WO2021086981A1 - Compositions and methods for treating cancer using anti-her2 antibody drug conjugate - Google Patents

Compositions and methods for treating cancer using anti-her2 antibody drug conjugate Download PDF

Info

Publication number
WO2021086981A1
WO2021086981A1 PCT/US2020/057761 US2020057761W WO2021086981A1 WO 2021086981 A1 WO2021086981 A1 WO 2021086981A1 US 2020057761 W US2020057761 W US 2020057761W WO 2021086981 A1 WO2021086981 A1 WO 2021086981A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
cancer
dose
her2
dp303c
Prior art date
Application number
PCT/US2020/057761
Other languages
French (fr)
Inventor
Lixin Feng
Lisha ALLEN
Original Assignee
Cspc Dophen Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cspc Dophen Corporation filed Critical Cspc Dophen Corporation
Priority to CN202080076131.1A priority Critical patent/CN114901308A/en
Publication of WO2021086981A1 publication Critical patent/WO2021086981A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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
    • A61K47/6855—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present application is in the field of cancer therapeutics, and relates to compositions and methods for treating cancers using an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the human epidermal growth factor receptor (HER) family plays an important role in the pathogenesis of many tumors.
  • the HER2 receptor is overexpressed in many tumors (25% of breast, 20% of ovarian, 30% of intestinal type gastric, 20% of lung cancers and such overexpression is associated with aggressive tumors and poor prognosis (Slamon DJ et al, Science, 1987, 235(4785): 177; Slamon DJ et al, Science, 1989, 244(495): 707; Morrison C et al, J. Clin. Oncol., 2006, 24(15):2376).
  • HER2 receptor is overexpressed in many tumors (25% of breast, 20% of ovarian, 30% of intestinal type gastric, 20% of lung cancers and such overexpression is associated with aggressive tumors and poor prognosis (Slamon DJ et al, Science, 1987, 235(4785): 177; Slamon DJ et al, Science, 1989, 244(495): 707; Morrison C e
  • the ado-trastuzumab emtansine group demonstrated statistically significant improvements in objective response rate (43.6%), progression-free survival (median 9.6 months) and overall survival (median 30.9 months).
  • the prescribing information includes boxed warnings for hepatotoxicity, cardiac toxicity, and embryo-fetal toxicity. Additional warnings and precautions are listed for pulmonary toxicity, infusion-related reactions, hemorrhage, thrombocytopenia, and neurotoxicity. As noted in an editorial that discussed results from a more recent trial of ado-trastuzumab emtansine (K.
  • the efficacy of ado-trastuzumab emtansine may be limited by “...poor internalization of the HER2-T-DM1 complex, defective intracellular and endosomal trafficking of the HER2–T-DM1 complex, defective lysosomal degradation of T-DM1, and increased expression of drug efflux pumps, such as multidrug resistance 1.”
  • a new ADC that improves on the efficacy and/or safety profile of ado- trastuzumab emtansine could provide a beneficial treatment alternative for patients with HER2-positive cancers.
  • the present invention in one aspect provides a method of treating a HER2-positive cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue.
  • the HER2-positive cancer is HER23+ as determined by an immunohistochemistry (IHC) test. In some embodiments, the HER2-positive cancer is HER2 2+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by a Fluorescence In Situ Hybridization (FISH) test.
  • FISH Fluorescence In Situ Hybridization
  • the HER2-positive cancer is resistant or refractory to a second HER2-targeted agent.
  • the second HER-2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib.
  • the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of no more than about 6 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg to about 8 mg/kg.
  • the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1 mg/kg to about 2 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 2 mg/kg to about 3 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 2.0 mg/kg.
  • the antibody- drug conjugate is administered at a dose of about 3.0 mg/kg.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg.
  • the antibody- drug conjugate is administered at a dose of no more than about 6 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg to about 8 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1 mg/kg to about 2 mg/kg.
  • the antibody-drug conjugate is administered at a dose of about 2 mg/kg to about 3 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 2.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 3.0 mg/kg. [0012] In some embodiments according to any one of the methods described above, the antibody-drug conjugate is administered intravenously. In some embodiments, the antibody- drug conjugate is administered about once every three weeks, about every other week, or about once per week. In some embodiments, the individual is human.
  • the cancer is a solid cancer.
  • the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer.
  • the cancer is an advanced-stage cancer.
  • the cancer is metastatic cancer.
  • the individual has failed a prior cancer therapy, such as an anti-Her2 antibody therapy (e.g., trastuzumab).
  • the anti-HER2 antibody is N-glycosylated in the Fc region.
  • the acceptor glutamine residue is Q295 in the heavy chain of the anti-HER2 antibody according to the EU numbering.
  • each heavy chain of the HER2 antibody is conjugated to the conjugation moiety.
  • the conjugation moiety is conjugated to the acceptor glutamine residue by transglutamination.
  • the anti-HER2 antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6.
  • VH heavy chain variable region
  • HC-CDR heavy chain complementarity-determining region
  • LC-CDR light chain complementarity-determining region
  • the anti-HER2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the Fc region is an IgG1 Fc.
  • the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10.
  • the conjugation moiety comprises a cleavable linker.
  • the toxin is a monomethyl auristatin E (MMAE).
  • MMAE monomethyl auristatin E
  • the conjugation moiety has the chemical structure of Formula (III):
  • the conjugation moiety has the chemical structure of Formula (I): In some embodiments, the conjugation moiety is LND1002. [0018] In some embodiments according to any one of the methods described above, the antibody-drug conjugate is DP303c. [0019] Also provided are kits and articles of manufacture for use in any of the methods described above. [0020] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art.
  • FIG.1 depicts a schematic structure of DP001.
  • FIG.2 shows the cell proliferation inhibition curves of SK-BR-3 cells (HER23+ cell line) following treatment with DP001 (blue circles and blue line), DP303c (green triangles and green line), or T-DM1 (brown squares and brown line).
  • FIG.3 shows the cell proliferation inhibition curves of JIMT-1 cells (HER22+ cell line) following treatment with DP001 (blue circles and blue line), DP303c (green triangles and green line), or T-DM1 (brown squares and brown line).
  • FIG.4 shows the cell proliferation inhibition curves of Hs746T cells (HER2 negative cell line) following treatment with DP001 (blue circles and blue line), DP303c (green triangles and green line), or T-DM1 (brown squares and brown line).
  • FIG.5 shows effect of DP303c on growth of human gastric cancer xenograft model NCI-N87. Athymic nude mice were implanted with the NCI-N87 cells (HER23+ cell line).
  • Animals were administered by a single intravenous dose of vehicle (PBS, grey closed circles and grey line), DP303c at 2 mg/kg (purple open diamonds and purple dotted line), DP303c at 4 mg/kg (purple closed diamonds and purple dash line), DP303c at 8 mg/kg (purple open diamonds and purple solid line), T-DM1 at 2 mg/kg (back open squares and black dotted line), T-DM1 at 4 mg/kg (black closed squares and black dash line), or T-DM1 at 8 mg/kg (black closed squares and black solid line),. Sizes of tumors were measured at designated time points. Data is present as mean of each treatment group.
  • FIG.6 shows the effect of DP303c on growth of human breast cancer xenograft model JIMT-1.
  • Athymic nude mice were implanted with the JIMT-1 cells. Animals were administered by a single intravenous dose of vehicle (Control), DP303c, T-DM1, or BP-ADC. Sizes of tumors were measured at designated time points. There were 5 animals in each group. Data is present as mean of each treatment group.
  • FIG.7 shows serum DP303c concentration-time profile following a single IV bolus injection of 3 mg/kg (blue diamonds and blue long-dashed line), 10 mg/kg (red squares and red short-dashed line), or 30 mg/kg (grey triangles and grey solid line) DP303c in SD rats. Data represents mean ⁇ standard deviation.
  • FIG.8 shows serum total antibody (DP001) concentration-time profile following a single IV bolus injection of 3 mg/kg (blue diamonds and blue long-dashed line), 10 mg/kg (red squares and red short-dashed line), or 30 mg/kg (green triangles and green solid line) DP303c in SD rats. Data represents mean ⁇ standard deviation.
  • FIG.9 shows free MMAE concentration-time profile following a single IV bolus injection of 3 mg/kg (blue diamonds and blue long-dashed line), 10 mg/kg (red squares and red short-dashed line), or 30 mg/kg (green triangles and green solid line) DP303c in SD rats. Data represents mean ⁇ standard deviation.
  • FIG.10 shows serum concentration-time profiles of DP303c following a single intravenous infusion injection of 1.2 mg/kg (squares and dotted line), 4 mg/kg (triangles and dashed line), or 12 mg/kg (diamonds and solid line) DP303c in cynomolgus monkeys.
  • FIG.11 shows serum concentration-time profiles of total antibody (DP001) following a single intravenous infusion injection of 1.2 mg/kg (squares and dotted line), 4 mg/kg (triangles and dashed line), or 12 mg/kg (diamonds and solid line) DP303c in cynomolgus monkeys. Data represents mean ⁇ standard deviation.
  • FIG.12 shows plasma concentration-time profiles of free MMAE following a single intravenous infusion injection of 1.2 mg/kg (squares and dotted line), 4 mg/kg (triangles and dashed line), or 12 mg/kg (diamonds and solid line) DP303c in cynomolgus monkeys.
  • FIG.13 shows plasma concentration-time profiles of free MMAE following two intravenous infusion injection of 6 mg/kg (blue squares and dotted line) or 20 mg/kg (red triangles and long-dashed line) DP303c in cynomolgus monkeys. Data represents mean ⁇ standard deviation.
  • FIG.14A and 14B show serum concentration-time profiles of DP303c following intravenous infusions of 0, 6, 20/12, 40/30 mg/kg DP303c in cynomolgus monkeys.
  • Serum DP303c concertation-time profile of the following groups and infusions are shown: group 2 after first dose (6.0 mg/kg, day 1, open circles), group 3 after first dose (20.0 mg/kg, day 1, open squares), group 4 after first dose (40.0 mg/kg, day 1, closed squares), group 2 after fourth dose (6.0 mg/kg, day 64, open triangles), group 3 after fourth dose (12.0 mg/kg, day 64, closed triangles), group 4 after third dose (30.0 mg/kg, day 43, closed circles).
  • FIG.14A shows serum concentration-time profiles of DP303c in male cynomolgus monkeys.
  • FIG.14B shows serum concentration-time profiles of DP303c in female cynomolgus monkeys.
  • FIG.15A and 15B show serum concentration-time profiles of total antibody (DP001) following intravenous infusions of 0, 6, 20/12, 40/30 mg/kg DP303c in cynomolgus monkeys.
  • Serum total antibody (DP001) concertation-time profile of the following groups and infusions are shown: group 2 after first dose (6.0 mg/kg, day 1, open circles), group 3 after first dose (20.0 mg/kg, day 1, open squares), group 4 after first dose (40.0 mg/kg, day 1, closed squares), group 2 after fourth dose (6.0 mg/kg, day 64, open triangles), group 3 after fourth dose (12.0 mg/kg, day 64, closed triangles), group 4 after third dose (30.0 mg/kg, day 43, closed circles).
  • FIG.15A shows serum concentration-time profiles of total antibody (DP001) in male cynomolgus monkeys.
  • FIG.15B shows serum concentration-time profiles of total antibody (DP001) in female cynomolgus monkeys.
  • FIG.16A and 16B show serum concentration-time profiles of free MMAE following intravenous infusions of 0 6 20/12 40/30 mg/kg DP303c in cynomolgus monkeys Serum free MMAE concertation-time profile of the following groups and infusions are shown: group 2 after first dose (6.0 mg/kg, day 1, open circles), group 3 after first dose (20.0 mg/kg, day 1, open squares), group 4 after first dose (40.0 mg/kg, day 1, open triangles), group 2 after fourth dose (6.0 mg/kg, day 64, closed circles), group 3 after fourth dose (12.0 mg/kg, day 64, closed squares), group 4 after third dose (30.0 mg/kg, day 43, closed triangles).
  • FIG.16A shows serum concentration-time profiles of free MMAE in male cynomolgus monkeys.
  • FIG.16B shows serum concentration-time profiles of free MMAE in female cynomolgus monkeys.
  • ADC antibody-drug conjugate
  • the ADC is DP303c.
  • the ADCs described herein have improved conjugation stability in vitro and in vivo, which contributes to increased efficacy against HER2-positive cancers, such as HER22+ and 3+ cancers, and reduced adverse effects.
  • the methods described herein can be used to treat a variety of HER2-positve solid cancers, including those resistant to standard HER2-targeted therapies.
  • the present application in one aspect provides a method of treating a HER2-expressing cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue.
  • the HER2-positive cancer is HER23+ as determined by an IHC test.
  • the HER2-positive cancer is HER22+ as determined by an IHC test.
  • the individual is resistant or refractory to a second HER2-targetd agent, such as trastuzumab, trastuzumab emtansine, pertuzumab, or lapatinib.
  • a second HER2-targetd agent such as trastuzumab, trastuzumab emtansine, pertuzumab, or lapatinib.
  • the antibody-drug conjugate is DP303c.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to 3 mg/kg).
  • the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin
  • the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue
  • the conjugation moiety is conjugated to the acceptor glutamine residue
  • the antibody-drug conjugate is administered intravenously. In some embodiments, the antibody- drug conjugate is administered once every three weeks, every other week, or once per week. In some embodiments, the antibody-drug conjugate is DP303c.
  • HER2 refers to human epidermal growth factor receptor 2.
  • HER2-positive cancer refers to a cancer that overexpresses HER2 on the cancer cells as compared to a non-cancerous, normal cell.
  • HER2 status can be determined using known HER2 tests, including ImmunoHistoChemistry (IHC) test, Fluorescence In Situ Hybridization (FISH) test, Subtraction Probe Technology Chromogenic In Situ Hybridization (SPoT-Light HER2 CISH) test, and Inform Dual In Situ Hybridization (Inform HER2 Dual ISH) test.
  • HER2-positive cancers include cancers that are tested 2+ (borderline) or 3+ (positive) in an IHC test.
  • HER2-positive cancers also include cancers that are tested positive in a HER2 FISH test, a SPoT-Light HER2 CISH test, and/or an Inform HER2 Dual ISH test.
  • HER22+ cancer refers to a cancer that is tested 2+ on an IHC test.
  • HER23+ cancer refers to a cancer that is tested 3+ on an IHC test.
  • treatment or “treating” is an approach for obtaining beneficial or desired results including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, reducing recurrence rate of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival.
  • the treatment reduces the severity of one or more symptoms associated with cancer by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to the corresponding symptom in the same subject prior to treatment or compared to the corresponding symptom in other subjects not receiving the treatment.
  • treatment is a reduction of pathological consequence of cancer.
  • the methods of the invention contemplate any one or more of these aspects of treatment.
  • adjuvant setting refers to a clinical setting in which an individual has had a history of cancer, and generally (but not necessarily) been responsive to therapy, which includes, but is not limited to, surgery (e.g., surgery resection), radiotherapy, and chemotherapy. However, because of their history of cancer, these individuals are considered at risk of development of the disease.
  • Treatment or administration in the “adjuvant setting” refers to a subsequent mode of treatment.
  • the degree of risk e.g., when an individual in the adjuvant setting is considered as “high risk” or “low risk” depends upon several factors, most usually the extent of disease when first treated.
  • “Neoadjuvant setting” refers to a clinical setting in which the method is carried out before the primary/definitive therapy.
  • “delaying” the development of cancer means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
  • a method that “delays” development of cancer is a method that reduces probability of disease development in a given time frame and/or reduces the extent of the disease in a given time frame, when compared to not using the method.
  • Cancer development can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT scan), Magnetic Resonance Imaging (MRI), ultrasound, clotting tests, arteriography, biopsy, urine cytology, and cystoscopy. Development may also refer to cancer progression that may be initially undetectable and includes occurrence, recurrence, and onset.
  • the term “effective amount” used herein refers to an amount of a compound or composition sufficient to treat a specified disorder condition or disease such as ameliorate palliate, lessen, and/or delay one or more of its symptoms.
  • an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation in cancer. In some embodiments, an effective amount is an amount sufficient to delay development of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. In some embodiments, an effective amount is an amount sufficient to reduce recurrence rate in the individual. An effective amount can be administered in one or more administrations.
  • the effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit ( ., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; (vii) reduce recurrence rate of tumor, and/or (viii) relieve to some extent one or more of the symptoms associated with the cancer.
  • an “effective amount” may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint.
  • an effective amount may be considered in the context of administering one or more therapeutic agents, and a therapeutic agent (e.g., an antibody-drug conjugate) may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved.
  • the components (e.g., the first and second therapies) in a combination therapy of the invention may be administered sequentially, simultaneously, or concurrently using the same or different routes of administration for each component.
  • an effective amount of a combination therapy includes an amount of the first therapy and an amount of the second therapy that when administered sequentially, simultaneously, or concurrently produces a desired outcome.
  • An “individual” or a “subject” is a mammal, more preferably a human.
  • Mammals also include, but are not limited to, farm animals, sport animals, pets (such as cats, dogs, horses), primates, mice and rats.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or function.
  • immunoglobulin Ig
  • antibody antibody
  • Fram length antibody refers to a molecule that constitutes the natural biological form of an antibody, including variable and constant regions.
  • the full length antibody of the IgG isotype is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, CH1, CH2, and CH3.
  • IgG antibodies may consist of only two heavy chains, each heavy chain comprising a variable domain attached to the Fc region.
  • Fc region refers to the polypeptide comprising the constant region of an antibody heavy chain excluding the first constant region immunoglobulin domain.
  • the Fc region may comprise immunoglobulin domains CH2 and CH3 and the hinge between CH1 and CH2.
  • the term “specifically recognizes” or “specifically binds” refers to measurable and reproducible interactions such as attraction or binding between a target and an antibody (or a molecule or a moiety), that is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • an antibody that specifically or preferentially binds to an epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other epitopes of the target or non-target epitopes.
  • an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.
  • “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding.
  • An antibody that specifically binds to a target may have an association constant of at least about 10 3 M -1 or 10 4 M -1 , sometimes about 10 5 M -1 or 10 6 M -1 , in other instances about 10 6 M -1 or 10 7 M -1 , about 10 8 M -1 to 10 9 M -1 , or about 10 10 M -1 to 10 11 M -1 or higher.
  • immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • constant domain refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site.
  • the constant domain contains the CH1, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
  • the “variable region” or “variable domain” of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody.
  • the variable domain of the heavy chain may be referred to as “VH.”
  • the variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.
  • the term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • variable domains of antibodies differ in three segments called hypervariable regions (HVRs, also referred to as CDRs) both in the light-chain and the heavy-chain variable domains.
  • HVRs hypervariable regions
  • CDRs CDRs
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • the “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“ ⁇ ) and lambda (“ ⁇ ”), based on the amino acid sequences of their constant domains.
  • ⁇ kappa
  • ⁇ lambda
  • IgG immunoglobulins
  • subclass any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • antibodies (immunoglobulins) can be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000).
  • an antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
  • CDR or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem.252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol.
  • Residue numbering follows the nomenclature of Kabat et al., supra 2Residue numbering follows the nomenclature of Chothia et al., supra 3Residue numbering follows the nomenclature of MacCallum et al., supra 4Residue numbering follows the nomenclature of Lefranc et al., supra 5Residue numbering follows the nomenclature of Honegger and Plückthun, supra [0060] “Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).
  • the “CH1 domain” of a human IgG Fc region usually extends from about amino acid 118 to about amino acid 215 (EU numbering system).
  • “Hinge region” is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol.22:161-206 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions.
  • the “CH2 domain” of a human IgG Fc region usually extends from about amino acid 231 to about amino acid 340.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain.
  • the “CH3 domain” (also referred to as “C2” or “H3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG).
  • amino acid modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a given position in a protein sequence with another amino acid.
  • a “variant” of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or “parent” polypeptide.
  • the polypeptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence.
  • Transglutaminase used interchangeably herein with “TGase,” refers to an enzyme capable of carrying out tranglutamination reactions.
  • transglutamination refers to a reaction where the ⁇ -glutaminyl of an acceptor glutamine residue from a protein/peptide is transferred to an amine group, such as a primary amine or the ⁇ -amino group of lysine.
  • acceptor glutamine residue when referring to an amino acid residue of a polypeptide or protein, refers to a glutamine residue that, under suitable conditions, is recognized by a TGase and can be crosslinked to a conjugation moiety comprising a donor amine group by a TGase through a reaction between the glutamine and a donor amine group (such as lysine or a structurally related primary amine such as amino pentyl group).
  • An “endogenous acceptor glutamine residue on an antibody” used herein refers to an acceptor glutamine residue in a naturally occurring antibody Fc region.
  • the endogenous acceptor glutamine residue is Q295 by the EU numbering and flanked by an N-glycosylation site at Asn297 position
  • ADC antibody-drug conjugate
  • ADCs Antibody-drug conjugates
  • a method of treating a HER2-positive (e.g., HER 2+ or HER23+ as determined by IHC) cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue.
  • the Fc region is N- glycosylated.
  • the acceptor glutamine residue is flanked by an N- glycosylation site at +2 position relative to the glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295 of a heavy chain of the anti-HER2 antibody, wherein the numbering is according to the EU numbering.
  • the HER2- positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2- positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2- positive cancer is positive as determined by FISH test.
  • the individual is unresponsive or ineligible for a standard therapy. In some embodiments, the individual has not previously received a second HER2-targeted agent.
  • the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer.
  • a method of treating a HER2-positive (e.g., HER 2+ or HER23+ as determined by IHC) cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety comprises an monomethyl auristatin E (MMAE), and wherein the anti-HER2 antibody is conjugated to the conjugation moiety via the acceptor glutamine residue.
  • MMAE monomethyl auristatin E
  • the conjugation moiety has the chemical structure of Formula (II), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the HER2-positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by FISH test. In some embodiments, the individual is unresponsive or ineligible for a standard therapy.
  • the individual has not previously received a second HER2-targeted agent.
  • the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer.
  • a method of treating a HER2-positive (e.g., HER 2+ or HER23+ as determined by IHC) cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the anti-HER2 antibody comprises a glyco
  • the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering.
  • the anti-HER2 antibody is N- glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering.
  • the anti-HER2 antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 7, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the conjugation moiety has the chemical structure of Formula (I).
  • the conjugation moiety is LND1002.
  • the HER2-positive cancer is HER23+ as determined by an IHC test.
  • the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by FISH test. In some embodiments, the individual is unresponsive or ineligible for a standard therapy. In some embodiments, the individual has not previously received a second HER2-targeted agent. In some embodiments, the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer.
  • a method of treating a HER2-positive (e.g., HER 2+ or HER23+ as determined by IHC) cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10, wherein the conjugation moiety has the chemical structure of Formula (I), and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue.
  • the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety
  • the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10
  • the conjugation moiety has the chemical structure of Formula (I)
  • the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering.
  • the anti- HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering.
  • the conjugation moiety is LND1002.
  • the anti-HER2 antibody is DP001.
  • the anti- HER2 antibody is trastuzumab.
  • the HER2-positive cancer is HER23+ as determined by an IHC test.
  • the HER2-positive cancer is HER22+ as determined by an IHC test.
  • the HER2-positive cancer is positive as determined by FISH test In some embodiments the individual is unresponsive or ineligible for a standard therapy. In some embodiments, the individual has not previously received a second HER2-targeted agent. In some embodiments, the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer.
  • a method of treating a HER2-positive cancer that is resistant or refractory to a second HER2-targeted agent in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue.
  • the Fc region is N-glycosylated.
  • the acceptor glutamine residue is flanked by an N- glycosylation site at +2 position relative to the glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295 of a heavy chain of the anti-HER2 antibody, wherein the numbering is according to the EU numbering.
  • the HER2- positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2- positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2- positive cancer is positive as determined by FISH test.
  • the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib.
  • the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer.
  • a method of treating a HER2-positive cancer that is resistant or refractory to a second HER2-targeted agent in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety comprises an MMAE, and wherein the anti-HER2 antibody is conjugated to the conjugation moiety via the acceptor glutamine residue.
  • the conjugation moiety has the chemical structure of Formula (II), wherein n is an integer between 1 and 12.
  • the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (I) In some embodiments the conjugation moiety is LND1002 In some embodiments, the HER2-positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by FISH test. In some embodiments, the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib.
  • the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer.
  • a method of treating a HER2-positive cancer that is resistant or refractory to a second HER2-targeted agent in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 compris
  • the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering.
  • the anti-HER2 antibody is N- glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering.
  • the anti-HER2 antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 7, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the conjugation moiety has the chemical structure of Formula (I).
  • the conjugation moiety is LND1002.
  • the HER2-positive cancer is HER23+ as determined by an IHC test.
  • the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by FISH test. In some embodiments, the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab or lapatinib In some embodiments the HER2-positive cancer is a solid cancer such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer.
  • a method of treating a HER2-positive cancer that is resistant or refractory to a second HER2-targeted agent in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10, wherein the conjugation moiety has the chemical structure of Formula (I), and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue.
  • the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering.
  • the anti- HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering.
  • the conjugation moiety is LND1002.
  • the anti-HER2 antibody is DP001.
  • the anti- HER2 antibody is trastuzumab.
  • the HER2-positive cancer is HER23+ as determined by an IHC test.
  • the HER2-positive cancer is HER22+ as determined by an IHC test.
  • the HER2-positive cancer is positive as determined by FISH test.
  • the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib.
  • the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of an antibody- drug conjugate (ADC), wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg).
  • ADC antibody- drug conjugate
  • the Fc region is N-glycosylated.
  • the acceptor glutamine residue is flanked by an N-glycosylation site at +2 position relative to the glutamine residue
  • the acceptor glutamine residue is at position 295 of a heavy chain of the anti-HER2 antibody, wherein the numbering is according to the EU numbering.
  • the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0 mg/kg or 8.0 mg/kg.
  • the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, such as about 1.0 mg/kg, or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, such as about 2.0 mg/kg, or about 3.0 mg/kg. In some embodiments, the ADC is administered intravenously. In some embodiments, the ADC is administered about once every three weeks, about every other week, or about once per week. In some embodiments, the cancer is a HER2-positive cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of an antibody- drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety comprises an MMAE, wherein the anti-HER2 antibody is conjugated to the conjugation moiety via the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg).
  • the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety
  • the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue
  • the conjugation moiety comprises an MMAE
  • the conjugation moiety has the chemical structure of Formula (II), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg.
  • the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, such as about 1.0 mg/kg, or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, such as about 2.0 mg/kg, or about 3.0 mg/kg. In some embodiments, the ADC is administered intravenously. In some embodiments the ADC is administered about once every three weeks about every other week, or about once per week. In some embodiments, the cancer is a HER2-positive cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of an antibody- drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises: (a) a VH comprising an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the anti-HER2 antibody comprises a glycosylated (e.g., N-glycosylated) Fc region comprising an endogen
  • the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering.
  • the anti-HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering.
  • the anti-HER2 antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 7, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002.
  • the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg.
  • the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, such as about 1.0 mg/kg, or about 2.0 mg/kg.
  • the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, such as about 2.0 mg/kg, or about 3.0 mg/kg.
  • the ADC is administered intravenously.
  • the ADC is administered about once every three weeks about every other week or about once per week.
  • the cancer is a HER2-positive cancer.
  • the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of an antibody- drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10, wherein the conjugation moiety has the chemical structure of Formula (I), wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg).
  • the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety
  • the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and
  • the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering.
  • the anti-HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering.
  • the conjugation moiety is LND1002.
  • the anti-HER2 antibody is DP001.
  • the anti-HER2 antibody is trastuzumab.
  • the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg.
  • the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, such as about 1.0 mg/kg, or about 2.0 mg/kg.
  • the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, such as about 2.0 mg/kg, or about 3.0 mg/kg.
  • the ADC is administered intravenously.
  • the ADC is administered about once every three weeks, about every other week, or about once per week.
  • the cancer is a HER2- positive cancer.
  • the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of DP303c, wherein the DP303c is administered intravenously about once every three weeks and at a dose of no more than about 8 mg/kg
  • the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of DP303c, wherein the DP303c is administered intravenously about once every three weeks and at a dose of about 1 mg/kg to about 2 mg/kg.
  • the ADC is administered at a dose of about 1.0 mg/kg, or about 2.0 mg/kg.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of DP303c, wherein the DP303c is administered intravenously about once every three weeks and at a dose of about 2 mg/kg to about 3 mg/kg.
  • the ADC is administered at a dose of about 2.0 mg/kg, or about 3.0 mg/kg.
  • the cancer is a HER2-positive cancer.
  • the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer.
  • the HER2-positive cancer is HER23+ as determined by an IHC test.
  • the HER2-positive cancer is HER22+ as determined by an IHC test.
  • the HER2-positive cancer is positive as determined by FISH test.
  • the individual is unresponsive or ineligible for a standard therapy.
  • the individual has not previously received a second HER2-targeted agent. In some embodiments, the individual has previously received a second HER2-targeted agent. In some embodiments, the cancer is resistant or refractory to a second HER2 targeted agent. In some embodiments, the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib.
  • DP303c is an antibody drug conjugate having a HER2-targeting monoclonal IgG1 antibody (DP001) with one cleavable LND1002 (toxin) attached site-specifically to Glutamine 295 in the constant region of each heavy chain of DP001.
  • DP303c has a DAR (drug antibody ratio) of about 1.8 to about 2.2, such as about 1.8, 1.9, 2.0, 2.1, or 2.2.
  • Cancer treatments can be evaluated by, e.g., tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, quality of life, protein expression and/or activity.
  • Exemplary routes of administration of the ADCs include, but are not limited to, oral, intravenous intracavitary intratumoral intraarterial intramuscular subcutaneous parenteral transmucosal, transdermal, ocular, topical, intraperitoneal, intracranial, intrapleural, and epidermal routes, or be delivered into lymph glands, body spaces, organs or tissues known to contain cancer cells.
  • the ADC is administered intravenously.
  • the ADC is administered by infusion.
  • the ADC is administered by injection.
  • the dosing regimen of the ADCs administered to the individual may vary with the particular ADC composition, the method of administration, and the particular type and stage of cancer being treated.
  • that effective amount of the ADC is below the level that induces a toxicological effect (i.e., an effect above a clinically acceptable level of toxicity) or is at a level where a potential side effect can be controlled or tolerated when the composition is administered to the individual.
  • the doses referred herein are determined with respect to the entire molecular weight of the ADC.
  • the ADC is administered at a dose of no more than about any one of 12 mg/kg, 11 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7.5 mg/kg, 7 mg/kg, 6.5 mg/kg, 6 mg/kg, 5.5 mg/kg, 5 mg/kg, 4.5 mg/kg, 4 mg/kg, 3.5 mg/kg, 3.25 mg/kg, 3 mg/kg, 2.9 mg/kg, 2.8 mg/kg, 2.75 mg/kg, 2.7 mg/kg, 2.6 mg/kg, 2.5 mg/kg, 2.4 mg/kg, 2.3 mg/kg, 2.25 mg/kg, 2.2 mg/kg, 2.1 mg/kg, 2 mg/kg, 1.9 mg/kg, 1.8 mg/kg, 1.75 mg/kg, 1.7 mg/kg, 1.6 mg/kg, 1.5 mg/kg, 1.4 mg/kg, 1.3 mg/kg, 1.25 mg/kg, 1.2 mg/kg, 1.1 mg/kg, 1 mg/kg, 0.8 mg/kg, 1.3 mg/kg
  • the dose of the ADC is within any of the following range, wherein the ranges have an upper limit of any of: 12 mg/kg, 11 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7.5 mg/kg, 7 mg/kg, 6.5 mg/kg, 6 mg/kg, 5.5 mg/kg, 5 mg/kg, 4.5 mg/kg, 4 mg/kg, 3.5 mg/kg, 3.25 mg/kg, 3 mg/kg, 2.9 mg/kg, 2.8 mg/kg, 2.75 mg/kg, 2.7 mg/kg, 2.6 mg/kg, 2.5 mg/kg, 2.4 mg/kg, 2.3 mg/kg, 2.25 mg/kg, 2.2 mg/kg, 2.1 mg/kg, 2 mg/kg, 1.9 mg/kg, 1.8 mg/kg, 1.75 mg/kg, 1.7 mg/kg, 1.6 mg/kg, 1.5 mg/kg, 1.4 mg/kg, 1.3 mg/kg, 1.25 mg/kg, 1.2 mg/kg, 1.1 mg/kg, 1 mg/kg, 1
  • the ADC is administered at a dose of any one of about 03 mg/kg to about 12 mg/kg about 06 mg/kg to about 8 mg/kg about 1 mg/kg to about 8 mg/kg, about 3 mg/kg to about 8 mg/kg, about 0.6 mg/kg to about 6 mg/kg, about 1 mg/kg to about 6 mg/kg, about 1 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 2.0 mg/kg, about 2.0 mg/kg to about 2.5 mg/kg, about 1 mg/kg to about 3 mg/kg, about 2 mg/kg to about 3 mg/kg, about 2.5 mg/kg to about 3 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 1.5 mg/kg to about 2.5 mg/kg, about 0.5 mg/kg to about 3.0 mg/kg, about 2 mg/kg to about 4 mg/kg, about 4 mg/kg to about 8 mg/kg, about 8 mg/kg to about 12 mg/kg, about 0.5 mg/kg to about 3.0 mg/
  • the doses described herein may refer to a suitable dose for cynomolgus monkeys, a human equivalent dose thereof, or an equivalent dose for the specific species of the individual.
  • the ADC is administered at a dose equivalent to about 0.3 mg/kg to about 8 mg/kg (such as such as about 0.3 mg/kg to about 6 mg/kg, about 0.6 mg/kg to about 4.5 mg/kg, about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg) for a cynomolgus monkey or a human individual.
  • the ADC is administered at a dose equivalent to no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1mg/kg to about 2 mg/kg, or about 2mg/kg to about 3 mg/kg) for a cynomolgus monkey or a human individual.
  • the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3, 0.6, 1, 2, 3, 4.5, 6 or 8 mg/kg.
  • the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, such as about 1.0 mg/kg, or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, such as about 2.0 mg/kg, or about 3.0 mg/kg.
  • the effective amount of the ADC may be administered in a single dose or in multiple doses.
  • exemplary dosing frequencies include, but are not limited to, weekly, weekly without break, weekly for two out of three weeks, weekly for three out of four weeks, once every three weeks, once every two weeks, monthly, every six months, yearly, etc.
  • the ADC is administered about weekly, once every 2 weeks, or once every 3 weeks.
  • the intervals between each administration are less than about any of 3 years, 2 years, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week.
  • the intervals between each administration are more than about any of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years. In some embodiments, there is no break in the dosing schedule.
  • the MSFP is administered at a low frequency, for example, any one of no more frequent than once per week, once every other week, once per three weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, once per 10 months, once per 11 months, once per year, or less.
  • the ADC is administered in a single dose. In some embodiments, the ADC is administered about once every three weeks.
  • the ADC is administered at a dose of no more than about 8 mg/kg, such as no more than any one of 6 mg/kg, 4.5 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg, per week, once every other week, or once every three weeks.
  • the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3, 0.6, 1, 2, 3, 4.5, 6 or 8 mg/kg, per week, once every other week, or once every three weeks.
  • the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, per week, once every other week, or once every three weeks.
  • the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, per week, once every other week, or once every three weeks. In some embodiments, the ADC is administered at a dose of about 1.0 mg/kg, per week, once every other week, or once every three weeks. In some embodiments, the ADC is administered at a dose of about 2.0 mg/kg, per week, once every other week, or once every three weeks. In some embodiments, the ADC is administered at a dose of about 3.0 mg/kg, per week, once every other week, or once every three weeks.
  • the administration of the ADC can be extended over an extended period of time, such as from about a week to about a month, from about a month to about a year, from about a year to about several years.
  • the MSFP is administered over a period of at least any of about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or more.
  • the methods described herein are suitable for treating a variety of cancers such as solid cancer.
  • the methods are applicable to cancers of all stages, including early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission.
  • the methods described herein may be used as a first therapy, second therapy third therapy or combination therapy with other types of cancer therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting.
  • the cancer has been resistant or refractory to a prior therapy.
  • the individual has relapsed from a prior therapy.
  • the individual has recurrent cancer. In some embodiments, the method is carried out before the primary/definitive therapy. In some embodiments, the method is used to treat an individual who has previously been treated. In some embodiments, the method is used to treat an individual who has not previously been treated. In some embodiments, the method is used as a first line therapy. In some embodiments, the method is used as a second line therapy. [0097] In some embodiments, the method is suitable for treating cancers that overexpress HER2 on the surface of the cancer cells, such as HER2-positive solid cancers. In some embodiments, the solid cancer is HER22+ as determined by an IHC test. In some embodiments, the solid cancer is HER23+ as determined by an IHC test.
  • the solid cancer is HER2 positive as determined by a FISH test.
  • the cancer cells in the individual express at least about any of more than 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more fold of HER2 compared to normal cells.
  • the cancer cells in the individual have no more than about 250,000, such as no more than about 200,000; 100,000; 75,000; 50,000; 25,000; 10,000; 7,500; or 5,000 relative HER2 density on cell, as determined using quantitative HER2 receptor density assays, e.g., Quantum 647 MESF (Bang Laboratories).
  • the cancer cells in the individual have HER2 receptor density comparable to or higher than that of JIMT-1 cells.
  • the HER2-positive solid cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer.
  • the breast cancer is early stage breast cancer non-metastatic breast cancer advanced breast cancer stage IV breast cancer, locally advanced breast cancer, metastatic breast cancer, breast cancer in remission, breast cancer in an adjuvant setting, or breast cancer in a neoadjuvant setting.
  • the breast cancer is in a neoadjuvant setting.
  • the breast cancer is at an advanced stage.
  • the breast cancer is HER2- positive breast cancer.
  • the breast cancer is HER22+ as determined by an IHC test. In some embodiments, the breast cancer is HER23+ as determined by an IHC test. In some embodiments, the breast cancer is HER2 positive as determined by a FISH test. In some embodiments, the breast cancer has metastasized to liver, lung, adrenal gland, lymph node, and/or peritoneum. [0099] In some embodiments, there is provided a method of treating ovarian cancer (e.g., HER2-positive ovarian cancer) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein. In some embodiments, the ovarian cancer is ovarian epithelial cancer.
  • ovarian cancer e.g., HER2-positive ovarian cancer
  • the ovarian cancer is stage I (e.g., stage IA, IB, or IC), stage II (e.g., stage IIA, IIB, or IIC), stage III (e.g., stage IIIA, HIB, or HIC), or stage IV.
  • the ovarian cancer is HER2-positive ovarian cancer.
  • the ovarian cancer is HER22+ as determined by an IHC test.
  • the ovarian cancer is HER23+ as determined by an IHC test.
  • the ovarian cancer is HER2 positive as determined by a FISH test.
  • a method of treating colorectal cancer comprising administering to the individual an effective amount of any one of the ADCs described herein.
  • the colorectal cancer is sigmoid colon cancer.
  • the colorectal cancer is stage I, stage II (e.g., stage IIA, IIB, or IIC), stage III (e.g., stage IIIA, IIIB, or IIIC), or stage IV (e.g., stage IVA, IVB, or IVC).
  • the ovarian cancer is HER2-positive ovarian cancer.
  • the colorectal cancer is HER22+ as determined by an IHC test. In some embodiments, the colorectal cancer is HER23+ as determined by an IHC test. In some embodiments, the colorectal cancer is HER2 positive as determined by a FISH test. [0101] In some embodiments, there is provided a method of treating gastric cancer (e.g., HER2-positive gastric cancer) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein.
  • gastric cancer e.g., HER2-positive gastric cancer
  • the gastric cancer is adenocarcinoma, lymphoma, astrointestinal stromal tumor (GIST), or carcinoid tumor
  • the gastric cancer is stage 0 (carcinoma in situ) Stage I stage II, stage III, or stage IV.
  • the gastric cancer is HER2-positive gastric cancer.
  • the gastric cancer is HER22+ as determined by an IHC test.
  • the gastric cancer is HER23+ as determined by an IHC test.
  • the gastric cancer is HER2 positive as determined by a FISH test.
  • urethral cancer e.g., HER2-positive urethral cancer
  • the urethral cancer is squamous cell carcinoma, transitional cell carcinoma, or adenocarcinoma.
  • the urethral cancer is distal urethral cancer or proximal urethral cancer.
  • the urethral cancer is HER2-positive urethral cancer.
  • the urethral cancer is HER22+ as determined by an IHC test.
  • the urethral cancer is HER23+ as determined by an IHC test. In some embodiments, the urethral cancer is HER2 positive as determined by a FISH test. [0103] In some embodiments, there is provided a method of treating lung cancer (e.g., HER2-positive lung cancer) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein.
  • the lung cancer is a non-small cell lung cancer (NSCLC). Examples of NSCLC include, but are not limited to, large-cell carcinoma, adenocarcinoma, neuroendocrine lung tumors, and squamous cell carcinoma.
  • the lung cancer is small cell lung cancer (SCLC).
  • the lung cancer is HER2-positive lung cancer. In some embodiments, the lung cancer is HER22+ as determined by an IHC test. In some embodiments, the lung cancer is HER23+ as determined by an IHC test. In some embodiments, the lung cancer is HER2 positive as determined by a FISH test. [0104] The methods described herein are useful for various aspects of cancer treatment.
  • a method of inhibiting cell proliferation comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2mg/kg, or about 2 mg/kg to about 3 mg/kg).
  • at least about 10% including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%
  • cell proliferation is inhibited.
  • a method of inhibiting tumor metastasis in an individual comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg).
  • at least about 10% including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%
  • metastasis is inhibited.
  • a method of reducing (such as eradicating) pre-existing tumor metastasis (such as metastasis to the lymph node) in an individual comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg).
  • a method of reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg).
  • a method of reducing tumor size in an individual comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg).
  • a method of prolonging time to disease progression of cancer in an individual comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg).
  • the method prolongs the time to disease progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 28, 32, 36, or more weeks [0110]
  • a method of prolonging survival of an individual having cancer comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg).
  • the method prolongs the survival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months.
  • a method of alleviating one or more symptoms in an individual having cancer comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg).
  • ADCs Antibody-drug conjugates
  • the present application also provides antibody-drug conjugates (ADCs) useful for the treatment methods described herein.
  • the ADCs may comprise any one of the anti-HER2 antibodies described herein, which is conjugated to any one of the conjugation moieties described herein via an endogenous acceptor glutamine residue in the Fc region of the anti- HER2 antibody.
  • an ADC comprising an anti-HER2 antibody and a conjugation moiety comprising a toxin
  • the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue.
  • the Fc region is N-glycosylated.
  • the acceptor glutamine residue is flanked by an N-glycosylation site at +2 position relative to the glutamine residue.
  • an ADC comprising a full-length anti- HER2 antibody and a conjugation moiety comprising a toxin
  • the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue
  • the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue at position 295 of a heavy chain of the anti-HER2 antibody wherein the numbering is according to the EU numbering.
  • the anti- HER2 antibody comprises an N-glycosylated Fc region.
  • the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue at position 295 of a heavy chain of the anti-HER2 antibody, and wherein the N- glycosylation is at position 297 of the heavy chain, wherein the numbering is according to the EU numbering.
  • an ADC comprising an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety comprises at least one MMAE (such as 1, 2, or more), wherein the anti-HER2 antibody is conjugated to the conjugation moiety via the acceptor glutamine residue.
  • the conjugation moiety has the chemical structure of Formula (II), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the average molar ratio between the conjugation moiety and the anti-HER2 antibody in the composition is about 1:1 to about 2:1. In some embodiments, at least about 80% (such as at least about any of 85%, 90%, 95% or more) of the ADC in the composition has a molar ratio between the conjugation moiety and the anti-HER2 antibody of about 2:1.
  • an ADC comprising an anti-HER2 antibody and a conjugation moiety
  • the anti-HER2 antibody comprises: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the anti-HER2 antibody comprises a glycosylated (e.g., N-glycosylated) Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12, and
  • the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering
  • the anti-HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering.
  • the conjugation moiety has the chemical structure of Formula (I).
  • the conjugation moiety is LND1002.
  • the average molar ratio between the conjugation moiety and the anti-HER2 antibody in the composition is about 1:1 to about 2:1.
  • an ADC comprising an anti-HER2 antibody and a conjugation moiety
  • the anti-HER2 antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 7; and (b) a VL comprising the amino acid sequence of SEQ ID NO: 8, wherein the anti-HER2 antibody comprises a glycosylated (e.g., N- glycosylated) Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue.
  • the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering.
  • the anti-HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering.
  • the conjugation moiety has the chemical structure of Formula (I).
  • the conjugation moiety is LND1002.
  • the average molar ratio between the conjugation moiety and the anti-HER2 antibody in the composition is about 1:1 to about 2:1.
  • At least about 80% (such as at least about any of 85%, 90%, 95% or more) of the ADC in the composition has a molar ratio between the conjugation moiety and the anti-HER2 antibody of about 2:1.
  • an ADC comprising an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10, wherein the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue.
  • the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering.
  • the anti-HER2 antibody is N- glycosylated at position 297 of the Fc region wherein the numbering is according to the EU numbering.
  • the conjugation moiety has the chemical structure of Formula (I).
  • the conjugation moiety is LND1002.
  • the anti-HER2 antibody is DP001.
  • the anti-HER2 antibody is trastuzumab.
  • the ADC is DP303c.
  • the average molar ratio between the conjugation moiety and the anti-HER2 antibody in the composition is about 1:1 to about 2:1.
  • the anti-HER2 antibody is a full-length antibody.
  • the anti-HER2 antibody is an antibody fragment comprising an Fc region.
  • the Fc region comprises part or all of the hinge region.
  • the anti-HER2 antibody comprises the Fc region of a naturally occurring immunoglobulin.
  • the anti-HER2 antibody comprises an Fc region of IgG1, IgG2, IgG3, IgG4 subtypes, or from IgA, IgE, IgD, or IgM.
  • the Fc region is from human IgG, and the Fc region is from an amino acid residue at position Glu216 or Ala231 to the carboxyl-terminus thereof according to the EU numbering system.
  • the Fc region in the anti-HER2 antibody is N-glycosylated.
  • the polysaccharide chain attached at the N-glycosylation site is at least about any of 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 units.
  • composition comprising any one of the ADCs described herein, wherein at least some (but not necessarily all) of the anti-HER2 antibody in the composition is glycosylated (for example N-glycosylated) in the Fc region.
  • a composition comprising an antibody- drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody conjugated to a conjugation moiety via an endogenous acceptor glutamine residue on the anti- HER2 antibody, and wherein at least some of (for example at least about any of 50%, 60%, 70%, 80%, 90%, or 95%) the antibody-drug conjugates in the composition is glycosylated (for example N-glycosylated) in the Fc region.
  • the N-glycosylation site flanks the acceptor glutamine residue to which the conjugation moiety is attached.
  • the N-glycosylation site and the acceptor glutamine residue are 5 or less amino acid residues apart. In some embodiments, the N-glycosylation site and the acceptor glutamine are 5, 4, 3, 2, or 1 amino acids apart In some embodiments the N-glycosylation site and the acceptor glutamine are next to each other. In some embodiments, the acceptor glutamine residue is flanked by an N- glycosylation site at +2 position relative to the glutamine residue. In some embodiments, the acceptor glutamine residue is flanked by an N-glycosylation site at +1, +2, +3, +4, or +5 position relative to the glutamine residue.
  • the acceptor glutamine residue is flanked by an N-glycosylation site at -1, -2, -3, -4, or -5 position relative to the glutamine residue.
  • the N-glycosylated Fc region comprises the amino acid sequence of SEQ ID NO: 11 (KPREEQX1NSTX2R, wherein X 1 is Y or F and X 2 is Y or F), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 6 of SEQ ID NO: 11, and wherein the N-glycosylation is at position 8 of SEQ ID NO: 11.
  • the N-glycosylated Fc region comprises amino acid sequence of SEQ ID NO: 12 (KPREEQYNSTYR), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 6 of SEQ ID NO: 12, and wherein the N-glycosylation is at position 8 of SEQ ID NO: 2.
  • the anti-HER2 antibody comprises an Fc region of human IgG1.
  • the anti-HER2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO: 13 (CH2 sequence of human IgG1), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 65 of SEQ ID NO: 13, and wherein the N-glycosylation is at position 67 of SEQ ID NO: 13.
  • the anti-HER2 antibody comprises an Fc region of human IgG2.
  • the anti-HER2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO: 14 (CH2 sequence of human IgG2), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 64 of SEQ ID NO: 14, and wherein the N-glycosylation is at position 66 of SEQ ID NO: 14.
  • the anti-HER2 antibody comprises an Fc region of human IgG3.
  • the anti-HER2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO: 15 (CH2 sequence of human IgG3), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 65 of SEQ ID NO: 15, and wherein the N-glycosylation is at position 67 of SEQ ID NO: 15.
  • the anti-HER2 antibody comprises an Fc region of human IgG4.
  • the anti-HER2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO: 16 (CH2 sequence of human IgG4), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 65 of SEQ ID NO: 16, and wherein the N-glycosylation is at position 67 of SEQ ID NO: 16.
  • the ADCs described herein have the anti-HER2 antibody component conjugated to the conjugation moiety in a specific and stoichiometrically controlled fashion, i.e., at the acceptor glutamine residue at the Fc region that is flanked by an N-glycosylation site.
  • the molar ratio of the conjugation moiety to the anti-HER2 antibody is about 1:1. In some embodiments, the molar ratio of the conjugation moiety to the anti-HER2 antibody is about 2:1. In some embodiments, at least about 80% (such as at least about any of 85%, 90%, 95% or more) of the ADC in the composition has the conjugation moiety to the anti-HER2 antibody molar ratio of about 1:1. In some embodiments, at least about 80% (such as at least about any of 85%, 90%, 95% or more) of the ADC in the composition has the conjugation moiety to the anti-HER2 antibody molar ratio of about 2:1.
  • At least about 80% (such as at least about any of 85% 90% 95% or more) of the ADC in the composition has the molar ratio of the conjugation moiety to the anti-HER2 antibody of about 1:1 or about 2:1.
  • the ADC is present in an individual (e.g., a mammal) at about 50% or more after at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more upon administration in vivo.
  • the ADC is present in an individual (e.g., a mammal) at about any of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more after at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, or more upon administration in vivo.
  • the free toxin (e.g., MMAE) exposure to an individual is no more than about any one of 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% or less of the ADC exposure upon administration of the ADC in vivo.
  • Anti-HER2 antibody [0130]
  • the ADCs described herein comprise an anti-HER2 antibody.
  • the anti-HER2 antibody specifically binds human HER2 expressed on the cell surface of a human cell (e.g., a human cancer cell).
  • the anti-HER2 antibody specifically binds a HER2 expressed on the cell surface of a human cancer cell (e.g., a breast cancer cell, an ovarian cancer cell, a gastric cancer cell, a urethral cancer cell, or a lung cancer cell.
  • a human cancer cell e.g., a breast cancer cell, an ovarian cancer cell, a gastric cancer cell, a urethral cancer cell, or a lung cancer cell.
  • the anti-HER2 antibody is trastuzumab.
  • the anti-HER2 antibody is not trastuzumab.
  • the anti- HER2 antibody specifically binds to the same epitope in HER2 as trastuzumab.
  • the anti-HER2 antibody comprises the same sequences, e.g., heavy chain CDRs, light chain CDRs, heavy chain variable region, light chain variable region, heavy chain and/or light chain sequences, as trastuzumab.
  • the anti-HER2 antibody is a biosimilar of trastuzumab.
  • the anti-HER2 antibody is DP001.
  • the anti-HER2 antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HC-CDR)1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
  • VH heavy chain variable region
  • HC-CDR heavy chain complementarity determining region
  • the anti-HER2 antibody comprises a light chain variable region (VL) comprising a light chain complementarity determining region (LC-CDR)1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
  • VL light chain variable region
  • LC-CDR light chain complementarity determining region
  • the anti-HER2 antibody comprises a VH comprising an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6.
  • the anti-HER2 antibody comprises: a) VH comprising the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3; and ii) a VL comprising the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO: 6.
  • the anti-HER2 antibody comprises: a) a VH comprising one, two or three CDRs of SEQ ID NO: 7, and/or b) a VL comprising one, two or three CDRs of SEQ ID NO: 8.
  • the anti-HER2 antibody comprises: a) a VH comprising HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain variable region of SEQ ID NO: 7, and/or b) a VL comprising LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain variable region of SEQ ID NO: 8.
  • the anti-HER2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 7.
  • the anti-HER2 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 8. [0137] In some embodiments, the anti-HER2 antibody comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 7; and b) a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 9.
  • the anti-HER2 antibody comprises a lambda light chain constant region.
  • the anti-HER2 antibody comprises a kappa light chain constant region.
  • the anti-HER2 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 10.
  • the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10.
  • Exemplary anti-HER2 antibody sequences are shown in Table B below. The exemplary CDR sequences are predicted using the IgBLAST algorithm. See, for example, Ye J. et al.
  • the anti-HER2 antibody is DP001.
  • DP001 is an anti-HER2 monoclonal antibody, which has the same amino acid sequence as trastuzumab (HERCEPTIN ® ). Specifically, it contains 1328 amino acids with two heavy chains (HC) of 450 amino acids (49284.65 Da, SEQ ID NO: 9), and two light chains (LC) of 214 amino acids (23443.10 Da, SEQ ID NO: 10).
  • DP001 is a heterotetramer of two HCs of the IgG1 subclass, and two LCs of the kappa subclass linked by 16 disulfide bonds (12 intra and 4 inter chain).
  • a schematic structure of DP001 is depicted in FIG.1.
  • the anti-HER2 antibodies described herein encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab’, F(ab’) 2 , Fv, Fc, etc.), chimeric antibodies, humanized antibodies, human antibodies (e.g., fully human antibodies), single chain (ScFv), bispecific antibodies, multispecific antibodies, mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.
  • the antibodies may be murine, rat, camel, human, or any other origin (including humanized antibodies).
  • Antibodies used in the present disclosure also include single domain antibodies, which are either the variable domain of an antibody heavy chain or the variable domain of an antibody light chain. Holt et al., Trends Biotechnol.21:484-490, 2003. Methods of making domain antibodies comprising either the variable domain of an antibody heavy chain or the variable domain of an antibody light chain, containing three of the six naturally occurring HVRs or CDRs from an antibody, are also known in the art. See, e.g., Muyldermans, Rev. Mol. Biotechnol.74:277-302, 2001. [0143] In some embodiments, the anti-HER2 antibody is a monoclonal antibody.
  • a monoclonal antibody refers to an antibody of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), monoclonal antibody is not a mixture of discrete antibodies.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies used in the present disclosure may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No.4,816,567.
  • the monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature, 348:552-554, for example.
  • the anti-HER2 antibody is a chimeric antibody.
  • a chimeric antibody refers to an antibody having a variable region or part of variable region from a first species and a constant region from a second species.
  • chimeric antibody comprises two copies of a chimeric light chain and two copies of a chimeric heavy chain.
  • the production of chimeric antibodies is known in the art (Cabilly et al. (1984), Proc. Natl. Acad. Sci. USA, 81:3273-3277; Harlow and Lane (1988), Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory).
  • the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to the sequences in antibodies derived from another.
  • variable regions can conveniently be derived from presently known sources using readily available hybridomas or B cells from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation, and the specificity is not affected by its source, the constant region being human is less likely to elicit an immune response from a human subject when the antibodies are injected than would the constant region from a non- human source. However, the definition is not limited to this particular example.
  • the anti-HER2 antibody is a humanized antibody.
  • humanized antibodies refer to forms of non-human (e.g.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a HVR or CDR of the recipient are replaced by residues from a HVR or CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported HVR or CDR or framework sequences, but are included to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVR or CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Antibodies may have Fc regions modified as described in WO 99/58572.
  • humanized antibodies have one or more HVRs or CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more HVRs or CDRs “derived from” one or more HVRs or CDRs from the original antibody.
  • the anti-HER2 antibody is a human antibody.
  • a human antibody means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies known in the art.
  • a human antibody used herein includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide.
  • Human antibodies can be produced using various techniques known in the art.
  • the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581).
  • Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Patent Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.
  • the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or may have been immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
  • anti-HER2 antibodies described herein may further include analogs and derivatives that are either modified, i.e., by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity.
  • the derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • Chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formulation, etc.
  • the analog or derivative can contain one or more unnatural amino acids.
  • amino acid sequence variants of the anti-HER2 antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis Such modifications include for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. In some embodiments, anti-HER2 antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Conservative substitutions are shown in Table C below. TABLE C: CONSERVATIVE SUBSTITITIONS Amino acids may be grouped into different classes according to common side-chain properties: a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp, Glu; d. basic: His, Lys, Arg; e. residues that influence chain orientation: Gly, Pro; f.
  • An exemplary substitutional variant is an affinity matured antibody moiety, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and the variant antibody moieties displayed on phage and screened for a particular biological activity (e.g., binding affinity). Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody moiety affinity.
  • HVR hotspots
  • SDRs specificity determining residues
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • one or more amino acid modifications may be introduced into the Fc region of a full-length anti-HER2 antibody provided herein, thereby generating an Fc region variant.
  • the Fc region variant has enhanced ADCC effector function, often related to binding to Fc receptors (FcRs).
  • FcRs Fc receptors
  • the Fc region variant has decreased ADCC effector function.
  • changes or mutations to Fc sequences that can alter effector function. For example, WO 00/42072 and Shields et al. J Biol. Chem.9(2): 6591-6604 (2001) describe antibody variants with improved or diminished binding to FcRs. The contents of those publications are specifically incorporated herein by reference.
  • the anti-HER2 antibody comprises an Fc region that possesses some but not all effector functions, which makes it a desirable candidate for applications in which the half-life of the anti-HER2 antibody in vivo is important yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fc ⁇ R binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No.5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
  • non-radioactive assay methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CYTOTOX 96TM non-radioactive cytotoxicity assay (Promega, Madison, Wis.).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).
  • C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol.
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759- 1769 (2006)).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat.
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).
  • DANA so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine
  • Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No.6,737,056; WO 2004/056312, and Shields et al., J. Biol.
  • alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No.6,194,551, WO 99/51642, and Idusogie et al., J. Immunol.164: 4178-4184 (2000).
  • the anti-HER2 antibody comprises a variant Fc region comprising one or more amino acid substitutions, which increase half-life and/or improve binding to the neonatal Fc receptor (FcRn).
  • Antibodies with increased half-lives and improved binding to FcRn are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).
  • the anti-HER2 antibody is altered to increase or decrease the extent to which the anti-HER2 antibody is glycosylated. Addition or deletion of glycosylation sites to an anti-HER2 antibody may be conveniently accomplished by altering the amino acid sequence of the anti-HER2 antibody or polypeptide portion thereof such that one or more glycosylation sites is created or removed.
  • the anti-HER2 antibody comprises an Fc region
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an anti-HER2 antibody of the invention may be made in order to create anti-HER2 antibody variants with certain improved properties.
  • the N-glycans attached to the CH2 domain of Fc is heterogeneous.
  • Antibodies or Fc fusion proteins generated in CHO cells are fucosylated by fucosyltransferase activity See Shoji-Hosaka et al., J. Biochem.2006, 140:777- 83. Normally, a small percentage of naturally occurring afucosylated IgGs may be detected in human serum.
  • Anti-HER2 antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the anti-HER2 antibody is bisected by GlcNAc. Such anti-HER2 antibody variants may have reduced fucosylation and/or improved ADCC function.
  • Anti-HER2 antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such anti-HER2 antibody variants may have improved CDC function.
  • the anti-HER2 antibody variants comprising an Fc region are capable of binding to an Fc ⁇ RIII.
  • the anti-HER2 antibody comprises a human wild-type IgG1 Fc region.
  • Also provided are one or more nucleic acids encoding the heavy chain and/or the light chain of the anti-HER2 antibody, vectors comprising the one or more nucleic acids, and methods of preparing the anti-HER2 antibody.
  • the conjugation moiety of the ADCs described herein comprise a toxin, such as a cytotoxic agent useful for cancer therapy.
  • the toxin is a chemotherapeutic agent.
  • the toxin is a small molecule drug.
  • cytotoxic agent examples include, but are not limited to, an anthracycline, an auristatin, a dolastatin, CC-1065, a duocarmycin, an enediyne, a geldanamycin, a maytansine, a puromycin, a taxane, a vinca alkaloid, SN-38, tubulysin, hemiasterlin, and stereoisomers, isosteres, analogs or derivatives thereof.
  • the conjugation moiety comprises monodansylcadaverine (MDC).
  • the conjugation moiety comprises TAM1.
  • the conjugation moiety comprises monomethyl auristatin E (MMAE).
  • the anthracyclines are derived from bacteria Streptomyces and have been used to treat a wide range of cancers, such as leukemias, lymphomas, breast, uterine, ovarian, and lung cancers.
  • Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.
  • Dolastatins and their peptidic analogs and derivatives, auristatins are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, e.g., U.S.
  • exemplary dolastatins and auristatins include, but are not limited to, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD, MMAF, MMAE, and 5-benzoylvaleric acid-AE ester (AEVB).
  • AEB auristatin E
  • AEFP auristatin EFP
  • MMAD MMAF
  • MMAE 5-benzoylvaleric acid-AE ester
  • Duocarmycin and CC-1065 are DNA alkylating agents with cytotoxic potency. See Boger and Johnson, PNAS 92:3642-3649 (1995).
  • Exemplary dolastatins and auristatins include, but are not limited to, (+)-docarmycin A and (+)-duocarmycin SA, and (+)-CC-1065.
  • Enediynes are a class of anti-tumor bacterial products characterized by either nine- and ten-membered rings or the presence of a cyclic system of conjugated triple-double-triple bonds.
  • Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, and dynemicin.
  • Geldanamycins are benzoquinone ansamycin antibiotic that bind to Hsp90 (Heat Shock Protein 90) and have been used antitumor drugs.
  • geldanamycins include, but are not limited to, 17-AAG (17-N-Allylamino-17-Demethoxygeldanamycin) and 17- DMAG (17-Dimethylaminoethylamino-17-demethoxygeldanamycin).
  • Maytansines or their derivatives maytansinoids inhibit cell proliferation by inhibiting the microtubules formation during mitosis through inhibition of polymerization of tubulin. See Remillard et al., Science 189:1002-1005 (1975).
  • Exemplary maytansines and maytansinoids include, but are not limited to, mertansine (DM1) and its derivatives as well as ansamitocin.
  • Taxanes are diterpenes that act as anti-tubulin agents or mitotic inhibitors.
  • Exemplary taxanes include, but are not limited to, paclitaxel (e.g., TAXOL ® ) and docetaxel (TAXOTERE ® ).
  • Vinca alkyloids are also anti-tubulin agents.
  • Exemplary vinca alkyloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.
  • One skilled in the art may make chemical modifications to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
  • the conjugation moiety comprises a toxin polypeptide (or a toxin protein).
  • a toxin polypeptide include, but are not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, tricothecenes, inhibitor cystine knot (ICK) peptides (e.g., ceratotoxins), and conotoxin (e.g., KIIIA or SmIIIa).
  • ICK inhibitor cystine knot
  • the conjugation moiety described herein comprises an amine donor group, which is conjugated to the acceptor glutamine residue in the anti-HER2 antibody. Any conjugation moiety not containing an amine donor group can be indirectly conjugated to the anti-HER2 antibody via a small molecule handle, which contains an amine donor group.
  • the term “amine donor group” as used herein refers to a reactive group containing one or more reactive amines (e.g., primary amines).
  • the conjugation moiety can comprise an amine donor group (e.g., primary amine -NH2), a linker, and a toxin (e.g., a small molecule).
  • the conjugation moiety can also be a polypeptide or a biocompatible polymer containing a reactive Lys (e.g., an endogenous Lys).
  • the amine donor group in some embodiments is a primary amine (-NH 2 ) that provides a substrate for transglutaminase to allow conjugation of the conjugation moiety to the anti-HER2 antibody via the acceptor glutamine. Accordingly, the linkage between the donor glutamine and the amine donor group can be of the formula -CH 2 - CH 2 -CO-NH- . [0176] In some embodiments, the anti-HER2 antibody and the conjugation moiety are linked through a linker.
  • linker refers to a structural element of a compound that links one structural element of said compound to one or more other structural elements of said same compound.
  • the linker is a non-cleavable linker.
  • Suitable non- cleavable linkers include, but are not limited to, NH 2 -R-X, NH 2 NH-R-X, and NH 2 -O-R-X, wherein R is alkyl or polyethylene glycol group (also referred to as PEG), wherein X is the toxin.
  • a polyethylene glycol group or PEG group may have a formula of --CH 2 CH 2 O) n -, wherein n is an integer of at least 1.
  • n is any of 2, 4 6 8 10 12 16 20 or 24 [0177]
  • the anti-HER2 antibody and the conjugation moiety are linked through a cleavable linker.
  • Suitable cleavable linkers include, but are not limited to, Lys-Phe-X, Lys-Val-Cit-PABC-X, NH 2 -(CH 2 CH 2 O) n -Val-Cit-PABC-X, and NH 2 -(CH 2 CH 2 O)n- (Val-Cit-PABC-X)2, wherein X is the toxin, and n is an integer of at least 1 (such as any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24).
  • PABC refers to p-aminobenzyloxycarbonyl.
  • Cit refers to citrulline.
  • Other exemplary amine donor group-linkers include, but are not limited to, Ac-Lys- Gly, aminocaproic acid, Ac-Lys-beta -Ala, amino-PEG2 (Polyethylene Glycol)-C2, amino- PEG3-C2, amino-PEG6-C2, Ac-Lys-Val (valine)-Cit (citrulline)-PABC (p- aminobenzyloxycarbonyl), aminocaproyl-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine.
  • the conjugation moiety is linked to the acceptor glutamine residue via a -NH-(C) n - linker, wherein the (C) n is a substituted or unsubstituted alkyl or heteroalkyl chain, wherein n is an integer from about 1 to about 60.
  • the carbon of the chain is substituted with an alkoxyl, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide, or alkylamide.
  • n is about 2 to about 20.
  • the linker is branched.
  • the linker is linear. In some embodiments, the linker has more than one (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) attachment sites for the attachment of active moieties. These active moieties can be the same or different from each other.
  • the conjugation moiety may comprise a polyacetal- or polyacetal derivative-based polymer linked to a plurality of toxins (such as chemotherapeutic agents).
  • the conjugation moiety comprises a toxin that is selected from the group consisting of Ac-Lys-Gly-MMAD, amino-PEG3-C2-MMAD, amino-PEG6- C2-MMAD, amino-PEG3-C2-amino-nonanoyl-MMAD], aminocaproyl-Val-Cit-PABC- MMAD, Ac-Lys-beta -Ala-MMAD, Aminocaproyl-MMAD, Ac-Lys-Val-Cit-PABC-MMAD, Aminocaproyl-MMAE, amino-PEG3-C2-MMAE, amino-PEG2-C2-MMAE, Aminocaproyl- MMAF, Aminocaproyl-Val-Cit-PABC-MMAE, Aminocaproyl-Val-Cit-PABC-MMAF, amino-PEG2-C2-MMAF, putrescinyl-geldana
  • the amine donor agent is aminocaproyl- Val-Cit-PABC-MMAE, aminocaproyl-Val-Cit-PABC-MMAF, Ac-Lys-putrescinyl- geldanamycin, Ac-Lys-beta -Ala-MMAD, Ac-Lys-Val-Cit-PABC-MMAD, aminocaproyl- Val-Cit-PABC-MMAD and amino-PEG6-C2-MMAD [0182]
  • the conjugation moiety is a maytansine derivative.
  • the conjugation moiety is an MMAE derivative comprising a non-cleavable linker (such as an amino-(CH 2 CH 2 O) n- linker).
  • the conjugation moiety has the chemical structure of Formula (II): (II), wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. [0183]
  • the conjugation moiety is an MMAE derivative comprising a cleavable linker (such as an amino-(CH 2 CH 2 O) n –Val-Cit-PABC-MMAE).
  • the conjugation moiety has the chemical structure of Formula (III): wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.
  • the conjugation moiety comprises a toxin that has the chemical structure of Formula (I): [0185]
  • the toxin is LND1002.
  • LND1002 is a toxin derived from MMAE, which has a PEG linker with a primary amine group for conjugation.
  • LND1002 has the chemical structure of formula (I).
  • the chemical compounds contemplated herein include salts, solvates, or stereoisomers thereof, which include all permutations of salts, solvates and stereoisomers, such as a solvate of a pharmaceutically acceptable salt of a stereoisomer of subject compound.
  • salt means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids.
  • “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.
  • salt thereof means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient.
  • salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.
  • “Solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the solute.
  • the solvent can be an organic compound, an inorganic compound, or a mixture of both.
  • solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.
  • “Stereoisomer” and “stereoisomers” refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.
  • the ADCs described herein can be made using any suitable methods in the art, for example, by using a transglutaminase to conjugate an amine group in the conjugation moiety to the endogenous acceptor glutamine residue in the antibody. See, for example, U.S. Pat. No. 10,357,472, which is incorporated herein by reference in its entirety.
  • the anti-HER2 ADC is prepared using a wildtype or an engineered transglutaminase.
  • Engineered transglutaminases suitable for preparing the ADCs described herein include those described in U.S. Pat. No.10,471,037, which is incorporated herein by reference in its entirety.
  • TGases catalyzes covalent protein crosslinking by forming proteinase resistant isopeptide bonds between a lysine donor residue of one protein and an acceptor glutamine residue of another protein, and is accompanied by the release of ammonia.
  • the catalytic mechanism of transglutaminases has been proposed as follows. After the glutamine- containing first substrate (acceptor or Q-substrate) binds to the enzyme, it forms a gamma - glutamylthioester with the cysteine residue in the active center of TGase, known as the acylenzyme intermediate, accompanied by the release of ammonia.
  • the second substrate (donor or K-substrate) then binds to the acylenzyme intermediate and attacks the thioester bond.
  • the product two proteins crosslinked by an Nepsilon (gamma -glutamyl)lysine isopetide bridge) is formed and released. This re-establishes the active-center Cys residue of the enzyme in its original form and allows it to participate in another cycle of catalysis.
  • the formation of the covalent acylenzyme intermediate is thought to be the rate-limiting step in these reactions.
  • the catalytic triad of many transglutaminases is papain-like, containing Cys- His-Asp (where His is histidine and Asp is aspartic acid) and, crucially, a tryptophan (Trp) residue located 36 residues away from the active-center Cys.
  • bacterial TGases isolated from Streptoverticillium sp has an atypical catalytic triad and shows no sequence homology with the papain-like catalytic triad of other TGases.
  • Several types of transglutaminases have been reported in various living organisms including microbial organisms.
  • TGase from guinea pig liver (GTGase), fish liver (FTGase) and microorganisms (mTGase) and any recombinant TGase (rTGase).
  • GTGase guinea pig liver
  • FGase fish liver
  • mTGase microorganisms
  • rTGase any recombinant TGase
  • useful TGases include microbial transglutaminases, such as e.g. from Streptomyces mobaraense, Streptomyces cinnamoneum and Streptomyces griseocarneum disclosed in U.S. Pat. No.5,156,956, and Streptomyces lavendulae disclosed in U.S. Pat.
  • transglutaminase from Bacilus lydicus
  • Useful non-microbial transglutaminases include guinea-pig liver transglutaminase, and transglutaminases from various marine sources like the flat fish Pagrus major (disclosed in EP-0555649), and the Japanese oyster Crassostrea gigas (disclosed in U.S. Pat. No.5,736,356).
  • An exemplary TGase is bacterial transglutaminase (BTG) (see, e.g. EC 2.3.2.13, protein-glutamine-gamma -glutamyltransferase).
  • the TGase is from S.
  • the TGase is a mutant (e.g., engineered) TGase having at least 80% sequence homology with native TGase.
  • An example is recombinant bacterial transglutaminase derived from Streptomyces mobaraensis (available from Zedira, Darmstadt, Germany).
  • Streptomyces ladakanum ATCC 27441 or NRRL3191 mTgase is expressed as Pre- Pro-mTgase (GenBank access number AY241675). There are 410 amino acid residues in pre- pro-mTGase, 331 in mature enzyme plus 30 of pre and 49 of pro. Pro peptide is a strong inhibitor of mature enzyme.
  • Primers designed according to AY241675 were used to clone the pro-mTgase and mature mTgase from ATCC 27441DNA into pET29b(+) vector’s Nde I and Xho I sites.
  • Active mTgase can be obtained either from enterokinase light chain (EKL) digestion of Pro-mTgase or refolding of mature mTgase.
  • EKL enterokinase light chain
  • TG_SL Strep Ladakanum
  • TG_SM mobaraensis
  • transglutaminase used in methods described herein can be obtained or made from a variety of sources.
  • the transglutaminase is a calcium dependent transglutaminase, which requires calcium to induce enzyme conformational changes and allow enzyme activity.
  • transglutaminase can be derived from guinea pig liver and obtained through commercial sources (e.g., Sigma-Aldrich (St Louis, Mo.) and MP Biomedicals (Irvine, Calif.)).
  • the transglutaminase is a calcium independent transglutaminase, which does not require calcium to induce enzyme conformational changes and allow enzyme activity.
  • the transglutaminase is a microbial transglutaminase derived from a microbial genome, such as transglutaminase from Streptoverticillium or Streptomices (e.g., Streptomyces mobarensis or Streptoverticillium mobarensis).
  • the transglutaminase is a mammalian protein (e.g., human transglutaminase), a bacterial protein, a plant protein, a fungi protein (e.g., Oomycetes and Actinomicetes transglutaminases), or a prokaryotic protein.
  • the transglutaminase is from Micrococcus, Clostridium, Turolpsis, Rhizopus, Monascus, or Bacillus. [0197] In some embodiments, the transglutaminase used in the methods described herein is a recombinant protein produced using recombinant techniques.
  • the TGase is prepared by: (a) culturing a host cell (such as a prokaryotic cell) comprising a vector comprising a nucleic acid encoding a pro-enzyme of TGase, and (b) obtaining mature TGase by cleavage of the pro-sequence of the pro-enzyme (for example by endokinase light chain).
  • a host cell such as a prokaryotic cell
  • the TGase is purified by chromatography (such as by affinity chromatography or ion exchange chromatography).
  • the TGase is tagged (such as his-tagged) to facilitate purification.
  • the anti-HER2 ADC is prepared by contacting the anti-HER2 antibody with the conjugation moiety in the presence of a transglutaminase under a condition that is sufficient to generate the ADC, wherein the anti-HER2 antibody comprises an N- glycosylated Fc region, wherein the N-glycosylated Fc region comprises an acceptor glutamine residue flanked by an N-glycosylation site, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue.
  • the anti-HER2 ADC is prepared by contacting a composition comprising the anti-HER2 antibody with the conjugation moiety in the presence of a transglutaminase under a condition that is sufficient to generate the ADC, wherein at least some (e.g., at least about 50%, 60%, 70%, 80%, 90%, or more) of the anti-HER2 antibody comprise an N-glycosylated Fc region, wherein the Fc region comprises an acceptor glutamine residue flanked by an N- glycosylation site, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue.
  • the anti-HER2 ADC is prepared in two steps.
  • a small molecule handle is conjugated to the anti-HER2 antibody via a TGase to create an intermediate conjugate.
  • a toxin is coupled to the intermediate conjugate via the small molecule handle, either covalently or non-covalently.
  • the small molecule handle can be specifically designed to tailor the coupling of the toxin, thus allows the conjugation of any kind of toxin to the anti-HER2 antibody.
  • the two-step method is particularly useful when the supply of the anti-HER2 antibody and/or the toxin is limited, and when the toxin has low water solubility and/or induces aggregation of the anti-HER2 antibody.
  • the small molecule handle described herein generally has the structure of -NH 2 -R, wherein R is a moiety that allows the attachment of the toxin.
  • R is a moiety that allows the attachment of the toxin.
  • the introduction of the small molecule handle in the methods described herein significantly increases the flexibility of the methods Specifically the structure of the small molecule handle can be tailored to the attachment of the desired toxin.
  • R is a ligand, which specifically binds to a binding partner. This allows attachment of any molecule (such as protein) that contains the binding partner.
  • Suitable ligand/binding partner pairs include, but are not limited to, antibody/antigen, antigen/antibody, avidin/biotin, biotin/avidin, streptavidin/biotin, biotin/streptavidin, glutathione/GST, GST/glutathione, maltose binding protein/amylose, amylose/maltose binding protein, cellulose binding protein and cellulose, cellulose/cellulose binding protein, etc.
  • These small molecule handles allow the attachment of the conjugation moiety through suitable linkers such as NH 2 -O-R-X, Maleimide-R-X, and Cyclooctyne-R-(R’-X) 2 , wherein X is the active moiety, and R and R’ are independently linker groups, such as linker groups comprising alkyl or polyethylene glycol groups.
  • the TGase-catalyzed reaction can be carried out from several hours to a day (e.g. overnight).
  • the conjugation moiety or the small molecule handle are allowed to react with anti-HER2 antibody (e.g., 1 mg/mL) at ligand concentrations between 400 and 600 Pmol/L, providing a 60 to 90-fold excess of the substrates over the anti-HER2 antibody, or at lower excess of substrates, e.g.1- to 20-fold, or 10-20 fold.
  • the reactions can be performed in potassium-free phosphate buffered saline (PBS; pH 8) at 37 °C. After 4 h to several days, steady-state conditions are achieved.
  • PBS potassium-free phosphate buffered saline
  • the resulting ADC can be analyzed using any suitable method.
  • the stoichiometry of the ADC can be characterized by liquid chromatography mass spectrometry (LC/MS) using a top-down approach in order to assess the number of conjugation moiety conjugated to antibodies, and in particular the homogeneity of the composition. Conjugates can be reduced before LC/MS analysis and light chains and heavy chains are measured separately.
  • the ADC is analyzed for drug loading (e.g. number of toxin in the conjugate per anti-HER2 antibody).
  • drug loading e.g. number of toxin in the conjugate per anti-HER2 antibody.
  • Such methods can be used to determine the mean number of conjugation moieties or toxins (such as MMAE) per anti-HER2 antibody as well as the distribution of number of conjugation moieties or toxins (such as MMAE) per antibody in a composition, i.e., the percentage of total antibody with any given level of drug loading or DAR.
  • HIC hydrophobic interaction chromatography
  • the molar ratio of the transglutaminase and the anti-HER2 antibody is about 10:1 to about 1:100.
  • the amount of the transglutaminase in the reaction mixture can be controlled to allow efficient transglutaminase reaction.
  • the concentration of the tranglutaminase in the reaction mixture is about any of about 0.01 mg/ml to about 5 mg/ml.
  • the concentration ratio between the conjugation moiety and the anti-HER2 antibody is from about 2:1 to about 800:1.
  • the conjugation efficiency of the anti-HER2 antibody and the conjugation moiety is at least about 30% (e.g., at least about 40%, 50%, 60%, 70%, 80%, 90% or more). Conjugation efficiency can also be measured at different temperature, such as room temperature or 37 °C.
  • the ADC is purified after the conjugation reaction.
  • the ADC may be purified using affinity chromatography, such as a protein A column, and/or a size exclusion column.
  • Pharmaceutical compositions, kits, and articles of manufacture [0207] Also provided are pharmaceutical compositions comprising any one of the antibody- drug conjugates described herein.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises about 50mM Sodium Citrate, about 10 mM Citric Acid, about 4.0% (w/v) Sucrose and about 0.02% (w/v) Polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 10 mg/mL of the ADC.
  • pharmaceutically acceptable carrier is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient(s) to a large extent depend on factors such as the particular mode of administration the effect of the excipient on solubility and stability, and the nature of the dosage form.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents including, but not limited to, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride are included in the pharmaceutical composition.
  • the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.
  • the pharmaceutical composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.
  • the pharmaceutical compositions to be used for in vivo administration are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Sterility is readily accomplished by filtration through sterile filtration membranes.
  • the composition is free of pathogen.
  • the pharmaceutical composition can be in the form of liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the pharmaceutical composition can be in a solid form and re-dissolved or suspended immediately prior to use. Lyophilized compositions are also included.
  • the pharmaceutical composition is suitable for administration to a human.
  • the pharmaceutical composition is contained in a single- use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved. [0212]
  • the pharmaceutical compositions described herein may be prepared, packaged, or sold in bulk as a single unit dose or as a plurality of single unit doses As used herein a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the pharmaceutical compositions described herein in some embodiments are suitable for parenteral administration.
  • Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ.
  • parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques.
  • the pharmaceutical composition is suitable for intravenous administration.
  • Formulations of a pharmaceutical composition suitable for parenteral administration may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi dose containers containing a preservative.
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e.
  • Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile pyrogen-free water
  • a suitable vehicle such as sterile pyrogen-free water
  • parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
  • compositions for parenteral administration may be formulated to be immediate and/or engineered release.
  • Engineered release formulations include controlled, delayed, sustained, pulsed, targeted and programmed release formulations.
  • sterile injectable solutions can be prepared by incorporating the ADC in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the exemplary methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. [0215] Dosage regimens may be adjusted to provide the optimum desired response.
  • kits for use in the treatment of cancers described herein.
  • the kits may include one or more containers comprising any one of the ADCs for treating a cancer (e.g., HER2-positive cancer).
  • kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein.
  • the kit comprises instructions for administration of the ADC to treat a cancer such as breast cancer colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer.
  • the kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the cancer and the stage of the cancer.
  • the instructions relating to the use of the ADC generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • kits may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • the kits are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an infusion device, e.g., a minipump.
  • a kit may have a sterile access port, for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.
  • the container may further comprise a second pharmaceutically active agent.
  • a method of treating a HER2-positive cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody- drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue.
  • the HER2-positive cancer is HER23+ as determined by an immunohistochemistry (IHC) test.
  • IHC immunohistochemistry
  • FISH Fluorescence In Situ Hybridization
  • the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib. 10. The method of any one of embodiments 1-9, wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg. 11.
  • a method of treating a cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg.
  • the cancer is a solid cancer.
  • the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancers, and lung cancer.
  • the antibody-drug conjugate is administered at a dose of no more than about 6 mg/kg.
  • the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg to about 8 mg/kg. 16.
  • the method of any one of embodiments 1-17, wherein the antibody-drug conjugate is administered about once every three weeks, about every other week, or about once per week 19.
  • the method of any one of embodiments 1-18, wherein the individual is human. 20.
  • the anti-HER2 antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity- determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6. 25.
  • VH heavy chain variable region
  • HC-CDR heavy chain complementarity- determining region
  • LC-CDR light chain complementarity-determining region
  • LC-CDR light chain complementarity-determining region
  • the anti-HER2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the Fc region is an IgG1 Fc.
  • the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10.
  • the toxin is a monomethyl auristatin E (MMAE).
  • MMAE monomethyl auristatin E
  • the conjugation moiety comprises a cleavable linker.
  • DP303c is an antibody drug conjugate, which is designed to target epidermal growth factor receptor 2 (HER2) positive cancer in humans. More specifically, the product is a HER2 targeting monoclonal IgG1 antibody (DP001) with one cleavable LND1002 (toxin) attached site-specifically to Glutamine 295 in the constant region of each heavy chain of DP001.
  • HER2 epidermal growth factor receptor 2
  • DP303c has a DAR (drug antibody ratio) of about 2.0 (such as 1.8 to 2.2).
  • DAR drug antibody ratio
  • DP001 is an anti-HER2 monoclonal antibody, which has the same amino acid sequence as trastuzumab (HERCEPTIN ® ). Specifically, it contains 1328 amino acids with two heavy chains (HC) of 450 amino acids (49284.65 Da, SEQ ID NO: 9), and two light chains (LC) of 214 amino acids (23443.10 Da, SEQ ID NO: 10).
  • HC heavy chains
  • LC light chains
  • DP001 is a heterotetramer of two HCs of the IgG1 subclass, and two LCs of the kappa subclass linked by 16 disulfide bonds (12 intra and 4 inter chain).
  • LND1002 is a toxin derived from MMAE, which has a PEG linker with a primary amine group for conjugation to the antibody.
  • LND1002 has the chemical structure of formula (I), shown below: [0223] DP303c was obtained by conjugating DP001 to LND1002, which was catalyzed by a microbial transglutaminase (mTgase).
  • mTgase microbial transglutaminase
  • DP001 reaction buffer (50mM Tris-Acetate, 2mM EDTA, 0.1% Tween-20) at pH 8.0 and LND1002 in DMSO were loaded to a sterile, flexible, ethylene-vinyl acetate bag under a laminar flow hood in a clean room. Once the mixture reached the desired temperature, mTGase was added to the bag through a sterile syringe to start the conjugation reaction. The bag was then incubated at 30°C for up to 120 hours. The reaction was monitored using RP-HPLC. The conjugation reaction was considered complete when the heavy chain conversion reached ⁇ 95%.
  • the completed reaction was loaded onto a sanitized CAPTIVA ® protein A column using a sanitized AKTA ® Ready system at 30-35g DP303c/L resin with a minimum residence time of 5 minutes to ensure complete binding of the DP303c product.
  • An in-line 0.2 ⁇ m depth filter was used to remove any particulates from the reaction and or buffers during the purification, and to maintain sterility.
  • the column was then washed with excess binding and wash buffers in order to remove mTGase, un-reacted LND1002, and any unwanted buffer components before low pH elution of the desired product.
  • the elution fraction was collected in a sterile collection bag pre-filled with neutralization buffer.
  • the purified DP303c was formulated as follows: 10mg/mL DP303c in 50mM Sodium Citrate, 10 mM Citric Acid, 4.0% (w/v) Sucrose and 0.02% (w/v) Polysorbate 20. Concentrated components of DP303c formulation were prepared in sterile containers and filtered through 0.2 ⁇ m sterile PES filters and added to the DP303c concentrated stock solution. The formulation step was conducted inside the laminar flow hood in a clean room. The final formulated solution was then filtered through a 0.2 ⁇ m sterile PES filter. The final filtered product was stored at -20 oC for long-term storage.
  • Example 2 The purified DP303c was formulated as follows: 10mg/mL DP303c in 50mM Sodium Citrate, 10 mM Citric Acid, 4.0% (w/v) Sucrose and 0.02% (w/v) Polysorbate 20. Concentrated components of DP303c formulation were prepared in sterile containers and
  • Cytotoxicity effects of DP303c compared to DP001 and T-DM1 against a HER2-high expressing cell line [0226] The cytotoxicity of DP303c along with DP001 and T-DM1 (also known as ado- trastuzumab emtansine (KADCYLA ® )) was evaluated in a cell-based assay with a HER23+ human breast cancer SK-BR-3 cell line (Li JY et al., Cancer Cell 29,117-129). [0227] SK-BR-3 cells were grown to about 70% confluence in a tissue-culture flask, then media was aspirated out of the flask and the cells were washed with a small amount of magnesium and calcium free DPBS.
  • the cells were treated with trypsin to lift adhered cells and re-suspended in fresh media to final concentration of about 1 x 10 5 cells/mL.
  • the re- suspended cells were transferred into wells of a 96-well plate and incubated overnight at 37°C, 5% CO2, 85% relative humidity to allow cells to adhere. After overnight incubation, DP001, DP303c, or T-DM1 was added to the wells at final concentrations of 500 ng/mL, 167 ng/mL, 55.6 ng/mL, 19 ng/mL, 6.2 ng/mL, 2.06 ng/mL, 0.69 ng/mL, or 0.229 ng/mL.
  • DP001, DP303c, or T-DM1 concentration of DP001, DP303c, or T-DM1 was added in triplicate. Fresh media was used as negative control. Then, the plate was shaken lightly on a plate shaker for 15-20 seconds and incubated for 3 days at 85% humidity. [0228] After incubation, Resazurin (Sigma, Cat# 199303, Lot# MKBP3801V) was added to every well to a final concentration of about 0.0005 ⁇ and mixed lightly with a plate shaker. The plate was further incubated for 3 hours.
  • the plate was assayed using a template for the cell based assay protocol on the SPECTRAMAX GEMINIXS ® plate reader with ⁇ ex 555 / ⁇ em 585nm (570nm cut-off).
  • the average RFU of blank medium wells were subtracted from the RFU readings of all the wells with DP001, DP303c, or T-DM1 treated cells.
  • Software XLFit Excel Version (4.3.2 Build 11) (ID Business Solutions Limited) Fit model 201 was used to calculate IC 50 . Table 1 below compares IC 50 of DP001, DP303c, and T-DM1 in SK-BR-3 cells.
  • FIG.2 shows the cell proliferation inhibition curves of SK-BR-3 cells following treatment with DP001 (blue circle and blue line), DP303c (green triangle and green line), or T-DM1 (brown square and brown line).
  • DP001, DP303c, and T-DM1 are cytotoxic against SK-BR-3 cells, a HER2 high-expression cell line.
  • DP303c has similar cytotoxic potency to that of T-DM1 against this HER2 high-expression cell line.
  • DP303c and T-DM1 are more potent in killing SK-BR-3 cells than DP001, the HER2 targeted antibody.
  • Example 3 Cytotoxicity effects of DP303c compared to DP001 and T-DM1 against a HER2 low-expressing cell line [0230] The cytotoxicity of DP303c along with DP001 and T-DM1 was evaluated in a cell- based assay with a HER22+ breast cancer JIMT-1cell line (Li JY et al., Cancer Cell 29,117- 129).
  • JIMT-1 cells were grown to about 70% confluence in a tissue-culture flask, then media was aspirated out of the flask and the cells were washed with a small amount of magnesium and calcium free DPBS. The cells were treated with trypsin to lift adhered cells and re-suspended in fresh media to final concentration of about 1 x 10 5 cells/mL. The re- suspended cells were transferred into wells of a 96-well plate and incubated overnight at 37°C, 5% CO2, 85% relative humidity to allow cells to adhere.
  • DP001, DP303c, or T-DM1 were added to each well at final concentrations of 10,000 ng/mL, 2,500 ng/mL, 625 ng/mL, 156 ng/mL, 39.1 ng/mL, 9.77 ng/mL, or 2.44 ng/mL. Each concentration of DP001, DP303c, or T-DM1 was added in triplicate. Fresh media was used as negative control. Then the plate was shaken lightly on a plate shaker for 15-20 seconds and incubated for 5 days at 85% humidity.
  • Resazurin (Sigma, Cat# 199303, Lot# MKBP3801V) was added to every well to final concentration of about 0.0005 ⁇ and mixed lightly with a plate shaker. The plate was further incubated for 3 hours. Subsequently, the plate was assayed using the template for the cell based assay protocol on the SPECTRAMAX GEMINIXS ® plate reader with ⁇ ex 555 / ⁇ em 585nm (570nm cut-off). The average RFU of blank medium wells were subtracted from the RFU readings of all the wells with DP001, DP303c, or T-DM1 treated cells.
  • FIG.3 shows the cell proliferation inhibition curves of JIMT-1 cells following treatment with DP001 (blue circle and blue line), DP303c (green triangle and green line), or T-DM1 (brown square and brown line). As shown in FIG.3, both DP303c and T-DM1 are cytotoxic against the JIMT-1 cell line, a HER2 low-expression cell line.
  • DP303c has higher cytotoxicity than TDM-1 against this HER2 low-expression cell line.
  • Example 4 Cytotoxicity effects of DP303c compared to DP001 and T-DM1 against a HER2 negative cell line.
  • the cytotoxicity of DP303c along with DP001 and T-DM1 was evaluated in a cell- based assay against Hs746T cell.
  • Hs746T is a human gastric carcinoma cell line that has little to no expression of HER2 (Corso S, et al., Mol Cancer 2010; 9:121).
  • Hs746T cells were grown to about 70% confluence in a tissue-culture flask, then media was aspirated out of the flask and the cells were washed with a small amount of magnesium and calcium free DPBS. The cells were treated with trypsin to life adhered cells and re-suspended in fresh media to final concentration of about 1 x 10 5 cells/mL. The re- suspended cells were transferred into wells of a 96-well plate and incubated overnight at 37°C, 5% CO2, 85% relative humidity to allow cells to adhere.
  • DP001, DP303c, or T-DM1 were added to each well at final concentrations of 10,000 ng/mL, 2,500 ng/mL, 625 ng/mL, 156 ng/mL, 39.1 ng/mL, 9.77 ng/mL, 2.44 ng/mL, or 0.610 ng/mL.
  • concentration of DP001, DP303c, or T-DM1 was added in triplicate. Fresh media was used as negative control. Then the plate was shaken lightly on a plate shaker for 15-20 seconds and incubated for 5 days at 85% humidity.
  • Resazurin (Sigma, Cat# 199303, Lot# MKBP3801V) was added to every well to final concentration of about 0.0005 ⁇ and mixed lightly with a plate shaker. The plate was further incubated for 3 hours. Subsequently, the plate was assayed using the template for the cell based assay protocol on the SPECTRAMAX GEMINIXS ® plate reader with ⁇ ex 555 / ⁇ em 585nm (570nm cut-off). The average RFU of blank medium wells were subtracted from the RFU readings of all the wells with DP001, DP303c, or T-DM1 treated cells.
  • FIG.4 shows the cell proliferation inhibition curves of Hs746T cells following treatment with DP001 (blue circle and blue line), DP303c (green triangle and green line), or T-DM1 (brown square and brown line). As shown in FIG.4, DP001, T-DM1, and DP303c did not show cytotoxic effects against Hs746T cell a HER2 negative cell line Example 5.
  • NCI-N87 is a gastric cancer cell line overexpressing HER2 (HER23+).
  • NCI-N87 cells were implanted subcutaneously into female athymic nude mice. The tumor- bearing mice were randomized into 7 treatment groups (6 mice/group) and treated with vehicle, DP303c, or T-DM1 by a single intravenous injection.
  • DP303c effectively inhibited the tumor growth in the NCI-N87 xenograft model (FIG.5).
  • DP303c A single dose treatment of tumor-bearing mice with 4 or 8 mg/kg of DP303c resulted in tumor regression (81.1% and 92.5% of TGI:) without regrowth for up to 40 days. At the low dose level (2 mg/kg), DP303c showed 47.9% of tumor growth inhibition. As comparison, T-DM1 did not cause tumor regression at all dose levels, and the percent tumor growth inhibition was -28.2%, 16.8%, and 39.9%, for 2, 4, and 8 mg/kg, respectively.
  • Example 6 A single dose treatment of tumor-bearing mice with 4 or 8 mg/kg of DP303c resulted in tumor regression (81.1% and 92.5% of TGI:) without regrowth for up to 40 days. At the low dose level (2 mg/kg), DP303c showed 47.9% of tumor growth inhibition. As comparison, T-DM1 did not cause tumor regression at all dose levels, and the percent tumor growth inhibition was -28.2%, 16.8%, and 39.9%, for 2, 4, and 8 mg/
  • JIMT-1 Tumor growth inhibition effects of DP303c compared to T-DM1 and an anti- HER2 Biparatopic single chain antibody ADC on a mouse xenograft JIMT-1 cancer model
  • the in vivo antitumor activity of DP303c was investigated in a mouse xenograft model of human breast cancer.
  • JIMT-1 is a HER2-positive breast cancer cell line.
  • the JIMT- 1 xenograft model is known for its insensitivity to current anti-HER2 therapeutic agents such as trastuzumab and T-DM1 (Li et al, 2016).
  • mice After tumor cell implantation, the tumor-bearing mice were randomized into 10 treatment groups (5 mice/group) for treatment with a single intravenous injection of vehicle, DP303c, T-DM1, or Biparatopic ADC.
  • administration of a single dose of DP303c induced complete tumor regression in the JIMT-1 xenograft model at all dose levels. Tumor regrowth was not found in some of these animals and they remained tumor-free for up to 4 weeks until the end of the study.
  • T-DM1 did not significantly inhibit tumor growth with TGI % of 4.9%, 16.9%, and 16.8% for 8, 116, and 32 mg/kg, respectively. No significant weight loss or mortalities were observed in all treatment groups in this study.
  • Example 7 Example 7.
  • DP303c Pharmacokinetic and toxicity studies of DP303c
  • This example describes the pharmacokinetic studies of DP303c in rats following a single dose and in cynomolgus monkeys following a single dose.
  • This example also describes nonclinical safety studies of DP303c, including a non-GLP single-dose toxicology study in rats, a non-GLP 29-day dose-range finding study in cynomolgus monkeys, and a GLP repeated dose toxicology study in cynomolgus monkeys.
  • this example describes in vitro evaluation of DP303c stability in human and cynomolgus monkey plasma as well as an in vitro GLP studies assessing the effects of DP303c formulation on hemolysis and erythrocyte aggregation.
  • Five-Week Pharmacokinetic Study of DP303c in Rats Following a Single Intravenous Injection of DP303c (non-GLP) [0244] The objective of this study was to determine pharmacokinetic profiles of DP303c, total anti-HER2 antibody, free (unconjugated) MMAE, and LND1002 (linker MMAE) following a single intravenous administration of DP303c in Sprague Dawley rats at dose levels of 3 mg/kg, 10 mg/kg, and 30 mg/kg (3/sex/group).
  • the AUC ⁇ values were 470 ⁇ g ⁇ day/mL at 3 mg/kg, 1331 ⁇ g ⁇ day/mL at 10 mg/kg, and 4498 ⁇ g ⁇ day/mL at 30 mg/kg.
  • the DP303C clearance (CL) was 6.44, 7.68, and 6.90 mL/day/kg and the terminal phase half-life (t 1/2 ) was 9.25, 9.79, and 9.42 days for 3, 10 mg/kg and 30 mg/kg, respectively.
  • Volume of distribution at steady state (V ) was 82.9 mL/kg at 3 mg/kg, 104.0 mL/kg at 10 mg/kg, and 90.2 mL/kg at 30 mg/kg.
  • the C max and AUC ⁇ of total antibody also increased dose-proportionally over the dose range of 3 to 30 mg/kg (FIG.8).
  • Peak concentrations (C max ) of total antibody were 71.2, 256, and 701 ⁇ g/mL at 3, 10, and 30 mg/kg (Table 3), respectively.
  • the AUC ⁇ values were 515, 1432, and 4428 ⁇ g ⁇ day/mL at 3, 10, and 30 mg/kg, respectively.
  • Terminal phase half-life (t 1/2 ) of total antibody was 9.67, 9.75, and 8.67 days, and clearance (CL) was 5.86, 7.07, and 6.90 mL/day/kg at does levels of 3, 20, and 30 mg/kg, respectively.
  • Volume of distribution of total antibody at stead state (V ) was 79.1 mL/kg at 3 mg/kg, 96.6 mL/kg at 10 mg/kg, and 83.1 mL/kg at 30 mg/kg.
  • the PK profile of total antibody was very similar to that of DP303c, suggesting limited de-conjugation of MMAE from DP303c in rats.
  • the serum concentration of MMAE reached the peak level at 6 hour postdose in the 3 and 10 mg/kg dose groups and at 24 hour in the 30 mg/kg dose group, followed by a slow elimination phase (FIG.9).
  • the peak concentrations of unconjugated MMAE were about 0.03, 0.08, and 0.3 ng/mL for 3, 10, and 30 mg/kg groups respectively.
  • the exposure of MMAE is about (0.011%, 0.014%, 0.016%) of the exposure of DP303c ( AUC 0-t , on a molar basis) at dose levels of 3, 10 and 30 mg/kg, respectively.
  • Serum concentrations of payload LND1002 were generally undetectable in most samples and PK analysis was not conducted.
  • the PK profiles such as exposure and elimination half-life, are very similar between DP303c and the total antibody following a single intravenous administration of DP303c in rats. Linear PK is suggested over the dose range of 3 to 30 mg/kg and no gender difference was observed. Serum exposure of free MMAE was ⁇ 0.03% of DP303c after a single-dose IV administration of D303c. Serum concentrations of LND1002 were generally undetectable.
  • the AUC ⁇ was 1300, 6417, and 24834 h ⁇ g/mL and the clearance (CL) was 0.96, 0.65, and 0.49 mL/h/kg at 1.2, 4.0, and 12 mg/kg.
  • the increase in DP303c AUC ⁇ was greater than dose-proportional while the DP303c clearance (CL) was decreased with increased dose levels, indicating nonlinear PK of DP303c over the dose range of 1.2 to 12 mg/kg in monkeys.
  • the terminal phase half-life (t 1/2 ) of DP303c was 130.1, 136.8, and 118.6 hours at 1.2, 4.0, and 12 mg/kg, respectively.
  • the total antibody PK was similar to the DP303c PK following a single IV infusion of DP303c in cynomolgus monkeys.
  • the total antibody C max increased in a dose-proportional manner over the dose range of 1.2 mg/kg to 12 mg/kg (FIG.11). Peak concentrations of total antibody were 30.8 ⁇ g/mL at 1.2 mg/kg, 103.7 ⁇ g/mL at 4 mg/kg, and 323.7 ⁇ g/mL at 12 mg/kg (Table 4).
  • the increase in mean AUC ⁇ of total antibody was greater than dose- proportional from 1.2 to 12 mg/kg.
  • the mean AUC ⁇ values were 1374 h ⁇ g/mL at 1.2 mg/kg, 7333 h ⁇ g/mL at 4 mg/kg, and 27854 h ⁇ g/mL at 12 mg/kg.
  • the mean total antibody AUC ⁇ was no more than 15% greater than the mean DP303c AUC ⁇ for all dose levels, suggesting limited de-conjugation of MMAE from DP303c.
  • the clearance (CL) of total antibody was decreased from 0.91 mL/day/kg at 1.2 mg/kg, 0.57 mL/day/kg at 4 mg/kg, to 0.44 mL/day/kg at 12 mg/kg.
  • the half-life (t1/2) of total antibody was 123.0, 138.4, and 124.0 hours at 1.2, 4, and 12 mg/kg, respectively.
  • t1/2 The half-life (t1/2) of total antibody was 123.0, 138.4, and 124.0 hours at 1.2, 4, and 12 mg/kg, respectively.
  • DP303c After dosing with DP303c, free MMAE in monkey plasma was not detected or was below the lower limit of quantification in most of the time points at the 1.2 mg/kg group. Therefore, the pharmacokinetic parameters of free MMAE were not estimated for the animals in the 1.2 mg/kg group (Table 4). Exposure of free MMAE was very low at both 4 mg/kg and 12 mg/kg. The observed medium time to reach the peak level of free MMAE in plasma was 48 hours (FIG.12).
  • the C max of free MMAE in plasma was 0.059 and 0.135 ng/mL and the AUC0-t were 7.56 and 32.0 h ⁇ ng/mL at 4 and 12 mg/kg, respectively. Exposure of free MMAE was only 0.01% of that of DP303c in corresponding dose groups of 4 mg/kg and 12 mg/kg. [0257] In conclusion, following a single IV administration of DP303c in cynomolgus monkeys, the mean AUC ⁇ of DP303c and total antibody increased more than dose- proportionally, indicating nonlinear PK of DP303c in cynomolgus monkeys over the dose range of 1.2 to 12 mg/kg.
  • DP303c exposure was ⁇ 15% lower than total antibody in monkeys, suggesting limited de-conjugation of MMAE from DP303c. Exposure of free MMAE was very low and no more than 0.03% of DP303c exposure. There was no gender difference observed in DP303c PK in this study. Pilot Toxicity Study of DP303c in Rats Following a Single Intravenous Injection (non-GLP) [0258] The objectives of this study were to evaluate the potential toxicity and determine the maximum tolerated dose (MTD) of DP303c by a single IV administration of DP303c in rats, followed by a 21-day follow-up period.
  • MTD maximum tolerated dose
  • Sprague Dawley rats (10/group) were administered with either vehicle (control) or DP303c at dose levels of 50, 100, and 200 mg/kg, respectively.
  • the parameters and end points evaluated in this study included clinical signs and observations, body weight, food consumption, and clinical pathology parameters (hematology) during the postdose period of 22 days.200 mg/kg DP303c caused mortality in rats. Edema/eschar/ulcer on chin or neck, temporary abnormal eye secretion, reduced weight gain and food consumption was observed in groups with 100 mg/kg DP303c dosage level or higher. Increase in monocytes and decrease in lymphocytes and eosinophils were observed in groups with 50 mg/kg DP303c dosage level or higher.
  • Toxicological end points evaluated in this study included veterinary physical observations, clinical signs, injection site observations, body weights, food consumption, clinical pathology (hematology, coagulation, clinical chemistry, urinalysis), toxicokinetics, gross necropsy findings, organ weights, and histopathologic examinations.
  • DP303c-associated mortality occurred in Groups 4 (60 mg/kg) and 5 (100 mg/kg). Two animals at the dose level of 60 mg/kg died on Day 10 and Day 12, respectively, after the first dose. In the 100 mg/kg group, one animal died on Day 8 and 3 animals were euthanized on Day 8 and Day 10, respectively, due to poor physical condition.
  • Concentrations of DP303c and total antibody DP001 in cynomolgus monkey serum collected from this study were determined by ELISA assays with a lower limit of quantification of 0.3125 ng/mL for both DP303c and total antibody DP001.
  • Concentrations of both free MMAE and linker-MMAE (LND002, free payload) in monkey plasma were analyzed by tandem liquid chromatography/mass spectrometry (LC- MS/MS) methods with a lower limit of quantification of 0.03 ng/mL for MMAE and 1.0 ng/mL for LND1002 for this study.
  • Table 5 shows pharmacokinetic parameters of DP303c in cynomolgus monkeys after IV infusion of first and second dose of DP303c. Values are presented as Mean ⁇ standard deviation; [n] indicates animal sample number; C max is maximum observed concentration; AUC 0-t is area under the concentration time curve up to the last measureable concentration; AUC 0- ⁇ is area under the concentration time curve from time zero to infinity; t 1/2 is half-life; CL is systemic clearance; Vss is steady-state volume of distribution. Table 5 [0270] Table 6 below shows mean pharmacokinetic parameters of free MMAE in cynomolgus monkeys following first and second dose of IV Infusion of DP303c.
  • MMAE concentrations reached the peak level between 48 and 96 hours after IV infusion (FIG.13).
  • MMAE AUC 0-t was 9.29 h ⁇ ng/mL at 6 mg/kg and 51.32 h ⁇ ng/mL at 20 mg/kg.
  • the AUC 0- ⁇ of DP303c and total antibody both increased more than dose-proportionally from the dose level of 6 to 20 mg/kg (Table 5).
  • the C max and AUC 0- ⁇ values of DP303c were similar between after the first dose and after the second dose.
  • the peak concentrations (C max ) of MMAE were 0.045 and 0.210 ng/mL at 6 and 20 mg/kg, respectively.
  • the PK of DP303c, total antibody, and free MMAE were analyzed in the dose groups of 6 and 30 mg/kg.
  • the AUC ⁇ of DP303c and total antibody increased greater than dose-proportionally following IV infusion of DP303c in cynomolgus monkeys, suggesting nonlinear kinetics.
  • the terminal half-life of DP303c was about 95.3 to 115 hours.
  • the MMAE exposure was very low in monkeys as compared with DP303c. Comparable plasma exposure of intact DP303c and total antibody, along with the low level of free MMAE, indicates negligible de-conjugation of MMAE from DP303c in monkeys.
  • a total of 20 male and 20 female na ⁇ ve cynomolgus monkeys were selected and enrolled in this study. These animals were randomly assigned to 4 groups based on body weight and gender. Group 1 was the vehicle control (saline, 0 mg/kg), and Groups 2, 3 and 4 were DP303c treatment groups, low (6.0 mg/kg), middle (20 mg/kg), and high (40 mg/kg) dose groups, respectively. Each group consisted of five animals per sex. DP303c and saline were administrated via 30-minute intravenous infusion once every three weeks. Three monkeys per sex per group were assigned to main study phase and the terminal necropsy was scheduled on Day 89, 4 days after the administration of the last dose.
  • saline and DP303c at 6 mg/kg were administered for five doses (on Days 1, 22, 43, 64, and 85) as scheduled.
  • DP303c at the middle dose group was administered with three doses at 20 mg/kg (Days 1, 22, and 43). The dose was reduced to 12 mg/kg for the remaining two doses (Days 64 and 85) due to one mortality that occurred following the third dose at 20mg/kg. The dose for this dose group was indicated as 20/12 mg/kg.
  • DP303c at the high dose group was administered with two doses at 40 mg/kg (Days 1 and 22).
  • the dose was reduced to 30 mg/kg at the third dose (Day 43) due to unscheduled animal death and no more doses were given after the third dose.
  • the dose for this dose group was indicated as 40/30 mg/kg. Necropsy of the survival animals in the 40/30 mg group was scheduled on Day 71, 4 weeks after the last dose (recovery phase).
  • Toxicological end points evaluated in this study included clinical signs and observations, local tolerance, body weights, food consumption, ophthalmoscopic examinations, body temperature, ECG, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), lymphocyte phenotypes, anti-drug antibody formation, and toxicokinetics (TK), gross necropsy findings, organ weights, and histopathological examinations.
  • the dose level in 40/30 mg/kg DP303c group was poorly tolerated. After the second dose at 40 mg/kg, three animals were euthanized on Day 20, Day 26, and Day 29, respectively, due to moribund conditions.
  • Hematological changes in the high dose group included mild to moderate decreases in white blood cells (WBC), lymphocytes, eosinophils, and severe decreases in neutrophils. Other changes included mildly to moderately decreased red blood cells (RBC), hemoglobin, hematocrit, and increased reticulocytes and platelets. These hematological changes were observed as early as on Day 8 in all animals of the high dose group. An increase in monocyte count was found at later time points of CP303c treatment. The hematologic manifestations of anemia and leukopenia returned to the baseline or alleviated in the survival animals after recovery.
  • WBC white blood cells
  • RBC red blood cells
  • hemoglobin hemoglobin
  • hematocrit hemoglobin
  • reticulocytes and platelets increased reticulocytes and platelets.
  • Coagulation changes comprised minimal prolongation of the prothrombin time and the activated partial thromboplastin time in the unscheduled death animals.
  • Clinical chemistry changes comprised mild to moderate decreases in albumin and increases in globulins along with a corresponding decreased albumin to globulin ratio.
  • Other findings included 2-3-fold increases in aspartate aminotransferase and minimal decreases in blood calcium, chloride, and creatine. The increased aspartate aminotransferase was back to normal after recovery.
  • Gross observation at the high dose group included purple or dark discoloration of lung sometimes along with lung adherence to the chest wall or fluid in the thoracic cavity in 4 of the 6 unscheduled death animals.
  • the hematological findings comprised minimally decreased hemoglobin.
  • Clinical chemistry changes included decreased albumin to globulin ratio due to mildly increased globulins.
  • Gross observation included discoloration of lung in 4 of 10 animals at terminal necropsy along with increased lung organ weights.
  • Major microscopic findings comprised minimally to moderately decreased lymphocytes in spleen and/or lymph nodes in 5 of the 6 terminal necropsy animals and minimal to moderate inflammation and fibrosis in the lungs in 4 of the 6 animals.
  • At recovery necropsy only mild depletion of lymphocytes in lymphoid organs was observed in 1 of the 3 animals, indicating that depletion of lymphocytes in spleen, thymus, and lymph nodes was partially reversed.
  • HNSTD non-severely toxic dose
  • the no observed adverse effect level (NOAEL) of DP303c was considered to be 6 mg/kg/dose (4 th dose interval mean C max 137.2 ⁇ g/mL, AUC 0-t 14149 h ⁇ g/mL) [0283]
  • the TK profiles of DP303c was evaluated by determining serum or plasma levels of DP303c (intact molecule), total antibody (conjugated and unconjugated antibodies) and MMAE over time. Concentrations of DP303c and total antibody DP001 in monkey serum were determined using ELISA assays with a lower limit of quantification of 0.3125 ng/mL for both DP303c and total antibody DP001.
  • Table 7 [0285] Table 8 below shows pharmacokinetic parameters of total antibody in cynomolgus monkeys after IV infusion of DP303c. Values are presented as Mean ⁇ standard deviation; [n] indicates animal sample number; M indicates male; F indicates female; C max is maximum observed concentration; AUC 0-t is area under the concentration time curve up to the last measureable concentration; AUC 0- ⁇ is area under the concentration time curve from time zero to infinity; t 1/2 is half-life; CL is systemic clearance; V ss is steady-state volume of distribution. Table 8 [0286] Table 9 below shows pharmacokinetic parameters of free MMAE in cynomolgus monkeys after IV infusion of DP303c.
  • DP303c C max max increased dose- proportionally from 131.7 (Male)/147.4 (Female) ⁇ g/mL at 6 mg/kg to 935.4 (M)/943.6 (F) ⁇ g/mL at 40 mg/mL (FIG.14A and FIG.14B).
  • Mean AUC 0-t increased more than dose- proportionally from 12668 (M)/13463 (F) ⁇ g ⁇ h/mL at 6 mg/kg to 126795 (M)/132131(F) ⁇ g ⁇ h/mL at 40 mg/kg (Table 7).
  • the clearance (CL) of DP303c was 0.46 (M)/0.44 (F), 0.33 (M) /0.34 (F), and 0.28 (M)/0.26 (F) mL/day/kg, and the half-life (t1 ⁇ 2) was 115.1 (M)/103.0 (F), 168.3 (M)/159.8 (F), and 158.3 (M)/162.3 (F) hours for the 6-, 20-, and 40-mg/kg dose levels, respectively.
  • the CL of total antibody was 0.38 (M)/0.38 (F), 0.29 (M) /0.27 (F), 0.28 (M)/0.30 (F) mL/day/kg, and the t 1 ⁇ 2 was 123.7 (M)/108.8 (F), 179.0 (M)/178.1 (F), and 158.3 (M)/177.6 (F) hours for the 6-, 12-, and 30-mg/kg dose levels, respectively.
  • the mean values of C max and AUC 0-t of total antibody after the first dose were comparable to those after the 4 th dose at 6 mg/kg, indicating no accumulation of total antibody exposure after multiple doses.
  • AUC 0-t was 7.11 (M)/14.15 (F) ng ⁇ h/mL at 6 mg/kg, 114.52 (M)/53.53 (F) at 12 mg/kg, and 151.37 (M)/143.39 (F) ng ⁇ h/mL at 30 mg/kg.
  • ADA anti-drug antibody
  • a bridging immunoassay was used to screen anti-drug antibody (ADA) against DP303c in serum samples from cynomolgus monkeys in the GLP repeated-dose toxicology study.
  • the biotin-labelled DP303c, ADA, and DP303c form a bridge complex that is quantified using streptavidin-HRP.
  • the signal to background ratio (S/B) for each sample was compared to the cutpoint factor. Any sample whose S/B was equal or higher than the cutpoint factor was considered as positive.
  • the sensitivity of the assay was determined to be 14.27 ng/mL. All samples were found negative and no ADA was detected. [0292]
  • plasma exposure of DP303c and total antibody was increased greater than dose-proportionally following IV infusion administration of DP303c in cynomolgus monkeys on Day 1 and Day43/Day64, suggesting nonlinear kinetics.
  • DP303c was incubated in human or monkey plasma at a concentration of 100 ⁇ g/mL at 37°C for 96 hours. Samples were collected at 0, 4, 24, 48, 72, and 96 hours after incubation and then analyzed for DP303c, free MMAE, and total DP001 (naked DP001 plus DP303c). [0294] Concentrations of DP303c and total antibody DP001 in cynomolgus monkey and human plasma were analyzed using ELISA assays with a lower limit of quantification of 0.3125 ng/mL for DP303c and total antibody DP001.
  • Table 11 shows formation of free MMAE in monkey and human plasma during 96 hours at 37°C. Values are presented as Mean ⁇ standard deviation; NC represent value not calculated; a indicates free MMAE concentrations were below lower level of qualification (LLOQ) in 2 of the 3 samples.
  • Table 11 [0297] The changes in the concentrations of DP303c and DP001 were very minimal in both monkey (no changes) and human (about 10% decrease) plasma during the testing period of 96 hours (Table 10). The ratio between DP303c and DP001 ranged from 0.96 to 1.11 across all time points.
  • DP303c Human or cynomolgus monkey erythrocytes were incubated with DP303c at concentrations of 0.2, 0.4, 0.6, 0.8, and 1mg/mL at 37°C for 3 hours. No hemolysis and coagulation were observed with DP303c treatment by either macroscopic observation or spectrophotometric analysis during the 3-hour testing period, indicating that DP303c did not cause hemolysis and aggregation of human and monkey red blood cells. Discussion [0299] DP303c demonstrated very good stability in vitro in human and monkey plasma. Ninety percent of DP303c were intact molecules after 96 hours incubation in human plasma.
  • DP303c displayed a linear PK with terminal half-life of 9 days in rats over the dose range of 3 to 30 mg/kg.
  • the PK parameters were very similar between DP303c and total antibody in rats across all doses, indicating limited de-conjugation of MMAE from DP303c.
  • DP303c displayed nonlinear PK with a terminal half-life of 4 to 7 days over the dose range of 1.2 to 40 mg/kg. Comparable exposure of DP303c and total antibody in cynomolgus monkeys also suggests no significant de- conjugation of MMAE in monkeys.
  • the plasma levels of MMAE were very low in monkeys after IV dose of DP303c.
  • the major dose limiting toxicities were dose-dependent lymphocyte depletion in spleen, thymus and lymph nodes and pulmonary toxicity.
  • the clinical symptoms of pulmonary toxicity comprised cough, dyspnea, and heavy breathing, together with gross observation of purple or dark discoloration of lung, increased lung organ weights, and lung inflammation with fibrosis.
  • pulmonary toxicity was not clearly noted in the 29-day non-GLP study as assessed by gross observation and lung organ weights. The likely explanation could be that it took a longer time to develop notable lung inflammation with fibrosis and pulmonary injury following DP303c treatment because unscheduled animal death occurred on Day 10 or earlier in the 29-day non-GLP dose range finding study.
  • HNSTD nonseverely toxic dose
  • DP303c PK assays for first-time-in-human (FTIH) studies [0304] Based on current assays validated for nonclinical studies, the following PK assays for first-time-in-human (FTIH) study is developed and validated: 1) an ELISA assay for quantifying the level of DP303c in human serum samples; 2) an ELISA assay for quantifying the level of DP001 (total antibody) in human serum samples; 3) a liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to quantify the level of free MMAE warhead in human serum samples.
  • LC-MS/MS liquid chromatography with tandem mass spectrometry
  • Dose escalation scheme for the DP303c FTIH study is 0.6, 1.2, 2.0, 3.0, and 4 mg/kg IV infusion once every 3 weeks (Q3W).
  • An optional escalation to a higher dose level achieved through an up to 25% increase from the previous dose level per cohort may be evaluated based on safety and clinical activity data from the study.
  • DP303c dose selection is based on safety margins from nonclinical safety studies and FDA publication (Saber and Leighton, 2015) and ICH S9. Safety margins were estimated based on 1/10th the no observed adverse effect level (NOAEL) at 6 mg/kg in cynomolgus monkeys using body weight for scaling.
  • NOAEL no observed adverse effect level
  • the safety margins were also calculated based on 1/6th the highest non-severely toxic dose (HNSTD) at 12 mg/kg in cynomolgus monkeys using body surface area for scaling.
  • the starting dose of 0.6 mg/kg is expected to have a safety margin of 10 based on the HED NOAEL of 6 mg/kg and a safety margin of 6.5 based on the HED HNSTD of 3.87 mg/kg (Table 12)
  • the highest dose of 4 mg/kg is anticipated to have a safety margin of 1.5 based on the HED NOAEL and a safety margin of 1 based on HED HNSTD.
  • Table 12 Predicted Safety Margins for Proposed DP303c Clinical Doses Example 9.
  • This example describes a Phase 1a/1b multicenter, open-label, dose-escalation/de- escalation and dose-expansion study to evaluate the safety, pharmacokinetics, immunogenicity, and antitumor activity of DP303c in subjects with select HER2-expressing advanced solid tumors.
  • the primary objectives of the study are assessment of the safety and tolerability of DP303c in subjects with HER2- positive advanced solid tumors refractory to standard therapy or for which no standard therapy exists and determination of the maximum tolerated dose (MTD) and/or recommended Phase 2 dose (RP2D) of DP303c.
  • MTD maximum tolerated dose
  • R2D Phase 2 dose
  • Phase 1a Dose Escalation/De-escalation [0307] Phase 1a portion of the study enrolls subjects with HER2-positive advanced solid tumors. Dose level cohorts of 3 subjects each are treated.
  • the dose levels (DL) to be studied in Phase 1a are: DL -1: 0.3 mg/kg; DL 1: 0.5 mg/kg or 0.6 mg/kg; DL 2: 1 mg/kg or 1.2 mg/kg; DL 3: 2.0 mg/kg; DL 4: 3.0 mg/kg; DL 5: 4.0 mg/kg.
  • the starting dose (DL 1) is 0.5 mg/kg or 0.6 mg/kg.
  • the first dose is administered by IV infusion over 60 ( ⁇ 10) minutes in normal saline for the first dose; if tolerated, subsequent infusions may be administered over 30 ( ⁇ 5) minutes.
  • This study uses a traditional 3 + 3 design, which is widely used in Phase 1 studies to determine MTD and as a basis for selection of the RP2D.
  • the starting dose is 0.6 mg/kg, one tenth of the NOAEL (no observed adverse effect level) in the monkey definitive repeated dose toxicity study.
  • the 4.0 mg/kg dose is below that which was associated with serious toxicity in the monkey (20 mg/kg, which is comparable to a human dose of 6.7 mg/kg after allometric scaling).
  • dose-limiting toxicity DLT is assessed over the initial 21-day period and beyond to determine dose escalation or de-escalation. If the 4.0 mg/kg dose level does not exceed the MTD, then dose escalation may continue with increases up to 25% from the previous dose level until the MTD is exceeded ( ⁇ 2 DLT) at a dose level.
  • a DLT is defined as a study drug-related AE by NCI CTCAE v 4.03 or study drug-related and clinically significant laboratory abnormality that occurs during the first 21-day cycle of DP303c and meets the criteria: grade 4 neutropenia (absolute neutrophil count [ANC] ⁇ 500/mm 3 ) lasting >7 days; febrile neutropenia (ANC ⁇ 1000/mm 3 ) with fever (temp >38.0° C) lasting >1 hour; grade 4 thrombocytopenia (platelet count ⁇ 250,000/mm 3 ) lasting >2 days; grade ⁇ 3 thrombocytopenia (platelet count ⁇ 50,000/mm 3 ) with clinically significant bleeding; grade ⁇ 2 pneumonitis; any ⁇ other Grade ⁇ 3 non-hematologic toxicity, excluding suboptimally treated nausea, vomiting, and diarrhea, alopecia, and/or brief ( ⁇ 1 week) Grade 3 fatigue; optimally treated nausea, vomiting, or diarrhea ⁇ Grade 3 that persists for more than 72 hours; LVEF ⁇
  • the following rules are used to determine if dose escalation is appropriate: 3 subjects will initially be enrolled in each cohort; if none of the first 3 subjects in a cohort experiences DLT, up to 3 new subjects will be enrolled into a cohort at the next dose level; if 1 of the first 3 subjects in a cohort experience DLT the cohort will be expanded up to 6 subjects If no additional subjects develop DLT in the 6 subjects in that cohort, up to 3 new subjects will be enrolled into a cohort at the next dose level; if 2 or more subjects in a 3- or 6-subject cohort experience DLT, that dose level is higher than the MTD and dose escalation will stop, 3 additional subjects will be enrolled and evaluated for DLT at the previous dose level, unless 6 subjects have already been evaluated at that dose level; when a dose higher than the MTD has been tested, the highest dose at which fewer than 2 of 6 subjects (ie, ⁇ 33%) experience DLT will be considered the MTD.
  • a dose-escalation decision for each cohort is made after the last subject has completed the DLT observation period, or experienced DLT, and all relevant safety data have been reviewed. Up to 3 subjects may be added to a cohort even though no DLT was observed, to more thoroughly evaluate a potential safety signal. Under some circumstances, treatment- related AEs that occur beyond the 21-day DLT evaluation period are considered for dose- escalation/de-escalation decision making.
  • Phase 1b Dose Expansion
  • the RP2D is a dose that is expected to be tolerable for repeated administration. The RP2D may be lower than the MTD, based on review of clinical safety data.
  • the Phase 1b dose-expansion part of the study will begin.
  • approximately 10 subjects each will enroll into 2 defined cancer types: breast cancer and gastric cancer (including adenocarcinomas of the gastroesophageal junction).
  • HER2-positive is defined as: HER2 IHC 2+ and ISH positive or IHC 3+.
  • the SMC will review all available safety data and assess whether any modification of the dosing regimen or study design is warranted.
  • Subjects are eligible to be included in the study only if all the following criteria apply: 1. Signed informed consent prior to study-related procedures; 2. Male or female 18-75 years of age; 3. Diagnosis of advanced, HER2-positive estrogen receptor (ER) malignancy that has progressed following standard therapy or for which no standard therapy exists. Subjects who have been previously treated with a HER2-targeted therapy such as trastuzumab, pertuzumab, lapatinib, or ado-trastuzumab emtansine are eligible.
  • a HER2-targeted therapy such as trastuzumab, pertuzumab, lapatinib, or ado-trastuzumab emtansine are eligible.
  • HER2-positive is defined as IHC 2+ and ISH positive or IHC 3+: for Phase 1a, subjects may have any type of solid tumor, provided it is positive for HER2 either IHC 2+ or 3+, or ISH-positive; for Phase 1b, subjects must have breast or gastric carcinomas that are positive for HER2 either IHC 2+ or 3+, or ISH-positive; HER2 testing must have been performed in a College of American Pathology (CAP) accredited laboratory using an FDA-approved or validated assay. 4. ECOG performance status 0 to 1, and the expected survival time is more than 3 months; 5.
  • CAP College of American Pathology
  • Subjects must have laboratory values within the limits described below: ANC ⁇ 1.5 x 10 9 /L; platelet count ⁇ 100 x 10 9 /L; hemoglobin ⁇ 9 g/dL; serum creatinine within normal limits OR creatinine clearance ⁇ 60 mL/minute; serum total bilirubin ⁇ 1.5 x ULN (up to 3 x ULN in subjects with Gilbert’s syndrome); AST (SGOT) and ALT (SGPT) ⁇ 2.5 x ULN (OR ⁇ 5 X ULN for subjects with liver metastases); PT/INR and APTT ⁇ 1.5 x ULN; 6.
  • Phase 1a measurable or evaluable disease
  • Phase 1b disease must be measurable (per RECIST 1.1); 7.
  • WOCBP must have a negative pregnancy test prior to study entry; 8.
  • WOCBP and male subjects must agree to use adequate contraception from study entry through at least 12 weeks after the last dose of study drug; 9.
  • a washout period is required for subjects who have recently received antitumor systemic therapy.
  • the period prior to the subject’s planned first dose of DP303c must be either at least 28 days or 5 half-lives, whichever is shorter. This applies for investigational and approved therapies.
  • Antitumor therapy includes chemotherapy, immunotherapy, targeted therapy, endocrine therapy, radiotherapy (except local radiotherapy for pain relief, 14 days after treatment).
  • Subjects are prohibited from receiving the following therapies during screening and the study treatment period: anti-cancer systemic chemotherapy, hormonal therapy, or immunotherapy; elective surgical/dental treatment after discussion with medical monitor as per consultation with the sponsor; investigational agents other than DP303c; radiotherapy (except palliative radiation therapy for disease-related pain with a consult with the sponsor's medical monitor); radio-/toxin-immunoconjugates.
  • Subjects who meet any of the following criteria will not be eligible to participate in the study: 1. Pregnant or breastfeeding women; 2. Refusal to use effective methods of contraception (see inclusion criteria for details); 3.
  • Subjects with evidence of an active infection including: subjects being treated with antibiotics for an active infection at the time of enrollment; subjects who have evidence of active hepatitis C or chronic active hepatitis B; subjects who have a known diagnosis of human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS); 15.
  • HIV human immunodeficiency virus
  • CYP3A inhibitors within 14 days of the first dose (drugs that increased specific CYP substrate AUC ⁇ 5 times, such as Mibefradil, verapamil, diltiazem, nefazodone, clarithromycin, Telithromycin, Troleandomycin, Erythromycin, fluconazole, itraconazole, ketoconazole, Posaconazole,VoriconazoleTablets, Elvitegravir, indinavir, lopinavir, Nelfinavir, Ritonavir, Saquinavir, Boceprevir, Incivo, telaprevir, Conivaptan, idelalisib) or strong CYP3A inducers (Avasimibe, phenobarbital, phenytoin, carbamazepine, Rifampicin, rifabutin,enzalutamide, mitotane, Hypericum perforatum); 16.
  • Dose Modification may be considered for the following circumstances: 1. In the event of DLT during cycle 1 or AE that meets DLT criteria or is considered unacceptable by the investigator during Cycle 2 or beyond, either discontinue the subject from the study or resuming dosing at a reduced dose level in subsequent cycles, after recovery from the DLT to Grade 0, 1, or baseline. 2. In the event of drug-related AE that has not recovered to Grade 0, 1, or baseline by the end of a 21-day cycle, if the AE recovers by Day 42 post-dose, dosing may resume at full or reduced dose.
  • AE has not recovered by Day 42, discontinue the subject from the study. 3.
  • dosing may resume at next feasible time. If dosing cannot resume by Day 42 post-dose, discontinue the subject from the study.
  • Dose reductions are permitted to the next-lower protocol-specified dose level. Patients who enrolled in the lowest dose level (DL 1: 0.6 mg/kg) may have their dose reduced to 0.3 mg/kg. If a subject is unable to tolerate the reduced dose level, then he/she should be discontinued from the study. Intra-subject dose escalation will not be permitted.
  • Efficacy is assessed by tumor response upon completion of Cycle 2 and at the end of every other cycle until study discontinuation. Safety is assessed by measurements of physical examinations, vital signs, electrocardiograms (ECGs), left ventricular ejection fraction (LVEF), pulmonary function testing, and high resolution CT of chest. [0321] Measurements of the primary objectives are: adverse events (AEs), serious adverse events (SAEs), dose limiting toxicity (DLT), changes from baseline in laboratory parameters, vital signs (VS), and electrocardiograms (ECGs). All AE and SAE will be collected from the signing of the ICF until 30 ( ⁇ 5) days after the last dose of DP303c. Events Meeting the AE Definition are: 1.
  • Any abnormal laboratory test results hematology, clinical chemistry, or urinalysis
  • other safety assessments e.g., ECG, radiological scans, vital signs measurements
  • ECG electrospray, radiological scans, vital signs measurements
  • Exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency and/or intensity of the condition.
  • Signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction 5. Signs, symptoms, or the clinical sequelae of a suspected overdose of either study treatment or a concomitant medication.
  • Measurements of the secondary objectives are: best overall response and disease control rates based on RECIST 1.1; duration of response and progression-free survival (PFS); individual subject DP303c serum concentrations and other analytes at specified time points after DP303c administration, and derived PK parameters; number (%) of subjects who develop detectable antidrug antibody (ADA).
  • Pharmacokinetics will be assessed by measurement of DP303c concentrations, total antibody in serum, and MMAE derivative and free MMAE concentration in plasma. Blood samples will be collected from all subjects for measurement of DP303c concentrations in serum or plasma at day 1, 2, 4, 8, 15 of during Cycle 1 and Cycle 2 and day 1 of all following cycles.
  • Antidrug antibody to DP303c in serum will be measured in blood samples collected at day1 of each treatment cycle. Antitumor activity will be analyzed by objective response rate (ORR), disease control rate (DCR), duration of response, and progression-free survival (PFS).
  • ORR is defined as a proportion ([confirmed complete response (CR) + confirmed partial response (PR)]/N evaluable) using RECIST 1.1 criteria. The proportion obtaining a CR or PR without the confirmation requirement will also be provided. ORR will be summarized using descriptive statistics.
  • Disease control rate (DCR) [CR + PR + stable disease (SD)/N evaluable] will be summarized similarly. Duration of response and PFS will be calculated using the Kaplan-Meier approach.
  • Phase 1a Initial Results 10 subjects were enrolled in a Phase 1a study to evaluate safety and efficacy of DP303c treatment in the subjects. All of the enrolled subjects had Her2 positive cancer and had received HERCEPTIN ® treatment before the study. To date, five subjects have dropped out and five subjects are still participating in the study. For the dose escalation study design, one subject was administered 0.5 mg/kg DP303c, and three subjects each were administered 1, 2, or 3 mg/kg DP303c. Doses were administered by intravenous infusion once every three weeks. [0325] DP303c related toxicity was assessed. At the 2 mg/kg dose, one subject showed grade 2 eye toxicity with blurry vision.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Toxicology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present application provides compositions and methods for treating cancers using an antibody-drug conjugate (ADC) comprising an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 62/927,623, filed on October 29, 2019, the content of which is hereby incorporated by reference in its entirety for all purposes.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE [0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 720692000240SEQLIST.TXT, date recorded: October 19, 2020, size: 14 KB).
FIELD OF THE INVENTION
[0003] The present application is in the field of cancer therapeutics, and relates to compositions and methods for treating cancers using an antibody-drug conjugate (ADC).
BACKGROUND
[0004] The human epidermal growth factor receptor (HER) family plays an important role in the pathogenesis of many tumors. The HER2 receptor is overexpressed in many tumors (25% of breast, 20% of ovarian, 30% of intestinal type gastric, 20% of lung cancers and such overexpression is associated with aggressive tumors and poor prognosis (Slamon DJ et al, Science, 1987, 235(4785): 177; Slamon DJ et al, Science, 1989, 244(495): 707; Morrison C et al, J. Clin. Oncol., 2006, 24(15):2376). Despite recent progress in the development of therapeutic agents that target HER2, patients with metastatic HER2-positive breast and gastric cancers are seldom cured of their diseases. The prototypic HER2 -directed antibody-drug conjugate (ADC), ado-trastuzumab emtansine (KADCYLA®), has been approved to treat HER2 -overexpressing cancer. However, its application has been restricted to breast cancer. [0005] The FDA-approved U.S. Prescribing Information for KADCYLA® includes data from a Phase 3 trial in women with advanced HER2-positive breast cancer who had received prior therapy with a taxane and trastuzumab. In comparison to a control group treated with lapatinib and capecitabine, the ado-trastuzumab emtansine group demonstrated statistically significant improvements in objective response rate (43.6%), progression-free survival (median 9.6 months) and overall survival (median 30.9 months). The prescribing information includes boxed warnings for hepatotoxicity, cardiac toxicity, and embryo-fetal toxicity. Additional warnings and precautions are listed for pulmonary toxicity, infusion-related reactions, hemorrhage, thrombocytopenia, and neurotoxicity. As noted in an editorial that discussed results from a more recent trial of ado-trastuzumab emtansine (K. Jhaveri, J Clin Oncol.2017; 35(2):127-130), the efficacy of ado-trastuzumab emtansine may be limited by “…poor internalization of the HER2-T-DM1 complex, defective intracellular and endosomal trafficking of the HER2–T-DM1 complex, defective lysosomal degradation of T-DM1, and increased expression of drug efflux pumps, such as multidrug resistance 1.” [0006] There remains a need for improved therapeutic agents to treat patients with HER2- positive cancers. A new ADC that improves on the efficacy and/or safety profile of ado- trastuzumab emtansine could provide a beneficial treatment alternative for patients with HER2-positive cancers. [0007] All publications, patents, and patent applications cited herein are hereby incorporated by reference herein in their entirety. BRIEF SUMMARY OF THE INVENTION [0008] The present invention in one aspect provides a method of treating a HER2-positive cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue. In some embodiments, the HER2-positive cancer is HER23+ as determined by an immunohistochemistry (IHC) test. In some embodiments, the HER2-positive cancer is HER2 2+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by a Fluorescence In Situ Hybridization (FISH) test. [0009] In some embodiments according to any one of the methods described above, the individual is unresponsive or ineligible for a standard therapy. In some embodiments, the individual has not previously received a second HER2-targeted agent. In some embodiments, the individual has previously received a second HER2-targeted agent. In some embodiments, the HER2-positive cancer is resistant or refractory to a second HER2-targeted agent. In some embodiments, the second HER-2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib. [0010] In some embodiments according to any one of the methods described above, the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of no more than about 6 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg to about 8 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1 mg/kg to about 2 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 2 mg/kg to about 3 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 2.0 mg/kg. In some embodiments, the antibody- drug conjugate is administered at a dose of about 3.0 mg/kg. [0011] In one aspect, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg. In some embodiments, the antibody- drug conjugate is administered at a dose of no more than about 6 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg to about 8 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1 mg/kg to about 2 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 2 mg/kg to about 3 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 2.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 3.0 mg/kg. [0012] In some embodiments according to any one of the methods described above, the antibody-drug conjugate is administered intravenously. In some embodiments, the antibody- drug conjugate is administered about once every three weeks, about every other week, or about once per week. In some embodiments, the individual is human. [0013] In some embodiments according to any one of the methods described above, the cancer is a solid cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer. In some embodiments, the cancer is an advanced-stage cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the individual has failed a prior cancer therapy, such as an anti-Her2 antibody therapy (e.g., trastuzumab). [0014] In some embodiments according to any one of the methods described above, the anti-HER2 antibody is N-glycosylated in the Fc region. In some embodiments, the acceptor glutamine residue is Q295 in the heavy chain of the anti-HER2 antibody according to the EU numbering. [0015] In some embodiments according to any one of the methods described above, each heavy chain of the HER2 antibody is conjugated to the conjugation moiety. In some embodiments, the conjugation moiety is conjugated to the acceptor glutamine residue by transglutamination. [0016] In some embodiments according to any one of the methods described above, the anti-HER2 antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the anti-HER2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the Fc region is an IgG1 Fc. In some embodiments, the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. [0017] In some embodiments according to any one of the methods described above, the conjugation moiety comprises a cleavable linker. In some embodiments, the toxin is a monomethyl auristatin E (MMAE). In some embodiments, the conjugation moiety has the chemical structure of Formula (III):
Figure imgf000006_0001
wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (I):
Figure imgf000006_0002
In some embodiments, the conjugation moiety is LND1002. [0018] In some embodiments according to any one of the methods described above, the antibody-drug conjugate is DP303c. [0019] Also provided are kits and articles of manufacture for use in any of the methods described above. [0020] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. BRIEF DESCRIPTION OF THE DRAWINGS [0021] FIG.1 depicts a schematic structure of DP001. [0022] FIG.2 shows the cell proliferation inhibition curves of SK-BR-3 cells (HER23+ cell line) following treatment with DP001 (blue circles and blue line), DP303c (green triangles and green line), or T-DM1 (brown squares and brown line). [0023] FIG.3 shows the cell proliferation inhibition curves of JIMT-1 cells (HER22+ cell line) following treatment with DP001 (blue circles and blue line), DP303c (green triangles and green line), or T-DM1 (brown squares and brown line). [0024] FIG.4 shows the cell proliferation inhibition curves of Hs746T cells (HER2 negative cell line) following treatment with DP001 (blue circles and blue line), DP303c (green triangles and green line), or T-DM1 (brown squares and brown line). [0025] FIG.5 shows effect of DP303c on growth of human gastric cancer xenograft model NCI-N87. Athymic nude mice were implanted with the NCI-N87 cells (HER23+ cell line). Animals were administered by a single intravenous dose of vehicle (PBS, grey closed circles and grey line), DP303c at 2 mg/kg (purple open diamonds and purple dotted line), DP303c at 4 mg/kg (purple closed diamonds and purple dash line), DP303c at 8 mg/kg (purple open diamonds and purple solid line), T-DM1 at 2 mg/kg (back open squares and black dotted line), T-DM1 at 4 mg/kg (black closed squares and black dash line), or T-DM1 at 8 mg/kg (black closed squares and black solid line),. Sizes of tumors were measured at designated time points. Data is present as mean of each treatment group. [0026] FIG.6 shows the effect of DP303c on growth of human breast cancer xenograft model JIMT-1. Athymic nude mice were implanted with the JIMT-1 cells. Animals were administered by a single intravenous dose of vehicle (Control), DP303c, T-DM1, or BP-ADC. Sizes of tumors were measured at designated time points. There were 5 animals in each group. Data is present as mean of each treatment group. [0027] FIG.7 shows serum DP303c concentration-time profile following a single IV bolus injection of 3 mg/kg (blue diamonds and blue long-dashed line), 10 mg/kg (red squares and red short-dashed line), or 30 mg/kg (grey triangles and grey solid line) DP303c in SD rats. Data represents mean ± standard deviation. [0028] FIG.8 shows serum total antibody (DP001) concentration-time profile following a single IV bolus injection of 3 mg/kg (blue diamonds and blue long-dashed line), 10 mg/kg (red squares and red short-dashed line), or 30 mg/kg (green triangles and green solid line) DP303c in SD rats. Data represents mean ± standard deviation. [0029] FIG.9 shows free MMAE concentration-time profile following a single IV bolus injection of 3 mg/kg (blue diamonds and blue long-dashed line), 10 mg/kg (red squares and red short-dashed line), or 30 mg/kg (green triangles and green solid line) DP303c in SD rats. Data represents mean ± standard deviation. [0030] FIG.10 shows serum concentration-time profiles of DP303c following a single intravenous infusion injection of 1.2 mg/kg (squares and dotted line), 4 mg/kg (triangles and dashed line), or 12 mg/kg (diamonds and solid line) DP303c in cynomolgus monkeys. Data represents mean ± standard deviation [0031] FIG.11 shows serum concentration-time profiles of total antibody (DP001) following a single intravenous infusion injection of 1.2 mg/kg (squares and dotted line), 4 mg/kg (triangles and dashed line), or 12 mg/kg (diamonds and solid line) DP303c in cynomolgus monkeys. Data represents mean ± standard deviation. [0032] FIG.12 shows plasma concentration-time profiles of free MMAE following a single intravenous infusion injection of 1.2 mg/kg (squares and dotted line), 4 mg/kg (triangles and dashed line), or 12 mg/kg (diamonds and solid line) DP303c in cynomolgus monkeys. Data represents mean ± standard deviation. [0033] FIG.13 shows plasma concentration-time profiles of free MMAE following two intravenous infusion injection of 6 mg/kg (blue squares and dotted line) or 20 mg/kg (red triangles and long-dashed line) DP303c in cynomolgus monkeys. Data represents mean ± standard deviation. [0034] FIG.14A and 14B show serum concentration-time profiles of DP303c following intravenous infusions of 0, 6, 20/12, 40/30 mg/kg DP303c in cynomolgus monkeys. Serum DP303c concertation-time profile of the following groups and infusions are shown: group 2 after first dose (6.0 mg/kg, day 1, open circles), group 3 after first dose (20.0 mg/kg, day 1, open squares), group 4 after first dose (40.0 mg/kg, day 1, closed squares), group 2 after fourth dose (6.0 mg/kg, day 64, open triangles), group 3 after fourth dose (12.0 mg/kg, day 64, closed triangles), group 4 after third dose (30.0 mg/kg, day 43, closed circles). FIG.14A shows serum concentration-time profiles of DP303c in male cynomolgus monkeys. FIG.14B shows serum concentration-time profiles of DP303c in female cynomolgus monkeys. [0035] FIG.15A and 15B show serum concentration-time profiles of total antibody (DP001) following intravenous infusions of 0, 6, 20/12, 40/30 mg/kg DP303c in cynomolgus monkeys. Serum total antibody (DP001) concertation-time profile of the following groups and infusions are shown: group 2 after first dose (6.0 mg/kg, day 1, open circles), group 3 after first dose (20.0 mg/kg, day 1, open squares), group 4 after first dose (40.0 mg/kg, day 1, closed squares), group 2 after fourth dose (6.0 mg/kg, day 64, open triangles), group 3 after fourth dose (12.0 mg/kg, day 64, closed triangles), group 4 after third dose (30.0 mg/kg, day 43, closed circles). FIG.15A shows serum concentration-time profiles of total antibody (DP001) in male cynomolgus monkeys. FIG.15B shows serum concentration-time profiles of total antibody (DP001) in female cynomolgus monkeys. [0036] FIG.16A and 16B show serum concentration-time profiles of free MMAE following intravenous infusions of 0 6 20/12 40/30 mg/kg DP303c in cynomolgus monkeys Serum free MMAE concertation-time profile of the following groups and infusions are shown: group 2 after first dose (6.0 mg/kg, day 1, open circles), group 3 after first dose (20.0 mg/kg, day 1, open squares), group 4 after first dose (40.0 mg/kg, day 1, open triangles), group 2 after fourth dose (6.0 mg/kg, day 64, closed circles), group 3 after fourth dose (12.0 mg/kg, day 64, closed squares), group 4 after third dose (30.0 mg/kg, day 43, closed triangles). FIG.16A shows serum concentration-time profiles of free MMAE in male cynomolgus monkeys. FIG.16B shows serum concentration-time profiles of free MMAE in female cynomolgus monkeys. DETAILED DESCRIPTION OF THE INVENTION [0037] The present application provides a method of treating a HER2-positive cancer in an individual using an antibody-drug conjugate (ADC) having its toxin component conjugated to an endogenous acceptor glutamine residue in a glycosylated Fc region of an anti-HER2 antibody. In some embodiments, the ADC is DP303c. The ADCs described herein have improved conjugation stability in vitro and in vivo, which contributes to increased efficacy against HER2-positive cancers, such as HER22+ and 3+ cancers, and reduced adverse effects. The methods described herein can be used to treat a variety of HER2-positve solid cancers, including those resistant to standard HER2-targeted therapies. [0038] Accordingly, the present application in one aspect provides a method of treating a HER2-expressing cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue. In some embodiments, the HER2-positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the individual is resistant or refractory to a second HER2-targetd agent, such as trastuzumab, trastuzumab emtansine, pertuzumab, or lapatinib. In some embodiments, the antibody-drug conjugate is DP303c. [0039] In another aspect, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to 3 mg/kg). In some embodiments, the antibody-drug conjugate is administered intravenously. In some embodiments, the antibody- drug conjugate is administered once every three weeks, every other week, or once per week. In some embodiments, the antibody-drug conjugate is DP303c. I. Definitions [0040] As used herein, “HER2” refers to human epidermal growth factor receptor 2. “HER2-positive cancer” refers to a cancer that overexpresses HER2 on the cancer cells as compared to a non-cancerous, normal cell. HER2 status can be determined using known HER2 tests, including ImmunoHistoChemistry (IHC) test, Fluorescence In Situ Hybridization (FISH) test, Subtraction Probe Technology Chromogenic In Situ Hybridization (SPoT-Light HER2 CISH) test, and Inform Dual In Situ Hybridization (Inform HER2 Dual ISH) test. HER2-positive cancers include cancers that are tested 2+ (borderline) or 3+ (positive) in an IHC test. HER2-positive cancers also include cancers that are tested positive in a HER2 FISH test, a SPoT-Light HER2 CISH test, and/or an Inform HER2 Dual ISH test. “HER22+ cancer” refers to a cancer that is tested 2+ on an IHC test. “HER23+ cancer” refers to a cancer that is tested 3+ on an IHC test. [0041] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, reducing recurrence rate of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival. In some embodiments, the treatment reduces the severity of one or more symptoms associated with cancer by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to the corresponding symptom in the same subject prior to treatment or compared to the corresponding symptom in other subjects not receiving the treatment. Also encompassed by "treatment" is a reduction of pathological consequence of cancer. The methods of the invention contemplate any one or more of these aspects of treatment. [0042] The terms “recurrence,” “relapse” or “relapsed” refers to the return of a cancer or disease after clinical assessment of the disappearance of disease. A diagnosis of distant metastasis or local recurrence can be considered a relapse. [0043] The term “refractory” or “resistant” refers to a cancer or disease that has not responded to treatment. [0044] “Adjuvant setting” refers to a clinical setting in which an individual has had a history of cancer, and generally (but not necessarily) been responsive to therapy, which includes, but is not limited to, surgery (e.g., surgery resection), radiotherapy, and chemotherapy. However, because of their history of cancer, these individuals are considered at risk of development of the disease. Treatment or administration in the “adjuvant setting” refers to a subsequent mode of treatment. The degree of risk (e.g., when an individual in the adjuvant setting is considered as “high risk” or “low risk”) depends upon several factors, most usually the extent of disease when first treated. [0045] “Neoadjuvant setting” refers to a clinical setting in which the method is carried out before the primary/definitive therapy. [0046] As used herein, “delaying” the development of cancer means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method that “delays” development of cancer is a method that reduces probability of disease development in a given time frame and/or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of subjects. Cancer development can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT scan), Magnetic Resonance Imaging (MRI), ultrasound, clotting tests, arteriography, biopsy, urine cytology, and cystoscopy. Development may also refer to cancer progression that may be initially undetectable and includes occurrence, recurrence, and onset. [0047] The term “effective amount” used herein refers to an amount of a compound or composition sufficient to treat a specified disorder condition or disease such as ameliorate palliate, lessen, and/or delay one or more of its symptoms. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation in cancer. In some embodiments, an effective amount is an amount sufficient to delay development of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. In some embodiments, an effective amount is an amount sufficient to reduce recurrence rate in the individual. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (
Figure imgf000012_0001
., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; (vii) reduce recurrence rate of tumor, and/or (viii) relieve to some extent one or more of the symptoms associated with the cancer. [0048] As is understood in the art, an “effective amount” may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and a therapeutic agent (e.g., an antibody-drug conjugate) may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable or beneficial result may be or is achieved. The components (e.g., the first and second therapies) in a combination therapy of the invention may be administered sequentially, simultaneously, or concurrently using the same or different routes of administration for each component. Thus, an effective amount of a combination therapy includes an amount of the first therapy and an amount of the second therapy that when administered sequentially, simultaneously, or concurrently produces a desired outcome. [0049] An “individual” or a “subject” is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets (such as cats, dogs, horses), primates, mice and rats. [0050] The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or function. As used herein, the terms “immunoglobulin” (Ig) and “antibody” are used interchangeably. [0051] “Full length antibody” as used herein refers to a molecule that constitutes the natural biological form of an antibody, including variable and constant regions. For example, in most mammals, including humans and mice, the full length antibody of the IgG isotype is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, CH1, CH2, and CH3. In some mammals, for example in camels and llamas, IgG antibodies may consist of only two heavy chains, each heavy chain comprising a variable domain attached to the Fc region. [0052] “Fc region” as used herein refers to the polypeptide comprising the constant region of an antibody heavy chain excluding the first constant region immunoglobulin domain. For IgG, the Fc region may comprise immunoglobulin domains CH2 and CH3 and the hinge between CH1 and CH2. [0053] As used herein, the term “specifically recognizes” or “specifically binds” refers to measurable and reproducible interactions such as attraction or binding between a target and an antibody (or a molecule or a moiety), that is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that specifically or preferentially binds to an epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other epitopes of the target or non-target epitopes. It is also understood that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. An antibody that specifically binds to a target may have an association constant of at least about 103 M -1 or 104 M -1, sometimes about 105 M -1 or 106 M -1 , in other instances about 106 M -1 or 107 M -1, about 108 M -1 to 109 M -1 , or about 1010 M -1 to 1011 M -1 or higher. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. [0054] The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CH1, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain. [0055] The “variable region” or “variable domain” of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites. [0056] The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs, also referred to as CDRs) both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. [0057] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“κ ) and lambda (“λ”), based on the amino acid sequences of their constant domains. [0058] The term IgG “isotype” or “subclass” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides. [0059] As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem.252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol. Biol.196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table A as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entireties for use in the present invention and for possible inclusion in one or more claims herein. TABLE A: CDR DEFINITIONS
Figure imgf000016_0001
1Residue numbering follows the nomenclature of Kabat et al., supra 2Residue numbering follows the nomenclature of Chothia et al., supra 3Residue numbering follows the nomenclature of MacCallum et al., supra 4Residue numbering follows the nomenclature of Lefranc et al., supra 5Residue numbering follows the nomenclature of Honegger and Plückthun, supra [0060] “Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004). [0061] The “CH1 domain” of a human IgG Fc region (also referred to as “C1” of “H1” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system). [0062] “Hinge region” is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol.22:161-206 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions. [0063] The “CH2 domain” of a human IgG Fc region (also referred to as “C2” of “H2” domain) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol.22:161- 206 (1985). [0064] The “CH3 domain” (also referred to as “C2” or “H3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG). [0065] By “amino acid modification” herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. By “amino acid substitution” or “substitution” herein is meant the replacement of an amino acid at a given position in a protein sequence with another amino acid. A “variant” of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or “parent” polypeptide. The polypeptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence. [0066] “Transglutaminase,” used interchangeably herein with “TGase,” refers to an enzyme capable of carrying out tranglutamination reactions. The term “transglutamination” as used herein refers to a reaction where the γ-glutaminyl of an acceptor glutamine residue from a protein/peptide is transferred to an amine group, such as a primary amine or the ε-amino group of lysine. [0067] The term “acceptor glutamine residue,” when referring to an amino acid residue of a polypeptide or protein, refers to a glutamine residue that, under suitable conditions, is recognized by a TGase and can be crosslinked to a conjugation moiety comprising a donor amine group by a TGase through a reaction between the glutamine and a donor amine group (such as lysine or a structurally related primary amine such as amino pentyl group). [0068] An “endogenous acceptor glutamine residue on an antibody” used herein refers to an acceptor glutamine residue in a naturally occurring antibody Fc region. In some embodiments, the endogenous acceptor glutamine residue is Q295 by the EU numbering and flanked by an N-glycosylation site at Asn297 position [0069] It is understood that aspects and embodiments of the invention described herein include “consisting of” and “consisting essentially of” aspects and embodiments. [0070] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.” The term “about X-Y” used herein has the same meaning as “about X to about Y.” [0071] As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. [0072] As used herein and in the appended claims, the singular forms “a,” “an,” or “the” include plural referents unless the context clearly dictates otherwise. II. Methods of treatment [0073] The present application provides methods for treating cancers such as HER2- positive cancer using an antibody-drug conjugate (ADC) comprising an anti-HER2 antibody conjugated to a conjugation moiety comprising a toxin via an endogenous acceptor glutamine residue in the Fc region of the anti-HER2 antibody. Any one of the ADCs described in Section III “Antibody-drug conjugates (ADCs)” may be used in the methods described herein. [0074] In some embodiments, there is provided a method of treating a HER2-positive (e.g., HER 2+ or HER23+ as determined by IHC) cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue. In some embodiments, the Fc region is N- glycosylated. In some embodiments, the acceptor glutamine residue is flanked by an N- glycosylation site at +2 position relative to the glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295 of a heavy chain of the anti-HER2 antibody, wherein the numbering is according to the EU numbering. In some embodiments, the HER2- positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2- positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2- positive cancer is positive as determined by FISH test. In some embodiments, the individual is unresponsive or ineligible for a standard therapy. In some embodiments, the individual has not previously received a second HER2-targeted agent. In some embodiments, the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer. [0075] In some embodiments, there is provided a method of treating a HER2-positive (e.g., HER 2+ or HER23+ as determined by IHC) cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety comprises an monomethyl auristatin E (MMAE), and wherein the anti-HER2 antibody is conjugated to the conjugation moiety via the acceptor glutamine residue. In some embodiments, the conjugation moiety has the chemical structure of Formula (II), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the HER2-positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by FISH test. In some embodiments, the individual is unresponsive or ineligible for a standard therapy. In some embodiments, the individual has not previously received a second HER2-targeted agent. In some embodiments, the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer. [0076] In some embodiments, there is provided a method of treating a HER2-positive (e.g., HER 2+ or HER23+ as determined by IHC) cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the anti-HER2 antibody comprises a glycosylated (e.g., N-glycosylated) Fc region comprising an endogenous acceptor glutamine residue wherein the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering. In some embodiments, the anti-HER2 antibody is N- glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering. In some embodiments, the anti-HER2 antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 7, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the HER2-positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by FISH test. In some embodiments, the individual is unresponsive or ineligible for a standard therapy. In some embodiments, the individual has not previously received a second HER2-targeted agent. In some embodiments, the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer. [0077] In some embodiments, there is provided a method of treating a HER2-positive (e.g., HER 2+ or HER23+ as determined by IHC) cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10, wherein the conjugation moiety has the chemical structure of Formula (I), and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering. In some embodiments, the anti- HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering. In some embodiments, the conjugation moiety is LND1002. In some embodiments, the anti-HER2 antibody is DP001. In some embodiments, the anti- HER2 antibody is trastuzumab. In some embodiments, the HER2-positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by FISH test In some embodiments the individual is unresponsive or ineligible for a standard therapy. In some embodiments, the individual has not previously received a second HER2-targeted agent. In some embodiments, the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer. [0078] In some embodiments, there is provided a method of treating a HER2-positive cancer that is resistant or refractory to a second HER2-targeted agent in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue. In some embodiments, the Fc region is N-glycosylated. In some embodiments, the acceptor glutamine residue is flanked by an N- glycosylation site at +2 position relative to the glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295 of a heavy chain of the anti-HER2 antibody, wherein the numbering is according to the EU numbering. In some embodiments, the HER2- positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2- positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2- positive cancer is positive as determined by FISH test. In some embodiments, the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib. In some embodiments, the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer. [0079] In some embodiments, there is provided a method of treating a HER2-positive cancer that is resistant or refractory to a second HER2-targeted agent in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety comprises an MMAE, and wherein the anti-HER2 antibody is conjugated to the conjugation moiety via the acceptor glutamine residue. In some embodiments, the conjugation moiety has the chemical structure of Formula (II), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (I) In some embodiments the conjugation moiety is LND1002 In some embodiments, the HER2-positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by FISH test. In some embodiments, the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib. In some embodiments, the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer. [0080] In some embodiments, there is provided a method of treating a HER2-positive cancer that is resistant or refractory to a second HER2-targeted agent in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC- CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the anti-HER2 antibody comprises a glycosylated (e.g., N-glycosylated) Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering. In some embodiments, the anti-HER2 antibody is N- glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering. In some embodiments, the anti-HER2 antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 7, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the HER2-positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by FISH test. In some embodiments, the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab or lapatinib In some embodiments the HER2-positive cancer is a solid cancer such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer. [0081] In some embodiments, there is provided a method of treating a HER2-positive cancer that is resistant or refractory to a second HER2-targeted agent in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10, wherein the conjugation moiety has the chemical structure of Formula (I), and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering. In some embodiments, the anti- HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering. In some embodiments, the conjugation moiety is LND1002. In some embodiments, the anti-HER2 antibody is DP001. In some embodiments, the anti- HER2 antibody is trastuzumab. In some embodiments, the HER2-positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by FISH test. In some embodiments, the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib. In some embodiments, the HER2-positive cancer is a solid cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer. [0082] In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of an antibody- drug conjugate (ADC), wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg). In some embodiments, the Fc region is N-glycosylated. In some embodiments, the acceptor glutamine residue is flanked by an N-glycosylation site at +2 position relative to the glutamine residue In some embodiments the acceptor glutamine residue is at position 295 of a heavy chain of the anti-HER2 antibody, wherein the numbering is according to the EU numbering. In some embodiments, the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0 mg/kg or 8.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, such as about 1.0 mg/kg, or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, such as about 2.0 mg/kg, or about 3.0 mg/kg. In some embodiments, the ADC is administered intravenously. In some embodiments, the ADC is administered about once every three weeks, about every other week, or about once per week. In some embodiments, the cancer is a HER2-positive cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer. [0083] In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of an antibody- drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety comprises an MMAE, wherein the anti-HER2 antibody is conjugated to the conjugation moiety via the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg). In some embodiments, the conjugation moiety has the chemical structure of Formula (II), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg. In some embodiments, the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, such as about 1.0 mg/kg, or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, such as about 2.0 mg/kg, or about 3.0 mg/kg. In some embodiments, the ADC is administered intravenously. In some embodiments the ADC is administered about once every three weeks about every other week, or about once per week. In some embodiments, the cancer is a HER2-positive cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer. [0084] In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of an antibody- drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises: (a) a VH comprising an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the anti-HER2 antibody comprises a glycosylated (e.g., N-glycosylated) Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12, wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg). In some embodiments, the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering. In some embodiments, the anti-HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering. In some embodiments, the anti-HER2 antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 7, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg. In some embodiments, the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, such as about 1.0 mg/kg, or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, such as about 2.0 mg/kg, or about 3.0 mg/kg. In some embodiments, the ADC is administered intravenously. In some embodiments, the ADC is administered about once every three weeks about every other week or about once per week. In some embodiments, the cancer is a HER2-positive cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer. [0085] In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of an antibody- drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10, wherein the conjugation moiety has the chemical structure of Formula (I), wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg). In some embodiments, the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering. In some embodiments, the anti-HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering. In some embodiments, the conjugation moiety is LND1002. In some embodiments, the anti-HER2 antibody is DP001. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg. In some embodiments, the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, such as about 1.0 mg/kg, or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, such as about 2.0 mg/kg, or about 3.0 mg/kg. In some embodiments, the ADC is administered intravenously. In some embodiments, the ADC is administered about once every three weeks, about every other week, or about once per week. In some embodiments, the cancer is a HER2- positive cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer. [0086] In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of DP303c, wherein the DP303c is administered intravenously about once every three weeks and at a dose of no more than about 8 mg/kg In some embodiments the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg. In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of DP303c, wherein the DP303c is administered intravenously about once every three weeks and at a dose of about 1 mg/kg to about 2 mg/kg. In some embodiments, the ADC is administered at a dose of about 1.0 mg/kg, or about 2.0 mg/kg. In some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of DP303c, wherein the DP303c is administered intravenously about once every three weeks and at a dose of about 2 mg/kg to about 3 mg/kg. In some embodiments, the ADC is administered at a dose of about 2.0 mg/kg, or about 3.0 mg/kg. In some embodiments, the cancer is a HER2-positive cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer. In some embodiments, the HER2-positive cancer is HER23+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is HER22+ as determined by an IHC test. In some embodiments, the HER2-positive cancer is positive as determined by FISH test. In some embodiments, the individual is unresponsive or ineligible for a standard therapy. In some embodiments, the individual has not previously received a second HER2-targeted agent. In some embodiments, the individual has previously received a second HER2-targeted agent. In some embodiments, the cancer is resistant or refractory to a second HER2 targeted agent. In some embodiments, the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib. [0087] DP303c is an antibody drug conjugate having a HER2-targeting monoclonal IgG1 antibody (DP001) with one cleavable LND1002 (toxin) attached site-specifically to Glutamine 295 in the constant region of each heavy chain of DP001. DP303c has a DAR (drug antibody ratio) of about 1.8 to about 2.2, such as about 1.8, 1.9, 2.0, 2.1, or 2.2. [0088] Cancer treatments can be evaluated by, e.g., tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, quality of life, protein expression and/or activity. Approaches to determining efficacy of therapy can be employed, including for example, measurement of response through radiological imaging. [0089] Exemplary routes of administration of the ADCs include, but are not limited to, oral, intravenous intracavitary intratumoral intraarterial intramuscular subcutaneous parenteral transmucosal, transdermal, ocular, topical, intraperitoneal, intracranial, intrapleural, and epidermal routes, or be delivered into lymph glands, body spaces, organs or tissues known to contain cancer cells. In some embodiments, the ADC is administered intravenously. In some embodiments, the ADC is administered by infusion. In some embodiments, the ADC is administered by injection. [0090] The dosing regimen of the ADCs administered to the individual may vary with the particular ADC composition, the method of administration, and the particular type and stage of cancer being treated. In some embodiments, that effective amount of the ADC is below the level that induces a toxicological effect (i.e., an effect above a clinically acceptable level of toxicity) or is at a level where a potential side effect can be controlled or tolerated when the composition is administered to the individual. The doses referred herein are determined with respect to the entire molecular weight of the ADC. In some embodiments, the ADC is administered at a dose of no more than about any one of 12 mg/kg, 11 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7.5 mg/kg, 7 mg/kg, 6.5 mg/kg, 6 mg/kg, 5.5 mg/kg, 5 mg/kg, 4.5 mg/kg, 4 mg/kg, 3.5 mg/kg, 3.25 mg/kg, 3 mg/kg, 2.9 mg/kg, 2.8 mg/kg, 2.75 mg/kg, 2.7 mg/kg, 2.6 mg/kg, 2.5 mg/kg, 2.4 mg/kg, 2.3 mg/kg, 2.25 mg/kg, 2.2 mg/kg, 2.1 mg/kg, 2 mg/kg, 1.9 mg/kg, 1.8 mg/kg, 1.75 mg/kg, 1.7 mg/kg, 1.6 mg/kg, 1.5 mg/kg, 1.4 mg/kg, 1.3 mg/kg, 1.25 mg/kg, 1.2 mg/kg, 1.1 mg/kg, 1 mg/kg, 0.8 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, or 0.3 mg/kg. In some embodiments, the dose of the ADC is within any of the following range, wherein the ranges have an upper limit of any of: 12 mg/kg, 11 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7.5 mg/kg, 7 mg/kg, 6.5 mg/kg, 6 mg/kg, 5.5 mg/kg, 5 mg/kg, 4.5 mg/kg, 4 mg/kg, 3.5 mg/kg, 3.25 mg/kg, 3 mg/kg, 2.9 mg/kg, 2.8 mg/kg, 2.75 mg/kg, 2.7 mg/kg, 2.6 mg/kg, 2.5 mg/kg, 2.4 mg/kg, 2.3 mg/kg, 2.25 mg/kg, 2.2 mg/kg, 2.1 mg/kg, 2 mg/kg, 1.9 mg/kg, 1.8 mg/kg, 1.75 mg/kg, 1.7 mg/kg, 1.6 mg/kg, 1.5 mg/kg, 1.4 mg/kg, 1.3 mg/kg, 1.25 mg/kg, 1.2 mg/kg, 1.1 mg/kg, 1 mg/kg, 0.8 mg/kg, 0.6 mg/kg, 0.5 mg/kg, or 0.4 mg/kg, and an independently selected lower limit of any of 11 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7.5 mg/kg, 7 mg/kg, 6.5 mg/kg, 6 mg/kg, 5.5 mg/kg, 5 mg/kg, 4.5 mg/kg, 4 mg/kg, 3.5 mg/kg, 3.25 mg/kg, 3 mg/kg, 2.9 mg/kg, 2.8 mg/kg, 2.75 mg/kg, 2.7 mg/kg, 2.6 mg/kg, 2.5 mg/kg, 2.4 mg/kg, 2.3 mg/kg, 2.25 mg/kg, 2.2 mg/kg, 2.1 mg/kg, 2 mg/kg, 1.9 mg/kg, 1.8 mg/kg, 1.75 mg/kg, 1.7 mg/kg, 1.6 mg/kg, 1.5 mg/kg, 1.4 mg/kg, 1.3 mg/kg, 1.25 mg/kg, 1.2 mg/kg, 1.1 mg/kg, 1 mg/kg, 0.8 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, or 0.3 mg/kg, and wherein the lower limit is less than the upper limit. In some embodiments, the ADC is administered at a dose of any one of about 03 mg/kg to about 12 mg/kg about 06 mg/kg to about 8 mg/kg about 1 mg/kg to about 8 mg/kg, about 3 mg/kg to about 8 mg/kg, about 0.6 mg/kg to about 6 mg/kg, about 1 mg/kg to about 6 mg/kg, about 1 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 2.0 mg/kg, about 2.0 mg/kg to about 2.5 mg/kg, about 1 mg/kg to about 3 mg/kg, about 2 mg/kg to about 3 mg/kg, about 2.5 mg/kg to about 3 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 1.5 mg/kg to about 2.5 mg/kg, about 0.5 mg/kg to about 3.0 mg/kg, about 2 mg/kg to about 4 mg/kg, about 4 mg/kg to about 8 mg/kg, about 8 mg/kg to about 12 mg/kg, about 0.5 mg/kg to about 5 mg/kg, or about 1 mg/kg to about 5 mg/kg. The doses described herein may refer to a suitable dose for cynomolgus monkeys, a human equivalent dose thereof, or an equivalent dose for the specific species of the individual. In some embodiments, the ADC is administered at a dose equivalent to about 0.3 mg/kg to about 8 mg/kg (such as such as about 0.3 mg/kg to about 6 mg/kg, about 0.6 mg/kg to about 4.5 mg/kg, about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg) for a cynomolgus monkey or a human individual. In some embodiments, the ADC is administered at a dose equivalent to no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1mg/kg to about 2 mg/kg, or about 2mg/kg to about 3 mg/kg) for a cynomolgus monkey or a human individual. [0091] In some embodiments, the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3, 0.6, 1, 2, 3, 4.5, 6 or 8 mg/kg. In some embodiments, the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, such as about 1.0 mg/kg, or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, such as about 2.0 mg/kg, or about 3.0 mg/kg. [0092] The effective amount of the ADC may be administered in a single dose or in multiple doses. For methods that comprises administration of the ADC in multiple doses, exemplary dosing frequencies include, but are not limited to, weekly, weekly without break, weekly for two out of three weeks, weekly for three out of four weeks, once every three weeks, once every two weeks, monthly, every six months, yearly, etc. In some embodiments, the ADC is administered about weekly, once every 2 weeks, or once every 3 weeks. In some embodiments, the intervals between each administration are less than about any of 3 years, 2 years, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week. In some embodiments, the intervals between each administration are more than about any of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years. In some embodiments, there is no break in the dosing schedule. [0093] In some embodiments, the MSFP is administered at a low frequency, for example, any one of no more frequent than once per week, once every other week, once per three weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, once per 10 months, once per 11 months, once per year, or less. In some embodiments, the ADC is administered in a single dose. In some embodiments, the ADC is administered about once every three weeks. [0094] In some embodiments, the ADC is administered at a dose of no more than about 8 mg/kg, such as no more than any one of 6 mg/kg, 4.5 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg, per week, once every other week, or once every three weeks. In some embodiments, the ADC is administered at a dose of about 0.3 mg/kg to about 8 mg/kg, such as about any one of 0.3, 0.6, 1, 2, 3, 4.5, 6 or 8 mg/kg, per week, once every other week, or once every three weeks. In some embodiments, the ADC is administered at a dose of about 1 mg/kg to about 2 mg/kg, per week, once every other week, or once every three weeks. In some embodiments, the ADC is administered at a dose of about 2 mg/kg to about 3 mg/kg, per week, once every other week, or once every three weeks. In some embodiments, the ADC is administered at a dose of about 1.0 mg/kg, per week, once every other week, or once every three weeks. In some embodiments, the ADC is administered at a dose of about 2.0 mg/kg, per week, once every other week, or once every three weeks. In some embodiments, the ADC is administered at a dose of about 3.0 mg/kg, per week, once every other week, or once every three weeks. [0095] The administration of the ADC can be extended over an extended period of time, such as from about a week to about a month, from about a month to about a year, from about a year to about several years. In some embodiments, the MSFP is administered over a period of at least any of about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or more. [0096] The methods described herein are suitable for treating a variety of cancers such as solid cancer. The methods are applicable to cancers of all stages, including early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission. The methods described herein may be used as a first therapy, second therapy third therapy or combination therapy with other types of cancer therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting. In some embodiments, the cancer has been resistant or refractory to a prior therapy. In some embodiments, the individual has relapsed from a prior therapy. In some embodiments, the individual has recurrent cancer. In some embodiments, the method is carried out before the primary/definitive therapy. In some embodiments, the method is used to treat an individual who has previously been treated. In some embodiments, the method is used to treat an individual who has not previously been treated. In some embodiments, the method is used as a first line therapy. In some embodiments, the method is used as a second line therapy. [0097] In some embodiments, the method is suitable for treating cancers that overexpress HER2 on the surface of the cancer cells, such as HER2-positive solid cancers. In some embodiments, the solid cancer is HER22+ as determined by an IHC test. In some embodiments, the solid cancer is HER23+ as determined by an IHC test. In some embodiments, the solid cancer is HER2 positive as determined by a FISH test. In some embodiments, the cancer cells in the individual express at least about any of more than 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more fold of HER2 compared to normal cells. In some embodiments, the cancer cells in the individual have no more than about 250,000, such as no more than about 200,000; 100,000; 75,000; 50,000; 25,000; 10,000; 7,500; or 5,000 relative HER2 density on cell, as determined using quantitative HER2 receptor density assays, e.g., Quantum 647 MESF (Bang Laboratories). In some embodiments, the cancer cells in the individual have HER2 receptor density comparable to or higher than that of JIMT-1 cells. HER2 density of various cancer cells lines have been described, see, for example, LI. JY et al. “A Biparatopic HER2-targeting antibody-drug conjugate induces tumor regression in primary models refractory to or ineligible for HER2-targeted therapy.” Cancer Cell, 29(1): 117-129, 2016, which is incorporated herein by reference. In some embodiments, the HER2-positive solid cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, and lung cancer. [0098] In some embodiments, there is provided a method of treating breast cancer (e.g., HER2-positive breast cancer) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein. In some embodiments, the breast cancer is early stage breast cancer non-metastatic breast cancer advanced breast cancer stage IV breast cancer, locally advanced breast cancer, metastatic breast cancer, breast cancer in remission, breast cancer in an adjuvant setting, or breast cancer in a neoadjuvant setting. In some embodiments, the breast cancer is in a neoadjuvant setting. In some embodiments, the breast cancer is at an advanced stage. In some embodiments, the breast cancer is HER2- positive breast cancer. In some embodiments, the breast cancer is HER22+ as determined by an IHC test. In some embodiments, the breast cancer is HER23+ as determined by an IHC test. In some embodiments, the breast cancer is HER2 positive as determined by a FISH test. In some embodiments, the breast cancer has metastasized to liver, lung, adrenal gland, lymph node, and/or peritoneum. [0099] In some embodiments, there is provided a method of treating ovarian cancer (e.g., HER2-positive ovarian cancer) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein. In some embodiments, the ovarian cancer is ovarian epithelial cancer. In some embodiments, the ovarian cancer is stage I (e.g., stage IA, IB, or IC), stage II (e.g., stage IIA, IIB, or IIC), stage III (e.g., stage IIIA, HIB, or HIC), or stage IV. In some embodiments, the ovarian cancer is HER2-positive ovarian cancer. In some embodiments, the ovarian cancer is HER22+ as determined by an IHC test. In some embodiments, the ovarian cancer is HER23+ as determined by an IHC test. In some embodiments, the ovarian cancer is HER2 positive as determined by a FISH test. [0100] In some embodiments, there is provided a method of treating colorectal cancer (e.g., HER2-positive colorectal cancer) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein. In some embodiments, the colorectal cancer is sigmoid colon cancer. In some embodiments, the colorectal cancer is stage I, stage II (e.g., stage IIA, IIB, or IIC), stage III (e.g., stage IIIA, IIIB, or IIIC), or stage IV (e.g., stage IVA, IVB, or IVC). In some embodiments, the ovarian cancer is HER2-positive ovarian cancer. In some embodiments, the colorectal cancer is HER22+ as determined by an IHC test. In some embodiments, the colorectal cancer is HER23+ as determined by an IHC test. In some embodiments, the colorectal cancer is HER2 positive as determined by a FISH test. [0101] In some embodiments, there is provided a method of treating gastric cancer (e.g., HER2-positive gastric cancer) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein. In some embodiments, the gastric cancer is adenocarcinoma, lymphoma, astrointestinal stromal tumor (GIST), or carcinoid tumor In some embodiments the gastric cancer is stage 0 (carcinoma in situ) Stage I stage II, stage III, or stage IV. In some embodiments, the gastric cancer is HER2-positive gastric cancer. In some embodiments, the gastric cancer is HER22+ as determined by an IHC test. In some embodiments, the gastric cancer is HER23+ as determined by an IHC test. In some embodiments, the gastric cancer is HER2 positive as determined by a FISH test. [0102] In some embodiments, there is provided a method of treating urethral cancer (e.g., HER2-positive urethral cancer) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein. In some embodiments, the urethral cancer is squamous cell carcinoma, transitional cell carcinoma, or adenocarcinoma. In some embodiments, the urethral cancer is distal urethral cancer or proximal urethral cancer. In some embodiments, the urethral cancer is HER2-positive urethral cancer. In some embodiments, the urethral cancer is HER22+ as determined by an IHC test. In some embodiments, the urethral cancer is HER23+ as determined by an IHC test. In some embodiments, the urethral cancer is HER2 positive as determined by a FISH test. [0103] In some embodiments, there is provided a method of treating lung cancer (e.g., HER2-positive lung cancer) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein. In some embodiments, the lung cancer is a non-small cell lung cancer (NSCLC). Examples of NSCLC include, but are not limited to, large-cell carcinoma, adenocarcinoma, neuroendocrine lung tumors, and squamous cell carcinoma. In some embodiments, the lung cancer is small cell lung cancer (SCLC). In some embodiments, the lung cancer is HER2-positive lung cancer. In some embodiments, the lung cancer is HER22+ as determined by an IHC test. In some embodiments, the lung cancer is HER23+ as determined by an IHC test. In some embodiments, the lung cancer is HER2 positive as determined by a FISH test. [0104] The methods described herein are useful for various aspects of cancer treatment. In some embodiments, there is provided a method of inhibiting cell proliferation (such as tumor growth) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2mg/kg, or about 2 mg/kg to about 3 mg/kg). In some embodiments, at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) cell proliferation is inhibited. [0105] In some embodiments, there is provided a method of inhibiting tumor metastasis in an individual comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg). In some embodiments, at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) metastasis is inhibited. [0106] In some embodiments, there is provided a method of reducing (such as eradicating) pre-existing tumor metastasis (such as metastasis to the lymph node) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg). In some embodiments, at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) metastasis is reduced. [0107] In some embodiments, there is provided a method of reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node) in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg). [0108] In some embodiments, there is provided a method of reducing tumor size in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg). [0109] In some embodiments, there is provided a method of prolonging time to disease progression of cancer in an individual, comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg). In some embodiments, the method prolongs the time to disease progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 28, 32, 36, or more weeks [0110] In some embodiments, there is provided a method of prolonging survival of an individual having cancer, comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg). In some embodiments, the method prolongs the survival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months. [0111] In some embodiments, there is provided a method of alleviating one or more symptoms in an individual having cancer, comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg). [0112] Also provided are compositions of any one of the ADCs described herein for use in the methods described in this section, and use of the ADCs in the manufacture of a medicament for treating cancer. III. Antibody-drug conjugates (ADCs) [0113] The present application also provides antibody-drug conjugates (ADCs) useful for the treatment methods described herein. The ADCs may comprise any one of the anti-HER2 antibodies described herein, which is conjugated to any one of the conjugation moieties described herein via an endogenous acceptor glutamine residue in the Fc region of the anti- HER2 antibody. [0114] In some embodiments, there is provided an ADC comprising an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue. In some embodiments, the Fc region is N-glycosylated. In some embodiments, the acceptor glutamine residue is flanked by an N-glycosylation site at +2 position relative to the glutamine residue. [0115] In some embodiments, there is provided an ADC comprising a full-length anti- HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue at position 295 of a heavy chain of the anti-HER2 antibody wherein the numbering is according to the EU numbering. In some embodiments, the anti- HER2 antibody comprises an N-glycosylated Fc region. In some embodiments, the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue at position 295 of a heavy chain of the anti-HER2 antibody, and wherein the N- glycosylation is at position 297 of the heavy chain, wherein the numbering is according to the EU numbering. [0116] In some embodiments, there is provided an ADC comprising an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety comprises at least one MMAE (such as 1, 2, or more), wherein the anti-HER2 antibody is conjugated to the conjugation moiety via the acceptor glutamine residue. In some embodiments, the conjugation moiety has the chemical structure of Formula (II), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the average molar ratio between the conjugation moiety and the anti-HER2 antibody in the composition is about 1:1 to about 2:1. In some embodiments, at least about 80% (such as at least about any of 85%, 90%, 95% or more) of the ADC in the composition has a molar ratio between the conjugation moiety and the anti-HER2 antibody of about 2:1. [0117] In some embodiments, there is provided an ADC comprising an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the anti-HER2 antibody comprises a glycosylated (e.g., N-glycosylated) Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering In some embodiments the anti-HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the average molar ratio between the conjugation moiety and the anti-HER2 antibody in the composition is about 1:1 to about 2:1. In some embodiments, at least about 80% (such as at least about any of 85%, 90%, 95% or more) of the ADC in the composition has a molar ratio between the conjugation moiety and the anti-HER2 antibody of about 2:1. [0118] In some embodiments, there is provided an ADC comprising an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 7; and (b) a VL comprising the amino acid sequence of SEQ ID NO: 8, wherein the anti-HER2 antibody comprises a glycosylated (e.g., N- glycosylated) Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering. In some embodiments, the anti-HER2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to the EU numbering. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the average molar ratio between the conjugation moiety and the anti-HER2 antibody in the composition is about 1:1 to about 2:1. In some embodiments, at least about 80% (such as at least about any of 85%, 90%, 95% or more) of the ADC in the composition has a molar ratio between the conjugation moiety and the anti-HER2 antibody of about 2:1. [0119] In some embodiments, there is provided an ADC comprising an anti-HER2 antibody and a conjugation moiety, wherein the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10, wherein the conjugation moiety has the chemical structure of Formula (III), wherein n is an integer between 1 and 12, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein the numbering is according to the EU numbering. In some embodiments, the anti-HER2 antibody is N- glycosylated at position 297 of the Fc region wherein the numbering is according to the EU numbering. In some embodiments, the conjugation moiety has the chemical structure of Formula (I). In some embodiments, the conjugation moiety is LND1002. In some embodiments, the anti-HER2 antibody is DP001. In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the ADC is DP303c. In some embodiments, the average molar ratio between the conjugation moiety and the anti-HER2 antibody in the composition is about 1:1 to about 2:1. In some embodiments, at least about 80% (such as at least about any of 85%, 90%, 95% or more) of the ADC in the composition has a molar ratio between the conjugation moiety and the anti-HER2 antibody of about 2:1. [0120] In some embodiments, the anti-HER2 antibody is a full-length antibody. In some embodiments, the anti-HER2 antibody is an antibody fragment comprising an Fc region. In some embodiments, the Fc region comprises part or all of the hinge region. In some embodiments, the anti-HER2 antibody comprises the Fc region of a naturally occurring immunoglobulin. In some embodiments, the anti-HER2 antibody comprises an Fc region of IgG1, IgG2, IgG3, IgG4 subtypes, or from IgA, IgE, IgD, or IgM. In some embodiments, the Fc region is from human IgG, and the Fc region is from an amino acid residue at position Glu216 or Ala231 to the carboxyl-terminus thereof according to the EU numbering system. [0121] In some embodiments, the Fc region in the anti-HER2 antibody is N-glycosylated. For example, in some embodiments, the polysaccharide chain attached at the N-glycosylation site is at least about any of 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 units. [0122] In some embodiments, there is provided a composition comprising any one of the ADCs described herein, wherein at least some (but not necessarily all) of the anti-HER2 antibody in the composition is glycosylated (for example N-glycosylated) in the Fc region. For example, in some embodiments, there is provided a composition comprising an antibody- drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody conjugated to a conjugation moiety via an endogenous acceptor glutamine residue on the anti- HER2 antibody, and wherein at least some of (for example at least about any of 50%, 60%, 70%, 80%, 90%, or 95%) the antibody-drug conjugates in the composition is glycosylated (for example N-glycosylated) in the Fc region. [0123] In some embodiments, the N-glycosylation site flanks the acceptor glutamine residue to which the conjugation moiety is attached. In some embodiments, the N-glycosylation site and the acceptor glutamine residue are 5 or less amino acid residues apart. In some embodiments, the N-glycosylation site and the acceptor glutamine are 5, 4, 3, 2, or 1 amino acids apart In some embodiments the N-glycosylation site and the acceptor glutamine are next to each other. In some embodiments, the acceptor glutamine residue is flanked by an N- glycosylation site at +2 position relative to the glutamine residue. In some embodiments, the acceptor glutamine residue is flanked by an N-glycosylation site at +1, +2, +3, +4, or +5 position relative to the glutamine residue. In some embodiments, the acceptor glutamine residue is flanked by an N-glycosylation site at -1, -2, -3, -4, or -5 position relative to the glutamine residue. In some embodiments, the N-glycosylated Fc region comprises the amino acid sequence of SEQ ID NO: 11 (KPREEQX1NSTX2R, wherein X1 is Y or F and X2 is Y or F), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 6 of SEQ ID NO: 11, and wherein the N-glycosylation is at position 8 of SEQ ID NO: 11. In some embodiments, the N-glycosylated Fc region comprises amino acid sequence of SEQ ID NO: 12 (KPREEQYNSTYR), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 6 of SEQ ID NO: 12, and wherein the N-glycosylation is at position 8 of SEQ ID NO: 2. [0124] In some embodiments, the anti-HER2 antibody comprises an Fc region of human IgG1. In some embodiments, the anti-HER2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO: 13 (CH2 sequence of human IgG1), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 65 of SEQ ID NO: 13, and wherein the N-glycosylation is at position 67 of SEQ ID NO: 13. [0125] In some embodiments, the anti-HER2 antibody comprises an Fc region of human IgG2. In some embodiments, the anti-HER2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO: 14 (CH2 sequence of human IgG2), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 64 of SEQ ID NO: 14, and wherein the N-glycosylation is at position 66 of SEQ ID NO: 14. [0126] In some embodiments, the anti-HER2 antibody comprises an Fc region of human IgG3. In some embodiments, the anti-HER2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO: 15 (CH2 sequence of human IgG3), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 65 of SEQ ID NO: 15, and wherein the N-glycosylation is at position 67 of SEQ ID NO: 15. [0127] In some embodiments, the anti-HER2 antibody comprises an Fc region of human IgG4. In some embodiments, the anti-HER2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO: 16 (CH2 sequence of human IgG4), and wherein the conjugation moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue at position 65 of SEQ ID NO: 16, and wherein the N-glycosylation is at position 67 of SEQ ID NO: 16.
Figure imgf000040_0001
[0128] The ADCs described herein have the anti-HER2 antibody component conjugated to the conjugation moiety in a specific and stoichiometrically controlled fashion, i.e., at the acceptor glutamine residue at the Fc region that is flanked by an N-glycosylation site. In some embodiments, the molar ratio of the conjugation moiety to the anti-HER2 antibody is about 1:1. In some embodiments, the molar ratio of the conjugation moiety to the anti-HER2 antibody is about 2:1. In some embodiments, at least about 80% (such as at least about any of 85%, 90%, 95% or more) of the ADC in the composition has the conjugation moiety to the anti-HER2 antibody molar ratio of about 1:1. In some embodiments, at least about 80% (such as at least about any of 85%, 90%, 95% or more) of the ADC in the composition has the conjugation moiety to the anti-HER2 antibody molar ratio of about 2:1. In some embodiments at least about 80% (such as at least about any of 85% 90% 95% or more) of the ADC in the composition has the molar ratio of the conjugation moiety to the anti-HER2 antibody of about 1:1 or about 2:1. [0129] In some embodiments, the ADC is present in an individual (e.g., a mammal) at about 50% or more after at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more upon administration in vivo. In some embodiments, the ADC is present in an individual (e.g., a mammal) at about any of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more after at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, or more upon administration in vivo. In some embodiments, the free toxin (e.g., MMAE) exposure to an individual is no more than about any one of 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% or less of the ADC exposure upon administration of the ADC in vivo. Anti-HER2 antibody [0130] The ADCs described herein comprise an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody specifically binds human HER2 expressed on the cell surface of a human cell (e.g., a human cancer cell). In some embodiments, the anti-HER2 antibody specifically binds a HER2 expressed on the cell surface of a human cancer cell (e.g., a breast cancer cell, an ovarian cancer cell, a gastric cancer cell, a urethral cancer cell, or a lung cancer cell. [0131] In some embodiments, the anti-HER2 antibody is trastuzumab. In some embodiments, the anti-HER2 antibody is not trastuzumab. In some embodiments, the anti- HER2 antibody specifically binds to the same epitope in HER2 as trastuzumab. In some embodiments, the anti-HER2 antibody comprises the same sequences, e.g., heavy chain CDRs, light chain CDRs, heavy chain variable region, light chain variable region, heavy chain and/or light chain sequences, as trastuzumab. In some embodiments, the anti-HER2 antibody is a biosimilar of trastuzumab. In some embodiments, the anti-HER2 antibody is DP001. [0132] In some embodiments, the anti-HER2 antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HC-CDR)1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the anti-HER2 antibody comprises a light chain variable region (VL) comprising a light chain complementarity determining region (LC-CDR)1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. [0133] In some embodiments, the anti-HER2 antibody comprises a VH comprising an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6. [0134] In some embodiments, the anti-HER2 antibody comprises: a) VH comprising the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3; and ii) a VL comprising the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO: 6. [0135] In some embodiments, the anti-HER2 antibody comprises: a) a VH comprising one, two or three CDRs of SEQ ID NO: 7, and/or b) a VL comprising one, two or three CDRs of SEQ ID NO: 8. In some embodiments, the anti-HER2 antibody comprises: a) a VH comprising HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain variable region of SEQ ID NO: 7, and/or b) a VL comprising LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain variable region of SEQ ID NO: 8. [0136] In some embodiments, the anti-HER2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 7. In some embodiments, the anti-HER2 antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 8. [0137] In some embodiments, the anti-HER2 antibody comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 7; and b) a VL comprising the amino acid sequence of SEQ ID NO: 8. [0138] In some embodiments, the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 9. In some embodiments, the anti-HER2 antibody comprises a lambda light chain constant region. In some embodiments, the anti-HER2 antibody comprises a kappa light chain constant region. In some embodiments, the anti-HER2 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 10. [0139] In some embodiments, the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. [0140] Exemplary anti-HER2 antibody sequences are shown in Table B below. The exemplary CDR sequences are predicted using the IgBLAST algorithm. See, for example, Ye J. et al. Nucleic Acids Research, 41:W34-W40 (2013), the disclosure of which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that many algorithms are known for prediction of CDR positions in antibody heavy chain and light chain variable regions, and antibody agents comprising CDRs from antibodies described herein, but based on prediction algorithms other than IgBLAST, are within the scope of this invention. The exemplary antibody heavy chain and light chain variable region sequences are delimited according to the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM® (IMGT). See, for example, Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015), the disclosure of which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that antibody agents comprising VH or VL sequences from antibodies described herein, but based on algorithms other than IMGT, are within the scope of this invention. Table B. Anti-HER2 antibody sequences
Figure imgf000044_0001
Figure imgf000044_0002
[0141] In some embodiments, the anti-HER2 antibody is DP001. DP001 is an anti-HER2 monoclonal antibody, which has the same amino acid sequence as trastuzumab (HERCEPTIN®). Specifically, it contains 1328 amino acids with two heavy chains (HC) of 450 amino acids (49284.65 Da, SEQ ID NO: 9), and two light chains (LC) of 214 amino acids (23443.10 Da, SEQ ID NO: 10). DP001 is a heterotetramer of two HCs of the IgG1 subclass, and two LCs of the kappa subclass linked by 16 disulfide bonds (12 intra and 4 inter chain). A schematic structure of DP001 is depicted in FIG.1. [0142] The anti-HER2 antibodies described herein encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab’, F(ab’)2, Fv, Fc, etc.), chimeric antibodies, humanized antibodies, human antibodies (e.g., fully human antibodies), single chain (ScFv), bispecific antibodies, multispecific antibodies, mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity. The antibodies may be murine, rat, camel, human, or any other origin (including humanized antibodies). Antibodies used in the present disclosure also include single domain antibodies, which are either the variable domain of an antibody heavy chain or the variable domain of an antibody light chain. Holt et al., Trends Biotechnol.21:484-490, 2003. Methods of making domain antibodies comprising either the variable domain of an antibody heavy chain or the variable domain of an antibody light chain, containing three of the six naturally occurring HVRs or CDRs from an antibody, are also known in the art. See, e.g., Muyldermans, Rev. Mol. Biotechnol.74:277-302, 2001. [0143] In some embodiments, the anti-HER2 antibody is a monoclonal antibody. As used herein, a monoclonal antibody refers to an antibody of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), monoclonal antibody is not a mixture of discrete antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in the present disclosure may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No.4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature, 348:552-554, for example. [0144] In some embodiments, the anti-HER2 antibody is a chimeric antibody. As used herein, a chimeric antibody refers to an antibody having a variable region or part of variable region from a first species and a constant region from a second species. An intact chimeric antibody comprises two copies of a chimeric light chain and two copies of a chimeric heavy chain. The production of chimeric antibodies is known in the art (Cabilly et al. (1984), Proc. Natl. Acad. Sci. USA, 81:3273-3277; Harlow and Lane (1988), Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory). Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to the sequences in antibodies derived from another. One clear advantage to such chimeric forms is that, for example, the variable regions can conveniently be derived from presently known sources using readily available hybridomas or B cells from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation, and the specificity is not affected by its source, the constant region being human is less likely to elicit an immune response from a human subject when the antibodies are injected than would the constant region from a non- human source. However, the definition is not limited to this particular example. [0145] In some embodiments, the anti-HER2 antibody is a humanized antibody. As used herein, humanized antibodies refer to forms of non-human (e.g. murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a HVR or CDR of the recipient are replaced by residues from a HVR or CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported HVR or CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVR or CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more HVRs or CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more HVRs or CDRs “derived from” one or more HVRs or CDRs from the original antibody. [0146] In some embodiments, the anti-HER2 antibody is a human antibody. As used herein, a human antibody means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies known in the art. A human antibody used herein includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Patent Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or may have been immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Patent No.5,750,373. [0147] The anti-HER2 antibodies described herein may further include analogs and derivatives that are either modified, i.e., by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity. For example, the derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formulation, etc. Additionally, the analog or derivative can contain one or more unnatural amino acids. [0148] In some embodiments, amino acid sequence variants of the anti-HER2 antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis Such modifications include for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. In some embodiments, anti-HER2 antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Conservative substitutions are shown in Table C below. TABLE C: CONSERVATIVE SUBSTITITIONS
Figure imgf000048_0001
Amino acids may be grouped into different classes according to common side-chain properties: a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp, Glu; d. basic: His, Lys, Arg; e. residues that influence chain orientation: Gly, Pro; f. aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. [0149] An exemplary substitutional variant is an affinity matured antibody moiety, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and the variant antibody moieties displayed on phage and screened for a particular biological activity (e.g., binding affinity). Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody moiety affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol.207:179-196 (2008)), and/or specificity determining residues (SDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) [0150] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody. [0151] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of a full-length anti-HER2 antibody provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC effector function, often related to binding to Fc receptors (FcRs). In some embodiments, the Fc region variant has decreased ADCC effector function. There are many examples of changes or mutations to Fc sequences that can alter effector function. For example, WO 00/42072 and Shields et al. J Biol. Chem.9(2): 6591-6604 (2001) describe antibody variants with improved or diminished binding to FcRs. The contents of those publications are specifically incorporated herein by reference. [0152] In some embodiments, the anti-HER2 antibody comprises an Fc region that possesses some but not all effector functions, which makes it a desirable candidate for applications in which the half-life of the anti-HER2 antibody in vivo is important yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No.5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No.5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CYTOTOX 96™ non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759- 1769 (2006)). [0153] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581). [0154] Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No.6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem.9(2): 6591-6604 (2001).) [0155] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No.6,194,551, WO 99/51642, and Idusogie et al., J. Immunol.164: 4178-4184 (2000). [0156] In some embodiments, the anti-HER2 antibody comprises a variant Fc region comprising one or more amino acid substitutions, which increase half-life and/or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-lives and improved binding to FcRn are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No.5,624,821; and WO 94/29351 concerning other examples of Fc region variants. [0157] In some embodiments, the anti-HER2 antibody is altered to increase or decrease the extent to which the anti-HER2 antibody is glycosylated. Addition or deletion of glycosylation sites to an anti-HER2 antibody may be conveniently accomplished by altering the amino acid sequence of the anti-HER2 antibody or polypeptide portion thereof such that one or more glycosylation sites is created or removed. [0158] Where the anti-HER2 antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an anti-HER2 antibody of the invention may be made in order to create anti-HER2 antibody variants with certain improved properties. [0159] The N-glycans attached to the CH2 domain of Fc is heterogeneous. Antibodies or Fc fusion proteins generated in CHO cells are fucosylated by fucosyltransferase activity See Shoji-Hosaka et al., J. Biochem.2006, 140:777- 83. Normally, a small percentage of naturally occurring afucosylated IgGs may be detected in human serum. N-glycosylation of the Fc is important for binding to FcJR; and afucosylation of the N-glycan increases Fc's binding capacity to FcJRIIIa. Increased FcJRIIIa binding can enhance ADCC, which can be advantageous in certain antibody therapeutic applications in which cytotoxicity is desirable. [0160] Anti-HER2 antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the anti-HER2 antibody is bisected by GlcNAc. Such anti-HER2 antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No.6,602,684 (Umana et al.); US 2005/0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5): 851-861 (2006). Anti-HER2 antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such anti-HER2 antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.). [0161] In some embodiments, the anti-HER2 antibody variants comprising an Fc region are capable of binding to an FcγRIII. In some embodiments, the anti-HER2 antibody comprises a human wild-type IgG1 Fc region. [0162] Also provided are one or more nucleic acids encoding the heavy chain and/or the light chain of the anti-HER2 antibody, vectors comprising the one or more nucleic acids, and methods of preparing the anti-HER2 antibody. Conjugation moiety [0163] The conjugation moiety of the ADCs described herein comprise a toxin, such as a cytotoxic agent useful for cancer therapy. In some embodiments, the toxin is a chemotherapeutic agent. In some embodiments, the toxin is a small molecule drug. Examples of a cytotoxic agent include, but are not limited to, an anthracycline, an auristatin, a dolastatin, CC-1065, a duocarmycin, an enediyne, a geldanamycin, a maytansine, a puromycin, a taxane, a vinca alkaloid, SN-38, tubulysin, hemiasterlin, and stereoisomers, isosteres, analogs or derivatives thereof. In some embodiments, the conjugation moiety comprises monodansylcadaverine (MDC). In some embodiments, the conjugation moiety comprises TAM1. In some embodiments, the conjugation moiety comprises monomethyl auristatin E (MMAE). [0164] The anthracyclines are derived from bacteria Streptomyces and have been used to treat a wide range of cancers, such as leukemias, lymphomas, breast, uterine, ovarian, and lung cancers. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone. [0165] Dolastatins and their peptidic analogs and derivatives, auristatins, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, e.g., U.S. Pat. No.5,663,149 and Pettit et al., Antimicrob. Agents Chemother. 42:2961-2965 (1998). Exemplary dolastatins and auristatins include, but are not limited to, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD, MMAF, MMAE, and 5-benzoylvaleric acid-AE ester (AEVB). [0166] Duocarmycin and CC-1065 are DNA alkylating agents with cytotoxic potency. See Boger and Johnson, PNAS 92:3642-3649 (1995). Exemplary dolastatins and auristatins include, but are not limited to, (+)-docarmycin A and (+)-duocarmycin SA, and (+)-CC-1065. [0167] Enediynes are a class of anti-tumor bacterial products characterized by either nine- and ten-membered rings or the presence of a cyclic system of conjugated triple-double-triple bonds. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, and dynemicin. [0168] Geldanamycins are benzoquinone ansamycin antibiotic that bind to Hsp90 (Heat Shock Protein 90) and have been used antitumor drugs. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-Allylamino-17-Demethoxygeldanamycin) and 17- DMAG (17-Dimethylaminoethylamino-17-demethoxygeldanamycin). [0169] Maytansines or their derivatives maytansinoids inhibit cell proliferation by inhibiting the microtubules formation during mitosis through inhibition of polymerization of tubulin. See Remillard et al., Science 189:1002-1005 (1975). Exemplary maytansines and maytansinoids include, but are not limited to, mertansine (DM1) and its derivatives as well as ansamitocin. [0170] Taxanes are diterpenes that act as anti-tubulin agents or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (e.g., TAXOL®) and docetaxel (TAXOTERE®). [0171] Vinca alkyloids are also anti-tubulin agents. Exemplary vinca alkyloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine. [0172] One skilled in the art may make chemical modifications to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention. [0173] In some embodiments, the conjugation moiety comprises a toxin polypeptide (or a toxin protein). Examples of a toxin polypeptide include, but are not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, tricothecenes, inhibitor cystine knot (ICK) peptides (e.g., ceratotoxins), and conotoxin (e.g., KIIIA or SmIIIa). [0174] The conjugation moiety described herein comprises an amine donor group, which is conjugated to the acceptor glutamine residue in the anti-HER2 antibody. Any conjugation moiety not containing an amine donor group can be indirectly conjugated to the anti-HER2 antibody via a small molecule handle, which contains an amine donor group. [0175] The term “amine donor group” as used herein refers to a reactive group containing one or more reactive amines (e.g., primary amines). For example, the conjugation moiety can comprise an amine donor group (e.g., primary amine -NH2), a linker, and a toxin (e.g., a small molecule). The conjugation moiety can also be a polypeptide or a biocompatible polymer containing a reactive Lys (e.g., an endogenous Lys). The amine donor group in some embodiments is a primary amine (-NH2) that provides a substrate for transglutaminase to allow conjugation of the conjugation moiety to the anti-HER2 antibody via the acceptor glutamine. Accordingly, the linkage between the donor glutamine and the amine donor group can be of the formula -CH2- CH2-CO-NH- . [0176] In some embodiments, the anti-HER2 antibody and the conjugation moiety are linked through a linker. The phrase “linker” refers to a structural element of a compound that links one structural element of said compound to one or more other structural elements of said same compound. In some embodiments, the linker is a non-cleavable linker. Suitable non- cleavable linkers include, but are not limited to, NH2-R-X, NH2NH-R-X, and NH2-O-R-X, wherein R is alkyl or polyethylene glycol group (also referred to as PEG), wherein X is the toxin. A polyethylene glycol group or PEG group may have a formula of --CH2CH2O) n -, wherein n is an integer of at least 1. In some embodiments, n is any of 2, 4 6 8 10 12 16 20 or 24 [0177] In some embodiments, the anti-HER2 antibody and the conjugation moiety are linked through a cleavable linker. Suitable cleavable linkers include, but are not limited to, Lys-Phe-X, Lys-Val-Cit-PABC-X, NH2-(CH2CH2O) n -Val-Cit-PABC-X, and NH2-(CH2CH2O)n- (Val-Cit-PABC-X)2, wherein X is the toxin, and n is an integer of at least 1 (such as any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24). PABC refers to p-aminobenzyloxycarbonyl. Cit refers to citrulline. [0178] Other exemplary amine donor group-linkers include, but are not limited to, Ac-Lys- Gly, aminocaproic acid, Ac-Lys-beta -Ala, amino-PEG2 (Polyethylene Glycol)-C2, amino- PEG3-C2, amino-PEG6-C2, Ac-Lys-Val (valine)-Cit (citrulline)-PABC (p- aminobenzyloxycarbonyl), aminocaproyl-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine. [0179] In some embodiments, the conjugation moiety is linked to the acceptor glutamine residue via a -NH-(C)n- linker, wherein the (C)n is a substituted or unsubstituted alkyl or heteroalkyl chain, wherein n is an integer from about 1 to about 60. In some embodiments, the carbon of the chain is substituted with an alkoxyl, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide, or alkylamide. In some embodiments, n is about 2 to about 20. [0180] In some embodiments, the linker is branched. In some embodiments, the linker is linear. In some embodiments, the linker has more than one (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) attachment sites for the attachment of active moieties. These active moieties can be the same or different from each other. For example, the conjugation moiety may comprise a polyacetal- or polyacetal derivative-based polymer linked to a plurality of toxins (such as chemotherapeutic agents). [0181] In some embodiments, the conjugation moiety comprises a toxin that is selected from the group consisting of Ac-Lys-Gly-MMAD, amino-PEG3-C2-MMAD, amino-PEG6- C2-MMAD, amino-PEG3-C2-amino-nonanoyl-MMAD], aminocaproyl-Val-Cit-PABC- MMAD, Ac-Lys-beta -Ala-MMAD, Aminocaproyl-MMAD, Ac-Lys-Val-Cit-PABC-MMAD, Aminocaproyl-MMAE, amino-PEG3-C2-MMAE, amino-PEG2-C2-MMAE, Aminocaproyl- MMAF, Aminocaproyl-Val-Cit-PABC-MMAE, Aminocaproyl-Val-Cit-PABC-MMAF, amino-PEG2-C2-MMAF, amino-PEG3-C2-MMAF, putrescinyl-geldanamycin, and Ac-Lys- putrescinyl-geldanamycin. In some embodiments, the amine donor agent is aminocaproyl- Val-Cit-PABC-MMAE, aminocaproyl-Val-Cit-PABC-MMAF, Ac-Lys-putrescinyl- geldanamycin, Ac-Lys-beta -Ala-MMAD, Ac-Lys-Val-Cit-PABC-MMAD, aminocaproyl- Val-Cit-PABC-MMAD and amino-PEG6-C2-MMAD [0182] In some embodiments, the conjugation moiety is a maytansine derivative. In some embodiments, the conjugation moiety is an MMAE derivative comprising a non-cleavable linker (such as an amino-(CH2CH2O) n- linker). In some embodiments, the conjugation moiety has the chemical structure of Formula (II):
Figure imgf000056_0001
(II), wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. [0183] In some embodiments, the conjugation moiety is an MMAE derivative comprising a cleavable linker (such as an amino-(CH2CH2O) n –Val-Cit-PABC-MMAE). In some embodiments, the conjugation moiety has the chemical structure of Formula (III):
Figure imgf000056_0002
wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. [0184] In some embodiments, the conjugation moiety comprises a toxin that has the chemical structure of Formula (I):
Figure imgf000056_0003
[0185] In some embodiments, the toxin is LND1002. LND1002 is a toxin derived from MMAE, which has a PEG linker with a primary amine group for conjugation. LND1002 has the chemical structure of formula (I). [0186] The chemical compounds contemplated herein include salts, solvates, or stereoisomers thereof, which include all permutations of salts, solvates and stereoisomers, such as a solvate of a pharmaceutically acceptable salt of a stereoisomer of subject compound. [0187] The term “pharmaceutically acceptable salt” means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like. [0188] The term “salt thereof” means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient. By way of example, salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt. [0189] “Solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate. [0190] “Stereoisomer” and “stereoisomers” refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers. Methods of making antibody-drug conjugates [0191] The ADCs described herein can be made using any suitable methods in the art, for example, by using a transglutaminase to conjugate an amine group in the conjugation moiety to the endogenous acceptor glutamine residue in the antibody. See, for example, U.S. Pat. No. 10,357,472, which is incorporated herein by reference in its entirety. [0192] In some embodiments, the anti-HER2 ADC is prepared using a wildtype or an engineered transglutaminase. Engineered transglutaminases suitable for preparing the ADCs described herein include those described in U.S. Pat. No.10,471,037, which is incorporated herein by reference in its entirety. [0193] TGases catalyzes covalent protein crosslinking by forming proteinase resistant isopeptide bonds between a lysine donor residue of one protein and an acceptor glutamine residue of another protein, and is accompanied by the release of ammonia. The catalytic mechanism of transglutaminases has been proposed as follows. After the glutamine- containing first substrate (acceptor or Q-substrate) binds to the enzyme, it forms a gamma - glutamylthioester with the cysteine residue in the active center of TGase, known as the acylenzyme intermediate, accompanied by the release of ammonia. The second substrate (donor or K-substrate) then binds to the acylenzyme intermediate and attacks the thioester bond. The product (two proteins crosslinked by an Nepsilon (gamma -glutamyl)lysine isopetide bridge) is formed and released. This re-establishes the active-center Cys residue of the enzyme in its original form and allows it to participate in another cycle of catalysis. The formation of the covalent acylenzyme intermediate is thought to be the rate-limiting step in these reactions. The catalytic triad of many transglutaminases is papain-like, containing Cys- His-Asp (where His is histidine and Asp is aspartic acid) and, crucially, a tryptophan (Trp) residue located 36 residues away from the active-center Cys. In contrast, bacterial TGases isolated from Streptoverticillium sp (vide supra) has an atypical catalytic triad and shows no sequence homology with the papain-like catalytic triad of other TGases. [0194] Several types of transglutaminases have been reported in various living organisms including microbial organisms. Examples are TGase from guinea pig liver (GTGase), fish liver (FTGase) and microorganisms (mTGase) and any recombinant TGase (rTGase). Other TGases than the ones listed here can also be used according to the invention. Examples of useful TGases include microbial transglutaminases, such as e.g. from Streptomyces mobaraense, Streptomyces cinnamoneum and Streptomyces griseocarneum disclosed in U.S. Pat. No.5,156,956, and Streptomyces lavendulae disclosed in U.S. Pat. No.5,252,469, and Streptomyces ladakanum disclosed in JP2003199569. Other useful microbial transglutaminases have been isolated from Bacillus subtilis (disclosed in U.S. Pat. No. 5,731,183) and from various Myxomycetes. Other examples of useful microbial transglutaminases are those disclosed in WO 96/06931 (e.g. transglutaminase from Bacilus lydicus) and WO 96/22366 Useful non-microbial transglutaminases include guinea-pig liver transglutaminase, and transglutaminases from various marine sources like the flat fish Pagrus major (disclosed in EP-0555649), and the Japanese oyster Crassostrea gigas (disclosed in U.S. Pat. No.5,736,356). An exemplary TGase is bacterial transglutaminase (BTG) (see, e.g. EC 2.3.2.13, protein-glutamine-gamma -glutamyltransferase). In another exemplary embodiment, the TGase is from S. mobaraense. In another embodiment, the TGase is a mutant (e.g., engineered) TGase having at least 80% sequence homology with native TGase. An example is recombinant bacterial transglutaminase derived from Streptomyces mobaraensis (available from Zedira, Darmstadt, Germany). [0195] Streptomyces ladakanum ATCC 27441 or NRRL3191 mTgase is expressed as Pre- Pro-mTgase (GenBank access number AY241675). There are 410 amino acid residues in pre- pro-mTGase, 331 in mature enzyme plus 30 of pre and 49 of pro. Pro peptide is a strong inhibitor of mature enzyme. Primers designed according to AY241675 were used to clone the pro-mTgase and mature mTgase from ATCC 27441DNA into pET29b(+) vector’s Nde I and Xho I sites. Active mTgase can be obtained either from enterokinase light chain (EKL) digestion of Pro-mTgase or refolding of mature mTgase. mTgase from Strep Ladakanum (TG_SL) is very similar to mTgase from Strep. mobaraensis (TG_SM, sold by Ajinomoto as ACTIVA®) with a few amino acid differences. [0196] The transglutaminase used in methods described herein can be obtained or made from a variety of sources. In some embodiments, the transglutaminase is a calcium dependent transglutaminase, which requires calcium to induce enzyme conformational changes and allow enzyme activity. For example, transglutaminase can be derived from guinea pig liver and obtained through commercial sources (e.g., Sigma-Aldrich (St Louis, Mo.) and MP Biomedicals (Irvine, Calif.)). In some embodiments, the transglutaminase is a calcium independent transglutaminase, which does not require calcium to induce enzyme conformational changes and allow enzyme activity. In some embodiments, the transglutaminase is a microbial transglutaminase derived from a microbial genome, such as transglutaminase from Streptoverticillium or Streptomices (e.g., Streptomyces mobarensis or Streptoverticillium mobarensis). In some embodiments, the transglutaminase is a mammalian protein (e.g., human transglutaminase), a bacterial protein, a plant protein, a fungi protein (e.g., Oomycetes and Actinomicetes transglutaminases), or a prokaryotic protein. In some embodiments, the transglutaminase is from Micrococcus, Clostridium, Turolpsis, Rhizopus, Monascus, or Bacillus. [0197] In some embodiments, the transglutaminase used in the methods described herein is a recombinant protein produced using recombinant techniques. In some embodiments, the TGase is prepared by: (a) culturing a host cell (such as a prokaryotic cell) comprising a vector comprising a nucleic acid encoding a pro-enzyme of TGase, and (b) obtaining mature TGase by cleavage of the pro-sequence of the pro-enzyme (for example by endokinase light chain). In some embodiments, the TGase is purified by chromatography (such as by affinity chromatography or ion exchange chromatography). In some embodiments, the TGase is tagged (such as his-tagged) to facilitate purification. [0198] In some embodiments, the anti-HER2 ADC is prepared by contacting the anti-HER2 antibody with the conjugation moiety in the presence of a transglutaminase under a condition that is sufficient to generate the ADC, wherein the anti-HER2 antibody comprises an N- glycosylated Fc region, wherein the N-glycosylated Fc region comprises an acceptor glutamine residue flanked by an N-glycosylation site, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue. In some embodiments, the anti-HER2 ADC is prepared by contacting a composition comprising the anti-HER2 antibody with the conjugation moiety in the presence of a transglutaminase under a condition that is sufficient to generate the ADC, wherein at least some (e.g., at least about 50%, 60%, 70%, 80%, 90%, or more) of the anti-HER2 antibody comprise an N-glycosylated Fc region, wherein the Fc region comprises an acceptor glutamine residue flanked by an N- glycosylation site, and wherein the conjugation moiety is conjugated to the anti-HER2 antibody via the acceptor glutamine residue. [0199] In some embodiments, the anti-HER2 ADC is prepared in two steps. First, a small molecule handle is conjugated to the anti-HER2 antibody via a TGase to create an intermediate conjugate. Subsequently, a toxin is coupled to the intermediate conjugate via the small molecule handle, either covalently or non-covalently. The small molecule handle can be specifically designed to tailor the coupling of the toxin, thus allows the conjugation of any kind of toxin to the anti-HER2 antibody. The two-step method is particularly useful when the supply of the anti-HER2 antibody and/or the toxin is limited, and when the toxin has low water solubility and/or induces aggregation of the anti-HER2 antibody. [0200] The small molecule handle described herein generally has the structure of -NH2-R, wherein R is a moiety that allows the attachment of the toxin. The introduction of the small molecule handle in the methods described herein significantly increases the flexibility of the methods Specifically the structure of the small molecule handle can be tailored to the attachment of the desired toxin. For example, in some embodiments, R is a ligand, which specifically binds to a binding partner. This allows attachment of any molecule (such as protein) that contains the binding partner. Suitable ligand/binding partner pairs include, but are not limited to, antibody/antigen, antigen/antibody, avidin/biotin, biotin/avidin, streptavidin/biotin, biotin/streptavidin, glutathione/GST, GST/glutathione, maltose binding protein/amylose, amylose/maltose binding protein, cellulose binding protein and cellulose, cellulose/cellulose binding protein, etc. [0201] Other suitable small molecule handles described herein include, but are not limited to, NH2-CH2-CH(OH)-CH2-NH2, NH2-R-(OR’)2, NH2-R=O, NH2-R-SH, NH2-R-Azide. These small molecule handles allow the attachment of the conjugation moiety through suitable linkers such as NH2-O-R-X, Maleimide-R-X, and Cyclooctyne-R-(R’-X)2, wherein X is the active moiety, and R and R’ are independently linker groups, such as linker groups comprising alkyl or polyethylene glycol groups. [0202] The TGase-catalyzed reaction can be carried out from several hours to a day (e.g. overnight). The conjugation moiety or the small molecule handle are allowed to react with anti-HER2 antibody (e.g., 1 mg/mL) at ligand concentrations between 400 and 600 Pmol/L, providing a 60 to 90-fold excess of the substrates over the anti-HER2 antibody, or at lower excess of substrates, e.g.1- to 20-fold, or 10-20 fold. The reactions can be performed in potassium-free phosphate buffered saline (PBS; pH 8) at 37 °C. After 4 h to several days, steady-state conditions are achieved. Excess ligand and enzyme are then removed using centrifugation-dialysis (VIVASPIN® MWCO 50 kDa, Vivascience, Winkel, Switzerland) or diafiltration (PELLICON® MWMCO 50kDa, Millipore). Reactions may be monitored by HPLC. [0203] The resulting ADC can be analyzed using any suitable method. For example, the stoichiometry of the ADC can be characterized by liquid chromatography mass spectrometry (LC/MS) using a top-down approach in order to assess the number of conjugation moiety conjugated to antibodies, and in particular the homogeneity of the composition. Conjugates can be reduced before LC/MS analysis and light chains and heavy chains are measured separately. [0204] In one embodiment, the ADC is analyzed for drug loading (e.g. number of toxin in the conjugate per anti-HER2 antibody). Such methods can be used to determine the mean number of conjugation moieties or toxins (such as MMAE) per anti-HER2 antibody as well as the distribution of number of conjugation moieties or toxins (such as MMAE) per antibody in a composition, i.e., the percentage of total antibody with any given level of drug loading or DAR. The portion of antibodies having a number (n) of conjugated acceptor glutamines (e.g. n=1, 2, 3, 4, 5, 6, etc.) can be determined. One technique adapted to such determination and more generally drug loading is hydrophobic interaction chromatography (HIC), HIC can be carried out as described for example in Hamblett et al. (2004) Cancer Res. 10: 7063-7070; Wakankar et al. (2011) mAbs 3(2): 161-172; and Lyon et al (2012) Methods in Enzymology, Vol.502: 123-138, the disclosure of which are incorporated herein by reference. [0205] The molar ratio between the transglutaminase and the anti-HER2 antibody in the conjugation reaction can be controlled to allow efficient transglutamination reaction. For example, in some embodiments, the molar ratio of the transglutaminase and the anti-HER2 antibody is about 10:1 to about 1:100. The amount of the transglutaminase in the reaction mixture can be controlled to allow efficient transglutaminase reaction. For example, in some embodiments, the concentration of the tranglutaminase in the reaction mixture is about any of about 0.01 mg/ml to about 5 mg/ml. In some embodiments, the concentration ratio between the conjugation moiety and the anti-HER2 antibody is from about 2:1 to about 800:1. In some embodiments, the conjugation efficiency of the anti-HER2 antibody and the conjugation moiety is at least about 30% (e.g., at least about 40%, 50%, 60%, 70%, 80%, 90% or more). Conjugation efficiency can also be measured at different temperature, such as room temperature or 37 °C. [0206] In some embodiments, the ADC is purified after the conjugation reaction. For example, the ADC may be purified using affinity chromatography, such as a protein A column, and/or a size exclusion column. IV. Pharmaceutical compositions, kits, and articles of manufacture [0207] Also provided are pharmaceutical compositions comprising any one of the antibody- drug conjugates described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises about 50mM Sodium Citrate, about 10 mM Citric Acid, about 4.0% (w/v) Sucrose and about 0.02% (w/v) Polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 10 mg/mL of the ADC. [0208] The term “pharmaceutically acceptable carrier” is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient(s) to a large extent depend on factors such as the particular mode of administration the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In some embodiments, isotonic agents, including, but not limited to, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride are included in the pharmaceutical composition. Additional examples of pharmaceutically acceptable substances include, but are not limited to, wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. [0209] In some embodiments, the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0. In some embodiments, the pharmaceutical composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol. [0210] The pharmaceutical compositions to be used for in vivo administration are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Sterility is readily accomplished by filtration through sterile filtration membranes. In some embodiments, the composition is free of pathogen. For injection, the pharmaceutical composition can be in the form of liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the pharmaceutical composition can be in a solid form and re-dissolved or suspended immediately prior to use. Lyophilized compositions are also included. [0211] In some embodiments, the pharmaceutical composition is suitable for administration to a human. In some embodiments, the pharmaceutical composition is contained in a single- use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved. [0212] The pharmaceutical compositions described herein may be prepared, packaged, or sold in bulk as a single unit dose or as a plurality of single unit doses As used herein a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. [0213] The pharmaceutical compositions described herein in some embodiments are suitable for parenteral administration. Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. For example, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusions; and kidney dialytic infusion techniques. In some embodiments, the pharmaceutical composition is suitable for intravenous administration. [0214] Formulations of a pharmaceutical composition suitable for parenteral administration may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile pyrogen-free water Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Other parentally administrable formulations which are useful include those, which comprise the active ingredient in microcrystalline form, or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and/or engineered release. Engineered release formulations include controlled, delayed, sustained, pulsed, targeted and programmed release formulations. For example, in one aspect, sterile injectable solutions can be prepared by incorporating the ADC in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the exemplary methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. [0215] Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, or several divided doses may be administered over time. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the patients/subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. [0216] The present application also provides kits (or articles of manufacture) for use in the treatment of cancers described herein. The kits may include one or more containers comprising any one of the ADCs for treating a cancer (e.g., HER2-positive cancer). The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein. In some embodiments, the kit comprises instructions for administration of the ADC to treat a cancer such as breast cancer colorectal cancer, ovarian cancer, gastric cancer, urethral cancer, or lung cancer. The kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the cancer and the stage of the cancer. The instructions relating to the use of the ADC generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. [0217] The kits are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an infusion device, e.g., a minipump. A kit may have a sterile access port, for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The container may further comprise a second pharmaceutically active agent. These articles of manufacture may further be sterilized and/or sealed. VIII. Exemplary embodiments [0218] Among the embodiments provided herein are: 1. A method of treating a HER2-positive cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody- drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugation moiety is conjugated to the acceptor glutamine residue. 2. The method of embodiment 1, wherein the HER2-positive cancer is HER23+ as determined by an immunohistochemistry (IHC) test. 3. The method of embodiment 1, wherein the HER2-positive cancer is HER22+ as determined by an IHC test. 4. The method of embodiment 1, wherein the HER2-positive cancer is positive as determined by a Fluorescence In Situ Hybridization (FISH) test. 5. The method of any one of embodiments 1-4, wherein the individual is unresponsive or ineligible for a standard therapy. 6. The method of embodiment 5, wherein the individual has not previously received a second HER2-targeted agent. 7. The method of any one of embodiments 1-4, wherein the individual has previously received a second HER2-targeted agent. 8. The method of embodiment 7, wherein the HER2-positive cancer is resistant or refractory to a second HER2-targeted agent. 9. The method of embodiment 7 or 8, wherein the second HER2 targeted agent is trastuzumab, ado-trastuzumab emtansine, pertuzumab, or lapatinib. 10. The method of any one of embodiments 1-9, wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg. 11. A method of treating a cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg. 12. The method of embodiment 11, wherein the cancer is a solid cancer. 13. The method of embodiment 12, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancers, and lung cancer. 14. The method of any one of embodiments 11-13, wherein the antibody-drug conjugate is administered at a dose of no more than about 6 mg/kg. 15. The method of any one of embodiments 11-13, wherein the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg to about 8 mg/kg. 16. The method of embodiment 15, wherein the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg or 8.0mg/kg . 17. The method of any one of embodiments 1-16, wherein the antibody-drug conjugate is administered intravenously. 18. The method of any one of embodiments 1-17, wherein the antibody-drug conjugate is administered about once every three weeks, about every other week, or about once per week 19. The method of any one of embodiments 1-18, wherein the individual is human. 20. The method of any one of embodiments 1-19, wherein the anti-HER2 antibody is N- glycosylated in the Fc region. 21. The method of any one of embodiments 1-20, wherein the acceptor glutamine residue is Q295 in the heavy chain of the anti-HER2 antibody according to the EU numbering. 22. The method of any one of embodiments 1-21, wherein each heavy chain of the HER2 antibody is conjugated to the conjugation moiety. 23. The method of any one of embodiments 1-22, wherein the conjugation moiety is conjugated to the acceptor glutamine residue by transglutamination. 24. The method of any one of embodiments 1-23, wherein the anti-HER2 antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity- determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6. 25. The method of embodiment 24, wherein the anti-HER2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8. 26. The method of any one of embodiments 1-25, wherein the Fc region is an IgG1 Fc. 27. The method of embodiment 26, wherein the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. 28. The method of any one of embodiments 1-27, wherein the toxin is a monomethyl auristatin E (MMAE). 29. The method of any one of embodiments 1-28, wherein the conjugation moiety comprises a cleavable linker. 30. The method of embodiment 29, wherein the conjugation moiety is a compound of Formula I:
Figure imgf000069_0001
31. The method of any one of embodiments 1-30, wherein the antibody-drug conjugate is DP303c. EXAMPLES [0219] The invention can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting. Example 1. Preparation and formulation of DP303c. [0220] DP303c is an antibody drug conjugate, which is designed to target epidermal growth factor receptor 2 (HER2) positive cancer in humans. More specifically, the product is a HER2 targeting monoclonal IgG1 antibody (DP001) with one cleavable LND1002 (toxin) attached site-specifically to Glutamine 295 in the constant region of each heavy chain of DP001. DP303c has a DAR (drug antibody ratio) of about 2.0 (such as 1.8 to 2.2). [0221] DP001 is an anti-HER2 monoclonal antibody, which has the same amino acid sequence as trastuzumab (HERCEPTIN®). Specifically, it contains 1328 amino acids with two heavy chains (HC) of 450 amino acids (49284.65 Da, SEQ ID NO: 9), and two light chains (LC) of 214 amino acids (23443.10 Da, SEQ ID NO: 10). DP001 is a heterotetramer of two HCs of the IgG1 subclass, and two LCs of the kappa subclass linked by 16 disulfide bonds (12 intra and 4 inter chain). A schematic structure of DP001 is depicted in FIG.1. [0222] LND1002 is a toxin derived from MMAE, which has a PEG linker with a primary amine group for conjugation to the antibody. LND1002 has the chemical structure of formula (I), shown below:
Figure imgf000069_0002
[0223] DP303c was obtained by conjugating DP001 to LND1002, which was catalyzed by a microbial transglutaminase (mTgase). Briefly, DP001, reaction buffer (50mM Tris-Acetate, 2mM EDTA, 0.1% Tween-20) at pH 8.0 and LND1002 in DMSO were loaded to a sterile, flexible, ethylene-vinyl acetate bag under a laminar flow hood in a clean room. Once the mixture reached the desired temperature, mTGase was added to the bag through a sterile syringe to start the conjugation reaction. The bag was then incubated at 30°C for up to 120 hours. The reaction was monitored using RP-HPLC. The conjugation reaction was considered complete when the heavy chain conversion reached ≥ 95%. [0224] The completed reaction was loaded onto a sanitized CAPTIVA® protein A column using a sanitized AKTA® Ready system at 30-35g DP303c/L resin with a minimum residence time of 5 minutes to ensure complete binding of the DP303c product. An in-line 0.2μm depth filter was used to remove any particulates from the reaction and or buffers during the purification, and to maintain sterility. The column was then washed with excess binding and wash buffers in order to remove mTGase, un-reacted LND1002, and any unwanted buffer components before low pH elution of the desired product. The elution fraction was collected in a sterile collection bag pre-filled with neutralization buffer. [0225] The purified DP303c was formulated as follows: 10mg/mL DP303c in 50mM Sodium Citrate, 10 mM Citric Acid, 4.0% (w/v) Sucrose and 0.02% (w/v) Polysorbate 20. Concentrated components of DP303c formulation were prepared in sterile containers and filtered through 0.2 μm sterile PES filters and added to the DP303c concentrated stock solution. The formulation step was conducted inside the laminar flow hood in a clean room. The final formulated solution was then filtered through a 0.2 μm sterile PES filter. The final filtered product was stored at -20 ºC for long-term storage. Example 2. Cytotoxicity effects of DP303c compared to DP001 and T-DM1 against a HER2-high expressing cell line [0226] The cytotoxicity of DP303c along with DP001 and T-DM1 (also known as ado- trastuzumab emtansine (KADCYLA®)) was evaluated in a cell-based assay with a HER23+ human breast cancer SK-BR-3 cell line (Li JY et al., Cancer Cell 29,117-129). [0227] SK-BR-3 cells were grown to about 70% confluence in a tissue-culture flask, then media was aspirated out of the flask and the cells were washed with a small amount of magnesium and calcium free DPBS. The cells were treated with trypsin to lift adhered cells and re-suspended in fresh media to final concentration of about 1 x 105 cells/mL. The re- suspended cells were transferred into wells of a 96-well plate and incubated overnight at 37°C, 5% CO2, 85% relative humidity to allow cells to adhere. After overnight incubation, DP001, DP303c, or T-DM1 was added to the wells at final concentrations of 500 ng/mL, 167 ng/mL, 55.6 ng/mL, 19 ng/mL, 6.2 ng/mL, 2.06 ng/mL, 0.69 ng/mL, or 0.229 ng/mL. Each concentration of DP001, DP303c, or T-DM1 was added in triplicate. Fresh media was used as negative control. Then, the plate was shaken lightly on a plate shaker for 15-20 seconds and incubated for 3 days at 85% humidity. [0228] After incubation, Resazurin (Sigma, Cat# 199303, Lot# MKBP3801V) was added to every well to a final concentration of about 0.0005‰ and mixed lightly with a plate shaker. The plate was further incubated for 3 hours. Subsequently, the plate was assayed using a template for the cell based assay protocol on the SPECTRAMAX GEMINIXS® plate reader with λex 555 / λem 585nm (570nm cut-off). The average RFU of blank medium wells were subtracted from the RFU readings of all the wells with DP001, DP303c, or T-DM1 treated cells. Software XLFit Excel Version (4.3.2 Build 11) (ID Business Solutions Limited) Fit model 201 was used to calculate IC50. Table 1 below compares IC50 of DP001, DP303c, and T-DM1 in SK-BR-3 cells. The IC50 suggests higher potency of cytotoxicity of DP303c and T- DM1 compared to DP001. Table 1
Figure imgf000071_0001
[0229] FIG.2 shows the cell proliferation inhibition curves of SK-BR-3 cells following treatment with DP001 (blue circle and blue line), DP303c (green triangle and green line), or T-DM1 (brown square and brown line). As shown in FIG.2, DP001, DP303c, and T-DM1 are cytotoxic against SK-BR-3 cells, a HER2 high-expression cell line. DP303c has similar cytotoxic potency to that of T-DM1 against this HER2 high-expression cell line. Both DP303c and T-DM1 are more potent in killing SK-BR-3 cells than DP001, the HER2 targeted antibody. Example 3. Cytotoxicity effects of DP303c compared to DP001 and T-DM1 against a HER2 low-expressing cell line [0230] The cytotoxicity of DP303c along with DP001 and T-DM1 was evaluated in a cell- based assay with a HER22+ breast cancer JIMT-1cell line (Li JY et al., Cancer Cell 29,117- 129). [0231] JIMT-1 cells were grown to about 70% confluence in a tissue-culture flask, then media was aspirated out of the flask and the cells were washed with a small amount of magnesium and calcium free DPBS. The cells were treated with trypsin to lift adhered cells and re-suspended in fresh media to final concentration of about 1 x 105 cells/mL. The re- suspended cells were transferred into wells of a 96-well plate and incubated overnight at 37°C, 5% CO2, 85% relative humidity to allow cells to adhere. After overnight incubation, DP001, DP303c, or T-DM1 were added to each well at final concentrations of 10,000 ng/mL, 2,500 ng/mL, 625 ng/mL, 156 ng/mL, 39.1 ng/mL, 9.77 ng/mL, or 2.44 ng/mL. Each concentration of DP001, DP303c, or T-DM1 was added in triplicate. Fresh media was used as negative control. Then the plate was shaken lightly on a plate shaker for 15-20 seconds and incubated for 5 days at 85% humidity. [0232] After incubation, Resazurin (Sigma, Cat# 199303, Lot# MKBP3801V) was added to every well to final concentration of about 0.0005‰ and mixed lightly with a plate shaker. The plate was further incubated for 3 hours. Subsequently, the plate was assayed using the template for the cell based assay protocol on the SPECTRAMAX GEMINIXS® plate reader with λex 555 / λem 585nm (570nm cut-off). The average RFU of blank medium wells were subtracted from the RFU readings of all the wells with DP001, DP303c, or T-DM1 treated cells. Software XLFit Excel Version (4.3.2 Build 11) (ID Business Solutions Limited) Fit model 201 was used to calculate IC50. Table 2 below compares IC50 of DP001, DP303c, and T-DM1 in JIMT-1 cells. The IC50 suggests higher potency of cytotoxicity of DP303c than T- DM1. Table 2
Figure imgf000072_0001
[0233] FIG.3 shows the cell proliferation inhibition curves of JIMT-1 cells following treatment with DP001 (blue circle and blue line), DP303c (green triangle and green line), or T-DM1 (brown square and brown line). As shown in FIG.3, both DP303c and T-DM1 are cytotoxic against the JIMT-1 cell line, a HER2 low-expression cell line. DP303c has higher cytotoxicity than TDM-1 against this HER2 low-expression cell line. Example 4. Cytotoxicity effects of DP303c compared to DP001 and T-DM1 against a HER2 negative cell line [0234] The cytotoxicity of DP303c along with DP001 and T-DM1 was evaluated in a cell- based assay against Hs746T cell. Hs746T is a human gastric carcinoma cell line that has little to no expression of HER2 (Corso S, et al., Mol Cancer 2010; 9:121). [0235] Hs746T cells were grown to about 70% confluence in a tissue-culture flask, then media was aspirated out of the flask and the cells were washed with a small amount of magnesium and calcium free DPBS. The cells were treated with trypsin to life adhered cells and re-suspended in fresh media to final concentration of about 1 x 105 cells/mL. The re- suspended cells were transferred into wells of a 96-well plate and incubated overnight at 37°C, 5% CO2, 85% relative humidity to allow cells to adhere. After overnight incubation, DP001, DP303c, or T-DM1 were added to each well at final concentrations of 10,000 ng/mL, 2,500 ng/mL, 625 ng/mL, 156 ng/mL, 39.1 ng/mL, 9.77 ng/mL, 2.44 ng/mL, or 0.610 ng/mL. Each concentration of DP001, DP303c, or T-DM1 was added in triplicate. Fresh media was used as negative control. Then the plate was shaken lightly on a plate shaker for 15-20 seconds and incubated for 5 days at 85% humidity. [0236] After incubation, Resazurin (Sigma, Cat# 199303, Lot# MKBP3801V) was added to every well to final concentration of about 0.0005‰ and mixed lightly with a plate shaker. The plate was further incubated for 3 hours. Subsequently, the plate was assayed using the template for the cell based assay protocol on the SPECTRAMAX GEMINIXS® plate reader with λex 555 / λem 585nm (570nm cut-off). The average RFU of blank medium wells were subtracted from the RFU readings of all the wells with DP001, DP303c, or T-DM1 treated cells. Software XLFit Excel Version (4.3.2 Build 11) (ID Business Solutions Limited) Fit model 201 was used to calculate IC50. [0237] FIG.4 shows the cell proliferation inhibition curves of Hs746T cells following treatment with DP001 (blue circle and blue line), DP303c (green triangle and green line), or T-DM1 (brown square and brown line). As shown in FIG.4, DP001, T-DM1, and DP303c did not show cytotoxic effects against Hs746T cell a HER2 negative cell line Example 5. Antitumor effects of DP303c compared to T-DM1 on the mouse xenograft NCI-N87 cancer model [0238] The in vivo antitumor activity of DP303c was investigated in the NCI-N87 mouse xenograft model. NCI-N87 is a gastric cancer cell line overexpressing HER2 (HER23+). NCI-N87 cells were implanted subcutaneously into female athymic nude mice. The tumor- bearing mice were randomized into 7 treatment groups (6 mice/group) and treated with vehicle, DP303c, or T-DM1 by a single intravenous injection. [0239] Relative Tumor Volume (RTV) was calculated according to the following formula: RTV = TVn/TV0, where TVn is the tumor volume at day n and TV0 is the tumor volume at day 0. The tumor growth inhibition (TGI) is calculated using the formula TGI (%) = (1 – T/C )×100, where T/C is determined by calculating T/C = (mean RTV of treated group)/(mean RTV of control group). [0240] DP303c effectively inhibited the tumor growth in the NCI-N87 xenograft model (FIG.5). A single dose treatment of tumor-bearing mice with 4 or 8 mg/kg of DP303c resulted in tumor regression (81.1% and 92.5% of TGI:) without regrowth for up to 40 days. At the low dose level (2 mg/kg), DP303c showed 47.9% of tumor growth inhibition. As comparison, T-DM1 did not cause tumor regression at all dose levels, and the percent tumor growth inhibition was -28.2%, 16.8%, and 39.9%, for 2, 4, and 8 mg/kg, respectively. Example 6. Tumor growth inhibition effects of DP303c compared to T-DM1 and an anti- HER2 Biparatopic single chain antibody ADC on a mouse xenograft JIMT-1 cancer model [0241] The in vivo antitumor activity of DP303c was investigated in a mouse xenograft model of human breast cancer. JIMT-1 is a HER2-positive breast cancer cell line. The JIMT- 1 xenograft model is known for its insensitivity to current anti-HER2 therapeutic agents such as trastuzumab and T-DM1 (Li et al, 2016). After tumor cell implantation, the tumor-bearing mice were randomized into 10 treatment groups (5 mice/group) for treatment with a single intravenous injection of vehicle, DP303c, T-DM1, or Biparatopic ADC. [0242] As shown in FIG.6, administration of a single dose of DP303c induced complete tumor regression in the JIMT-1 xenograft model at all dose levels. Tumor regrowth was not found in some of these animals and they remained tumor-free for up to 4 weeks until the end of the study. By contrast, T-DM1 did not significantly inhibit tumor growth with TGI % of 4.9%, 16.9%, and 16.8% for 8, 116, and 32 mg/kg, respectively. No significant weight loss or mortalities were observed in all treatment groups in this study. Example 7. Pharmacokinetic and toxicity studies of DP303c [0243] This example describes the pharmacokinetic studies of DP303c in rats following a single dose and in cynomolgus monkeys following a single dose. This example also describes nonclinical safety studies of DP303c, including a non-GLP single-dose toxicology study in rats, a non-GLP 29-day dose-range finding study in cynomolgus monkeys, and a GLP repeated dose toxicology study in cynomolgus monkeys. Additionally, this example describes in vitro evaluation of DP303c stability in human and cynomolgus monkey plasma as well as an in vitro GLP studies assessing the effects of DP303c formulation on hemolysis and erythrocyte aggregation. Five-Week Pharmacokinetic Study of DP303c in Rats Following a Single Intravenous Injection of DP303c (non-GLP) [0244] The objective of this study was to determine pharmacokinetic profiles of DP303c, total anti-HER2 antibody, free (unconjugated) MMAE, and LND1002 (linker MMAE) following a single intravenous administration of DP303c in Sprague Dawley rats at dose levels of 3 mg/kg, 10 mg/kg, and 30 mg/kg (3/sex/group). [0245] Concentrations of DP303c and total antibody DP001 in rat serum collected from this study were analyzed using ELISA assays with a lower limit of quantification of 0.3125 for both DP303c and total antibody DP001. Concentrations of free MMAE and linker-MMAE (LND002, free payload) were analyzed by a tandem liquid chromatography/mass spectrometry (LC-MS/MS) method with a lower limit of quantification of 5.0 pg/mL for MMAE and 0.170 ng/mL for LND1002. [0246] Table 3 below shows mean pharmacokinetic parameters of DP303c and total antibody. Values are presented as Mean± standard deviation; [n] indicates animal sample number; Cmax is maximum observed concentration; AUC0-t is area under the concentration time curve up to the last measureable concentration; AUC0-∞ is area under the concentration time curve from time zero to infinity; t1/2 is half-life; CL is systemic clearance; Vss is steady- state volume of distribution. Table 3
Figure imgf000076_0001
[0247] Following a single IV bolus of DP303c, both DP303c Cmax and AUC∞ increased in a dose-proportional manner over the dose range of 3 mg/kg to 30 mg/kg (FIG.7). Peak concentrations (Cmax) were 73.5, 238, and 630 μg/mL for 3, 10, and 30 mg/kg dose levels (Table 3), respectively. The AUC∞ values were 470 μg·day/mL at 3 mg/kg, 1331 μg·day/mL at 10 mg/kg, and 4498 μg·day/mL at 30 mg/kg. The DP303C clearance (CL) was 6.44, 7.68, and 6.90 mL/day/kg and the terminal phase half-life (t1/2 ) was 9.25, 9.79, and 9.42 days for 3, 10 mg/kg and 30 mg/kg, respectively. Volume of distribution at steady state (V ) was 82.9 mL/kg at 3 mg/kg, 104.0 mL/kg at 10 mg/kg, and 90.2 mL/kg at 30 mg/kg. The results indicate a linear PK of DP303c in rats over a dose range from 3 to 30 mg/kg. [0248] Following a single IV bolus injection of DP303c, the Cmax and AUC∞ of total antibody also increased dose-proportionally over the dose range of 3 to 30 mg/kg (FIG.8). Peak concentrations (Cmax ) of total antibody were 71.2, 256, and 701 μg/mL at 3, 10, and 30 mg/kg (Table 3), respectively. The AUC∞ values were 515, 1432, and 4428 μg·day/mL at 3, 10, and 30 mg/kg, respectively. Terminal phase half-life (t1/2 ) of total antibody was 9.67, 9.75, and 8.67 days, and clearance (CL) was 5.86, 7.07, and 6.90 mL/day/kg at does levels of 3, 20, and 30 mg/kg, respectively. Volume of distribution of total antibody at stead state (V ) was 79.1 mL/kg at 3 mg/kg, 96.6 mL/kg at 10 mg/kg, and 83.1 mL/kg at 30 mg/kg. The PK profile of total antibody was very similar to that of DP303c, suggesting limited de-conjugation of MMAE from DP303c in rats. [0249] Following IV dose of DP303c, the serum concentration of MMAE (Cmax ) reached the peak level at 6 hour postdose in the 3 and 10 mg/kg dose groups and at 24 hour in the 30 mg/kg dose group, followed by a slow elimination phase (FIG.9). The peak concentrations of unconjugated MMAE were about 0.03, 0.08, and 0.3 ng/mL for 3, 10, and 30 mg/kg groups respectively. The exposure of MMAE is about (0.011%, 0.014%, 0.016%) of the exposure of DP303c ( AUC0-t, on a molar basis) at dose levels of 3, 10 and 30 mg/kg, respectively. Serum concentrations of payload LND1002 were generally undetectable in most samples and PK analysis was not conducted. [0250] In conclusion, the PK profiles, such as exposure and elimination half-life, are very similar between DP303c and the total antibody following a single intravenous administration of DP303c in rats. Linear PK is suggested over the dose range of 3 to 30 mg/kg and no gender difference was observed. Serum exposure of free MMAE was <0.03% of DP303c after a single-dose IV administration of D303c. Serum concentrations of LND1002 were generally undetectable. Pharmacokinetics Study of DP303c Following A Single Intravenous Administration in Cynomolgus Monkeys [0251] The objective of this study was to determine pharmacokinetic profiles of DP303c following a single intravenous administration of DP303c in cynomolgus monkeys at dose levels of 1.2, 4.0, and 12 mg/kg (3/sex/group). In addition, the PK of total antibody and free MMAE were also evaluated. [0252] Concentrations of DP303c and total antibody DP001 in cynomolgus monkey serum collected from this study were determined by ELISA assays with a lower limit of quantification of 0.3125 ng/mL for both DP303c and total antibody DP001. Concentrations of both free MMAE and linker-MMAE (LND002, free payload) in monkey plasma were analyzed by tandem liquid chromatography/mass spectrometry (LC-MS/MS) methods with a lower limit of quantification of 0.03 ng/mL for MMAE and 1.0 ng/mL for LND1002 for this study. [0253] Table 4 below shows mean pharmacokinetic parameters of free MMAE in cynomolgus monkeys following a single IV infusion of DP303c. Values are presented as Mean± standard deviation; [n] indicates animal sample number; Cmax is maximum observed concentration; AUC0-t is area under the concentration time curve up to the last measureable concentration; AUC0-∞ is area under the concentration time curve from time zero to infinity; NC indicates value not calculated. Table 4
Figure imgf000078_0001
[0254] Following a single IV infusion of DP303c, the Cmax of DP303c increased in a dose- proportional manner over the dose range of 1.2 mg/kg to 12 mg/kg (FIG.10). Peak concentrations were 30.1 μg/mL at 1.2 mg/kg, 93.5 μg/mL at 4 mg/kg, and 297.4 μg/mL at 12 mg/kg (Table 4). The AUC∞ was 1300, 6417, and 24834 h·μg/mL and the clearance (CL) was 0.96, 0.65, and 0.49 mL/h/kg at 1.2, 4.0, and 12 mg/kg. The increase in DP303c AUC∞ was greater than dose-proportional while the DP303c clearance (CL) was decreased with increased dose levels, indicating nonlinear PK of DP303c over the dose range of 1.2 to 12 mg/kg in monkeys. The terminal phase half-life (t1/2) of DP303c was 130.1, 136.8, and 118.6 hours at 1.2, 4.0, and 12 mg/kg, respectively. [0255] The total antibody PK was similar to the DP303c PK following a single IV infusion of DP303c in cynomolgus monkeys. The total antibody Cmax increased in a dose-proportional manner over the dose range of 1.2 mg/kg to 12 mg/kg (FIG.11). Peak concentrations of total antibody were 30.8 μg/mL at 1.2 mg/kg, 103.7 μg/mL at 4 mg/kg, and 323.7 μg/mL at 12 mg/kg (Table 4). The increase in mean AUC∞ of total antibody was greater than dose- proportional from 1.2 to 12 mg/kg. The mean AUC∞ values were 1374 h·μg/mL at 1.2 mg/kg, 7333 h·μg/mL at 4 mg/kg, and 27854 h·μg/mL at 12 mg/kg. The mean total antibody AUC∞ was no more than 15% greater than the mean DP303c AUC∞ for all dose levels, suggesting limited de-conjugation of MMAE from DP303c. The clearance (CL) of total antibody was decreased from 0.91 mL/day/kg at 1.2 mg/kg, 0.57 mL/day/kg at 4 mg/kg, to 0.44 mL/day/kg at 12 mg/kg. The half-life (t1/2) of total antibody was 123.0, 138.4, and 124.0 hours at 1.2, 4, and 12 mg/kg, respectively. [0256] After dosing with DP303c, free MMAE in monkey plasma was not detected or was below the lower limit of quantification in most of the time points at the 1.2 mg/kg group. Therefore, the pharmacokinetic parameters of free MMAE were not estimated for the animals in the 1.2 mg/kg group (Table 4). Exposure of free MMAE was very low at both 4 mg/kg and 12 mg/kg. The observed medium time to reach the peak level of free MMAE in plasma was 48 hours (FIG.12). The Cmax of free MMAE in plasma was 0.059 and 0.135 ng/mL and the AUC0-t were 7.56 and 32.0 h·ng/mL at 4 and 12 mg/kg, respectively. Exposure of free MMAE was only 0.01% of that of DP303c in corresponding dose groups of 4 mg/kg and 12 mg/kg. [0257] In conclusion, following a single IV administration of DP303c in cynomolgus monkeys, the mean AUC∞ of DP303c and total antibody increased more than dose- proportionally, indicating nonlinear PK of DP303c in cynomolgus monkeys over the dose range of 1.2 to 12 mg/kg. DP303c exposure was <15% lower than total antibody in monkeys, suggesting limited de-conjugation of MMAE from DP303c. Exposure of free MMAE was very low and no more than 0.03% of DP303c exposure. There was no gender difference observed in DP303c PK in this study. Pilot Toxicity Study of DP303c in Rats Following a Single Intravenous Injection (non-GLP) [0258] The objectives of this study were to evaluate the potential toxicity and determine the maximum tolerated dose (MTD) of DP303c by a single IV administration of DP303c in rats, followed by a 21-day follow-up period. [0259] Female Sprague Dawley rats (5/group) were administered with either vehicle (control) or DP303c at dose levels of 10, 20, 40, 60, and 100 mg/kg, respectively. The parameters and end points evaluated in this study included clinical signs and observations, body weight, food consumption, and clinical pathology parameters (hematology) during the postdose period of 21 days. There was no mortality or moribund across all dose groups in this study. Clinical observation was limited to reduced activity in animals at the highest dose group of 100 mg/kg. The reduced activity started 5 days after dosing, became most pronounced around Day 8, and then gradually returned to normal. Mild body weight loss (about 4% on Day 4) was observed at the 100 mg/kg group, and a trend of lower gain of body weight was found in other dose groups. [0260] Hematology analysis demonstrated moderate decreases in red blood cells (RBC), hematocrit, and hemoglobin content in animals treated with 60 and 100 mg/kg of DP303c. The numbers of total leukocytes and macrophage/monocyte also decreased following DP303c treatment in the two high dose groups. Platelet count slightly decreased but was not statistically significant. All the observed adverse effects on hematologic parameters at the two high dose levels recovered to normal 21 days after DP303c treatment. There were no significant treatment-related hematological changes in animals treated with 10, 20, or 40 mg/kg of DP303c. [0261] An additional single dose study was conducted to evaluate toxicity of additional dose levels. Sprague Dawley rats (10/group) were administered with either vehicle (control) or DP303c at dose levels of 50, 100, and 200 mg/kg, respectively. The parameters and end points evaluated in this study included clinical signs and observations, body weight, food consumption, and clinical pathology parameters (hematology) during the postdose period of 22 days.200 mg/kg DP303c caused mortality in rats. Edema/eschar/ulcer on chin or neck, temporary abnormal eye secretion, reduced weight gain and food consumption was observed in groups with 100 mg/kg DP303c dosage level or higher. Increase in monocytes and decrease in lymphocytes and eosinophils were observed in groups with 50 mg/kg DP303c dosage level or higher. [0262] In conclusion, the major toxicity of single IV administration of DP303c in rats was anemia and leukopenia at high doses (60 and 100 mg/kg), which were reversible after 21 days. The maximum tolerated dose of DP303c in rats was not reached in this study and would not be lower than 100 mg/kg after a single intravenous administration. 29-Day Dose Range Finding and Toxicological Study of DP303c Following Two Intravenous Injections in Cynomolgus Monkeys (non-GLP) [0263] The objectives of this study were to evaluate the potential toxicity and toxicokinetics (TK) of DP303c in cynomolgus monkeys after IV administration of DP303c once every 3 weeks for 2 doses. This study served as a dose range finding to help the dose selection for a subsequent GLP study in monkeys. Male and female cynomolgus monkeys were IV administered with either vehicle (control, 1/sex/group) or DP303c (2/sex/group) at dose levels of 6 (group 2), 20 (group 3), 60 (group 4), and 100 mg/kg (group 5) by IV infusion every 3 weeks for 2 doses. Terminal necropsy was scheduled on Day 29, 7 days after the second dose. Due to mortality and moribundity, only one dose of DP303c was administrated in the two high-dose groups (60 and 100 mg/kg, group 4 and group 5) by protocol amendment. [0264] Toxicological end points evaluated in this study included veterinary physical observations, clinical signs, injection site observations, body weights, food consumption, clinical pathology (hematology, coagulation, clinical chemistry, urinalysis), toxicokinetics, gross necropsy findings, organ weights, and histopathologic examinations.. [0265] DP303c-associated mortality occurred in Groups 4 (60 mg/kg) and 5 (100 mg/kg). Two animals at the dose level of 60 mg/kg died on Day 10 and Day 12, respectively, after the first dose. In the 100 mg/kg group, one animal died on Day 8 and 3 animals were euthanized on Day 8 and Day 10, respectively, due to poor physical condition. These early death animals presented with clinical signs of decreased activity, self-mutilation, hunched posture, cold skin, prostrate, inappetence, muscular weakness, watery feces, decreased startle reflex, heavy breathing, and skin necrosis, starting on Day 7. Body weight loss (up to 18%) was found in all these unscheduled death animals. Hematology changes included severe decreases in white blood cells (WBC), neutrophils, lymphocytes, monocytes, eosinophils, and reticulocytes on D8 in both 60 and 100 mg/kg groups. Increased platelets were found in 2 survival animals at 60 mg/kg on Day 15 and later time points. Gross observation included moderate black discoloration of the lungs in one unscheduled necropsy animal at 60 mg/kg and vagina hyperplasia in one animal at 100 mg/kg. [0266] No mortality or moribund was found in animals treated with low dose levels of DP303c at 6 mg/kg and 20 mg/kg. DP303c-related clinical signs were not observed in either groups during the treatment period. Mild decreases in white blood cells (WBC), neutrophils, and lymphocytes were observed on Day 15 in the animals at 20mg/kg, but not at 6 mg/kg. These adverse changes were reversed at later time points. There were no DP303c-related effects on body weights, organ weights, food consumption, coagulation, and clinical chemistry parameters in these 2 treatment groups. [0267] In conclusion, a single dose of DP303c resulted in mortality or morbidity in all 6 animals at the dose level of 100 mg/kg and in half of the animals at the dose level of 60 mg/kg. Animals in these 2 high-dose groups suffered from leukopenia characterized by severely reduced white blood cells (WBC), neutrophils, lymphocytes, monocytes, and eosinophils. Moderate black discoloration of lung was found in one of the unscheduled death animals at 60 mg/kg. The cause of unscheduled animal death was not very clear as histopathological analysis was not conducted in this study At low dose levels of 6 mg/kg and 20 mg/kg for 2 doses, all animals survived until the end of the study and no significant DP303c-related clinical signs and clinical pathologic changes were found. Based on these results, DP303c ≤20 mg/kg administrated by IV for two doses is thought to be well tolerated in cynomolgus monkeys. [0268] The TK profile of DP303c was assessed by determining plasma concentrations of DP303c, total antibody and free MMAE. Concentrations of DP303c and total antibody DP001 in cynomolgus monkey serum collected from this study were determined by ELISA assays with a lower limit of quantification of 0.3125 ng/mL for both DP303c and total antibody DP001. Concentrations of both free MMAE and linker-MMAE (LND002, free payload) in monkey plasma were analyzed by tandem liquid chromatography/mass spectrometry (LC- MS/MS) methods with a lower limit of quantification of 0.03 ng/mL for MMAE and 1.0 ng/mL for LND1002 for this study. [0269] Table 5 below shows pharmacokinetic parameters of DP303c in cynomolgus monkeys after IV infusion of first and second dose of DP303c. Values are presented as Mean± standard deviation; [n] indicates animal sample number; Cmax is maximum observed concentration; AUC0-t is area under the concentration time curve up to the last measureable concentration; AUC0-∞is area under the concentration time curve from time zero to infinity; t1/2 is half-life; CL is systemic clearance; Vss is steady-state volume of distribution. Table 5
Figure imgf000082_0001
Figure imgf000083_0001
[0270] Table 6 below shows mean pharmacokinetic parameters of free MMAE in cynomolgus monkeys following first and second dose of IV Infusion of DP303c. Values are presented as Mean± standard deviation; [n] indicates animal sample number; Cmax is maximum observed concentration; AUC0-t is area under the concentration time curve up to the last measureable concentration. Table 6
Figure imgf000083_0002
[0271] The analysis of pharmacokinetic parameters was conducted in group 2 and group 3 in this study because the PK sample collection was incomplete for designated time points in group 4 and 5. Following an IV infusion of DP303c on Day 1, the AUC∞ of DP303c increased higher than dose-proportionally from 12076 h·μg/mL at 6 mg/kg to 78466 h·μg/mL at 20 mg/kg. The half-life (t1/2) of DP303c increased from 86.4 to 152 hours while the clearance (CL) decreased from 0.525 to 0.275 mL/h/kg at 6 and 20 mg/kg, respectively. These data suggested nonlinear PK of DP303c over the dose range of 6 to 20 mg/kg in monkeys (Table 5). A very similar nonlinear PK profile was observed for total antibody (Table 5). The exposure (AUC∞ ) of total antibody and DP303c was comparable. The AUC0-∞ of total antibody was <10% higher than that of DP303c following IV infusion of DC303c. The Cmax of MMAE was 0.078 and 0.173 ng/mL at the dose levels of 6 and 20 mg/kg, respectively (Table 6). The plasma MMAE concentrations reached the peak level between 48 and 96 hours after IV infusion (FIG.13). MMAE AUC0-t was 9.29 h·ng/mL at 6 mg/kg and 51.32 h·ng/mL at 20 mg/kg. [0272] Following an IV infusion of DP303c on Day 22, the AUC0-∞ of DP303c and total antibody both increased more than dose-proportionally from the dose level of 6 to 20 mg/kg (Table 5). The Cmax and AUC0-∞ values of DP303c were similar between after the first dose and after the second dose. The peak concentrations (Cmax ) of MMAE were 0.045 and 0.210 ng/mL at 6 and 20 mg/kg, respectively. [0273] In conclusion, The PK of DP303c, total antibody, and free MMAE were analyzed in the dose groups of 6 and 30 mg/kg. The AUC∞ of DP303c and total antibody increased greater than dose-proportionally following IV infusion of DP303c in cynomolgus monkeys, suggesting nonlinear kinetics. The terminal half-life of DP303c was about 95.3 to 115 hours. The MMAE exposure was very low in monkeys as compared with DP303c. Comparable plasma exposure of intact DP303c and total antibody, along with the low level of free MMAE, indicates negligible de-conjugation of MMAE from DP303c in monkeys. Repeated Dose Intravenous Toxicity Study of DP303c Administered to Cynomolgus Monkeys Once Every 3 Weeks for Five Doses, with a 4-Week Recovery Period (GLP) [0274] The objectives of this study were to evaluate the potential toxicity, toxicokinetics (TK) and immunogenicity of DP303c after IV administration of DP303c once every 3 weeks for a total of 5 doses in cynomolgus monkeys. This study also evaluated the reversibility of any adverse effects and possible delayed toxicity during a 4 week treatment-free period. This GLP study is intended to provide the pivotal nonclinical safety data to support the first-time- in-human (FTIH) Phase 1 clinical study of DP303c. [0275] A total of 20 male and 20 female naïve cynomolgus monkeys were selected and enrolled in this study. These animals were randomly assigned to 4 groups based on body weight and gender. Group 1 was the vehicle control (saline, 0 mg/kg), and Groups 2, 3 and 4 were DP303c treatment groups, low (6.0 mg/kg), middle (20 mg/kg), and high (40 mg/kg) dose groups, respectively. Each group consisted of five animals per sex. DP303c and saline were administrated via 30-minute intravenous infusion once every three weeks. Three monkeys per sex per group were assigned to main study phase and the terminal necropsy was scheduled on Day 89, 4 days after the administration of the last dose. Two monkeys per sex per group were assigned to the recovery phase and the recovery necropsy was scheduled on Day 113, 4 weeks after the last dose. During the proceeding of the study, saline and DP303c at 6 mg/kg were administered for five doses (on Days 1, 22, 43, 64, and 85) as scheduled. DP303c at the middle dose group was administered with three doses at 20 mg/kg (Days 1, 22, and 43). The dose was reduced to 12 mg/kg for the remaining two doses (Days 64 and 85) due to one mortality that occurred following the third dose at 20mg/kg. The dose for this dose group was indicated as 20/12 mg/kg. DP303c at the high dose group was administered with two doses at 40 mg/kg (Days 1 and 22). The dose was reduced to 30 mg/kg at the third dose (Day 43) due to unscheduled animal death and no more doses were given after the third dose. The dose for this dose group was indicated as 40/30 mg/kg. Necropsy of the survival animals in the 40/30 mg group was scheduled on Day 71, 4 weeks after the last dose (recovery phase). [0276] Toxicological end points evaluated in this study included clinical signs and observations, local tolerance, body weights, food consumption, ophthalmoscopic examinations, body temperature, ECG, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), lymphocyte phenotypes, anti-drug antibody formation, and toxicokinetics (TK), gross necropsy findings, organ weights, and histopathological examinations. [0277] The dose level in 40/30 mg/kg DP303c group was poorly tolerated. After the second dose at 40 mg/kg, three animals were euthanized on Day 20, Day 26, and Day 29, respectively, due to moribund conditions. After the third dose at 30 mg/kg, one animal died on Day 54 and two others were euthanized on Day 43 and Day 53, respectively. The observed symptoms included decreased activity, hunched posture, prostate, cold skin, inappetence, salivation, transparent and/or red discharges in nose, and scabbed area at multiple sites of the skin, body stiffness, starting between Day 10 to Day 14 in the majority of the early death animals. Other signs included lacrimation in eyes, soft and watery feces, rale, bradypnea, dyspnea, and heavy breathing found in some of the animals. Body weight loss was observed starting at the dosing period. [0278] Hematological changes in the high dose group included mild to moderate decreases in white blood cells (WBC), lymphocytes, eosinophils, and severe decreases in neutrophils. Other changes included mildly to moderately decreased red blood cells (RBC), hemoglobin, hematocrit, and increased reticulocytes and platelets. These hematological changes were observed as early as on Day 8 in all animals of the high dose group. An increase in monocyte count was found at later time points of CP303c treatment. The hematologic manifestations of anemia and leukopenia returned to the baseline or alleviated in the survival animals after recovery. Coagulation changes comprised minimal prolongation of the prothrombin time and the activated partial thromboplastin time in the unscheduled death animals. Clinical chemistry changes comprised mild to moderate decreases in albumin and increases in globulins along with a corresponding decreased albumin to globulin ratio. Other findings included 2-3-fold increases in aspartate aminotransferase and minimal decreases in blood calcium, chloride, and creatine. The increased aspartate aminotransferase was back to normal after recovery. [0279] Gross observation at the high dose group included purple or dark discoloration of lung sometimes along with lung adherence to the chest wall or fluid in the thoracic cavity in 4 of the 6 unscheduled death animals. Increased organ weights of the lungs (associated with bronchi) were also found in these animals. Major microscopic findings included marked decreased cellularity in thymus, spleen, and lymph nodes and lung inflammation with fibrosis in unscheduled death animals. The cause of unscheduled death was attributed to DP303c- related lung inflammation and fibrosis, and depletion of lymphocytes in thymus, spleen and lymph nodes. Other significant findings included thrombi in small vessels and glomeruli and slight tubule degeneration with or without cast in the kidneys, minimal to mild degeneration/necrosis in the liver, and some single cell necrosis in the skin. After recovery, those histopathological changes in the lymphoid organs, lung, and liver were still observed in some survival animals, but the incidence and severity were lower. [0280] At the middle dose level of 20/12 mg/kg, one animal died on Day 60 after the third dose at 20 mg/kg. All other animals survived until scheduled necropsy on Day 89 or Day 113. The cause of the unscheduled death was attributed to depletion of lymphocytes in spleen and lymph nodes and lung inflammation and fibrosis. The clinical signs in the 20/12 mg/kg group included decreased activity, inappetence, cold skin, transparent or red nose discharges, soft feces, cough, and tachypnea in a few animals. The hematological findings comprised minimally decreased hemoglobin. Clinical chemistry changes included decreased albumin to globulin ratio due to mildly increased globulins. Gross observation included discoloration of lung in 4 of 10 animals at terminal necropsy along with increased lung organ weights. Major microscopic findings comprised minimally to moderately decreased lymphocytes in spleen and/or lymph nodes in 5 of the 6 terminal necropsy animals and minimal to moderate inflammation and fibrosis in the lungs in 4 of the 6 animals. At recovery necropsy, only mild depletion of lymphocytes in lymphoid organs was observed in 1 of the 3 animals, indicating that depletion of lymphocytes in spleen, thymus, and lymph nodes was partially reversed. Lung inflammation and fibrosis were still found in the animals after recovery. [0281] All animals treated with DP303c at the low dose level of 6 mg/kg survived in good health until scheduled necropsy on Day 89 and Day 113. No significant DP303c-related clinical signs were observed during the main and recovery periods. There were no DP303c- related effects on body weight, food consumption, ocular examinations, body temperature, injection site observations, ECG, hematology parameters, coagulation, and clinical chemistry parameters. In addition, there were no DP303c-related organ weight changes and gross findings at the terminal or recovery necropsy. [0282] In conclusion, intravenous administration of DP303c at 20 mg/mL for 3 doses led to one mortality, and other animals survived to scheduled necropsy after changing the dose from 20mg/kg to 12 mg/kg. Intravenous administration of DP303c at 40/30 mg/mg for 3 doses resulted in mortality or morbidity in 6 of 10 animals. The causes of unscheduled death were attributed to DP303c-related lung inflammation and fibrosis, and depletion of lymphocytes in thymus, spleen and lymph nodes. Lung inflammation with fibrosis and minimal to moderate depletion of lymphocytes in spleen and/or lymph node were also observed in most terminal necropsy animals, but the incidence and severity of these changes were lower after recovery. Under the conditions of this study, the highest non-severely toxic dose (HNSTD) was suggested to be 12 mg/kg. Administration of DP303c at 6 mg/mL for 5 doses did not induce any DP303c-related clinical signs and abnormal changes in clinical pathology parameters. Therefore, the no observed adverse effect level (NOAEL) of DP303c was considered to be 6 mg/kg/dose (4th dose interval mean Cmax 137.2 μg/mL, AUC0-t 14149 h·μg/mL) [0283] The TK profiles of DP303c was evaluated by determining serum or plasma levels of DP303c (intact molecule), total antibody (conjugated and unconjugated antibodies) and MMAE over time. Concentrations of DP303c and total antibody DP001 in monkey serum were determined using ELISA assays with a lower limit of quantification of 0.3125 ng/mL for both DP303c and total antibody DP001. Concentrations of MMAE in monkey plasma were analyzed by a tandem liquid chromatography/mass spectrometry (LC-MS/MS) method with a lower limit of quantification of 0.03 ng/mL for MMAE. [0284] Table 7 below shows pharmacokinetic parameters of DP303c in cynomolgus monkeys after IV infusion of DP303c. Values are presented as Mean± standard deviation; [n] indicates animal sample number; M indicates male; F indicates female; Cmax is maximum observed concentration; AUC0-t is area under the concentration time curve up to the last measureable concentration; AUC0-∞ is area under the concentration time curve from time zero to infinity; t1/2 is half-life; CL is systemic clearance; Vss is steady-state volume of distribution.
Table 7
Figure imgf000088_0001
[0285] Table 8 below shows pharmacokinetic parameters of total antibody in cynomolgus monkeys after IV infusion of DP303c. Values are presented as Mean± standard deviation; [n] indicates animal sample number; M indicates male; F indicates female; Cmax is maximum observed concentration; AUC0-t is area under the concentration time curve up to the last measureable concentration; AUC0-∞ is area under the concentration time curve from time zero to infinity; t1/2 is half-life; CL is systemic clearance; Vss is steady-state volume of distribution. Table 8
Figure imgf000088_0002
Figure imgf000089_0001
[0286] Table 9 below shows pharmacokinetic parameters of free MMAE in cynomolgus monkeys after IV infusion of DP303c. Values are presented as Mean± standard deviation; [n] indicates animal sample number; M indicates male; F indicates female; Tmax is time to present at the maximum concentration; AUC0-t is area under the concentration time curve up to the last measureable concentration. Table 9
Figure imgf000089_0002
[0287] Following an IV infusion of DP303c on Day 1, DP303c Cmax max increased dose- proportionally from 131.7 (Male)/147.4 (Female) μg/mL at 6 mg/kg to 935.4 (M)/943.6 (F) μg/mL at 40 mg/mL (FIG.14A and FIG.14B). Mean AUC0-t increased more than dose- proportionally from 12668 (M)/13463 (F) μg·h/mL at 6 mg/kg to 126795 (M)/132131(F) μg·h/mL at 40 mg/kg (Table 7). The clearance (CL) of DP303c was 0.46 (M)/0.44 (F), 0.33 (M) /0.34 (F), and 0.28 (M)/0.26 (F) mL/day/kg, and the half-life (t½) was 115.1 (M)/103.0 (F), 168.3 (M)/159.8 (F), and 158.3 (M)/162.3 (F) hours for the 6-, 20-, and 40-mg/kg dose levels, respectively. The decrease in CL with increasing dose of DP303c was paralleled by an increase in t½. These data indicated nonlinear PK of DP303c and suggested the presence of a sink that becomes saturated between single IV doses of 6 and 40 mg/kg. [0288] After IV dose of DP303c on Day 64, DP303c Cmax and AUC0-t were 134.3 (M)/140.1 (F) μg/mL and 14031 (M)/14266 (F) μg·h/mL in the 6 mg/kg dose level group (FIG.14A and 14B). The mean values of DP303c Cmax and AUC0-t were similar after the first dose and after the 4th dose on Day 64, indicating no accumulation of DP303c exposure following its Q3W administration. The t1/2 and CL values were generally comparable between the first dose interval and the 4th dose interval. In addition, the similar concentration-time profiles of DP303c were observed in males and females suggesting similar systemic exposure in both genders (Table 7). [0289] Total antibody PK was generally similar to DP303c PK. After a single IV infusion of DP303c on Day 1, the increase in mean total antibody Cmax was dose-proportional from 144.5 (M)/ 157.2 (F) μg/mL at 6 mg/kg to 935.4 (M)/ 833.7 (F) μg/mL at 40 mg/mL (FIG.15A and 15B). The mean AUC0-t values of total antibody increased slightly more than dose- proportionally from 15039 (M)/15338 (F) μg·h/mL at 6 mg/kg to 126795/113875 μg·h/mL at 40 mg/kg (Table 8). The CL of total antibody was 0.38 (M)/0.38 (F), 0.29 (M) /0.27 (F), 0.28 (M)/0.30 (F) mL/day/kg, and the t½ was 123.7 (M)/108.8 (F), 179.0 (M)/178.1 (F), and 158.3 (M)/177.6 (F) hours for the 6-, 12-, and 30-mg/kg dose levels, respectively. The mean values of Cmax and AUC0-t of total antibody after the first dose were comparable to those after the 4th dose at 6 mg/kg, indicating no accumulation of total antibody exposure after multiple doses. [0290] Following an IV infusion administration of DP303c on Day 1, peak concentrations of free MMAE in plasma were observed between 72-317 hours across all dose levels (FIG. 16A and 16B). The mean Cmax of free MMAE increased from 0.0635 (M)/0.0641 (F) ng/kg at 6 mg/kg to 0.3838 (M)/0.4878 (F) ng/kg at 40 mg/kg (Table 9). The AUC0-t of free MMAE also increased with the dose from 13.08 (M)/8.94 (F) to 135.12 (M)/141.12 ng·h/mL. After an IV infusion of DP303c on Day 43 or Day 64, the peak concentrations of free MMAE in plasma were found between 24-96 hours across all dose levels (FIG.16A and 16B). The Cmax values of free MMAE were 0.0529 (M)/ 0.0616 (F), 0.3845 (M)/0.1727 (F), and 0.6787 (M)/0.8637 (F) ng/kg at 6-, 12-, and 30- mg/kg dose levels of DP303c, respectively (Table 9). The mean AUC0-t was 7.11 (M)/14.15 (F) ng·h/mL at 6 mg/kg, 114.52 (M)/53.53 (F) at 12 mg/kg, and 151.37 (M)/143.39 (F) ng·h/mL at 30 mg/kg. [0291] An anti-drug antibody (ADA) test was conducted in this study to assess ADA against DP303c. A bridging immunoassay was used to screen anti-drug antibody (ADA) against DP303c in serum samples from cynomolgus monkeys in the GLP repeated-dose toxicology study. In the assay, the biotin-labelled DP303c, ADA, and DP303c form a bridge complex that is quantified using streptavidin-HRP. The signal to background ratio (S/B) for each sample was compared to the cutpoint factor. Any sample whose S/B was equal or higher than the cutpoint factor was considered as positive. The sensitivity of the assay was determined to be 14.27 ng/mL. All samples were found negative and no ADA was detected. [0292] In conclusion, plasma exposure of DP303c and total antibody was increased greater than dose-proportionally following IV infusion administration of DP303c in cynomolgus monkeys on Day 1 and Day43/Day64, suggesting nonlinear kinetics. No accumulation of DP303c or total antibody was observed following Q3W administration of DP303c. The terminal half-life of DP303c was from 120.6 to 162.5 hours. Comparable plasma exposure of intact DP303c and total antibody along with the very low levels of free MMAE indicate negligible de-conjugation of MMAE from DP303c in monkeys. Male and female cynomolgus monkeys exhibited similar concentration-time profiles of all analytes. Plasma Stability Study of DP303c in Human and Cynomolgus Monkey Plasma (GLP) [0293] The objective of this study was to evaluate the in vitro stability of DP303c in pooled plasma from human and cynomolgus monkeys. DP303c was incubated in human or monkey plasma at a concentration of 100 μg/mL at 37°C for 96 hours. Samples were collected at 0, 4, 24, 48, 72, and 96 hours after incubation and then analyzed for DP303c, free MMAE, and total DP001 (naked DP001 plus DP303c). [0294] Concentrations of DP303c and total antibody DP001 in cynomolgus monkey and human plasma were analyzed using ELISA assays with a lower limit of quantification of 0.3125 ng/mL for DP303c and total antibody DP001. Concentrations of MMAE in monkey plasma were analyzed by a tandem liquid chromatography/mass spectrometry (LC-MS/MS) method with a lower limit of quantification of 0.03 ng/mL for MMAE. [0295] Table 10 below shows DP303c stability in monkey and human plasma at 37°C. Table 10
Figure imgf000091_0001
Figure imgf000092_0001
[0296] Table 11 below shows formation of free MMAE in monkey and human plasma during 96 hours at 37°C. Values are presented as Mean ± standard deviation; NC represent value not calculated; a indicates free MMAE concentrations were below lower level of qualification (LLOQ) in 2 of the 3 samples. Table 11
Figure imgf000092_0002
[0297] The changes in the concentrations of DP303c and DP001 were very minimal in both monkey (no changes) and human (about 10% decrease) plasma during the testing period of 96 hours (Table 10). The ratio between DP303c and DP001 ranged from 0.96 to 1.11 across all time points. Consistent with this result, the free MMAE concentrations in monkey and human plasma were very low by the end of the study: 1.318 and 0.752 ng/ml in monkey and human plasma, respectively (Table 11). Free MMAE was about 0.132% and 0.073% of the DP303c concentrations in the monkey and human plasma. All these data suggest that DP303c has very good conjugation stability in plasma. Hemolytic Potential Study of DP303c in Human and Cynomolgus Monkey Blood (GLP) [0298] The objective of this study was to evaluate the potential effect of DP303c formulation on hemolysis and erythrocyte aggregation in human and cynomolgus monkey blood. Human or cynomolgus monkey erythrocytes were incubated with DP303c at concentrations of 0.2, 0.4, 0.6, 0.8, and 1mg/mL at 37°C for 3 hours. No hemolysis and coagulation were observed with DP303c treatment by either macroscopic observation or spectrophotometric analysis during the 3-hour testing period, indicating that DP303c did not cause hemolysis and aggregation of human and monkey red blood cells. Discussion [0299] DP303c demonstrated very good stability in vitro in human and monkey plasma. Ninety percent of DP303c were intact molecules after 96 hours incubation in human plasma. DP303c displayed a linear PK with terminal half-life of 9 days in rats over the dose range of 3 to 30 mg/kg. The PK parameters were very similar between DP303c and total antibody in rats across all doses, indicating limited de-conjugation of MMAE from DP303c. In all 3 single- dose and repeated-dose cynomolgus monkey studies, DP303c displayed nonlinear PK with a terminal half-life of 4 to 7 days over the dose range of 1.2 to 40 mg/kg. Comparable exposure of DP303c and total antibody in cynomolgus monkeys also suggests no significant de- conjugation of MMAE in monkeys. The plasma levels of MMAE were very low in monkeys after IV dose of DP303c. There was no gender difference observed in the PK profiles of DP303c, total antibody or free MMAE in all animal studies. No accumulation of DP303c or total antibody was observed following Q3W administration of DP303c in the repeated dose GLP study. [0300] Dose levels ≤ 40/30 mg/kg DP303c were not tolerated in cynomolgus monkeys, resulting in mortality and moribund animals in both the non-GLP and the GLP studies. Dose- dependent leukopenia and anemia were also found in both these 2 studies, characterized by decreased RBC mass, WBC, neutrophils, eosinophils, and lymphocytes. In the GLP repeated dose toxicology study, the major dose limiting toxicities were dose-dependent lymphocyte depletion in spleen, thymus and lymph nodes and pulmonary toxicity. The clinical symptoms of pulmonary toxicity comprised cough, dyspnea, and heavy breathing, together with gross observation of purple or dark discoloration of lung, increased lung organ weights, and lung inflammation with fibrosis. However, pulmonary toxicity was not clearly noted in the 29-day non-GLP study as assessed by gross observation and lung organ weights. The likely explanation could be that it took a longer time to develop notable lung inflammation with fibrosis and pulmonary injury following DP303c treatment because unscheduled animal death occurred on Day 10 or earlier in the 29-day non-GLP dose range finding study. Other significant histopathological findings in the GLP repeated dose toxicology study included slight tubule degeneration with or without cast in kidney, minimal or mild degeneration/necrosis in the liver, and slight single cell necrosis in the epidermis in the skin or injection sites. [0301] The dose level of 20 mg/kg of DP303c was well tolerated in the 29-day non-GLP dose range finding study. In the GLP repeated dose toxicology study, all animals survived at 20/12 mg/kg of DP303c except one unscheduled animal death that happened before the dose was changed from 20 to 12 mg/kg. The clinical signs at 20/12 mg/mL were restricted to red discharge in noses, inappetence, soft feces, cold skin, decreased activity, cough, and tachypnea in a few animals. Histopathological findings were similar to those at 40/30mg/kg DP303c, but were generally milder and partially reversible after recovery. Based on these data, the highest nonseverely toxic dose (HNSTD) of DP303c in monkeys was considered to be 12 mg/kg/dose IV once every 3 weeks. At the low dose level of 6 mg/kg of DP303c, all animals survived without DP303c-related clinical signs and clinical pathology findings in both the 29-day non-GLP dose range finding study and the GLP repeated dose toxicology study. In addition, no signs of DP303c-related cardiac toxicity were observed at any doses as assessed by ECG. Therefore, the no observed adverse effect level (NOAEL) of DP303c in monkeys was determined to be 6 mg/kg IV administrated once every three weeks. [0302] Besides cynomolgus monkeys, the nonclinical safety of DP303c was also assessed in rats in a non-GLP single-dose toxicology study (Study DP-15-0818). There was no mortality or moribund in all groups in this study. The major adverse effects included decreases in leukocytes, macrophage/monocyte, hematocrit, and hemoglobin at high doses (60 and 100 mg/kg) of DP303c. The observed hematology changes returned to normal levels 21 days after DP303c treatment. Therefore, the maximum tolerated dose of DP303c was not reached in rats in this study and was determined to be no less than 100 mg/kg following a single intravenous administration. The difference in tolerability of DP303c in monkeys and rats may be due to the difference in the antigen distribution and binding. [0303] In summary, the major toxicity of DP303c in nonclinical safety studies was anemia, leukopenia, depletion of lymphocytes in thymus, spleen and lymph nodes and lung inflammation and fibrosis These adverse effects were dose-dependent reversible and monitorable in animals. Therefore, based on the completed nonclinical safety package, ICH S9 Guidance and FDA publication (Saber and Leighton, 2015), a starting dose of 0.6 mg/kg is proposed for the first human trial with DP303c. The starting dose was calculated based on 1/10th NOAEL at 6 mg/kg in cynomolgus monkeys using body weight for scaling. Example 8. DP303c PK assays for first-time-in-human (FTIH) studies [0304] Based on current assays validated for nonclinical studies, the following PK assays for first-time-in-human (FTIH) study is developed and validated: 1) an ELISA assay for quantifying the level of DP303c in human serum samples; 2) an ELISA assay for quantifying the level of DP001 (total antibody) in human serum samples; 3) a liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to quantify the level of free MMAE warhead in human serum samples. Experiments are conducted to evaluate the assay parameters of 1) upper and lower limits of quantitation; 2) accuracy and precision; 3) specificity; 4) selectivity; 5) dilutional linearity; and 6) sample stability. Immunoassays and a tiered immunogenicity testing approach are employed for ADA detection in support of the FTIH clinical trial. Samples will be first screened, and once a test sample is confirmed positive for the presence of the ADA, the specificity of the ADA will be assessed. The ADA assays will be evaluated for 1) limit of detection; 2) precision; 3) cut points; and 4) drug tolerance. The sample matrix will be serum. [0305] Dose escalation scheme for the DP303c FTIH study is 0.6, 1.2, 2.0, 3.0, and 4 mg/kg IV infusion once every 3 weeks (Q3W). An optional escalation to a higher dose level achieved through an up to 25% increase from the previous dose level per cohort may be evaluated based on safety and clinical activity data from the study. DP303c dose selection is based on safety margins from nonclinical safety studies and FDA publication (Saber and Leighton, 2015) and ICH S9. Safety margins were estimated based on 1/10th the no observed adverse effect level (NOAEL) at 6 mg/kg in cynomolgus monkeys using body weight for scaling. The safety margins were also calculated based on 1/6th the highest non-severely toxic dose (HNSTD) at 12 mg/kg in cynomolgus monkeys using body surface area for scaling. The starting dose of 0.6 mg/kg is expected to have a safety margin of 10 based on the HED NOAEL of 6 mg/kg and a safety margin of 6.5 based on the HED HNSTD of 3.87 mg/kg (Table 12) The highest dose of 4 mg/kg is anticipated to have a safety margin of 1.5 based on the HED NOAEL and a safety margin of 1 based on HED HNSTD. Table 12. Predicted Safety Margins for Proposed DP303c Clinical Doses
Figure imgf000096_0001
Example 9. Safety, Pharmacokinetics, Immunogenicity, and Antitumor Activity studies for DP303c [0306] This example describes a Phase 1a/1b multicenter, open-label, dose-escalation/de- escalation and dose-expansion study to evaluate the safety, pharmacokinetics, immunogenicity, and antitumor activity of DP303c in subjects with select HER2-expressing advanced solid tumors. The primary objectives of the study are assessment of the safety and tolerability of DP303c in subjects with HER2- positive advanced solid tumors refractory to standard therapy or for which no standard therapy exists and determination of the maximum tolerated dose (MTD) and/or recommended Phase 2 dose (RP2D) of DP303c. The secondary objectives of the study are evaluation of the antitumor activity of DP303c in subjects with HER2-positive breast or gastric cancer, determination of the pharmacokinetic (PK) profile of DP303c, and determination of the immunogenicity of DP303c. Phase 1a: Dose Escalation/De-escalation [0307] Phase 1a portion of the study enrolls subjects with HER2-positive advanced solid tumors. Dose level cohorts of 3 subjects each are treated. The dose levels (DL) to be studied in Phase 1a are: DL -1: 0.3 mg/kg; DL 1: 0.5 mg/kg or 0.6 mg/kg; DL 2: 1 mg/kg or 1.2 mg/kg; DL 3: 2.0 mg/kg; DL 4: 3.0 mg/kg; DL 5: 4.0 mg/kg. The starting dose (DL 1) is 0.5 mg/kg or 0.6 mg/kg. The first dose is administered by IV infusion over 60 (±10) minutes in normal saline for the first dose; if tolerated, subsequent infusions may be administered over 30 (±5) minutes. [0308] This study uses a traditional 3 + 3 design, which is widely used in Phase 1 studies to determine MTD and as a basis for selection of the RP2D. The starting dose is 0.6 mg/kg, one tenth of the NOAEL (no observed adverse effect level) in the monkey definitive repeated dose toxicity study. The 4.0 mg/kg dose is below that which was associated with serious toxicity in the monkey (20 mg/kg, which is comparable to a human dose of 6.7 mg/kg after allometric scaling). [0309] In the dose-de-escalation design, dose-limiting toxicity (DLT) is assessed over the initial 21-day period and beyond to determine dose escalation or de-escalation. If the 4.0 mg/kg dose level does not exceed the MTD, then dose escalation may continue with increases up to 25% from the previous dose level until the MTD is exceeded (≥2 DLT) at a dose level. A DLT is defined as a study drug-related AE by NCI CTCAE v 4.03 or study drug-related and clinically significant laboratory abnormality that occurs during the first 21-day cycle of DP303c and meets the criteria: grade 4 neutropenia (absolute neutrophil count [ANC] <500/mm3) lasting >7 days; febrile neutropenia (ANC <1000/mm3) with fever (temp >38.0° C) lasting >1 hour; grade 4 thrombocytopenia (platelet count <250,000/mm3) lasting >2 days; grade ≥3 thrombocytopenia (platelet count <50,000/mm3) with clinically significant bleeding; grade ≥2 pneumonitis; any ≥other Grade ≥3 non-hematologic toxicity, excluding suboptimally treated nausea, vomiting, and diarrhea, alopecia, and/or brief (<1 week) Grade 3 fatigue; optimally treated nausea, vomiting, or diarrhea ≥Grade 3 that persists for more than 72 hours; LVEF <40% or LVEF decrease ≥10% from baseline with LVEF <45%; treatment delay of >21 days because of a DP303c-related toxicity. [0310] The occurrence of DLT-like toxicities beyond Cycle 1 (C1) is assessed continuously and incorporated into future decision-making on further dose escalations/de-escalations. AEs are not be considered as possible DLT if available evidence indicates that a relationship to study treatment is not logically possible, not medically plausible, or highly unlikely because of a clear alternative explanation. [0311] Decisions concerning dose escalations are made based on cumulative safety data including reports of adverse events, vital signs, laboratory results, ECGs, and results of LVEF and pulmonary function testing. During the DLT evaluation period in C1, the following rules are used to determine if dose escalation is appropriate: 3 subjects will initially be enrolled in each cohort; if none of the first 3 subjects in a cohort experiences DLT, up to 3 new subjects will be enrolled into a cohort at the next dose level; if 1 of the first 3 subjects in a cohort experience DLT the cohort will be expanded up to 6 subjects If no additional subjects develop DLT in the 6 subjects in that cohort, up to 3 new subjects will be enrolled into a cohort at the next dose level; if 2 or more subjects in a 3- or 6-subject cohort experience DLT, that dose level is higher than the MTD and dose escalation will stop, 3 additional subjects will be enrolled and evaluated for DLT at the previous dose level, unless 6 subjects have already been evaluated at that dose level; when a dose higher than the MTD has been tested, the highest dose at which fewer than 2 of 6 subjects (ie, <33%) experience DLT will be considered the MTD. [0312] A dose-escalation decision for each cohort is made after the last subject has completed the DLT observation period, or experienced DLT, and all relevant safety data have been reviewed. Up to 3 subjects may be added to a cohort even though no DLT was observed, to more thoroughly evaluate a potential safety signal. Under some circumstances, treatment- related AEs that occur beyond the 21-day DLT evaluation period are considered for dose- escalation/de-escalation decision making. Phase 1b: Dose Expansion [0313] The RP2D is a dose that is expected to be tolerable for repeated administration. The RP2D may be lower than the MTD, based on review of clinical safety data. Upon determination of the MTD and selection of the RP2D, the Phase 1b dose-expansion part of the study will begin. During the expansion, approximately 10 subjects each will enroll into 2 defined cancer types: breast cancer and gastric cancer (including adenocarcinomas of the gastroesophageal junction). For both tumor types, HER2-positive is defined as: HER2 IHC 2+ and ISH positive or IHC 3+. [0314] After 10 subjects (aggregated across both dose-expansion cancer types) have received the first cycle of study treatment (3 weeks/21 days), the SMC will review all available safety data and assess whether any modification of the dosing regimen or study design is warranted. [0315] For each expansion cancer type, if at any time ≥3 subjects experience DLT-like AEs or other unacceptable toxicity, then further enrollment will be suspended pending SMC review. The SMC may recommend that subsequent subjects will be enrolled at the next lower dose level or an intermediate dose level. Inclusion and Exclusion Criteria [0316] Approximately 54 subjects from various sites are enrolled, up to 15-30 during dose- escalation/de-escalation (Phase 1a) and up to 20-24 during dose-expansion (Phase 1b) with approximately 10 subjects in each cancer type. Additional subjects may be enrolled to account for dropouts and to ensure a minimum number of DLT-evaluable subjects during dose- escalation. Subjects are eligible to be included in the study only if all the following criteria apply: 1. Signed informed consent prior to study-related procedures; 2. Male or female 18-75 years of age; 3. Diagnosis of advanced, HER2-positive estrogen receptor (ER) malignancy that has progressed following standard therapy or for which no standard therapy exists. Subjects who have been previously treated with a HER2-targeted therapy such as trastuzumab, pertuzumab, lapatinib, or ado-trastuzumab emtansine are eligible. HER2-positive is defined as IHC 2+ and ISH positive or IHC 3+: for Phase 1a, subjects may have any type of solid tumor, provided it is positive for HER2 either IHC 2+ or 3+, or ISH-positive; for Phase 1b, subjects must have breast or gastric carcinomas that are positive for HER2 either IHC 2+ or 3+, or ISH-positive; HER2 testing must have been performed in a College of American Pathology (CAP) accredited laboratory using an FDA-approved or validated assay. 4. ECOG performance status 0 to 1, and the expected survival time is more than 3 months; 5. Subjects must have laboratory values within the limits described below: ANC ≥1.5 x 109/L; platelet count ≥100 x 109/L; hemoglobin ≥9 g/dL; serum creatinine within normal limits OR creatinine clearance ≥60 mL/minute; serum total bilirubin ≥1.5 x ULN (up to 3 x ULN in subjects with Gilbert’s syndrome); AST (SGOT) and ALT (SGPT) ≥2.5 x ULN (OR ≥5 X ULN for subjects with liver metastases); PT/INR and APTT ≥1.5 x ULN; 6. Disease measurability: Phase 1a: measurable or evaluable disease; Phase 1b: disease must be measurable (per RECIST 1.1); 7. WOCBP must have a negative pregnancy test prior to study entry; 8. WOCBP and male subjects must agree to use adequate contraception from study entry through at least 12 weeks after the last dose of study drug; 9. A washout period is required for subjects who have recently received antitumor systemic therapy. The period prior to the subject’s planned first dose of DP303c must be either at least 28 days or 5 half-lives, whichever is shorter. This applies for investigational and approved therapies. Antitumor therapy includes chemotherapy, immunotherapy, targeted therapy, endocrine therapy, radiotherapy (except local radiotherapy for pain relief, 14 days after treatment). [0317] Subjects are prohibited from receiving the following therapies during screening and the study treatment period: anti-cancer systemic chemotherapy, hormonal therapy, or immunotherapy; elective surgical/dental treatment after discussion with medical monitor as per consultation with the sponsor; investigational agents other than DP303c; radiotherapy (except palliative radiation therapy for disease-related pain with a consult with the sponsor's medical monitor); radio-/toxin-immunoconjugates. [0318] Subjects who meet any of the following criteria will not be eligible to participate in the study: 1. Pregnant or breastfeeding women; 2. Refusal to use effective methods of contraception (see inclusion criteria for details); 3. Has not recovered (ie, ≤Grade 1 or at baseline) from AEs, except alopecia, due to previously administered agent(s) or radiotherapy; 4. History of cardiac dysfunction <40% while on trastuzumab therapy; 5. Subjects with a history of allergy to any components of DP303c (trastuzumab analogs, MMAE, sodium citrate dihydrate, citrate monohydrate, polysorbitol 20, sucrose, etc.); 6. History of unstable central nervous system (CNS) metastases or seizure disorder related to the malignancy; however, those subjects who were treated for prior CNS metastases and who have been asymptomatic for a minimum period of four weeks while off steroids and anticonvulsants may participate in the study; 7. History of interstitial lung disease, previous or active lung infection, or inflammation (pneumonitis); 8. Requires supplemental oxygen; 9. History of congestive heart failure, unstable angina pectoris, unstable atrial fibrillation, or cardiac arrhythmia. Subjects who have the following types of cardiac impairment at the time of enrollment: New York Heart Association class III or IV heart disease; uncontrolled angina, congestive heart failure, or myocardial infarction within 6 months prior to enrollment; an LVEF by echocardiogram (ECHO) or multi-gated acquisition (MUGA) scan <50%; QT interval prolongation (>450 ms in males, >470 ms in females); 10. In the first 90 days of the study, cumulative anthracycline dose ≥360 mg/m2 doxorubicin or equivalent; 11. Peripheral neuropathy ≥Grade 2 or greater (NCI CTCAE v 4.03); 12. Non-manageable electrolyte imbalances including hypokalemia, hypocalcemia, or hypomagnesemia (≥Grade 2 or greater based on NCI CTCAE v 403); 13. Any uncontrollable intercurrent illness, infection, or other conditions that could limit study compliance or interfere with assessments; 14. Subjects with evidence of an active infection including: subjects being treated with antibiotics for an active infection at the time of enrollment; subjects who have evidence of active hepatitis C or chronic active hepatitis B; subjects who have a known diagnosis of human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS); 15. Patients were treated with CYP3A inhibitors within 14 days of the first dose (drugs that increased specific CYP substrate AUC ≥ 5 times, such as Mibefradil, verapamil, diltiazem, nefazodone, clarithromycin, Telithromycin, Troleandomycin, Erythromycin, fluconazole, itraconazole, ketoconazole, Posaconazole,VoriconazoleTablets, Elvitegravir, indinavir, lopinavir, Nelfinavir, Ritonavir, Saquinavir, Boceprevir, Incivo, telaprevir, Conivaptan, idelalisib) or strong CYP3A inducers (Avasimibe, phenobarbital, phenytoin, carbamazepine, Rifampicin, rifabutin,enzalutamide, mitotane, Hypericum perforatum); 16. Other severe or poorly controlled illness or circumstance that would interfere with evaluation of key study endpoints or which would put the subject at risk from participating in the study in the opinion of the Investigator. Dose Modification [0319] Dose reductions and/or delays may be considered for the following circumstances: 1. In the event of DLT during cycle 1 or AE that meets DLT criteria or is considered unacceptable by the investigator during Cycle 2 or beyond, either discontinue the subject from the study or resuming dosing at a reduced dose level in subsequent cycles, after recovery from the DLT to Grade 0, 1, or baseline. 2. In the event of drug-related AE that has not recovered to Grade 0, 1, or baseline by the end of a 21-day cycle, if the AE recovers by Day 42 post-dose, dosing may resume at full or reduced dose. If the AE has not recovered by Day 42, discontinue the subject from the study. 3. In the event of dose missed for administrative reasons (e.g., site closure during holidays, subject vacation, intercurrent illness), dosing may resume at next feasible time. If dosing cannot resume by Day 42 post-dose, discontinue the subject from the study. Dose reductions are permitted to the next-lower protocol-specified dose level. Patients who enrolled in the lowest dose level (DL 1: 0.6 mg/kg) may have their dose reduced to 0.3 mg/kg. If a subject is unable to tolerate the reduced dose level, then he/she should be discontinued from the study. Intra-subject dose escalation will not be permitted. Study Assessments [0320] Efficacy is assessed by tumor response upon completion of Cycle 2 and at the end of every other cycle until study discontinuation. Safety is assessed by measurements of physical examinations, vital signs, electrocardiograms (ECGs), left ventricular ejection fraction (LVEF), pulmonary function testing, and high resolution CT of chest. [0321] Measurements of the primary objectives are: adverse events (AEs), serious adverse events (SAEs), dose limiting toxicity (DLT), changes from baseline in laboratory parameters, vital signs (VS), and electrocardiograms (ECGs). All AE and SAE will be collected from the signing of the ICF until 30 (±5) days after the last dose of DP303c. Events Meeting the AE Definition are: 1. Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or other safety assessments (e.g., ECG, radiological scans, vital signs measurements), including those that worsen from baseline, considered clinically significant in the medical and scientific judgment of the investigator (i.e., not related to progression of underlying disease). 2. Exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency and/or intensity of the condition. 3. New conditions detected or diagnosed after study treatment administration even though it may have been present before the start of the study. 4. Signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction. 5. Signs, symptoms, or the clinical sequelae of a suspected overdose of either study treatment or a concomitant medication. Overdose per se will not be reported as an AE/SAE unless it is an intentional overdose taken with possible suicidal/self-harming intent. Such overdoses should be reported regardless of sequelae. 6. "Lack of efficacy" or "failure of expected pharmacological action" per se will not be reported as an AE or SAE. Such instances will be captured in the efficacy assessments. However, the signs, symptoms, and/or clinical sequelae resulting from lack of efficacy will be reported as AE or SAE if they fulfil the definition of an AE or SAE. [0322] Events not Meeting the AE Definition are: 1. Any clinically significant abnormal laboratory findings or other abnormal safety assessments, which are associated with the underlying disease, unless judged by the investigator to be more severe than expected for the subject’s condition. 2. The disease/disorder being studied or expected progression, signs, or symptoms of the disease/disorder being studied, unless more severe than expected for the subject’s condition. 3. Medical or surgical procedure (eg, endoscopy, appendectomy): the condition that leads to the procedure is the AE. 4. Situations in which an untoward medical occurrence did not occur (social and/or convenience admission to a hospital). 5. Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or detected at the start of the study that do not worsen. [0323] Measurements of the secondary objectives are: best overall response and disease control rates based on RECIST 1.1; duration of response and progression-free survival (PFS); individual subject DP303c serum concentrations and other analytes at specified time points after DP303c administration, and derived PK parameters; number (%) of subjects who develop detectable antidrug antibody (ADA). Pharmacokinetics will be assessed by measurement of DP303c concentrations, total antibody in serum, and MMAE derivative and free MMAE concentration in plasma. Blood samples will be collected from all subjects for measurement of DP303c concentrations in serum or plasma at day 1, 2, 4, 8, 15 of during Cycle 1 and Cycle 2 and day 1 of all following cycles. Antidrug antibody to DP303c in serum will be measured in blood samples collected at day1 of each treatment cycle. Antitumor activity will be analyzed by objective response rate (ORR), disease control rate (DCR), duration of response, and progression-free survival (PFS). ORR is defined as a proportion ([confirmed complete response (CR) + confirmed partial response (PR)]/N evaluable) using RECIST 1.1 criteria. The proportion obtaining a CR or PR without the confirmation requirement will also be provided. ORR will be summarized using descriptive statistics. Disease control rate (DCR) [CR + PR + stable disease (SD)/N evaluable] will be summarized similarly. Duration of response and PFS will be calculated using the Kaplan-Meier approach. Phase 1a Initial Results [0324] To date, 10 subjects were enrolled in a Phase 1a study to evaluate safety and efficacy of DP303c treatment in the subjects. All of the enrolled subjects had Her2 positive cancer and had received HERCEPTIN® treatment before the study. To date, five subjects have dropped out and five subjects are still participating in the study. For the dose escalation study design, one subject was administered 0.5 mg/kg DP303c, and three subjects each were administered 1, 2, or 3 mg/kg DP303c. Doses were administered by intravenous infusion once every three weeks. [0325] DP303c related toxicity was assessed. At the 2 mg/kg dose, one subject showed grade 2 eye toxicity with blurry vision. At the 3 mg/kg dose, one subject showed grade 3 eye toxicity with blurry vision. After a pause dose in these two patients (i.e., they both skipped one scheduled dose), all the observed toxicity resolved and treatment with DP303c resumed. After the pause, the patient who had received 3 mg/kg before was lowered to 2 mg/kg. [0326] Efficacy was assessed using RECIST 1.1 criteria (see Eisenhauer, E.A. et al. Eur J Cancer.2009 Jan;45(2):228-47). At the 1, 2 and 3 mg/kg dose, one patient from each dose level exhibited a partial response (30% or higher reduction in tumor size). Summaries of the toxicity and efficacy results are provided in Table 13, below, with “PD” indicating progressive disease, “PR” indicating partial response, and “SD” indicating stable disease. Table 13. Summary of Phase 1a efficacy and toxicity results
Figure imgf000104_0001
Figure imgf000105_0001

Claims

CLAIMS What is claimed is: 1. A method of treating a cancer in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER2 antibody and a conjugation moiety comprising a toxin, wherein the anti-HER2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugation moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg. 2. The method of claim 1, wherein the cancer is a solid cancer. 3. The method of claim 2, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancers, and lung cancer. 4. The method of any one of claims 1-3, wherein the antibody-drug conjugate is administered at a dose of no more than about 6 mg/kg. 5. The method of any one of claims 1-3, wherein the antibody-drug conjugate is administered at a dose of about 1 mg/kg to about 2 mg/kg, or about 2 mg/kg to about 3 mg/kg 6. The method of claim 5, wherein the antibody-drug conjugate is administered at a dose of about 1.0 mg/kg, about 2.0 mg/kg, or about 3.0 mg/kg. 7. The method of any one of claims 1-6, wherein the antibody-drug conjugate is administered intravenously. 8. The method of any one of claims 1-7, wherein the antibody-drug conjugate is administered about once every three weeks, about every other week, or about once per week. 9. The method of any one of claims 1-8, wherein the individual is human. 10. The method of any one of claims 1-9, wherein the anti-HER2 antibody is N-glycosylated in the Fc region. 11. The method of any one of claims 1-10, wherein the acceptor glutamine residue is Q295 in the heavy chain of the anti-HER2 antibody according to the EU numbering. 12. The method of any one of claims 1-11, wherein each heavy chain of the HER2 antibody is conjugated to the conjugation moiety. 13. The method of any one of claims 1-12, wherein the conjugation moiety is conjugated to the acceptor glutamine residue by transglutamination. 14. The method of any one of claims 1-13, wherein the anti-HER2 antibody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:2, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable region (VL) comprising a light chain complementarity-determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:6. 15. The method of claim 14, wherein the anti-HER2 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8. 16. The method of any one of claims 1-15, wherein the Fc region is an IgG1 Fc. 17. The method of claim 16, wherein the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. 18. The method of any one of claims 1-17, wherein the toxin is a monomethyl auristatin E (MMAE). 19. The method of any one of claims 1-18, wherein the conjugation moiety comprises a cleavable linker. 20. The method of claim 19, wherein the conjugation moiety is a compound of Formula I:
Figure imgf000107_0001
21. The method of any one of claims 1-20, wherein the antibody-drug conjugate is DP303c.
PCT/US2020/057761 2019-10-29 2020-10-28 Compositions and methods for treating cancer using anti-her2 antibody drug conjugate WO2021086981A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202080076131.1A CN114901308A (en) 2019-10-29 2020-10-28 Compositions and methods for treating cancer using anti-HER 2 antibody drug conjugates

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962927623P 2019-10-29 2019-10-29
US62/927,623 2019-10-29

Publications (1)

Publication Number Publication Date
WO2021086981A1 true WO2021086981A1 (en) 2021-05-06

Family

ID=75716304

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2020/057761 WO2021086981A1 (en) 2019-10-29 2020-10-28 Compositions and methods for treating cancer using anti-her2 antibody drug conjugate

Country Status (2)

Country Link
CN (1) CN114901308A (en)
WO (1) WO2021086981A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022242692A1 (en) * 2021-05-21 2022-11-24 Remegen Co., Ltd. Use of antibody-drug conjugate in combination with immune checkpoint inhibitor in treatment of urothelial cancer
WO2023044016A3 (en) * 2021-09-17 2023-04-20 Cspc Dophen Corporation Antibody immune-stimulant conjugates and use thereof
WO2023070125A1 (en) * 2021-10-22 2023-04-27 Academia Sinica Antibody-drug conjugate for reducing glycosylation of membrane glycoprotein
WO2023231942A1 (en) * 2022-05-31 2023-12-07 石药集团巨石生物制药有限公司 Pharmaceutical composition of recombinant anti-human cldn18.2 monoclonal antibody-mmae conjugate

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015191883A1 (en) * 2014-06-12 2015-12-17 Dophen Biomedical Homogenous antibody drug conjugates via enzymatic methods
WO2017093844A1 (en) * 2015-11-30 2017-06-08 Pfizer Inc. Site specific her2 antibody drug conjugates

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015191883A1 (en) * 2014-06-12 2015-12-17 Dophen Biomedical Homogenous antibody drug conjugates via enzymatic methods
WO2017093844A1 (en) * 2015-11-30 2017-06-08 Pfizer Inc. Site specific her2 antibody drug conjugates

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022242692A1 (en) * 2021-05-21 2022-11-24 Remegen Co., Ltd. Use of antibody-drug conjugate in combination with immune checkpoint inhibitor in treatment of urothelial cancer
WO2023044016A3 (en) * 2021-09-17 2023-04-20 Cspc Dophen Corporation Antibody immune-stimulant conjugates and use thereof
WO2023070125A1 (en) * 2021-10-22 2023-04-27 Academia Sinica Antibody-drug conjugate for reducing glycosylation of membrane glycoprotein
WO2023231942A1 (en) * 2022-05-31 2023-12-07 石药集团巨石生物制药有限公司 Pharmaceutical composition of recombinant anti-human cldn18.2 monoclonal antibody-mmae conjugate

Also Published As

Publication number Publication date
CN114901308A (en) 2022-08-12

Similar Documents

Publication Publication Date Title
US11279764B2 (en) Antibodies specific for epidermal growth factor receptor variant III and their uses
JP6475277B2 (en) Antibodies specific for TROP-2 and uses thereof
JP6521959B2 (en) Engineered polypeptide conjugates
TWI671317B (en) Anti-cxcr4 antibodies and antibody-drug conjugates
WO2021086981A1 (en) Compositions and methods for treating cancer using anti-her2 antibody drug conjugate
JP6979950B2 (en) Site-specific HER2 antibody drug conjugate
JP2020521481A (en) Antibodies specific for FLT3 and uses thereof
JP6326137B2 (en) Anti-HER2 antibody and conjugate thereof
BR112020005212A2 (en) antibody conjugate, kit, pharmaceutical composition, and methods of treatment or prevention and diagnosis of a disease or condition.
KR20160047567A (en) Engineered anti-dll3 conjugates and methods of use
JP2020502271A (en) Anti-CUB domain containing protein 1 (CDCP1) antibodies, antibody drug conjugates, and methods of using the same
EP2970443A2 (en) Cancer treatment using antibodies that bind cell surface grp78
JP2020504114A (en) Anti-SEZ6L2 antibody and antibody drug conjugate
CN116251196A (en) anti-EDB antibodies and antibody-drug conjugates
TW201713363A (en) Calicheamicin constructs and methods of use
CN112020519A (en) anti-HER 2 biparatopic antibody-drug conjugates and methods of use
JP6936399B2 (en) Anti-SEZ6 antibody drug conjugate and usage
WO2023232140A1 (en) Cancer treatment with a pd-1 or pd-l1 inhibitor and an antibody-drug conjugates targeting claudin 18.2
RU2806049C2 (en) Anti-her2 biparatopic antibody-drug conjugates and methods of their application
RU2806049C9 (en) Anti-her2 biparatopic antibody-drug conjugates and methods of their application
US20230330241A1 (en) Humanized anti-liv1 antibodies for the treatment of cancer
WO2024050031A2 (en) Novel camptothecin derivatves as antibody-drug conjugates (adc) payloads
KR20220082846A (en) B-lymphocyte-specific amatoxin antibody conjugate
CA3165135A1 (en) Treatment with site specific her2 antibody-drug conjugates

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20880406

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20880406

Country of ref document: EP

Kind code of ref document: A1