and fluorescent properties of rhodamine-123 make it a convenient marker to use in the evaluation of ABCB5-mediated efflux, the inhibition of ABCB5-mediated efflux by an antibody of the invention may also be studied using clinically-relevant chemotherapeutic drugs (e.g., doxorubicin).
  • chemotherapeutic drugs e.g., doxorubicin
  • chemotherapeutics such as doxorubicin, fluoresce, enabling the quantification of the concentration of the chemotherapeutic in the cell and viability in the same experiment.
  • ADCC and CDC is evaluated in vitro (Kroesen et al., J. Immunol. Methods, 156: 47-54, 1992, incorporated by reference in its entirety) or in vivo, using histological analysis to quantify the infiltration of immune cells into a tumor.
  • the effect of targeting ABCB5 in vivo is evaluated by xenografting human ABCB5+ cells (e.g., melanoma cells) into NOD/SCID mice.
  • Mice are injected intraperitonealy with anti-ABCB5 antibody ⁇ e.g., about 500 ⁇ g per injection) about bi-weekly, starting 24 hours before the xenografting or 14 days post-tumor inoculation, when tumors are established. Tumor formation and growth is measured over about 8 weeks.
  • tumors are removed and histological analysis of the tumor for infiltrating immune cells is performed, to evaluate the mechanism of tumor cell death (Schatton et al., Nature, 451 : 345-349, incorporated by reference in its entirety).
  • antibodies of the invention are labeled with a radioactive moiety ⁇ e.g., 99m Tc) and the diagnostic and imaging potential of the radiolabeled antibodies are evaluated.
  • Animals are prepared and treated as outlined above. After injection of the radiolabeled antibodies, the animals are scintigraphically imaged over a period of 48 hours, to evaluate accumulation of the antibody at the site of the xenografted tumor and in other regions of the body.
  • the full-length ABCB5 protein (SEQ ID NO: 10; 1 ,257 amino acids) consists of two primary domains, N- and C-terminus, each with six transmembrane (TM) helices.
  • the C-terminal portion of the full-length protein (discussed above), starting at position 446 of the full-length protein, contains six C-terminal helices and was initially believed to be active as a homodimer (see SEQ ID NO: 1 for C-terminal portion alone) (Frank et al., J. Biol. Chem., 278: 47156-47165, 2003; Frank et al., Cancer Res., 65: 4320-4333, 2005, each incorporated by reference in its entirety).
  • Example 2 The set of peptide antigens proposed in Example 2 were all designed based on the C-terminal region of ABCB5 (SEQ ID NO: 1 ; NB: positions 525 in that polypeptide has been reported as both E and K). As shown in Example 2, the extracellular loops in the C-terminal portion of the protein (including novel Loop 1 ) were identified using a combination of TM domain predictions based on the primary polypeptide sequence, and three-dimensional homology modeling based on the structure of the MsbA transporter of Salmonella (Protein Data Bank No.
  • Figure 5 shows the resulting structure and the locations of the six extracellular loops - three from the N-terminus of the full-length protein (designated Loops 4-6, to maintain consistency with previous nomenclature) and three from the C-terminus of the full-length protein (Loops 1 -3, including newly-identified Loop 1 ).
  • Equation 4 The probability that a worm-like chain of N residues has an end-to- end distance of r (p( ⁇ ) was calculated using Equation 4 from Zhou, J. MoI. Biol., 329: 1 -8, 2003, incorporated by reference in its entirety. The equation is reproduced below:
  • Kr // c +2r 2 // c 2 -33r 4 /80y c 3 - 79///160/, -329r 2 / /120/ c 3 +6799/ /1600/ c 4 - 3441/ /2800/ // +1089r 8 /12800/ 2 lf)
  • p(N) QIA ⁇ l p bNf n exp(-3 J 0 2 14l p bN)( ⁇ -5l p I bN + 2J 0 2 /[bN] 2 - 33J 0 4 /80/ p [&N] 3 - 79// /160[6N] 2 -329r% /120[ ⁇ N]3 +6799J 0 4 /1600[ ⁇ N] 4 -344 W 0 6 /2800/ ⁇ N] 5 +1089J 0 8 /12800//[ ⁇ N] 6 )
  • Tables 4 and 5 show the primary sequences of each loop of the six loops displayed in Figure 5, and the corresponding end-to-end distance, N * based on the worm-like chain model, and ideal number of ⁇ - and C-terminal glycine residues based on the end-to-end distance and N * .
  • peptide sequences that will function as antigens for the identification of antibodies to the corresponding loops are provided (sixth column in Tables 4 and 5).
  • Peptide sequences based on ideal theoretical predictions are provided in bold font.
  • Alternative peptide sequences, which account for potential imperfections in the three dimensional and worm-like chain models, are provided below the ideal (bolded) sequences.
  • Peptides were obtained from New England Peptide, Garnder, MA.
  • LCBiot-(mPEG2) refers to a biotin - polyethylene glycol-based moiety that can be used for detection purposes (see Figure 7).
  • Loops and Corresponding Peptides from the C-terminal Domain (Loops 1-3).
  • Loops and Corresponding Peptides from the N-terminal Domain (Loops 4-6).
  • loop 4 designated by the following underlined region QTNTTNYQNCTQ (SEQ ID NO: 45) is flanked by two potential N-linked glycosylation sites (NTT / NCT; bolded). These glycosylation sites could be useful, for example, to produce peptide antigens that contain sugar structures similar to any naturally-occurring sugar structures in these regions.

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Abstract

La présente invention porte sur une boucle extracellulaire d'ABCB5, sur des anticorps connexes et sur leurs procédés d'utilisation.
PCT/US2009/066539 2008-12-04 2009-12-03 Épitope abcb5 et anticorps connexes pour le traitement du cancer WO2010065711A1 (fr)

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WO2019210109A1 (fr) * 2018-04-25 2019-10-31 Children's Medical Center Corporation Ligands abcb5 et substrats
US11129854B2 (en) 2013-02-19 2021-09-28 Schepens Eye Research Institute ABCB5(+) stem cells for treating ocular disease
WO2021211761A1 (fr) * 2020-04-15 2021-10-21 Children's Medical Center Corporation Ciblage d'abcb5 dans le glioblastome multiforme
WO2021222861A1 (fr) * 2020-04-30 2021-11-04 Children's Medical Center Corporation Anticorps spécifiques à abcb5 et leurs utilisations
US11446331B2 (en) 2013-05-10 2022-09-20 The Brigham And Women's Hospital, Inc. Wound healing and tissue engineering
US11542328B2 (en) 2008-11-14 2023-01-03 The Brigham And Women's Hospital, Inc. Therapeutic and diagnostic methods relating to cancer stem cells
US11564986B2 (en) 2015-07-16 2023-01-31 Onkosxcel Therapeutics, Llc Approach for treatment of cancer via immunomodulation by using talabostat

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11542328B2 (en) 2008-11-14 2023-01-03 The Brigham And Women's Hospital, Inc. Therapeutic and diagnostic methods relating to cancer stem cells
US11129854B2 (en) 2013-02-19 2021-09-28 Schepens Eye Research Institute ABCB5(+) stem cells for treating ocular disease
US11446331B2 (en) 2013-05-10 2022-09-20 The Brigham And Women's Hospital, Inc. Wound healing and tissue engineering
US11564986B2 (en) 2015-07-16 2023-01-31 Onkosxcel Therapeutics, Llc Approach for treatment of cancer via immunomodulation by using talabostat
WO2019210109A1 (fr) * 2018-04-25 2019-10-31 Children's Medical Center Corporation Ligands abcb5 et substrats
CN112423765A (zh) * 2018-04-25 2021-02-26 儿童医学中心公司 Abcb5配体和底物
WO2021211761A1 (fr) * 2020-04-15 2021-10-21 Children's Medical Center Corporation Ciblage d'abcb5 dans le glioblastome multiforme
WO2021222861A1 (fr) * 2020-04-30 2021-11-04 Children's Medical Center Corporation Anticorps spécifiques à abcb5 et leurs utilisations

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