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WO2018102212A1 - Thérapie pour le cancer urothélial métastatique avec le conjugué anticorps-médicament, sacituzumab govitécan (immu-132) - Google Patents

Thérapie pour le cancer urothélial métastatique avec le conjugué anticorps-médicament, sacituzumab govitécan (immu-132) Download PDF

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Publication number
WO2018102212A1
WO2018102212A1 PCT/US2017/062976 US2017062976W WO2018102212A1 WO 2018102212 A1 WO2018102212 A1 WO 2018102212A1 US 2017062976 W US2017062976 W US 2017062976W WO 2018102212 A1 WO2018102212 A1 WO 2018102212A1
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antibody
cancer
antibodies
immu
group
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Serengulam V. Govindan
David M. Goldenberg
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Immunomedics Inc
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Immunomedics Inc
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Priority to CN201780084389.4A priority Critical patent/CN110248680A/zh
Priority to RU2019119320A priority patent/RU2757395C2/ru
Priority to EP17876335.5A priority patent/EP3548088A4/fr
Priority to CA3044096A priority patent/CA3044096A1/fr
Publication of WO2018102212A1 publication Critical patent/WO2018102212A1/fr
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • 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
    • A61K47/6861—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 kidney or bladder cancer cell

Definitions

  • the present invention relates to therapeutic use of immunoconjugates of antibodies or antigen-binding antibody fragments and camptothecins, such as SN-38, with improved ability to target various cancer cells in human subjects.
  • the antibodies and therapeutic moieties are linked via an intracellularly-cleavable linkage that increases therapeutic efficacy.
  • the immunoconjugates are administered at specific dosages and/or specific schedules of administration that optimize the therapeutic effect.
  • the optimized dosages and schedules of administration of SN-38-conjugated antibodies for human therapeutic use disclosed herein show unexpected superior efficacy that could not have been predicted from animal model studies, allowing effective treatment of cancers that are resistant to standard anti-cancer therapies, including irinotecan (CPT-11), the parent compound of SN-38.
  • the methods and compositions are of use to treat Trop-2 positive cancer, particularly urothelial cancer, using an anti-Trop-2 hRS7-SN-38 immunoconjugate.
  • the immunoconjugate may be administered to a human subject with a Trop-2 positive cancer at a dosage of between 3 and 18 mg/kg, more preferably between 4 and 12 mg/kg, most preferably between 8 and 10 mg/kg.
  • the methods and compositions may be used to treat Trop-2 positive cancer that is relapsed from or refractory to other standard anti-cancer therapies, such as chemotherapeutic drugs.
  • the anti-Trop-2-SN38 antibody drug conjugates are effective to treat Trop-2 positive cancers in patients who had relapsed from or shown resistance to therapy with standard anti-cancer agents, including irinotecan.
  • an anti-Trop-2-SN-38 ADC such as IMMU-132, may be
  • ADC a synergistic effect with the ADC
  • other therapeutic agents that may exhibit a synergistic effect with the ADC, such as microtubule inhibitors, PARP inhibitors, Bruton kinase inhibitors or PI3K inhibitors.
  • MAbs monoclonal antibodies
  • the toxic agent is most commonly a chemotherapeutic drug, although particle-emitting radionuclides, or bacterial or plant toxins, have also been conjugated to MAbs, especially for the therapy of cancer (Sharkey and Goldenberg, CA Cancer J Clin. 2006 Jul-Aug;56(4):226-243).
  • MAb-chemotherapeutic drug conjugates are that (a) the chemotherapeutic drug itself is structurally well defined; (b) the chemotherapeutic drug is linked to the MAb protein using very well-defined conjugation chemistries, often at specific sites remote from the MAbs' antigen binding regions; (c) MAb-chemotherapeutic drug conjugates can be made more reproducibly and usually with less immunogenicity than chemical conjugates involving MAbs and bacterial or plant toxins, and as such are more amenable to commercial development and regulatory approval; and (d) the MAb- chemotherapeutic drug conjugates are orders of magnitude less toxic systemically than radionuclide MAb conjugates, particularly to the radiation-sensitive bone marrow.
  • Camptothecin (CPT) and its derivatives are a class of potent antitumor agents.
  • Irinotecan also referred to as CPT-11
  • topotecan are CPT analogs that are approved cancer therapeutics (Iyer and Ratain, Cancer Chemother. Phamacol. 42: S31-S43 (1998)).
  • CPTs act by inhibiting topoisomerase I enzyme by stabilizing topoisomerase I-DNA complex (Liu, et al. in The Camptothecins: Unfolding Their Anticancer Potential, Liehr J.G.,
  • CPTs present specific issues in the preparation of conjugates.
  • One issue is the insolubility of most CPT derivatives in aqueous buffers.
  • CPTs provide specific challenges for structural modification for conjugating to macromolecules. For instance, CPT itself contains only a tertiary hydroxyl group in ring-E. The hydroxyl functional group in the case of CPT must be coupled to a linker suitable for subsequent protein conjugation; and in potent CPT
  • the conjugation protocol is performed such that it is carried out at a pH of 7 or lower to avoid the lactone ring opening.
  • conjugation of a bifunctional CPT possessing an amine- reactive group such as an active ester would typically require a pH of 8 or greater.
  • an intracellularly-cleavable moiety preferably is incorporated in the linker/spacer connecting the CPTs and the antibodies or other binding moieties.
  • the methods comprise optimized dosing and administration schedules that maximize efficacy and minimize toxicity of the antibody-CPT conjugates for therapeutic use in human patients.
  • CPT may refer to camptothecin or any of its derivatives, such as SN-38, unless expressly stated otherwise.
  • the present invention resolves an unfulfilled need in the art by providing improved methods and compositions for preparing and administering CPT-antibody immunoconjugates.
  • the camptothecin is SN-38.
  • the disclosed methods and compositions are of use for the treatment of a variety of diseases and conditions which are refractory or less responsive to other forms of therapy, and can include diseases against which suitable antibodies or antigen-binding antibody fragments for selective targeting can be developed, or are available or known.
  • the targeting moiety is an antibody, antibody fragment, bispecific or other multivalent antibody, or other antibody -based molecule or compound.
  • the antibody can be of various isotypes, preferably human IgGl, IgG2, IgG3 or IgG4, more preferably comprising human IgGl hinge and constant region sequences.
  • the antibody or fragment thereof can be a chimeric human-mouse, a chimeric human-primate, a humanized (human framework and murine hypervariable (CDR) regions), or fully human antibody, as well as variations thereof, such as half-IgG4 antibodies (referred to as "unibodies"), as described by van der Neut Kolfschoten et al. ⁇ Science 2007; 317: 1554-1557). More preferably, the antibody or fragment thereof may be designed or selected to comprise human constant region sequences that belong to specific allotypes, which may result in reduced immunogenicity when the immunoconjugate is administered to a human subject.
  • Preferred allotypes for administration include a non-Glml allotype (nGlml), such as Glm3, Glm3, l, Glm3,2 or Glm3,l,2. More preferably, the allotype is selected from the group consisting of the nGlml, Glm3, nGlml, 2 and Km3 allotypes.
  • nGlml non-Glml allotype
  • Antibodies of use may bind to any disease-associated antigen known in the art.
  • the disease state is cancer
  • many antigens expressed by or otherwise associated with tumor cells are known in the art, including but not limited to, carbonic anhydrase IX, alpha-fetoprotein (AFP), a-actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCL19, CCL21, CD1, CDla, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70
  • the antibody binds to CEACAM5, CEACAM6, Trop-2, AFP, MUC5ac, CD74, CD19, CD20, CD22 or HLA-DR. Most preferably, the antibody binds to Trop-2.
  • anti-cancer antibodies that may be utilized include, but are not limited to, hRl (anti-IGF-lR, U.S. Patent No. 9,441,043), hPAM4 (anti-MUC-5ac, U.S. Patent No. 7,282,567), hA20 (anti-CD20, U.S. Patent No. 7, 151,164), hA19 (anti-CD19, U.S. Patent No. 7, 109,304), hFMMU31 (anti-AFP, U.S. Patent No. 7,300,655), hLLl (anti-CD74, U.S. Patent No. 7,312,318), hLL2 (anti-CD22, U.S. Patent No.
  • hMu-9 anti-CSAp, U.S. Patent No. 7,387,772
  • hL243 anti-HLA-DR, U.S. Patent No. 7,612,180
  • hMN-14 anti- CEACAM5, U.S. Patent No. 6,676,924
  • hMN-15 anti-CEACAM6, U.S. Patent No.
  • the antibody is IMMU-31 (anti-AFP), hRS7 (anti-Trop-2), hMN-14 (anti- CEACAM5), hMN-3 (anti-CEACAM6), hMN-15 (anti-CEACAM6), hLLl (anti-CD74), hLL2 (anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20 (anti- CD20).
  • the antibody is hRS7 (anti-Trop-22).
  • Alternative antibodies of use include, but are not limited to, abciximab (anti- glycoprotein Ilb/IIIa), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab (anti-CD20), panitumumab (anti- EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti- CTLA-4), abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA), abc
  • the chemotherapeutic moiety is selected from
  • camptothecin CPT and its analogs and derivatives and is more preferably SN-38.
  • chemotherapeutic moieties include taxanes (e.g, baccatin III, taxol), epothilones, anthracyclines (e.g., doxorubicin (DOX), epirubicin, morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2- pyrrolinodoxorubicin (2-PDOX) or a prodrug form of 2-PDOX (pro-2-PDOX); see, e.g., Priebe W (ed.), ACS symposium series 574, published by American Chemical Society, Washington D.C., 1995 (332pp) and Nagy et al, Proc.
  • the antibody or fragment thereof links to at least one chemotherapeutic moiety; preferably 1 to about 5 chemotherapeutic moieties; more preferably 6 or more chemotherapeutic moieties, most preferably about 6 to about 12 chemotherapeutic moieties.
  • CPT-11 An example of a water soluble CPT derivative is CPT-11.
  • Extensive clinical data are available concerning CPT-l l's pharmacology and its in vivo conversion to the active SN-38 (Iyer and Ratain, Cancer Chemother Pharmacol. 42: S31-43 (1998); Mathijssen et al, Clin Cancer Res. 7:2182-2194 (2002); Rivory, Ann NY Acad Sci. 922:205-215, 2000)).
  • the active form SN-38 is about 2 to 3 orders of magnitude more potent than CPT-11.
  • the immunoconjugate may be an hMN-14-SN-38, hMN-3- SN-38, hMN-15-SN-38, IMMU-31 -SN-38, hRS7-SN-38, hA20-SN-38, hL243-SN-38, hLLl-SN-38 or hLL2-SN-38 conjugate.
  • Various embodiments may concern use of the subject methods and compositions to treat a cancer, including but not limited to non-Hodgkin's lymphomas, B-cell acute and chronic lymphoid leukemias, Burkitt lymphoma, Hodgkin's lymphoma, acute large B-cell lymphoma, hairy cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, T-cell lymphomas and leukemias, multiple myeloma, Waldenstrom's macroglobulinemia, carcinomas, melanomas, sarcomas, gliomas, bone, and skin cancers.
  • the carcinomas may include carcinomas of the oral cavity, esophagus, gastrointestinal tract, pulmonary tract, lung, stomach, colon, breast, ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas, bone, brain, connective tissue, liver, gall bladder, urinary bladder, kidney, skin, central nervous system and testes.
  • the cancer is urothelial cancer, more preferably metastatic urothelial cancer, most preferably metastatic urothelial cancer that is relapsed from or refractory to standard anticancer therapy, such as treatment with chemotherapeutic drugs.
  • the ADCs may be used in combination with surgery, radiation therapy, chemotherapy, immunotherapy with naked antibodies, radioimmunotherapy, immunomodulators, vaccines, and the like.
  • combination therapies can allow lower doses of each therapeutic to be given in such combinations, thus reducing certain severe side effects, and potentially reducing the courses of therapy required. When there is no or minimal overlapping toxicity, full doses of each can also be given.
  • combination therapy with antibody-SN38 immunoconjugates and microtubule inhibitors or PARP inhibitors shows unexpected synergistic effects.
  • Preferred optimal dosing of ADCs may include a dosage of between 3 mg/kg and 18 mg/kg, preferably given either weekly, twice weekly or every other week.
  • the optimal dosing schedule may include treatment cycles of two consecutive weeks of therapy followed by one, two, three or four weeks of rest, or alternating weeks of therapy and rest, or one week of therapy followed by two, three or four weeks of rest, or three weeks of therapy followed by one, two, three or four weeks of rest, or four weeks of therapy followed by one, two, three or four weeks of rest, or five weeks of therapy followed by one, two, three, four or five weeks of rest, or administration once every two weeks, once every three weeks or once a month.
  • Treatment may be extended for any number of cycles, preferably at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, or at least 16 cycles.
  • the dosage may be up to 24 mg/kg.
  • Exemplary dosages of use may include 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, and 18 mg/kg.
  • Preferred dosages are 4, 6, 8, 9, 10, 12, 14, 16 or 18 mg/kg.
  • the person of ordinary skill will realize that a variety of factors, such as age, general health, specific organ function or weight, as well as effects of prior therapy on specific organ systems (e.g., bone marrow) may be considered in selecting an optimal dosage of immunoconjugate, and that the dosage and/or frequency of administration may be increased or decreased during the course of therapy.
  • the dosage may be repeated as needed, with evidence of tumor shrinkage observed after as few as 4 to 8 doses.
  • the optimized dosages and schedules of administration disclosed herein show unexpected superior efficacy and reduced toxicity in human subjects, which could not have been predicted from animal model studies. Surprisingly, the superior efficacy allows treatment of tumors that were previously found to be resistant to one or more standard anti-cancer therapies, including the parental compound, CPT-11, from which SN-38 is derived in vivo.
  • the subject methods may include use of CT and/or PET/CT, or MRI, to measure tumor response at regular intervals.
  • CA19-9 (carcinoembryonic antigen), CA19-9, AFP, CA 15.3, or PSA, may also be monitored.
  • Dosages and/or administration schedules may be adjusted as needed, according to the results of imaging and/or marker blood levels.
  • a surprising result with the instant claimed compositions and methods is the unexpected tolerability of high doses of antibody-drug conjugate, even with repeated infusions, with only relatively low-grade toxicities of nausea and vomiting observed, or manageable neutropenia.
  • a further surprising result is the lack of accumulation of the antibody-drug conjugate, unlike other products that have conjugated SN-38 to albumin, PEG or other carriers. The lack of accumulation is associated with improved tolerability and lack of serious toxicity even after repeated or increased dosing.
  • the claimed methods provide for shrinkage of solid tumors, in individuals with previously resistant cancers, of 15% or more, preferably 20% or more, preferably 30% or more, more preferably 40% or more in size (as measured by longest diameter).
  • tumor size may be measured by a variety of different techniques, such as total tumor volume, maximal tumor size in any dimension or a combination of size measurements in several dimensions. This may be with standard radiological procedures, such as computed tomography, ultrasonography, and/or positron- emission tomography.
  • the means of measuring size is less important than observing a trend of decreasing tumor size with immunoconjugate treatment, preferably resulting in elimination of the tumor.
  • the immunoconjugate may be administered as a periodic bolus injection, in alternative embodiments the immunoconjugate may be administered by continuous infusion of antibody-drug conjugates.
  • a continuous infusion may be administered for example by indwelling catheter.
  • indwelling catheter Such devices are known in the art, such as HICKMAN®, BROVIAC® or PORT-A-C ATH® catheters (see, e.g., Skolnik et al., Ther Drug Monit 32:741-48, 2010) and any such known indwelling catheter may be used.
  • a variety of continuous infusion pumps are also known in the art and any such known infusion pump may be used.
  • the dosage range for continuous infusion may be between 0.1 and 3.0 mg/kg per day. More preferably, these immunoconjugates can be administered by intravenous infusions over relatively short periods of 2 to 5 hours, more preferably 2-3 hours.
  • the immunoconjugates and dosing schedules may be efficacious in patients resistant to standard therapies.
  • an anti-Trop-2 hRS7-SN-38 immunoconjugate may be administered to a patient who has not responded to prior therapy with irinotecan, the parent agent of SN-38.
  • the irinotecan-resistant patient may show a partial or even a complete response to hRS7-SN-38.
  • the ability of the immunoconjugate to specifically target the tumor tissue may overcome tumor resistance by improved targeting and enhanced delivery of the therapeutic agent.
  • the ADC may also be efficacious to treat cancers resistant to other therapeutic agents, such as platinum-based anticancer agents.
  • a specific preferred subject may be a metastatic colon cancer patient, a triple- negative breast cancer patient, a HER+, ER+, progesterone+ breast cancer patient, a metastatic non-small-cell lung cancer (NSCLC) patient, a metastatic pancreatic cancer patient, a metastatic renal cell carcinoma patient, a metastatic gastric cancer patient, a metastatic prostate cancer patient, a metastatic urothelial cancer patient or a metastatic small- cell lung cancer patient.
  • NSCLC metastatic non-small-cell lung cancer
  • an antibody or immunoconjugate such as sacituzumab govitecan
  • at least one microtubule inhibitor may be used in combination therapy with at least one microtubule inhibitor.
  • microtubule inhibitors are known in the art, such as vinca alkaloids (e.g., vincristine, vinblastine), taxanes (e.g., paclitaxel), maytansinoids (e.g., mertansine) and auristatins.
  • Other known microtubule inhibitors include demecolcine, nocodazole, epothilone, docetaxel, discodermolide, colchicine, combrestatin, podophyllotoxin, CI-980,
  • microtubule inhibitor may be used in combination with an antibody or antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the microtubule inhibitor is one that exhibits synergistic effects when used in combination with an antibody or ADC.
  • One potent example is SN-38- conjugated antibody, such as sacituzumab govitecan or labetuzumab govitecan (targeting CEACAM5) expressed by many solid cancers.
  • the microtubule inhibitor is paclitaxel or eribulin mesylate.
  • the antibody or ADC may be used in combination therapy with at least one PARP inhibitor.
  • PARP inhibitors are known in the art, such as olaparib, talazoparib (BMN-673), rucaparib, veliparib, niraparib, iniparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699, BSI-201, CEP-8983 and 3-aminobenzamide (see, e.g., Rouleau et al., 2010, Nat Rev Cancer 10:293-301, Bao et al., 2015, Oncotarget [Epub ahead of print, September 22, 2015]).
  • any such known PARP inhibitor may be used in combination with an antibody or ADC, such as, for example, an SN-38-antibody conjugate.
  • the PARP inhibitor is one that exhibits synergistic effects when used in combination with the antibody or ADC. This has been validated when using an SN-38- conjugated antibody, such as sacituzumab govitecan.
  • the PARP inhibitor is olaparib or rucaparib.
  • an antibody or immunoconjugate may be used in combination with a Bruton kinase inhibitor or PI3K inhibitor.
  • a Bruton kinase inhibitor or PI3K inhibitor include, but are not limited to, ibrutinib (PCI-32765), PCI-45292, CC-292 (AVL- 292), ONO-4059, GDC-0834, LFM-A13 or RN486.
  • exemplary PI3K inhibitors include, but are not limited to, idelalisib, Wortmannin, demethoxyviridin, perifosine, PX-866, IPI-145
  • FIG. 2A Assessment of target lesions on computed tomography (CT) scans using response evaluation criteria in solid tumors, version 1.1, in patient (Pt) 6 before and after sacituzumab govitecan treatment.
  • CT computed tomography
  • Patient 6 initially presented with 4 target lesions (2 Liver, 1 Sigmoid Colon, 1 Peritoneum of Pelvis), with a sum of the largest diameters of 138 mm. Additional nontarget lesions were present in the liver and lymph node in the pelvis.
  • Treatment was initiated at a dose level of 8 mg/kg.
  • White arrows highlight target lesions 1-3 in axial slices obtained at baseline.
  • Axial slices of the same region 6 months later after 9 cycles of sacituzumab govitecan treatment demonstrated reduction in sum diameter of target lesions to 86 mm (-38%) and stable disease in nontarget lesions.
  • FIG. 3 IMMU-132 phase I/II data for best response by RECIST criteria.
  • FIG. 4 Responses in 52 human TNBC patients treated with 10 mg/kg IMMU-132, after failing numerous prior therapies.
  • FIG. 5 Progression-free survival in TNBC patients treated with 10 mg/kg IMMU- 132.
  • FIG. 6 Best response in 29 assessable human NSCLC patients treated with 8 to 10 mg/kg IMMU-132.
  • FIG. 7 Time to progression in NSCLC patients treated with 8- 10 mg/kg IMMU- 132.
  • FIG. 8 Progression-free survival in NSCLC patients treated with 8 or 10 mg/kg IMMU-132.
  • FIG. 9A Tumor growth inhibition of combined IMMU-132 and Olaparib in TNBC: BRCAl/2 and PTEN defective tumors.
  • Tumor-bearing mice (TV-0.3 cm 3 ) were treated with Olaparib (1 mg; -50 mg/kg, i.p. on a M-F schedule; red arrows) or IMMU-132 (i.v. weekly, black arrows).
  • a non-tumor-targeting anti-CD20 SN-38-ADC was used as a control.
  • HCC1806 is a BRCAl/2-defechve TNBC tumor line. Olaparib alone had no significant antitumor effects. IMMU-132 alone significantly inhibited tumor growth compared to all control groups (PO.0106, AUC). EVIMU-132 plus olaparib further improved anti-tumor responses significantly compared to all groups (PO.0019; AUC). Mice in the combination group have yet to reach median survival (>80.5 days) which is more than 2- and 4-fold longer than IMMU-132 or olaparib monotherapy, respectively (. ⁇ 0.0083).
  • FIG. 9B Tumor growth inhibition of combined IMMU-132 and Olaparib in TNBC: BRCAl/2 and PTEN defective tumors.
  • Tumor-bearing mice (TV-0.3 cm 3 ) were treated with Olaparib (1 mg; -50 mg/kg, i.p. on a M-F schedule; light arrows) or IMMU-132 (i.v. weekly, dark arrows).
  • a non-tumor-targeting anti-CD20 SN-38-ADC was used as a control.
  • IMMU-132 alone had significant anti-tumor effects compared to all control groups (P ⁇ 0.0098; AUC).
  • an antibody refers to a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecule ⁇ e.g., an IgG antibody) or an antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment.
  • An antibody or antibody fragment may be conjugated or otherwise derivatized within the scope of the claimed subject matter.
  • Such antibodies include but are not limited to IgGl, IgG2, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes.
  • MAb may be used interchangeably to refer to an antibody, antibody fragment, monoclonal antibody or multispecific antibody.
  • An antibody fragment is a portion of an antibody such as F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv (single chain Fv), single domain antibodies (DABs or VHHs) and the like, including the half-molecules of IgG4 cited above (van der Neut Kolfschoten et al. (Science 2007;
  • an antibody fragment of use binds with the same antigen that is recognized by the intact antibody.
  • the term "antibody fragment” also includes synthetic or genetically engineered proteins that act like an antibody by binding to a specific antigen to form a complex.
  • antibody fragments include isolated fragments consisting of the variable regions, such as the "Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins").
  • the fragments may be constructed in different ways to yield multivalent and/or multispecific binding forms.
  • a naked antibody is generally an entire antibody that is not conjugated to a therapeutic agent.
  • a naked antibody may exhibit therapeutic and/or cytotoxic effects, for example by Fc-dependent functions, such as complement fixation (CDC) and ADCC
  • Naked antibodies include polyclonal and monoclonal antibodies, naturally occurring or recombinant antibodies, such as chimeric, humanized or human antibodies and fragments thereof. In some cases a “naked antibody” may also refer to a “naked” antibody fragment. As defined herein, “naked” is synonymous with “unconjugated,” and means not linked or conjugated to a therapeutic agent.
  • a chimeric antibody is a recombinant protein that contains the variable domains of both the heavy and light antibody chains, including the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, more preferably a murine antibody, while the constant domains of the antibody molecule are derived from those of a human antibody.
  • CDRs complementarity determining regions
  • the constant domains of the chimeric antibody may be derived from that of other species, such as a primate, cat or dog.
  • a humanized antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a murine antibody, are transferred from the heavy and light variable chains of the murine antibody into human heavy and light variable domains (framework regions).
  • the constant domains of the antibody molecule are derived from those of a human antibody.
  • specific residues of the framework region of the humanized antibody particularly those that are touching or close to the CDR sequences, may be modified, for example replaced with the corresponding residues from the original murine, rodent, subhuman primate, or other antibody.
  • a human antibody is an antibody obtained, for example, from transgenic mice that have been "engineered” to produce human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for various antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al, Nature Genet. 7: 13 (1994), Lonberg et al, Nature 3(55:856 (1994), and Taylor et al, Int. Immun. 6:579 (1994).
  • a fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art. See for example, McCafferty et al, Nature 348:552-553 (1990) for the production of human antibodies and fragments thereof in vitro, from immunoglobulin variable domain gene repertoires from unimmunized donors.
  • human antibody variable domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, and displayed as functional antibody fragments on the surface of the phage particle.
  • the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of the B cell.
  • Phage display can be performed in a variety of formats, for their review, see e.g. Johnson and Chiswell, Current Opinion in Structural Biology 3 :5564-571 (1993).
  • Human antibodies may also be generated by in vitro activated B cells. See U.S. Patent Nos. 5,567,610 and 5,229,275, the Examples section of each of which is incorporated herein by reference.
  • a therapeutic agent is an atom, molecule, or compound that is useful in the treatment of a disease.
  • therapeutic agents include, but are not limited to, antibodies, antibody fragments, immunoconjugates, drugs, cytotoxic agents, pro-apopoptotic agents, toxins, nucleases (including DNAses and RNAses), hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes, radionuclides, oligonucleotides, interference RNA, siRNA, RNAi, anti-angiogenic agents, chemotherapeutic agents, cyokines,
  • chemokines prodrugs, enzymes, binding proteins or peptides or combinations thereof.
  • An immunoconjugate is an antibody, antigen-binding antibody fragment, antibody complex or antibody fusion protein that is conjugated to a therapeutic agent. Conjugation may be covalent or non-covalent. Preferably, conjugation is covalent.
  • a particular form of immunoconjugate, in which the antibody component is conjugated to a drug, is referred to herein as an antibody-drug conjugate (ADC).
  • the term antibody fusion protein is a recombinantly-produced antigen- binding molecule in which one or more natural antibodies, single-chain antibodies or antibody fragments are linked to another moiety, such as a protein or peptide, a toxin, a cytokine, a hormone, etc.
  • the fusion protein may comprise two or more of the same or different antibodies, antibody fragments or single-chain antibodies fused together, which may bind to the same epitope, different epitopes on the same antigen, or different antigens.
  • An immunomodulator is a therapeutic agent that when present, alters, suppresses or stimulates the body's immune system.
  • an immunomodulator of use stimulates immune cells to proliferate or become activated in an immune response cascade, such as macrophages, dendritic cells, B-cells, and/or T-cells.
  • an immune response cascade such as macrophages, dendritic cells, B-cells, and/or T-cells.
  • an immunomodulator may suppress proliferation or activation of immune cells.
  • An example of an immunomodulator as described herein is a cytokine, which is a soluble small protein of approximately 5-20 kDa that is released by one cell population (e.g., primed T-lymphocytes) on contact with specific antigens, and which acts as an intercellular mediator between cells.
  • cytokines include lymphokines, monokines, interleukins, and several related signaling molecules, such as tumor necrosis factor (TNF) and interferons.
  • TNF tumor necrosis factor
  • Chemokines are a subset of cytokines.
  • Certain interleukins and interferons are examples of cytokines that stimulate T cell or other immune cell proliferation.
  • Exemplary interferons include interferon-a, interferon- ⁇ , interferon- ⁇ and interferon- ⁇ .
  • CPT is an abbreviation for camptothecin, and as used in the present application CPT represents camptothecin itself or an analog or derivative of camptothecin, such as SN-38.
  • Anti-Trop-2 Antibodies are an abbreviation for camptothecin, and as used in the present application CPT represents camptothecin itself or an analog or derivative of camptothecin, such as SN-38.
  • the subject ADCs include at least one antibody or fragment thereof that binds to Trop-2.
  • the anti-Trop-2 antibody may be a humanized RS7 antibody (see, e.g., U.S. Patent No. 7,238,785, incorporated herein by reference in its entirety), comprising the light chain CDR sequences CDR1
  • the RS7 antibody was a murine IgGi raised against a crude membrane preparation of a human primary squamous cell lung carcinoma. (Stein et al., Cancer Res. 50: 1330, 1990) The RS7 antibody recognizes a 46-48 kDa glycoprotein, characterized as cluster 13. (Stein et al., Int. J. Cancer Supp. 8:98-102, 1994) The antigen was designated as EGP-1 (epithelial glycoprotein- 1), but is also referred to as Trop-2.
  • Trop-2 is a type-I transmembrane protein and has been cloned from both human (Fornaro et al., Int J Cancer 1995; 62:610-8) and mouse cells (Sewedy et al., Int J Cancer 1998; 75:324-30).
  • human Trop-2 In addition to its role as a tumor-associated calcium signal transducer (Ripani et al., Int J Cancer 1998;76:671-6), the expression of human Trop-2 was shown to be necessary for tumorigenesis and invasiveness of colon cancer cells, which could be effectively reduced with a polyclonal antibody against the extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008;7:280-5).
  • RS7 MAb detects antigen on a variety of tumor types, with limited binding to normal human tissue (Stein et al., 1990).
  • Trop-2 is expressed primarily by carcinomas such as carcinomas of the lung, stomach, urinary bladder, breast, ovary, uterus, and prostate.
  • Localization and therapy studies using radiolabeled murine RS7 MAb in animal models have demonstrated tumor targeting and therapeutic efficacy (Stein et al., 1990; Stein et al., 1991). Strong RS7 staining has been demonstrated in tumors from the lung, breast, bladder, ovary, uterus, stomach, and prostate (Stein et al., Int. J.
  • the lung cancer cases comprised both squamous cell carcinomas and adenocarcinomas (Stein et al., Int. J. Cancer 55:938, 1993). Both cell types stained strongly, indicating that the RS7 antibody does not distinguish between histologic classes of non-small-cell carcinoma of the lung.
  • the RS7 MAb is rapidly internalized into target cells (Stein et al., 1993).
  • the internalization rate constant for RS7 MAb is intermediate between the internalization rate constants of two other rapidly internalizing MAbs, which have been demonstrated to be useful for immunoconjugate production. ⁇ Id.)
  • hRS7 antibody is preferred
  • other anti-Trop-2 antibodies are known and/or publicly available and in alternative embodiments may be utilized in the subject ADCs.
  • humanized or human antibodies are preferred for reduced immunogenicity, in alternative embodiments a chimeric antibody may be of use.
  • methods of antibody humanization are well known in the art and may be utilized to convert an available murine or chimeric antibody into a humanized form.
  • Anti-Trop-2 antibodies are commercially available from a number of sources and include LS-C126418, LS-C178765, LS-C126416, LS-C126417 (LifeSpan Biosciences, Inc., Seattle, WA); 10428-MMOl, 10428-MM02, 10428-ROOl, 10428-R030 (Sino Biological Inc., Beijing, China); MR54 (eBioscience, San Diego, CA); sc-376181, sc-376746, Santa Cruz Biotechnology (Santa Cruz, CA); MM0588-49D6, (Novus Biologicals, Littleton, CO);
  • U.S. Patent No. 7,420,041 disclosed an anti-Trop-2 antibody produced by hybridoma cell line AR52A301.5, deposited with the IDAC as accession number 141205-03.
  • U.S. Publ. No. 2013/0122020 disclosed anti-Trop-2 antibodies 3E9, 6G11, 7E6, 15E2, 18B1. Hybridomas encoding a representative antibody were deposited with the American Type Culture Collection (ATCC), Accession Nos. PTA-12871 and PTA-12872.
  • Immunoconjugate PF 06263507 comprising an anti-5T4 (anti-Trop-2) antibody attached to the tubulin inhibitor monomethylauristatin F (MMAF) is available from Pfizer, Inc. (Groton, CT) (see, e.g., Sapra et al., 2013, Mol Cancer Ther 12:38-47).
  • U.S. Patent No. 8,715,662 discloses anti-Trop-2 antibodies produced by hybridomas deposited at the AID-ICLC (Genoa, Italy) with deposit numbers PD 08019, PD 08020 and PD 08021.
  • U.S. Patent Application Publ. No. 20120237518 discloses anti-Trop-2 antibodies 77220, KM4097 and KM4590.
  • Patent No. 8,309,094 discloses antibodies Al and A3, identified by sequence listing. The Examples section of each patent or patent application cited above in this paragraph is incorporated herein by reference.
  • Numerous anti-Trop-2 antibodies are known in the art and/or publicly available. As discussed below, methods for preparing antibodies against known antigens were routine in the art.
  • the sequence of the human Trop-2 protein was also known in the art (see, e.g., GenBank Accession No. CAA54801.1). Methods for producing humanized, human or chimeric antibodies were also known. The person of ordinary skill, reading the instant disclosure in light of general knowledge in the art, would have been able to make and use the genus of anti-Trop-2 antibodies in the subject ADCs.
  • Non-limiting methods and compositions for preparing immunoconjugates comprising a camptothecin therapeutic agent attached to an antibody or antigen-binding antibody fragment are described below.
  • the solubility of the drug is enhanced by placing a defined polyethyleneglycol (PEG) moiety (i.e., a PEG containing a defined number of monomeric units) between the drug and the antibody, wherein the defined PEG is a low molecular weight PEG, preferably containing 1-30 monomeric units, more preferably containing 1-12 monomeric units, most preferably containing 6-8 monomeric units.
  • PEG polyethyleneglycol
  • a first linker connects the drug at one end and may terminate with an acetylene or an azide group at the other end.
  • This first linker may comprise a defined PEG moiety with an azide or acetylene group at one end and a different reactive group, such as carboxylic acid or hydroxyl group, at the other end.
  • Said bifunctional defined PEG may be attached to the amine group of an amino alcohol, and the hydroxyl group of the latter may be attached to the hydroxyl group on the drug in the form of a carbonate.
  • non- azide(or acetylene) moiety of said defined bifunctional PEG is optionally attached to the N- terminus of an L-amino acid or a polypeptide, with the C-terminus attached to the amino group of amino alcohol, and the hydroxy group of the latter is attached to the hydroxyl group of the drug in the form of carbonate or carbamate, respectively.
  • a second linker comprising an antibody-coupling group and a reactive group complementary to the azide (or acetylene) group of the first linker, namely acetylene (or azide), may react with the drug-(first linker) conjugate via acetylene-azide cycloaddition reaction to furnish a final bifunctional drug product that is useful for conjugating to disease- targeting antibodies.
  • the antibody-coupling group is preferably either a thiol or a thiol- reactive group.
  • bifunctional CPT is conjugated to an antibody without prior deprotection of this protecting group.
  • the protecting group is readily deprotected under physiological pH conditions after the bioconjugate is administered.
  • the azide part may be on L2 with the acetylene part on L3.
  • L2 may contain acetylene, with L3 containing azide.
  • 'Click chemistry' refers to a copper (+l)-catalyzed cycloaddition reaction between an acetylene moiety and an azide moiety (Kolb HC and Sharpless KB, DrugDiscov Today 2003; 8: 1128-37), although alternative forms of click chemistry are known and may be used. Click chemistry takes place in aqueous solution at near-neutral pH conditions, and is thus amenable for drug conjugation. The advantage of click chemistry is that it is a copper (+l)-catalyzed cycloaddition reaction between an acetylene moiety and an azide moiety (Kolb HC and Sharpless KB, DrugDiscov Today 2003; 8: 1128-37), although alternative forms of click chemistry are known and may be used. Click chemistry takes place in aqueous solution at
  • An exemplary preferred embodiment is directed to a conjugate of a drug derivative and an antibody of the general formula (1) shown below.
  • MAb is a disease-targeting antibody
  • L2 is a component of the cross-linker comprising an antibody-coupling moiety and one or more of acetylene (or azide) groups
  • LI comprises a defined PEG with azide (or acetylene) at one end, complementary to the acetylene (or azide) moiety in L2, and a reactive group such as carboxylic acid or hydroxyl group at the other end
  • AA is an L-amino acid
  • m is an integer with values of 0, 1, 2, 3, or 4
  • A' is an additional spacer, selected from the group of ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol, or substituted or unsubstituted ethylenediamine.
  • the L amino acids of ' AA' are selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. If the A' group contains hydroxyl, it is linked to the hydroxyl group or amino group of the drug in the form of a carbonate or carbamate, respectively.
  • A' is a substituted ethanolamine derived from an L-amino acid, wherein the carboxylic acid group of the amino acid is replaced by a hydroxymethyl moiety.
  • A' may be derived from any one of the following L-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • m is 0, A' is L-valinol, and the drug is exemplified by SN-38.
  • m is 1 and represented by a derivatized L-lysine, A' is L-valinol, and the drug is exemplified by SN- 38.
  • an amide bond is first formed between the carboxylic acid of an amino acid such as lysine and the amino group of valinol, using orthogonal protecting groups for the lysine amino groups.
  • the protecting group on the N-terminus of lysine is removed, keeping the protecting group on the side chain of lysine intact, and the N-terminus is coupled to the carboxyl group on the defined PEG with azide (or acetylene) at the other end.
  • the hydroxyl group of valinol is then attached to the 20-chloroformate derivative of 10-hydroxy- protected SN-38, and this intermediate is coupled to an L2 component carrying the antibody- binding moiety as well as the complementary acetylene (or azide) group involved in the click cycloaddition chemistry.
  • removal of protecting groups at both lysine side chain and SN-38 gives the product of this example.
  • the small MW SN-38 product namely valinol-SN-38 carbonate, generated after intracellular proteolysis, has the additional pathway of liberation of intact SN-38 through intramolecular cyclization involving the amino group of valinol and the carbonyl of the carbonate.
  • A' of the general formula 1 is A-OH, whereby A- OH is a collapsible moiety such as 4-aminobenzyl alcohol or a substituted 4-aminobenzyl alcohol substituted with a Ci-Cio alkyl group at the benzylic position, and the latter, via its amino group, is attached to an L-amino acid or a polypeptide comprising up to four L-amino acid moieties; wherein the N-terminus is attached to a cross-linker terminating in the antibody-binding group.
  • A- OH is a collapsible moiety such as 4-aminobenzyl alcohol or a substituted 4-aminobenzyl alcohol substituted with a Ci-Cio alkyl group at the benzylic position, and the latter, via its amino group, is attached to an L-amino acid or a polypeptide comprising up to four L-amino acid moieties; wherein the N-terminus is attached to a cross-
  • Single amino acid of AA may be selected from any one of the following L-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • the substituent R on 4-aminobenzyl alcohol moiety is hydrogen or an alkyl group selected from C1-C10 alkyl groups.
  • the structure differs from the linker MAb-CL2-SN-38 in the substitution of a single lysine residue for a Phe-Lys dipeptide found in the CL2 linker.
  • the Phe-Lys dipeptide was designed as a cathepsin B cleavage site for lysosomal enzyme, which was considered to be important for intracellular release of bound drug.
  • immunoconjugates comprising a CL2A linker are apparently more efficacious in vivo than those comprising a CL2 linker.
  • AA comprises a polypeptide moiety, preferably a di, tri or tetrapeptide, that is cleavable by intracellular peptidase.
  • polypeptide moiety preferably a di, tri or tetrapeptide, that is cleavable by intracellular peptidase. Examples are: Ala-Leu, Leu-Ala- Leu, and Ala-Leu- Ala-Leu (SEQ ID NO: 7) (Trouet et al., 1982).
  • the LI component of the conjugate contains a defined polyethyleneglycol (PEG) spacer with 1-30 repeating monomeric units.
  • PEG polyethyleneglycol
  • PEG is a defined PEG with 1-12 repeating monomeric units.
  • the introduction of PEG may involve using heterobifunctionalized PEG derivatives which are available commercially.
  • the heterobifunctional PEG may contain an azide or acetylene group.
  • L2 has a plurality of acetylene (or azide) groups, ranging from 2-40, but preferably 2-20, and more preferably 2-5, and a single antibody-binding moiety.
  • the 'L2' component is appended to 2 acetylenic groups, resulting in the attachment of two azide-appended SN-38 molecules.
  • the bonding to MAb may involve a succinimide.
  • the thiols on the antibody are generated on the lysine groups of the antibody using a thiolating reagent.
  • Methods for introducing thiol groups onto antibodies by modifications of MAb's lysine groups are well known in the art (Wong in Chemistry of protein conjugation and cross-linking, CRC Press, Inc., Boca Raton, FL (1991), pp 20-22).
  • mild reduction of interchain disulfide bonds on the antibody (Willner et al., Bioconjugate Chem.
  • reducing agents such as dithiothreitol (DTT)
  • DTT dithiothreitol
  • attachment of SN-38 to reduced disulfide sulfhydryl groups results in formation of an antibody-SN-38 immunoconjugate with 6 SN-38 moieties covalently attached per antibody molecule.
  • cysteine residues for attachment of drugs or other therapeutic agents are known, such as the use of cysteine engineered antibodies (see U.S. Patent No. 7,521,541, the Examples section of which is incorporated herein by reference.)
  • the chemotherapeutic moiety is selected from the group consisting of doxorubicin (DOX), epirubicin, morpholinodoxorubicin (morpholino- DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2- PDOX), Pro-2PDOX, CPT, 10-hydroxy camptothecin, SN-38, topotecan, lurtotecan, 9- aminocamptothecin, 9-nitrocamptothecin, taxanes, geldanamycin, ansamycins, and epothilones.
  • the chemotherapeutic moiety is SN-38.
  • the antibody links to at least one chemotherapeutic moiety; preferably 1 to about 12 chemotherapeutic moieties; most preferably about 6 to about 12 chemotherapeutic moieties.
  • the linker component 'L2' comprises a thiol group that reacts with a thiol -reactive residue introduced at one or more lysine side chain amino groups of said antibody.
  • the antibody is pre-derivatized with a thiol- reactive group such as a maleimide, vinylsulfone, bromoacetamide, or iodoacetamide by procedures well described in the art.
  • CPT drug-linkers can be prepared wherein CPT additionally has a 10-hydroxyl group.
  • This process involves, but is not limited to, the protection of the 10-hydroxyl group as a t- butyloxycarbonyl (BOC) derivative, followed by the preparation of the penultimate intermediate of the drug-linker conjugate.
  • BOC t- butyloxycarbonyl
  • removal of the BOC group requires treatment with strong acid such as trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the CPT 20-O-linker carbonate, containing protecting groups to be removed is also susceptible to cleavage, thereby giving rise to unmodified CPT.
  • An alternative approach involves protecting the CPT analog's 10-hydroxy position with a group other than 'BOC, such that the the final product is ready for conjugation to antibodies without a need for deprotecting the 10-OH protecting group.
  • the 10-hydroxy protecting group which converts the 10-OH into a phenolic carbonate or a phenolic ester, is readily deprotected by physiological pH conditions or by esterases after in vivo
  • a 10-hydroxy protecting group on SN-38 can be 'COR' where R can be a substituted alkyl such as "N(CH 3 ) 2 -(CH 2 ) n -" where n is 1-10 and wherein the terminal amino group is optionally in the form of a quaternary salt for enhanced aqueous solubility, or a simple alkyl residue such as "CH 3 -(CH 2 ) n -" where n is 0-10, or it can be an alkoxy moiety such as "CH 3 -(CH 2 )n-0-" where n is 0-10, or "N(CH 3 ) 2 -(CH 2 ) n -0-" where n is 2-10, or "RiO-(CH 2 -CH 2 -0) n -CH 2 -CH 2 -0-" where Ri is ethyl or methyl and n is an integer with values of 0-10.
  • 10-hydroxy derivatives are readily prepared by treatment with the chloroformate of the chosen reagent, if the final derivative is to be a carbonate.
  • the 10-hydroxy-containing camptothecin such as SN-38 is treated with a molar equivalent of the chloroformate in dimethylformamide using triethylamine as the base. Under these conditions, the 20-OH position is unaffected.
  • the acid chloride of the chosen reagent is used.
  • cross-linker [Ll]-[AA] m -[A-OH]
  • the cross-linker [Ll]-[AA] m -[A-OH]
  • the antibody is a monoclonal antibody (MAb).
  • the antibody may be a multivalent and/or multispecific MAb.
  • the antibody may be a murine, chimeric, humanized, or human monoclonal antibody, and said antibody may be in intact, fragment (Fab, Fab', F(ab) 2 , F(ab') 2 ), or sub-fragment (single-chain constructs) form, or of an IgGl, IgG2a, IgG3, IgG4, IgA isotype, or submolecules therefrom.
  • the antibody binds to an antigen or epitope of an antigen expressed on a cancer or malignant cell.
  • the cancer cell is preferably a cell from a hematopoietic tumor, carcinoma, sarcoma, melanoma or a glial tumor.
  • the antibody moiety is an anti-Trop-2 and the anti-Trop-2-SN-38 ADC is of use to treat any Trop-2-expressing cancer.
  • the intracellularly- cleavable moiety may be cleaved after it is internalized into the cell upon binding by the MAb-drug conjugate to a receptor thereof.
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, removing the spleen to obtain B- lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • the person of ordinary skill will realize that where antibodies are to be administered to human subjects, the antibodies will bind to human antigens.
  • MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A or Protein-G Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al., "Purification of Immunoglobulin G (IgG)," in METHODS IN
  • the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art, as discussed below.
  • Antibodies of use may be commercially obtained from a wide variety of known sources.
  • a variety of antibody secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, VA).
  • a large number of antibodies against various disease targets, including but not limited to tumor-associated antigens, have been deposited at the ATCC and/or have published variable region sequences and are available for use in the claimed methods and compositions. See, e.g., U.S. Patent Nos. 7,312,318; 7,282,567; 7,151, 164; 7,074,403; 7,060,802;
  • antibody sequences or antibody-secreting hybridomas against almost any disease-associated antigen may be obtained by a simple search of the ATCC, NCBI and/or USPTO databases for antibodies against a selected disease-associated target of interest.
  • the antigen binding domains of the cloned antibodies may be amplified, excised, ligated into an expression vector, transfected into an adapted host cell and used for protein production, using standard techniques well known in the art.
  • Isolated antibodies may be conjugated to therapeutic agents, such as camptothecins, using the techniques disclosed herein.
  • a chimeric antibody is a recombinant protein in which the variable regions of a human antibody have been replaced by the variable regions of, for example, a mouse antibody, including the complementarity-determining regions (CDRs) of the mouse antibody. Chimeric antibodies exhibit decreased immunogenicity and increased stability when administered to a subject. Methods for constructing chimeric antibodies are well known in the art ⁇ e.g., Leung et al., 1994, Hybridoma 13 :469).
  • a chimeric monoclonal antibody may be humanized by transferring the mouse CDRs from the heavy and light variable chains of the mouse immunoglobulin into the
  • Humanized monoclonal antibodies may be used for therapeutic treatment of subjects. Techniques for production of humanized monoclonal antibodies are well known in the art. (See, e.g., Jones et al., 1986, Nature, 321 :522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen et al., 1988, Science, 239: 1534; Carter et al., 1992, Proc. Natl Acad. Sci. USA, 89:4285; Sandhu, Crit. Rev.
  • an antibody may be a human monoclonal antibody. Such antibodies may be obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge, as discussed below.
  • the phage display technique may be used to generate human antibodies ⁇ e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4: 126-40, incorporated herein by reference).
  • Human antibodies may be generated from normal humans or from humans that exhibit a particular disease state, such as cancer (Dantas-Barbosa et al., 2005).
  • the advantage to constructing human antibodies from a diseased individual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.
  • RNAs were converted to cDNAs and used to make Fab cDNA libraries using specific primers against the heavy and light chain immunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97, incorporated herein by reference).
  • bacteriophage genome to make the phage display library.
  • libraries may be screened by standard phage display methods.
  • This technique is exemplary only and any known method for making and screening human antibodies or antibody fragments by phage display may be utilized.
  • transgenic animals that have been genetically engineered to produce human antibodies may be used to generate antibodies against essentially any immunogenic target, using standard immunization protocols as discussed above.
  • Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7: 13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).
  • a non-limiting example of such a system is the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods 231 : 11-23, incorporated herein by reference) from Abgenix (Fremont, CA), in which).
  • the mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the remainder of the mouse immune system remains intact.
  • the transgenic mice were transformed with germline-configured YACs (yeast artificial chromosomes) that contained portions of the human IgH and Ig kappa loci, including the majority of the variable region sequences, along accessory genes and regulatory sequences.
  • the human variable region repertoire may be used to generate antibody producing B cells, which may be processed into hybridomas by known techniques.
  • a XENOMOUSE® immunized with a target antigen will produce human antibodies by the normal immune response, which may be harvested and/or produced by standard techniques discussed above.
  • a variety of strains of genetically engineered mice are available, each of which is capable of producing a different class of antibody.
  • Transgenically produced human antibodies have been shown to have therapeutic potential, while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999).
  • the skilled artisan will realize that the claimed compositions and methods are not limited to use of the XENOMOUSE® system but may utilize any transgenic animal that has been genetically engineered to produce human antibodies.
  • Some embodiments of the claimed methods and/or compositions may concern antibody fragments.
  • Such antibody fragments may be obtained, for example, by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments may be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment may be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment.
  • the two Fab fragments may be covalently conjugated to generate a F(ab) 2 antibody fragment.
  • Fv fragments comprise an association of V H and V L chains. This association can be noncovalent, as described in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.
  • the variable chains may be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See Sandhu, 1992, Crit. Rev. Biotech, 12:437.
  • the Fv fragments comprise V H and V L chains connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains, connected by an oligonucleotides linker sequence. The structural gene is inserted into an expression vector that is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are well-known in the art.
  • Another form of an antibody fragment is a single-domain antibody (dAb), sometimes referred to as a single chain antibody.
  • Techniques for producing single-domain antibodies are well known in the art (see, e.g., Cossins et al., Protein Expression and Purification, 2007, 51 :253-59; Shuntao et al., Mole c Immunol 2006, 43 : 1912-19; Tanha et al., J. Biol. Chem. 2001, 276:24774-780).
  • Other types of antibody fragments may comprise one or more complementarity-determining regions (CDRs).
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest.
  • Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See Larrick et al., 1991, Methods: A Companion to Methods in Enzymology 2: 106; Ritter et al. (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (Cambridge University Press); Birch et al., (eds.), 1995, MONOCLONAL
  • the sequences of antibodies may be varied to optimize the physiological characteristics of the conjugates, such as the half-life in serum.
  • Methods of substituting amino acid sequences in proteins are widely known in the art, such as by site-directed mutagenesis (e.g. Sambrook et al., Molecular Cloning, A laboratory manual, 2 nd Ed, 1989).
  • the variation may involve the addition or removal of one or more glycosylation sites in the Fc sequence (e.g., U.S. Patent No. 6,254,868, the Examples section of which is incorporated herein by reference).
  • specific amino acid substitutions in the Fc sequence may be made (e.g., Hornick et al., 2000, J NuclMed 41 :355-62; Hinton et al., 2006, J Immunol 176:346-56; Petkova et al. 2006, Int Immunol 18: 1759-69; U.S. Patent No.
  • antibodies are used that recognize and/or bind to antigens that are expressed at high levels on target cells and that are expressed predominantly or exclusively on diseased cells versus normal tissues. More preferably, the antibodies internalize rapidly following binding.
  • An exemplary rapidly internalizing antibody is the LLl (anti-CD74) antibody, with a rate of internalization of approximately 8 x 10 6 antibody molecules per cell per day (e.g., Hansen et al., 1996, Biochem J. 320:293-300).
  • a "rapidly internalizing" antibody may be one with an internalization rate of about 1 x 10 6 to about 1 x 10 7 antibody molecules per cell per day.
  • Antibodies of use in the claimed compositions and methods may include MAbs with properties as recited above.
  • Exemplary antibodies of use for therapy of, for example, cancer include but are not limited to LLl (anti- CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20), obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1 receptor), nivolumab (anti- PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-Trop-2), PAM4 or KC4 (both anti- mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known as CD66e or
  • CEACAM5 ), MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu 31 (an anti-alpha-fetoprotein), Rl (anti-IGF-lR), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA (prostate-specific membrane antigen)), AB-PGl- XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IX MAb), L243 (anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20);
  • panitumumab (anti-EGFR); tositumomab (anti-CD20); PAM4 (aka clivatuzumab, anti- MUC5ac) and trastuzumab (anti-ErbB2).
  • anti-EGFR panitumumab
  • anti-CD20 tositumomab
  • PAM4 aka clivatuzumab, anti- MUC5ac
  • trastuzumab anti-ErbB2
  • hMN-14 U.S. Patent No. 6,676,924
  • hMN-15 U.S. Patent No. 8,287,865)
  • hRl U.S. Patent No. 9,441,043
  • hRS7 U.S. Patent No. 7,238,785
  • hMN-3 U.S. Patent No. 7,541,440
  • AB-PGl-XGl-026 U.S. Patent Application 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO 2009/130575)
  • the text of each recited patent or application is incorporated herein by reference with respect to the Figures and Examples sections.
  • the antibody is hRS7.
  • Other useful antigens that may be targeted using the described conjugates include carbonic anhydrase IX, B7, CCL19, CCL21, CSAp, ⁇ -2/neu, BrE3, CD 1, CD la, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM6, CTLA-4, alpha-fetoprotein (AFP), VEGF (e.g., bevacizumab, fibronectin splic
  • CD Cluster Designation
  • the CD66 antigens consist of five different glycoproteins with similar structures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) gene family members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66 antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normal epithelial cells of the digestive tract and tumor cells of various tissues. Also included as suitable targets for cancers are cancer testis antigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007; 120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al., Cancer Genet.
  • CEACAM6 carcinoembryonic antigen
  • Cancer Inst. 2007; 99: 1435-40 have antigens that can be targeted in certain cancer types, such as CD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos. Proc. 2006; 5: 155-79), non-small-cell lung cancer (Donnenberg et al., J. Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al., Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerba er al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreatic cancer (Li et al., Cancer Res.
  • Another useful target for breast cancer therapy is the LIV-1 antigen described by Taylor et al. (Biochem. J. 2003; 375:51-9).
  • the CD47 antigen is a further useful target for cancer stem cells (see, e.g., Naujokat et al., 2014, Immunotherapy 6:290-308; Goto et al., 2014, Eur J Cancer 50: 1836- 46; Unanue, 2013, Proc Natl Acad Sci USA 110: 10886-7).
  • CTL4 also known as CD 152
  • programmed cell death protein 1 also known as CD279
  • programmed cell death 1 ligand 1 PD-Ll, also known as CD274
  • exemplary anti-PD-1 antibodies include lambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK), and pidilizumab (CT-011, CURETECH LTD.).
  • Anti-PD-1 antibodies are commercially available, for example from ABCAM® (AB 137132),
  • anti-PD-Ll antibodies include MDX-1105 (MEDAREX), MEDI4736 (MEDFMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB). Anti-PD-Ll antibodies are also commercially available, for example from
  • anti-CTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER).
  • Anti-PDl antibodies are commercially available, for example from ABCAM® (AB 134090), SINO BIOLOGICAL INC. (11159-H03H, 11159-H08H), and THERMO SCIENTIFIC PIERCE (PA5-29572, PA5- 23967, PA5-26465, MA1-12205, MA1-35914).
  • Ipilimumab has recently received FDA approval for treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 11 :89).
  • Macrophage migration inhibitory factor is an important regulator of innate and adaptive immunity and apoptosis. It has been reported that CD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med 197: 1467-76).
  • the therapeutic effect of antagonistic anti-CD74 antibodies on MIF-mediated intracellular pathways may be of use for treatment of a broad range of disease states, such as cancers of the bladder, prostate, breast, lung, colon and chronic lymphocytic leukemia (e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran, 2011, Leuk Lymphoma 52: 1446-54); autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (Morand & Leech, 2005, Front Biosci 10: 12-22; Shachar & Haran, 2011, Leuk Lymphoma 52: 1446-54); kidney diseases such as renal allograft rejection (Lan, 2008, Nephron Exp Nephrol.
  • a broad range of disease states such as cancers of the bladder, prostate, breast, lung, colon and chronic lymphocytic leukemia (e.g., Meyer-Siegler et al., 2004, BMC Cancer 12
  • Anti-T F- ⁇ antibodies are known in the art and may be of use to treat various diseases.
  • Known antibodies against T F- ⁇ include the human antibody CDP571 (Ofei et al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI, M3TNFAI,
  • M3TNFABI, M302B and M303 Thermo Scientific, Rockford, IL
  • infliximab Centocor, Malvern, PA
  • certolizumab pegol UB, Brussels, Belgium
  • adalimumab Abbott, Abbott Park, IL
  • antibodies are used that internalize rapidly and are then re-expressed, processed and presented on cell surfaces, enabling continual uptake and accretion of circulating conjugate by the cell.
  • An example of a most-preferred antibodies are used that internalize rapidly and are then re-expressed, processed and presented on cell surfaces, enabling continual uptake and accretion of circulating conjugate by the cell.
  • an anti-CD74 MAb invariant chain, class II-specific chaperone, Ii
  • the CD74 antigen is highly expressed on B-cell lymphomas (including multiple myeloma) and leukemias, certain T-cell lymphomas, melanomas, colonic, lung, and renal cancers, glioblastomas, and certain other cancers (Ong et al., Immunology 95:296-302 (1999)).
  • a review of the use of CD74 antibodies in cancer is contained in Stein et al., Clin Cancer Res. 2007 Sep 15; 13(18 Pt 2):5556s-5563s,
  • the diseases that are preferably treated with anti-CD74 antibodies include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's disease, melanoma, lung, renal, colonic cancers, glioblastome multiforme, histiocytomas, myeloid leukemias, and multiple myeloma.
  • Continual expression of the CD74 antigen for short periods of time on the surface of target cells, followed by internalization of the antigen, and re-expression of the antigen enables the targeting LL1 antibody to be internalized along with any chemotherapeutic moiety it carries. This allows a high, and therapeutic, concentration of LL1 -chemotherapeutic drug conjugate to be accumulated inside such cells. Internalized LL1 -chemotherapeutic drug conjugates are cycled through lysosomes and endosomes, and the chemotherapeutic moiety is released in an active form within the target cells.
  • the antibodies discussed above and other known antibodies against disease- associated antigens may be used as CPT-conjugates, more preferably SN-38-conjugates, in the practice of the claimed methods and compositions.
  • the drug-conjugated antibody is an anti-Trop-2-SN-38 (e.g., hRS7-SN-38) conjugate.
  • Bispecific antibodies are useful in a number of biomedical applications. For instance, a bispecific antibody with binding sites for a tumor cell surface antigen and for a T-cell surface receptor can direct the lysis of specific tumor cells by T cells. Bispecific antibodies recognizing gliomas and the CD3 epitope on T cells have been successfully used in treating brain tumors in human patients (Nitta, et al. Lancet. 1990; 355:368-371). A preferred bispecific antibody is an anti-CD3 X anti-Trop-2 antibody.
  • an anti-CD3 antibody or fragment thereof may be attached to an antibody or fragment against a B-cell associated antigen, such as anti-CD3 X anti-CD 19, anti-CD3 X anti-CD20, anti-CD3 X anti-CD22, anti-CD3 X anti-HLA-DR or anti-CD3 X anti-CD74.
  • a B-cell associated antigen such as anti-CD3 X anti-CD 19, anti-CD3 X anti-CD20, anti-CD3 X anti-CD22, anti-CD3 X anti-HLA-DR or anti-CD3 X anti-CD74.
  • the techniques and compositions for therapeutic agent conjugation disclosed herein may be used with bispecific or multispecific antibodies as the targeting moieties.
  • Bispecific antibodies can be produced by the quadroma method, which involves the fusion of two different hybridomas, each producing a monoclonal antibody recognizing a different antigenic site (Milstein and Cuello, Nature, 1983; 305:537- 540).
  • bispecific antibodies uses heterobifunctional cross- linkers to chemically tether two different monoclonal antibodies (Staerz, et al. Nature, 1985; 314:628-631; Perez, et al. Nature, 1985; 316:354-356). Bispecific antibodies can also be produced by reduction of each of two parental monoclonal antibodies to the respective half molecules, which are then mixed and allowed to reoxidize to obtain the hybrid structure (Staerz and Bevan. Proc Natl Acad Sci USA. 1986; 83 : 1453-1457). Another alternative involves chemically cross-linking two or three separately purified Fab' fragments using appropriate linkers. (See, e.g., European Patent Application 0453082).
  • Other methods include improving the efficiency of generating hybrid hybridomas by gene transfer of distinct selectable markers via retrovirus-derived shuttle vectors into respective parental hybridomas, which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA. 1990, 87:2941-2945); or transfection of a hybridoma cell line with expression plasmids containing the heavy and light chain genes of a different antibody.
  • Cognate V H and V L domains can be joined with a peptide linker of appropriate composition and length (usually consisting of more than 12 amino acid residues) to form a single-chain Fv (scFv) with binding activity.
  • a peptide linker of appropriate composition and length usually consisting of more than 12 amino acid residues
  • Methods of manufacturing scFvs are disclosed in U.S. Pat. No. 4,946,778 and U.S. Pat. No. 5, 132,405, the Examples section of each of which is incorporated herein by reference. Reduction of the peptide linker length to less than 12 amino acid residues prevents pairing of V H and V L domains on the same chain and forces pairing of V H and V L domains with complementary domains on other chains, resulting in the formation of functional multimers.
  • Polypeptide chains of V H and V L domains that are joined with linkers between 3 and 12 amino acid residues form predominantly dimers (termed diabodies). With linkers between 0 and 2 amino acid residues, trimers (termed triabody) and tetramers (termed tetrabody) are favored, but the exact patterns of oligomerization appear to depend on the composition as well as the orientation of V-domains (V H -linker-V L or V L - linker-V H ), in addition to the linker length.
  • the technique utilizes complementary protein binding domains, referred to as anchoring domains (AD) and dimerization and docking domains (DDD), which bind to each other and allow the assembly of complex structures, ranging from dimers, trimers, tetramers, quintamers and hexamers. These form stable complexes in high yield without requirement for extensive purification.
  • DNL® technique allows the assembly of monospecific, bispecific or multispecific antibodies. Any of the techniques known in the art for making bispecific or multispecific antibodies may be utilized in the practice of the presently claimed methods.
  • Immunogenicity of therapeutic antibodies is associated with increased risk of infusion reactions and decreased duration of therapeutic response (Baert et al., 2003, N Engl J Med 348 :602-08).
  • the extent to which therapeutic antibodies induce an immune response in the host may be determined in part by the allotype of the antibody (Stickler et al., 201 1 , Genes and Immunity 12:213-21).
  • Antibody allotype is related to amino acid sequence variations at specific locations in the constant region sequences of the antibody.
  • the allotypes of IgG antibodies containing a heavy chain ⁇ -type constant region are designated as Gm allotypes (1976, J Immunol 111: 1056-59).
  • Glml For the common IgGl human antibodies, the most prevalent allotype is Glml (Stickler et al., 2011, Genes and Immunity 12:213-21). However, the Glm3 allotype also occurs frequently in Caucasians ⁇ Id.). It has been reported that Glml antibodies contain allotypic sequences that tend to induce an immune response when administered to non-Glml (nGlml) recipients, such as Glm3 patients ⁇ Id.). Non-Glml allotype antibodies are not as immunogenic when administered to Glml patients ⁇ Id.).
  • the human Glml allotype comprises the amino acids aspartic acid at Kabat position 356 and leucine at Kabat position 358 in the CH3 sequence of the heavy chain IgGl .
  • the nGlml allotype comprises the amino acids glutamic acid at Kabat position 356 and methionine at Kabat position 358.
  • Both Glml and nGlml allotypes comprise a glutamic acid residue at Kabat position 357 and the allotypes are sometimes referred to as DEL and EEM allotypes.
  • a non-limiting example of the heavy chain constant region sequences for Glml and nGlml allotype antibodies is shown for the exemplary antibodies rituximab (SEQ ID NO: 8) and veltuzumab (SEQ ID NO: 9).
  • veltuzumab and rituximab are, respectively, humanized and chimeric IgGl antibodies against CD20, of use for therapy of a wide variety of hematological malignancies.
  • Table 1 compares the allotype sequences of rituximab vs. veltuzumab.
  • rituximab (Glml7, l) is a DEL allotype IgGl, with an additional sequence variation at Kabat position 214 (heavy chain CHI) of lysine in rituximab vs. arginine in veltuzumab. It has been reported that veltuzumab is less immunogenic in subjects than rituximab ⁇ see, e.g., Morchhauser et al., 2009, J Clin Oncol 27:3346-53;
  • the allotype of the antibody In order to reduce the immunogenicity of therapeutic antibodies in individuals of nGlml genotype, it is desirable to select the allotype of the antibody to correspond to the Glm3 allotype, characterized by arginine at Kabat 214, and the nGlml,2 null-allotype, characterized by glutamic acid at Kabat position 356, methionine at Kabat position 358 and alanine at Kabat position 431. Surprisingly, it was found that repeated subcutaneous administration of Glm3 antibodies over a long period of time did not result in a significant immune response.
  • the human IgG4 heavy chain in common with the Glm3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356, methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicity appears to relate at least in part to the residues at those locations, use of the human IgG4 heavy chain constant region sequence for therapeutic antibodies is also a preferred embodiment. Combinations of Glm3 IgGl antibodies with IgG4 antibodies may also be of use for therapeutic administration.
  • Antibodies or fragments thereof may be conjugated to one or more therapeutic or diagnostic agents.
  • the therapeutic agents do not need to be the same but can be different, e.g. a drug and a radioisotope.
  • 131 I can be incorporated into a tyrosine of an antibody or fusion protein and a drug attached to an epsilon amino group of a lysine residue.
  • Therapeutic and diagnostic agents also can be attached, for example to reduced SH groups and/or to carbohydrate side chains.
  • Many methods for making covalent or non-covalent conjugates of therapeutic or diagnostic agents with antibodies or fusion proteins are known in the art and any such known method may be utilized.
  • a therapeutic or diagnostic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation.
  • such agents can be attached using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu et al, Int. J. Cancer 56: 244 (1994).
  • SPDP N-succinyl 3-(2-pyridyldithio)propionate
  • the therapeutic or diagnostic agent can be conjugated via a carbohydrate moiety in the Fc region of the antibody.
  • the carbohydrate group can be used to increase the loading of the same agent that is bound to a thiol group, or the carbohydrate moiety can be used to bind a different therapeutic or diagnostic agent.
  • the general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
  • the Fc region may be absent if the antibody used as the antibody component of the immunoconjugate is an antibody fragment.
  • a carbohydrate moiety into the light chain variable region of a full length antibody or antibody fragment. See, for example, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S. Patent No. 5,443,953 (1995), Leung et al, U.S. patent No. 6,254,868, incorporated herein by reference in their entirety.
  • the engineered carbohydrate moiety is used to attach the therapeutic or diagnostic agent.
  • the preferred conjugation protocol is based on a thiol-maleimide, a thiol- vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamide reaction that is facile at neutral or acidic pH. This obviates the need for higher pH conditions for conjugations as, for instance, would be necessitated when using active esters. Further details of exemplary conjugation protocols are described below in the Examples section.
  • the invention relates to a method of treating a subject, comprising administering to a subject a therapeutically effective amount of an antibody-drug conjugate (ADC) as described herein.
  • ADC antibody-drug conjugate
  • Diseases that may be treated with the ADCs described herein include, but are not limited to B-cell malignancies (e.g., non-Hodgkin's lymphoma, mantle cell lymphoma, multiple myeloma, Hodgkin's lymphoma, diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia) using, for example an anti-CD22 antibody such as the hLL2 MAb (epratuzumab, see U.S.
  • B-cell malignancies e.g., non-Hodgkin's lymphoma, mantle cell lymphoma, multiple
  • Patent No. 6, 183,744 against another CD22 epitope (hRFB4) or antibodies against other B cell antigens, such as CD19, CD20, CD21, CD22, CD23, CD37, CD40, CD40L, CD52, CD74, CD80 or HLA-DR.
  • Other diseases include, but are not limited to, adenocarcinomas of endodermally-derived digestive system epithelia, cancers such as breast cancer and non-small cell lung cancer, and other carcinomas, sarcomas, glial tumors, myeloid leukemias, etc.
  • antibodies against an antigen e.g., an oncofetal antigen, produced by or associated with a malignant solid tumor or hematopoietic neoplasm, e.g., a gastrointestinal, stomach, colon, esophageal, liver, lung, breast, pancreatic, liver, prostate, ovarian, testicular, brain, bone, urothelial or lymphatic tumor, a sarcoma or a melanoma, are advantageously used.
  • an antigen e.g., an oncofetal antigen
  • a malignant solid tumor or hematopoietic neoplasm e.g., a gastrointestinal, stomach, colon, esophageal, liver, lung, breast, pancreatic, liver, prostate, ovarian, testicular, brain, bone, urothelial or lymphatic tumor, a sarcoma or a melanoma
  • Such therapeutics can be given once or repeatedly, depending on the disease state and tolerability of the conjugate, and can also be used optionally in combination with other therapeutic modalities, such as surgery, external radiation, radioimmunotherapy, immunotherapy, chemotherapy, anti sense therapy, interference RNA therapy, gene therapy, and the like. Each combination will be adapted to the tumor type, stage, patient condition and prior therapy, and other factors considered by the managing physician.
  • the term "subject” refers to any animal (i.e., vertebrates and invertebrates) including, but not limited to mammals, including humans. It is not intended that the term be limited to a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are encompassed by the term. Doses given herein are for humans, but can be adjusted to the size of other mammals, as well as children, in accordance with weight or square meter size.
  • therapeutic conjugates comprising an anti-Trop-2 antibody such as the hRS7 MAb can be used to treat carcinomas such as carcinomas of the esophagus, pancreas, lung, stomach, colon and rectum, urinary bladder, breast, ovary, uterus, kidney and prostate, as disclosed in U.S. Patent No. 7,238,785; 7,517,964 and 8,084,583, the Examples section of which is incorporated herein by reference.
  • An hRS7 antibody is a humanized antibody that comprises light chain complementarity-determining region (CDR) sequences CDR1 (KASQDVSIAVA, SEQ ID NO: l); CDR2 (SASYRYT, SEQ ID NO:2); and CDR3 (QQHYITPLT, SEQ ID NO:3) and heavy chain CDR sequences CDR1
  • CDR1 KASQDVSIAVA, SEQ ID NO: l
  • CDR2 SASYRYT, SEQ ID NO:2
  • CDR3 QQHYITPLT, SEQ ID NO:3
  • the antibodies that are used in the treatment of human disease are human or humanized (CDR-grafted) versions of antibodies; although murine and chimeric versions of antibodies can be used.
  • Same species IgG molecules as delivery agents are mostly preferred to minimize immune responses. This is particularly important when considering repeat treatments.
  • a human or humanized IgG antibody is less likely to generate an anti-IgG immune response from patients.
  • Antibodies such as hLLl and hLL2 rapidly internalize after binding to internalizing antigen on target cells, which means that the chemotherapeutic drug being carried is rapidly internalized into cells as well.
  • antibodies that have slower rates of internalization can also be used to effect selective therapy.
  • a therapeutic agent used in combination with the camptothecin conjugate of this invention may comprise one or more isotopes. Radioactive
  • 212 isotopes useful for treating diseased tissue include, but are not limited to- In, Lu, Bi, 213 Bi, 211 At, 62 Cu, 67 Cu, 90 Y, 125 I, 131 1, 32 P, 33 P, 47 Sc, lu Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, 227 Th and 211 Pb.
  • the therapeutic radionuclide preferably has a decay-energy in the range of 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter, and 4,000- 6,000 keV for an alpha emitter.
  • Maximum decay energies of useful beta-particle-emitting nuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, and most preferably 500-2,500 keV. Also preferred are radionuclides that substantially decay with Auger-emitting particles.
  • beta-particle-emitting nuclides are preferably ⁇ 1,000 keV, more preferably ⁇ 100 keV, and most preferably ⁇ 70 keV. Also preferred are radionuclides that substantially decay with generation of alpha-particles.
  • radionuclides include, but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po- 215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227 and Fm-255. Decay energies of useful alpha-particle-emitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV. Additional potential radioisotopes
  • Radionuclides and other metals may be delivered, for example, using chelating groups attached to an antibody or conjugate.
  • Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with
  • radionuclides of gallium, yttrium and copper are radionuclides of gallium, yttrium and copper, respectively.
  • metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest.
  • Other ring-type chelates, such as macrocyclic polyethers for complexing 223 Ra, may be used.
  • Therapeutic agents of use in combination with the camptothecin conjugates described herein also include, for example, chemotherapeutic drugs such as vinca alkaloids,
  • cancer chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, and the like. Suitable chemotherapeutic agents are described in REMINGTON'S
  • Exemplary drugs of use include, but are not limited to, 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, biyostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxy camptothecin, carmustine, Celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin,
  • therapeutic naked antibodies as are known in the art may be used in combination with the described conjugates. Exemplary therapeutic naked antibodies are described above.
  • a therapeutic agent to be used in combination with a DNA- breaking antibody conjugate is a microtubule inhibitor, such as a vinca alkaloid, a taxanes, a maytansinoid or an auristatin.
  • Exemplary known microtubule inhibitors include paclitaxel, vincristine, vinblastine, mertansine, epothilone, docetaxel, discodermolide, combrestatin, podophyllotoxin, CI-980, phenylahistins, steganacins, curacins, 2-methoxy estradiol, E7010, methoxy benzenesuflonamides, vinorelbine, vinflunine, vindesine, dolastatins, spongistatin, rhizoxin, tasidotin, halichondrins, hemiasterlins, cryptophycin 52, MMAE and eribulin mesylate.
  • a therapeutic agent to be used in combination with a DNA-breaking ADC is a PARP inhibitor, such as olaparib, talazoparib (BMN-673), rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699, BSI-201, CEP-8983 or 3-aminobenzamide.
  • a PARP inhibitor such as olaparib, talazoparib (BMN-673), rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699, BSI-201, CEP-8983 or 3-aminobenzamide.
  • a therapeutic agent used in combination with an antibody or immunoconjugate is a Bruton kinase inhibitor, such as such as ibrutinib (PCI-32765), PCI- 45292, CC-292 (AVL-292), ONO-4059, GDC-0834, LFM-A13 or RN486.
  • a Bruton kinase inhibitor such as such as ibrutinib (PCI-32765), PCI- 45292, CC-292 (AVL-292), ONO-4059, GDC-0834, LFM-A13 or RN486.
  • a therapeutic agent used in combination with an antibody or immunoconjugate is a PI3K inhibitor, such as idelalisib, Wortmannin, demethoxyviridin, perifosine, PX-866, IPI-145 (duvelisib), BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120, XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, PI-103, G E477, CUDC-907, AEZS-136 or LY294002.
  • PI3K inhibitor such as idelalisib, Wortmannin, demethoxyviridin, perifosine, PX-866, IPI-145 (duvelisib), BAY 80-6946, BEZ235, RP6530, TGR120
  • Therapeutic agents that may be used in concert with the camptothecin conjugates also may comprise toxins conjugated to targeting moieties.
  • Toxins that may be used in this regard include ricin, abrin, ribonuclease (RNase), DNase I, ranpirnase, Staphylococcal enterotoxin- A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and
  • Pseudomonas endotoxin See, e.g., Pastan. et al., Cell (1986), 47:641, and Sharkey and Goldenberg, CA Cancer J Clin. 2006 Jul-Aug;56(4):226-43.
  • Additional toxins suitable for use herein are known to those of skill in the art and are disclosed in U.S. 6,077,499.
  • Immunomodulators of use may be selected from a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and a combination thereof. Specifically useful are
  • lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors, such as interleukin (IL), colony stimulating factor, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF), interferon, such as
  • interferons-a, - ⁇ , - ⁇ or - ⁇ interferons-a, - ⁇ , - ⁇ or - ⁇ , and stem cell growth factor, such as that designated "SI factor”.
  • cytokines include growth hormones such as human growth hormone, N- methionyl human growth hormone, and bovine growth hormone; parathyroid hormone;
  • thyroxine insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-a and - B; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor;
  • FSH follicle stimulating hormone
  • TSH thyroid stimulating hormone
  • LH luteinizing hormone
  • integrin thrombopoietin
  • TPO nerve growth factors
  • NGF-B platelet-growth factor
  • TGFs transforming growth factors
  • TGFs transforming growth factors
  • EPO erythropoietin
  • osteoinductive factors interferons such as interferon-a, - ⁇ , - ⁇ and - ⁇
  • colony stimulating factors CSFs
  • M-CSF macrophage-CSF
  • ILs interleukins
  • ILs interleukins
  • ILs interleukins
  • ILs such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3, angiostatin, thrombospondin,
  • Chemokines of use include RANTES, MCAF, MlPl-alpha, MIPl-Beta and IP-10.
  • the subject immunoconjugates comprising a camptothecin conjugated to an antibody or antibody fragment
  • chemotherapeutic agent radiation therapy, chemokine, cytokine, immunomodulator, enzyme, hormone, oligonucleotide, RNAi or siRNA.
  • additional therapeutic agents may be administered separately, in combination with, or attached to the subject antibody-drug immunoconjugates.
  • Suitable routes of administration of the conjugates include, without limitation, oral, parenteral, subcutaneous, rectal, transmucosal, intestinal administration, intramuscular, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injections.
  • the preferred routes of administration are parenteral.
  • Immunoconjugates can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the immunoconjugate is combined in a mixture with a pharmaceutically suitable excipient.
  • a pharmaceutically suitable excipient Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient.
  • Other suitable excipients are well-known to those in the art. See, for example, Ansel et al, PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990), and revised editions thereof.
  • the immunoconjugate is formulated in Good's biological buffer (pH 6-7), using a buffer selected from the group consisting of N-(2-acetamido)-2- aminoethanesulfonic acid (ACES); N-(2-acetamido)iminodiacetic acid (ADA); N,N-bis(2- hydroxyethyl)-2-aminoethanesulfonic acid (BES); 4-(2-hydroxyethyl)piperazine-l- ethanesulfonic acid (HEPES); 2-(N-morpholino)ethanesulfonic acid (MES); 3-(N- morpholino)propanesulfonic acid (MOPS); 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); and piperazine-N,N'-bis(2-ethanesulfonic acid) [Pipes].
  • a buffer selected from the group consisting of N-(2-acetamido)-2- aminoethane
  • More preferred buffers are MES or MOPS, preferably in the concentration range of 20 to 100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH 6.5.
  • the formulation may further comprise 25 mM trehalose and 0.01% v/v polysorbate 80 as excipients, with the final buffer concentration modified to 22.25 mM as a result of added excipients.
  • the preferred method of storage is as a lyophilized formulation of the conjugates, stored in the temperature range of -20 °C to 2 °C, with the most preferred storage at 2 °C to 8 °C.
  • the immunoconjugate can be formulated for intravenous administration via, for example, bolus injection, slow infusion or continuous infusion.
  • the antibody of the present invention is infused over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours.
  • the first 25-50 mg could be infused within 30 minutes, preferably even 15 min, and the remainder infused over the next 2-3 hrs.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi- dose containers, with an added preservative.
  • compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Control release preparations can be prepared through the use of polymers to complex or adsorb the immunoconjugate.
  • biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. Sherwood et al, Bio/Technology 10: 1446 (1992). The rate of release of an immunoconjugate from such a matrix depends upon the molecular weight of the immunoconjugate, the amount of immunoconjugate within the matrix, and the size of dispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);
  • the dosage of an administered immunoconjugate for humans will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. It may be desirable to provide the recipient with a dosage of immunoconjugate that is in the range of from about 1 mg/kg to 24 mg/kg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate.
  • the dosage may be repeated as needed, for example, once per week for 4-10 weeks, once per week for 8 weeks, or once per week for 4 weeks.
  • Preferred dosages may include, but are not limited to, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22 mg/kg and 24 mg/kg. Any amount in the range of 1 to 24 mg/kg may be used.
  • the dosage is preferably administered multiple times, once or twice a week.
  • a minimum dosage schedule of 4 weeks, more preferably 8 weeks, more preferably 16 weeks or longer may be used.
  • the schedule of administration may comprise administration once or twice a week, on a cycle selected from the group consisting of: (i) weekly; (ii) every other week; (iii) one week of therapy followed by two, three or four weeks off; (iv) two weeks of therapy followed by one, two, three or four weeks off; (v) three weeks of therapy followed by one, two, three, four or five week off; (vi) four weeks of therapy followed by one, two, three, four or five week off; (vii) five weeks of therapy followed by one, two, three, four or five week off; and (viii) monthly.
  • the cycle may be repeated 4, 6, 8, 10, 12, 16 or 20 times or more.
  • an immunoconjugate may be administered as one dosage every 2 or 3 weeks, repeated for a total of at least 3 dosages. Or, twice per week for 4-6 weeks. If the dosage is lowered to approximately 200-300 mg/m 2 (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), it may be administered once or even twice weekly for 4 to 10 weeks. Alternatively, the dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3 months. It has been determined, however, that even higher doses, such as 12 mg/kg once weekly or once every 2-3 weeks can be administered by slow i.v. infusion, for repeated dosing cycles. The dosing schedule can optionally be repeated at other intervals and dosage may be given through various parenteral routes, with appropriate adjustment of the dose and schedule
  • the immunoconjugates are of use for therapy of cancer.
  • cancers include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies.
  • squamous cell cancer e.g., epithelial squamous cell cancer
  • Ewing sarcoma e.g., Ewing sarcoma
  • Wilms tumor astrocytomas
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors, medullary thyroid cancer, differentiated thyroid carcinoma, breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, anal carcinoma, penile carcinoma, as well as head-and-neck cancer.
  • squamous cell cancer e.g.,
  • cancer includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).
  • primary malignant cells or tumors e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor
  • secondary malignant cells or tumors e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor.
  • cancers or malignancies include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary)
  • Mesothelioma Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma,
  • Metastatic Occult Primary Squamous Neck Cancer Metastatic Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, O
  • Pheochromocytoma Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma
  • compositions described and claimed herein may be used to treat malignant or premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above.
  • Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).
  • Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia. It is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplasia characteristically occurs where there exists chronic irritation or inflammation.
  • Dysplastic disorders which can be treated include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epi
  • Additional pre-neoplastic disorders which can be treated include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
  • benign dysproliferative disorders e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps or adenomas, and esophageal dysplasia
  • leukoplakia keratoses
  • Bowen's disease keratoses
  • Farmer's Skin Farmer's Skin
  • solar cheilitis solar keratosis
  • the method of the invention is used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
  • Additional hyperproliferative diseases, disorders, and/or conditions include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias; e.g., acute lymphocytic leukemia, acute myelocytic leukemia [including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia]) and chronic leukemias (e.g., chronic myelocytic [granulocytic] leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, lipos
  • lymphangioendotheliosarcoma synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
  • Autoimmune diseases that may be treated with immunoconjugates may include acute and chronic immune thrombocytopenias, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, poststreptococcal nephritis, erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thrombo
  • kits containing components suitable for treating cancer in a patient may contain at least one drug-conjugated antibody as described herein. If the composition containing components for administration is not formulated for delivery via the alimentary canal, such as by oral delivery, a device capable of delivering the kit components through some other route may be included.
  • a device capable of delivering the kit components through some other route may be included.
  • the kit components may be packaged together or separated into two or more containers.
  • the containers may be vials that contain sterile, lyophilized formulations of a composition that are suitable for reconstitution.
  • a kit may also contain one or more buffers suitable for reconstitution and/or dilution of other reagents.
  • Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers.
  • Another component that can be included is instructions to a person using a kit for its use.
  • Trop-2 is widely expressed in ⁇ 83% of urothelial carcinomas. Of the 6 patients, 3 had a clinically significant response (progression-free survival, 6.7 to 8.2 months; overall survival, 7.5+ to 11.4+ months). Sacituzumab govitecan was well tolerated. Because of these results, a phase II trial has been initiated. The present report demonstrates the utility of anti- Trop-2 antibody-drug conjugates, such as sacituzumab govitecan, as a novel therapeutic strategy for the treatment of PRUC.
  • Urothelial bladder carcinoma is the sixth most frequent form of cancer (e.g., Sharma et al., 2009, Am Fam Physician 80:717-23). Cisplatin-based combination
  • ADCs Antibody-drug conjugates (ADCs) targeting cell-surface antigens represent an attractive therapeutic strategy for chemotherapy -refractory tumors, including PRUC (Cardillo et al., 2015, Bioconjug Chem 26:919-31).
  • Sacituzumab govitecan IMMU-132 is a second- generation ADC comprising a humanized anti-Trop-2 monoclonal antibody (hRS7) conjugated with the active metabolite of irinotecan, SN-38 (Goldenberg et al., 2015,
  • Oncotarget 6:22496-5120 It has demonstrated acceptable toxicity and excellent therapeutic activity in several solid tumors, both preclinically and clinically (Cardillo et al., 2015, Bioconjug Chem 26:919-31; Starodub et al., 2015, Clin Cancer Res 21 :3870-78), and is a rational choice for targeting UC.
  • Trop-2 (TACSTD2) protein is known to be expressed in normal urothelium (Stepan et al., 2011, J Histochem Cytochem 59:701-10) and in ⁇ 83% of urothelial carcinomas (Faltas et al., 2016, Clin Genitourin Cancer 14:e75-9).
  • hRS7 humanized RS7
  • SN-38 attached to a CL2A linker was produced and conjugated to hRS7 (anti- Trop-2) according to U.S. Patent 7,999,083 (Example 10 and 12 of which are incorporated herein by reference).
  • the conjugation protocol resulted in a ratio of between about 6 to 8 SN- 38 molecules attached per antibody molecule.
  • the median patient age was 72.5 years (range, 42-80 years). All patients had metastatic disease and had been previously treated with platinum-containing regimens and other lines of therapy (median number of previous therapies, 3; Table 2). Of the 6 patients, 5 were in the poor or intermediate-risk groups according to the prognostic model for patients with UC receiving salvage systemic therapy (Sonpavde et al., 2015, J Clin Oncol 33 (abstract 311). All 6 patients with PRUC were available for the response assessment. Two achieved a partial response, with the best responder having a 38% reduction in target lesions, including liver metastases (FIG. 1).
  • Sacituzumab govitecan was generally well tolerated. Two patients experienced grade 3 toxicities (flank pain and bacteremia). No grade 4 nonhematologic toxicities were observed. Immunohistochemical analysis of archival PRUC tumor tissue from patients treated with sacituzumab govitecan showed significant cell surface expression of Trop-2 protein (FIG. 2).
  • BCG bacille Calmette-Guerin
  • Hb hemoglobin
  • LDH lactate
  • the overall response rate for patients treated with second-line therapy has usually been ⁇ 20%, with a median overall survival of only 4 to 9 months (Bellmunt et al., 2009, J Clin Oncol 27:4454-61 ; Sweeney et al., 2006, J Clin Oncol 24:3451-57; Galsky et al., 2007, Invest New Drugs 25 :265-70).
  • Example 1 Following Example 1, further studies were performed in patients with mUC pre- treated with platinum-containing chemotherapy. Such patients have limited therapeutic options, with checkpoint-inhibitor immunotherapy (10) responses in a minority of patients. We provide further evidence of the safety and activity of sacituzumab govitecan (IMMU- 132) as therapy for chemotherapy-pretreated mUC pts (ClinicalTrials.gov, NCT01631552).
  • IMMU- 132 sacituzumab govitecan
  • the present Example reports results from a phase I clinical trial and ongoing phase II extension with IMMU-132, an ADC of the internalizing, humanized, hRS7 anti-Trop-2 antibody conjugated by a pH-sensitive linker to SN-38 (mean drug-antibody ratio 7.6).
  • Trop-2 is a type I transmembrane, calcium-transducing, protein expressed at high density ( ⁇ 1 x 10 5 ), frequency, and specificity by many human carcinomas, with limited normal tissue expression.
  • IMMU-132 is capable of delivering as much as 120-fold more SN- 38 to tumor than derived from a maximally tolerated irinotecan therapy.
  • the TTP for the remaining 48 patients was 12.6+ wks (range 6.0 to 51.4 wks).
  • Plasma CEA and CA19-9 correlated with responses who had elevated titers of these antigens in their blood. No anti-hRS7 or anti-SN-38 antibodies were detected despite dosing over months.
  • Best Response data of 8 assessable patients with TNBC (triple-negative breast cancer), there were 2 PR (partial response), 4 SD (stable disease) and 2 PD
  • Exemplary partial responses to the anti-Trop-2 ADC were confirmed by CT data (not shown).
  • CT data As an exemplary PR in CRC, a 62 year-old woman first diagnosed with CRC underwent a primary hemicolectomy. Four months later, she had a hepatic resection for liver metastases and received 7 mos of treatment with FOLFOX and 1 mo 5FU. She presented with multiple lesions primarily in the liver (3+ Trop-2 by immunohistology), entering the hRS7-SN-38 trial at a starting dose of 8 mg/kg about 1 year after initial diagnosis. On her first CT assessment, a PR was achieved, with a 37% reduction in target lesions (not shown). The patient continued treatment, achieving a maximum reduction of 65% decrease after 10 months of treatment (not shown) with decrease in CEA from 781 ng/mL to 26.5 ng/mL), before progressing 3 months later.
  • Trop-2 is a type-I transmembrane, calcium-transducing protein expressed at high density, frequency, and specificity in many epithelial cancers, including pancreatic ductal adenocarcinoma, with limited normal tissue expression. All 29 pancreatic tumor microarray specimens tested were Trop-2-positive by immunohistochemistry, and human pancreatic cancer cell lines were found to express 115k-891k Trop-2 copies on the cell membrane.
  • IMMU-132 Five had progressive disease by RECIST; 1 withdrew after just 1 dose due to clinical progression and was not assessable. Serum CA19-9 titers decreased in 3 of the patients with stable disease by 23 to 72%. Despite multiple administrations, none of the patients developed an antibody response to IMMU-132 or SN-38. Peak and trough serum samples showed that IMMU-132 cleared more quickly than the IgG, which is expected based on the known local release of SN-38 within the tumor cell. Concentrations of SN-38-bound to IgG in peak samples from one patient given 12 mg/kg of IMMU-132 showed levels of -4000 ng/mL, which is 40-times higher than the SN-38 titers reported in patients given irinotecan therapy.
  • IMMU-132 is active (long-term stable disease) in 62% (8/13) of PDC patients who failed multiple prior therapies, with manageable neutropenia and little GI toxicity.
  • Advanced PDC patients can be given repeated treatment cycles (>6) of 8-10 mg/kg IMMU-132 on days 1 and 8 of a 21-day cycle, with some dose adjustments or growth factor support for neutropenia in subsequent treatment cycles.
  • monotherapy IMMU-132 is a novel, efficacious treatment regimen for patients with PDC, including those with tumors that were previously resistant to other therapeutic regimens for PDC.
  • Trop-2 expression The expression of Trop-2 on the surface of various cancer cell lines was determined by flow cytometry using QUANTBRITE® PE beads. The results for number of Trop-2 molecules detected in the different cell lines was: BxPC-3 pancreatic cancer (891,000); NCI-N87 gastric cancer (383,000); MDA-MB-468 breast cacner (341,000); SK-MES-1 squamous cell lung cancer (27,000); Capan-1 pancreatic cancer (115,000); AGS gastric cancer (78,000) COLO 205 colon cancer (52,000). Trop-2 expression was also observed in 29 of 29 (100%) tissue microarrays of pancreatic adenocarcinoma (not shown).
  • Necropsies were performed on 3 animals per interval, in irinotecan injected mice at 5 min, 1, 2, 6 and 24 hours or in IMMU-132 injected mice at 1, 6, 24, 48 and 72 h.
  • Tissues were extracted and analyzed by reversed-phase HPLC analysis for SN-38, SN-38G, and irinotecan. Extracts from EVIMU-132-treated animals also were acid hydrolyzed to release SN-38 from the conjugate (i.e., SN-38 (TOTAL]).
  • TOTAL conjugate
  • the results demonstrate that the EVIMU-132 ADC has the potential to deliver 120 times more SN-38 to the tumor compared to irinotecan, even though 22-fold less SN-38 equivalents were administered with the ADC.
  • IMMU-132 clinical protocol The protocol used in the phase I/II study was as indicated in Table 5 below.
  • IMMU-132 Patients were administered IMMU-132 according to the protocol summarized above.
  • An exemplary case study is as follows.
  • a 34 y/o white male initially diagnosed with metastatic pancreatic cancer (liver) had progressed on multiple chemotherapy regimens, including gemcitabine/ Erlotinib/FG-3019, FOLFIRINOX and GTX prior to introduction of IMMU-132 (8 mg/kg dose given days 1 and 8 of a 21 day cycle).
  • the patient received the drug for 4 mo with good symptomatic tolerance, an improvement in pain, a 72% maximum decline in CA19-9 (from 15885 U/mL to 4418 U/mL) and stable disease by CT RECIST criteria along with evidence of tumor necrosis.
  • Therapy had to be suspended due to a liver abscess; the patient expired ⁇ 6 weeks later, 6 mo following therapy initiation.
  • TNBC Triple-Negative Breast Cancer
  • CR complete responses
  • PR partial responses
  • SD stable disease
  • IMMU-132 treated patients not shown.
  • the median time to progression in this heavily pretreated population of TNBC patients was 9.4 months, with a range of 2.9 to 14.2 months to date.
  • the progression-free survival in this group of patients is shown in FIG. 5.
  • Metastatic NSCLC Metastatic non-small cell lung cancer
  • NSCLC metastatic non-small cell lung cancer
  • FIG. 7 The time to progression for NSCLC patients is shown in FIG. 7, which shows that 21/33 (64%) of NSCLC patients exhibited PR or SD.
  • the median time to progression was 9/4 months, with a range from 1.8 to 15.5+ months and 47% of patients still undergoing treatment.
  • Progression-free survival in NSCLC patients treated with 8 or 10 mg/kg EVIMU-132 is shown in FIG. 8.
  • Median PFS was 3.4 months at 8 mg/kg and 3.8 months at 10 mg/kg.
  • studies are still ongoing and the median progression- free survival numbers are likely to improve.
  • the continuing phase I/II clinical trial shows superior efficacy of IMMU- 132, when administered at the recited dosages of ADC, in at least TNBC, NSCLC, SCLC and urothelial cancers.
  • the superior therapeutic effect in these heavily pretreated and resistant metastatic cancers occurred without inducing severe toxicities that might preclude clinical use.
  • EVIMU-132 showed an acceptable safety profile in heavily pretreated patients with diverse solid cancers, and a median of 2-5 prior therapies. Only neutropenia showed an incidence of greater than 20% of the patient population for Grade 3 or higher adverse reactions.
  • IMMU-132 may be administered to human patients, at therapeutic dosages, without evoking interfering host anti- IMMU-132 antibodies.
  • Synthetic lethality is a concept in which a cell harboring one out of two possible gene or protein defects is viable, while a cell containing both defects is nonviable.
  • BRCAl/2 mutations are linked to deficiencies in DNA repair and are associated with TNBC.
  • Other repair mechanisms involve poly(adenosine diphosphoribose) polymerase (PARP), which can be used by cancer cells to overcome loss of BRACAl/2.
  • PARP poly(adenosine diphosphoribose) polymerase
  • Treatment of TNBC cells with either IMMU-132 or paclitaxel results in cleavage and deactivation of PARP, whereas the small molecule olaparib directly inhibits PARP. Therefore, the rationale of combining IMMU-132 with either paclitaxel or olaparib to effectively knock-out PARP activity was investigated in TNBC xenografts to ascertain if these combinations will result in synthetic lethality.
  • the purpose of this study was to determine whether combining an antibody-drug conjugate that induces DNA strand breaks, such as sacituzumab govitecan (also known as IMMU-132, an anti-Trop-2 hRS7-CL2A-SN-38), with microtubule inhibitors (e.g., paclitaxel or eribulin mesylate) or poly(adenosine diphosphoribose) polymerase (PARP) inhibitors (e.g., olaparib) in cancer (e.g., nude mice bearing TNBC xenografts) improves antitumor effects.
  • sacituzumab govitecan also known as IMMU-132, an anti-Trop-2 hRS7-CL2A-SN-38
  • microtubule inhibitors e.g., paclitaxel or eribulin mesylate
  • PARP poly(adenosine diphosphoribose) polymerase
  • mice bearing human TNBC (triple negative breast cancer) xenografts (MDA-MB-468 or HCC1806; -0.3 cm 3 ) were treated with the maximum tolerated dose of paclitaxel (15 mg/kg weekly x 5 wks) and IMMU-132 at either 10 mg/kg or 12.5 mg/kg on days 1, 8, 22, and 29.
  • Mice bearing HCC1806 tumors (-0.28 cm 3 ) were treated for 2 cycles with IMMU-132 (12.5 mg/kg) and 0.5 mg/kg of eribulin mesylate (equivalent to human dose of 1.4 mg/m 2 ) weekly for 2 weeks on a 21 -day cycle.
  • Olaparib was administered as i.p. injections daily for 5 days in a row with two day's rest before repeating (qdx5). This was done for four weeks.
  • EVIMU- 132 was administered i.p. twice weekly for four weeks.
  • the primary endpoint was the median survival time (MST), defined as the time for tumors to progress to 1.0 cm 3 .
  • MST median survival time
  • assay for synergistic effects may be determined by in vitro assay.
  • a clonogenic assay may be used to determine survival fraction of cells (Ibrahim et al., 2012, Cancer Discovery 2: 1036-47). Briefly, 350-800 cells are plated in 6-well flat bottom cell culture plates in duplicates. Twenty-four hours after plating, cells are washed and fresh medium is added in the presence or absence of increasing doses of ADC and/or PARP or microtubule inhibitor (e.g., olaparib) alone and in combination. Media containing the drug and/or is refreshed on day 4. Colonies are fixed and stained after 7 days of treatment with 1.5 ml of 6.0 % glutaraldehyde and 0.5 % crystal violet and colonies are counted by standard procedures. The surviving fraction (SF) of cells is calculated as follows:
  • This method quantitatively describes the interaction between two or more drugs, with values less than 1 indicating synergistic interactions, values greater than 1 indicating antagonistic interactions, and values equal to 1 indicating additive interactions.
  • IMMU-132 has been stable for several years.
  • mice bearing Trop-2 + human gastric carcinoma xenografts were given 2 treatments 7 days apart, each with equal protein (0.5 mg) doses of IMMU-132 having DARs of 6.89, 3.28, or 1.64.
  • mice bearing NCI-N87 gastric tumors were administered 0.5 mg IMMU-132 with a DAR of 6.89 twice weekly for two weeks (not shown).
  • treatment with the lower DAR was not significantly different than the untreated controls.
  • IMMU-132 Mechanism of action of IMMU-132 in T BC.
  • the apoptotic pathway utilized by IMMU-132 was examined in the TNBC cell line, MDA-MB-468, and in the HER2 + SK-BR- 3 cell line, in order to confirm that the ADC functions on the basis of its incorporated SN-38.
  • Cells were exposed to 1 ⁇ SN-38, the SN-38-equivalent of IMMU-132, or protein equivalent of hRS7. Cells were harvested and Western blots were performed.
  • FMMU-132 mediated a greater degree of pro-caspase-3 cleavage, with the highest level observed after 48 h when compared to cells exposed to SN-38 (not shown).
  • SN-38 and IMMU-132 both mediated poly ADP ribose polymerase (PARP) cleavage, starting at 24 h, with near complete cleavage after 48 h (not shown).
  • PARP poly ADP ribose polymerase
  • IMMU-132 is a humanized anti-Trop-2 antibody conjugated with 7.6 molecules of SN-38, the active metabolite of irinotecan, a topoisomerase I inhibitor. Clinically, IMMU- 132 has shown manageable toxicity and encouraging responses in patients with
  • IMMU-132 can be combined with two different microtubule- inhibitors or a PARP-inhibitor with significantly enhanced anti-tumor activity
  • ADCs antibody-drug conjugates
  • a preferred ADC class is represented by anti-Trop-2 antibody conjugates in patients with Trop-2 positive cancers, including but not limited to TNBC, metastatic colon cancer, SCLC, NSCLC and urothelial cancer, since this is a target that is expressed in high amounts in a large number of cancers, and is localized on the cell surface and cytoplasmically in the cancer cells.
  • IMMU-132 was combined with PARP-inhibitors (e.g., olaparib) in TNBC tumor lines that had BRCAl/2 defects, as well as wild-type expression, including one with only a PTEN defect. This suggests that IMMU-132 may synergize with any tumor that has any kind of disruption in DNA homologous recombination pathways. Combined with olaparib, EVIMU-132 therapy achieved significant anti -tumor effects above that observed with monotherapy with each, resulting in a significant survival benefit. IMMU- 132 combined with microtubule inhibitors, (e.g., paclitaxel or eribulin mesylate) also enhanced efficacy significantly compared to monotherapy with each agent.
  • PARP-inhibitors e.g., olaparib

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Abstract

L'invention concerne des ADC thérapeutiques comprenant la SN-38 fixée à un anticorps anti-Trop2 ou un fragment d'anticorps à liaison d'antigène. L'ADC peut être administré à une dose comprise entre 4 mg/kg et 18 mg/kg, de préférence 4, 6, 8, 9, 10, 12, 16 ou 18 mg/kg, mieux encore 8 ou 10 mg/kg. Lorsqu'administré selon des dosages et des posologies spécifiés, l'ADC peut réduire la taille de tumeurs solides, réduire ou éliminer les métastases et est efficace pour traiter les cancers résistant aux thérapies standard, telles que la radiothérapie, la chimiothérapie ou l'immunothérapie. De préférence, l'ADC est administré en combinaison avec un ou plusieurs autres agents thérapeutiques, tels qu'un inhibiteur de PARP, un inhibiteur de microtubules, un inhibiteur de la kinase de Bruton ou un inhibiteur de PI3K. Idéalement, l'ADC est utile pour traiter un cancer exprimant Trop-2, tel qu'un cancer urothélial métastatique.
PCT/US2017/062976 2016-12-01 2017-11-22 Thérapie pour le cancer urothélial métastatique avec le conjugué anticorps-médicament, sacituzumab govitécan (immu-132) Ceased WO2018102212A1 (fr)

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