EP1817059A2 - Conjugues medicament anticorps et procedes correspondants - Google Patents

Conjugues medicament anticorps et procedes correspondants

Info

Publication number
EP1817059A2
EP1817059A2 EP05852595A EP05852595A EP1817059A2 EP 1817059 A2 EP1817059 A2 EP 1817059A2 EP 05852595 A EP05852595 A EP 05852595A EP 05852595 A EP05852595 A EP 05852595A EP 1817059 A2 EP1817059 A2 EP 1817059A2
Authority
EP
European Patent Office
Prior art keywords
antibody
compound
genbank accession
receptor
cancer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05852595A
Other languages
German (de)
English (en)
Inventor
Lewis J. Gazzard
Edward Hyungsuk Ha
David Y. Jackson
Joann Um
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genentech Inc
Original Assignee
Genentech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genentech Inc filed Critical Genentech Inc
Publication of EP1817059A2 publication Critical patent/EP1817059A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents

Definitions

  • the invention relates generally to compounds with anti-cancer activity and more specifically to antibodies conjugated with chemotherapeutic drugs or toxins.
  • the invention also relates to methods of using antibody drug conjugate compounds for in vitro, in situ, and in vivo diagnosis or treatment of mammalian cells, or associated pathological conditions.
  • Trastuzumab is an IgGl kappa antibody that contains human framework regions with the complementarity-determining regions (cdr) of a murine antibody (4D5) that binds to HER2. Trastuzumab binds to the HER2 antigen and thus inhibits the growth of cancerous cells. Because Trastuzumab is a humanized antibody, it minimizes any HAMA (Human Anti-Mouse Antibody) response in patients.
  • HAMA Human Anti-Mouse Antibody
  • Trastuzumab has been shown, in both in vitro assays and in animals, to inhibit the proliferation of human tumor cells that overexpress HER2 (Hudziak RM, et al (1989) MoI Cell Biol 9:1165-72; Lewis GD, et al (1993) Cancer Immunol Immunother; 37:255-63; Baselga J, et al (1998) Cancer Res. 58:2825-2831).
  • Trastuzumab is a mediator of antibody-dependent cellular cytotoxicity, ADCC (Hotaling TE, et al (1996) [abstract]. Proc. Annual Meeting Am Assoc Cancer Res; 37:471; Pegram MD, et al (1997) [abstract].
  • HERCEPTIN® as a single agent is indicated for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein and who have received one or more chemotherapy regimens for their metastatic disease.
  • HERCEPTIN® in combination with paclitaxel is indicated for treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein and who have not received chemotherapy for their metastatic disease.
  • HERCEPTIN® is clinically active in patients with ErbB2- overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al, (1996) J. Ciin. Oncol. 14:737-744).
  • ADC antibody-drug conjugates
  • cytotoxic or cytostatic agents for the local delivery of cytotoxic or cytostatic agents to kill or inhibit tumor cells in the treatment of cancer
  • Cyrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; US 4975278) theoretically allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.
  • Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).
  • Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) Jour, of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.
  • cytotoxic drugs may effect their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
  • ZEVALIN® is an antibody-radioisotope conjugate composed of a murine IgGl kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and 111 In or 90 Y radioisotope bound by a thiourea linker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol.
  • ZEVALIN has activity against B-cell non-Hodgkin's Lymphoma (NHL), administration results in severe and prolonged cytopenias in most patients.
  • MYLOTARGTM (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):686; US 4970198; US 5079233; US 5585089; US 5606040; US 5693762; US 5739116; US 5767285; US 5773001).
  • Cantuzumab mertansine (Immunogen, Inc.), an antibody drug conjugate composed of the huC242 antibody linked via the disulfide linker SPP to the maytansinoid drug moiety, DMl, is advancing into Phase II trials for the treatment of cancers that express Can Ag, such as colon, pancreatic, gastric, and others.
  • MLN-2704 (Millennium Pharm., BZL Biologies, Immunogen Inc.)
  • PSMA anti-prostate specific membrane antigen
  • auristatin peptides auristatin E (AE) and monomethylauristatin (MMAE) synthetic analogs of dolastatin, were conjugated to chimeric monoclonal antibodies including: cBR96 (specific to Lewis Y on carcinomas); cAClO (specific to CD30 on hematological malignancies); and other antibodies (US 20050238649 Al) and are under therapeutic development (Doronina et al (2003) Nature Biotechnology 21(7):778-784).
  • Bis-1,8 naphthalimide compounds have been investigated for their anti-cancer properties (Brana et al (2004) Jour. Med. Chem. 47(6):1391-1399; Bailly et al (2003) Biochemistry 42:4136-4150; Carrasco et al (2003) 42:11751-11761; Brana, M.F. and Ramos, A. (2001) Current Med. Chem. - Anti-Cancer Agents 1:237- 255; Mekapati et al (2001) Bioorganic & Med. Chem. 9:2757-2762).
  • the investigational antitumor drug bis 1,8 naphthalimide mesylate (LU79553, N,N-bis[l,8-naphthalimido)ethyl]-1,3-diaminopropane bismethane sulfonate; N,N'-Bis[2-(l,3-dioxo-2,3-dihydro-lH-benz[de]isoquinolin-2-yl)ethyl]-1,3-diaminopropane dimethanesulfonate; 2,2'-Propane-1,3-diylbis(iminoethylene)bis(2,3-dihydro-lH-benz[de]isoquinoline-1,3- dione) dimethanesulfonate, Abbott Laboratories, Knoll AG, Ludwigshafen, DE), is composed of two tricyclic 1,8-naphthalimide chromophores separated by an aminoalkyl linker chain and designed to permit bisinter
  • the present invention provides novel compounds with biological activity against cancer cells.
  • the compounds of the invention may inhibit tumor growth in mammals.
  • the compounds of the. invention may be useful for treating human cancer patients.
  • ADC antibody drug conjugate
  • Ab binds specifically to a tumor-associated antigen or cell-surface receptor.
  • the antibody of the Formula I ADC of the invention specifically binds to a receptor encoded by an ErbB gene such as, but not limited to, EGFR, HER2, HER3 and HER4.
  • the antibody may bind specifically to an HER2 receptor.
  • the antibody of the antibody-drug conjugate is a humanized antibody selected from huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (Trastuzumab).
  • the invention provides pharmaceutical compositions comprising an effective amount of a Formula I ADC and a pharmaceutically acceptable carrier or vehicle.
  • the invention includes a method of treating cancer comprising administering to a mammal, such as a patient with a hyperproliferative disorder, a formulation of a Formula I ADC and a pharmaceutically acceptable diluent, carrier or excipient.
  • a mammal such as a patient with a hyperproliferative disorder
  • a formulation of a Formula I ADC and a pharmaceutically acceptable diluent, carrier or excipient.
  • the invention provides methods for preventing the multiplication of a tumor cell or cancer cell including administering to a mammal, such as a patient with a hyperproliferative disorder, an effective amount of a Formula I ADC.
  • the invention provides methods for preventing cancer including administering to a patient with a hyperproliferative disorder, an effective amount of a Formula I ADC.
  • the invention includes a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a Formula I ADC, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier or excipient.
  • the composition may further comprise a therapeutically effective amount of chemotherapeutic agent such as a tubulin-forming inhibitor, a topoisomerase inhibitor, or a DNA binder.
  • the invention includes a method for killing or inhibiting the proliferation of tumor cells or cancer cells comprising treating tumor cells or cancer cells with an amount of a Formula I ADC, or a pharmaceutically acceptable salt or solvate thereof, being effective to kill or inhibit the proliferation of the tumor cells or cancer cells.
  • the invention includes a method of inhibiting cellular proliferation comprising exposing mammalian cells in a cell culture medium to an ADC of the invention.
  • the invention includes a method for treating an autoimmune disease, comprising administering to a patient, for example a human with a hyperproliferative disorder, an amount of the ADC of Formula I or a pharmaceutically acceptable salt or solvate thereof, said amount being effective to treat an autoimmune disease.
  • the invention includes a method for killing or inhibiting the multiplication of a tumor cell or cancer cell comprising administering to a patient, for example a human, with a hyperproliferative disorder, an amount of the ADC of Formula I or a pharmaceutically acceptable salt or solvate thereof, said amount being effective to kill or inhibit the multiplication of a tumor cell or cancer cell.
  • the invention includes a method for treating cancer comprising administering to a patient, for example a human, with a hyperproliferative disorder, an amount of the ADC of Formula I or a pharmaceutically acceptable salt or solvate thereof, said amount being effective to treat cancer, alone or together with an effective amount of an additional anticancer agent.
  • the invention includes a method of inhibiting the growth of tumor cells that overexpress a growth factor receptor selected from the group consisting of HER2 receptor and EGF receptor comprising administering to a patient an antibody drug conjugate compound of the invention which binds specifically to said growth factor receptor and a chemotherapeutic agent wherein said antibody drug conjugate and said chemotherapeutic agent are each administered in amounts effective to inhibit growth of tumor cells in the patient.
  • a growth factor receptor selected from the group consisting of HER2 receptor and EGF receptor
  • the invention includes a method for the treatment of a human patient susceptible to or diagnosed with a disorder characterized by overexpression of ErbB2 receptor, comprising administering an effective amount of a combination of an ADC and a chemotherapeutic agent.
  • the invention includes an assay for detecting cancer cells comprising:
  • the present invention provides assays for identifying ADC which specifically target and bind the overexpressed HER2 protein, the presence of which is correlated with abnormal cellular function, and in the pathogenesis of cellular proliferation and/or differentiation of mammary gland that is causally related to the development of breast tumors.
  • the invention includes an article of manufacture comprising an antibody-drug conjugate compound of the invention; a container; and a package insert or label indicating that the compound can be used to treat cancer characterized by the overexpression of an ErbB receptor.
  • the invention includes a method for the treatment of cancer in a mammal, wherein the cancer is characterized by the overexpression of an ErbB receptor and does not respond, or responds poorly, to treatment with an anti-ErbB antibody, comprising administering to the mammal a therapeutically effective amount of a Formula I ADC.
  • the invention includes a method of making an antibody drug conjugate compound comprising conjugating a 1,8 bis naphthalimide drug moiety and an antibody.
  • Figure 1 shows an in vitro, cell proliferation assay with SK-BR-3 cells treated with: -o- trastuzumab and —•- trastuzumab-MC-vc-PAB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 202, measured in Relative Fluorescence Units (RLU) versus ⁇ g/ml concentration of antibody or ADC.
  • RLU Relative Fluorescence Units
  • Figure 2 shows an in vitro, cell proliferation assay with SK-BR-3 cells treated with: -•- Trastuzumab and - ⁇ - trastuzumab-MC-ala-phe-P AB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N- imidazolyl)-1,8 naphthalimide) 203, measured in Relative Fluorescence Units (RLU) versus ⁇ g/ml concentration of antibody or ADC.
  • RLU Relative Fluorescence Units
  • H trastuzumab, where H is linked via a cysteine [cys].
  • Figure 3 shows an in vitro, cell proliferation assay with BT-474 cells treated with: -•- trastuzumab, and -o- trastuzumab-(succinate-gly-ala-phe)-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 204, measured in Relative Fluorescence Units (RLU) versus ⁇ g/ml concentration of antibody or ADC.
  • RLU Relative Fluorescence Units
  • Figure 4 shows an in vitro, cell proliferation assay with BT-474 cells treated with: -•— trastuzumab, and — ⁇ — trastuzumab-(MC-val-cit-PAB-(N, N'-( N, N'-(bis-aminoethyl-1,3-propanediamine)-3-nitro, 4-amino- 1,8 naphthalimide) 205, measured in Relative Fluorescence Units (RLU) versus ⁇ g/ml concentration of antibody or ADC.
  • RLU Relative Fluorescence Units
  • H trastuzumab, where H is linked via a cysteine [cys].
  • Figure 5 shows an in vitro, cell proliferation assay with SK-BR-3 cells treated with: -•- trastuzumab, - ⁇ - trastuzumab-MC-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 206, and -T- trastuzumab-N'-cyclopropylmethyl, N 2 -maleimidopropyl-gly-val-cit- PAB-(N, N'-(bis-aminoethyI-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 207, measured in Relative Fluorescence Units (RLU) versus ⁇ g/ml concentration of antibody or ADC.
  • H trastuzumab, where H is linked via a cysteine [cys].
  • Figure 6 shows a method for preparing a valine-citrulline (val-cit or vc) dipeptide Linker having a maleimide
  • Stretcher and optionally a p-aminobenzyloxycarbonyl (PAB) self-immolative Spacer where Q is -Ci-C 8 alkyl, -0-(C1-C 8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
  • Figure 7 shows a method for preparing a phe-lys(Mtr) dipeptide linker reagent having a maleimide Stretcher unit and a p-aminobenzyl self-immolative Spacer unit, where Q is -C 1 -C 8 alkyl, -O-(C 1 -C 8 alkyl), - halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
  • Figure 8 shows three exemplary strategies for covalent attachment of the amino group of a drug moiety to a linker reagent to form a bis 1,8 naphthalimide-linker reagent.
  • Figure 9 shows a method for synthesis of a bis 1,8 naphthalimide-linker reagent.
  • Figure 10 shows a method for the synthesis of a branched linker reagent containing a BHMS group.
  • antibody herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, tnultispecific antibodies ⁇ e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.
  • Antibodies may be murine, human, humanized, chimeric, or derived from other species.
  • Antibody fragments comprise a portion of a full length antibody, generally the antigen binding or variable region thereof.
  • Examples of antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • an “intact antibody” herein is one comprising a VL and VH domains, as well as complete light and heavy chain constant domains.
  • An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York).
  • a target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.
  • antibody also refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease.
  • the immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • the immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin.
  • monoclonal antibody 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 polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
  • the modifier "monoclonal” 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 et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, US 4816567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. MoI. Biol., 222:581-597; for example.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4816567; and Morrison et al (1984) Proc. Natl. Acad. ScL USA, 81:6851- 6855).
  • Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc) and human constant region sequences.
  • MAbs monoclonal antibodies
  • Hybridoma technology which refers to a cloned cell line that produces a single type of antibody, uses the cells of various species, including mice (murine), hamsters, rats, and humans.
  • Another method to prepare MAbs uses genetic engineering including recombinant DNA techniques.
  • Monoclonal antibodies made from these techniques nclude, among others, chimeric antibodies and humanized antibodies.
  • a chimeric antibody combines DNA ⁇ ncoding regions from more than one type of species. For example, a chimeric antibody may derive the variable region from a mouse and the constant region from a human.
  • a humanized antibody comes predominantly from a. human, even though it contains nonhuman portions.
  • a humanized antibody may contain a completely human constant region. But unlike a chimeric antibody, the variable region may be partially derived from a human. The nonhuman, synthetic portions of a humanized antibody often come from CDRs in murine antibodies. In any event, these regions are crucial to allow the antibody to recognize and bind to a specific antigen. While useful for diagnostics and short-term therapies, murine antibodies cannot be administered to people long-term without increasing the risk of a deleterious immunogenic response. This response, called Human Anti-Mouse Antibody (HAMA), occurs when a human immune system recognizes the murine antibody as foreign and attacks it. A HAMA response can cause toxic shock or even death.
  • HAMA Human Anti-Mouse Antibody
  • Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the nonhuman portions of administered antibodies. Furthermore, chimeric and humanized antibodies can have the additional benefit of activating secondary human immune responses, such as antibody dependent cellular cytotoxicity.
  • Antibody fragments comprise a portion of an intact antibody, e.g. comprising the antigen-binding or variable region thereof.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragment(s).
  • an “intact” antibody is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CHl, CH2 and CH3.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.
  • the intact antibody may have one or more "effector functions" which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody.
  • effector functions include CIq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
  • intact antibodies can be assigned to different "classes.” There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Useful non-immunoreactive protein, polypeptide, or peptide antibodies include, but are not limited to, transferrin, epidermal growth factors ("EGF"), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors ("TGF"), such as TGF- ⁇ and TGF- ⁇ , vaccinia growth factor (“VGF”), insulin and insulin-like growth factors I and ⁇ , lectins and apoprotein from low density lipoprotein.
  • EGF epidermal growth factors
  • TGF transforming growth factors
  • VGF vaccinia growth factor
  • I and ⁇ vaccinia growth factor
  • lectins and apoprotein from low density lipoprotein lectins and apoprotein from low density lipoprotein.
  • Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Various procedures well known in the art may be used for the production of polyclonal
  • various host animals can be immunized by injection with an antigen of interest or derivative thereof, including but not limited to rabbits, mice, rats, and guinea pigs.
  • Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete) adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • corynebacterium parvum Such adjuvants are also well known in the art.
  • Useful monoclonal antibodies are homogeneous populations of antibodies to a particular antigenic determinant (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments thereof).
  • a monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof.
  • the hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.
  • Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. ScL U.S.A. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).
  • the antibody can also be a bispecific antibody.
  • Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al., 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually performed using affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion may be with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, C H 2, and C H 3 regions.
  • the first heavy-chain constant region (C H 1) may contain the site necessary for light chain binding, present in at least one of the fusions.
  • Nucleic acids with sequences encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co- transfected into a suitable host organism.
  • Bispecific antibodies may have a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm.
  • This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation (WO 94/04690; Suresh et al., Methods in Enzymology, 1986, 121:210; Rodrigues et al., 1993, J.
  • bispecific antibodies can be prepared for conjugation as ADC in the treatment or prevention of disease as defined herein.
  • Hybrid or bifunctional antibodies can be derived either biologically, i.e., by cell fusion techniques, or chemically, especially with cross-linking agents or disulfide-bridge forming reagents, and may comprise whole antibodies or fragments thereof (EP 105360; WO 83/03679; EP 217577).
  • the antibody can be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to cancer cell antigens, viral antigens, or microbial antigens or other antibodies bound to tumor cells or matrix.
  • “functionally active” means that the fragment, derivative or analog is able to elicit anti-anti-idiotype antibodies that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived recognized.
  • the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen.
  • synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay) (See, for e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md; Kabat E et al., 1980, J. of Immunology 125(3):961-969).
  • Other useful antibodies include fragments of antibodies such as, but not limited to, F(ab')2 fragments, which contain the variable region, the light chain constant region and the CHl domain of the heavy chain can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Other useful antibodies are heavy chain and light chain dimers of antibodies, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in US 4946778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., (1989) Nature 334:544-54), or any other molecule with the same specificity as the antibody.
  • SCAs single chain antibodies
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal and human immunoglobulin constant regions.
  • Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in WO 87/02671; EP 184,187; EP 171496; EP 173494; WO 86/01533; US 4816567; EP 12023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol.
  • Completely human antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies.
  • Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et al., J. MoI. Biol, 222:581 (1991)).
  • the antibody may be a fusion protein of an antibody, or a functionally active fragment thereof, for example in which the antibody is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, such as at least 10, 20 or 50 amino acid portion of the protein) that is not the antibody.
  • a covalent bond e.g., a peptide bond
  • the antibody or fragment thereof may be covalently linked to the other protein at the N-terminus of the constant domain.
  • Antibodies include analogs and derivatives that are either modified, Le., by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity.
  • the derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, the analog or derivative can contain one or more unnatural amino acids.
  • the antibodies in ADC include antibodies having modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors.
  • antibodies include antibodies having modifications in amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., WO 97/34631, which is incorporated herein by reference in its entirety).
  • Antibodies immunospecific for a cancer cell antigen can be obtained commercially, for example, from Genentech (San Francisco, CA) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques.
  • the nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
  • the antibody of the antibody-drug conjugates (ADC) of the invention may specifically bind to a receptor encoded by an ErbB gene.
  • the antibody may bind specifically to an ErbB receptor selected from EGFR, HER2, HER3 and HER4.
  • the ADC may specifically bind to the extracellular domain of the HER2 receptor and inhibit the growth of tumor cells which overexpress HER2 receptor.
  • the antibody of the ADC may be a monoclonal antibody, e.g. a murine monoclonal antibody, a chimeric antibody, or a humanized antibody.
  • a humanized antibody may be huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8 (Trastuzumab).
  • the antibody may be an antibody fragment, e.g. a Fab fragment.
  • Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., recombinant expression techniques.
  • the nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
  • antibodies available for the treatment of cancer include, but are not limited to, humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; RlTUXAN® (rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA) which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murine IgG 2a antibody for the treatment of colorectal cancer; Cetuximab Erbitux (Imclone Systems Inc., NY) which is an anti-EGFR IgG chimeric antibody for the treatment of epidermal growth factor positive cancers, such as head and neck cancer; Vitaxin (Medlmmune, Inc., MD) which is a humanized antibody for the treatment of sarcoma; Campath I/H (Leukosite,
  • antibodies useful in the treatment of cancer include, but are not limited to, antibodies against the following antigens: CA125 (ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline phosphatase (carcinomas), prostate specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), MAGE-I (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE - 4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MUCl-KLH (breast cancer), CEA (colorectal), gplOO (melanoma), MARTl (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphomas), CD
  • Some specific, useful antibodies include, but are not limited to, BR96 mAb (Trail, P. A., et al Science (1993) 261, 212-215), BR64 (Trail, PA, et al Cancer Research (1997) 57, 100-105, mAbs against the CD40 antigen, such as S2C6 mAb (Francisco, J. A., et al Cancer Res. (2000) 60:3225-3231), mAbs against the CD70 antigen, such as 1F6 mAb, and mAbs against the CD30 antigen, such as AClO (Bowen, M. A., et al (1993) J.
  • autoimmune disorders include systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjogren's syndrome, immune thromobocytopenia, and multiple sclerosis.
  • Antibodies immunospecific for an antigen of a cell that is responsible for producing autoimmune antibodies can be obtained by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques.
  • SLE is marked by the overexpression of interferon-alpha (IFN- ⁇ ) cytokine genes (Bennett et al (2003) Jour. Exp. Med. 197:711-723).
  • IFN- ⁇ interferon-alpha
  • Type-1 interferons (IFN- ⁇ / ⁇ ) play a significant role in the pathogenesis of lupus (Santiago-Raber (2003) Jour. Exp. Med. 197:777-788).
  • Knockout mice (-IFN- ⁇ / ⁇ ) showed significantly reduced anti-erythrocyte autoantibodies, erythroblastosis, hemolytic anemia, anti-DNA autobodies, kidney disease, and mortality.
  • Ty ⁇ e-1 IFNs mediate murine lupus, and that reducing their activity in the human counterpart may be beneficial.
  • Anti-IFN Ab conjugated to bis 1,8 naphthalimide drug moieties may be effective therapeutic agents against SLE and other autoimmune disorders.
  • useful antibodies in ADC are immunospecific for the treatment of autoimmune diseases include, but are not limited to, Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; Anti Phospholipid Antibody IgM, IgG; Anti SM Antibody; Anti Mitochondrial Antibody; Thyroid Antibody; Microsomal Antibody; Thyroglobulin Antibody; Anti SCL-70; Anti- Jo; Anti-U 1 RNP; Anti- La/SSB; Anti SSA; Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti-RNP; C-ANCA; P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti-GBM Antibody.
  • Antibodies of an ADC can bind to both a receptor or a receptor complex expressed on an activated lymphocyte, such as one associated with an autoimmune disease.
  • the receptor or receptor complex can comprise an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein.
  • suitable immunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD28, CD79, CD90, CD152/CTLA-4, PD-I, and ICOS.
  • TNF receptor superfamily members are CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, TNF-Rl, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAEL-Rl, TRAIL-R2, TRAIL-R3, TRABL-R4, and APO-3.
  • suitable integrins are CDl Ia, CDlIb, CDlIc, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, and CD104.
  • suitable lectins are C-type, S- type, and I-type lectin.
  • viral antigen includes, but is not limited to, any viral peptide, polypeptide protein ⁇ e.g., HIV gpl20, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein ⁇ e.g., Gb, Gc, Gd, and Ge) and hepatitis B surface antigen) that is capable of eliciting an immune response.
  • any viral peptide polypeptide protein ⁇ e.g., HIV gpl20, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein ⁇ e.g., Gb, Gc, Gd, and Ge) and hepatitis B surface antigen
  • microbial antigen includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule ⁇ e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptide including, e.g., LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response.
  • Antibodies immunospecific for a viral or microbial antigen can be obtained commercially, for example, from BD Biosciences (San Francisco, CA), Chemicon International, Inc. (Temecula, CA), or Vector Laboratories,Inc. (Burlingame, CA) or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques.
  • the nucleotide sequence encoding antibodies that are immunospecific for a viral or microbial antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
  • useful antibodies in ADC are those that treat or prevent viral or microbial infection in accordance with the methods disclosed herein.
  • Examples of antibodies available useful for the treatment of viral infection or microbial infection include, but are not limited to, SYNAGIS (Medlmmune, Inc., MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody useful for the treatment of patients with RSV infection; PRO542 (Progenies) which is a CD4 fusion antibody useful for the treatment of HIV infection; OsTAVIR (Protein Design Labs, Inc., CA) which is a human antibody useful for the treatment of hepatitis B virus; PROTOVIR. (Protein Design Labs, Inc., CA) which is a humanized IgG 1 antibody useful for the treatment of cytomegalovirus (CMV); and anti-LPS antibodies.
  • SYNAGIS Medlmmune, Inc., MD
  • RSV humanized anti-respiratory syncytial virus
  • antibodies useful in ADC for the treatment of infectious diseases include, but are not limited to, antibodies against the antigens from pathogenic strains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pest
  • antibodies useful in ADC for treatment of viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including as examples and not by limitation: Poxviridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus.
  • Herpesviridae Herpes Simplex virus 1
  • ErbB receptor is a receptor protein tyrosine kinase which belongs to the ErbB receptor family whose members are mediators of cell growth, differentiation and survival.
  • the ErbB receptor family includes four distinct members including epidermal growth factor receptor (EGFR, ErbB1, HER1), HER2 (ErbB2 or pl85 neu ), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
  • EGFR epidermal growth factor receptor
  • HER2 ErbB2 or pl85 neu
  • HER3 ErbB3
  • HER4 ErbB4 or tyro2
  • a panel of anti-ErbB2 antibodies has been characterized using the human breast tumor cell line SKBR3 (Hudziak ef al, (1989) MoI. Cell. Biol. 9(3):1165-1172. Maximum inhibition was obtained with the antibody called 4D5 which inhibited cellular proliferation by 56%.
  • the antibody 4D5 was further found to sensitize ErbB2-overexpressing breast tumor cell lines to the cytotoxic effects of TNF- ⁇ (US 5677171).
  • the anti-ErbB2 antibodies discussed in Hudziak et al. are further characterized in Fendly et al (1990) Cancer Research 50:1550-1558; Kotts et al. (1990) In Vitro 26(3):59A; Sarup et al. (1991) Growth Regulation 1:72-82; Shepard et al. J. (1991) Clin. Immunol. 11(3):117-127; Kumar et al. (1991) MoI. Cell. Biol.
  • the ErbB receptor will generally comprise an extracellular domain, which may bind an ErbB ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain harboring several tyrosine residues which can be phosphorylated.
  • the ErbB receptor may be a "native sequence” ErbB receptor or an "amino acid sequence variant" thereof.
  • the ErbB receptor may be a native sequence human ErbB receptor. Accordingly, a "member of the ErbB receptor family" is EGFR (ErbBl), ErbB2, ErbB3, ErbB4 or any other ErbB receptor currently known or to be identified in the future.
  • ErbBl "epidermal growth factor receptor", “EGFR” and “HERl” are used interchangeably herein and refer to EGFR as disclosed, for example, in Carpenter et al (1987) Ann. Rev. Biochem., 56:881-914, including naturally occurring mutant forms thereof (e.g., a deletion mutant EGFR as in Humphrey et al., PNAS (USA), 87:4207-4211 (1990)).
  • the term erbBl refers to the gene encoding the EGFR protein product. Antibodies against HERl are described, for example, in Murthy et al (1987) Arch. Biochem. Biophys., 252:549-560 and in WO 95/25167.
  • ERRP epidermal growth factor receptor
  • EGF epidermal growth factor receptor
  • ErbB2 and "HER2” are used interchangeably herein and refer to human HER2 protein described, for example, in Semba et al., PNAS (USA), 82:6497-6501 (1985) and Yamamoto et al., (1986) Nature, 319:230-234 (Genebank accession number X03363).
  • the term “erbB2” refers to the gene encoding human ErbB2 and "neu” refers to the gene encoding rat pl85neu.
  • ErbB2 may be a native sequence human ErbB2.
  • ErbB3 and HER3 refer to the receptor polypeptide as disclosed, for example, in US 5183884 and US 5480968 as well as Kraus et al., PNAS (USA), 86:9193-9197 (1989).
  • Antibodies against ErbB3 are known in the art and are described, for example, in U.S. Patent Nos. 5183884, 5480968 and in WO 97/35885.
  • ErbB4 and "HER4" herein refer to the receptor polypeptide as disclosed, for example, in EP Pat Application No 599,274; Plowman et al., Proc. Natl. Acad. ScL USA, 90:1746-1750 (1993); and Plowman et al., Nature, 366:473-475 (1993), including isoforms thereof, e.g., as disclosed in WO 99/19488.
  • Antibodies against HER4 are described, for example, in WO 02/18444.
  • Antibodies to ErbB receptors are available commercially from a number of sources, including, for example, Santa Cruz Biotechnology, Inc., California, USA.
  • amino acid sequence variant refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 70% sequence identity with at least one receptor binding domain of a native antibody or with at least one ligand binding domain of a native receptor, and preferably, they will be at least about 80%, more preferably, at least about 90% homologous by sequence with such receptor or ligand binding domains. The amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Amino acids are designated by the conventional names, one-letter and three-letter codes.
  • Sequence identity is defined as the percentage of residues in the amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for the alignment are well known in the art. One such computer program is "Align 2,” authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, DC 20559, on December 10, 1991.
  • Fc receptor or “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
  • An exemplary FcR is a native sequence human FcR.
  • a FcR may be one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RH, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RUA (an “activating receptor”) and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • FcR FcR
  • FcRn neonatal receptor
  • “Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (CIq) to a molecule (e.g., an antibody) complexed with a cognate antigen.
  • CIq first component of the complement system
  • a CDC assay e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), may be performed.
  • “Native 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 among 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. 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 chain and heavy chain variable domains.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hyper variable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs).
  • the variable domains of native heavy and light chains each comprise four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al supra) and/or those residues from a "hypervariable loop” ⁇ e.g., residues 26-32 (Ll), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk (1987) J. MoI. Biol., 196:901-917).
  • CDR complementarity determining region
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and antigen- binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • scFv see Pl ⁇ ckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).
  • Anti-ErbB2 antibody scFv fragments are described in WO 93/16185; US Patent Nos. 5571894; and 5587458.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH - VL).
  • VH variable heavy domain
  • VL variable light domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.
  • “Humanized” forms of non-human ⁇ e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Humanization is a method to transfer the murine antigen binding information to a non-immunogenic human antibody acceptor, and has resulted in many therapeutically useful drugs. The method of humanization generally begins by transferring all six murine complementarity determining regions (CDRs) onto a human antibody framework (Jones et al, (1986) Nature 321:522-525). These CDR-grafted antibodies generally do not retain their original affinity for antigen binding, and in fact, affinity is often severely impaired.
  • CDRs complementarity determining regions
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine 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 hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Humanized anti-ErbB2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN®) as described in Table 3 of US 5821337, expressly incorporated herein by reference; humanized 520C9 (WO 93/21319) and humanized 2C4 antibodies as described herein below.
  • a “parent antibody” is an antibody comprising an amino acid sequence from which one or more amino acid residues are replaced by one or more cysteine residues.
  • the parent antibody may comprise a native or wild type sequence.
  • the parent antibody may have pre-existing amino acid sequence modifications (such as additions, deletions and/or substitutions) relative to other native, wild type, or modified forms of an antibody.
  • a parent antibody is directed against a target antigen of interest.
  • Antibodies directed against nonpolypeptide antigens are also contemplated.
  • exemplary parent antibodies include those selected from, and without limitation, anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-HER-2/neu antibody, anti-EGFR antibody, anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti- CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24 antibody, anti-CDIO antibody, anti-CDl lc antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, or more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a gas phase protein sequencer, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • an antibody "which binds" a molecular target or an antigen of interest is one capable of binding that antigen with sufficient affinity such that the antibody is useful in targeting a cell expressing the antigen.
  • the antibody is one which binds ErbB2
  • it will usually preferentially bind ErbB2 as opposed to other ErbB receptors, and may be one which does not significantly cross-react with other proteins such as EGFR, ErbB 3 or ErbB4.
  • the extent of binding of the antibody to these non- ErbB2 proteins will be less than 10% as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).
  • FACS fluorescence activated cell sorting
  • RIA radioimmunoprecipitation
  • the anti- ErbB2 antibody will not significantly cross-react with the rat neu protein, e.g., as described in Schecter et al., Nature 312:513 (1984) and Drebin et al., Nature 312:545-548 (1984).
  • Molecular targets for the antibody drug conjugates (ADC) encompassed by the present invention include: (i) tumor-associated antigens; (ii) cell surface receptors, (iii) CD proteins and their ligands, such as CD3, CD4, CD8, CD19, CD20, CD22, CD34, CD40, CD79 ⁇ (CD79a), and CD79 ⁇ (CD79b); (iv) members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; (v) cell adhesion molecules such as LFA-I, Macl, pl50,95, VLA-4, ICAM-I, VCAM and ⁇ v/ ⁇ 3 integrin including either alpha or beta subunits thereof (e.g.
  • anti-CDlla, anti-CD18 or anti-CDllb antibodies include growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; inpl receptor; CTLA-4; protein C, BR3, c- met, tissue factor, ⁇ 7 etc.
  • growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; inpl receptor; CTLA-4; protein C, BR3, c- met, tissue factor, ⁇ 7 etc.
  • the term “monoclonal antibody 4D5" refers to an antibody that has antigen binding residues of, or derived from, the murine 4D5 antibody (ATCC CRL 10463).
  • the monoclonal antibody 4D5 may be murine monoclonal antibody 4D5 or a variant thereof, such as a humanized 4D5.
  • Exemplary humanized 4D5 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5- 4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (Trastuzumab, HERCEPTIN®) as in US Patent No. 5821337.
  • the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • the term "therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal.
  • the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
  • bioavailability refers to the systemic availability (i.e., blood/plasma levels) of a given amount of drug administered to a patient. Bioavailability is an absolute term that indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the general circulation from an administered dosage form.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • a “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • squamous cell cancer e.g., epithelial squamous cell cancer
  • lung cancer including small- cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • NSCLC non-small cell lung cancer
  • adenocarcinoma of the lung and squamous carcinoma of the lung cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer
  • An "ErbB-expressing cancer” is one comprising cells which have ErbB protein present at their cell surface.
  • An "ErbB2-expressing cancer” is one which produces sufficient levels of ErbB2 at the surface of cells thereof, such that an anti-ErbB2 antibody can bind thereto and have a therapeutic effect with respect to the cancer.
  • a cancer "characterized by excessive activation" of an ErbB receptor is one in which the extent of ErbB receptor activation in cancer cells significantly exceeds the level of activation of that receptor in noncancerous cells of the same tissue type. Such excessive activation may result from overexpression of the ErbB receptor and/or greater than normal levels of an ErbB ligand available for activating the ErbB receptor in the cancer cells. Such excessive activation may cause and/or be caused by the malignant state of a cancer cell. In some embodiments, the cancer will be subjected to a diagnostic or prognostic assay to determine whether amplification and/or overexpression of an ErbB receptor is occurring which results in such excessive activation of the ErbB receptor.
  • the cancer may be subjected to a diagnostic or prognostic assay to determine whether amplification and/or overexpression an ErbB ligand is occurring in the cancer which attributes to excessive activation of the receptor.
  • a diagnostic or prognostic assay to determine whether amplification and/or overexpression an ErbB ligand is occurring in the cancer which attributes to excessive activation of the receptor.
  • excessive activation of the receptor may result from an autocrine stimulatory pathway.
  • a cancer which "overexpresses" an ErbB receptor is one which has significantly higher levels of an ErbB receptor, such as ErbB2, at the cell surface thereof, compared to a noncancerous cell of the same tissue type.
  • Such overexpression may be caused by gene amplification or by increased transcription or translation.
  • ErbB receptor overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the ErbB protein present on the surface of a cell (e.g., via an immunohistochemistry assay; IHC).
  • FISH fluorescent in situ hybridization
  • PCR polymerase chain reaction
  • Overexpression of the ErbB ligand may be determined diagnostically by evaluating levels of the ligand (or nucleic acid encoding it) in the patient, e.g., in a tumor biopsy or by various diagnostic assays such as the IHC, FISH, southern blotting, PCR or in vivo assays described above.
  • shed antigen e.g., ErbB extracellular domain
  • serum see, e.g., US 4933294; WO 91/05264; US 5401638; and Sias et al., (1990) /. Immunol. Methods, 132: 73-80.
  • various other in vivo assays are available to the skilled practitioner.
  • a detectable label e.g., a radioactive isotope
  • 0 0-10,000 copies/cell
  • 1+ at least about 200,000 copies/cell
  • 2+ at least about 500,000 copies/cell
  • 3+ about 1-2 x 10 6 copies/cell.
  • Overexpression of HER2 at the 3+ level which leads to ligand-independent activation of the tyrosine kinase (Hudziak et al., (1987) Proc. Natl. Acad.
  • a cancer which is "not characterized by overexpression of the ErbB2 receptor" is one which, in a diagnostic assay, does not express higher than normal levels of ErbB2 receptor compared to a noncancerous cell of the same tissue type.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., 211 At, 131 I, 125 1, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 60 C, and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.
  • radioactive isotopes e.g., 211 At, 131 I, 125 1, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 60 C, and radioactive isotopes of Lu
  • chemotherapeutic agents e.g., chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal
  • chemotherapeutic agent and “anticancer agent” are terms that denote a chemical compound useful in the treatment of cancer, and which may be administered in combination therapy with the antibody drug conjugate compounds of the invention.
  • chemotherapeutic agents include Erlotinib (TARCEV A®, Genentech/OSI Pharm.), Bortezomib (VELCADE®, Millenium Pharm.), Fulvestrant (FASLODEX®, Astrazeneca), Sutent (SUl 1248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib
  • calicheamicin especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Intl. Ed. Engl, 33: 183-186 (1994)) and anthracyclines such as annamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX- 100, idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactin
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • SERMs selective estrogen receptor modulators
  • tamoxifen including NOLVADEX® tamoxifen
  • raloxifene including NOLVADEX® tamoxifen
  • droloxifene 4-hydroxytamoxifen
  • trioxifene keoxifene
  • LYl 17018, onapristone and FARESTON® toremifene
  • aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARHVIIDEX® anastrozole
  • anti-androgens such as fiutamide
  • EGFR-targeted drug refers to a therapeutic agent that binds to EGFR and, optionally, inhibits EGFR activation.
  • agents include antibodies and small molecules that bind to EGFR.
  • antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US 4943533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBITUX®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); antibodies that bind type II mutant EGFR (US 5212290); humanized and chimeric antibodies that bind EGFR as described in US 5891996; and human antibodies that bind EGFR, such as
  • the anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP 659,439A2, Merck Patent GmbH).
  • a cytotoxic agent such as a cytotoxic agent, thus generating an immunoconjugate.
  • small molecules that bind to EGFR include ZD 1839 or Gefitinib (IRESS ATM; Astra Zeneca), Erlotinib HCl (CP-358774, TARCEVATM; Genentech/OSI) and AG1478, AG1571 (SU 5271; Sugen).
  • a "tyrosine kinase inhibitor” is a molecule which inhibits to some extent tyrosine kinase activity of a tyrosine kinase such as an ErbB receptor.
  • examples of such inhibitors include the EGFR-targeted drugs noted in the preceding paragraph as well as quinazolines such as PD 153035,4-(3-chloroanilino) quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines, curcumin (diferuloyl methane, 4,5- bis (4-fluoroanilino) ⁇ hthalimide), tyrphostines containing nitrothiophene moieties; PD-0183
  • an "anti-angiogenic agent” refers to a compound which blocks, or interferes with to some degree, the development of blood vessels.
  • the anti-angiogenic factor may, for instance, be a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis.
  • An exemplary anti- angiogenic factor herein is an antibody that binds to Vascular Endothelial Growth Factor (VEGF).
  • VEGF Vascular Endothelial Growth Factor
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N- methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet-growth factor;
  • a “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as the anti-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal.
  • a drug such as the anti-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • Phage display is a technique by which variant polypeptides are displayed as fusion proteins to a coat protein on the surface of phage, e.g., filamentous phage, particles.
  • phage display One utility of phage display lies in the fact that large libraries of randomized protein variants can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide and protein libraries on phage has been used for screening millions of polypeptides for ones with specific binding properties. Polyvalent phage display methods have been used for displaying small random peptides and small proteins, typically through fusions to either PIII or PV ⁇ i of filamentous phage. Wells and Lowman, Curr. Opin. Struct.
  • phage display In monovalent phage display, a protein or peptide library is fused to a phage coat protein or a portion thereof, and expressed at low levels in the presence of wild type protein. Avidity effects are reduced relative to polyvalent phage so that sorting is on the basis of intrinsic ligand affinity, and phagemid vectors are used, which simplify DNA manipulations. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991). Phage display includes techniques for producing antibody-like molecules (Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York, p627-628).
  • a "phagemid” is a plasmid vector having a bacterial origin of replication, e.g., CoIEl, and a copy of an intergenic region of a bacteriophage.
  • the phagemid may be used on any known bacteriophage, including filamentous bacteriophage and lambdoid bacteriophage.
  • the plasmid will also generally contain a selectable marker for antibiotic resistance. Segments of DNA cloned into these vectors can be propagated as plasmids. When cells harboring these vectors are provided with all genes necessary for the production of phage particles, the mode of replication of the plasmid changes to rolling circle replication to generate copies of one strand of the plasmid DNA and package phage particles.
  • the phagemid may form infectious or non-infectious phage particles.
  • This term includes phagemids which contain a phage coat protein gene or fragment thereof linked to a heterologous polypeptide gene as a gene fusion such that the heterologous polypeptide is displayed on the surface of the phage particle.
  • Alkyl is a C 1 -C 18 hydrocarbon moiety containing normal, secondary, tertiary or cyclic carbon atoms.
  • alkyl radicals include C 1 -C 8 hydrocarbon moieties such as: methyl (Me, -CH3), ethyl (Et, - CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n- butyl, -CH2CH2CH2CH3), 2-methyl-l-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, - CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3
  • Alkenyl is a C 2 -C 18 hydrocarbon moiety containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon, sp 2 double bond.
  • Alkynyl is a C 2 -C 18 hydrocarbon moiety containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon, sp triple bond. Examples include, but are not limited to: acetylenic (-C ⁇ CH) and propargyl (-CH 2 C ⁇ CH),
  • Alkylene refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane.
  • Typical alkylene radicals include, but are not limited to: methylene (-CH 2 -) 1,2-ethyl (-CH 2 CH 2 -), 1,3-propyl (-CH 2 CH 2 CH 2 -), 1,4-butyl (-CH 2 CH 2 CH 2 CH 2 -), and the like.
  • alkenylene refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkene.
  • Alkynylene refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne.
  • Aryl means a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented in the exemplary structures as "Ar”. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like.
  • Arylalkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with an aryl radical.
  • Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-l-yl, 2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2- naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like.
  • the arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.
  • Heteroarylalkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with a heteroaryl radical.
  • Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like.
  • the heteroarylalkyl group comprises 6 to 20 carbon atoms, e.g.
  • the alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to 6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S.
  • the heteroaryl moiety of the heteroarylalkyl group may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.
  • Substituted alkyl mean alkyl, aryl, and arylalkyl respectively, in which one or more hydrogen atoms are each independently replaced with a substituent.
  • Heteroaryl “heterocyclyl”, and “heterocycle” all refer to a ring system in which one or more ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur.
  • the heterocycle radical comprises 1 to 20 carbon atoms and 1 to
  • a heterocycle may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.
  • Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W.A.
  • heterocycles include by way of example and not limitation pyridyl, dihydroypyridyl, tetrahydropyridyl Cpiperidyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2- ⁇ yrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, bis-
  • carbon bonded heterocycles are bonded at position 2, 3, 4, 5, or
  • carbon bonded heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5- pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.
  • nitrogen bonded heterocycles are bonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3- imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, IH- indazole, position 2 of a isoindole, or isoindoline, position 4 of a morpholine, and position 9 of a carbazole, or ⁇ - carboline.
  • nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1- imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
  • Carbocycle and “carbocyclyl” mean a saturated or unsaturated ring having 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle.
  • Monocyclic carbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ring atoms.
  • Bicyclic carbocycles have 7 to 12 ring atoms, e.g. arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as a bicyclo [5,6] or [6,6] system.
  • Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-l-enyl, l-cyclo ⁇ ent-2-enyl, 1-cyclopent- 3-enyl, cyclohexyl, 1-cyclohex-l-enyl, l-cyclohex-2-enyl, l-cyclohex-3-enyl, cycloheptyl, and cyclooctyl.
  • Linker means a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety.
  • a linker is specified as L.
  • Linkers include, but are not limited to, a divalent radical such as an alkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as: — (CR 2 ) n O(CR 2 ) n - , repeating units of alkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino, JeffamineTM); and diacid ester and amides including maleimide, succinate, succinamide, diglycolate, malonate, and caproamide.
  • label means any moiety which can be covalently attached to an antibody and that functions to: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g. FRET (fluorescence resonance energy transfer); (iii) stabilize interactions or increase affinity of binding, with antigen or ligand; (iv) affect mobility, e.g. electrophoretic mobility, or cell- permeability, by charge, hydrophobicity, shape, or other physical parameters, or (v) provide a capture moiety, to modulate ligand affinity, antibody/antigen binding, or ionic complexation.
  • FRET fluorescence resonance energy transfer
  • chiral refers to molecules which have the property of non-superimposability of the mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner.
  • stereoisomers refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
  • Diastereomer refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography. “Enantiomers” refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.
  • d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory.
  • a compound prefixed with (+) or d is dextrorotatory.
  • these stereoisomers are identical except that they are mirror images of one another.
  • a specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
  • a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
  • racemic mixture and racemate refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
  • phrases "pharmaceutically acceptable salt,” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of an ADC.
  • Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis -(2-hydroxy-3
  • a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion.
  • the counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.
  • “Pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and an ADC.
  • solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
  • Boc is N-(t-butoxycarbonyl), cit is citrulline (2-amino-5-ureido pentanoic acid), dap is dolaproine, DCC is 1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA is diethylamine, DEAD is diethylazodicarboxylate, DEPC is diethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA is ⁇ iV-diisopropylethylamine, dil is dolaisoleuine, DMAP is 4-dimethylaminopyridine, DME is ethyleneglycol dimethyl ether (or 1,2-dimethoxyethane), DMF is N,N-dimethylformamide, DMSO is dimethylsulfoxide, doe is dolaphenine,N dov is N,N-dimethylvaline, DTNB is 5,5'-dithiobis(2-
  • HERCEPTIN® trastuzumab
  • trastuzumab F(ab')2 derived from antiHER2 enzymatically (MW 100000)
  • 4D5 full-length, murine antiHER2, from hybridoma
  • rhu4D5 transiently expressed, full-length humanized antibody
  • rhuFab4D5 recombinant humanized Fab (MW 47738)
  • 4D5Fc8 full-length, murine antiHER2, with mutated FcRn binding domain
  • the compounds of the invention include those with potential utility for anticancer activity, treatment of hyperproliferative disorders, autoimmune disorders, and infectious disease.
  • the compounds include an antibody conjugated, i.e. covalently attached by a linker, to a 1,8 bis-naphthalimide drug moiety where the corresponding drug when not conjugated to an antibody has a cytotoxic or cytostatic effect.
  • the biological activity of the drug is thus modulated by conjugation to an antibody.
  • the antibody drug conjugates (ADC) of the invention may selectively deliver an effective dose of a cytotoxic agent to tumor tissue whereby greater selectivity, i.e. a lower efficacious dose may be achieved, than upon delivery of the same dose of the 1,8 bis-naphthalimide compound not conjugated to an antibody.
  • the bioavailability of the ADC of the invention, or an intracellular metabolite of the ADC is improved in a mammal when compared to a 1,8 bis-naphthalimide compound comprising the 1,8 bis-naphthalimide moiety of the ADC. Also, the bioavailability of the ADC, or an intracellular metabolite of the ADC is improved in a mammal when compared to the analog of the ADC not having the 1,8 bis-naphthalimide drug moiety.
  • the drug moiety of the ADC is not cleaved from the antibody until the antibody- drug conjugate enters a cell with a cell-surface receptor specific for the antibody of the antibody-drug conjugate, and the drug moiety is cleaved from the antibody when the antibody-drug conjugate does enter the cell.
  • the 1,8 bis-naphthalimide drug moiety may be intracellularly cleaved in a mammal from the antibody of the compound, or an intracellular metabolite of the compound, by enzymatic action, hydrolysis, oxidation, or other mechanism.
  • An antibody-drug conjugate compound comprises an antibody covalently attached by a linker to one or more 1,8 bis-naphthalimide drug moieties, the compound having Formula I or a pharmaceutically acceptable salt or solvate thereof, wherein Ab is an antibody;
  • L is a linker covalently attached to an Ab, and L is covalently attached to D;
  • D is a 1,8 bis-naphthalimide drug moiety selected from Formulas Ila and llb:
  • Y is N(R b ), C(R a ) 2 , O, or S;
  • R b is independently selected from H, C 1 -C 8 alkyl, C 1 -C 8 substituted alkyl, C 2 -C 8 alkenyl, C 2 -C 8 substituted alkenyl, C 2 -C 8 alkynyl, C 2 -C 8 substituted alkynyl, C 6 -C 20 aryl, C 6 -C 20 substituted aryl, C 1 -C 20 heterocycle, and C 1 -C 20 substituted heterocycle; where C 1 -C 8 substituted alkyl, C 2 -C 8 substituted alkenyl, C 2 -C 8 substituted alkynyl, C 6 -C 20 substituted aryl, and C 2 -C 20 substituted heterocycle are independently substituted with one or more substituents selected from F, Cl, Br, I, OH, -N(R b ) 2 , -N(R b ) 3 + , C 1 -C 8 alkylhalide, carboxylate, s
  • X 1 , X 2 , X 3 , and X 4 are independently selected from F, Cl, Br, I, OH, -N(R b ) 2 , -N(R b ) 3 + ,
  • X 1 and X 2 together, and X 3 and X 4 together, independently form -CH 2 CH 2 - or -CH 2 CH 2 CH 2 -;
  • D may independently have more than one X 1 , X 2 , X 3 , or X 4 ; and where D has more than one X 1 , X 2 , X 3 , or X 4 , then two X 1 , X 2 , X 3 , or X 4 may form a fused C 6 -C 20 aryl, C 6 -C 20 substituted aryl, C 1 -C 20 heterocycle, or C 1 -C 20 substituted heterocycle; and p is an integer from 1 to 20.
  • the drug loading is represented by p, the average number of drugs per antibody in a molecule of Formula I.
  • Drug loading may range from 1 to 20 drugs (D) per antibody (Ab or mAb).
  • Compositions of ADC of Formula I include collections of antibodies conjugated with a range of drugs, from 1 to 20.
  • the average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC.
  • the quantitative distribution of ADC in terms of p may also be determined.
  • separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
  • p may be limited by the number of attachment sites on the antibody.
  • an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.
  • Higher drug loading, e.g. p >5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody drug conjugates.
  • an antibody may contain, for example, many lysine residues that do not react with the drug-linker intermediate or linker reagent. Only the most reactive lysine groups may react with an amine- reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety.
  • cysteine thiol residues in the antibodies of the compounds of the invention exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT), under partial or total reducing conditions. Additionally, the antibody must be subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.
  • the loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.
  • the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody.
  • the average number of drugs per antibody may be calculated from the mixture by dual ELISA antibody assay, specific for antibody and specific for the drug.
  • Individual ADC molecules may be identified in the mixture by mass spectroscopy, and separated by HPLC, e.g. hydrophobic interaction chromatography ("Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate", Hamblett, K.J., et al, Abstract No.
  • a homogeneous ADC with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.
  • Drug moieties (D) are the 1,8 bis-naphthalimide type and have Formulas Ila and llb.
  • One embodiment of a bis 1,8 naphthalimide drug moiety is the unsubstituted bis 1,8 naphthalimide, "elinafide", drug moiety (E) having the structure:
  • Y is N(R b ), R b is H, m is 3, and n is 2.
  • the 1,8 naphthalimide aromatic carbon atoms D moieties IIa and llb may be independently substituted with a range of substituents (X'-X 4 ) besides H.
  • X'-X 4 substituents
  • X 1 and X 2 together, or X 3 and X 4 together, independently may form -CH 2 CH 2 - or -CH 2 CH 2 CH 2 - .
  • Two X 1 , X 2 , X 3 , or X 4 on adjacent carbon atoms may form a fused C 6 -C 20 aryl, C 6 -C 2O substituted aryl, C 1 -C 20 heterocycle, or C 1 -C 20 substituted heterocycle.
  • Y is N(R b )
  • the bis-amino alkyl group that attaches the two 1,8 naphthalimide groups may bear a range of substituents besides H on the carbon atoms (R a ) and the nitrogen atom not linked to L (R b ).
  • exemplary embodiments of D where Y is N(R b ), m is 3 and n is 2 in the bis-amino alkyl group include the D moiety Ha structures:
  • the two non-equivalent alkylene groups between each 1,8 naphthalimide group and a nitrogen atom (n) are independently 1, 2, or 3 carbons in length.
  • the alkylene group between the nitrogen atoms (m) is 1, 2, 3, 4, 5, or 6 carbons in length.
  • Exemplary embodiments of Ila and Ilb where Y is 0 or S include the following structures:
  • 1,8 naphthalimide intermediates may be prepared from 1,8-naphthalic anhydride compounds (Chem. Rev. (1970) 70:439-469; US Patent Nos. 4146720; 5616589; 5416089; 5585382; 5552544).
  • Various substituted 1,8-naphthalic anhydride compounds are commercially available, such as 4-Bromo-1,8-naphthalic anhydride (Aldrich, Milwaukee, WI). Reaction of a 1,8-naphthalic anhydride compound with a primary amine gives the 1,8 naphthalimide. Displacement of bromine from the 4 position occurs with various nucleophilic reagents.
  • amine reagent is a bis-amino compound
  • two 1,8-naphthalic anhydride react to form bis 1,8 naphthalimide intermediates
  • the 1,8 naphthalimide groups may be attached to the polyamine unit sequentially (WO 94/02466) by protecting one of the terminal amino groups of the polyamine reagent during reaction with the first 1,8 naphthalic anhydride reagent. After deprotection of the terminal amino group of the mono 1,8 naphthalimide intermediate, a second 1,8 naphthalic anhydride reagent may be reacted to form the bis 1,8 naphthalimide product.
  • asymmetric bis 1,8 naphthalimide compounds can be prepared, i.e. where X 1 and X 2 are different than X 3 and X 4 .
  • Suitable amino protecting groups include mesitylenesulfonyl, dinitrobenzenesulfonyl, BOC (tert-butyloxycarbonyl), CBz (carbobenzoxy), or those detailed in Protective Groups in Organic Chemistry, Theodora W. Greene (1991) John Wiley & Sons, Inc., New York, or later editions thereto.
  • the terminal amino group for coupling to the second 1,8 naphthalic anhydride reagent may be generated by reductive amination of a carbonyl group such as aldehyde or ester, or by reduction of a nitrile group.
  • the linker (L) is a bifunctional or multifunctional moiety which is covalently attached to one or more Drug moieties (D) and an antibody unit (Ab) to form Antibody Drug Conjugates (ADC) of the invention.
  • the linker L of an ADC has the formula:
  • -A- is a Stretcher unit; a is 0 or 1; each -W- is independently an Amino Acid unit; w is independently an integer ranging from 0 to 12;
  • -SP- is a Spacer unit; and y is 0, 1 or 2.
  • the ADC may be represented by Formula Ia:
  • the linker may be a dendritic type linker for covalent attachment of more than one drug moiety through a branching, multifunctional linker moiety to an antibody (Sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003) Bioorganic & Medicinal Chemistry 11:1761-1768).
  • Dendritic linkers can increase the molar ratio of drug to antibody, i.e. loading, which is related to the potency of the ADC.
  • an antibody bears only one reactive group, e.g. a lysine amino or a cysteine thiol, a multitude of drug moieties may be attached through a dendritic linker.
  • the Stretcher unit (-A-), when present, is capable of linking an antibody (Ab) to an amino acid unit (- W-).
  • an antibody (Ab) has a functional group that can form a bond with a functional group of a Stretcher.
  • Useful functional groups that can be present on an antibody, either naturally or via chemical manipulation include, but are not limited to, sulfhydryl (-SH), amino, hydroxyl, carboxy, the anomeric hydroxyl group of a carbohydrate, and carboxyl.
  • the reactive functional groups on the antibody are sulfhydryl and amino. Sulfhydryl groups can be generated by reduction of an intramolecular cysteine disulfide bond of an antibody.
  • sulfhydryl groups can be generated by reaction of an amino group of a lysine moiety of an antibody using 2-iminothiolane (Traut's reagent) or another sulfhydryl generating reagent.
  • the Stretcher unit forms a bond with a sulfur atom, e.g. a cysteine amino acid residue, of the Antibody unit.
  • the sulfur atom can be derived from a sulfhydryl group of an antibody.
  • Representative Stretcher units of this embodiment are depicted in Formulas Illa and HIb, wherein Ab-, -W-, - SP-, -D, w and y are as defined above and wherein R 17 is selected from (CH 2 ) r , C 3 -C 8 carbocyclyl, O-(CH 2 ) r , arylene, (CH 2 ) r -arylene, -arylene-(CH 2 ) r -, (CH 2 ) -(C 3 -C 8 carbocyclyl), (C 3 -C 8 carbocyclyl)-(CH 2 ) r , C 3 -C 8 heterocyclyl, (CH 2 ) r -(C 3 -C 8 hetero
  • An illustrative Stretcher unit is that of Formula Illa is derived from maleimido-caproyl (MC) wherein R 17 is -(CH 2 ) 5 -:
  • An illustrative Stretcher unit is that of Formula Illa is derived from maleimido-propanoyl (MP) wherein R 17 is -(CH 2 ) 2 -:
  • Stretcher unit is that of Formula Illa wherein R 17 is -(CH 2 CH 2 O) r -CH 2 - and r is 2:
  • Stretcher unit is that of Formula Illa wherein R 17 is -(CH 2 ) r C(O)NR b (CH 2 CH 2 O) r -CH 2 - where R b is H and each r is 2:
  • Stretcher unit is that of Formula Illb wherein R 17 is -(CH 2 ) 5 -:
  • the Stretcher unit is linked to the Antibody unit via a disulfide bond between a sulfur atom of the Antibody unit and a sulfur atom of the Stretcher unit.
  • a representative Stretcher unit of this embodiment is depicted within the square brackets of Formula IV, wherein R 17 , Ab-, -W-, -SP-, -D, w and y are as defined above.
  • the reactive group of the Stretcher contains a reactive site that can form a bond with a primary or secondary amino group of an antibody.
  • these reactive sites include, but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates.
  • Representative Stretcher units of this embodiment are depicted within the square brackets of Formulas Va, Vb and Vc, wherein -R 17 -, Ab-, -W-, -SP-, -D, w and y are as defined above;
  • the reactive group of the Stretcher is reactive with an aldehyde, acetal, or ketal group on a sugar (carbohydrate) of a glycosylated antibody.
  • a carbohydrate can be mildly oxidized using a reagent such as sodium periodate and the resulting (-CHO) unit of the oxidized carbohydrate can be condensed with a Stretcher that contains a functionality such as a hydrazide, an oxime, a primary or secondary amine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and an arylhydrazide such as those described by Kaneko, T.
  • Amino Acid unit - W w - is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit.
  • Each -W- unit independently has the formula denoted below in the square brackets, and w is an integer ranging from 0 to 12:
  • R 19 includes all naturally occurring amino acid side chains, and analogs thereof.
  • the Amino Acid unit can be enzymatically cleaved by one or more enzymes, including a tumor- associated protease or apoptotic-related enzyme such as cathepsin B, C, and D, or a plasmin protease, to liberate the drug moiety (-D).
  • a tumor- associated protease or apoptotic-related enzyme such as cathepsin B, C, and D, or a plasmin protease
  • W w units are represented by Formulas (VII)-(IX):
  • R 20 and R 21 are as follows:
  • R 20 R 21 benzyl (CH 2 ) 4 NH 2 methyl (CH 2 ) 4 NH 2 methyl benzyl isopropyl (CH 2 ) 4 NH 2 isopropyl (CH 2 ) 3 NHCONH 2 benzyl (CH 2 ) 3 NHCONH 2 isobutyl (CH 2 ) 3 NHCONH 2 sec-butyl (CH 2 ) 3 NHCONH 2
  • R 20 , R 21 and R 22 are as follows:
  • R 20 , R 21 , R 22 and R 23 are as follows:
  • Exemplary Amino Acid units include, but are not limited to, units of Formula (VII) where: R 20 is benzyl and R 21 is -(CH 2 ) 4 NH 2 ; R 20 isopropyl and R 21 is -(CH 2 ) 4 NH 2 ; R 20 isopropyl and R 21 is - (CH 2 ) 3 NHCONH 2 .
  • Another exemplary Amino Acid unit is a unit of Formula (VIII) wherein R 20 is benzyl, R 21 is benzyl, and R 22 is -(CH 2 ) 4 NH 2 .
  • Exemplary -W w - Amino Acid units include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
  • Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe).
  • Exemplary tripeptides include: glycine- valine-citrulline and glycine-glycine-glycine-glycine.
  • R 19 , R 20 , R 21 , R 22 or R 23 is other than hydrogen
  • the carbon atom to which R 19 , R 20 , R 21 , R 22 or R 23 is attached is chiral.
  • the Spacer unit when present: (i) links an Amino Acid unit to the Drug unit when an Amino Acid unit is present, (ii) links the Stretcher unit to the Drug moiety when the Amino Acid unit is absent, or (iii) links the Drug moiety to the antibody unit when both the Amino Acid unit and Stretcher unit are absent.
  • Spacer units are of two general types: self-immolative and non self-immolative.
  • a non self-immolative Spacer unit is one in which part or all of the Spacer unit remains bound to the Drug moiety after cleavage, particularly enzymatic, of an Amino Acid unit from the Drug-Linker-antibody Conjugate or the Drug-Linker Compound.
  • Examples of a non self-immolative Spacer unit include, but are not limited to a (glycine-glycine) Spacer unit and a glycine Spacer unit.
  • a (glycine-glycine) Spacer unit When an Exemplary Compound containing a glycine-glycine Spacer unit or a glycine Spacer unit undergoes enzymatic cleavage via a tumor-cell associated-protease, a cancer-cell-associated protease or a lymphocyte-associated protease, a glycine-glycine-Drug moiety or a glycine-Drug moiety is cleaved from Ab-A a -Ww-.
  • an independent hydrolysis reaction takes place within the target cell, cleaving the glycine-Drug moiety bond and liberating the Drug.
  • -SP y - is a para-aminobenzyloxycarbonyl (PAB) unit whose phenylene portion is substituted with Q m wherein Q is -C 1 -C 8 alkyl, -O-(C 1 -C 8 alkyl), -halogen,- nitro or -cyano; and m is an integer ranging from 0-4.
  • PAB para-aminobenzyloxycarbonyl
  • Exemplary embodiments of a non self-immolative Spacer unit are: -Gly-Gly- ; -GIy- ; -Ala-Phe- ; -Val-Cit- .
  • an ADC containing a self-immolative Spacer unit can release -D.
  • - SP- is a PAB group that is linked to -W w - via the amino nitrogen atom of the PAB group, and connected directly to -D via a carbonate, carbamate or ether group, where the ADC has the exemplary structure:
  • Q is -C 1 -C 8 alkyl, -O-( C 1 -C 8 alkyl), -halogen, -nitro or -cyano;
  • m is an integer ranging from 0- 4; and
  • p ranges from 1 to 4.
  • self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group such as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals.
  • Self-immolative spacers also include where the PAB group is substituted by a heterocyclic group (WO 2005/082023).
  • Spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995) Chemistry Biology 2:223), appropriately substituted bicyclo [2.2.1] and bicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry et al (1990) J. Org. Chem., 55:5867). Elimination of amine-containing drugs that are substituted at glycine (Kingsbury et al (1984) J. Med. Chem., , 27, 1447) are also examples of self-immolative spacer useful in ADCs.
  • the Spacer unit is a branched bis(hydroxymethyl)styrene (BHMS), which can be used to incorporate and release multiple drugs, having the structure: wherein Q is -C 1 -C 8 alkyl, -O-(C 1 -C 8 alkyl), -halogen, -nitro or -cyano; m is an integer ranging from 0- 4; n is 0 or 1; and p ranges raging from 1 to 4.
  • BHMS branched bis(hydroxymethyl)styrene
  • the -D moieties are the same.
  • Spacer units (-SP y -) are represented by Formulas (X)-(XII):
  • Q is -C 1 -C 8 alkyl, -0-(C 1 -C 8 alkyl), -halogen, -nitro or -cyano; and m is an integer ranging fro
  • Embodiments of the Formula I antibody-drug conjugate compounds include XIIIa (val-cit), XIIIb (MC-val-cit), XIHc (MC-val-cit-PAB):
  • R is independently H or C 1 -C 6 alkyl; and n is 1 to 12.
  • Intermediates or reagents which include a bis 1,8 naphthalimide drug moiety and a reactive linker unit may comprise any combination of the bis 1,8 naphthalimide drug moieties and linker units.
  • Bis 1,8 naphthalimide-linker reagents bear functionality which is reactive with an antibody so as to allow covalent attachment, i.e. conjugation, of the reagent to the antibody to prepare an antibody drug conjugate (ADC) of the invention.
  • ADC antibody drug conjugate
  • Exemplary embodiments include the following bis 1,8 naphthalimide-linker reagents:
  • MC maleimido-caproyl
  • vc valine-citrulline amino acid subunit
  • PAB para- aminobenzyloxycarbonyl
  • E is the bis 1,8 naphthalimide drug moiety Ha where X 1 , X 2 , X 3 , and X 4 are H, R b
  • af is the alanine-phenylalanine amino acid subunit.
  • Another exemplary bis 1,8 naphthalimide drug -linker reagent is MC-vc-PAB-(N, N'-(bis-aminoethyl- l,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) Illa:
  • the antibody unit (Ab-) includes within its scope any unit of an antibody (Ab) that binds or reactively associates or complexes with a receptor, antigen or other receptive moiety associated with a given target-cell population.
  • An antibody can be any protein or protein-like molecule that binds to, complexes with, or reacts with a moiety of a cell population sought to be therapeutically or otherwise biologically modified.
  • the antibody unit acts to deliver the Drug unit to the particular target cell population with which the antibody unit reacts.
  • Such antibodies include, but are not limited to, large molecular weight proteins such as, for example, full-length antibodies, antibody fragments.
  • An antibody unit can form a bond to either a linker, a Stretcher unit, an Amino Acid unit, a Spacer Unit, or a Drug moiety directly.
  • An antibody unit can form a bond to a Linker unit via a heteroatom of the antibody.
  • the linking heteroatoms of the antibody may be a reactive nucleophilic group on any amino acid side chain, such as a cysteine thiol, a lysine amine, an aspartic acid or glutamic acid carboxyl, a serine, threonine, or tyrosine hydroxyl, or an arginine.
  • Heteroatoms that may be present on an antibody unit include sulfur (in one embodiment, from a sulfhydryl group of an antibody such as a cysteine thiol), oxygen (in one embodiment, from a carbonyl, carboxyl or hydroxyl group of an antibody) and nitrogen (in one embodiment, from a primary or secondary amino group of an antibody). These heteroatoms can be present on the antibody in the antibody's natural state, for example a naturally occurring antibody, or can be introduced into the antibody via chemical modification.
  • the antibody has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups.
  • the antibody unit then may bond to a linker reagent or drug-linker moiety via the sulfhydryl group's sulfur atom.
  • the reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-Iminothiolane hydrochloride (Traut's Reagent).
  • the antibody can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups.
  • the antibody unit bonds to the linker reagent or drug-linker moiety, such as the Stretcher Unit, via the sulfhydryl group's sulfur atom.
  • the antibody can have one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) group suitable for conjugation with a linker reagent or drug-linker moiety (see, for e.g., Laguzza, et al., J. Med. Chenu 1989, 32(3), 548-55).
  • Suitable oxidizing reagents include periodate reagents.
  • the corresponding aldehyde can form a bond with a Reactive Site on a Stretcher. The reaction may proceed through a Schiffs base intermediate and undergo subsequent reduction to a stable amine linkage.
  • Reactive sites on a Stretcher that can react with a carbonyl group on an antibody include, but are not limited to, hydrazine and hydroxylamine.
  • Other protocols for the modification of proteins for the attachment or association of Drug Units are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), incorporated herein by reference.
  • a tyrosine residue of the antibody may undergo diazotization by electrophilici aromatic substitution to form a diazo linkage with a linker reagent or drug-linker moiety.
  • tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies.
  • Antibodies which comprise Ab in Formula I antibody drug conjugates (ADC) and which may be useful in the treatment of cancer include, but are not limited to, antibodies against tumor-associated antigens (TAA).
  • TAA tumor-associated antigens
  • TAA include (l)-(35), but are not limited to TAA (l)-(36) listed below.
  • TAA is listed below and includes names, alternative names, Genbank accession numbers and primary reference(s).
  • Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to the sequences identified in the cited references, or which exhibit substantially the same biological properties or characteristics as a TAA having a sequence found in the cited references.
  • a TAA having a variant sequence generally is able to bind specifically to an antibody that binds specifically to the TAA with the corresponding sequence listed.
  • BMPRlB bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203
  • BMPRlB bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203
  • WO2004063362 Claim 2
  • WO2003042661 Claim 12
  • US2003134790-A1 Page 38-39
  • WO2002102235 Claim 13; Page 296
  • WO2003055443 Page 91-92
  • WO200299122 Example 2; Page 528-530
  • WO2003029421 (Claim 6); WO2003024392 (Claim 2; Fig 112); WO200298358 (Claim 1; Page 183); WO200254940 (Page 100-101); WO200259377(Page 349-350); WO200230268 (Claim 27; Page 376); WO200148204 (Example;
  • NP_036581 six transmembrane epithelial antigen of the prostate Cross-references: MM:604415; NP_036581.1; NM_012449_l
  • MPF MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823
  • Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b,Genbank accession no. NM_006424)
  • Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878)
  • PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628);
  • ETBR Endothelin type B receptor, Genbank accession no. AY275463
  • MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763); WO2003104275 (Claim 1); WO2004046342 (Example 2); WO2003042661 (Claim 12); WO2003083074
  • STEAP2 (HGNC_8639, IPCA-I, PCANAPl, STAMPl, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138)
  • TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636)
  • CRIPTO (CR, CRl, CRGF, CRIPTO, TDGFl, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212)
  • CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004)
  • FcRH2 (IFGP4, IRTA4, SPAPlA (SH2 domain containing phosphatase anchor protein Ia), SPAPlB, SPAPlC, Genbank accession no. NM_030764)
  • HER2 (ErbB2, Genbank accession no. Ml 1730)
  • NCA (CEACAM6, Genbank accession no. M18728);
  • MDP DPEPl, Genbank accession no. BC017023
  • WO2003016475 (Claim 1); WO200264798 (Claim 33; Page 85-87); JP05003790 (Fig 6-8); WO9946284 (Fig 9); Cross-references: MIM:179780; AAH17023.1; BC017023_l
  • IL20R ⁇ (IL20Ra, ZCYTOR7, Genbank accession no. AF184971);
  • EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no. NM_004442)
  • PSCA Prostate stem cell antigen precursor, Genbank accession no. AJ297436
  • Reiter R.E. et al Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740, 1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun.
  • AAP14954 lipoma HMGIC fusion-partner-like protein /pid AAP14954.1 - Homo sapiens
  • WO2003054152 (Claim 20); WO2003000842 (Claim 1); WO2003023013 (Example 3, Claim 20);
  • BAFF-R B cell -activating factor receptor, BLyS receptor 3, BR3, Genbank accession No. NP_443177.1
  • CD22 B-cell receptor CD22-B isoform, Genbank accession No. NP-OO 1762.1); StamenkovicJ. and Seed,B., Nature 345 (6270), 74-77 (1990); US2003157113; US2003118592; WO2003062401 (Claim 9); WO2003072036 (Claim 1; Fig 1); WO200278524 (Example 2); Cross-references: MM:107266; NP_001762.1; NM_001771_l
  • CD79a (CD79A, CD79cc, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation) 226 aa, pi: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19ql3.2, Genbank accession No. NP_001774.10)
  • CXCR5 Breast Cancer's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia 372 aa, pi: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 1 Iq23.3, Genbank accession No. NP_001707.1)
  • HLA-DOB Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+ T lymphocytes 273 aa, pi: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No. NP_002111.1)
  • P2X5 Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability) 422 aa, pi: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17pl3.3, Genbank accession No. NP_002552.2)
  • CD72 B-cell differentiation antigen CD72, Lyb-2) 359 aa, pi: 8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9pl3.3, Genbank accession No. NP_001773.1)
  • WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655 (pages 105-106); Von Hoegen et al (1990) J. Immunol. 144(12):4870-4877; Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99:16899-16903;
  • LY64 Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis) 661 aa, pi: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5ql2, Genbank accession No. NP_005573.1)
  • IRTA2 Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies 977 aa, pi: 6.88 MW: 106468 TM: 1 [P] Gene Chromosome: Iq21, Genbank accession No. NP_112571.1)
  • WO2003024392 (claim 2, Fig 97); Nakayama et al (2000) Biochem. Biophys. Res. Commun. 277(1): 124-127; WO2003077836; WO200138490 (claim 3, Fig 18B-1-18B-2);
  • TENB2 (TMEFF2, tomoregulin, TPEF, HPPl, TR, putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin); 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession No. AF179274; AY358907, CAF85723, CQ782436
  • WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ ID NOS 2, 4, 8); WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID NO 411); EP1295944 (pages 69-70); WO200230268 (page 329); WO200190304 (SEQ ID NO 2706); US2004249130; US2004022727; WO2004063355; US2004197325; US2003232350; US2004005563; US2003124579; Horie et al (2000) Genomics 67:146-152; Uchida et al (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang et al (2000) Cancer Res. 60:4907-12; Glynne- Jones et al (2001) Int J Cancer. Oct 15;94(2):178-84.
  • WO04/045516 (03 Jun 2004); WO03/000113 (03 Jan 2003); WO02/016429 (28 Feb 2002); WO02/16581 (28 Feb 2002); WO03/024392 (27 Mar 2003); WO04/016225 (26 Feb 2004); WO01/40309 (07 Jun 2001); US 20050238650 Al; all of which are incorporated herein by reference in their entirety.
  • Antibodies of the invention can be produced using any method known in the art to be useful for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques.
  • Recombinant expression of antibodies, or fragment, derivative or analog thereof may be conducted by assembling a nucleic acid encoding the antibody, if the nucleotide sequence of the antibody is known, from chemically synthesized oligonucleotides ⁇ e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242). This method involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a nucleic acid molecule encoding an antibody can be generated from a suitable source. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody is known, a nucleic acid encoding the antibody can be obtained from a suitable source ⁇ e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.
  • a suitable source e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin
  • antibodies specific for a particular antigen can be generated by any method known in the art, for example, by immunizing a patient, such as a rabbit, to generate polyclonal antibodies or, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • a clone encoding at least the Fab portion of the antibody can be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).
  • nucleic acid sequence encoding at least the variable domain of the antibody can be introduced into a vector containing the nucleotide sequence encoding the constant regions of the antibody (see, e.g., WO 86/05807; WO 89/01036; and US 5122464).
  • Vectors containing the complete light or heavy chain that allow for the expression of a complete antibody molecule are available.
  • the nucleic acid encoding the antibody can be used to introduce the nucleotide substitutions or deletion necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydyl group.
  • Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis and in vitro site directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551).
  • chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. ScL 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038-1041).
  • Antibody fragments that recognize specific epitopes can be generated by known techniques.
  • such fragments include, but are not limited to, the F(ab')2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • the vector for the production of the antibody can be produced by recombinant DNA technology using techniques well known in the art. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the antibody coding sequences and appropriate transcriptional and translational control signals.
  • a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyrog
  • Monoclonal antibodies are 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.
  • the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
  • the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (US 4816567).
  • Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1986). Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al, Curr. Opinion in Immunol, 5:256-262 (1993) and Pl ⁇ ckthun, Immunol. Revs., 130:151-188 (1992).
  • a suitable fusing agent such as polyethylene glycol
  • Monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al (1990) Nature 348:552-554; Clackson et al (1991) Nature, 352:624-628; and Marks et al (1991) J. MoI. Biol. 222:581-597. Subsequent publications describe the production of high affinity (nm range) human antibodies by chain shuffling (Marks et al (1992) Bio/Technology, 10:779-783) as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al (1993) Nuc. Acids. Res., 21:2265-2266). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences (US 4816567; and Morrison, et al (1984) Proc. Natl Acad. Sci. USA 81:6851), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • Humanization can be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody (Jones et al (1986) Nature 321:522-525; Riechmann et al (1988) Nature 332:323-327; Verhoeyen et al (1988) Science 239:1534-1536). Accordingly, such "humanized" antibodies are chimeric antibodies (US 4816567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies (Sims et al (1993) J.
  • the humanized antibody may be an antibody fragment, such as a Fab.
  • the humanized antibody may be an intact antibody, such as an intact IgGl antibody.
  • the murine monoclonal antibody 4D5 which specifically binds the extracellular domain of ErbB2 is produced, as described in Fendly et al (1990) Cancer Research 50:1550-1558, from NIH 3T3/HER2-3 400 cells (expressing approximately 1 x 10 5 ErbB2 molecules/cell), as described in Hudziak et al (1987) Proc. Natl. Acad. Sci.
  • human antibodies can be generated (Jakobovits et al (1993) Proc. Natl. Acad. Sci. USA, 90:2551; Jakobovits et al (1993) Nature, 362:255-258; Bruggermann et al (1993) Year in Immuno. 7:33; and US 5591669, US 5589369, US 5545807).
  • phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors (Johnson, Kevin S. and Chiswell, David J., (1993) Current Opinion in Structural Biology 3:564-571; Clackson et al (1991) Nature, 352:624-628).
  • Human antibodies may also be generated by in vitro activated B cells (see US 5567610 and US 5229275). Human anti-ErbB2 antibodies are described in US 5772997 and WO 97/00271.
  • Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants are prepared by introducing appropriate nucleotide changes into the antibody expressing nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide.
  • Other insertional variants include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • Peptide sequences which specifically bind to albumin may be fused or conjugated to the antibody which comprises the antibody drug conjugates (ADC).
  • ADC antibody drug conjugates
  • Plasma-protein binding can be an effective means of improving the pharmacokinetic properties of short lived molecules, such as antibodies or ADC.
  • Serum albumin binding peptides (ABP) can alter the pharmacodynamics of fused active domain proteins, including alteration of tissue uptake, clearance, penetration, and diffusion, and increase serum half life. These pharmacodynamic parameters can be modulated by specific selection of the appropriate serum albumin binding peptide sequence (US 20040001827 at [0076]).
  • a series of albumin binding peptides were identified by phage display screening (Dennis et al. (2002) J Biol Chem. 277:35035-35043 at Tables III and IV, page 35038; WO 01/45746); and WO 01/45746 at pages 12-13, all of which are incorporated herein by reference.
  • variants are amino acid substitution variants.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated.
  • Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
  • ADCC antigen-dependent cell-mediated cyotoxicity
  • CDC complement dependent cytotoxicity
  • This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody.
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC). See Caron et al J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research 53:2560-2565 (1993).
  • an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).
  • a salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule (e.g., IgGi, IgG 2 , IgG 3 , or IgG 4 ) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • Some antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, (1997) Chem. Immunol. 65:111-128; Wright and Morrison, (1997) TibTECH 15:26-32).
  • the oligosaccharide side chains of the immunoglobulins affect protein function (Boyd et al., (1996) MoI. Immunol. 32:1311-1318; Wittwe and Howard, (1990) Biochem. 29:4175-4180), and the intramolecular interaction between portions of the glycoprotein which can affect the conformation and presented three-dimensional surface of the glycoprotein (Hefferis and Lund, supra; Wyss and Wagner, (1996) Current Opin. Biotech.
  • the cytotoxic or cytostatic activity of an antibody drug conjugate is measured by: exposing mammalian cells having receptor proteins, e.g. EER2, to the antibody of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 5 days; and measuring cell viability.
  • EER2 antibody drug conjugate
  • Cell- based in vitro assays were used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the ADC of the invention.
  • the in vitro potency of antibody drug conjugates was measured by a cell proliferation assay ( Figures 1- 5).
  • the CellTiter-Glo ® Luminescent Cell Viability Assay is a commercially available (Promega Corp., Madison, WI), homogeneous assay method based on the recombinant expression of Coleoptera luciferase (US 5583024; US 5674713 and US 5700670).
  • This cell proliferation assay determines the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells (Crouch et al (1993) J. Immunol. Meth. 160:81-88, US 6602677).
  • the CellTiter-Glo ® Assay was conducted in 96 well format, making it amenable to automated high-throughput screening (HTS) (Cree et al (1995) Anticancer Drugs 6:398- 404).
  • the homogeneous assay procedure involves adding the single reagent (CellTiter-Glo ® Reagent) directly to cells cultured in serum-supplemented medium. Cell washing, removal of medium and multiple pipetting steps are not required.
  • the system detects as few as 15 cells/well in a 384-well format in 10 minutes after adding reagent and mixing.
  • the cells may be treated continuously with ADC, or they may be treated and separated from ADC. Generally, cells treated briefly, i.e. 3 hours, showed the same potency effects as continuously treated cells.
  • the homogeneous "add-mix-measure” format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present.
  • the amount of ATP is directly proportional to the number of cells present in culture.
  • the CellTiter-Glo ® Assay generates a "glow-type" luminescent signal, produced by the luciferase reaction, which has a half-life generally greater than five hours, depending on cell type and medium used. Viable cells are reflected in relative luminescence units (RLU).
  • the substrate, Beetle Luciferin is oxidatively decarboxylated by recombinant firefly luciferase with concomitant conversion of ATP to AMP and generation of photons.
  • This cell proliferation assay can be used with various multiwell formats, e.g. 96 or 384 well format. Data can be recorded by luminometer or CCD camera imaging device. The luminescence output is presented as relative light units (RLU), measured over time.
  • RLU relative light units
  • Figure 1 shows an in vitro, cell proliferation assay with SK-BR-3 cells treated with: -o- trastuzumab and -•- trastuzumab-MC-vc-P AB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 202, measured in Relative Fluorescence Units (RLU) versus ⁇ g/ml concentration of antibody or ADC.
  • RLU Relative Fluorescence Units
  • Figure 2 shows an in vitro, cell proliferation assay with SK-BR-3 cells treated with: -•- Trastuzumab and — ⁇ — trastuzumab-MC-ala-phe-P AB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N- imidazolyl)-1,8 naphthalimide) 203, measured in Relative Fluorescence Units (RLU) versus ⁇ g/ml concentration of antibody or ADC.
  • RLU Relative Fluorescence Units
  • H trastuzumab, where H is linked via a cysteine [cys].
  • Figure 3 shows an in vitro, cell proliferation assay with BT-474 cells treated with: — •- trastuzumab, and -o- trastuzumab-(succinate-gly-ala-phe)-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 204, measured in Relative Fluorescence Units (RLU) versus ⁇ g/ml concentration of antibody or ADC.
  • RLU Relative Fluorescence Units
  • Figure 4 shows an in vitro, cell proliferation assay with BT-474 cells treated with: -•- trastuzumab, and -A- trastuzumab-(MC-val-cit-PAB-(N, N'-( N, N'-(bis-aminoethyl-1,3-propanediamine)-3-nitro, 4- amino-1,8 naphthalimide) 205, measured in Relative Fluorescence Units (RLU) versus ⁇ g/ml concentration of antibody or ADC.
  • RLU Relative Fluorescence Units
  • Figure 5 shows an in vitro, cell proliferation assay with SK-BR-3 cells treated with: — •— trastuzumab, - ⁇ - trastuzumab-MC-(N, N' -(bis-aminoethyl- 1 ,3-propanediamine)-bis-(4-N-imidazolyl)- 1,8 naphthalimide) 206, and - ⁇ - trastuzumab-N'-cyclopropylmethyl, N 2 -maleimidopropyl-gly-val-cit-PAB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 207, measured in Relative Fluorescence Units (RLU) versus ⁇ g/ml concentration of antibody or ADC.
  • H trastuzumab, where H is linked via a cysteine [cys]. Table 2
  • H trastuzumab linked via a cysteine [cys] except where noted.
  • Antibody drug conjugates of Formula I were prepared where Ab included anti-EphB2R and anti-CD22 antibodies. These conjugates also showed in vitro cytotoxic or cytostatic activity.
  • Serum clearance and stability of ADC may be investigated in nude, naive (without tumors received by exogenous grafts) mice.
  • a difference in the amount of total antibody and ADC indicates cleavage of the linker and separation of the antibody from its drug moiety.
  • Efficacy of the antibody-drug conjugates of the invention was measured in vivo by implanting allografts or xenografts of cancer cells in rodents and treating the tumors with ADC. Variable results are to be expected depending on the cell line, the specificity of antibody binding of the ADC to receptors present on the cancer cells, dosing regimen, and other factors.
  • the in vivo efficacy of anti-HER2 ADC was measured by a high expressing HER2 transgenic explant mouse model. An allograft may be propagated from the Fo5 MMTV transgenic mouse which does not respond to, or responds poorly to, HERCEPTIN therapy.
  • Subjects were treated once with ADC and monitored over 3-6 weeks to measure the time to tumor doubling, log cell kill, and tumor shrinkage.
  • the ADC of the invention showed only modest efficacy in slowing the progression of tumor growth.
  • an IV administration of 10 mg H-MC-af-PAB-(bis 4-imidazolyl E) 203 per kg animal showed only a slight increase in the time for mean MMTV-HER2 Fo5 tumor volume doubling in athymic nude mice relative to control (injection vehicle, PBS buffer).
  • Antibody-drug conjugates and an ADC-minus control, "Vehicle” may be evaluated in an acute toxicity rat model (Brown et al (2002) Cancer Chemother. Pharmacol. 50:333-340). Toxicity of ADC may be investigated by treatment of rats with the ADC and subsequent inspection and analysis of the effects on various organs. Based on gross observations (body weights), clinical pathology parameters (serum chemistry and hematology) and histopathology, the toxicity of ADC may be observed, characterized, and measured. Clinical chemistry, serum enzymes and hematology analysis may also be conducted periodically; concluding with complete necropsy with histopathological assessment.
  • Toxicity signals included the clinical observation of weight loss, considering that weight loss, or weight change relative to animals dosed only with Vehicle in animals after dosing with ADC, is a gross and general indicator of systemic or localized toxicity.
  • Hepatotoxicity may be measured by: (i) elevated liver enzymes such as AST (aspartate aminotransferase), ALT (alanine aminotransferase), GGT (g-glutamyl transferase); (ii) increased numbers of mitotic and apoptotic figures; and (iii) hepatocyte necrosis.
  • Hematolymphoid toxicity is observed by depletion of leukocytes, primarily granuloctyes (neutrophils), and/or platelets, and lymphoid organ involvement, i.e. atrophy or apoptotic activity. Toxicity is also noted by gastrointestinal tract lesions such as increased numbers of mitotic and apoptotic figures and degenerative entercolitis.
  • ADC Antibody Drug Conjugates
  • ADC can be made using the synthetic procedures outlined below.
  • ADC can be conveniently prepared using a Linker having a reactive site for binding to the Drug and Antibody.
  • a Linker has a reactive site which has an electrophilic group that is reactive to a nucleophilic group present on an antibody.
  • Useful nucleophilic groups on an antibody include but are not limited to, sulfhydryl, hydroxyl and amino groups.
  • the heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group on a Linker and forms a covalent bond to a Linker unit.
  • Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. The electrophilic group provides a convenient site for Antibody attachment.
  • a Linker has a reactive site which has a nucleophilic group that is reactive to an electrophilic group present on an antibody.
  • Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups.
  • the heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit.
  • Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • the electrophilic group on an antibody provides a convenient site for attachment to a Linker.
  • Carboxylic acid functional groups and chloroformate functional groups are also useful reactive sites for a Linker because they can react with secondary amino groups of a Drug to form an amide linkage.
  • a reactive site is a carbonate functional group on a Linker, such as but not limited to p-nitrophenyl carbonate, which can react with an amino group of a Drug, such as but not limited to N-methyl valine, to form a carbamate linkage.
  • peptide-based Drugs can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method ⁇ see E. Schroder and K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press) that is well known in the field of peptide chemistry.
  • a Linker has an electrophilic group that is reactive with a nucleophilic group present on an antibody.
  • Useful nucleophilic groups on a Antibody include but are not limited to, sulfhydryl, hydroxyl and amino groups.
  • the heteroatom of the nucleophilic group of an antibody is reactive to an electrophilic group on a Linker and forms a covalent bond to a Linker unit.
  • Useful electrophilic groups include, but are not limited to, maleimide, carbonate, and haloacetamide groups. The electrophilic group provides a convenient site for Antibody attachment.
  • a Linker has a reactive functional group which has a nucleophilic group that is reactive to an electrophilic group present on an antibody.
  • Useful electrophilic groups on an Antibody include, but are not limited to, aldehyde and ketone carbonyl groups.
  • the heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit.
  • Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • the electrophilic group on an antibody provides a convenient site for attachment to a Linker.
  • peptide-type Linkers can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments.
  • Such peptide bonds can be prepared, for example, according to the liquid phase synthesis method ⁇ see E. Schroder and K. L ⁇ bke, "The Peptides", volume 1, pp 76-136, (1965), Academic Press) that is well known in the field of peptide chemistry.
  • Linker intermediates may be assembled with any combination or sequence of reactions including Spacer, Stretcher, and Amino Acid units.
  • the Spacer, Stretcher, and Amino Acid units may employ reactive functional groups which are electrophilic, nucleophilic, free radical in nature.
  • Reactive functional groups include, but are not limited to:
  • X is a leaving group, e.g. O-mesyl, O-tosyl, -Cl, -Br, -I, an alkyldisulfide or aryldisulfide (RSS- ), or a maleimide group.
  • the Linker may be substituted with groups which modulated solubility or reactivity.
  • a sulfonate substituent may increase water solubility of the reagent and facilitate the coupling reaction of the linker reagent with the antibody or the drug moiety, or facilitate the coupling reaction of Ab-L with D, or D-L with Ab, depending on the synthetic route employed to prepare the ADC.
  • the compounds of the invention expressly contemplate, but are not limited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo- SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available from Pierce Biotechnology, Inc., Rockford, II.
  • ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH,
  • Useful Stretchers may be incorporated into a Linker using the commercially available intermediates from Molecular Biosciences (Boulder, CO) described below by utilizing known techniques of organic synthesis.
  • Stretchers of formula (HTa) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:
  • n is an integer ranging from 1-10 and T is -H or -SO 3 Na;
  • n is an integer ranging from 0-3;
  • Stretcher units of can be introduced into a Linker by reacting the following bifunctional reagents with the N-terminus of an Amino Acid unit: where X is Br or I.
  • Stretcher units of Formula IlIa and IIIb can also be introduced into a Linker by reacting the following bifunctional reagents with the N-terminus of an Amino Acid unit:
  • Stretcher units of formula (Va) can be introduced into a Linker by reacting the following intermediates with the N-terminus of an Amino Acid unit:
  • Stretchers may be synthesized according to known procedures.
  • Aminooxy Stretchers (H 2 N-O-R 17 -C(O)- ) can be prepared by treating alkyl halides with N-Boc-hydroxylamine according to procedures described in Jones, D.S. et al., Tetrahedron Letters, 2000, 41(10), 1531-1533; and Gilon, C.
  • -R 17 - is selected from -C 1 -C 10 alkylene-, -C 3 -C 8 carbocyclo-, - 0-(C 1 -C 8 alkyl)-, -arylene-, -C1-C 10 alkylene-arylene-, -arylene-Ci-Cio alkylene-, -C r Ci 0 alkylene-(C 3 -C 8 carbocyclo)-, -(C 3 -C 8 carbocyclo) -C1-C 10 alkylene-, -C 3 -C 8 heterocyclo-, -C 1 -Ci 0 alkylene-(C 3 -C 8 heterocyclo)-, -(C 3 -C 8 heterocyclo)-C r C 10 alkylene-, -(CH 2 CH 2 O),-, -(CH 2 CH 2 O) 1 -CH
  • Figure 6 shows a method for preparing a valine-citrulline (val-cit or vc) dipeptide Linker having a maleimide Stretcher and optionally a p-aminobenzyloxycarbonyl (PAB) self-imr ⁇ olative Spacer
  • Q is -C 1 - C 8 alkyl, -O-(C 1 -C 8 alkyl), -halogen, -nitro or -cyano
  • m is an integer ranging from 0-4.
  • Figure 7 illustrates the synthesis of a phe-lys(Mtr) dipeptide Linker unit having a maleimide Stretcher unit and a p-aminobenzyloxycarbonyl self-immolative Spacer unit, where Q is -C 1 -C 8 alkyl, -0-(C 1 -C 8 alkyl), - halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
  • Starting material, lys(Mtr) is commercially available (Bachem, Torrance, CA) or can be prepared according to Dubowchik, et al. (1997) Tetrahedron Letters, 38:5257-60.
  • Figure 8 shows a Linker reacted with an amino group of a Drug moiety to form an ADC that contains an amide or carbamate group, linking the Drug unit to the Linker unit.
  • a linker intermediate has a carboxylic acid group, as in Linker AJ
  • the coupling reaction can be performed using HATU or PyBrop and an appropriate amine base, resulting in a Drug-Linker Compound AK, containing an amide bond between the Drug unit and the Linker unit.
  • the Linker can be coupled to the Drug using HOBt in a mixture of DMF/pyridine to provide a Drug-Linker Compound AM, containing a carbamate bond between the Drug unit and the Linker unit
  • the reactive functional group is a good leaving group, such as halide in Linker AN
  • the Linker can be coupled with an amine group of a Drug via a nucleophilic substitution process to provide a Drug-Linker Compound having an amine linkage (AO) between the Drug unit and the Linker unit.
  • Illustrative methods useful for linking a Drug to an antibody to form a Drug- Linker Compound are depicted in Figure 8 and are outlined in General Procedures G-H.
  • Drug-Linker Compounds containing either a bromoacetamide Stretcher AR or a PEG maleimide Stretcher AS is illustrated in Figure 9 where Q is -C 1 -C 8 alkyl, -0-(C 1 -C 8 alkyl), - halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
  • Figure 10 shows the preparation of a Linker unit containing a branched spacer is shown in, which illustrates the synthesis of a val-cit dipeptide linker having a maleimide Stretcher unit and a bis(4- hydroxymethyl)styrene (BHMS) unit.
  • BHMS bis(4- hydroxymethyl)styrene
  • One exemplary method of preparing an antibody for conjugation with a bis 1,8 naphthalimide drug moiety of the invention entails treating the antibody with a reducing agent, such as dithiothreitol (DTT) to reduce some or all of the cysteine disulfide residues to form highly nucleophilic cysteine thiol groups (- CH 2 SH).
  • DTT dithiothreitol
  • the partially reduced antibody thus reacts with bis 1,8 naphthalimide drug-linker compounds, or linker reagents with electrophilic functional groups such as maleimide or ⁇ -halo carbonyl, according to the conjugation method at page 766 of Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773.
  • an antibody e.g. trastuzumab
  • 50OmM sodium borate and 500 mM sodium chloride at pH 8.0 is treated with an excess of 10OmM dithiothreitol (DTT).
  • DTT dithiothreitol
  • the buffer is exchanged by elution over Sephadex G25 resin and eluted with PBS with 1mM DTPA.
  • the thiol/ Ab value is checked by determining the reduced antibody concentration from the absorbance at 280 nm of the solution and the thiol concentration by reaction with DTNB (Aldrich, Milwaukee, WI) and determination of the absorbance at 412 nm.
  • the reduced antibody dissolved in PBS is chilled on ice.
  • the drug linker e.g. MC-val-cit-PAB-bis 1,8 naphthalimide in DMSO, dissolved in acetonitrile and water at known concentration, is added to the chilled reduced antibody in PBS. After about one hour, an excess of maleimide is added to quench the reaction and cap any unreacted antibody thiol groups.
  • the reaction mixture is concentrated by centrifugal ultrafiltration and the ADC, e.g. trastuzumab-MC-vc-PAB-bis 1,8 naphthalimide, is purified and desalted by elution through G25 resin in PBS, filtered through 0.2 ⁇ m filters under sterile conditions, and frozen for storage.
  • the antibody drug conjugates (ADC) of the invention may be administered by any route appropriate to the condition to be treated.
  • the ADC will typically be administered parenterally, i.e. infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.
  • compositions of therapeutic antibody drug conjugates (ADC) of the invention are typically prepared for parenteral administration, i.e. bolus, intravenous, intratumor injection with a pharmaceutically acceptable parenteral vehicle and in a unit dosage injectable form.
  • An antibody-drug conjugate (ADC) having the desired degree of purity is optionally mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.), in the form of a lyophilized formulation or an aqueous solution.
  • Acceptable diluents, carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparag
  • Zn-protein complexes Zn-protein complexes
  • non- ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
  • TWEENTM TWEENTM
  • PLURONICSTM polyethylene glycol
  • PEG polyethylene glycol
  • the active pharmaceutical ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the ADC, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (US 3773919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and pory-D-(-)-3-hydroxybutyric acid.
  • polyesters for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)
  • polylactides US 3773919
  • copolymers of L-glutamic acid and gamma-ethyl-L-glutamate non-degradable ethylene- vinyl acetate
  • the formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.
  • the formulations include those suitable for the foregoing administration routes.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients include a suspending agent, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono
  • the aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.
  • compositions of ADC may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
  • a sterile injectable preparation such as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile fixed oils may conventionally be employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic
  • an aqueous solution intended for intravenous infusion may contain from about 3 to 500 ⁇ g of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • formulations of ADC suitable for oral administration may be prepared as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the ADC.
  • the formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injection immediately prior to use.
  • sterile liquid carrier for example water
  • Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described.
  • Exemplary unit dosage formulations include a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.
  • the invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefore.
  • Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by any other desired route.
  • the antibody drug conjugates (ADC) of the present invention may be used to treat various diseases or disorders, e.g. characterized by the overexpression of a tumor antigen.
  • exemplary conditions or disorders include benign or malignant tumors; leukemia and lymphoid malignancies; other disorders such as neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic disorders.
  • the ADC compounds which are identified in the animal models and cell-based assays can be further tested in tumor-bearing higher primates and human clinical trials.
  • Human clinical trials can be designed similar to the clinical trials testing the efficacy of the anti-HER2 monoclonal antibody HERCEPTIN in patients with HER2 overexpressing metastatic breast cancers that had received extensive prior anti-cancer therapy as reported by Baselga et al. (1996) J. Clin. Oncol. 14:737-744.,The clinical trial may be designed to evaluate the efficacy of an ADC in combinations with known therapeutic regimens, such as radiation and/or chemotherapy involving known chemotherapeutic and/or cytotoxic agents.
  • known therapeutic regimens such as radiation and/or chemotherapy involving known chemotherapeutic and/or cytotoxic agents.
  • cancer the disease or disorder to be treated is cancer.
  • cancer to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,
  • the cancer will generally comprise HER2-expressing cells, such that the ADC of the present invention are able to bind to the cancer cells.
  • various diagnostic/prognostic assays are available.
  • ErbB2 overexpression may be analyzed by IHC, e.g. using the HERCEPTEST (Dako).
  • Parrafin embedded tissue sections from a tumor biopsy may be subjected to the IHC assay and accorded a ErbB2 protein staining intensity criteria as follows: Score 0, no staining is observed or membrane staining is observed in less than 10% of tumor cells; Score 1+, a faint/barely perceptible membrane staining is detected in more than 10% of the tumor cells, the cells are only stained in part of their membrane; Score 2+, a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells; Score 3+, a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells.
  • Those tumors with 0 or 1+ scores for ErbB2 overexpression assessment may be characterized as not overexpressing ErbB2, whereas those tumors with 2+ or 3+ scores may be characterized as overexpressing ErbB2.
  • FISH assays such as the INFORMTM (Ventana Co., Ariz.) or PATHVISIONTM (Vysis, IU.) may be carried out on formalin-fixed, paraffin-embedded tumor tissue to determine the extent (if any) of ErbB2 overexpression in the tumor.
  • the cancer to be treated herein may be one characterized by excessive activation of an ErbB receptor, e.g. HER2. Such excessive activation may be attributable to overexpression or increased production of the ErbB receptor or an ErbB ligand.
  • a diagnostic or prognostic assay will be performed to determine whether the patient's cancer is characterized by excessive activation of an ErbB receptor. For example, ErbB gene amplification and/or overexpression of an ErbB receptor in the cancer may be determined.
  • Assays for determining such amplification/overexpression are available in the art and include the IHC, FISH and shed antigen assays described above.
  • levels of an ErbB ligand, such as TGF-alpha., in or associated with the tumor may be determined according to known procedures. Such assays may detect protein and/or nucleic acid encoding it in the sample to be tested. In one embodiment, ErbB ligand levels in the tumor may be determined using immunohistochemistry (IHC); see, for example, Seller et al. (1995) Clin. Cancer Research 1:545-550. Alternatively, or additionally, one may evaluate levels of ErbB ligand-encoding nucleic acid in the sample to be tested; e.g. via FISH, southern blotting, or PCR techniques.
  • IHC immunohistochemistry
  • ErbB receptor or ErbB ligand overexpression or amplification may be evaluated using an in vivo diagnostic assay, e.g. by administering a molecule (such as an antibody) which binds the molecule to be detected and is tagged with a detectable label (e.g. a radioactive isotope) and externally scanning the patient for localization of the label.
  • a detectable label e.g. a radioactive isotope
  • an ADC for the prevention or treatment of disease, the appropriate dosage of an ADC will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the molecule is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • An exemplary dosage of ADC to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight.
  • An exemplary dosing regimen comprises administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the anti-ErbB2 antibody. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • An antibody drug conjugate (ADC) of the invention may be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anti-cancer properties.
  • the second compound of the pharmaceutical combination formulation or dosing regimen may have complementary activities to the ADC of the combination such that they do not adversely affect each other.
  • the second compound may be a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant.
  • chemotherapeutic agent such as a tubulin- forming inhibitor, a topoisomerase inhibitor, or a DNA binder.
  • the second compound may be an antibody which binds ErbB2 and blocks ligand activation of an ErbB receptor.
  • the second antibody may be monoclonal antibody 2C4 or humanized 2C4.
  • the second antibody may be conjugated with a cytotoxic or chemotherapeutic agent, e.g., a 1,8 bis- naphthalimide moiety.
  • a cytotoxic or chemotherapeutic agent e.g., a 1,8 bis- naphthalimide moiety.
  • An exemplary combination therapy of the invention is a Formula I ADC and bevacizumab (AvastinTM, Genentech, South San Francisco, CA).
  • the combination therapy may be administered as a simultaneous or sequential regimen.
  • the combination may be administered in two or more administrations.
  • the combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • treatment with an ADC of the present invention involves the combined administration of an anticancer agent identified herein, and one or more chemotherapeutic agents or growth inhibitory agents, including coadministration of cocktails of different chemotherapeutic agents, optionally along with treatment with an anti-ErbB2 antibody, such as trastuzumab.
  • Chemotherapeutic agents include taxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md. (1992).
  • the anticancer agent may be combined with an anti-hormonal compound; e.g., an anti-estrogen compound such as tamoxifen; an anti-progesterone such as onapristone (EP 616812); or an anti-androgen such as flutamide, in dosages known for such molecules.
  • an anti-hormonal compound e.g., an anti-estrogen compound such as tamoxifen; an anti-progesterone such as onapristone (EP 616812); or an anti-androgen such as flutamide
  • an anti-hormonal compound such as tamoxifen
  • an anti-progesterone such as onapristone (EP 616812)
  • an anti-androgen such as flutamide
  • Suitable dosages for any of the above coadministered agents are those presently used and may be lowered due to the combined action (synergy) of the newly identified agent and other chemotherapeutic agents or treatments.
  • the combination therapy may provide "synergy” and prove “synergistic", i.e. the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately.
  • a synergistic effect may be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen.
  • a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g. by different injections in separate syringes.
  • an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.
  • the invention includes novel and unobvious compounds produced by a process comprising contacting a compound of this invention with a mammal for a period of time sufficient to yield a metabolic product thereof.
  • Metabolite products typically may be identified by preparing a radiolabeled (e.g. C 14 or H 3 ) ADC, administering it parenterally in a detectable dose (e.g. greater than about 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples.
  • a detectable dose e.g. greater than about 0.5 mg/kg
  • an article of manufacture, or "kit”, containing materials useful for the treatment of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, blister pack, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds an antibody-drug conjugate (ADC) composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an ADC.
  • ADC antibody-drug conjugate
  • the label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.
  • the article of manufacture may comprise (a) a first container with a compound contained therein, wherein the compound comprises an ADC of the present invention in which the antibody of the ADC is a first antibody inhibits growth of cancer cells; and (b) a second container with a compound contained therein, wherein the compound comprises a second compound, composition, or formulation having biological activity.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the first and second compounds can be used to treat cancer, or other disorder.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • Carbonyl diimidazole (CDI, 0.71 gm, 4.40 mmoles) was added to a mixture of 10 ml dichloromethane and BOC-glycine (0.73gm, 4.19 mmoles) at 0 °C under nitrogen. After 2 hr at 0 °C, 1,3 propanediamine (0.18 ml, 2.0 mmoles) was added and the mixture was warmed to room temperature and stirred overnight. The mixture was diluted with dichloromethane and extracted with sat. NaHCO 3 . The aqueous phase was extracted 2X with dichloromethane. The combined organic phases were washed with sat.
  • CDI Carbonyl diimidazole
  • N-hydoxyethyl-3-nitro-1,8 naphthalimide was prepared from 3-nitro-1,8 naphthalimide and ethanolamine in ethanol by microwave heating at 150 °C for 5 minutes, and precipitation from boiling toluene.
  • Methanesulfonyl chloride (1.05 ml, 13 mmole) was added to a solution of N-hydoxyethyl-3-nitro-1,8 naphthalimide (1.70 gm, 5.94 mmole) and 100 ml pyridine.
  • N-methanesulfonyloxyethyl-(3-nitro-1,8 naphthalimide 13 (2.0 gm, 5.49 mmole) was dissolved in 250 ml 2-butanone and treated with sodium iodide (5.15 gm, 33.9 mmole) and stirred overnight at room temperature under nitrogen. The precipitate was filtered and the eluate washed with sat. NaCl, dried over MgSO4, and concentrated under vacuum to give N-iodoethyl-(3-nitro-1,8 naphthalimide 14.
  • N,N-bis(aminoethyl)-1,3-pro ⁇ anediamine (0.91 gm, 5.29 mmole) in 5 ml N- methylmorpholine (NMM) was added to a solution of 4-nitro-1,8-naphthalic anhydride (2.54 gm, 9.92 mmole) in 10 ml NMM.
  • the reaction was stirred at room temperature under nitrogen for 5 minutes, then heated at 38 °C for one hour, then at 120 °C (reflux) for 2 hours.
  • N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-3- nitro-1,8 naphthalimide 16b was prepared from 3-nitro-1,8-naphthalic anhydride (1.00 gm, 3.91 mmole) and N,N-bis(aminoethyl)-1,3-propanediamine (11.7 mmole, 3 equiv) in 3 ml, at 100 °C for 5 minutes.
  • N,N-bis(aminoethyl)-1,3-propanediamine also: 1,4,8,11-tetraazaundecane, 0.87 ml, 5.03 mmole
  • 4-chloro-1,8-naphthalic anhydride (2.35 gm, 10.10 mmole) in 12 ml NMM.
  • the reaction was stirred at room temperature under nitrogen for 5 minutes, then heated at 38 °C for 45 minutes, the heat was increased slowly to 115 °C and held for 1.5 hours.
  • N,N-bis(aminoethyl)-1,3-propanediamine also: 1,4,8,11-tetraazaundecane, 0.081 ml, 0.47 mmole
  • dioxane 5 ml dioxane
  • 3-bromo-1,8-naphthalic anhydride 0.267 gm, 0.93 mmole
  • the reaction was stirred at room temperature under nitrogen for 5 minutes, then heated at 38 °C for 45 minutes, the heat was increased slowly to 115 °C and held for 1.5 hours.
  • N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-4-bromo-1,8 naphthalimide (19 mg, 0.021 mmole) and 1 ml NMM was heated in a sealed tube to 70 °C and held for 3 hours, then increased to 100 °C and held for 2 hours. The mixture was cooled, dissolved in 5 ml acetic acid and 1 ml 0.1% aqueous TFA and purified by prep. HPLC to give N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-4-morpholino-1,8 naphthalimide 20 as the bis-TFA salt. MS m/z 691(M + H) + .
  • N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-4-bromo-1,8 naphthalimide (16 mg, 0.017 mmole) and 1.5 ml 40% aqueous dimethylamine was placed in a sealed tube for one hour at room temperature, then heated to 60 °C and held for 1 hour, then increased to 70 °C and held for 30 minutes. Dimethylformamide (0.75 ml) was added and heating at 70 °C was continued for 1.5 hours, then let stand at room temperature for 48 hours. The mixture was cooled, dissolved in 5 ml acetic acid and 1 ml 0.1% aqueous TFA and purified by prep.
  • N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N- imidazolyl)-1,8 naphthalimide 24b was prepared from N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-4- bromo-1,8 naphthalimide, imidazole, potassium carbonate, in DMF with heating.
  • N 1 -methyl, N, N'-(bis-aminoethyl-1,3- propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide 24c was prepared from N'-methyl, N, N'-(bis- aminoethyl-1,3-propanediamine)-bis-4-bromo-1,8 naphthalimide and imidazole.
  • N, N'-(bis-aminoethyl -1,3-propanediamine)-bis-(4- azido)-1,8 naphthalimide 24d was prepared from N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-4-bromo-1,8 naphthalimide, sodium azide, potassium carbonate, in DMF with heating at 150 °C for 5 minutes.
  • Example 26 Synthesis of N, N'-(N-benzyl-aminoethyl-1,3-propanediamine)-bis-3-nitro-1,8 naphthalimide 26a and N, N'-(bis-N-benzyl-aminoethyl-1,3-propanediamine)-bis-3-nitro-1,8 naphthalimide 26b
  • Example 27 Synthesis of N. N'-(N-allyl-aminoethyl- 1,3, -propanediamine)-bis-3-nitro-1,8 naphthalimide 27a and N, N'-(bis-N-allyl-aminoethyl-1,3-propanediamine)-bis-3-nitro-1,8 naphthalimide 27b
  • N, N'-(N-cyclopropylmethyl, bis-aminoethyl-1,3-propanediamine)-bis-4-N- imidazolyl-1,8 naphthalimide 30b was prepared from (bromomethyl)cyclopropane and N, N'-(bis-aminoethyl- l,3-propanediamine)-bis-4-N-imidazolyl-1,8 naphthalimide 24b.
  • N, N'-(N-cyclopropylmethyl, bis-aminoethyH,3-propanediamine)-bis-3- nitro-1,8 naphthalimide 30c was prepared from (bromomethyl)cyclopropane and N, N'-(bis-aminoethyl-1,3- propanediamine)-bis-3-nitro-1,8 naphthalimide 16b.
  • Example 31 Synthesis of N, N'-(N-(4-acetylbenzamide), bis-aminoethyl-1,3-propanediamineVbis-3-nitro- 1 ,8 naphthalimide 31a and N, N'-(bis-N-(4-acetylbenzamide'), bis-aminoethyl-1,3-propanediamine)-bis-3-nitro-1,8 naphthalimide 31b
  • 3-benzoylpropionic acid (0.037 gm, 0.20 mmole), diisopropylethylamine (0.07 ml, 0.40 mmole), HATU (0.077 gm, 0.20 mmole), and 2 ml DMF were stirred under nitrogen for 10 minutes at room temperature, then it was added to a solution of N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-3-nitro-1,8 naphthalimide (0.162 gm, 0.35 mmole), diisopropylethylamine (0.17 ml, 0.90 mmole) and 6 ml DMF was added.
  • Levulinic acid (0.015 ml, 0.15 mmole), diisopropylethylamine (0.04 ml, 0.25 mmole), HATU (0.054 gm, 0.15 mmole), and 1 ml DMF were stirred under nitrogen for 15 minutes at room temperature, then it was added to a solution of N, N'-(N-F ⁇ BOC-aminoethyl-1,3-propanediamine)-bis-3-nitro-1,8 naphthalimide (62 mg, 0.049 mmole), having the structure:
  • N-(tert-butylmalonamide), bis-aminoethyl-1,3- propanediamine)-bis-4-N-imidazolyl-1,8 naphthalimide 33b was prepared from mono-tert-butyl malonic acid and N, N'-(N-Fi 7 BOC-aminoethyl-1,3-propanediamine)-bis-4-N-imidazolyl-1,8 naphthalimide, followed by hydrolysis of the N-Fi 7 BOC group.
  • N, N'-(4-aza-octanyl)-bis-4-bromo-1,8 naphthalimide 44 (0.029 gm, 0.031 mmole) and 3 ml morpholine was heated at 80 °C for 16 hours. LC/MS analysis showed the reaction was complete. The mixture was cooled, concentrated, dissolved in 2.5 ml acetic acid and 0.5 ml 0.1% aqueous TFA, and purified by prep. HPLC to give N, N'-(4-aza-octanyl)-bis-4-morpholino-1,8 naphthalimide 45. MS ⁇ n/z 676 (M + H) + .
  • N, N'-(2-ethoxy-N-(2,4-dinitrobenzensulfonyl)-ethylethanamine)-bis-3-nitro-1,8 naphthalimide 46 36 mg, 0.043 mmole
  • cesium carbonate 0.045 gm, 0.14 mmole
  • thiophenol 0.045 ml, 0.043 mmole
  • 2 ml DMF was stirred under nitrogen at room temperature for 20 minutes. The mixture was concentrated, and purified by prep. HPLC to give N, N'-(2-ethoxy-N-ethylethanamine)-bis-3-nitro-1,8 naphthalimide 47.
  • N, N'-(Bis-2-acetamido-1,3-propanediamine)- 4-piperazinyl, 4'-(4N-(3-mercaptopropyl)-piperazinyl- 1,8 naphthalimide 51 was prepared following the protocol of Example 50.
  • N, N'-2-acetamido-1,3-ethanediamine-propyl)-bis-4-morpholino-1,8 naphthalimide 38 (9 mg, 0.011 mmole), DIEA (0.0044 ml, 0.05 mmole) and 1.2 ml DMF was stirred at room temperature under nitrogen for 10 minutes, then maleimido-caproyl-valine-citrulline-para-aminobenzyl-4-nitrophenylcarbonate (MC-vc-PAB-OPNP, 18 mg, 0.025 mmole) and DIEA (0.005 ml) were added, and the mixture was stirred at room temperature overnight.
  • MC-vc-PAB-OPNP maleimido-caproyl-valine-citrulline-para-aminobenzyl-4-nitrophenylcarbonate
  • DIEA 0.005 ml
  • N, N'-2-acetamido-1,2-propanediamine-ethyl)-bis-4-morpholino-1,8 naphthalimide 41 (9 mg, 0.011 mmole), DIEA (0.0044 ml, 0.05 mmole) and 1.2 ml DMF was stirred at room temperature under nitrogen for 10 minutes, then maleimido-caproyl-valine-citrulline-para-aminobenzyl-4-nitrophenylcarbonate (MC-vc-PAB-OPNP, 7 mg, 0.011 mmole) and DIEA (0.005 ml) were added, and the mixture was stirred at room temperature overnight.
  • MC-vc-PAB-OPNP maleimido-caproyl-valine-citrulline-para-aminobenzyl-4-nitrophenylcarbonate
  • N, N'-2-acetamido-1,3-ethanediamine-propyl)-bis-4-amino-1,8 naphthalimide 39 (10 mg, 0.013 mmole), DIEA (0.003 ml, 0.03 mmole) and 0.7 ml DMF was stirred at room temperature under nitrogen for 10 minutes, then maleimido-caproyl-valine-citrulline-para-aminobenzyl-4-nitrophenylcarbonate (MC-vc- PAB-OPNP, 10 mg, 0.013 mmole) and DIEA (0.005 ml) were added, and the mixture was stirred at room temperature overnight.
  • MC-vc- PAB-OPNP maleimido-caproyl-valine-citrulline-para-aminobenzyl-4-nitrophenylcarbonate
  • the TFA salt of N, N'-(N-(levulinamide), bis-aminoethyl-1,3-propanediamine)-bis-3-nitro-1,8 naphthalimide 33a (15 mg, 0.018 mmole), N-[6-maleimidocaproic acid]hydrazide (EMCH, Pierce Biotechnology, 20 mg, 0.088 mmole), acetic acid (0.010 ml, 0.002 mmole), and 3 ml DMF were stirred at room temperature for about 48 hours. The mixture was diluted with DMF and purified by prep.
  • MC-val-cit-P AB-OPNP and N, N'-(bis-aminoethyl-1,3- propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide 24b were reacted to give MC-VC-PAB-(N, N'-(bis- aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) IHa.
  • MC-ala-phe-P AB -OPNP and N, N'-(bis-aminoethyl-1,3- propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide 24b were reacted to give MC-ala-phe-P AB -(N, N'- (bis-aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-l ,8 naphthalimide) IHb,
  • N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N- imidazolyl)-1,8 naphthalimide 24b was converted to the maleimidocaproyl amide, MC-(N, N'-(bis-aminoethyl- l,3- ⁇ ropanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 113a.
  • N'-acetyl, N 2 -Adip-vc-PAB- N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N-methylpiperidine)-1,8 naphthalimide
  • N'-acetyl, N 2 -Adip-vc-P AB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N- methylpiperidine)-1,8 naphthalimide) 115 (about 2 mg, 0.001 mmole), N,N'-disuccinimidyl carbonate (DSC, 10 mg, 0.037 mmole), 0.1 ml acetonitrile, and 0.1 ml DMF was stirred at room temperature for about 1.5 hours, then quenched with acetic acid and dilute aqueous TFA, concentrated under vacuum, and purified by prep.
  • N ⁇ methyl, N 2 -MC-af-P AB-(N, N'-(bis- aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 117 was prepared.
  • N'-methyl, N 2 -(MC-vc-PAB-N-methylglycyl)-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 118 was prepared.
  • N 1 -methyl, N 2 -(MC-af-PAB-N-methylglycyl)-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 119 was prepared.
  • N 1 -methyl, N 2 -(MC-vc-PAB-(3-N- methylpropanamide))-(N, N' -(bis-aminoethyl- 1 ,3-propanediamine)-bis-(4-N-imidazolyl)- 1 ,8 naphthalimide) 120 was prepared.
  • N 1 -methyl, N 2 -(MC-af-PAB-(3-N- methylpropanamide))-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 121 was prepared.
  • N, N'-(bis-aminoethyl-1,3-bis N-methyl- propanediamine)-(4-N-imidazolyl, 4'-N-(MC-vc-PAB)-1,8 naphthalimide) 122 was prepared.
  • N, N'-(bis-aminoethyl-1,3-bis N-methyl- propanediamine)-(4-N-imidazolyl, 4'-N-(MC-af-PAB)-1,8 naphthalimide) 122a was prepared.
  • N, N'-(bis-aminoethyl-1,3-bis N-methyl- propanediamine)-(4-N-imidazolyl, 4'-N-(MC-vc)-1,8 naphthalimide) 123 was prepared.
  • N, N'-(bis-aminoethyl-1,3-bis N-methyl- propanediamine)-(4-N-imidazolyl, 4'-N-(MC-vc)-1,8 naphthalimide) 123a was prepared.
  • N 1 -ethyl, N 2 -MC-af-PAB-(N, N'-(bis-aminoethyl- l,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 124 was prepared.
  • N 1 -ethyl, N 2 -MC-af-PAB-(N-methylvaline)-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 125 was prepared.
  • N'-H, N 2 -MC- VC-PAB-(N, N'-(bis-aminoethyl- 1 ,3-propanediamine)-bis 3-nitro-1,8 naphthaliinide) 126 was prepared.
  • N 1 -H, N 2 -MC- Vc-PAB-(N, N'-(bis-aminoethyl- l,3-propanediamine)-3-nitro, 4-amino-1,8 naphthalimide) 126a was prepared.
  • N 1 -H, N 2 -MC-af-PAB-(N, N'-(bis-aminoethyl- l,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 127 was prepared.
  • N 1 -H, N 2 -(tert-butyladipate-gly-gly-gly-P AB)-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 128 was prepared.
  • N 1 -H, N 2 -(MC-val-cit)-(N, N'-(bis-aminoethyl- l,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 129 was prepared.
  • N'-H, N 2 -(MC-vc-gly)-(N, N'-(bis-aminoethyl- l,3-propanediamine)-bis 3-nitro-1, 8 naphthalimide) 130 was prepared.
  • N 1 -H, N 2 -(MC-af)-(N, N'-(bis-aminoethyl-1,3- propanediamine)-bis 3-nitro-1,8 naphthalimide) 131 was prepared.
  • N 1 -H, N 2 -(MC-ala-phe-gly)-(N, N'-(bis- aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 132 was prepared.
  • N'-H, N 2 -(succinic-gly-val-cit)-(N, N'-(bis- aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 133 was prepared.
  • N 1 -H, N 2 -(succinic-gly-val-cit-gly)-(N, N'-(bis- aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 134 was prepared.
  • N 1 - H, N 2 -(succinic-gly-ala-phe)-(N, N'-(bis- aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 135 was prepared.
  • N 1 -H, N 2 -(succinic-gly-ala-phe-gly)-(N, N'-(bis- aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 136 was prepared.
  • N'-ethyl, N 2 -(MC-vc-PAB-N-methylvaline)-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis 2-nitro-1,8 naphthalimide) 137 was prepared.
  • N 1 -ethyl, N 2 -(maleimido-4-oxo-caproyl-vc-PAB- N- methyl valine)-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis 2-nitro-1,8 naphthalimide) 138 was prepared.
  • N'-methyl, N 2 -(N-methylglycyl)-(N, N'-(bis- aminoethyl-1,3-propanediamine)-bis 4-N-imidazolyl-1,8 naphthalimide) 139 was prepared.
  • N'-H, N 2 -(methoxyethoxyethoxyacetamide)-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis 4-N-imidazolyl-1,8 naphthalimide) 140 was prepared.
  • MP-gvc-P AB-OPNP and N, N' -(N- cyclopropylmethyl, bis-aminoethyl-1,3-propanediamine)-bis-3-nitro-1,8 naphthalimide 30c were reacted to give N ⁇ cyclopropylmethyl, N 2 -maleimidopropyl-gly-val-cit-P AB-(N, N'-(bis-aminoethyl-1,3- ⁇ ropanediamine)-bis 3-nitro-1,8 naphthalimide) 143.
  • HERCEPTIN® (huMAb4D5-8, rhuMAb HER2, US5821337) antibody) was dissolved in 50 mL MES buffer (25 mM MES, 50 mM NaCl, pH 5.6) and loaded on a cation exchange column (Sepharose S, 15 cm x 1.7 cm) that had been equilibrated in the same buffer. The column was then washed with the same buffer (5 column volumes). Trastuzumab was eluted by raising the NaCl concentration of the buffer to 200 mM. Fractions containing the antibody were pooled, diluted to 10 mg/mL, and dialyzed into a buffer containing 50 mM potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5.
  • Trastuzumab dissolved in 50OmM sodium borate and 500 mM sodium chloride at pH 8.0 is treated with an excess of 10OmM dithiothreitol (DTT). After incubation at 37 °C for about 30 minutes, the buffer is exchanged by elution over Sephadex G25 resin and eluted with PBS with ImM DTPA. The thiol/ Ab value is checked by determining the reduced antibody concentration from the absorbance at 280 nm of the solution and the thiol concentration by reaction with DTNB (Aldrich, Milwaukee, WI) and determination of the absorbance at 412 nm. The reduced antibody dissolved in PBS is chilled on ice.
  • DTT dithiothreitol
  • trastuzumab- MC-vc-PAB-(N, N'- (bis-aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 202 was prepared by conjugation of trastuzumab and MC-Vc-PAB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N- imidazolyl)-1,8 naphthalimide) 111a.
  • trastuzumab-MC-ala-phe-P AB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 203 was prepared by conjugation of trastuzumab and MC-ala-phe-P AB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N- imidazolyl)-1,8 naphthalimide) 111b.
  • trastuzumab-(succinate-gly-ala-phe)-(N, N' -(bis-aminoethyl- 1 ,3-propanediamine)-bis 3-nitro- 1,8 naphthalimide) 204 was prepared by conjugation of trastuzumab and N ⁇ H, N 2 -(N-hydroxysuccinimide- succinate-gly-ala-phe)-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 135a.
  • Example 107 Preparation of trastuzumab-MC-val-cit-PAB-(N, N'-(bis-aminoethyl-1,3- ⁇ ropanediamine)-3- nitro, 4-amino-1,8 naphthalimide) 205
  • antibody drug conjugate, trastuzumab-MC-val-cit-PAB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-3-nitro, 4-amino-1,8 naphthalimide) 205 was prepared by conjugation of trastuzumab and (N 1 -H, N 2 -MC- vc-PAB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-3-nitro, 4-amino-1,8 naphthalimide) 126a.
  • trastuzumab-MC-(N, N'-(bis- aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 206 was prepared by conjugation of trastuzumab and MC-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis-(4-N-imidazolyl)-1,8 naphthalimide) 113a.
  • antibody drug conjugate trastuzumab-N'-cyclopropylmethyl, N 2 -maleimidopropyl-gly-val-cit-P AB-(N, N'-(bis-aminoethyl-1,3-propanediamine)-bis 3-nitro-1,8 naphthalimide) 207, was prepared by conjugation of trastuzumab and N 1 -cyclopropylmethyl, N 2 - maleimidopropyl-gly-val-cit-PAB -(N, N' -(bis-aminoethyl- 1 ,3-propanediamine)-bis 3-nitro- 1 ,8 naphthalimide) 143.
  • Example 110 In vitro cell proliferation assay
  • Control wells were prepared containing medium and without cells.
  • ADC was added to the experimental wells and incubated for 3-5 days.
  • the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal.

Abstract

L'invention concerne des composés conjugués de médicament anticorps (antibody drug conjugate -ADC) représentés par la formule (I) où au moins des fractions médicement 1,8 bis-naphthalimide (D) ayant les formules (IIa) et (Ilb) sont liées par covalence à un anticorps (Ab) par un lieur (L). L'invention concerne également des compositions pharmaceutiques ayant une quantité efficace d'un ADC de la formule (I) pour le traitement de troubles hyperprolifératifs et autres troubles. L'invention concerne également des procédés permettant de tuer ou d'inhiber la multiplication d'une cellule tumorale ou d'une cellule cancéreuse comportant l'administration à un patient d'une quantité efficace d'un ADC de formule (I).
EP05852595A 2004-12-01 2005-11-29 Conjugues medicament anticorps et procedes correspondants Withdrawn EP1817059A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US63261304P 2004-12-01 2004-12-01
PCT/US2005/043409 WO2006060533A2 (fr) 2004-12-01 2005-11-29 Conjugues medicament anticorps et procedes correspondants

Publications (1)

Publication Number Publication Date
EP1817059A2 true EP1817059A2 (fr) 2007-08-15

Family

ID=36263731

Family Applications (1)

Application Number Title Priority Date Filing Date
EP05852595A Withdrawn EP1817059A2 (fr) 2004-12-01 2005-11-29 Conjugues medicament anticorps et procedes correspondants

Country Status (3)

Country Link
EP (1) EP1817059A2 (fr)
JP (1) JP4993645B2 (fr)
WO (1) WO2006060533A2 (fr)

Families Citing this family (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070134243A1 (en) * 2004-12-01 2007-06-14 Gazzard Lewis J Antibody drug conjugates and methods
US7947839B2 (en) 2004-12-01 2011-05-24 Genentech, Inc. Heterocyclic-substituted bis-1,8 naphthalimide compounds, antibody drug conjugates, and methods of use
FR2898903B1 (fr) 2006-03-24 2012-08-31 Oreal Composition de teinture comprenant un colorant disulfure fluorescent, procede d'eclaircissement des matieres keratiniques a partir de ce colorant
FR2898902B1 (fr) * 2006-03-24 2010-09-10 Oreal Colorants particuliers de type naphtalimide, composition tinctoriale comprenant au moins un tel colorant, procede de mise en oeuvre et utilisations
ATE518916T1 (de) 2006-03-24 2011-08-15 Oreal Färbezusammensetzung mit einem thiol/disulfid- leuchtfarbstoff mit einem heterozyklus und einer internen kationischen ladung sowie verfahren zur aufhellung von keratinmaterial mit diesem farbstoff
EP2024444B1 (fr) 2006-03-24 2011-07-06 L'Oréal Composition de teinture comprenant un colorant fluorescent naphtylimide thiol/disulfure, procede d'eclaircissement des matieres keratiniques a partir de ce colorant
EP2069308B1 (fr) * 2006-09-12 2017-03-29 Government of the United States of America, Represented by the Secretary, Department of Health and Human Services Composés dérivés d'azonafide pour le ciblage de tumeurs et de cancers
JP5469600B2 (ja) 2007-07-16 2014-04-16 ジェネンテック， インコーポレイテッド 抗ＣＤ７９ｂ抗体及びイムノコンジュゲートとその使用方法
TW200918089A (en) 2007-07-16 2009-05-01 Genentech Inc Humanized anti-CD79b antibodies and immunoconjugates and methods of use
FR2921373B1 (fr) 2007-09-21 2009-10-30 Oreal Colorant derive d'indole styryle a linker alkylene, composition tinctoriale comprenant ce colorant, procede d'eclaircissement des matieres keratiniques a partir de ce colorant
FR2921381B1 (fr) 2007-09-21 2009-10-30 Oreal Colorant hemicyanine styryle thiol/disulfure, composition tinctoriale comprenant ce colorant, procede d'eclaircissement des matieres keratiniques a partir de ce colorant
FR2921376B1 (fr) 2007-09-21 2009-10-30 Oreal Compose styryl tetrahydroquinolinium thiol/disulfure, procede d'eclaircissement des matieres keratiniques a partir de ce colorant
FR2921377B1 (fr) 2007-09-21 2009-10-30 Oreal Compose styryl a motif hydroxy(cyclo)alkylamino thiol/disulfure, procede d'eclaircissement des matieres keratiniques a partir de ce colorant
US8119656B2 (en) 2007-12-07 2012-02-21 The Board Of Regents Of The University Of Texas System Inhibitors of the influenza virus non-structural 1 protein
US8420665B2 (en) 2008-01-11 2013-04-16 Northwestern University Anti-cancer compounds
SI2657253T1 (sl) 2008-01-31 2017-10-30 Genentech, Inc. Protitelesa proti CD79b in imunokonjugati in postopki za uporabo
WO2009144205A1 (fr) 2008-05-30 2009-12-03 Basf Se Matières semi-conductrices à base de rylène et leurs procédés de fabrication et d'utilisation
RU2636046C2 (ru) 2009-01-12 2017-11-17 Сайтомкс Терапьютикс, Инк Композиции модифицированных антител, способы их получения и применения
MX2012009215A (es) 2010-02-23 2012-11-23 Genentech Inc Composiciones y metodos para el diagnostico y tratamiento de tumores.
MX345519B (es) 2010-12-20 2017-02-01 Genentech Inc Anticuerpos anti-mesotelina e inmunoconjugados.
AR090549A1 (es) 2012-03-30 2014-11-19 Genentech Inc Anticuerpos anti-lgr5 e inmunoconjugados
RU2014148162A (ru) 2012-05-01 2016-06-20 Дженентек, Инк. Анти-pmel17 антитела и их иммуноконъюгаты
JP6642940B2 (ja) * 2012-05-02 2020-02-12 ルブリゾル アドバンスド マテリアルズ， インコーポレイテッド 芳香族分散剤組成物
US9981046B2 (en) 2012-05-15 2018-05-29 Concortis Biosystems, Corp., a wholly owned Subsidiary of Sorrento Therapeutics, Inc. Drug-conjugates, conjugation methods, and uses thereof
CA2872327A1 (fr) 2012-05-21 2013-11-28 Genentech, Inc. Anticorps et immunoconjugues anti-ly6e et procedes d'utilisation
EP2879711A4 (fr) 2012-08-02 2016-03-16 Genentech Inc Anticorps et immunoconjugués anti-etbr
WO2014022679A2 (fr) 2012-08-02 2014-02-06 Genentech, Inc. Anticorps et immunoconjugués anti-etbr
CN103013497A (zh) * 2012-12-21 2013-04-03 武汉大学 一种巯基荧光探针及其制备方法
GB201309807D0 (en) 2013-05-31 2013-07-17 Pharma Mar Sau Antibody drug conjugates
CN105518027A (zh) 2013-09-17 2016-04-20 豪夫迈·罗氏有限公司 使用抗lgr5抗体的方法
WO2015061209A1 (fr) 2013-10-21 2015-04-30 Genentech, Inc. Anticorps anti-ly6e et procédés d'utilisation
PL3262071T3 (pl) 2014-09-23 2020-08-10 F. Hoffmann-La Roche Ag Sposób stosowania immunokoniugatów anty-CD79b
US10618935B2 (en) 2015-11-03 2020-04-14 Industrial Technology Research Institute Antibody-drug conjugate (ADC) and method for forming the same
TR201618160A2 (tr) * 2016-12-09 2018-06-21 T C Istanbul Medipol Ueniversitesi Parazi̇ti̇k ve neoplasti̇k hastaliklarin tedavi̇si̇nde kullanima uygun özgün i̇laç molekülleri̇ ve bunlarin hazirlanmasinda kullanilacak usuller
JOP20190254A1 (ar) 2017-04-27 2019-10-27 Pharma Mar Sa مركبات مضادة للأورام
CN107625771B (zh) * 2017-09-27 2018-12-14 徐州玖胜医疗器械有限公司 一种miR-17基因抑制剂及用于治疗胃癌的用途
TWI824043B (zh) 2018-10-25 2023-12-01 西班牙商瑪製藥股份有限公司 藥物抗體共軛物
JP2022548530A (ja) 2019-09-05 2022-11-21 ファルマ、マール、ソシエダード、アノニマ 薬物抗体コンジュゲート
AR121894A1 (es) 2020-04-21 2022-07-20 Pharma Mar Sa Conjugados de anticuerpos de fármacos

Family Cites Families (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4970198A (en) 1985-10-17 1990-11-13 American Cyanamid Company Antitumor antibiotics (LL-E33288 complex)
US5079233A (en) 1987-01-30 1992-01-07 American Cyanamid Company N-acyl derivatives of the LL-E33288 antitumor antibiotics, composition and methods for using the same
DE3707651A1 (de) 1987-03-10 1988-09-22 Knoll Ag Bis-naphthalimide, ihre herstellung und verwendung
US4975278A (en) 1988-02-26 1990-12-04 Bristol-Myers Company Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells
US5606040A (en) 1987-10-30 1997-02-25 American Cyanamid Company Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methyl-trithio group
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
ES2206447T3 (es) 1991-06-14 2004-05-16 Genentech, Inc. Anticuerpo humanizado para heregulina.
WO1994004679A1 (fr) 1991-06-14 1994-03-03 Genentech, Inc. Procede pour fabriquer des anticorps humanises
US5416089A (en) 1993-06-24 1995-05-16 The Du Pont Merck Pharmaceutical Company Polycyclic and heterocyclic chromophores for bis-imide tumoricidals
IL110460A (en) 1993-08-18 2001-01-11 Basf Ag Bite-naphthalimides, their preparation and pharmaceutical preparations containing them
US5773001A (en) 1994-06-03 1998-06-30 American Cyanamid Company Conjugates of methyltrithio antitumor agents and intermediates for their synthesis
US5561042A (en) 1994-07-22 1996-10-01 Cancer Therapy And Research Center Diamine platinum naphthalimide complexes as antitumor agents
US5641782A (en) 1995-02-16 1997-06-24 The Dupont Merck Pharmaceutical Company 3-aromatic and 3-heteroaromatic substituted bisnaphthalimides
JPH11510170A (ja) 1995-07-27 1999-09-07 ジェネンテック インコーポレーテッド タンパク質の処方
JP3646191B2 (ja) 1996-03-19 2005-05-11 大塚製薬株式会社 ヒト遺伝子
WO2002016429A2 (fr) 2000-08-24 2002-02-28 Genentech, Inc. Compositions et procedes de diagnostic et traitement de tumeurs
IL149116A0 (en) 1999-10-29 2002-11-10 Genentech Inc Anti-prostate stem cell antigen (psca) antibody compositions and methods of use
EP2418212B1 (fr) 1999-11-29 2016-06-15 The Trustees of Columbia University in the City of New York Isolation de cinq nouveaux gènes codant pour des nouveaux récepteurs Fc de type mélanome intervenant dans la pathogénèse du lymphome malin et du mélanome
ATE337403T1 (de) 1999-12-24 2006-09-15 Genentech Inc Verfahren und verbindungen zur verlängerung der halbwertzeiten bei der ausscheidung von biowirksamen verbindungen
US20040001827A1 (en) 2002-06-28 2004-01-01 Dennis Mark S. Serum albumin binding peptides for tumor targeting
US20040005561A1 (en) 2000-03-01 2004-01-08 Corixa Corporation Compositions and methods for the detection, diagnosis and therapy of hematological malignancies
AU2001274888A1 (en) 2000-05-19 2001-12-03 Human Genome Sciences, Inc. Nucleic acids, proteins, and antibodies
WO2002016581A2 (fr) 2000-08-24 2002-02-28 Genentech, Inc. Compositions et methodes pour le diagnostic et le traitement de tumeurs
MXPA03003151A (es) 2000-10-13 2003-08-19 Eos Biotechnology Inc Metodos de diagnostico de cancer de prostata, composiciones y metodos para seleccionar moduladores de cancer de prostata.
AU2002243495A1 (en) 2001-01-12 2002-07-24 University Of Medicine And Dentistry Of New Jersey Bone morphogenetic protein-2 in the treatment and diagnosis of cancer
AU2002314901A1 (en) 2001-06-04 2002-12-16 Eos Biotechnology, Inc. Methods of diagnosis and treatment of androgen-dependent prostate cancer, prostate cancer undergoing androgen-withdrawal, and androgen-independent prostate cancer
ATE483976T1 (de) 2001-06-05 2010-10-15 Exelixis Inc Gfats als modifikatoren des p53-wegs und verwendungsverfahren
CA2451465A1 (fr) 2001-06-18 2002-12-27 Eos Biotechnology Inc. Procedes de diagnostic du cancer ovarien, compositions et procedes de criblage de modulateurs du cancer ovarien
US7189507B2 (en) 2001-06-18 2007-03-13 Pdl Biopharma, Inc. Methods of diagnosis of ovarian cancer, compositions and methods of screening for modulators of ovarian cancer
EP1992643A3 (fr) 2001-06-20 2008-12-10 Genentech, Inc. Compositions et procédés pour le traitement et le diagnostic d'une tumeur
US20030108963A1 (en) 2001-07-25 2003-06-12 Millennium Pharmaceuticals, Inc. Novel genes, compositions, kit, and methods for identification, assessment, prevention and therapy of prostate cancer
US20030124579A1 (en) 2001-09-05 2003-07-03 Eos Biotechnology, Inc. Methods of diagnosis of ovarian cancer, compositions and methods of screening for modulators of ovarian cancer
WO2003024392A2 (fr) 2001-09-18 2003-03-27 Genentech, Inc. Compositions et procedes pour le diagnostic et le traitement des tumeurs
US20040249144A1 (en) 2001-10-03 2004-12-09 Zairen Sun Regulated breast cancer genes
AU2002365258A1 (en) 2001-10-12 2003-09-02 University Of Vermont And State Agricultural College Binding peptides specific for the extracellular domain of erbb2 and uses therefor
US20050123925A1 (en) 2002-11-15 2005-06-09 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
US20050238650A1 (en) 2002-04-17 2005-10-27 Genentech, Inc. Compositions and methods for the treatment of tumor of hematopoietic origin
KR20100013352A (ko) 2001-10-31 2010-02-09 알콘, 인코퍼레이티드 뼈 형태발생 단백질(ｂｍｐ), ｂｍｐ 수용체 및 ｂｍｐ 결합 단백질 및 녹내장 진단 및 치료에서 그의 용도
WO2003042661A2 (fr) 2001-11-13 2003-05-22 Protein Design Labs, Inc. Methodes de diagnostic du cancer, compositions et methodes de criblage des modulateurs du cancer
US20030232350A1 (en) 2001-11-13 2003-12-18 Eos Biotechnology, Inc. Methods of diagnosis of cancer, compositions and methods of screening for modulators of cancer
US20030134790A1 (en) 2002-01-11 2003-07-17 University Of Medicine And Dentistry Of New Jersey Bone Morphogenetic Protein-2 And Bone Morphogenetic Protein-4 In The Treatment And Diagnosis Of Cancer
US7193069B2 (en) 2002-03-22 2007-03-20 Research Association For Biotechnology Full-length cDNA
EP1490085A2 (fr) 2002-03-25 2004-12-29 Uab Research Foundation Homologues des recepteurs de fc, reactifs associes et leurs utilisations
US20040022727A1 (en) 2002-06-18 2004-02-05 Martin Stanton Aptamer-toxin molecules and methods for using same
US20040249130A1 (en) 2002-06-18 2004-12-09 Martin Stanton Aptamer-toxin molecules and methods for using same
AU2003259913A1 (en) 2002-08-19 2004-03-03 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
EP1391213A1 (fr) 2002-08-21 2004-02-25 Boehringer Ingelheim International GmbH Compositions et méthodes pour le traitement du cancer en utilisant un conjugué d'un anticorps contre le CD44 avec un maytansinoide et des agents chimiothérapeutiques
JP2004113151A (ja) 2002-09-27 2004-04-15 Sankyo Co Ltd 癌遺伝子及びその用途
WO2004058171A2 (fr) 2002-12-20 2004-07-15 Protein Design Labs, Inc. Anticorps anti-gpr64 et utilisations
US20050227301A1 (en) 2003-01-10 2005-10-13 Polgen Cell cycle progression proteins
US20050181375A1 (en) 2003-01-10 2005-08-18 Natasha Aziz Novel methods of diagnosis of metastatic cancer, compositions and methods of screening for modulators of metastatic cancer
EP1594893A2 (fr) 2003-02-14 2005-11-16 Sagres Discovery, Inc. Cibles therapeutiques pour le cancer
US8088387B2 (en) 2003-10-10 2012-01-03 Immunogen Inc. Method of targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker, said conjugates, and methods of making said conjugates
KR101520209B1 (ko) 2003-11-06 2015-05-13 시애틀 지네틱스, 인크. 리간드에 접합될 수 있는 모노메틸발린 화합물

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
None *
See also references of WO2006060533A2 *

Also Published As

Publication number Publication date
WO2006060533A2 (fr) 2006-06-08
WO2006060533A3 (fr) 2007-07-26
JP2008521920A (ja) 2008-06-26
JP4993645B2 (ja) 2012-08-08

Similar Documents

Publication Publication Date Title
US7947839B2 (en) Heterocyclic-substituted bis-1,8 naphthalimide compounds, antibody drug conjugates, and methods of use
AU2006320945B2 (en) Heterocyclic-substituted bis-1,8 naphthalimide compounds, antibody drug conjugates, and methods of use
JP4993645B2 (ja) 抗体薬剤結合体および方法
US10808039B2 (en) Monomethylvaline compounds capable of conjugation to ligands

Legal Events

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

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20070620

AK Designated contracting states

Kind code of ref document: A2

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

AX Request for extension of the european patent

Extension state: AL BA HR MK YU

R17D Deferred search report published (corrected)

Effective date: 20070726

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 35/00 20060101ALI20070802BHEP

Ipc: A61K 47/48 20060101AFI20070802BHEP

RIN1 Information on inventor provided before grant (corrected)

Inventor name: UM, JOANN

Inventor name: JACKSON, DAVID, Y.

Inventor name: GAZZARD, LEWIS, J.

Inventor name: HA, EDWARD, HYUNGSUK

17Q First examination report despatched

Effective date: 20071004

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

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

18D Application deemed to be withdrawn

Effective date: 20170822