EP3049443A1 - Conjugués anticorps anti-trop-2-médicament et leurs utilisations - Google Patents

Conjugués anticorps anti-trop-2-médicament et leurs utilisations

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Publication number
EP3049443A1
EP3049443A1 EP14847616.1A EP14847616A EP3049443A1 EP 3049443 A1 EP3049443 A1 EP 3049443A1 EP 14847616 A EP14847616 A EP 14847616A EP 3049443 A1 EP3049443 A1 EP 3049443A1
Authority
EP
European Patent Office
Prior art keywords
antibody
trop
cancer
seq
drug
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.)
Pending
Application number
EP14847616.1A
Other languages
German (de)
English (en)
Other versions
EP3049443A4 (fr
Inventor
Serengulam V. Govindan
David M. Goldenberg
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.)
Immunomedics Inc
Original Assignee
Immunomedics 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
Priority claimed from US14/040,024 external-priority patent/US8758752B2/en
Priority claimed from US14/258,228 external-priority patent/US9138485B2/en
Application filed by Immunomedics Inc filed Critical Immunomedics Inc
Publication of EP3049443A1 publication Critical patent/EP3049443A1/fr
Publication of EP3049443A4 publication Critical patent/EP3049443A4/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • 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
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    • 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
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
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    • 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
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    • A61K47/6855—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 the tumour determinant being from breast cancer cell
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    • 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
    • A61K47/6857—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 the tumour determinant being from lung cancer cell
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
    • A61K47/6859—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 the tumour determinant being from liver or pancreas cancer cell
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/6863—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 the tumour determinant being from stomach or intestines cancer cell
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C07K—PEPTIDES
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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Definitions

  • This invention relates to antibody-drug conjugates (ADCs) comprising one or more cytotoxic drug moieties conjugated to an antibody or antigen-binding antibody fragment that binds to Trop-2 antigen (also known as EGP- 1, TACSTD2, M1 S1, GP50, or GA733-1).
  • the antibody may be a humanized RS7 antibody and the drug may be SN-38 or pro-2PDox.
  • the embodiments are not limiting and any other known anti- Trop-2 antibody or cytotoxic drug may be utilized.
  • a linker such as CL2A may be used to attach the drug to the antibody or antibody fragment.
  • other linkers and other known methods of conjugating drugs to antibodies may be utilized.
  • the antibody or fragment may be attached to 1-12, 1-6, 1-5 or about six copies of drug moiety or drug- linker moiety per antibody or fragment. More preferably, the drug to antibody ratio may vary between 1.5: 1 to 8: 1.
  • the anti-Trop-2 ADCs are of use for therapy of Trop-2 expressing cancers, such as breast, ovarian, cervical, endometrial, lung, prostate, colorectal, stomach, esophageal, bladder, renal, pancreatic, thyroid, and head-and-neck cancer.
  • the ADC may be of particular use for treatment of cancers that are resistant to one or more standard anti-cancer therapies, such as colorectal cancer, pancreatic ductal cancer, triple-negative breast cancer or small-cell lung cancer.
  • the anti-Trop-2 ADCs may be used alone or as a combination therapy, along with one or more therapeutic modalities selected from the group consisting of surgery, radiation therapy, chemotherapy, immunomodulators, cytokines, chemotherapeutic agents, pro-apoptotic agents, anti-angiogenic agents, cytotoxic agents, drugs, toxins, radionuclides, RNAi, siRNA, a second antibody or antibody fragment, and an
  • the combination of ADC and other therapeutic modalities exhibits a synergistic effect or an additive effect without increased host toxicities, and is more effective to induce cancer cell death than either ADC or other therapeutic modality alone, or the sum of the effects of ADC and other therapeutic modality administered individually.
  • the combination may include one or more therapies directed against Trop-2, such as PROXINIUM® (VB4-845, Viventia), IGN- 101 (Aphton), adecatumumab (MT201 , Micromet), ING- 1 (Xoma) or EMD 273 066 (Lexigen).
  • the combination may also include administering an immunotherapy subsequent to tumor reduction with the ADC, such as subsequent administration of check point inhibiting agents (including antibodies) or T-cell (or NK-cell) redirecting bispecific antibodies.
  • the combination may be directed to different target antigens expressed on the same cancer, such as the combination of anti-Trop- 2 ADC and a radiolabeled anti-MUC5ac antibody, for example 90 Y-hPAM4.
  • the anti-Trop-2 ADC can be administered to human cancer patients at therapeutically effective dosages with only limited toxicity, more preferably ⁇ Grade 3 neutropenia, nausea, diarrhea, alopecia and vomiting and no more serious side effects.
  • the anti-Trop-2 ADC can be administered to human cancer patients with tumors that were previously resistant to one or more standard anti-cancer therapies, with only limited toxicity and without inducing a fatal immune response to the ADC.
  • the anti-Trop-2 ADC can also be effective against tumors that are refractory to topoisomerase- 1 or topoisomerase-2 inhibitors, such as irinotecan, the parent compound of SN-38.
  • administration of the anti-Trop-2 ADC to human cancer patients is capable of inducing partial response or stable disease of such tumors.
  • Trop-2 human trophoblast-cell-surface marker
  • Trop-2 is a cell surface glycoprotein that was originally identified in normal and malignant trophoblast cells (Lipinski et al., 1981, Proc Natl. Acad Sci USA 78:5147-50).
  • Trop-2 is highly expressed in most human carcinomas, particularly in epithelial carcinomas and adenocarcinomas, with reported low to restricted expression in normal tissues (see, e.g., Cubas et al., 2010, Molec Cancer 9:253; Stepan et al., 201 1, J Histochem Cytochem 59:701-10; Varughese et al., 201 1, Am J Obst Gyn 205:567e- e7).
  • Trop-2 is associated with metastasis, increased tumor aggressiveness and decreased patient survival (Cubas et al., 2010; Varughese et al., 201 1). Pathogenic effects of Trop-2 have been reported to be mediated, at least in part, by the ER 1/2 MAPK pathway (Cubas et al., 2010).
  • Trop-2 Overexpression of Trop-2 in many different types of human carcinomas and adenocarcinomas, squamous cell carcinomas, as well as its transmembrane location, render it a potential target for anti-cancer immunotherapy.
  • the present invention concerns treatment of Trop-2 expressing cancers with anti-Trop-2 antibody-drug conjugates (ADCs).
  • ADCs anti-Trop-2 antibody-drug conjugates
  • various anti-Trop-2 antibodies can be conjugated to a variety of drugs, all having selective efficacy against Trop-2-expressing cancers.
  • the anti-Trop-2 ADC may be used alone or as a combination therapy with one or more other therapeutic modalities, such as surgery, radiation therapy, chemotherapy, immunomodulators, cytokines, chemotherapeutic agents, pro-apoptotic agents, anti-angiogenic agents, cytotoxic agents, drugs, toxins, radionuclides, RNAi, siRNA, a second antibody or antibody fragment, or an
  • the anti-Trop-2 ADC may be of use for treatment of cancers for which standard therapies are not effective or to which the cancers have become refractive, such as colorectal cancer, small-cell lung cancer, pancreatic ductal and non-ductal (e.g., neuroendocrine), cancers or triple-negative breast cancer, but including also non-small cell lung cancers and endocrine- and Her2- responsive breast cancers. More preferably, the combination of ADC and other therapeutic modality is more efficacious than either alone, or the sum of the effects of individual treatments, especially without a concomitant increase in toxic side effects.
  • the anti-Trop-2 antibody may be a humanized RS7 antibody (see, e.g., U.S. Patent No. 7,238,785, the Figures and Examples section of which are incorporated herein by reference), comprising the light chain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO: l); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3
  • GGFGS S YWYFDV SEQ ID NO:6
  • anti-Trop-2 antibodies are known and may be used in the subject ADCs.
  • a number of cytotoxic drugs of use for cancer treatment are well-known in the art and any such known drug may be conjugated to the antibody of interest, so long as the conjugation method does not compromise the anti-Trop-2 antibody binding property by more than 65%, preferably not more than 50%, more preferably not more than 33%.
  • the drug conjugated to the antibody is a camptothecin or anthracycline, most preferably SN-38 or a pro-drug form of 2-pyrrolinodoxorubicin (2-PDox) (see, e.g., U.S. Patent Application Serial Nos. 14/175,089 and 14/204,698, the Figures and Examples section of each incorporated herein by reference).
  • the anti-Trop-2 antibody moiety may be a monoclonal antibody, an antigen-binding antibody fragment, a bispecific or other multivalent antibody, or other antibody -based molecule.
  • the antibody can be of various isotypes, preferably human IgGl, IgG2, IgG3 or IgG4, more preferably comprising human IgGl hinge and constant region sequences.
  • the antibody or fragment thereof can be a chimeric, a humanized, or a human antibody, as well as variations thereof, such as half-IgG4 antibodies (referred to as "unibodies"), as described by van der Neut Kolfschoten et al. (Science 2007; 317: 1554- 1557).
  • the antibody or fragment thereof may be designed or selected to comprise human constant region sequences that belong to specific allotypes, which may result in reduced immunogenicity when the ADC is administered to a human subject.
  • Preferred allotypes for administration include a non-Glml allotype (nGlml), such as Glm3, Glm3,l, Glm3,2 or Glm3,l,2. More preferably, the allotype is selected from the group consisting of the nGlml, Glm3, nGlml, 2 and Km3 allotypes.
  • the drug to be conjugated to the anti-Trop-2 antibody or antibody fragment may be selected from the group consisting of an anthracycline, a camptothecin, a tubulin inhibitor, a maytansinoid, a calicheamycin, an auristatin, a nitrogen mustard, an ethylenimine derivative, an alkyl sulfonate, a nitrosourea, a triazene, a folic acid analog, a taxane, a COX-2 inhibitor, a pyrimidine analog, a purine analog, an antibiotic, an enzyme inhibitor, an
  • epipodophyllotoxin a platinum coordination complex, a vinca alkaloid, a substituted urea, a methyl hydrazine derivative, an adrenocortical suppressant, a hormone antagonist, an antimetabolite, an alkylating agent, an antimitotic, an anti- angiogenic agent, a tyrosine kinase inhibitor, an mTOR inhibitor, a heat shock protein (HSP90) inhibitor, a proteosome inhibitor, an HDAC inhibitor, a pro-apoptotic agent, and a combination thereof.
  • HSP90 heat shock protein
  • drug does not include protein or peptide toxins, such as ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, onconase, gelonin, diphtheria toxin, Pseudomonas exotoxin or Pseudomonas endotoxin.
  • protein or peptide toxins such as ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, onconase, gelonin, diphtheria toxin, Pseudomonas exotoxin or Pseudomonas endotoxin.
  • Specific drugs of use may be selected from the group consisting of 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin- 1 , busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatinum, COX-2 inhibitors, irinotecan (CPT-1 1), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubi
  • Preferred optimal dosing of the subject ADCs may include a dosage of between 1 mg/kg and 20 mg/kg, preferably given either weekly, twice weekly, every other week, or every third week.
  • the optimal dosing schedule may include treatment cycles of two consecutive weeks of therapy followed by one, two, three or four weeks of rest, or alternating weeks of therapy and rest, or one week of therapy followed by two, three or four weeks of rest, or three weeks of therapy followed by one, two, three or four weeks of rest, or four weeks of therapy followed by one, two, three or four weeks of rest, or five weeks of therapy followed by one, two, three, four or five weeks of rest, or administration once every two weeks, once every three weeks, or once a month.
  • Treatment may be extended for any number of cycles, preferably at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, or at least 16 cycles. This depends on tolerability of the dose as well as status of the patient's disease; the less toxic the therapy and the better the control of the disease, the longer the therapy can be given in repeated cycles.
  • the dosage may be up to 24 mg/kg.
  • Exemplary dosages of use may include 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 1 1 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22 mg/kg and 24 mg/kg.
  • Preferred dosages are 1 , 2, 4, 6, 8, 9, 10, or 12 mg/kg.
  • the person of ordinary skill will realize that a variety of factors, such as age, general health, specific organ function or weight, as well as effects of prior therapy on specific organ systems (e.g., bone marrow), may be considered in selecting an optimal dosage of ADC, and that the dosage and/or frequency of administration may be increased or decreased during the course of therapy.
  • the dosage may be repeated as needed, with evidence of tumor shrinkage observed after as few as 3 to 8 doses.
  • the optimized dosages and schedules of administration disclosed herein show unexpected superior efficacy and reduced toxicity in human subjects, which could not have been predicted from animal model studies, especially in murine xenograft models where a toxic dose of the ADC is not readily established. Surprisingly, the superior efficacy allows treatment of tumors that were previously found to be resistant to one or more standard anti-cancer therapies.
  • the anti-Trop-2 ADCs are of use for therapy of Trop-2 expressing cancers, such as breast, ovarian, cervical, endometrial, lung, prostate, colorectal, stomach, esophageal, urinary bladder, renal, pancreatic, thyroid, or head-and-neck cancer.
  • the ADC may be of particular use for treatment of cancers that are resistant to one or more standard anti-cancer therapies, such as a metastatic colorectal cancer, triple-negative breast cancer, a HER+, ER+, progesterone+ breast cancer, metastatic non- small- cell lung cancer (NSCLC), metastatic pancreatic cancer, metastatic renal cell carcinoma, metastatic gastric cancer, metastatic prostate cancer, or metastatic small-cell lung cancer.
  • standard anti-cancer therapies such as a metastatic colorectal cancer, triple-negative breast cancer, a HER+, ER+, progesterone+ breast cancer, metastatic non- small- cell lung cancer (NSCLC), metastatic pancreatic cancer, metastatic
  • the anti-Trop-2 ADC can be administered to human cancer patients at therapeutically effective dosages with only limited toxicity, more preferably ⁇ Grade 3 neutropenia, nausea, diarrhea, alopecia and vomiting and no more serious side effects.
  • the anti-Trop-2 ADC can be administered to human cancer patients with tumors that were previously resistant to one or more standard anti-cancer therapies, with only limited toxicity and without inducing a fatal immune response to the ADC.
  • administration of the anti-Trop-2 ADC to human cancer patients is capable of inducing partial response or stable disease of such tumors.
  • FIG. 1 Preclinical in vivo therapy of athymic nude mice, bearing Capan 1 human pancreatic carcinoma, with SN-38 conjugates of hRS7 (anti-Trop-2), hPAM4 (anti-MUC5ac), hMN-14 (anti-CEACAM5) or non-specific control hA20 (anti-CD20).
  • FIG. 2 Preclinical in vivo therapy of athymic nude mice, bearing BxPC3 human pancreatic carcinoma, with anti-TROP2-CL2A-SN-38 conjugates compared to controls.
  • FIG. 3A Structure of doxorubicin. "Me” is a methyl group.
  • FIG. 3B Structure of 2-pyrrolinodoxorubicin,(2-PDox). "Me” is a methyl group.
  • FIG. 3C Structure of a prodrug form of 2-pyrrolinodoxorubicin,(pro-2-PDox).
  • Me is a methyl group and
  • Ac is an acetyl group.
  • FIG. 3D Structure of a maleimide- activated form of pro-2-PDox, for antibody coupling.
  • "Me” is a methyl group and "Ac” is an acetyl group.
  • FIG. 6A In vivo efficacy of pro-2-PDox conjugates in nude mice with NCI-N87 human gastric cancer xenografts. Mice were administered a saline control.
  • FIG. 6B In vivo efficacy of pro-2-PDox conjugates in nude mice with NCI-N87 human gastric cancer xenografts. Mice were administered 45 ⁇ g of hA20-pro-2-PDox as indicated by arrows.
  • FIG. 6C In vivo efficacy of pro-2-PDox conjugates in nude mice with NCI-N87 human gastric cancer xenografts. Mice were administered 45 ⁇ g of hMN-15-pro-2-PDox as indicated by arrows.
  • FIG. 6D In vivo efficacy of pro-2-PDox conjugates in nude mice with NCI-N87 human gastric cancer xenografts. Mice were administered 45 ⁇ g of hRS7-pro-2-PDox as indicated by arrows.
  • FIG. 6E In vivo efficacy of pro-2-PDox conjugates in nude mice with NCI-N87 human gastric cancer xenografts. Mice were administered 45 ⁇ g of hLLl-pro-2-PDox as indicated by arrows.
  • FIG. 6F In vivo efficacy of pro-2-PDox conjugates in nude mice with NCI-N87 human gastric cancer xenografts. Mice were administered 45 ⁇ g of hMN-14-pro-2-PDox as indicated by arrows.
  • FIG. 7 Effect of different dosing schedules of hRS7-pro-2-PDox on survival in nude mice with NCI-N87 human gastric carcinoma xenografts.
  • FIG. 8A Dosing schedule study in mice injected with NCI-N87 human gastric cancer. Mice were administered a saline control.
  • FIG. 8B Dosing schedule study in mice injected with NCI-N87 human gastric cancer. Mice were administered 45 ⁇ g q4dx4 of hRS7-pro-2-PDox.
  • FIG. 8C Dosing schedule study in mice injected with NCI-N87 human gastric cancer. Mice were administered 90 ⁇ g weekly x 2 of hRS7-pro-2-PDox.
  • FIG. 8D Dosing schedule study in mice injected with NCI-N87 human gastric cancer. Mice were administered a single dose of 180 ⁇ g hRS7-pro-2-PDox.
  • FIG. 8E Dosing schedule study in mice injected with NCI-N87 human gastric cancer. Mice were administered 45 ⁇ g q4dx4 of hA20-pro-2-PDox.
  • FIG. 8F Dosing schedule study in mice injected with NCI-N87 human gastric cancer. Mice were administered 90 ⁇ g weekly x 2 of hA20-pro-2-PDox.
  • FIG. 8G Dosing schedule study in mice injected with NCI-N87 human gastric cancer. Mice were administered a single dose of 180 ⁇ g hA20-pro-2-PDox.
  • FIG. 9 Effect of different single doses of hRS7-pro-2-PDox on growth of human gastric carcinoma xenografts.
  • FIG. 10 Effect of different single doses of hRS7-pro-2-PDox on survival of mice bearing human gastric carcinoma xenografts.
  • FIG. 11 ADCC of various hRS7-ADCs vs. hRS7 IgG.
  • FIG. 12A Structures of CL2-SN-38 and CL2A-SN-38.
  • FIG. 14A Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice. Fifty-six Swiss- Webster mice were administered 2 i.p. doses of buffer or the hRS7-CL2A-SN-38 3 days apart (4, 8, or 12 mg/kg of SN-38 per dose; 250, 500, or 750 mg conjugate protein/kg per dose). Seven and 15 days after the last injection, 7 mice from each group were euthanized, with blood counts and serum chemistries performed. Graphs show the percent of animals in each group that had elevated levels of AST.
  • FIG. 14B Tolerability of hRS7-CL2A-SN-38 in Swiss- Webster mice. Fifty-six Swiss- Webster mice were administered 2 i.p. doses of buffer or the hRS7-CL2A-SN-38 3 days apart (4, 8, or 12 mg/kg of SN-38 per dose; 250, 500, or 750 mg conjugate protein/kg per dose). Seven and 15 days after the last injection, 7 mice from each group were euthanized, with blood counts and serum chemistries performed. Graphs show the percent of animals in each group that had elevated levels of ALT.
  • FIG. 14C Tolerability of hRS7-CL2A-SN-38 in Cynomolgus monkeys.
  • Six monkeys per group were injected twice 3 days apart with buffer (control) or hRS7-CL2A-SN-38 at 0.96 mg/kg or 1.92 mg/kg of SN-38 equivalents per dose (60 and 120 mg/kg conjugate protein). All animals were bled on day -1, 3, and 6.
  • Four monkeys were bled on day 11 in the 0.96 mg/kg group, 3 in the 1.92 mg/kg group. Changes in neutrophil counts in Cynomolgus monkeys.
  • FIG. 14D Tolerability of hRS7-CL2A-SN-38 in Cynomolgus monkeys.
  • Six monkeys per group were injected twice 3 days apart with buffer (control) or hRS7-CL2A-SN-38 at 0.96 mg/kg or 1.92 mg/kg of SN-38 equivalents per dose (60 and 120 mg/kg conjugate protein). All animals were bled on day -1, 3, and 6.
  • Four monkeys were bled on day 1 1 in the 0.96 mg/kg group, 3 in the 1.92 mg/kg group. Changes in platelet counts in Cynomolgus monkeys.
  • FIG. 15 In vitro efficacy of anti-Trop-2-paclitaxel ADC against MDA-MB-468 human breast adenocarcinoma.
  • FIG. 16 In vitro efficacy of anti-Trop-2-paclitaxel ADC against BxPC-3 human pancreatic adenocarcinoma.
  • FIG. 17A Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38 versus MAB650-SN-38) in Capan-1 human pancreatic adenocarcinoma.
  • FIG. 17B Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38 versus MAB650-SN-38) in BxPC-3 human pancreatic adenocarcinoma.
  • FIG. 17C Comparison of in vitro efficacy of anti-Trop-2 ADCs (hRS7-SN-38 versus
  • FIG. 19 IMMU- 132 phase I/II data for best response by RECIST criteria.
  • FIG. 20 IMMU- 132 phase I/II data for time to progression and best response
  • FIG. 21 Therapeutic efficacy of murine anti-Trop-2-SN-38 ADC (162-46.2-SN-38) compared to hRS7-SN-38 in mice bearing NCI-N87 human gastric carcinoma xenografts.
  • FIG. 22 Therapeutic efficacy of murine anti-Trop-2-pro-2-PDox ADC (162-46.2- pro-2-PDox) compared to hRS7-pro-2-PDox in mice bearing NCI-N87 human gastric carcinoma xenografts.
  • FIG. 23 Accumulation of SN-38 in tumors of nude mice with Capan- 1 human pancreatic cancer xenografts, when administered as free irinotecan vs. IMMU- 132 ADC.
  • FIG. 24 Individual patient demographics and prior treatment for phase I/II IMMU- 132 anti-Trop-2 ADC in pancreatic cancer patients.
  • FIG. 25 Response assessment to IMMU-132 anti-Trop-2 ADC in pancreatic cancer patients.
  • FIG. 26 Summary of time to progression (TTP) results in human pancreatic cancer patients administered IMMU-132 anti-Trop-2 ADC.
  • An antibody refers to a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes)
  • immunoglobulin molecule e.g., an IgG antibody
  • immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule like an antibody fragment.
  • an antibody fragment is a portion of an antibody such as F(ab')2, Fab', Fab, Fv, sFv and the like. Antibody fragments may also include single domain antibodies and IgG4 half- molecules, as discussed below. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the full-length antibody.
  • the term "antibody fragment” also includes isolated fragments consisting of the variable regions of antibodies, such as the "Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins").
  • a chimeric antibody is a recombinant protein that contains the variable domains including the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, while the constant domains of the antibody molecule are derived from those of a human antibody.
  • the constant domains of the chimeric antibody may be derived from that of other species, such as a cat or dog.
  • a humanized antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains (e.g., framework region sequences).
  • the constant domains of the antibody molecule are derived from those of a human antibody.
  • a limited number of framework region amino acid residues from the parent (rodent) antibody may be substituted into the human antibody framework region sequences.
  • a human antibody is, e.g., an antibody obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous murine heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for particular antigens, and the mice can be used to produce human antibody-secreting hybridomas.
  • Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7: 13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun.
  • a fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art. See for example, McCafferty et al., Nature 348:552-553 (1990) for the production of human antibodies and fragments thereof in vitro, from immunoglobulin variable domain gene repertoires from unimmunized donors. In this technique, antibody variable domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, and displayed as functional antibody fragments on the surface of the phage particle.
  • the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of the B cell.
  • Phage display can be performed in a variety of formats, for review, see e.g. Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).
  • Human antibodies may also be generated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, the Examples section of which is incorporated herein by reference.
  • a therapeutic agent is a compound, molecule or atom which is administered separately, concurrently or sequentially with an antibody moiety or conjugated to an antibody moiety, i.e., antibody or antibody fragment, or a subfragment, and is useful in the treatment of a disease.
  • therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes and radioisotopes. Therapeutic agents of use are described in more detail below.
  • An immunoconjugate is an antibody, antibody fragment or fusion protein conjugated to at least one therapeutic and/or diagnostic agent.
  • a multispecific antibody is an antibody that can bind simultaneously to at least two targets that are of different structure, e.g., two different antigens, two different epitopes on the same antigen, or a hapten and/or an antigen or epitope.
  • Multispecific, multivalent antibodies are constructs that have more than one binding site, and the binding sites are of different specificity.
  • a bispecific antibody is an antibody that can bind simultaneously to two different targets.
  • Bispecific antibodies bsAb
  • bispecific antibody fragments bsFab
  • bsAb bispecific antibodies
  • bsFab bispecific antibody fragments
  • a variety of bispecific fusion proteins can be produced using molecular engineering.
  • the subject ADCs include at least one antibody or fragment thereof that binds to Trop-2.
  • the anti-Trop-2 antibody may be a humanized RS7 antibody (see, e.g., U.S. Patent No. 7,238,785, incorporated herein by reference in its entirety), comprising the light chain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO: l); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3) and the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:4); CDR2
  • the RS7 antibody was a murine IgGi raised against a crude membrane preparation of a human primary squamous cell lung carcinoma. (Stein et al., Cancer Res. 50: 1330, 1990) The RS7 antibody recognizes a 46-48 kDa glycoprotein, characterized as cluster 13. (Stein et al., Int. J. Cancer Supp. 8:98-102, 1994) The antigen was designated as EGP-1 (epithelial glycoprotein- 1), but is also referred to as Trop-2.
  • Trop-2 is a type-I transmembrane protein and has been cloned from both human (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse cells (Sewedy et al., Int J Cancer 1998; 75:324-30).
  • human Trop-2 In addition to its role as a tumor-associated calcium signal transducer (Ripani et al., Int J Cancer 1998;76:671-6), the expression of human Trop-2 was shown to be necessary for tumorigenesis and invasiveness of colon cancer cells, which could be effectively reduced with a polyclonal antibody against the extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008;7:280-5).
  • RS7 MAb detects antigen on a variety of tumor types, with limited binding to normal human tissue (Stein et al., 1990).
  • Trop-2 is expressed primarily by carcinomas such as carcinomas of the lung, stomach, urinary bladder, breast, ovary, uterus, and prostate.
  • Localization and therapy studies using radiolabeled murine RS7 MAb in animal models have demonstrated tumor targeting and therapeutic efficacy (Stein et al., 1990; Stein et al., 1991).
  • the RS7 MAb is rapidly internalized into target cells (Stein et al., 1993).
  • the internalization rate constant for RS7 MAb is intermediate between the internalization rate constants of two other rapidly internalizing MAbs, which have been demonstrated to be useful for immunoconjugate production. ⁇ Id.)
  • Internalization of drug immunoconjugates has been described as a major factor in anti-tumor efficacy. (Yang et al., Proc. Nat'l Acad. Sci. USA 85: 1 189, 1988)
  • the RS7 antibody exhibits several important properties for therapeutic applications.
  • hRS7 antibody is preferred
  • other anti-Trop-2 antibodies are known and/or publicly available and in alternative embodiments may be utilized in the subject ADCs.
  • humanized or human antibodies are preferred for reduced immunogenicity, in alternative embodiments a chimeric antibody may be of use.
  • methods of antibody humanization are well known in the art and may be utilized to convert an available murine or chimeric antibody into a humanized form.
  • Anti-Trop-2 antibodies are commercially available from a number of sources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417 (LifeSpan Biosciences, Inc., Seattle, WA); 10428-MMOl, 10428-MM02, 10428-ROOl, 10428-R030 (Sino Biological Inc., Beijing, China); MR54 (eBioscience, San Diego, CA); sc-376181, sc-376746, Santa Cruz Biotechnology (Santa Cruz, CA); MM0588-49D6, (Novus Biologicals, Littleton, CO);
  • anti-Trop-2 antibodies have been disclosed in the patent literature.
  • U.S. Publ. No. 2013/0089872 discloses anti-Trop-2 antibodies K5-70 (Accession No. FERM BP-1 1251), K5- 107 (Accession No. FERM BP- 1 1252), K5-1 16-2-1 (Accession No. FERM BP-1 1253), T6-16 (Accession No. FERM BP-1 1346), and T5-86 (Accession No. FERM BP- 1 1254), deposited with the International Patent Organism Depositary, Tsukuba, Japan.
  • U.S. Patent No. 7,420,040 disclosed an anti-Trop-2 antibody produced by hybridoma cell line AR47A6.4.2, deposited with the ID AC (International Depository Authority of Canada, Winnipeg, Canada) as accession number 141205-05.
  • U.S. Patent No. 7,420,041 disclosed an anti-Trop-2 antibody produced by hybridoma cell line AR52A301.5, deposited with the ID AC as accession number 141205-03.
  • U.S. Publ. No. 2013/0122020 disclosed anti-Trop-2 antibodies 3E9, 6G1 1, 7E6, 15E2, 18B 1.
  • Hybridomas encoding a representative antibody were deposited with the American Type Culture Collection (ATCC), Accession Nos. PTA-12871 and PTA-12872.
  • U.S. Patent No. 8,715,662 discloses anti-Trop-2 antibodies produced by hybridomas deposited at the AID-ICLC (Genoa, Italy) with deposit numbers PD 08019, PD 08020 and PD 08021.
  • 20120237518 discloses anti-Trop-2 antibodies 77220, KM4097 and KM4590.
  • U.S. Patent No. 8,309,094 discloses antibodies Al and A3, identified by sequence listing. The Examples section of each patent or patent application cited above in this paragraph is incorporated herein by reference.
  • anti-Trop-2 antibodies are known in the art and/or publicly available. As discussed below, methods for preparing antibodies against known antigens were routine in the art. The sequence of the human Trop-2 protein was also known in the art (see, e.g., GenBank Accession No. CAA54801.1). Methods for producing humanized, human or chimeric antibodies were also known. The person of ordinary skill, reading the instant disclosure in light of general knowledge in the art, would have been able to make and use the genus of anti-Trop-2 antibodies in the subject ADCs.
  • the drug to be conjugated to the anti-Trop-2 antibody or antibody fragment may be selected from the group consisting of an anthracycline, a camptothecin, a tubulin inhibitor, a maytansinoid, a calicheamycin, an auristatin, a nitrogen mustard, an ethylenimine derivative, an alkyl sulfonate, a nitrosourea, a triazene, a folic acid analog, a taxane, a COX-2 inhibitor, a pyrimidine analog, a purine analog, an antibiotic, an enzyme inhibitor, an
  • epipodophyllotoxin a platinum coordination complex, a vinca alkaloid, a substituted urea, a methyl hydrazine derivative, an adrenocortical suppressant, a hormone antagonist, an antimetabolite, an alkylating agent, an antimitotic, an anti- angiogenic agent, a tyrosine kinase inhibitor, an mTOR inhibitor, a heat shock protein (HSP90) inhibitor, a proteosome inhibitor, an HDAC inhibitor, a pro-apoptotic agent, and a combination thereof.
  • HSP90 heat shock protein
  • Specific drugs of use may be selected from the group consisting of 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin- 1 , busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatinum, COX-2 inhibitors, irinotecan (CPT-1 1), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubi
  • the murine anti-Trop-1 IgG2a antibody edrecolomab (PANOREX®) has been used for treatment of colorectal cancer, although the murine antibody is not well suited for human clinical use (Baeuerle & Gires, 2007, Br. J Cancer 96:417-423). Low-dose subcutaneous administration of ecrecolomab was reported to induce humoral immune responses against the vaccine antigen (Baeuerle & Gires, 2007).
  • Adecatumumab (MT201), a fully human anti-Trop-1 antibody, has been used in metastatic breast cancer and early-stage prostate cancer and is reported to act through ADCC and CDC activity (Baeuerle & Gires, 2007).
  • MT1 10 a single-chain anti-Trop-1 /anti-CD3 bispecific antibody construct has reported efficacy against ovarian cancer (Baeuerle & Gires, 2007).
  • Proxinium an immunotoxin comprising anti-Trop-1 single-chain antibody fused to
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A or Protein-G Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al., "Purification of Immunoglobulin G (IgG)," in METHODS IN
  • the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art, as discussed below.
  • a chimeric antibody is a recombinant protein in which the variable regions of a human antibody have been replaced by the variable regions of, for example, a mouse antibody, including the complementarity- determining regions (CDRs) of the mouse antibody.
  • Chimeric antibodies exhibit decreased immunogenicity and increased stability when administered to a subject.
  • CDRs complementarity- determining regions
  • a chimeric or murine monoclonal antibody may be humanized by transferring the mouse CDRs from the heavy and light variable chains of the mouse immunoglobulin into the
  • variable domains of a human antibody The mouse framework regions (FR) in the chimeric monoclonal antibody are also replaced with human FR sequences.
  • additional modification might be required in order to restore the original affinity of the murine antibody. This can be accomplished by the replacement of one or more human residues in the FR regions with their murine counterparts to obtain an antibody that possesses good binding affinity to its epitope. See, for example, Tempest et al., Biotechnology 9:266 (1991) and Verhoeyen et al., Science 239: 1534 (1988).
  • Preferred residues for substitution include FR residues that are located within 1 , 2, or 3 Angstroms of a CDR residue side chain, that are located adjacent to a CDR sequence, or that are predicted to interact with a CDR residue.
  • the phage display technique may be used to generate human antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4: 126-40).
  • Human antibodies may be generated from normal humans or from humans that exhibit a particular disease state, such as cancer (Dantas-Barbosa et al., 2005).
  • the advantage to constructing human antibodies from a diseased individual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.
  • Fab fragment antigen binding protein
  • RNAs were converted to cDNAs and used to make Fab cDNA libraries using specific primers against the heavy and light chain immunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).
  • Library construction was performed according to Andris-Widhopf et al. (2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1 st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp. 9.1 to 9.22).
  • Fab fragments were digested with restriction endonucleases and inserted into the bacteriophage genome to make the phage display library.
  • libraries may be screened by standard phage display methods, as known in the art.
  • Phage display can be performed in a variety of formats, for their review, see e.g. Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).
  • Human antibodies may also be generated by in vitro activated B-cells. See U.S. Patent Nos. 5,567,610 and 5,229,275, incorporated herein by reference in their entirety. The skilled artisan will realize that these techniques are exemplary and any known method for making and screening human antibodies or antibody fragments may be utilized.
  • transgenic animals that have been genetically engineered to produce human antibodies may be used to generate antibodies against essentially any immunogenic target, using standard immunization protocols.
  • Methods for obtaining human antibodies from transgenic mice are disclosed by Green et al, Nature Genet. 7: 13 (1994), Lonberg et al, Nature 355:856 (1994), and Taylor et al, Int. Immun. 6:579 (1994).
  • a non- limiting example of such a system is the XenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231 : 1 1-23, incorporated herein by reference) from Abgenix (Fremont, CA).
  • the mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the remainder of the mouse immune system remains intact.
  • the XenoMouse® was transformed with germline- configured YACs (yeast artificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences, along with accessory genes and regulatory sequences.
  • the human variable region repertoire may be used to generate antibody producing B-cells, which may be processed into hybridomas by known techniques.
  • a XenoMouse® immunized with a target antigen will produce human antibodies by the normal immune response, which may be harvested and/or produced by standard techniques discussed above.
  • a variety of strains of XenoMouse® are available, each of which is capable of producing a different class of antibody.
  • Transgenically produced human antibodies have been shown to have therapeutic potential, while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999).
  • the skilled artisan will realize that the claimed compositions and methods are not limited to use of the XenoMouse® system but may utilize any transgenic animal that has been genetically engineered to produce human antibodies.
  • the ADCs are of use for treatment of Trop-2-expressing cancer.
  • the target cancer may express one or more additional tumor-associated antigens (TAAs).
  • TAAs tumor-associated antigens
  • Particular antibodies that may be of use for therapy of cancer within the scope of the claimed methods and compositions include, but are not limited to, LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (bA20, anti-CD20), rituxumab (anti-CD20), obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD- 1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-epithelial glycoprotein- 1 (EGP-1, also known as Trop-2)), PAM4 or KC4 (both anti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known as
  • hPAM4 U.S. Patent No. 7,282,567
  • hA20 U.S. Patent No. 7,251, 164
  • hA19 U.S. Patent No. 7, 109,304
  • hIMMU-31 U.S. Patent No. 7,300,655
  • hLLl U.S. Patent No. 7,312,318
  • hLL2 U.S. Patent No. 7,074,403
  • hMu-9 U.S. Patent No. 7,387,773
  • hL243 U.S. Patent No.
  • tumor-associated antigens include carbonic anhydrase IX, B7, CCL19, CCL21, CSAp, UER-2/neu, BrE3, CD1, CDla, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD 18, CD 19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD 154, CEACAM5, CEACAM6, CTLA-4, alpha-fetoprotein (AFP), VEGF (e.g., AVASTIN®, fibronectin splice
  • AFP alpha-fe
  • Cancer stem cells which are ascribed to be more therapy-resistant precursor malignant cell populations (Hill and Perris, J. Natl. Cancer Inst. 2007; 99: 1435-40), have antigens that can be targeted in certain cancer types, such as CD 133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos. Proc. 2006; 5: 155-79), non-small-cell lung cancer (Donnenberg et al., J. Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al., Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerba er al., Proc. Natl.
  • the CD47 antigen is a further useful target for cancer stem cells (see, e.g., Naujokat et al., 2014, Immunotherapy 6:290-308; Goto et al., 2014, Eur J Cancer 50: 1836-46; Unanue, 2013, Proc Natl Acad Sci USA 1 10: 10886-7).
  • Immune checkpoints refer to inhibitory pathways in the immune system that are responsible for maintaining self-tolerance and modulating the degree of immune system response to minimize peripheral tissue damage.
  • tumor cells can also activate immune system checkpoints to decrease the effectiveness of immune response against tumor tissues.
  • Exemplary checkpoint inhibitor antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD 152), programmed cell death protein 1 (PD 1, also known as CD279) and programmed cell death 1 ligand 1 (PD-Ll, also known as CD274), may be used in combination with one or more other agents to enhance the effectiveness of immune response against disease cells, tissues or pathogens.
  • Exemplary anti-PD 1 antibodies include lambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK), and pidilizumab (CT-01 1, CURETECH LTD.).
  • Anti-PDl antibodies are commercially available, for example from ABCAM® (AB 137132),
  • BIOLEGEND® EH12.2H7, RMPl-14
  • AFFYMETRIX EBIOSCIENCE J105, Jl 16, MIH4
  • Exemplary anti-PD-Ll antibodies include MDX-1 105 (MEDAREX), MEDI4736 (MED IMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB).
  • Anti-PD-Ll antibodies are also commercially available, for example from AFFYMETRIX EBIOSCIENCE (MIH1).
  • Exemplary anti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER).
  • Anti-PDl antibodies are commercially available, for example from ABCAM® (AB 134090), SINO BIOLOGICAL INC. (11 159-H03H, 1 1 159-H08H), and THERMO SCIENTIFIC PIERCE (PA5-29572, PA5- 23967, PA5-26465, MAl-12205, MAl-35914). Ipilimumab has recently received FDA approval for treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 1 1 :89).
  • Macrophage migration inhibitory factor is an important regulator of innate and adaptive immunity and apoptosis. It has been reported that CD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med 197: 1467-76).
  • the therapeutic effect of antagonistic anti-CD74 antibodies on MIF -mediated intracellular pathways may be of use for treatment of a broad range of disease states, such as cancers of the bladder, prostate, breast, lung, and colon (e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran, 201 1, Leuk Lymphoma 52: 1446-54).
  • Milatuzumab hLLl
  • Antibodies of use may be commercially obtained from a wide variety of known sources.
  • a variety of antibody secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, VA).
  • ATCC American Type Culture Collection
  • VA Manassas
  • a large number of antibodies against various disease targets, including tumor-associated antigens, have been deposited at the ATCC and/or have published variable region sequences and are available for use in the claimed methods and compositions. See, e.g., U.S. Patent Nos. 7,312,318; 7,282,567;
  • Immunogenicity of therapeutic antibodies is associated with increased risk of infusion reactions and decreased duration of therapeutic response (Baert et al., 2003, N Engl J Med 348:602-08).
  • the extent to which therapeutic antibodies induce an immune response in the host may be determined in part by the allotype of the antibody (Stickler et al., 201 1, Genes and Immunity 12:213-21).
  • Antibody allotype is related to amino acid sequence variations at specific locations in the constant region sequences of the antibody.
  • the allotypes of IgG antibodies containing a heavy chain ⁇ -type constant region are designated as Gm allotypes (1976, J Immunol 1 17: 1056-59).
  • Glml For the common IgGl human antibodies, the most prevalent allotype is Glml (Stickler et al., 201 1, Genes and Immunity 12:213-21). However, the Glm3 allotype also occurs frequently in Caucasians (Stickler et al., 201 1). It has been reported that Glml antibodies contain allotypic sequences that tend to induce an immune response when administered to non-Glml (nGlml) recipients, such as Glm3 patients (Stickler et al., 201 1). Non-Glml allotype antibodies are not as immunogenic when administered to Glml patients (Stickler et al, 201 1).
  • the human Glml allotype comprises the amino acids aspartic acid at Kabat position 356 and leucine at Kabat position 358 in the CH3 sequence of the heavy chain IgGl .
  • the nGlml allotype comprises the amino acids glutamic acid at Kabat position 356 and methionine at Kabat position 358.
  • Both Glml and nGlml allotypes comprise a glutamic acid residue at Kabat position 357 and the allotypes are sometimes referred to as DEL and EEM allotypes.
  • a non-limiting example of the heavy chain constant region sequences for Glml and nGlml allotype antibodies is shown below for the exemplary antibodies rituximab (SEQ ID NO: 7) and veltuzumab (SEQ ID NO: 8).
  • veltuzumab and rituximab are, respectively, humanized and chimeric IgGl antibodies against CD20, of use for therapy of a wide variety of hematological malignancies and/or autoimmune diseases.
  • Table 1 compares the allotype sequences of rituximab vs. veltuzumab.
  • rituximab (Glml7, l) is a DEL allotype IgGl, with an additional sequence variation at Kabat position 214 (heavy chain CHI) of lysine in rituximab vs. arginine in veltuzumab.
  • veltuzumab is less immunogenic in subjects than rituximab ⁇ see, e.g., Morchhauser et al., 2009, J Clin Oncol 27:3346-53; Goldenberg et al., 2009, Blood 1 13: 1062-70; Robak & Robak, 201 1, BioDrugs 25: 13-25), an effect that has been attributed to the difference between humanized and chimeric antibodies.
  • the difference in allotypes between the EEM and DEL allotypes likely also accounts for the lower immunogenicity of veltuzumab.
  • the allotype of the antibody In order to reduce the immunogenicity of therapeutic antibodies in individuals of nGlml genotype, it is desirable to select the allotype of the antibody to correspond to the Glm3 allotype, characterized by arginine at Kabat 214, and the nGlml, 2 null-allotype, characterized by glutamic acid at Kabat position 356, methionine at Kabat position 358 and alanine at Kabat position 431. Surprisingly, it was found that repeated subcutaneous administration of Glm3 antibodies over a long period of time did not result in a significant immune response.
  • the human IgG4 heavy chain in common with the Glm3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356, methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicity appears to relate at least in part to the residues at those locations, use of the human IgG4 heavy chain constant region sequence for therapeutic antibodies is also a preferred embodiment. Combinations of Glm3 IgGl antibodies with IgG4 antibodies may also be of use for therapeutic administration.
  • Nanobodies are single-domain antibodies of about 12-15 kDa in size (about 1 10 amino acids in length). Nanobodies can selectively bind to target antigens, like full-size antibodies, and have similar affinities for antigens. However, because of their much smaller size, they may be capable of better penetration into solid tumors. The smaller size also contributes to the stability of the nanobody, which is more resistant to pH and temperature extremes than full size antibodies (Van Der Linden et al., 1999, Biochim Biophys Act 1431 :37-46).
  • Single-domain antibodies were originally developed following the discovery that camelids (camels, alpacas, llamas) possess fully functional antibodies without light chains (e.g., Hamsen et al., 2007, Appl Microbiol Biotechnol 77: 13-22).
  • the heavy-chain antibodies consist of a single variable domain (VHH) and two constant domains (CH2 and CH3).
  • VHH variable domain
  • CH2 and CH3 constant domains
  • nanobodies may be developed and used as multivalent and/or bispecific constructs.
  • Target antigens such as IL-6R, vWF, TNF, RSV, RANKL, IL- 17A & F and IgE (e.g., ABLYNX®, Ghent, Belgium), with potential clinical use in cancer and other disorders (e.g., Saerens et al., 2008, Curr Opin Pharmacol 8:600-8; Muyldermans, 2013, Ann Rev Biochem 82:775-97; Ibanez et al., 201 1, J Infect Dis 203: 1063-72).
  • target antigens such as IL-6R, vWF, TNF, RSV, RANKL, IL- 17A & F and IgE (e.g., ABLYNX®, Ghent, Belgium)
  • nanobodies are shorter than that of full-size antibodies, with elimination primarily by the renal route. Because they lack an Fc region, they do not exhibit complement dependent cytotoxicity.
  • Nanobodies may be produced by immunization of camels, llamas, alpacas or sharks with target antigen, following by isolation of mRNA, cloning into libraries and screening for antigen binding.
  • Nanobody sequences may be humanized by standard techniques (e.g., Jones et al., 1986, Nature 321 : 522, Riechmann et al., 1988, Nature 332: 323, Verhoeyen et al., 1988, Science 239: 1534, Carter et al., 1992, Proc. Nat'l Acad. Sci. USA 89: 4285, Sandhu, 1992, Crit. Rev. Biotech. 12: 437, Singer et al., 1993, J. Immun. 150: 2844). Humanization is relatively straight- forward because of the high homology between camelid and human FR sequences.
  • the subject ADCs may comprise nanobodies for targeted delivery of conjugated drug to targeted cancer cells.
  • Nanobodies of use are disclosed, for example, in U.S. Patent Nos. 7,807,162; 7,939,277; 8,188,223; 8,217,140; 8,372,398;
  • Antibody fragments are antigen binding portions of an antibody, such as F(ab') 2, Fab', F(ab)2, Fab, Fv, sFv, scFv and the like. Antibody fragments which recognize specific epitopes can be generated by known techniques. F(ab')2 fragments, for example, can be produced by pepsin digestion of the antibody molecule. These and other methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 and references contained therein. Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J.
  • Fab' expression libraries can be constructed (Huse et al., 1989, Science, 246: 1274-1281) to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity.
  • a single chain Fv molecule comprises a VL domain and a VH domain.
  • the VL and VH domains associate to form a target binding site. These two domains are further covalently linked by a peptide linker (L).
  • L peptide linker
  • a scFv molecule is denoted as either VL-L-VH if the VL domain is the N-terminal part of the scFv molecule, or as VH-L-VL if the VH domain is the N-terminal part of the scFv molecule.
  • VHH Single domain antibodies
  • Single domain antibodies may be obtained, for example, from camels, alpacas or llamas by standard immunization techniques.
  • the VHH may have potent antigen-binding capacity and can interact with novel epitopes that are inaccessible to conventional VH-VL pairs.
  • Alpaca serum IgG contains about 50% camelid heavy chain only IgG antibodies (HCAbs) (Maass et al., 2007).
  • Alpacas may be immunized with known antigens, such as TNF-a, and VHHs can be isolated that bind to and neutralize the target antigen (Maass et al., 2007).
  • PCR primers that amplify virtually all alpaca VHH coding sequences have been identified and may be used to construct alpaca VHH phage display libraries, which can be used for antibody fragment isolation by standard biopanning techniques well known in the art (Maass et al., 2007).
  • An antibody fragment can also be prepared by proteolytic hydrolysis of a full-length antibody or by expression in E. coli or another host of the DNA coding for the fragment.
  • An antibody fragment can be obtained by pepsin or papain digestion of full-length antibodies by conventional methods.
  • an antibody fragment can be produced by enzymatic cleavage of antibodies with pepsin to provide an approximate 100 kD fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce an approximate 50 Kd Fab' monovalent fragment.
  • an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly.
  • VK variable light chain
  • VH variable heavy chain sequences for an antibody of interest
  • the V genes of a MAb from a cell that expresses a murine MAb can be cloned by PCR amplification and sequenced.
  • the cloned VL and VH genes can be expressed in cell culture as a chimeric Ab as described by Orlandi et al. , (Proc. Natl.
  • a humanized MAb can then be designed and constructed as described by Leung et al. (Mol. Immunol, 32: 1413 (1995)).
  • cDNA can be prepared from any known hybridoma line or transfected cell line producing a murine MAb by general molecular cloning techniques (Sambrook et al., Molecular Cloning, A laboratory manual, 2 nd Ed (1989)).
  • the VK sequence for the MAb may be amplified using the primers VK1BACK and VK1FOR (Orlandi et al, 1989) or the extended primer set described by Leung et al. (BioTechniques, 15: 286 (1993)).
  • VH sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandi et al, 1989) or the primers annealing to the constant region of murine IgG described by Leung et al. (Hybridoma, 13:469 (1994)).
  • Humanized V genes can be constructed by a combination of long oligonucleotide template syntheses and PCR amplification as described by Leung et al. (Mol. Immunol, 32: 1413 (1995)).
  • PCR products for VK can be subcloned into a staging vector, such as a pBR327-based staging vector, VKpBR, that contains an Ig promoter, a signal peptide sequence and convenient restriction sites.
  • PCR products for VH can be subcloned into a similar staging vector, such as the pBluescript-based VHpBS.
  • Expression cassettes containing the VK and VH sequences together with the promoter and signal peptide sequences can be excised from VKpBR and VHpBS and ligated into appropriate expression vectors, such as pKh and pGlg, respectively (Leung et al., Hybridoma, 13:469 (1994)).
  • the expression vectors can be co- transfected into an appropriate cell and supernatant fluids monitored for production of a chimeric, humanized or human MAb.
  • the VK and VH expression cassettes can be excised and subcloned into a single expression vector, such as pdHL2, as described by Gillies et al. (J. Immunol. Methods 125: 191 (1989) and also shown in Losman et al., Cancer, 80:2660 (1997)).
  • expression vectors may be transfected into host cells that have been pre-adapted for transfection, growth and expression in serum- free medium.
  • Exemplary cell lines that may be used include the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Patent Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each of which is incorporated herein by reference). These exemplary cell lines are based on the Sp2/0 myeloma cell line, transfected with a mutant Bcl-EEE gene, exposed to methotrexate to amplify transfected gene sequences and pre-adapted to serum- free cell line for protein expression.
  • the anti-Trop-2 ADC and one or more other therapeutic antibodies may be administered as separate antibodies, either sequentially or concurrently.
  • antibodies or antibody fragments may be administered as a single bispecific or multispecific antibody.
  • Numerous methods to produce bispecific or multispecific antibodies are known, as disclosed, for example, in U.S. Patent No. 7,405,320, the Examples section of which is incorporated herein by reference.
  • Bispecific antibodies can be produced by the quadroma method, which involves the fusion of two different hybridomas, each producing a monoclonal antibody recognizing a different antigenic site (Milstein and Cuello, Nature, 1983; 305:537-540).
  • bispecific antibodies Another method for producing bispecific antibodies uses heterobifunctional cross- linkers to chemically tether two different monoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631 ; Perez, et al. Nature. 1985; 316:354-356). Bispecific antibodies can also be produced by reduction of each of two parental monoclonal antibodies to the respective half molecules, which are then mixed and allowed to reoxidize to obtain the hybrid structure (Staerz and Bevan. Proc Natl Acad Sci U S A. 1986; 83 : 1453- 1457).
  • Other methods include improving the efficiency of generating hybrid hybridomas by gene transfer of distinct selectable markers via retrovirus-derived shuttle vectors into respective parental hybridomas, which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci U S A. 1990, 87:2941 - 2945); or transfection of a hybridoma cell line with expression plasmids containing the heavy and light chain genes of a different antibody.
  • Cognate VH and VL domains can be joined with a peptide linker of appropriate composition and length (usually consisting of more than 12 amino acid residues) to form a single-chain Fv (scFv), as discussed above. Reduction of the peptide linker length to less than 12 amino acid residues prevents pairing of VH and VL domains on the same chain and forces pairing of VH and VL domains with complementary domains on other chains, resulting in the formation of functional multimers. Polypeptide chains of VH and VL domains that are joined with linkers between 3 and 12 amino acid residues form predominantly dimers (termed diabodies).
  • trimers with linkers between 0 and 2 amino acid residues, trimers (termed triabody) and tetramers (termed tetrabody) are favored, but the exact patterns of oligomerization appear to depend on the composition as well as the orientation of V-domains (VH-linker-VL or VL- linker-VH), in addition to the linker length.
  • Bispecific or multispecific antibodies or other constructs may be produced using the DOCK-AND-LOCKTM technology (see, e.g., U.S. Patent Nos. 7,550, 143; 7,521 ,056;
  • the technique takes advantage of the specific and high-affinity binding interactions that occur between a dimerization and docking domain (DDD) sequence of the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequence derived from any of a variety of AKAP proteins (Baillie et ah, FEBS Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959).
  • DDD and AD peptides may be attached to any protein, peptide or other molecule. Because the DDD sequences spontaneously dimerize and bind to the AD sequence, the technique allows the formation of complexes between any selected molecules that may be attached to DDD or AD sequences.
  • the standard DNLTM complex comprises a trimer with two DDD-linked molecules attached to one AD-linked molecule
  • variations in complex structure allow the formation of dimers, trimers, tetramers, pentamers, hexamers and other multimers.
  • the DNLTM complex may comprise two or more antibodies, antibody fragments or fusion proteins which bind to the same antigenic determinant or to two or more different antigens.
  • the DNLTM complex may also comprise one or more other effectors, such as proteins, peptides, immunomodulators, cytokines, interleukins, interferons, binding proteins, peptide ligands, carrier proteins, toxins, ribonucleases such as onconase, inhibitory oligonucleotides such as siRNA, antigens or xenoantigens, polymers such as PEG, enzymes, therapeutic agents, hormones, cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents or any other molecule or aggregate.
  • effectors such as proteins, peptides, immunomodulators, cytokines, interleukins, interferons, binding proteins, peptide ligands, carrier proteins, toxins, ribonucleases such as onconase, inhibitory oligonucleotides such as siRNA, antigens or xenoantigens, polymers such as PEG, enzymes, therapeutic agents, hormones,
  • PKA which plays a central role in one of the best studied signal transduction pathways triggered by the binding of the second messenger cAMP to the R subunits, was first isolated from rabbit skeletal muscle in 1968 (Walsh et ah, J. Biol. Chem. 1968;243:3763).
  • the structure of the holoenzyme consists of two catalytic subunits held in an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RI and RII), and each type has a and ⁇ isoforms (Scott, Pharmacol. Ther. 1991 ;50: 123).
  • the four isoforms of PKA regulatory subunits are RIa, R3 ⁇ 4 Rlla and RI ⁇ .
  • the R subunits have been isolated only as stable dimers and the dimerization domain has been shown to consist of the first 44 amino-terminal residues of Rlla (Newlon et ah, Nat. Struct. Biol. 1999; 6:222).
  • similar portions of the amino acid sequences of other regulatory subunits are involved in dimerization and docking, each located near the N-terminal end of the regulatory subunit.
  • Binding of cAMP to the R subunits leads to the release of active catalytic subunits for a broad spectrum of serine/threonine kinase activities, which are oriented toward selected substrates through the compartmentalization of PKA via its docking with AKAPs (Scott et al, J. Biol. Chem. 1990;265;21561)
  • AKAP microtubule-associated protein-2
  • the amino acid sequences of the AD are quite varied among individual AKAPs, with the binding affinities reported for RII dimers ranging from 2 to 90 nM (Alto et al, Proc. Natl. Acad. Sci. USA. 2003; 100:4445). AKAPs will only bind to dimeric R subunits.
  • the AD binds to a hydrophobic surface formed by the 23 amino -terminal residues (Colledge and Scott, Trends Cell Biol. 1999; 6:216).
  • the dimerization domain and AKAP binding domain of human Rlla are both located within the same N-terminal 44 amino acid sequence (Newlon et ah, Nat. Struct. Biol. 1999;6:222; Newlon et al, EMBO J. 2001 ;20: 1651), which is termed the DDD herein.
  • A would thus be composed of &2- Entity B is constructed by linking an AD sequence to a precursor of B, resulting in a second component hereafter referred to as b.
  • the dimeric motif of DDD contained in & will create a docking site for binding to the AD sequence contained in b, thus facilitating a ready association of & and b to form a binary, trimeric complex composed of a2b.
  • This binding event is made irreversible with a subsequent reaction to covalently secure the two entities via disulfide bridges, which occurs very efficiently based on the principle of effective local concentration because the initial binding interactions should bring the reactive thiol groups placed onto both the DDD and AD into proximity (Chmura et al, Proc. Natl. Acad. Sci. USA. 2001 ;98:8480) to ligate site-specifically.
  • linkers, adaptor modules and precursors a wide variety of DNLTM constructs of different stoichiometry may be produced and used (see, e.g., U.S. Nos. 7,550, 143; 7,521,056;
  • fusion proteins A variety of methods are known for making fusion proteins, including nucleic acid synthesis, hybridization and/or amplification to produce a synthetic double-stranded nucleic acid encoding a fusion protein of interest.
  • double-stranded nucleic acids may be inserted into expression vectors for fusion protein production by standard molecular biology techniques (see, e.g. Sambrook et al., Molecular Cloning, A laboratory manual, 2 nd Ed, 1989).
  • the AD and/or DDD moiety may be attached to either the N- terminal or C-terminal end of an effector protein or peptide.
  • site of attachment of an AD or DDD moiety to an effector moiety may vary, depending on the chemical nature of the effector moiety and the part(s) of the effector moiety involved in its physiological activity.
  • Site-specific attachment of a variety of effector moieties may be performed using techniques known in the art, such as the use of bivalent cross-linking reagents and/or other chemical conjugation techniques.
  • AD or DDD sequences may be utilized. Exemplary DDD and AD sequences are provided below.
  • SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA SEQ ID NO: 9
  • DDD2 CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA SEQ ID NO: 10.
  • DDD l and DDD2 are based on the DDD sequence of the human Rlla isoform of protein kinase A.
  • the DDD and AD moieties may be based on the DDD sequence of the human RIa form of protein kinase A and a corresponding AKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.
  • AD and/or DDD moieties may be utilized in construction of the DNLTM complexes.
  • Rlla DDD sequence is the basis of DDDl and DDD2 disclosed above.
  • the four human PKA DDD sequences are shown below.
  • the DDD sequence represents residues 1-44 of Rlla, 1-44 of RIip, 12-61 of RIa and 13-66 of Rip. (Note that the sequence of DDD 1 is modified slightly from the human PKA Rlla DDD moiety.)
  • DDD moiety sequences are shown in SEQ ID NO:20 to SEQ ID NO:39 below.
  • the skilled artisan will realize that an almost unlimited number of alternative species within the genus of DDD moieties can be constructed by standard techniques, for example using a commercial peptide synthesizer or well known site-directed mutagenesis techniques.
  • the effect of the amino acid substitutions on AD moiety binding may also be readily determined by standard binding assays, for example as disclosed in Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50).
  • THIQIPPGLTELLQGYTVEVLPvQQPPDLVEFAVEYFTRLREAPvA SEQ ID NO:20
  • SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA SEQ ID NO:21
  • SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA SEQ ID NO:22
  • SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA SEQ ID NO:23
  • SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA SEQ ID NO:24
  • SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA SEQ ID NO:25
  • SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA SEQ ID NO:26
  • SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA
  • Alto et al. performed a bioinformatic analysis of the AD sequence of various AKAP proteins to design an RII selective AD sequence called AKAP-IS (SEQ ID NO: 1 1), with a binding constant for DDD of 0.4 nM.
  • the AKAP-IS sequence was designed as a peptide antagonist of AKAP binding to PKA. Residues in the AKAP-IS sequence where substitutions tended to decrease binding to DDD are underlined in SEQ ID NO: 1 1 below.
  • the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moiety sequence to prepare DNLTM constructs.
  • Other alternative sequences that might be substituted for the AKAP-IS AD sequence are shown in SEQ ID NO:59-61. Substitutions relative to the AKAP-IS sequence are underlined. It is anticipated that, as with the AD2 sequence shown in SEQ ID NO: 12, the AD moiety may also include the additional N-terminal residues cysteine and glycine and C-terminal residues glycine and cysteine.
  • Figure 2 of Gold et al. disclosed additional DDD-binding sequences from a variety of AKAP proteins, shown below.
  • AKAP5-pep QYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO:86)
  • AKAP9-pep LEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO:87)
  • AKAP10-pep NTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO:88)
  • AKAP12-pep NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO:90)
  • Residues that were highly conserved among the AD domains of different AKAP proteins are indicated below by underlining with reference to the AKAP IS sequence (SEQ ID NO: 1 1). The residues are the same as observed by Alto et al. (2003), with the addition of the C-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated herein by reference.)
  • the sequences of peptide antagonists with particularly high affinities for the RII DDD sequence were those of AKAP-IS, AKAP78-wt-pep, AKAP78-L304T-pep and AKAP78-L308D-pep.
  • Carr et al. (2001, J Biol Chem 276: 17332-38) examined the degree of sequence homology between different AKAP-binding DDD sequences from human and non-human proteins and identified residues in the DDD sequences that appeared to be the most highly conserved among different DDD moieties. These are indicated below by underlining with reference to the human PKA Rlla DDD sequence of SEQ ID NO: 9. Residues that were particularly conserved are further indicated by italics. The residues overlap with, but are not identical to those suggested by Kinderman et al. (2006) to be important for binding to AKAP proteins.
  • DNLTM constructs may be formed using alternatively constructed antibodies or antibody fragments, in which an AD moiety may be attached at the C-terminal end of the kappa light chain (C k ), instead of the C-terminal end of the Fc on the heavy chain.
  • the alternatively formed DNLTM constructs may be prepared as disclosed in Provisional U.S. Patent Application Serial Nos. 61/654,310, filed June 1, 2012, 61/662,086, filed June 20, 2012, 61/673,553, filed July 19, 2012, and 61/682,531, filed August 13, 2012, the entire text of each incorporated herein by reference.
  • the light chain conjugated DNLTM constructs exhibit enhanced Fc-effector function activity in vitro and improved pharmacokinetics, stability and anti-lymphoma activity in vivo (Rossi et al., 2013, Bioconjug Chem 24:63-71).
  • Ck-conjugated DNLTM constructs may be prepared as disclosed in Provisional U.S. Patent Application Serial Nos. 61/654,310, 61/662,086, 61/673,553, and 61/682,531. Briefly, C k -AD2-IgG, was generated by recombinant engineering, whereby the AD2 peptide was fused to the C-terminal end of the kappa light chain. Because the natural C-terminus of CK is a cysteine residue, which forms a disulfide bridge to CHI, a 16-amino acid residue "hinge" linker was used to space the AD2 from the CK-VH1 disulfide bridge.
  • the mammalian expression vectors for C k -AD2-IgG-veltuzumab and C k -AD2-IgG-epratuzumab were constructed using the pdHL2 vector, which was used previously for expression of the homologous Cn3-AD2-IgG modules.
  • a 2208-bp nucleotide sequence was synthesized comprising the pdHL2 vector sequence ranging from the Bam HI restriction site within the VK/CK intron to the Xho / restriction site 3' of the Ck intron, with the insertion of the coding sequence for the hinge linker (EFPKPSTPPGSSGGAP, SEQ ID NO:93) and AD2, in frame at the 3 'end of the coding sequence for CK- This synthetic sequence was inserted into the IgG-pdHL2 expression vectors for veltuzumab and epratuzumab via Bam HI and Xho I restriction sites.
  • C k -AD2-IgG- veltuzumab and C k -AD2-IgG- epratuzumab were produced by stably-transfected production clones in batch roller bottle culture, and purified from the supernatant fluid in a single step using MabSelect (GE Healthcare) Protein A affinity chromatography.
  • Ck-AD2-IgG-epratuzumab was conjugated with CH1-DDD2- Fab-veltuzumab, a Fab-based module derived from veltuzumab, to generate the bsHexAb 22*-(20)-(20), where the 22* indicates the C k -AD2 module of epratuzumab and each (20) symbolizes a stabilized dimer of veltuzumab Fab.
  • C k -AD2-IgG-veltuzumab was conjugated with IFNa2b-DDD2, a module of IFNa2b with a DDD2 peptide fused at its C-terminal end, to generate 20*-2b, which comprises veltuzumab with a dimeric IFNa2b fused to each light chain.
  • the properties of 20*-2b were compared with those of 20-2b, which is the homologous Fc- IgG-IFNa.
  • Each of the bsHexAbs and IgG-IFNa were isolated from the DNLTM reaction mixture by MabSelect affinity chromatography.
  • the disclosed methods and compositions may involve production and use of proteins or peptides with one or more substituted amino acid residues.
  • the DDD and/or AD sequences used to make DNLTM constructs may be modified as discussed above.
  • amino acid substitutions typically involve the replacement of an amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions).
  • conservative amino acid substitutions The properties of the various amino acids and effect of amino acid substitution on protein structure and function have been the subject of extensive study and knowledge in the art.
  • the hydropathic index of amino acids may be considered (Kyte & Doolittle, 1982, J. Mol. Biol., 157: 105-132).
  • the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the use of amino acids whose hydropathic indices are within ⁇ 2 is preferred, within ⁇ 1 are more preferred, and within ⁇ 0.5 are even more preferred.
  • Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 .+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of amino acids with others of similar hydrophilicity is preferred.
  • amino acid side chain For example, it would generally not be preferred to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine.
  • a compact side chain such as glycine or serine
  • an amino acid with a bulky side chain e.g., tryptophan or tyrosine.
  • tryptophan or tyrosine The effect of various amino acid residues on protein secondary structure is also a
  • arginine and lysine glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent exposed.
  • conservative substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp.
  • conservative substitutions would include: Asp and Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr.
  • amino acid substitutions In determining amino acid substitutions, one may also consider the existence of intermolecular or intramolecular bonds, such as formation of ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between nearby cysteine residues.
  • ionic bonds salt bridges
  • positively charged residues e.g., His, Arg, Lys
  • negatively charged residues e.g., Asp, Glu
  • disulfide bonds between nearby cysteine residues.
  • Bispecific or multispecific antibodies may be of use in pretargeting techniques.
  • one or more therapeutic agent may be conjugated to a targetable construct that comprises one or more haptens.
  • the hapten is recognized by at least one arm of a bispecific or multispecific antibody that also binds to a tumor-associated antigen or other disease- associated antigen.
  • the therapeutic agent binds indirectly to the antibodies, via the binding of the targetable construct. This process is referred to as pretargeting.
  • Pre-targeting is a multistep process originally developed to resolve the slow blood clearance of directly targeting antibodies, which contributes to undesirable toxicity to normal tissues such as bone marrow.
  • a therapeutic agent is attached to a small delivery molecule (targetable construct) that is cleared within minutes from the blood.
  • a pretargeting bispecific or multispecific antibody which has binding sites for the targetable construct as well as a target antigen, is administered first, free antibody is allowed to clear from circulation and then the targetable construct is administered.
  • a pre-targeting method of treating or diagnosing a disease or disorder in a subject may be provided by: (1) administering to the subject a bispecific antibody or antibody fragment; (2) optionally administering to the subject a clearing composition, and allowing the composition to clear the antibody from circulation; and (3) administering to the subject the targetable construct, containing one or more chelated or chemically bound therapeutic or diagnostic agents.
  • targetable construct peptides labeled with one or more therapeutic or diagnostic agents for use in pre-targeting may be selected to bind to a bispecific antibody with one or more binding sites for a targetable construct peptide and one or more binding sites for a target antigen associated with a disease or condition.
  • Bispecific antibodies may be used in a pretargeting technique wherein the antibody may be administered first to a subject. Sufficient time may be allowed for the bispecific antibody to bind to a target antigen and for unbound antibody to clear from circulation. Then a targetable construct, such as a labeled peptide, may be administered to the subject and allowed to bind to the bispecific antibody and localize at the diseased cell or tissue.
  • Such targetable constructs can be of diverse structure and are selected not only for the availability of an antibody or fragment that binds with high affinity to the targetable construct, but also for rapid in vivo clearance when used within the pre-targeting method and bispecific antibodies (bsAb) or multispecific antibodies.
  • Hydrophobic agents are best at eliciting strong immune responses, whereas hydrophilic agents are preferred for rapid in vivo clearance.
  • hydrophilic chelating agents to offset the inherent hydrophobicity of many organic moieties.
  • sub-units of the targetable construct may be chosen which have opposite solution properties, for example, peptides, which contain amino acids, some of which are hydrophobic and some of which are hydrophilic.
  • Peptides having as few as two amino acid residues, preferably two to ten residues, may be used and may also be coupled to other moieties, such as chelating agents.
  • the linker should be a low molecular weight conjugate, preferably having a molecular weight of less than 50,000 daltons, and advantageously less than about 20,000 daltons, 10,000 daltons or 5,000 daltons. More usually, the targetable construct peptide will have four or more residues and one or more haptens for binding, e.g., to a bispecific antibody.
  • Exemplary haptens may include In-DTPA (indium- diethylene triamine pentaacetic acid) or HSG (histamine succinyl glycine).
  • the targetable construct may also comprise one or more chelating moieties, such as DOTA (l,4,7,10-tetraazacyclododecanel,4,7,10-tetraacetic acid), NOTA (1,4,7-triaza- cyclononane-l,4,7-triacetic acid), TETA (p-bromoacetamido-benzyl- tetraethylaminetetraacetic acid), NETA ([2-(4,7-biscarboxymethyl[l,4,7]triazacyclononan- l- yl-ethyl]-2-carbonylmethyl-amino]acetic acid) or other known chelating moieties.
  • DOTA l,4,7,10-tetraazacyclododecanel,4,7,10-tetraacetic acid
  • NOTA 1,4,7-triaza- cyclononane-l,4,7-triacetic acid
  • TETA p-bromoace
  • Chelating moieties may be used, for example, to bind to a therapeutic and or diagnostic radionuclide, paramagnetic ion or contrast agent.
  • the targetable construct may also comprise unnatural amino acids, e.g., D-amino acids, in the backbone structure to increase the stability of the peptide in vivo.
  • unnatural amino acids e.g., D-amino acids
  • other backbone structures such as those constructed from non-natural amino acids or peptoids may be used.
  • the peptides used as targetable constructs are conveniently synthesized on an automated peptide synthesizer using a solid-phase support and standard techniques of repetitive orthogonal deprotection and coupling. Free amino groups in the peptide, that are to be used later for conjugation of chelating moieties or other agents, are advantageously blocked with standard protecting groups such as a Boc group, while N-terminal residues may be acetylated to increase serum stability. Such protecting groups are well known to the skilled artisan. See Greene and Wuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, N.Y.). When the peptides are prepared for later use within the bispecific antibody system, they are advantageously cleaved from the resins to generate the corresponding C- terminal amides, in order to inhibit in vivo carboxypeptidase activity.
  • the antibody will contain a first binding site for an antigen produced by or associated with a target tissue and a second binding site for a hapten on the targetable construct.
  • haptens include, but are not limited to, HSG and In-DTPA.
  • Antibodies raised to the HSG hapten are known (e.g. 679 antibody) and can be easily incorporated into the appropriate bispecific antibody (see, e.g., U.S. Patent Nos. 6,962,702; 7,138,103 and 7,300,644, incorporated herein by reference with respect to the Examples sections).
  • haptens and antibodies that bind to them are known in the art and may be used, such as In-DTPA and the 734 antibody (e.g., U.S. Patent No.7,534,431, the Examples section incorporated herein by reference).
  • a cytotoxic drug or other therapeutic or diagnostic agent may be covalently attached to an antibody or antibody fragment to form an immunoconjugate.
  • a drug or other agent may be attached to an antibody or fragment thereof via a carrier moiety.
  • Carrier moieties may be attached, for example to reduced SH groups and/or to carbohydrate side chains.
  • a carrier moiety can be attached at the hinge region of a reduced antibody component via disulfide bond formation.
  • such agents can be attached using a heterobifunctional cross-linker, such as N-succinyl 3-(2- pyridyldithio)propionate (SPDP). Yu et al, Int. J. Cancer 56: 244 (1994).
  • the carrier moiety can be conjugated via a carbohydrate moiety in the Fc region of the antibody.
  • the Fc region may be absent if the antibody component of the ADC is an antibody fragment.
  • a carbohydrate moiety into the light chain variable region of a full length antibody or antibody fragment. See, for example, Leung et al, J. Immunol. 154: 5919 (1995); U.S. Patent Nos. 5,443,953 and 6,254,868, the Examples section of which is incorporated herein by reference.
  • the engineered carbohydrate moiety is used to attach the therapeutic or diagnostic agent.
  • An alternative method for attaching carrier moieties to a targeting molecule involves use of click chemistry reactions.
  • the click chemistry approach was originally conceived as a method to rapidly generate complex substances by joining small subunits together in a modular fashion.
  • Various forms of click chemistry reaction are known in the art, such as the Huisgen 1,3-dipolar cycloaddition copper catalyzed reaction (Tornoe et al., 2002, J Organic Chem 67:3057-64), which is often referred to as the "click reaction.”
  • Other alternatives include cycloaddition reactions such as the Diels-Alder, nucleophilic substitution reactions (especially to small strained rings like epoxy and aziridine compounds), carbonyl chemistry formation of urea compounds and reactions involving carbon-carbon double bonds, such as alkynes in thi
  • the azide alkyne Huisgen cycloaddition reaction uses a copper catalyst in the presence of a reducing agent to catalyze the reaction of a terminal alkyne group attached to a first molecule.
  • a second molecule comprising an azide moiety
  • the azide reacts with the activated alkyne to form a 1 ,4-disubstituted 1,2,3-triazole.
  • the copper catalyzed reaction occurs at room temperature and is sufficiently specific that purification of the reaction product is often not required.
  • a copper-free click reaction has been proposed for covalent modification of biomolecules.
  • the copper- free reaction uses ring strain in place of the copper catalyst to promote a [3 + 2] azide-alkyne cycloaddition reaction ⁇ Id.
  • cyclooctyne is an 8-carbon ring structure comprising an internal alkyne bond.
  • the closed ring structure induces a substantial bond angle deformation of the acetylene, which is highly reactive with azide groups to form a triazole.
  • cyclooctyne derivatives may be used for copper- free click reactions ⁇ Id.
  • Agard et al. (2004, J Am Chem Soc 126: 15046-47) demonstrated that a recombinant glycoprotein expressed in CHO cells in the presence of peracetylated N- azidoacetylmannosamine resulted in the bioincorporation of the corresponding N-azidoacetyl sialic acid in the carbohydrates of the glycoprotein.
  • the azido-derivatized glycoprotein reacted specifically with a biotinylated cyclooctyne to form a biotinylated glycoprotein, while control glycoprotein without the azido moiety remained unlabeled (Id.) Laughlin et al.
  • the TCO-labeled CC49 antibody was administered to mice bearing colon cancer xenografts, followed 1 day later by injection of 1 "in-labeled tetrazine probe (Id.)
  • the reaction of radiolabeled probe with tumor localized antibody resulted in pronounced radioactivity localization in the tumor, as demonstrated by SPECT imaging of live mice three hours after injection of radiolabeled probe, with a tumor- to-muscle ratio of 13: 1 (Id.)
  • the results confirmed the in vivo chemical reaction of the TCO and tetrazine-labeled molecules.
  • the landscaped antibodies were subsequently reacted with agents comprising a ketone-reactive moiety, such as hydrazide, hydrazine, hydroxylamino or thiosemicarbazide groups, to form a labeled targeting molecule.
  • agents attached to the landscaped antibodies included chelating agents like DTPA, large drug molecules such as doxorubicin-dextran, and acyl-hydrazide containing peptides.
  • the landscaping technique is not limited to producing antibodies comprising ketone moieties, but may be used instead to introduce a click chemistry reactive group, such as a nitrone, an azide or a cyclooctyne, onto an antibody or other biological molecule.
  • Reactive targeting molecule may be formed either by either chemical conjugation or by biological incorporation.
  • the targeting molecule such as an antibody or antibody fragment, may be activated with an azido moiety, a substituted cyclooctyne or alkyne group, or a nitrone moiety.
  • the targeting molecule comprises an azido or nitrone group
  • the corresponding targetable construct will comprise a substituted cyclooctyne or alkyne group, and vice versa.
  • Such activated molecules may be made by metabolic incorporation in living cells, as discussed above.
  • a wide variety of therapeutic reagents can be administered concurrently or sequentially with the subject ADCs.
  • such agents may be conjugated to the antibodies of the invention, for example, drugs, toxins, oligonucleotides, immunomodulators, hormones, hormone antagonists, enzymes, enzyme inhibitors, radionuclides, angiogenesis inhibitors, etc.
  • the therapeutic agents recited here are those agents that also are useful for administration separately with an ADC as described above.
  • Therapeutic agents include, for example, cytotoxic drugs such as vinca alkaloids, anthracyclines such as doxorubicin, 2- PDox or pro-2-PDox, gemcitabine, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2 inhibitors, antimitotics, anti- angiogenic and pro-apoptotic agents, particularly doxorubicin, methotrexate, taxol, CPT- 1 1 , camptothecans, proteosome inhibitors, mTOR inhibitors, HDAC inhibitors, tyrosine kinase inhibitors, and others.
  • cytotoxic drugs such as vinca alkaloids, anthracyclines such as doxorubicin, 2- PDox or pro-2-PDox, gemcitabine, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2 inhibitors, antimitotic
  • Suitable cytotoxic agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co.
  • conjugates of camptothecins and related compounds such as SN-38
  • hRS7 or other anti-Trop-2 antibodies may be conjugated to hRS7 or other anti-Trop-2 antibodies.
  • gemcitabine is administered to the subject in conjunction with SN-38-hRS7 and/or 90 Y-hPAM4.
  • a toxin can be of animal, plant or microbial origin.
  • Toxins of use include ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, onconase, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
  • RNase ribonuclease
  • DNase I DNase I
  • Staphylococcal enterotoxin-A Staphylococcal enterotoxin-A
  • pokeweed antiviral protein pokeweed antiviral protein
  • onconase gelonin
  • diphtheria toxin diphtheria toxin
  • Pseudomonas exotoxin Pseudomonas endotoxin.
  • Additional toxins suitable for use are known to those of skill in the art and are disclosed
  • the term "immunomodulator” includes a cytokine, a lymphokine, a monokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, a transforming growth factor (TGF), TGF-a, TGF- ⁇ , insulin-like growth factor (ILGF), erythropoietin, thrombopoietin, tumor necrosis factor (TNF), TNF- a, TNF- ⁇ , a mullerian-inhibiting substance, mouse
  • gonadotropin-associated peptide gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, interleukin (IL), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage- colony stimulating factor (GM-CSF), interferon- a, interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , SI factor, IL-1, IL-lcc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12, IL- 13, IL-14, IL- 15, IL-16, IL- 17, IL-18 IL-21 and IL-25, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin, lymphotoxin, and the like.
  • Particularly useful therapeutic radionuclides include, but are not limited to 11 'in, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 1, 32 P, 33 P, 47 Sc, m Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 227 Th, and 211 Pb.
  • the therapeutic radionuclide preferably has a decay energy in the range of 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter, and 4,000- 6,000 keV for an alpha emitter.
  • Maximum decay energies of useful beta-particle-emitting nuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, and most preferably 500-2,500 keV. Also preferred are radionuclides that substantially decay with Auger-emitting particles.
  • beta-particle-emitting nuclides are preferably ⁇ 1,000 keV, more preferably ⁇ 100 keV, and most preferably ⁇ 70 keV. Also preferred are radionuclides that substantially decay with generation of alpha-particles.
  • Such radionuclides include, but are not limited to: Dy- 152, At-21 1, Bi-212, Ra-223, Rn-219, Po- 215, Bi-21 1, Ac-225, Fr-221, At-217, Bi-213, Fm-255 and Th-227. Decay energies of useful alpha-particle-emitting radionuclides are preferably 2,000- 10,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.
  • 90 Y which emits an energetic beta particle
  • DTP A diethylenetriaminepentaacetic acid
  • Methods of conjugating 90 Y to antibodies or targetable constructs are known in the art and any such known methods may be used.
  • any such known methods may be used.
  • Additional potential therapeutic radioisotopes include C, N, O, Br, Au,
  • a radiosensitizer can be used in combination with a naked or conjugated antibody or antibody fragment.
  • the radiosensitizer can be used in combination with a radiolabeled antibody or antibody fragment.
  • the addition of the radiosensitizer can result in enhanced efficacy when compared to treatment with the radiolabeled antibody or antibody fragment alone.
  • Radiosensitizers are described in D. M. Goldenberg (ed.), CANCER THERAPY WITH RADIOLABELED ANTIBODIES, CRC Press (1995).
  • Other typical radionsensitizers of interest for use with this technology include gemcitabine, 5-fluorouracil, and cisplatin, and have been used in combination with external irradiation in the therapy of diverse cancers.
  • Antibodies or fragments thereof that have a boron addend-loaded carrier for thermal neutron activation therapy will normally be affected in similar ways. However, it will be advantageous to wait until non-targeted immunoconjugate clears before neutron irradiation is performed. Clearance can be accelerated using an anti- idiotypic antibody that binds to the anti-cancer antibody. See U.S. Pat. No. 4,624,846 for a description of this general principle.
  • boron addends such as carboranes, can be attached to antibodies. Carboranes can be prepared with carboxyl functions on pendant side chains, as is well-known in the art.
  • Attachment of carboranes to a carrier can be achieved by activation of the carboxyl groups of the carboranes and condensation with amines on the carrier.
  • the intermediate conjugate is then conjugated to the antibody.
  • a boron addend is activated by thermal neutron irradiation and converted to radioactive atoms which decay by alpha-emission to produce highly toxic, short-range effects.
  • Suitable routes of administration of ADCs include, without limitation, oral, parenteral, rectal, transmucosal, intestinal administration, intramedullary, intrathecal, direct
  • intraventricular, intravenous, intravitreal, intracavitary, intraperitoneal, or intratumoral injections are preferred routes of administration.
  • the preferred routes of administration are parenteral, more preferably intravenous.
  • ADCs can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the ADC is combined in a mixture with a pharmaceutically suitable excipient.
  • a pharmaceutically suitable excipient Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient.
  • Other suitable excipients are well-known to those in the art. See, for example, Ansel et al, PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S
  • the ADC is formulated in Good's biological buffer (pH 6- 7), using a buffer selected from the group consisting of N-(2-acetamido)-2- aminoethanesulfonic acid (ACES); N-(2-acetamido)iminodiacetic acid (ADA); N,N-bis(2- hydroxyethyl)-2-aminoethanesulfonic acid (BES); 4-(2-hydroxyethyl)piperazine- l- ethanesulfonic acid (HEPES); 2-(N-morpholino)ethanesulfonic acid (MES); 3-(N- morpholino)propanesulfonic acid (MOPS); 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); and piperazine-N,N'-bis(2-ethanesulfonic acid) [Pipes].
  • a buffer selected from the group consisting of N-(2-acetamido)-2- aminoethanes
  • More preferred buffers are MES or MOPS, preferably in the concentration range of 20 to 100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH 6.5.
  • the formulation may further comprise 25 mM trehalose and 0.01% v/v polysorbate 80 as excipients, with the final buffer concentration modified to 22.25 mM as a result of added excipients.
  • the preferred method of storage is as a lyophilized formulation of the conjugates, stored in the temperature range of -20 °C to 2 °C, with the most preferred storage at 2 °C to 8 °C.
  • the ADC can be formulated for intravenous administration via, for example, bolus injection, slow infusion or continuous infusion.
  • the antibody of the present invention is infused over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours.
  • the first 25-50 mg could be infused within 30 minutes, preferably even 15 min, and the remainder infused over the next 2-3 hrs.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi- dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Control release preparations can be prepared through the use of polymers to complex or adsorb the ADC.
  • biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. Sherwood et al, Bio/Technology 10: 1446 (1992). The rate of release of an ADC from such a matrix depends upon the molecular weight of the ADC, the amount of ADC within the matrix, and the size of dispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al, supra. Other solid dosage forms are described in Ansel et al, PHARMACEUTICAL DOSAGE FORMS AND DRUG
  • the dosage of an administered ADC for humans will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. It may be desirable to provide the recipient with a dosage of ADC that is in the range of from about 0.3 mg/kg to 5 mg/kg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate.
  • a dosage of 0.3-5 mg/kg for a 70 kg patient, for example, is 21-350 mg, or 12-206 mg/m 2 for a 1.7-m patient. The dosage may be repeated as needed, for example, once per week for 2- 10 weeks, once per week for 8 weeks, or once per week for 4 weeks.
  • Preferred dosages may include, but are not limited to, 0.3 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1.0 mg/kg, 1.2 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, and 5.0 mg/kg. More preferred dosages are 0.6 mg/kg for weekly administration and 1.2 mg/kg for less frequent dosing. Any amount in the range of 0.3 to 5 mg/kg may be used. The dosage is preferably administered multiple times, once a week.
  • a minimum dosage schedule of 4 weeks, more preferably 8 weeks, more preferably 16 weeks or longer may be used, with the dose frequency dependent on toxic side-effects and recovery therefrom, mostly related to hematological toxicities.
  • the schedule of administration may comprise administration once or twice a week, on a cycle selected from the group consisting of: (i) weekly; (ii) every other week; (iii) one week of therapy followed by two, three or four weeks off; (iv) two weeks of therapy followed by one, two, three or four weeks off; (v) three weeks of therapy followed by one, two, three, four or five week off; (vi) four weeks of therapy followed by one, two, three, four or five week off; (vii) five weeks of therapy followed by one, two, three, four or five week off; and (viii) monthly.
  • the cycle may be repeated 2, 4, 6, 8, 10, or 12 times or more.
  • an ADC may be administered as one dosage every 2 or 3 weeks, repeated for a total of at least 3 dosages. Or, twice per week for 4-6 weeks.
  • the dosage may be administered once every other week or even less frequently, so the patient can recover from any drug-related toxicities.
  • the dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3 months.
  • the dosing schedule can optionally be repeated at other intervals and dosage may be given through various parenteral routes, with appropriate adjustment of the dose and schedule.
  • compositions described and claimed herein may be used to treat malignant or premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above.
  • Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).
  • Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia. It is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplasia characteristically occurs where there exists chronic irritation or inflammation.
  • the method of the invention is used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
  • kits containing components suitable for treating cancer tissue in a patient may contain at least one anti-Trop-2 ADC as described herein. If the composition containing components for administration is not formulated for delivery via the alimentary canal, such as by oral delivery, a device capable of delivering the kit components through some other route may be included.
  • a device capable of delivering the kit components through some other route may be included.
  • an anti-Trop-2 antibody or antigen binding fragment thereof may be provided in the form of a prefilled syringe or autoinjection pen containing a sterile, liquid formulation or lyophilized preparation of antibody (e.g., Kivitz et al., Clin. Ther. 2006, 28: 1619-29).
  • the kit components may be packaged together or separated into two or more containers.
  • the containers may be vials that contain sterile, lyophilized formulations of a composition that are suitable for reconstitution.
  • a kit may also contain one or more buffers suitable for reconstitution and/or dilution of other reagents.
  • Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers. Another component that can be included is instructions for use of the kit.
  • the humanized RS7 (hRS7) anti-Trop-2 antibody was produced as described in U.S. Patent No. 7,238,785, the Figures and Examples section of which are incorporated herein by reference.
  • SN-38 attached to a CL2A linker was produced and conjugated to hRS7 (anti- Trop-2), hPAM4 (anti-MUC5ac), hA20 (anti-CD20) or hMN- 14 (anti-CEACAM5) antibodies according to U.S. Patent 7,999,083 (Example 10 and 12 of which are incorporated herein by reference).
  • the conjugation protocol resulted in a ratio of about 6 SN-38 molecules attached per antibody molecule.
  • FIG. 1 shows a Capan 1 pancreatic tumor model, wherein specific CL2A-SN-38 conjugates of hRS7 (anti-Trop-2), hPAM4 (anti-MUC-5ac), and hMN- 14 (anti-CEACAM5) antibodies showed better efficacies than control hA20-CL2A-SN-38 conjugate (anti-CD20) and untreated control.
  • the specific hRS7-CL2A-SN-38 showed better therapeutic efficacy than control treatments (FIG. 2).
  • pro-2-PDox A prodrug form of 2-PDox (referred to as pro-2-PDox) was prepared and conjugated to antibodies as disclosed in U.S. Patent Application 14/175,089 (Example 1 of which is incorporated herein by reference).
  • the structures of doxorubicin, 2-PDox, pro-2-PDox and a maleimide activated form of pro-2-PDox that is suitable for conjugation to sulfhydryl groups on antibodies or other proteins are shown in FIG. 3.
  • the number of drug moieties per antibody molecule was in the range of about 6.5 to about 7.5.
  • Serum stability - Serum stability of anti-Trop-2 ADC was determined by incubation in human serum at a concentration of 0.2 mg/mL at 37°C. The incubate was analyzed by HPLC using butyl hydrophobic interaction chromatography (HIC). The analysis showed that there was no release of free drug from the conjugate, suggesting high serum stability of the conjugate.
  • the pro-2-PDox conjugate was held tightly to the antibody because it cross-linked the peptide chains of the antibody together.
  • the cross- linking stabilizes the attachment of the drug to the antibody so that the drug is only released intracellularly after the antibody is metabolized.
  • the cross-linking assists in minimizing toxicity, for example cardiotoxicity, that would result from release of free drug in circulation.
  • Previous use of 2-PDox peptide conjugates failed because the drug cross-linked the peptide to other proteins or peptides in vivo.
  • the pro-2-PDox is attached to interchain disulfide thiol groups while in the prodrug form.
  • the prodrug protection is rapidly removed in vivo soon after injection and the resulting 2-PDox portion of the conjugate cross-links the peptide chains of the antibody, forming intramolecular cross- linking within the antibody molecule. This both stabilizes the ADC and prevents cross- linking to other molecules in circulation.
  • Tumor size was determined by caliper measurements of length (L) and width (W) with tumor volume calculated as (LxW )/2. Tumors were measured and mice weighed twice a week. Mice were euthanized if their tumors reached >1 cm 3 in size, lost greater than 15% of their starting body weight, or otherwise became moribund.
  • Statistical analysis for the tumor growth data was based on area under the curve (AUC) and survival time. Profiles of individual tumor growth were obtained through linear curve modeling. An test was employed to determine equality of variance between groups prior to statistical analysis of growth curves. A two-tailed i-test was used to assess statistical significance between all the various treatment groups and non-specific controls.
  • a single i.v. dose of > 300 ⁇ g [ ⁇ 10 ⁇ g of pro-2-PDox] of the anti-Trop-2 ADC was lethal, but 4 doses of 45 ⁇ g given in 2 weeks were tolerated by all animals.
  • PK and toxicity of hRS7-pro-2-PDox with substitutions of 6.8 or 3.7 drug/IgG - Antibody-drug conjugates (ADCs) carrying as much as 8 ultratoxic drugs/MAb are known to clear faster than unmodified MAb and to increase off-target toxicity, a finding that has led to the current trends to use drug substitutions of ⁇ 4 (Hamblett et al., 2004, Clin Cancer Res 10:7063-70).
  • ADCs were prepared and evaluated with mean drug/MAb substitution ratios (MSRs) of -6: 1 and -3: 1.
  • MED Minimum Effective Dose
  • hRS7- pro-2-PDox Anti-Trop-2 ADC
  • mTV in the saline control group was 0.801 ⁇ 0.181 cm 3 which was significantly larger than that in mice treated with 9, 6.75, 4.5, or 2.25 mg/kg dose with mTV of 0.21 1 ⁇ 0.042 cm 3 , 0.239 ⁇ 0.0.054 cm 3 , 0.264 ⁇ 0.087 cm 3 , and 0.567 ⁇ 0.179 cm 3 , respectively ( ⁇ 0.0047, one tailed t-test). From these, the minimum effective dose was estimated to be 2.25 mg/kg, while 9 mg/kg represented MTD.
  • FIG. 6A-F Further in vivo efficacy studies were performed in nude mice implanted with NCI- N87 human gastric cancer xenografts (FIG. 6A-F).
  • mice receiving 45 ⁇ g q4dx4 of hRS7-pro-2-PDox 8 of 9 mice were alive at day 94 (FIG. 8B).
  • mice receiving 90 ⁇ g weekly x 2 of hRS7-pro-2-PDox 9 of 9 mice were alive at day 94 (FIG. 8C).
  • mice receiving a single dose of 180 ⁇ g of hRS7-pro-2-PDox 7 of 9 mice were alive at day 94 (FIG. 8D).
  • the hRS7-pro-2-PDox conjugate was also effective in Capan- 1 pancreatic cancer (not shown) and was more effective at inhibiting tumor growth than a hRS7-SN-38 conjugate (not shown).
  • the hPAM4-pro-2-PDox conjugate was also more effective at inhibiting growth of Capan-1 human pancreatic cancer than an hPAM4-SN-38 conjugate (not shown).
  • mice were alive in the saline control, 10 of 10 mice were alive in mice treated twice weekly x 2 weeks with 45 ⁇ g of hPAM4-pro-2-PDox, 2 of 10 mice were alive in mice treated twice weekly x 2 weeks with 45 ⁇ g of hA20-pro-2-PDox, 0 of 10 mice were alive in mice treated twice weekly x 4 weeks with 250 ⁇ g of hPAM4-SN-38, and 0 of 10 mice were alive in mice treated twice weekly x 4 weeks with 250 ⁇ g of h20-SN-38.
  • hRS7-pro-2-PDox was substantially more effective than hRS7-SN-38 at inhibiting growth of PxPC-3 pancreatic cancer (not shown) and was slightly more effective than hRS7- SN-38 at inhibiting growth of MDA-MB-468 breast cancer (not shown).
  • FIG. 10 Survival curves for mice bearing NCI-N87 human gastric carcinoma xenografts and administered a single dose of anti-Trop-2 ADC are shown in FIG. 10.
  • a single dose of 45 ⁇ g was the minimum required to see a significant survival benefit compared to saline control (FIG. 10).
  • Mice administered single doses of 90 ⁇ g or higher showed 100% survival to the termination of the experiment.
  • hRS7-ADC conjugates were determined in comparison to hRS7 IgG (FIG. 11).
  • PBMCs were purified from blood purchased from the Blood Center of New Jersey.
  • a Trop-2-positive human pancreatic adenocarcinoma cell line (BxPC-3) was used as the target cell line with an effector to target ratio of 100: 1.
  • ADCC mediated by hRS7 IgG was compared to hRS7-Pro-2-PDox, hRS7-CL2A-SN-38, and the reduced and capped hRS7-NEM. All were used at 33.3 nM.
  • Results are shown in FIG. 11. Overall activity was low, but significant. There was 8.5% specific lysis for the hRS7 IgG which was not significantly different from hRS7-Pro-2- PDox. Both were significantly better than hLL2 control and hRS7-NEM and hRS7-SN-38 ( ⁇ 0.02, two-tailed i-test). There was no difference between hRS7-NEM and hRS7-SN-38.
  • hRS7 SN-38-anti-Trop-2
  • CL2-SN-38 and CL2A-SN-38 were conjugated to the anti-Trop-2-humanized antibody, hRS7.
  • the immunoconjugates were characterized in vitro for stability, binding, and cytotoxicity. Efficacy was tested in five different human solid tumor-xenograft models that expressed Trop-2 antigen. Toxicity was assessed in mice and in Cynomolgus monkeys.
  • the hRS7 conjugates of the two SN-38 derivatives were equivalent in drug substitution ( ⁇ 6), cell binding (K d ⁇ 1.2 nmol/L), cytotoxicity (IC S0 ⁇ 2.2 nmol/L), and serum stability in vitro (t/. /z ⁇ 20 hours). Exposure of cells to the ADC demonstrated signaling pathways leading to PARP cleavage, but differences versus free SN-38 in p53 and p21 upregulation were noted.
  • Cynomolgus monkeys infused with 2 ⁇ 0.96 mg/kg exhibited only transient decreases in blood counts, although, importantly, the values did not fall below normal ranges.
  • the anti-Trop-2 hRS7-CL2A-SN-38 ADC provided significant and specific antitumor effects against a range of human solid tumor types. It was well tolerated in monkeys, with tissue Trop-2 expression similar to humans, at clinically relevant doses.
  • human cancer xenograft models including non-small cell lung carcinoma, pancreatic, colorectal, and squamous cell lung carcinomas, all at nontoxic doses (e.g., ⁇ 3.2 mg/kg cumulative SN-38 equivalent dose).
  • Trop-2 is widely expressed in many epithelial cancers, but also some normal tissues, and therefore a dose escalation study in Cynomolgus monkeys was performed to assess the clinical safety of this conjugate.
  • Monkeys tolerated 24 mg SN-38 equivalents/kg with only minor, reversible, toxicities. Given its tumor-targeting and safety profile, hRS7-SN-38 provides a significant improvement in the management of solid tumors responsive to irinotecan.
  • immunoconjugates and irinotecan are shown in SN-38 equivalents. Based on a mean SN- 38/IgG substitution ratio of 6, a dose of 500 ⁇ g ADC to a 20-g mouse (25 mg/kg) contains 0.4 mg/kg of SN-38. Irinotecan doses are likewise shown as SN-38 equivalents (i.e., 40 mg irinotecan/kg is equivalent to 24 mg/kg of SN-38).
  • Tumor volume was determined by measurements in 2 dimensions using calipers, with volumes defined as: L x w 2 12, where L is the longest dimension of the tumor and w is the shortest. Tumors ranged in size between 0.10 and 0.47 cm 3 when therapy began. Treatment regimens, dosages, and number of animals in each experiment are described in the Results.
  • the lyophilized hRS7-CL2A-SN-38 and control ADC were reconstituted and diluted as required in sterile saline. All reagents were administered intraperitoneally (0.1 mL), except irinotecan, which was administered intravenously.
  • the dosing regimen was influenced by our prior investigations, where the ADC was given every 4 days or twice weekly for varying lengths of time (Moon et al., 2008, J Med Chem 51 :6916-26; Govindan et al., 2009, Clin Chem Res 15:6052-61). This dosing frequency reflected a consideration of the conjugate's serum half-life in vitro, to allow a more continuous exposure to the ADC. [0242] Statistics - Growth curves are shown as percent change in initial TV over time.
  • AUC area under the curve
  • mice were sorted into 4 groups each to receive 2-mL i.p. injections of either a sodium acetate buffer control or 3 different doses of hRS7-CL2A-SN-38 (4, 8, or 12 mg/kg of SN-38) on days 0 and 3 followed by blood and serum collection, as described in Results.
  • Cynomolgus monkeys (3 male and 3 female; 2.5-4.0 kg) were administered 2 different doses of hRS7-CL2A-SN- 38. Dosages, times, and number of monkeys bled for evaluation of possible hematologic toxicities and serum chemistries are described in the Results.
  • hRS7-CL2A and hRS7-CL2-SN-38 were compared in mice bearing COLO 205 (FIG. 12B) or Capan-1 tumors (FIG. 12C), using 0.4 mg or 0.2 mg/kg SN-38 twice weekly 4 weeks, respectively, and with starting tumors of 0.25 cm 3 size in both studies. Both the hRS7-CL2A and CL2-SN-38 conjugates significantly inhibited tumor growth compared to untreated (AUC W plague S P ⁇ 0.002 vs.
  • SN-38 is known to activate several signaling pathways in cells, leading to apoptosis (e.g., Cusack et al., 2001, Cancer Res 61 :3535-40; Liu et al. 2009, Cancer Lett 274:47-53; Lagadec et al., 2008, Br J Cancer 98:335-44).
  • Our initial studies examined the expression of 2 proteins involved in early signaling events (p21 Wafl/Cipl and p53) and 1 late apoptotic event [cleavage of poly-ADP-ribose polymerase (PARP)] in vitro (not shown).
  • PARP poly-ADP-ribose polymerase
  • hRS7 IgG had no appreciable effect on p21 Wafl/Cipl expression, it did induce the upregulation of p53 in both BxPC-3 and Calu-3, but only after a 48-hour exposure (not shown).
  • cleavage of PARP was evident in both cell lines when incubated with either SN-38 or the conjugate (not shown).
  • the presence of the cleaved PARP was higher at 24 hours in BxPC-3 (not shown), which correlates with high expression of p21 and its lower IC 50 .
  • the higher degree of cleavage with free SN-38 over the ADC was consistent with the cytotoxicity findings.
  • the MTD of irinotecan (24 mg SN-38/kg, q2dx5) was as effective as hRS7-CL2-SN- 38 in COLO 205 cells, because mouse serum can more efficiently convert irinotecan to SN- 38 (Morton et al., 2000, Cancer Res 60:4206-10) than human serum, but the SN-38 dose in irinotecan (2,400 ⁇ g cumulative) was 37.5-fold greater than with the conjugate (64 ⁇ g total).
  • mice bearing BxPC-3 human pancreatic tumors (FIG. 13D)
  • tumor uptake in a group of animals that first received a predose of 0.2 mg/kg (250 ⁇ g protein) of the hRS7 ADC 3 days before the injection of the "'In-labeled antibody was examined.
  • AST aspartate transaminase
  • ALT alanine transaminase
  • mice do not express Trop-2 identified by hRS7, a more suitable model was required to determine the potential of the hRS7 conjugate for clinical use.
  • Immunohistology studies revealed binding in multiple tissues in both humans and Cynomolgus monkeys (breast, eye, gastrointestinal tract, kidney, lung, ovary, fallopian tube, pancreas, parathyroid, prostate, salivary gland, skin, thymus, thyroid, tonsil, ureter, urinary bladder, and uterus; not shown). Based on this cross-reactivity, a tolerability study was performed in monkeys.
  • Histopathology of the animals necropsied on day 11 (8 days after last injection) showed microscopic changes in hematopoietic organs (thymus, mandibular and mesenteric lymph nodes, spleen, and bone marrow), gastrointestinal organs (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum), female reproductive organs (ovary, uterus, and vagina), and at the injection site. These changes ranged from minimal to moderate and were fully reversed at the end of the recovery period (day 32) in all tissues, except in the thymus and gastrointestinal tract, which were trending towards full recovery at this later timepoint (not shown).
  • Trop-2 is a protein expressed on many epithelial tumors, including lung, breast, colorectal, pancreas, prostate, and ovarian cancers, making it a potentially important target for delivering cytotoxic agents (Ohmachi et al., 2006, Clin Cancer Res 12:3057-63; Fong et al., 2008, Br J Cancer 99: 1290-95; Cubas et al., 2009, Biochim Biophys Acta 1796:309- 14).
  • the RS7 antibody internalizes when bound to Trop-2 (Shih et al., 1995, Cancer Res
  • SN-38 is a potent topoisomerase-I inhibitor, with IC S0 values in the nanomolar range in several cell lines. It is the active form of the prodrug, irinotecan, that is used for the treatment of colorectal cancer, and which also has activity in lung, breast, and brain cancers. We reasoned that a directly targeted SN-38, in the form of an ADC, would be a significantly improved therapeutic over CPT- 1 1 , by overcoming the latter's low and patient- variable bioconversion to active SN-38 (Mathijssen et al., 2001, Clin Cancer Res 7:2182-94).
  • ENZ-2208 utilizes a branched PEG to link about 3.5 to 4 molecules of SN-38 per PEG, whereas NK012 is a micelle nanoparticle containing 20% SN- 38 by weight.
  • this disparity i.e., ratio of potency with free vs.
  • both the CL2- and CL2A-SN-38 forms of hRS7-SN-38 yielded a t/. /z of ⁇ 20 hours, which is in contrast to the short f/ K of 12.3 minutes reported for ENZ-2208 (Zhao et al., 2008, Bioconjug Chem 19:849- 59), but similar to the 57% release of SN-38 from NK012 under physiological conditions after 24 hours (Koizumi et al., 2006, Cancer Res 66: 10048-56).
  • hRS7-SN-38 Treatment of tumor-bearing mice with hRS7-SN-38 (either with CL2-SN-38 or CL2A-SN-38) significantly inhibited tumor growth in 5 different tumor models. In 4 of them, tumor regressions were observed, and in the case of Calu-3, all mice receiving the highest dose of hRS7-SN-38 were tumor-free at the conclusion of study. Unlike in humans, irinotecan is very efficiently converted to SN-38 by a plasma esterase in mice, with a greater than 50% conversion rate, and yielding higher efficacy in mice than in humans (Morton et al., 2000, Cancer Res 60:4206-10; Furman et al., 1999, J Clin Oncol 17: 1815-24).
  • hRS7-SN-38 was significantly better in controlling tumor growth. Only when irinotecan was administered at its MTD of 24 mg/kg q2dx5 (37.5-fold more SN-38) did it equal the effectiveness of hRS7-SN- 38. In patients, we would expect this advantage to favor hRS7-CL2A-SN-38 even more, because the bioconversion of irinotecan would be substantially lower.
  • Trop-2 recognized by hRS7 is not expressed in mice, it was important to perform toxicity studies in monkeys that have a similar tissue expression of Trop-2 as humans. Monkeys tolerated 0.96 mg/kg/dose ( ⁇ 12 mg/m 2 ) with mild and reversible toxicity, which extrapolates to a human dose of ⁇ 0.3 mg/kg/dose ( ⁇ 1 1 mg/m 2 ). In a Phase I clinical trial of NK012, patients with solid tumors tolerated 28 mg/m 2 of SN-38 every 3 weeks with Grade 4 neutropenia as dose-limiting toxicity (DLT; Hamaguchi et al., 2010, Clin Cancer Res 16:5058-66).
  • Phase I clinical trials with ENZ-2208 revealed dose-limiting febrile neutropenia, with a recommendation to administer 10 mg/m 2 every 3 weeks or 16 mg/m 2 if patients were administered G-CSF (Kurzrock et al., AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2009 Nov 15-19; Boston, MA; Poster No C216; Patnaik et al., AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2009 Nov 15-19; Boston, MA; Poster No C221).
  • ADC antibody-drug conjugate
  • TAXOL® hRS7 anti-human Trop-2 antibody
  • the final product had a mean drug to antibody substitution ratio of 2.2.
  • This ADC was tested in vitro using two different Trop-2-postive cell lines as targets: BxPC-3 (human pancreatic adenocarcinoma) and MDA-MB-468 (human triple negative breast carcinoma).
  • BxPC-3 human pancreatic adenocarcinoma
  • MDA-MB-468 human triple negative breast carcinoma
  • hRS7-SN-38 and free SN-38 were also tested at a range of 3.84 x 10 "12 to 2.5 x 10 "7 M. Plates were incubated at 37°C for 96 h. After this incubation period, an MTS substrate was added to all of the plates and read for color development at half-hour intervals until untreated control wells had an OD492nm reading of approximately 1.0. Growth inhibition was measured as a percent of growth relative to untreated cells using Microsoft Excel and Prism software (non-linear regression to generate sigmoidal dose response curves which yield ICso-values).
  • the hRS7-paclitaxel ADC exhibited cytotoxic activity in the MDA-MB-468 breast cell line (FIG. 15), with an ICso-value approximately 4.5-fold higher than hRS7-SN-38.
  • the free paclitaxel was much more potent than the free SN-38 (FIG. 15). While the IC 50 for free SN-38 was 1.54xl0 "9 M, the IC 50 for free paclitaxel was less than 6.1xl0 "n M. Similar results were obtained for the BxPC-3 pancreatic cell line (FIG.
  • Example 7 Cytotoxicity of Anti-Trop-2 ADC (MAB650-SN-38) [0276] A novel anti-Trop-2 ADC was made with SN-38 and MAB650, yielding a mean drug to antibody substitution ratio of 6.89. Cytotoxicity assays were performed to compare the MAB650-SN-38 and hRS7-SN-38 ADCs using two different human pancreatic
  • adenocarcinoma cell lines BxPC-3 and Capan-1
  • MDA-MB-468 human triple negative breast carcinoma cell line
  • hRS7-SN-38 and MAB650-SN-38 had similar growth- inhibitory effects with ICso-values in the low nM range which is typical for SN-38-ADCs in these cell lines.
  • the hRS7-SN-38 ADC showed an IC 50 of 3.5 nM, compared to 4.1 nM for the MAB650-SN-38 ADC and 1.0 nM for free SN-38.
  • the human BxPC-3 pancreatic adenocarcinoma cell line FIG. 17A
  • the hRS7-SN-38 ADC showed an IC 50 of 2.6 nM, compared to 3.0 nM for the MAB650-SN-38 ADC and 1.0 nM for free SN-38.
  • the hRS7-SN-38 ADC showed an IC 50 of 3.6 nM, compared to 4.1 nM for the MAB650-SN-38 ADC and 4.3 nM for free SN-38.
  • a novel anti-Trop-2 ADC was made with SN-38 and 162-46.2, yielding a drug to antibody substitution ratio of 6.14. Cytotoxicity assays were performed to compare the 162- 46.2-SN-38 and hRS7-SN-38 ADCs using two different Trop-2-postive cell lines as targets, the BxPC-3 human pancreatic adenocarcinoma and the MDA-MB-468 human triple negative breast carcinoma.
  • the 162-46.2-SN-38 ADC had a similar IC 50 - values when compared to hRS7-SN-38.
  • hRS7-SN-38 had an IC50 of 5.8 nM, compared to 10.6 nM for 162-46.2-SN-38 and 1.6 nM for free SN-38.
  • MDA-MB-468 human breast adenocarcinoma cell line
  • hRS7-SN-38 had an IC 50 of 3.9 nM, compared to 6.1 nM for 162-46.2-SN-38 and 0.8 nM for free SN-38.
  • the free antibodies alone showed little cytotoxicity to either Trop-2 positive cancer cell line.
  • the present Example reports results from a phase I clinical trial and ongoing phase II extension with IMMU-132, an ADC of the internalizing, humanized, hRS7 anti-Trop-2 antibody conjugated by a pH-sensitive linker to SN-38 (mean drug-antibody ratio 7.6).
  • Trop-2 is a type I transmembrane, calcium- transducing, protein expressed at high density ( ⁇ 1 x 10 5 ), frequency, and specificity by many human carcinomas, with limited normal tissue expression.
  • IMMU-132 is capable of delivering as much as 120-fold more SN- 38 to tumor than derived from a maximally tolerated irinotecan therapy.
  • the present Example reports the initial Phase I trial of 25 patients who had failed multiple prior therapies (some including topoisomerase-I/II inhibiting drugs), and the ongoing Phase II extension now reporting on 69 patients, including in colorectal (CRC), small-cell and non-small cell lung (SCLC, NSCLC, respectively), triple-negative breast (TNBC), pancreatic (PDC), esophageal, and other cancers.
  • CRC colorectal
  • SCLC small-cell and non-small cell lung
  • NSCLC non-small cell lung
  • TNBC triple-negative breast
  • PDC pancreatic
  • esophageal esophageal
  • Trop-2 was not detected in serum, but was strongly expressed (>2 + immunohistochemical staining) in most archived tumors.
  • IMMU- 132 was given on days 1 and 8 in repeated 21 -day cycles, starting at 8 mg/kg/dose, then 12 and 18 mg/kg before dose-limiting neutropenia.
  • neutropenia >Grade 3 occurred in 28% (4% Grade 4).
  • Best Response data of 8 assessable patients with TNBC (triple-negative breast cancer), there were 2 PR (partial response), 4 SD (stable disease) and 2 PD
  • Exemplary partial responses to the anti-Trop-2 ADC were confirmed by CT data (not shown).
  • CT data As an exemplary PR in CRC, a 62 year-old woman first diagnosed with CRC underwent a primary hemicolectomy. Four months later, she had a hepatic resection for liver metastases and received 7 mos of treatment with FOLFOX and 1 mo 5FU. She presented with multiple lesions primarily in the liver (3+ Trop-2 by immunohistology), entering the hRS7-SN-38 trial at a starting dose of 8 mg/kg about 1 year after initial diagnosis. On her first CT assessment, a PR was achieved, with a 37% reduction in target lesions (not shown). The patient continued treatment, achieving a maximum reduction of 65% decrease after 10 months of treatment (not shown) with decrease in CEA from 781 ng/mL to 26.5 ng/mL), before progressing 3 months later.
  • IMMU-132 showed evidence of activity (PR and durable SD) in relapsed/refractory patients with triple-negative breast cancer, small cell lung cancer, non-small cell lung cancer, colorectal cancer and esophageal cancer, including patients with a previous history of relapsing on topoisomerase-I inhibitor therapy.
  • mice Female athymic nude mice were injected s.c. with 200 of NCI-N87 cell suspension mixed 1 : 1 with matrigel such that lxl 0 7 cells was administered to each mouse. Once tumors reached approximately 0.25 cm 3 in size (6 days later), the animals were divided up into seven different treatment groups of nine mice each.
  • mice received 500 ⁇ g i.v. injections once a week for two weeks.
  • Control mice received the non- tumor targeting hA20-SN-38 ADC at the same dose/schedule.
  • mice were administered 45 ⁇ g i.v. twice weekly for two weeks.
  • mice received hA20- Pro-2-PDox ADC at the same dose/schedule.
  • a final group of mice received only saline and served as the untreated control. Tumors were measured and mice weighed twice a week. Mice were euthanized for disease progression if their tumor volumes exceeded 1.0 cm 3 in size.
  • mice treated with hRS7-Pro-2-PDox experienced tumor regressions and an overall anti-tumor response that was significantly better than 162-46.2-Pro-2-PDox treatment ( ⁇ 0.0001 ; AUC). While the tumors did not immediately respond to the 162-46.2-Pro-2-PDox therapy, they did eventually regress. This regression did not occur until approximate 1 1 days after the start of therapy.
  • IMMU-132 (hRS7-SN-38) is an anti-Trop-2 ADC comprising the cancer cell internalizing, humanized, anti-Trop-2 hRS7 antibody, conjugated by a pH-sensitive linker to SN-38, the active metabolite of irinotecan, at a mean drug-antibody ratio of 7.6.
  • Trop-2 is a type-I transmembrane, calcium-transducing protein expressed at high density, frequency, and specificity in many epithelial cancers, including pancreatic ductal adenocarcinoma, with limited normal tissue expression.
  • pancreatic tumor microarray specimens tested were Trop-2-positive by immunohistochemistry, and human pancreatic cancer cell lines were found to express 1 15k-891k Trop-2 copies on the cell membrane.
  • IMMU-132 Phase I study enrolling patients with 13 different tumor types using a 3+3 design.
  • the Phase I dose-limiting toxicity was neutropenia.
  • Over 80% of 24 assessable patients in this study had long-term stable disease, with partial responses (RECIST) observed in patients with colorectal (CRC), triple -negative breast (TNBC), small-cell and non-small cell lung (SCLC, NSCLC), and esophageal (EAC) cancers.
  • CRC colorectal
  • TNBC triple -negative breast
  • SCLC small-cell and non-small cell lung
  • EAC esophageal
  • the present Example reports the results from the IMMU-132 Phase I/II study cohort of patients with metastatic PDC. Patients with PDC who failed a median of 2 prior therapies (range 1-5) were given IMMU-132 on days 1 and 8 in repeated 21 -day cycles.
  • IMMU- 132 is active (long-term stable disease) in 62% (8/13) of PDC patients who failed multiple prior therapies, with manageable neutropenia and little GI toxicity.
  • Advanced PDC patients can be given repeated treatment cycles (>6) of 8-10 mg/kg IMMU- 132 on days 1 and 8 of a 21 -day cycle, with some dose adjustments or growth factor support for neutropenia in subsequent treatment cycles.
  • monotherapy IMMU-132 is a novel, efficacious treatment regimen for patients with PDC, including those with tumors that were previously resistant to other therapeutic regimens for PDC.
  • Trop-2 expression The expression of Trop-2 on the surface of various cancer cell lines was determined by flow cytometry using QUANTBRITE® PE beads. The results for number of Trop-2 molecules detected in the different cell lines was: BxPC-3 pancreatic cancer (891,000); NCI-N87 gastric cancer (383,000); MDA-MB-468 breast cacner (341,000); SK-MES-1 squamous cell lung cancer (27,000); Capan-1 pancreatic cancer (115,000); AGS gastric cancer (78,000) COLO 205 colon cancer (52,000). Trop-2 expression was also observed in 29 of 29 (100%) tissue microarrays of pancreatic adenocarcinoma (not shown).
  • Necropsies were performed on 3 animals per interval, in irinotecan injected mice at 5 min, 1, 2, 6 and 24 hours or in IMMU-132 injected mice at 1, 6, 24, 48 and 72 h. Tissues were extracted and analyzed by reversed-phase HPLC analysis for SN-38, SN-38G, and irinotecan. Extracts from IMMU- 132-treated animals also were acid hydrolyzed to release SN-38 from the conjugate (i.e., SN-38 (TOTAL]). The results, shown in FIG. 23, demonstrate that the IMMU- 132 ADC has the potential to deliver 120 times more SN-38 to the tumor compared to irinotecan, even though 22-fold less SN-38 equivalents were administered with the ADC.
  • IMMU- 132 clinical protocol The protocol used in the phase I/II study was as indicated in Table 9 below.
  • Standard Phase I [3+3] design includes 15 patients in select cancers.
  • Grade 4 ANC > 7 d; >Grade 3 febrile neutropenia of any duration; Grade 4 Platelets > 5 d; Grade 4 Hgb; Grade 4 N/V/D of any duration or any Grade 3 N/V/D for > 48 h; Grade 3 infusion-related reactions; > Grade 3 non- heme toxicity at least possibly due to study drug.
  • An exemplary case study is as follows.
  • a 34 y/o white male initially diagnosed with metastatic pancreatic cancer (liver) had progressed on multiple chemotherapy regimens, including gemcitabine/ Erlotinib/FG-3019, FOLFIRINOX and GTX prior to introduction of IMMU- 132 (8 mg/kg dose given days 1 and 8 of a 21 day cycle).
  • the patient received the drug for 4 mo with good symptomatic tolerance, an improvement in pain, a 72% maximum decline in CA19-9 (from 15885 U/mL to 4418 U/mL) and stable disease by CT RECIST criteria along with evidence of tumor necrosis.
  • Therapy had to be suspended due to a liver abscess; the patient expired ⁇ 6 weeks later, 6 mo following therapy initiation.
  • IMMU- 132 delivers 120-times the amount of SN-38 to a human pancreatic tumor xenograft than when irinotecan is given.
  • Phase 2 dose of IMMU- 132 was determined to be 8 to 10 mg/kg, based on manageable neutropenia and diarrhea as the major side effects. No anti-antibody or anti-SN-38 antibodies have been detected to-date, even with repeated therapeutic cycles.
  • pro-2-PDox-hRS7 ADC is prepared as described in the Examples above. Patients with triple -negative breast cancer who had failed at least two standard therapies receive 3 cycles of 70 mg pro-2-PDox-hRS7 injected i.v. every 3 weeks. Objective responses are observed at this dose level of pro-2-PDox- hRS7, with an average decrease in tumor volume of 35%, after two cycles of therapy. All serum samples evaluated for human anti-hRS7 antibody (HAHA) are negative.
  • a 52-year old man with metastatic colon cancer (3-5 cm diameters) to his left and right liver lobes, as well as a 5 cm metastasis to his right lung, and an elevated blood CEA value of 130 ng/mL, is treated with a 100 mg dose of hRS7 anti-Trop-2 conjugated with pro- 2-PDox at 4 drug molecules per IgG, administered by slow intravenous infusion every other week for 4 doses.
  • a 62-year-old man with metastatic ductal adenocarcinoma of the pancreas who has relapsed after prior therapies with FOLFIRINOX followed by Nab-taxol (Abraxane®) plus gemcitabine is given hRS7-pro-2-PDox ADC at a dose of 120 mg every third week for 4 courses, and after a 3 -week delay, another course of 2 injections 2 weeks apart are given intravenously.
  • the patient shows some nausea and transient diarrhea with the therapy, and also Grade 3 neutropenia after the first course, which recovers before the second course of therapy.
  • CT measurements made at 8 weeks following start of therapy show an 18% shrinkage of the sum of the 3 target lesions in the liver, as compared to the pretreatment baseline measurements, constituting stable disease by RECIST 1.1 criteria. Also, the patient's CA19-9 blood titer is reduced by 55% from a baseline value of 12,400. His general symptoms of weakness, fatigue and abdominal discomfort also improve considerably, including regaining his appetite and a weight increase of 2 kg during the following 6 weeks.
  • an anti-Trop-2 ADC composed of hRS7 IgG linked to SN-38 shows anti-tumor activity in various solid tumors.
  • This ADC is very well tolerated in mice (e.g., > 60 mg), yet just 4.0 mg (0.5 mg, twice-weekly x 4) is significantly therapeutic.
  • Trop-2 is also expressed in most pancreatic cancers.
  • mice bearing 0.35 cm 3 subcutaneous xenografts of the human pancreatic cancer cell line, Capan-1.
  • MTD maximum tolerated dose
  • the SN-38 conjugated hRS7 antibody was prepared as described above and according to previously described protocols (Moon et al. J Med Chem 2008, 51 :6916-6926; Govindan et al., Clin Cancer Res 2009. 15:6052-6061).
  • a reactive bifunctional derivative of SN-38 (CL2A-SN-38) was prepared.
  • the formula of CL2A-SN-38 is (maleimido-[x]-Lys- PABOCO-20-O-SN-38, where PAB is p-aminobenzyl and 'x' contains a short PEG).
  • the CL2A-SN-38 was reacted with reduced antibody to generate the SN-38 conjugated RS7.
  • 90 Y-hPAM4 is prepared as previously described (Gold et al., Clin Cancer Res 2003, 9:3929S-37S; Gold et al., Int J Cancer 2004, 109:618-26).
  • the Trop-2 antigen is expressed in most epithelial cancers (lung, breast, prostate, ovarian, colorectal, pancreatic) and hRS7-SN-38 conjugates are being examined in various human cancer-mouse xenograft models.
  • Initial clinical trials with 90 Y-hPAM4 IgG plus radiosensitizing amounts of GEM are encouraging, with evidence of tumor shrinkage or stable disease.
  • therapy of pancreatic cancer is very challenging. Therefore, a combination therapy was examined to determine whether it would induce a better response.
  • administration ofhRS7-SN-38 at effective, yet non-toxic doses was combined with RAIT with 90 Y-hPAM4 IgG.

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Abstract

Cette invention concerne des compositions et des méthodes d'utilisation de conjugués anticorps-médicament (ADC) comprenant un anticorps anti-Trop-2 ou un fragment de celui-ci se liant à l'antigène, conjugué à un ou à plusieurs médicaments cytotoxiques. De préférence, l'anticorps est un anticorps RS7, 162-46.2 ou MAB650, de préférence, humanisé. De préférence le médicament est un SN-38, une pro-2-pyrrolino-doxorubicine, le paclitaxel, la calichemicine, un DM1, DM3, DM4, MMAE, MMAD ou MMAF. Les compositions et les méthodes selon l'invention sont utiles pour traiter les cancers exprimant Trop-2, tels que le cancer du sein, de l'ovaire, du col de l'utérus, de l'endomètre, du poumon, de la prostate, du côlon, de l'estomac, de l'œsophage, de la vessie, du rein, du pancréas, de la thyroïde, de l'épithélium ou de la tête et du cou. De préférence, le cancer est un cancer résistant à une ou plusieurs thérapies anticancéreuses standards. De préférence encore, l'anticorps anti-Trop-2 se lie à la protéine Trop-2 exprimée à la surface de cellules normales, mais l'administration de l'ADC anti-Trop-2 à des patients humains atteints de cancer à une dose thérapeutiquement efficace ne produit qu'une toxicité limitée.
EP14847616.1A 2013-09-27 2014-07-01 Conjugués anticorps anti-trop-2-médicament et leurs utilisations Pending EP3049443A4 (fr)

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US14/040,024 US8758752B2 (en) 2002-03-01 2013-09-27 RS7 antibodies
US14/258,228 US9138485B2 (en) 2002-12-13 2014-04-22 Immunoconjugates with an intracellularly-cleavable linkage
US14/259,469 US9833511B2 (en) 2002-03-01 2014-04-23 RS7 antibodies
PCT/US2014/045074 WO2015047510A1 (fr) 2013-09-27 2014-07-01 Conjugués anticorps anti-trop-2-médicament et leurs utilisations

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10422788B2 (en) 2016-12-19 2019-09-24 Tolero Pharmaceuticals, Inc. Profiling peptides and methods for sensitivity profiling
US10568887B2 (en) 2015-08-03 2020-02-25 Tolero Pharmaceuticals, Inc. Combination therapies for treatment of cancer
US10624880B2 (en) 2015-04-20 2020-04-21 Tolero Pharmaceuticals, Inc. Predicting response to alvocidib by mitochondrial profiling
US11279694B2 (en) 2016-11-18 2022-03-22 Sumitomo Dainippon Pharma Oncology, Inc. Alvocidib prodrugs and their use as protein kinase inhibitors
US11497756B2 (en) 2017-09-12 2022-11-15 Sumitomo Pharma Oncology, Inc. Treatment regimen for cancers that are insensitive to BCL-2 inhibitors using the MCL-1 inhibitor alvocidib

Families Citing this family (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10058621B2 (en) 2015-06-25 2018-08-28 Immunomedics, Inc. Combination therapy with anti-HLA-DR antibodies and kinase inhibitors in hematopoietic cancers
US9707302B2 (en) 2013-07-23 2017-07-18 Immunomedics, Inc. Combining anti-HLA-DR or anti-Trop-2 antibodies with microtubule inhibitors, PARP inhibitors, bruton kinase inhibitors or phosphoinositide 3-kinase inhibitors significantly improves therapeutic outcome in cancer
RU2605390C2 (ru) 2011-08-23 2016-12-20 Рош Гликарт Аг Биспецифические антитела, специфичные к антигенам, активирующим т-клетки, и опухолевому антигену, и способы их применения
US11253606B2 (en) 2013-07-23 2022-02-22 Immunomedics, Inc. Combining anti-HLA-DR or anti-Trop-2 antibodies with microtubule inhibitors, PARP inhibitors, Bruton kinase inhibitors or phosphoinositide 3-kinase inhibitors significantly improves therapeutic outcome in cancer
JP2018500930A (ja) 2014-12-04 2018-01-18 アブルッツォ・セラノスティックス・エッセ・エッレ・エッレ ヒト化抗ｔｒｏｐ−２モノクローナル抗体及びその使用
EP3286224A4 (fr) * 2015-04-22 2018-11-14 Immunomedics, Inc. Isolement, détection, diagnostic et/ou caractérisation de cellules cancéreuses trop-2 positives
ES2739749T3 (es) 2015-05-18 2020-02-03 Tolero Pharmaceuticals Inc Profármacos de alvocidib que tienen biodisponibilidad aumentada
JP6980980B2 (ja) * 2015-06-25 2021-12-15 イミューノメディクス、インコーポレイテッドImmunomedics, Inc. 抗ｈｌａ‐ｄｒまたは抗ｔｒｏｐ‐２抗体と微小管阻害剤、ｐａｒｐ阻害剤、ブルトンキナーゼ阻害剤またはホスホイノシチド３‐キナーゼ阻害剤との併用は癌の治療効果を有意に改善する
US10195175B2 (en) 2015-06-25 2019-02-05 Immunomedics, Inc. Synergistic effect of anti-Trop-2 antibody-drug conjugate in combination therapy for triple-negative breast cancer when used with microtubule inhibitors or PARP inhibitors
CN106729743B (zh) * 2015-11-23 2021-09-21 四川科伦博泰生物医药股份有限公司 抗ErbB2抗体-药物偶联物及其组合物、制备方法和应用
CN109310385A (zh) * 2016-04-27 2019-02-05 免疫医疗公司 抗trop-2-sn-38抗体药物缀合物用于检查点抑制剂复发/难治的肿瘤的疗法的功效
AU2017274442B2 (en) 2016-06-02 2021-08-19 Abbvie Inc. Glucocorticoid receptor agonist and immunoconjugates thereof
WO2018036438A1 (fr) * 2016-08-23 2018-03-01 四川科伦博泰生物医药股份有限公司 Conjugué anticorps-médicament et son procédé de préparation et d'application
CN108066772B (zh) * 2016-11-14 2021-07-13 中国科学院上海药物研究所 靶向tacstd2的抗体与药物偶联体(adc)分子
US10918734B2 (en) * 2017-03-27 2021-02-16 Immunomedics, Inc. Treatment of high Trop-2 expressing triple negative breast cancer (TNBC) with sacituzumab govitecan (IMMU-132) overcomes homologous recombination repair (HRR) rescue mediated by Rad51
CN107446050A (zh) * 2017-08-11 2017-12-08 百奥泰生物科技(广州)有限公司 Trop2阳性疾病治疗的化合物及方法
CN107674051B (zh) * 2017-09-20 2021-01-08 南京医科大学 4-羟基他莫昔酚环状衍生物乏氧活化前药及其药物用途
EP3884962A1 (fr) 2017-12-01 2021-09-29 AbbVie Inc. Agoniste du récepteur aux glucocorticoïdes et immunoconjugués correspondants
AU2019304175A1 (en) * 2018-07-09 2021-03-04 Genequantum Healthcare (Suzhou) Co., Ltd. Antibodies specific to trophoblast antigen 2 (TROP2)
EP3890749A4 (fr) 2018-12-04 2022-08-03 Sumitomo Dainippon Pharma Oncology, Inc. Inhibiteurs de cdk9 et leurs polymorphes destinés à être utilisés en tant qu'agents pour le traitement du cancer
US11793802B2 (en) 2019-03-20 2023-10-24 Sumitomo Pharma Oncology, Inc. Treatment of acute myeloid leukemia (AML) with venetoclax failure
CN112390885B (zh) * 2019-08-12 2024-01-19 凯惠科技发展(上海)有限公司 一种trop2抗体及其制备方法、其偶联物和应用
CN114901694A (zh) * 2019-12-31 2022-08-12 启德医药科技（苏州）有限公司 抗trop2抗体、抗体-药物缀合物及其应用
EP4095148A4 (fr) * 2020-01-22 2023-07-26 Jiangsu Hengrui Medicine Co., Ltd. Conjugué anticorps anti-trop-2-analogue d'exatécan et son utilisation médicale
CN115398010A (zh) * 2020-03-20 2022-11-25 免疫医疗公司 用于戈沙妥珠单抗疗法的生物标志物
IT202000031838A1 (it) * 2020-12-22 2022-06-22 Saverio Alberti Piattaforma per ottenere anticorpi monoclonali diretti contro antigeni processati tumore-specifici
US20240207432A1 (en) * 2021-04-21 2024-06-27 Biosion Inc. Antibodies binding trop2 and uses thereof
CN113480651B (zh) * 2021-07-19 2022-07-08 华东师范大学 一种靶向人cd133的纳米抗体及其制备方法和应用
WO2024012566A2 (fr) * 2022-07-15 2024-01-18 Genequantum Healthcare (Suzhou) Co., Ltd. Anticorps, lieurs, charge utile, conjugués et leurs applications

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067038A1 (fr) * 2003-01-24 2004-08-12 Immunomedics, Inc. Conjugues d'anticorps et d'anthracycline
US7238785B2 (en) * 2002-03-01 2007-07-03 Immunomedics, Inc. RS7 antibodies
WO2010093395A1 (fr) * 2009-02-13 2010-08-19 Immunomedics, Inc. Immunoconjugués avec une liaison intracellulaire clivable
US20110070156A1 (en) * 2002-06-14 2011-03-24 Immunomedics, Inc. Combining Radioimmunotherapy and Antibody-Drug Conjugates for Improved Cancer Therapy
WO2015012904A2 (fr) * 2012-12-13 2015-01-29 Immunomedics, Inc. Immunoconjugués anticorps-sn-38 avec un lieur cl2a

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10204792A1 (de) * 2002-02-06 2003-08-14 Merck Patent Gmbh Lyophilisierte Zubereitung enthaltend Immuncytokine
US8551480B2 (en) * 2004-02-13 2013-10-08 Immunomedics, Inc. Compositions and methods of use of immunotoxins comprising ranpirnase (Rap) show potent cytotoxic activity

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7238785B2 (en) * 2002-03-01 2007-07-03 Immunomedics, Inc. RS7 antibodies
US20110070156A1 (en) * 2002-06-14 2011-03-24 Immunomedics, Inc. Combining Radioimmunotherapy and Antibody-Drug Conjugates for Improved Cancer Therapy
WO2004067038A1 (fr) * 2003-01-24 2004-08-12 Immunomedics, Inc. Conjugues d'anticorps et d'anthracycline
WO2010093395A1 (fr) * 2009-02-13 2010-08-19 Immunomedics, Inc. Immunoconjugués avec une liaison intracellulaire clivable
WO2015012904A2 (fr) * 2012-12-13 2015-01-29 Immunomedics, Inc. Immunoconjugués anticorps-sn-38 avec un lieur cl2a

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
C.-H. CHANG ET AL: "Combining ABCG2 Inhibitors with IMMU-132, an Anti-Trop-2 Antibody Conjugate of SN-38, Overcomes Resistance to SN-38 in Breast and Gastric Cancers", MOLECULAR CANCER THERAPEUTICS, vol. 15, no. 8, 1 August 2016 (2016-08-01), US, pages 1910 - 1919, XP055349069, ISSN: 1535-7163, DOI: 10.1158/1535-7163.MCT-16-0219 *
See also references of WO2015047510A1 *
T. M. CARDILLO ET AL: "Humanized Anti-Trop-2 IgG-SN-38 Conjugate for Effective Treatment of Diverse Epithelial Cancers: Preclinical Studies in Human Cancer Xenograft Models and Monkeys", CLINICAL CANCER RESEARCH, vol. 17, no. 10, 3 March 2011 (2011-03-03), pages 3157 - 3169, XP055044596, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-10-2939 *

Cited By (7)

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US10624880B2 (en) 2015-04-20 2020-04-21 Tolero Pharmaceuticals, Inc. Predicting response to alvocidib by mitochondrial profiling
US10568887B2 (en) 2015-08-03 2020-02-25 Tolero Pharmaceuticals, Inc. Combination therapies for treatment of cancer
US10682356B2 (en) 2015-08-03 2020-06-16 Tolero Pharmaceuticals, Inc. Combination therapies for treatment of cancer
US10835537B2 (en) 2015-08-03 2020-11-17 Sumitomo Dainippon Pharma Oncology, Inc. Combination therapies for treatment of cancer
US11279694B2 (en) 2016-11-18 2022-03-22 Sumitomo Dainippon Pharma Oncology, Inc. Alvocidib prodrugs and their use as protein kinase inhibitors
US10422788B2 (en) 2016-12-19 2019-09-24 Tolero Pharmaceuticals, Inc. Profiling peptides and methods for sensitivity profiling
US11497756B2 (en) 2017-09-12 2022-11-15 Sumitomo Pharma Oncology, Inc. Treatment regimen for cancers that are insensitive to BCL-2 inhibitors using the MCL-1 inhibitor alvocidib

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