WO2018160794A1 - Liposomes générant du docétaxel ciblant epha2 en combinaison avec un agent qui entrave l'activité des lymphocytes t régulateurs pour le traitement du cancer - Google Patents

Liposomes générant du docétaxel ciblant epha2 en combinaison avec un agent qui entrave l'activité des lymphocytes t régulateurs pour le traitement du cancer Download PDF

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WO2018160794A1
WO2018160794A1 PCT/US2018/020381 US2018020381W WO2018160794A1 WO 2018160794 A1 WO2018160794 A1 WO 2018160794A1 US 2018020381 W US2018020381 W US 2018020381W WO 2018160794 A1 WO2018160794 A1 WO 2018160794A1
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docetaxel
epha2
cancer
ils
46scfv
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PCT/US2018/020381
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Daryl C. Drummond
Walid KAMOUN
Andrew J. SAWYER
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Merrimack Pharmaceuticals, Inc.
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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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • This disclosure relates to nanoliposomes that deliver docetaxel, useful in the treatment of cancer in combination with immunotherapies.
  • Ephrin receptors are cell to cell adhesion molecules that mediate signaling and are implicated in neuronal repulsion, cell migration and angiogenesis.
  • EphA2 is part of the Ephrin family of cell-cell junction proteins highly overexpressed in several solid tumors.
  • Ephrin receptor A2 (EphA2) is overexpressed in several solid tumors including prostate, pancreatic, ovarian, gastric and lung cancer, and is associated with poor prognosis in certain cancer conditions.
  • Eph receptors are comprised of a large family of tyrosine kinase receptors divided into two groups (A and B) based upon homology of the N-terminal ligand binding domain.
  • Eph receptors are involved several key signaling pathways that control cell growth, migration and differentiation. These receptors are unique in that their ligands bind to the surface of neighboring cells.
  • the Eph receptors and their ligands display specific patterns of expression during development.
  • the EphA2 receptor is expressed in the nervous system during embryonic development and also on the surface of proliferating epithelial cells in adults. EphA2 also plays an important role in angiogenesis and tumor vascularization, mediated through the ligand ephrin Al .
  • EphA2 is overexpressed in a variety of human epithelial tumors including breast, colon, ovarian, prostate and pancreatic carcinomas. Expression of EphA2 can also be detected in tumor blood vessels stromal cells as well.
  • Immunotherapeutics have rapidly become an important option for patients and oncologists in the treatment of malignant diseases.
  • Immunotherapy uses the immune system to fight cancer and disease. When the immune system is working properly, it is able to identify foreign and harmful components within the body and eliminate them. For example, when patients are infected with the flu virus or a bacterial infection, the immune system will recruit white blood cells and other immune cells to the site of the infection. Once there, the different components of the immune system work together to target the foreign viruses or bacteria and remove them from the body.
  • Cancer cells use a variety of mechanisms to remain invisible to the immune system.
  • the cancer cells down-regulate cell surface markers or proteins that would normally be used by the immune system to recognize the cells as being foreign and therefore targeted for destruction.
  • Cancer cells and other cells within its microenvironment can also secrete soluble proteins, called cytokines or chemokines, which limit the function of immune cells.
  • cancer cells can recruit other cells to make the immune system think they are normal or "host" cells that should not be destroyed.
  • Tregs regulatory T cells
  • Tregs have modified this paradigm by recruiting Tregs to its microenvironment in order to make them invisible to the immune system.
  • Many studies have noted that elevated levels of Tregs within the tumor microenvironment are associated with poor responses to therapy and poor overall survival. Immunotherapeutics targeting Tregs have led to improvements in clinical response rates, thus suggesting the importance of this pathway in a subset of cancer types.
  • Cancer immunotherapy irrespective of treatment modality (i.e., adoptive cellular therapy, vaccines, monoclonal antibodies, and checkpoint inhibitors) and target (i.e., CD19, GD2, PD1, PD-L1, and Tregs), has shown promise within a subset of patients.
  • treatment modality i.e., adoptive cellular therapy, vaccines, monoclonal antibodies, and checkpoint inhibitors
  • target i.e., CD19, GD2, PD1, PD-L1, and Tregs
  • Applicants have discovered novel EphA2 targeted nanoliposomes for delivering docetaxel to tumors, capable of leveraging organ specificity through enhanced permeability and retention, as well as leveraging cellular specificity through an EphA2 targeting moiety covalently bound to the nanoliposome membrane.
  • novel docetaxel- based nanoliposomes targeted against Ephrin receptor A2 (EphA2) which is overexpressed in a wide range of tumors.
  • EphA2 -targeted docetaxel-generating liposomes provide sustained release of docetaxel following accumulation in solid tumors.
  • EphA2 -targeted docetaxel-generating liposomes leverage tumor-specific accumulation through the enhanced permeability and retention effect, and cellular specificity through active targeting of EphA2 with specific scFv antibody fragments conjugated to the surface of the liposomes.
  • EphA2 targeted docetaxel-generating nanoliposomes and second agent that impedes regulatory T cell activity wherein the cancer is treated.
  • the invention is a method of treating cancer in a patient, comprising administering to the patient an effective amount of a first agent that is a liposome- encapsulated taxane or taxane prodrug and a second agent that impedes regulatory T cell activity.
  • the liposome is targeted to a tumor antigen, e.g., EphA2.
  • the liposome is an EphA2-targeted docetaxel-generating liposome.
  • the EphA2-targeted docetaxel-generating liposome comprises a docetaxel prodrug.
  • the EphA2-targeted docetaxel-generating liposome comprising a docetaxel prodrug is encapsulated within a lipid vesicle comprising one or more lipids, a PEG lipid derivative and an EphA2 binding moiety on the outside of the lipid vesicle.
  • the docetaxel prodrug is a compound of Formula (I)
  • Rl and R2 are each independently H or lower alkyl, and n is an integer 2-3. In some embodiments, Rl and R2 are C1-C3 alkyl, and n is 3.
  • the EphA2 binding moiety is a scFv moiety comprising the CDRs of SEQ ID NO:40. In some embodiments, the EphA2 binding moiety is a scFv moiety comprising the sequence of SEQ ID NO:41 or SEQ ID NO: 46. [0015] In some embodiments, the lipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG. In some embodiments, the lipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG in a weight ratio of about 4.4: 1.6: 1.
  • the EphA2 -targeted docetaxel-generating liposome is 46scFv- ILs-DTXp3 or 46scFv-ILs-DTXp6.
  • the second agent is anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody.
  • the second agent is atezolizumab, durvalumab, or avelumab.
  • the second agent is selected from the group consisting of nivolumab, pembrolizumab, spartalizumab, camrelizuab, tiselizumab, cemiplimab and pidilizumab.
  • the second agent is ipilimumab, or tremelimumab, or lirilumab.
  • the cancer is treated. In some embodiments, wherein the cancer is breast, esophageal, prostate, ovarian, bladder, gastric, pancreatic, or lung cancer. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a sarcoma.
  • the invention is a method of treating cancer in a patient, comprising administering to the patient an effective amount of a first agent that is a taxane or taxane prodrug generating composition and a second agent that impedes regulatory T cell activity.
  • the taxane or taxane prodrug generating composition has a taxane or taxane prodrug release half-life in mouse of 10 hours or greater (e.g., 20 hours or greater, 30 hours or greater, 40 hours or greater, 50 hours or greater, 60 hours or greater, 70 hours or greater, or 80 hours or greater).
  • the taxane or taxane prodrug generating composition is a liposome-encapsulated taxane or taxane prodrug.
  • the liposome is targeted to a tumor antigen, e.g., EphA2.
  • the liposome is an EphA2-targeted docetaxel-generating liposome.
  • the EphA2-targeted docetaxel-generating liposome comprises a docetaxel prodrug.
  • the EphA2-targeted docetaxel-generating liposome comprising a docetaxel prodrug is encapsulated within a lipid vesicle comprising one or more lipids, a PEG lipid derivative and an EphA2 binding moiety on the outside of the lipid vesicle.
  • the docetaxel prodrug is a compound of Formula (I)
  • Rl and R2 are each independently H or lower alkyl, and n is an integer 2-3. In some embodiments, Rl and R2 are C1-C3 alkyl, and n is 3.
  • the EphA2 binding moiety is a scFv moiety comprising the CDRs of SEQ ID NO:40. In some embodiments, the EphA2 binding moiety is a scFv moiety comprising the sequence of SEQ ID NO:41 or SEQ ID NO: 46.
  • the lipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG. In some embodiments, the lipid vesicle comprises sphingomyelin, cholesterol and PEG-DSG in a weight ratio of about 4.4: 1.6: 1.
  • the EphA2-targeted docetaxel-generating liposome is 46scFv- ILs-DTXp3 or 46scFv-ILs-DTXp6.
  • the EphA2-targeted docetaxel-generating liposome comprises a binding moiety that competes for binding to EphA2 with an scFv consisting of SEQ ID NO:40.
  • FIG. 1 A is a schematic of a docetaxel-generating liposome comprising an EphA2 binding moiety (anti-EphA2 scFv PEG-DSPE).
  • FIG. IB is a schematic showing the processes of docetaxel prodrug loading into a liposome comprising sucrose octasulfate (SOS) as a trapping agent, and the process of docetaxel generation.
  • SOS sucrose octasulfate
  • the insolubility of the salt in the liposome interior when combined with a low pH environment can stabilize the prodrug to reduce or prevent premature conversion to the active docetaxel.
  • FIG. 2A is a chemical reaction scheme for the synthesis of certain docetaxel prodrugs.
  • FIG. 2B is a chart showing selected examples of docetaxel prodrugs.
  • FIG. 3 A shows various CDR sequences useful in EphA2 binding moieties that can be used to prepare an EphA2 targeted docetaxel-generating nanoliposome composition.
  • FIG. 3B is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare an EphA2 targeted docetaxel-generating nanoliposome composition.
  • the DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.
  • FIG. 3C is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes.
  • the DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.
  • FIG. 3D is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes.
  • the DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.
  • FIG. 3E is an amino acid sequence and corresponding encoding DNA sequence for the scFv EphA2 binding moiety in an EphA2 targeted docetaxel-generating nanoliposome composition EphA2-ILs, used in Examples 2-9.
  • FIG. 3F is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes.
  • the DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.
  • FIG. 3G is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes.
  • the DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.
  • FIG. 3H is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes.
  • the DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.
  • FIG. 31 is an amino acid sequence and corresponding encoding DNA sequence for the scFv that can be used to prepare EphA2 -targeted docetaxel-generating liposomes.
  • the DNA sequence further encodes an N-terminal leader sequence that is cleaved off by mammalian (e.g., human or rodent) cells expressing the encoded scFv.
  • FIGS 4A and 4B are graphs showing single tumor growth curves from all the experimental groups in EMT-6 tumor model.
  • FIG. 4 A shows graphs showing saline control and 46scFv-ILs-DTXp3
  • FIG. 4B shows graphs of PD-1 monotherapy and the combination of 46scFv-ILs-DTXp3 with PD-1 and also a rechallenge saline control.
  • FIG. 4C is a Kaplan-Meier plot illustrating time to regrowth for all the experimental groups in EMT-6 tumor model comparing 46scFv-ILs-DTXp3, PD-1 monotherapy to
  • FIG. 5A is a graph showing single tumor growth curves from all the experimental groups in EMT-6 tumor model comparing 46scFv-ILs-DTXp3, PD-1, docetaxel monotherapy to combination with PD-1
  • FIG. 5B is a graph showing maximum tumor regression from all the experimental groups in EMT-6 tumor model comparing 46scFv-ILs-DTXp3, PD-1, docetaxel monotherapy to combination with PD-1
  • FIG. 6A is a chart showing the percentage of CD8+ T cells, out of all CD45+ cells, in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3, PD-1, docetaxel, PD- l/46scFv-ILs-DTXp3 or PD-1 /docetaxel.
  • FIG. 6B is a chart showing the percentage of CD8+ifny+ T cells, out of all CD45+ cells, in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3, PD-1, docetaxel, PD-l/46scFv-ILs-DTXp3 or PD-1 /docetaxel.
  • FIG. 6C is a chart showing the ratio of CD8+ T cells to Tregs in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3, PD-1, docetaxel, PD-l/46scFv-ILs-DTXp3 or PD-1 /docetaxel.
  • FIG. 7A is a chart showing the TGI of EMT-6 tumors from mice treated with 46scFv- ILs-DTXp3 or docetaxel given weekly (DTX-MTD) or three times a week (DTX-DD).
  • FIG. 7B is a chart showing the percentage of CD8+ T cells, out of all CD45+ cells, in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3 or docetaxel given weekly (DTX-MTD) or three times a week (DTX-DD). ** pO.01, *** pO.001
  • FIG. 7C is a chart showing the percentage of CD8+ifny+ T cells, out of all CD45+ cells, in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3 or docetaxel given weekly (DTX-MTD) or three times a week (DTX-DD).
  • FIG. 7D is a chart showing the ratio of CD8+ T cells to Tregs in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3 or docetaxel given weekly (DTX-MTD) or three times a week (DTX-DD).
  • FIG. 7E is a chart showing the percentage of CD4+ifny+ T cells, out of all CD45+ cells, in EMT-6 tumors from mice previously treated with 46scFv-ILs-DTXp3 or docetaxel given weekly (DTX-MTD) or three times a week (DTX-DD). * p ⁇ 0.05, ** p ⁇ 0.01
  • FIG. 8 is a chart showing the in vivo efficacy of treatment of A/J mice implanted with a SAIN murine cancer cell line with saline, 46scFv-ILs-DTXp3, PD-1, and 46scFv-ILs-DTXp3 in combination with PD-1.
  • FIG. 9A is a chart showing the in vivo efficacy of treatment of C57BL6/J mice implanted with MC38 murine cancer cells with saline, 46scFv-ILs-DTXp3 were treated with four tail vein injections (every 7 days), and mice receiving PD-1, PD-Ll, 46scFv-ILs-DTXp3 + PD-1 or 46scFv-ILs-DTXp3 + PD-Ll (dosed IP twice weekly).
  • FIG. 9B is a chart showing the in vivo efficacy of treatment of C57BL6/J mice implanted with MC38 murine cancer cells with saline, 46scFv-ILs-DTXp3, and DTX were treated with four tail vein injections (every 7 days), and mice receiving PD-1, DTX+ PD-1, 46scFv-ILs-DTXp3 + PD-1 or 46scFv-ILs-DTXp3 + PD-Ll (dosed IP twice weekly).
  • FIG. 10A is a chart showing the ratio of CD8+ T cells to Tregs in BALB/c mice implanted with murine breast cancer cell line-EMT-6 after treatment with saline, 46scFv-ILs DTXp3, NT-ILs-DTXp3, and 46scFv-ILs dosed to match the lipid content of the 46scFv-ILs- DTXp3 group.
  • FIG. 10B is a chart showing the percent of NK cells of CD45+ in BALB/c mice implanted with murine breast cancer cell line-EMT-6 after treatment with saline, 46scFv-ILs DTXp3, NT-ILs-DTXp3, and 46scFv-ILs dosed to match the lipid content of the 46scFv-ILs- DTXp3 group. *p ⁇ 0.05.
  • the efficacy of the current class of PD-1/PD-L1 antagonists can be limited by the immunogenicity of the tumor microenvironment.
  • chemotherapeutic agents including taxanes and anthracyclines can increase immunogenicity, resulting in therapeutic synergy with immune checkpoint inhibitors.
  • treatment with a taxane has been shown to increase the recruitment of CTLs and decrease immunosuppressive cells such as MDSCs and T-regs.
  • the immune-modulatory activity of paclitaxel has been shown to increase with prolonged exposure of the taxane at the tumor level, achieved through metronomic dosing.
  • MM-310 an Ephrin Receptor A2 (EphA2)-targeted antibody-directed nanotherapeutic (ADN) encapsulates a docetaxel prodrug.
  • EphA2 Ephrin Receptor A2
  • ADN antibody-directed nanotherapeutic
  • Combination therapy refers to simultaneous administration of at least two therapeutic agents to a patient or their sequential administration within a time period during which the first administered therapeutic agent is still present in the patient (e.g., in the patient's plasma or serum) when the second administered therapeutic agent is administered.
  • effective doses refers to an amount (administered in one or more doses) of an antibody, protein or additional therapeutic agent, which amount is sufficient to provide effective treatment to a subject in need thereof.
  • Effective treatment refers to a decrease or cessation in the symptoms of a disorder in the subject.
  • effective treatment may comprise, but is not limited to, one or more of the following results: reduction in size of one or more tumors, reduction in number of tumors in a subject, reduction in number of cancerous cells in a subject, inhibition of tumor growth in a subject, or prolongation of survival time for an animal with cancer.
  • compositions of the present disclosure for the treatment of the described conditions vary depending upon many different factors, including (but not limited to) means of administration, target site, physiological state of the patient, whether the patient is human or an animal, and other medications administered. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
  • EphA2 refers to Ephrin type-A receptor 2, also referred to as “epithelial cell kinase (ECK),” a receptor tyrosine kinase that can bind and be activated by Ephrin-A ligands.
  • ECK epihelial cell kinase
  • EphA2 can refer to any naturally occurring isoforms of EphA2.
  • the amino acid sequence of human EphA2 is recorded as GenBank Accession No. NP_004422.2.
  • mpk refers to mg per kg body weight in a dose
  • “Lower alkyl” means a Ci-C 6 linear or branched alkyl chain. EphA2-Targeted Liposomes for Delivery of Taxanes
  • non-targeted liposomes can be designated as "Ls" or "NT-Ls.”
  • Ls can refer to non-targeted liposomes with or without a docetaxel prodrug.
  • Ls-DTX refers to liposomes containing any suitable docetaxel prodrug, including equivalent or alternative embodiments to those docetaxel prodrugs disclosed herein.
  • NT -Ls-DTX refers to liposomes without a targeting moiety that encapsulate any suitable docetaxel prodrug, including equivalent or alternative embodiments to those docetaxel prodrugs disclosed herein.
  • Examples of non- targeted liposomes including a particular docetaxel prodrug can be specified in the format "Ls- DTXp[y]” or "NT-DTXp[y]” where [y] refers to a particular compound number specified herein.
  • Ls-DTXpl is a liposome containing the docetaxel prodrug of compound 1 herein, without an antibody targeting moiety.
  • targeted immunoliposomes can be designated as "ILs.”
  • ILs-DTXp refers to any embodiments or variations of the targeted docetaxel-generating immunoliposomes comprising a targeting moiety, such as a scFv.
  • the ILs disclosed herein refer to immunoliposomes comprising a moiety for binding a biological epitope, such as an epitope- binding scFv portion of the immunoliposome.
  • ILs recited herein refer to EphA2 binding immunoliposomes (alternatively referred to as "EphA2-ILs").
  • EphA2-ILs refers herein to immunoliposomes enabled by the present disclosure with a moiety targeted to bind to EphA2.
  • ILs include EphA2-ILs having a moiety that binds to EphA2 (e.g., using any scFv sequences that bind EphA2).
  • immunoliposomes include ILs-DTXp3, ILs-DTXp4, and ILs-DTXp6. Absent indication to the contrary, these include immunoliposomes with an EphA2 binding moiety and encapsulating docetaxel prodrugs of compound 3, compound 4 or compound 6, respectively ( Figure 2B).
  • EphA2-ILs can refer to and include immunoliposomes with or without a docetaxel prodrug (e.g., immunoliposomes encapsulating a trapping agent such as sucrose octasulfate without a docetaxel prodrug).
  • a docetaxel prodrug e.g., immunoliposomes encapsulating a trapping agent such as sucrose octasulfate without a docetaxel prodrug.
  • ILs immune-liposomes
  • DTXp docetaxel prodrug
  • the EphA2-targeted docetaxel-generating liposome is 46scFv- ILs-DTXp3 or 46scFv-ILs-DTXp6.
  • the EphA2 -targeted docetaxel- generating liposome is 46scFv-ILs-DTXp3 (also known as MM-310).
  • Figure 1 A is a schematic showing the structure of a PEGylated EphA2 targeted, nano- sized liposome (nanoliposome) encapsulating a docetaxel prodrug (e.g., having a liposome size on the order of about 100 nm).
  • the immunoliposome can include an Ephrin A2 (EphA2) targeted moiety, such as a scFv, bound to the liposome (e.g., through a covalently bound PEG- DSPE moiety).
  • EphA2 Ephrin A2
  • the PEGylated EphA2 targeted liposome encapsulating a docetaxel prodrug can be created by covalently conjugating single chain Fv (scFv) antibody fragments that recognize the EphA2 receptor to pegylated liposomes, containing docetaxel in the form of a prodrug described herein, resulting in an immunoliposomal drug product.
  • scFv single chain Fv
  • the lipid membrane can be composed of egg sphingomyelin, cholesterol, and 1,2-distearoyl-sn-glyceryl methoxypolyethylene glycol ether (PEG-DSG).
  • nanoliposomes can be dispersed in an aqueous buffered solution, such as a sterile pharmaceutical composition formulated for parenteral administration to a human.
  • the EphA2 targeted nanoliposome is preferably a unilamellar lipid bilayer vesicle, approximately 110 nm in diameter, which encapsulates an aqueous space which contains a compound of disclosed herein in a gelated or precipitated state, as sucrosofate (sucrose octasulfate) salt.
  • a docetaxel prodrug can be loaded at mildly acidic pH and entrapped in the acidic interior of liposomes, using an electrochemical gradient where it is stabilized in a non- soluble form. Upon release from the liposome, the docetaxel prodrug is subsequently converted to active docetaxel by simple base-mediated hydrolysis at neutral pH.
  • the PEGylated EphA2 targeted liposome encapsulating a Taxane or Taxane prodrug can encapsulate one or more suitable Taxane or taxane prodrugs (e.g., a docetaxel prodrugs).
  • a docetaxel prodrug comprises a weak base such as tertiary amine introduced to the 2' or 7 position hydroxyl group of docetaxel through ester bond to form a docetaxel prodrug.
  • Preferred 2'- docetaxel prodrugs suitable for loading into a liposome are characterized by comparatively high stability at acidic pH but convert to docetaxel at
  • the chemical environment of the 2'-ester bond can be tuned systematically to obtain docetaxel prodrugs that are stable at relatively low pH but will release free docetaxel rapidly at physiologic pH through hydrolysis.
  • Docetaxel prodrugs are loaded into liposome at relatively low pH by forming stable complexes with trapping agents such as polysulfated polyols, for example, sucrose octasulfate.
  • the trapping agent sucrose octasulfate can be included in the liposome interior, as a solution of its amine salt, such as diethylamine salt (DEA-SOS), or triethylamine salt (TEA-SOS).
  • amine salts of the trapping agents helps to create a transmembrane ion gradient that aids the prodrug loading into the liposome and also to maintain the acidic intraliposomal environment favorable for keeping the prodrug from premature conversion to docetaxel before the prodrug-loaded liposome reaches its anatomical target.
  • Encapsulation of docetaxel prodrugs inside liposome in such a way allows the practical application of pH triggered release of docetaxel upon release from the liposome within the body of a patient.
  • the liposome that encapsulates docetaxel-prodrug can be called docetaxel nanogenerator.
  • the docetaxel prodrug is a compound of formula (I), including
  • the docetaxel prodrug (DTX') compounds can form a pharmaceutically acceptable salt within the liposome (e.g., a salt with a suitable trapping agent such as a sulfonated polyol).
  • a suitable trapping agent such as a sulfonated polyol.
  • docetaxel prodrugs also include docetaxel analog compounds having a 2' substituents -0-(CO)-(CH2)nN(Rl)(R2) in formula (I) that are substituted in the manner disclosed in formula (III) of U.S. Patent 4,960,790 to Stella et al. (filed March 9, 1989), incorporated herein by reference in its entiret .
  • the docetaxel prodrugs can be prepared using the reaction Scheme in Figure 2A. Specific Examples of docetaxel prodrugs are shown in Figure 2B. Other examples of docetaxel prodrugs include 2'-(2-(N,N'-diethylamino)propionyl)- docetaxel or 7-(2-(N,N'-diethylamino)propionyl)-docetaxel.
  • Preferred docetaxel prodrug compounds of formula (I) include compounds where (n) is 2 or 3, to provide a rapid hydrolysis rate at pH 7.5 and a sufficiently high relative hydrolysis rate for the compound at pH 7.5 compared to pH 2.5 (e.g., selecting docetaxel prodrugs with maximum hydrolysis rate of the docetaxel prodrug to docetaxel at pH 7.5 compared to the hydrolysis rate at pH 2.5).
  • the docetaxel-generating liposome can comprise a tumor antigen targeting moiety, e.g., an EphA2 targeting moiety.
  • tumor antigens include alpha-actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733, BAGE, bcl-2, bcl-6, BCMA, BrE3- antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX (CAIX), 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, CD70L
  • the EphA2 targeting moiety is a binding agent that competes for binding with the scFv D2-1 A7 (SEQ ID NO:41).
  • the targeting moiety can be a single chain Fv ("scFv"), a protein that can be covalently bound to a liposome to target the docetaxel-producing liposomes disclosed herein.
  • the scFv can be comprised of a single polypeptide chain in which a VH and a VL are covalently linked to each other, typically via a linker peptide that allows the formation of a functional antigen binding site comprised of VH and VL CDRs.
  • An Ig light or heavy chain variable region is composed of a plurality of "framework" regions (FR) alternating with three hypervariable regions, also called “complementarity determining regions” or “CDRs".
  • the extent of the framework regions and CDRs can be defined based on homology to sequences found in public databases. See, for example, "Sequences of Proteins of Immunological Interest," E. Kabat et al., Sequences of proteins of immunological interest, 4th ed. U.S. Dept. Health and Human Services, Public Health Services, Bethesda, MD (1987). All scFv sequence numbering used herein is as defined by Kabat et al.
  • anti-EphA2 scFv refers to an scFv that immunospecifically binds to EphA2, preferably the ECD of EphA2.
  • An EphA2-specific scFv does not immunospecifically bind to antigens not present in EphA2 protein.
  • an scFv disclosed herein includes one or any combination of VH FR1, VH FR2, VH FR3, VL FR1, VL FR2, and VL FR3 set forth in Table A. In one embodiment, the scFv contains all of the frameworks of Table A below.
  • VH FR1 (SEQ ID NO: 1) Q VQL VQ S GGGL VQP GGSLRL S C A AS GF TF S
  • VH FR2 (SEQ ID NO: 2) WVRQAPGKGLEWVT
  • VH FR3 (SEQ ID NO: 3) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
  • VL FR1 (SEQ ID NO: 5) SSELTQPPSVSVAPGQTVTITC
  • VL FR2 (SEQ ID NO: 6) WYQQKPGTAPKLLIY
  • VL FR3 (SEQ ID NO: ⁇ GVPDRF SGS S SGTS ASLTITGAQ AEDEAD YYC
  • an scFv disclosed herein is thermostable, e.g., such that the scFv is well-suited for robust and scalable manufacturing.
  • a "thermostable" scFv is an scFv having a melting temperature (Tm) of at least about 70°C, e.g., as measured using differential scanning fluorimetry (DSF).
  • a preferred anti-EphA2 scFv binds to the extracellular domain of EphA2 polypeptide, i.e., the part of the EphA2 protein spanning at least amino acid residues 25 to 534 of the sequence set forth in GenBank Accession No. NP_004422.2 or UniProt Accession No. P29317.
  • an anti-EphA2 scFv disclosed herein includes a VH CDRl, VH CDR2, VH CDR3, VL CDRl, VL CDR2, and VL CDR3 each with a sequence as set forth in Table B.
  • VH CDR2 sequence also referred to as CDRH2
  • CDRH2 will be any one selected from the 18 different VH CDR2 sequences set forth in Table B.
  • VH CDRl (SEQ ID NO: 10) SYAMH
  • VH CDR2 (SEQ ID NO: 11) VISPAGNNTYYADSVK
  • VH CDR2 (SEQ ID NO: 12) VISPAGRNKYYADSVK
  • VH CDR2 (SEQ ID NO: 13) VISPDGHNTYYADSVKG
  • VH CDR2 (SEQ ID NO: 14) VISPHGRNKYYADSVK
  • VH CDR2 (SEQ ID NO: 15) VISRRGDNK YYAD S VK
  • VH CDR2 (SEQ ID NO: 16) VISNNGHNK YYAD S VK
  • VH CDR2 (SEQ ID NO: 17) VISPAGPNTYYADSVK
  • VH CDR2 (SEQ ID NO: 18) VISPSGHNTYYADSVK
  • VH CDR2 (SEQ ID NO: 19) VISPNGHNT YYAD S VK
  • VH CDR2 (SEQ ID NO: 20) AISPPGHNTYYADSVK
  • VH CDR2 (SEQ ID NO: 21) VISPTGANT YYAD S VK
  • VH CDR2 (SEQ ID NO: 22) VISPHGSNKYYADSVK
  • VH CDR2 (SEQ ID NO: 23) VISNNGHNT YYAD S VK
  • VH CDR2 (SEQ ID NO: 24) VISP AGTNT YYAD S VK VH CDR2 (SEQ ID NO: 25) VISPPGHNTYYADSVK VH CDR2 (SEQ ID NO: 26) VISHDGTNTYYADSVK VH CDR2 (SEQ ID NO: 27) VISRHGNNK YYAD S VK VH CDR2 (SEQ ID NO: 28) VIS YDGSNK YYAD SVKG VH CDR3 (SEQ ID NO: 29) ASVGATGPFDI VL CDR1 (SEQ ID NO: 30) QGDSLRSYYAS VL CDR2 (SEQ ID NO: 31) GENNRPS VL CDR3 (SEQ ID NO: 39) NSRDSSGTHLTV
  • an scFv disclosed herein is an internalizing anti-EphA2 scFv. Binding of such an scFv to the ECD of and EphA2 molecule present on the surface of a living cell under appropriate conditions results in internalization of the scFv. Internalization results in the transport of an scFv contacted with the exterior of the cell membrane into the cell-membrane- bound interior of the cell. Internalizing scFvs find use, e.g., as vehicles for targeted delivery of drugs, toxins, enzymes, nanoparticles (e.g., liposomes), DNA, etc., e.g., for therapeutic applications.
  • scFvs described herein are single chain Fv scFvs e.g., scFvs or (scFv')2s.
  • the VH and VL polypeptides are joined to each other in either of two orientations (i.e., the VH N-terminal to the VL, or the VL N-terminal to the VH) either directly or via an amino acid linker.
  • a linker may be, e.g., from 1 to 50, 5 to 40, 10 to 30, or 15 to 25 amino acids in length.
  • 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100% of the residues of the amino acid linker are serine (S) and/or glycine (G).
  • Suitable exemplary scFv linkers comprise or consist of the sequence:
  • An exemplary internalizing anti-EphA2 scFv is scFv TS1 (SEQ ID NO:40 ( Figure 3 A)).
  • VH of the scFv is at the amino terminus of the scFv and is linked to the VL by a linker indicated in italics.
  • the CDRs of the scFvs are underlined and are presented in the following order: VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3.
  • the docetaxel-generating EphA2 -targeted liposomes can also include one or more
  • VH CDR2 is selected from any of the 18 different CDRH2 sequences set forth above in Table B.
  • the scFvs disclosed herein may be prepared using standard techniques.
  • the amino acid sequences provided herein can be used to determine appropriate nucleic acid sequences encoding the scFvs and the nucleic acids sequences then used to express one or more of the scFvs .
  • the nucleic acid sequence(s) can be optimized to reflect particular codon "preferences" for various expression systems according to standard methods.
  • nucleic acids may be synthesized according to a number of standard methods. Oligonucleotide synthesis, is conveniently carried out on commercially available solid phase oligonucleotide synthesis machines or manually synthesized using, for example, the solid phase phosphoramidite triester method. Once a nucleic acid encoding an scFv disclosed herein is synthesized, it can be amplified and/or cloned according to standard methods.
  • Expression of natural or synthetic nucleic acids encoding the scFvs disclosed herein can be achieved by operably linking a nucleic acid encoding the scFv to a promoter (which may be constitutive or inducible), and incorporating the construct into an expression vector to generate a recombinant expression vector.
  • the vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both.
  • Typical cloning vectors contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the scFv.
  • the vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.
  • the EphA2 Targeted scFv Amino Acid Sequence can be attached to the liposome using an EphA2 (scFv) to maleimide-activated PEG-DSPE.
  • the scFv-PEG-DSPE drug substance can be a fully humanized single chain antibody fragment (scFv) conjugated to maleimide PEG-DSPE via the C-terminal cysteine residue of scFv.
  • the EphA2 targeted scFv is conjugated covalently through a stable thioether bond to a lipopolymer lipid, Mal-PEG-DSPE, which interacts to form a micellular structure.
  • the scFv is not glycosylated.
  • agents that impede regulatory T cell activity include small molecule immunomodulatory agents, radiation, anticancer vaccines, and
  • Tregs formerly known as suppressor T cells
  • Tregs are a subpopulation of T cells which modulate the immune system, suppress immune responses against other cells and maintain tolerance to self-antigens. Generally, these cells suppress or downregulate the induction and proliferation of effector T cells. Therefore, when regulatory T cell activity is impeded, the immune response suppressive activities of these cells will be lessened or removed.
  • Immunomodulatory antibodies include, but are not limited to, a human cytotoxic T- lymphocyte antigen 4 (CTLA-4)-blocking antibody such as ipilimumab, and a human T- lymphocyte antigen 4 (CTLA-4)-blocking antibody such as ipilimumab, and a human TLA-4 (CTLA-4)-blocking antibody such as ipilimumab, and a human TLA-4 (CTLA-4)-blocking antibody such as ipilimumab, and a human T- lymphocyte antigen 4 (CTLA-4)-blocking antibody such as ipilimumab, and a human T- lymphocyte antigen 4 (CTLA-4)-blocking antibody such as ipilimumab, and a human T- lymphocyte antigen 4 (CTLA-4)-blocking antibody such as ipilimumab, and a human T- lymphocyte antigen 4 (CTLA-4)-blocking antibody such as ipilimumab
  • PD-1 antibody such as nivolumab.
  • Other Immunomodulatory antibodies include but are not limited to, the group consisting of (a) an agonistic anti-receptor antibody that immunospecifically binds human OX40, CD40,GITR, CD27, ICOS, or 4- IBB; (b) an antagonistic anti-receptor antibody that immunospecifically binds human CTLA-4 (cytotoxic neutrophils)
  • T-lymphocyte-associated protein 4 also known as CD 152
  • PD-1 programmed cell death protein 1, also known as CD279
  • PD-Ll programmeed death-ligand 1, also known as CD274 or B7-H1
  • TIM-3 BTLA
  • VISTA LAG-3
  • KIR KIR
  • an anti-ligand antibody that blocks the function of IL-6, IL-10, TGFP, angiopoetin-2, VEGF, IL-17, IL-23, or TNF alpha
  • atezolizumab avelimumab, , pembrolizumab, tremelimumab and/or durvalumab.
  • the anti-CTLA-4 antibody is ipilimumab or tremelimumab.
  • the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, spartalizumab, camrelizuab, tiselizumab, cemiplimab, and pidilizumab.
  • the anti-PD-Ll antibody is selected from the group consisting of atezolizumab, durvalumab, and avelumab.
  • the anti-KIR antibody is lirilumab.
  • the immunotherapy can include molecules that bind to CTLA4, PDL1, PD1, 41BB and/or OX40 including the publicly available compounds in the table below or other compounds that bind to the same epitope or have the same or similar biological functions.
  • the agents that impede regulatory T cell activity are anticancer vaccines selected from, but is not limited to, the group consisting of OncoVex, MAGE- A3, PROSTVAC, GVAX, CDX110, CDX1307, CDX1401, CV9104, BIOVAXID, IMA 901, and ADXS 11-001.
  • the use of a combination of an EphA2 -targeted docetaxel-generating liposome and an immunotherapy can be used for the treatment of cancer in a host in need thereof, in an amount and in a schedule of administration that is therapeutically synergistic in the treatment of the cancer.
  • the cancer is breast, esophageal, prostate, ovarian, bladder, gastric, pancreatic or lung cancer.
  • the cancer is triple negative breast cancer (TNBC).
  • the cancer is a sarcoma.
  • the cancer is a solid tumor.
  • the cancer is gastric cancer, gastroesophageal junction cancer, or esophageal carcinoma.
  • the cancer is esophageal cancer.
  • the cancer is non-small cell lung cancer, or small cell lung cancer.
  • the cancer is ovarian cancer.
  • the cancer is prostate cancer.
  • the cancer is ovarian cancer, endometrial carcinoma, or urothelial carcinoma.
  • the cancer is prostate adenocarcinoma.
  • the cancer is soft tissue carcinoma or squamous cell carcinoma of the head and neck (SCCHN).
  • the cancer is pancreatic ductal adenocarcinoma
  • EMT-6 (ATCC CRL-2755) is a mouse mammary carcinoma cell line
  • CT-26 (ATCC CRL-2638) is a mouse colon carcinoma cell line
  • LLC (ATCC CRL-1642) is a lung carcinoma cell line.
  • In vivo activity studies and immune-phenotype studies were performed comparing 46scFv-ILs-DTXp3 + anti-PD-1 combination to the monotherapies.
  • 46scFv-ILs-DTXp3 administration was initiated two days prior to anti-PD-1 therapy and consisted of four weekly doses, while anti-PD-1 was dosed twice weekly for four weeks.
  • 46scFv-ILs-DTXp3 was administered intravenously at 50 mg/kg (docetaxel equivalent) Q7D x 4.
  • the anti-PDl antibody was administered at 10 mg/kg twice weekly for four weeks.
  • the response to 46scFv-ILs-DTXp3 or anti-PD-1 as monotherapies varied between the models.
  • Example 2 In vivo antitumor efficacy of 46scFv-ILs-DTXp3 in combination with a murine PD-1 antibody in EMT-6 murine breast cancer tumor model in mice
  • mice of strain BALB/c were implanted with murine breast cancer cell line EMT-6 (3.6x10 ⁇ 5 cells/mouse). Animals were randomized into the following experimental groups: Saline, anti-PD-1 murine antibody at 10 mg/kg twice a week, 46scFv-ILs- DTXp3 at 50 mg/kg, and PD-l/46scFv-ILs-DTXp3. For the combination group doses used for the monotherapy arms were combined and PD-1 dosing was initiated 2 days following 46scFv- ILs-DTXp3.
  • Tumor volume (TV) [(length) x (width)2] / 2
  • Max tumor regression [(TVmin - TVdayO) / TVdayO] x 100
  • Maximum tumor regression was classified as complete tumor regression (100% regression with no palpable tumor), partial tumor regression (max tumor regression more than 30%) or no tumor regression.
  • PD-1 showed minimal growth arrest and no tumor regression when given as monotherapies.
  • 46scFv-ILs-DTXp3 did induce partial regression in most animals with complete regression in 20% (2/10) animals.
  • a combination of 46scFv-ILs-DTXp3 and PD-1 significantly increased the tumor doubling time, in addition to inducing complete regression in 60% (6/10) of treated animals.
  • Time to regrowth shows that the complete regression seen in the animals treated with 46scFv-ILs-DTXp3 monotherapy or 46scFv-ILs-DTXp3 combined with PD-1 was durable with no regrowth up to day 98.
  • mice that had completely regressed tumors were rechallenged with a inoculation of EMT-6 cells (3.6X10 ⁇ 5 cells), identical to the initial induction dose. Additionally, a cohort of naive littermate mice were also given an injection of EMT-6 cells. Mice from the littermate control group rapidly grew tumors. However, both mice from the 46scFv-ILs-DTXp3 monotherapy group and 5 of the 6 mice in the PD-l/46scFv-ILs-DTXp3 group were resistant to rechallenge. The resistance to rechallenge indicates immune involvement and potential immunity.
  • Example 3 In vivo antitumor efficacy of 46scFv-ILs-DTXp3 when compared to free docetaxel monotherapy and in combination with a murine PD-1 antibody in EMT-6 murine breast cancer tumor model in mice
  • mice of strain BALB/c were implanted with murine breast cancer cell line EMT-6 (3.5x10 ⁇ 5). Animals were randomized into the following experimental groups: Saline, anti-PD-1 murine antibody at 10 mg/kg twice weekly, free docetaxel at 10 mg/kg qlw, 46scFv-ILs-DTXp3 at 50 mg/kg qlw, PD-l/docetaxel, and PD-l/46scFv-ILs-DTXp3. For the combination group doses used for the monotherapy arms were combined and PD-1 dosing was initiated 2 days following docetaxel or 46scFv-ILs-DTXp3.
  • Tumor volume (TV) [(length) x (width)2] / 2
  • Max tumor regression [(TVmin - TVdayO) / TVdayO] x 100
  • PD-1 and DTX showed minimal growth arrest and no tumor regression when given as monotherapies.
  • PD-1 and docetaxel showed improved activity, but limited tumor regression.
  • 46scFv-ILs-DTXp3 did induce partial regression in most animals.
  • a combination of 46scFv-ILs-DTXp3 and PD-1 significantly increased the tumor doubling time, in addition to inducing partial regression in 60% of treated animals and complete regression in 20% (2/10) of treated animals.
  • This study shows that 46scFv-ILs-DTXp3 has superior in vivo activity in comparison to an equitoxic dose of docetaxel both in monotherapy or in combination with PD-1 when evaluated against EMT-6 tumors.
  • Example 4 In vivo antitumor efficacy of 46scFv-ILs-DTXp3 in combination with a murine PD-1 antibody in murine models of lung and colon cancer in mice
  • mice of strains C57BL6/J and BALB/c were implanted with murine cancer cell lines LLC (5x 10 ⁇ 5 cells/mouse, lung cancer) or CT26 (lx 10 ⁇ 6 cells/mouse, colon cancer) respectively.
  • Animals were randomized into the following experimental groups: Saline, anti-PD-1 murine antibody at 10 mg/kg twice a week, 46scFv-ILs-DTXp3 at 50 mg/kg, and PD-l/46scFv-ILs-DTXp3.
  • For the combination group doses used for the monotherapy arms were combined and PD-1 dosing was initiated 2 days following 46scFv-ILs-DTXp3.
  • Tumor volume (TV) [(length) x (width)2] / 2
  • TGI Tumor growth inhibition
  • the activity of the monotherapy groups varied between the two models.
  • PD-1 inhibition had a negative effect on tumor growth, and 46scFv-ILs-DTXp3 had a moderately positive effect.
  • PD-1 inhibition was able to inhibit the growth of CT26 tumors, and the effect of 46scFv-ILs-DTXp3 was smaller in comparison.
  • PD-l/46scFv-ILs- DTXp3 was superior in both models, which illustrates the activity of the combination in multiple models regardless of the activity of the monotherapies.
  • Example 5 Effects of 46scFv-ILs-DTXp3, free docetaxel monotherapy and PD-1 combination on immune cell infiltrate in EMT-6 mure breast tumor model
  • combination group doses used for the monotherapy arms were combined and PD-1 dosing was initiated 2 days following docetaxel or 46scFv-ILs-DTXp3.
  • Animals receiving saline or 46scFv- ILs-DTXp3 received two tail vein injections, at an interval of 7 days.
  • Mice receiving PD-1 were dosed IP twice a week. All mice completed two treatment cycles before the tumors were harvested and prepared for analysis using fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • mice 48 hourse after the second dose of either saline, docetaxel, or 46scFv-ILs-DTXp3 the mice were euthanized and the tumors were removed. The tumors were homogenized using a combination of mechanical and enzymatic cell dissociation before being filtered into a suspension of single cells. Antibodies were used to identify total immune cells (CD45), T cells (CD3), T cell subsets (CD8, CD4, and FOXP3), as well as the inflammatory capacity of the T cells (IFNy).
  • CD45 total immune cells
  • T cells CD3
  • T cell subsets CD8, CD4, and FOXP3
  • IFNy the inflammatory capacity of the T cells
  • CD8+ T cells are also referred to as cytoxic T cells and are primarily responsible for cell killing.
  • T cells also express interferony (IFN réelle) it indicates that they are responding to antigen-specific immune signals and, in this context, acting to kill tumor cells.
  • IFN dilemma interferony
  • An additional measure that indicates the creation of an antitumor immune response is the ratio of CD8+ T cells to regulatory T cells (Tregs). Increases in the ratio show that the balance is shifted towards antitumor immunity and away from immune protection. The ratio of CD8+ T cells to Tregs is increased in mice that received 46scFv-ILs-DTXp3, indicating the enhancement of an antitumor immune response (Figure 6C).
  • mice of strain BALB/c were implanted with murine breast cancer cell line EMT-6 (3.5x10 ⁇ 5). Animals were randomized in cohorts of 10 into the following experimental groups: Saline, docetaxel 10mg/kg once a week (DTX-MTD), docetaxel 3.33mg/kg three times a week (DTX-DD), and 46scFv-ILs-DTXp3 at 50 mg/kg.
  • DTX-MTD docetaxel 10mg/kg once a week
  • DTX-DD docetaxel 3.33mg/kg three times a week
  • 46scFv-ILs-DTXp3 at 50 mg/kg.
  • Tumor volume (TV) [(length) x (width)2] / 2
  • TGI Tumor growth inhibition
  • mice 48 hours after the third dosing cycle of either saline, docetaxel, or 46scFv-ILs-DTXp3 the mice were euthanized and the tumors were removed. The tumors were homogenized using a combination of mechanical and enzymatic cell dissociation before being filtered into a suspension of single cells. Antibodies were used to identify total immune cells (CD45), T cells (CD3), T cell subsets (CD8, CD4, and F0XP3), as well as the inflammatory capacity of the T cells (ifny).
  • Figure 7 A shows the tumor growth inhibition for all groups in this study. Mice receiving 46scFv-ILs-DTXp3 showed more growth inhibition than mice receiving docetaxel in both dosing schedules. DTX-DD improved growth inhibition relative to DTX-MTD, but it did not recapitulate the inhibition observed in 46scFv-ILs-DTXp3 treatment.
  • FACS was used to compare the immune response generated after treatment with 46scFv-ILs-DTXp3 or docetaxel given in two different dosing schemes.
  • treatment with 46scFv-ILs-DTXp3 increased CD8+ T cell and CD8+ ifny+ T cell populations in the tumor, in addition to increasing the CD8+/Treg ratio ( Figure 7B, 7C, and 7D).
  • treatment with 46scFv-ILs-DTXp3 also increased CD4+ ⁇ FNy+ T cells in the tumor ( Figure 7E). Neither the dose-dense (DTX-DD) nor the weekly (DTX-MTD) docetaxel treatment groups were observed to affect these intratumoral immune cell populations in this study.
  • Example 7 In vivo antitumor efficacy of 46scFV-ILs-DTXp3 in combination with a murine PD-1 antibody in a murine model of sarcoma cancer in mice
  • Tumor volume (TV) [(length) x (width)2] / 2
  • Max tumor regression [(TVmin - TVdayO) / TVdayO] x 100 [00133] Maximum tumor regression was classified as complete tumor regression (100% regression with no palpable tumor), partial tumor regression (max tumor regression more than 30%) or no tumor regression.
  • TGI Tumor growth inhibition
  • Tumor growth inhibition is shown in the table below.
  • the PD-l/46scFv-ILs-DTXp3 group showed increased activity when compared to the saline and monotherapy groups.
  • PD-1 inhibition and treatment with 46scFv-ILs-DTXp3 as a monotherapy showed similar growth inhibition.
  • Example 8 In vivo antitumor efficacy of 46scFV-ILs-DTXp3 when compared to free docetaxel and in combination with murine PD-1 and PD-L1 antibodies in a murine model of colon cancer in mice
  • Tumor volume (TV) [(length) x (width)2] / 2
  • Tumor growth inhibition is defined as the average tumor size relative to the control group at the last time point before any mouse was sacrificed due to tumor burden. Animals sacrificed prior to tumor volume doubling are censored.
  • Tumor growth inhibition is shown in the table below.
  • the PD-l/46scFv-ILs-DTXp3 group showed increased activity when compared to the saline and all monotherapy groups.
  • 46scFv-ILs-DTXp3 treatment both alone and in combination with PD-1 improved growth inhibition relative to DTX and PD-l/DTX groups.
  • Combinations with 46scFv-ILs-DTXp3 and PD-1 or PD-L1 also improved survival relative to PD-l/46scFv-ILs-DTXp3 monotherapy.
  • the PD-Ll/46scFv-ILs-DTXp3 treatment resulted in a 10% complete response rate.
  • mice of strain BALB/c were implanted with murine breast cancer cell line EMT-6 (3.5x10 ⁇ 5).
  • Animals were randomized in cohorts of 10 into the following experimental groups: Saline, 46scFv-ILs-DTXp3 at 50 mg/kg, NT-ILs-DTXp3 at 50 mg/kg, and 46scFv-ILs dosed to match the lipid content of the 46scFv-ILs-DTXp3 group.
  • the NT-ILs- DTXp3 group is identical to 46scFv-ILs-DTXp3 but is lacking the antibody fragment.
  • the 46scFv-ILs group is identical to the 46scFv-ILs-DTXp3 but does not contain the docetaxel prodrug.
  • Tumor volume (TV) [(length) x (width)2] / 2
  • TGI Tumor growth inhibition
  • mice 48 hours after the third dosing cycle of either saline, docetaxel, or 46scFv-ILs-DTXp3 the mice were euthanized and the tumors were removed. The tumors were homogenized using a combination of mechanical and enzymatic cell dissociation before being filtered into a suspension of single cells. Antibodies were used to identify total immune cells (CD45), T cells (CD3), T cell subsets (CD8, CD4, and FOXP3).
  • FACS was used to compare the immune response generated after treatment with 46scFv-ILs-DTXp3, NT-ILs-DTXp3, or 46scFv-ILs.
  • treatment with 46scFv-ILs-DTXp3 increased CD8+ T cell and CD8+ ifny+ T cell populations in the tumor, in addition to increasing the CD8+/Treg ratio (see Figures 7B, 7C, and 7D).
  • the increase in CD8+/Treg ratio was observed only in the 46scFv-ILs-DTXp3 and the NT-ILs- DTXp3 groups ( Figure 10A).
  • An increase the in natural killer T cell (NK) population was also only observed in the 46scFv-ILs-DTXp3 and the NT-ILs-DTXp3 groups ( Figure 10B).

Abstract

L'invention concerne des méthodes de traitement d'un patient atteint d'un cancer comprenant l'administration au patient d'une quantité efficace d'un premier agent qui est un taxane encapsulé dans des liposomes ciblé par un anticorps et d'un second agent qui entrave l'activité des lymphocytes T régulateurs.
PCT/US2018/020381 2017-03-01 2018-03-01 Liposomes générant du docétaxel ciblant epha2 en combinaison avec un agent qui entrave l'activité des lymphocytes t régulateurs pour le traitement du cancer WO2018160794A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4960790A (en) 1989-03-09 1990-10-02 University Of Kansas Derivatives of taxol, pharmaceutical compositions thereof and methods for the preparation thereof
US8790691B2 (en) 2008-05-23 2014-07-29 The University Of British Columbia Modified drugs for use in liposomal nanoparticles
WO2015095404A2 (fr) * 2013-12-17 2015-06-25 Genentech, Inc. Procédés de traitement de cancers à l'aide d'antagonistes se liant à l'axe pd-1 et de taxanes
WO2015153345A1 (fr) * 2014-04-03 2015-10-08 Invictus Oncology Pvt. Ltd. Agents thérapeutiques combinatoires supramoléculaires
WO2017161069A1 (fr) 2016-03-16 2017-09-21 Merrimack Pharmaceuticals, Inc Ciblage nanoliposomal des récepteurs a2 de l'éphrine (epha2) et diagnostics associés
WO2017161071A1 (fr) * 2016-03-16 2017-09-21 Merrimack Pharmaceuticals, Inc Traitement du cancer positif au récepteur de l'éphrine a2 (epha2) avec des compositions de nanoliposomes ciblés de génération de docétaxel
US20180021294A1 (en) 2016-03-16 2018-01-25 Merrimack Pharmaceuticals, Inc. Ephrin Receptor A2 (EPHA2)-Targeted Docetaxel-Generating Nano-Liposome Compositions

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4960790A (en) 1989-03-09 1990-10-02 University Of Kansas Derivatives of taxol, pharmaceutical compositions thereof and methods for the preparation thereof
US8790691B2 (en) 2008-05-23 2014-07-29 The University Of British Columbia Modified drugs for use in liposomal nanoparticles
WO2015095404A2 (fr) * 2013-12-17 2015-06-25 Genentech, Inc. Procédés de traitement de cancers à l'aide d'antagonistes se liant à l'axe pd-1 et de taxanes
WO2015153345A1 (fr) * 2014-04-03 2015-10-08 Invictus Oncology Pvt. Ltd. Agents thérapeutiques combinatoires supramoléculaires
WO2017161069A1 (fr) 2016-03-16 2017-09-21 Merrimack Pharmaceuticals, Inc Ciblage nanoliposomal des récepteurs a2 de l'éphrine (epha2) et diagnostics associés
WO2017161071A1 (fr) * 2016-03-16 2017-09-21 Merrimack Pharmaceuticals, Inc Traitement du cancer positif au récepteur de l'éphrine a2 (epha2) avec des compositions de nanoliposomes ciblés de génération de docétaxel
US20180021294A1 (en) 2016-03-16 2018-01-25 Merrimack Pharmaceuticals, Inc. Ephrin Receptor A2 (EPHA2)-Targeted Docetaxel-Generating Nano-Liposome Compositions

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ANDREW J. SAWYER ET AL: "Abstract 4711: Immunomodulatory effects of an EphA2-targeted docetaxel antibody-directed nanotherapeutic and synergistic combination with PD-1 inhibitor | Cancer Research", CANCER RESEARCH, vol. 77, no. 13 Suppl, 1 July 2017 (2017-07-01), US, pages 4711, XP055482966, ISSN: 0008-5472, DOI: 10.1158/1538-7445.AM2017-4711 *
DARYL C DRUMMOND ET AL: "Abstract 2069: Activity of an EphA2-targeted docetaxel nanoliposome in pancreatic patient-derived models as monotherapy and in combination with gemcitabine | Cancer Research", CANCER RESEARCH, vol. 76, no. 14 suppl, 15 July 2016 (2016-07-15), US, pages 2069, XP055482934, ISSN: 0008-5472, DOI: 10.1158/1538-7445.AM2016-2069 *
DARYL C. DRUMMOND: "Activity of an EphA2-targeted docetaxel nanoliposome in pancreatic patient-derived models as monotherapy and in combination with gemcitabine", 14 April 2016 (2016-04-14), XP055377910, Retrieved from the Internet <URL:http://files.shareholder.com/downloads/AMDA-IWDM2/0x0x886055/6A524EF8-BE65-400D-8381-B4E7F23473BE/AACR2016-_Activity_of_an_EphA2-targeted_Docetaxel_Nanoliposome_in_pancreatic_patient-derived_models.pdf> [retrieved on 20170601] *
DATABASE Gene [O] retrieved from ncbi Database accession no. NP_004422.2
DATABASE UniProt [O] Database accession no. P29317
DMITRI B KIRPOTIN ET AL: "Abstract 3912: MM-310, a novel EphA2-targeted docetaxel nanoliposome | Cancer Research", CANCER RESEARCH, vol. 76, no. 14 Suppl, 15 July 2016 (2016-07-15), US, pages 3912, XP055482943, ISSN: 0008-5472, DOI: 10.1158/1538-7445.AM2016-3912 *
DMITRI B. KIRPOTIN ET AL: "MM-310, a Novel EphA2-Targeted Docetaxel Antibody Directed Nanotherapeutic (ADN)", PROCEEDINGS: AACR 107TH ANNUAL MEETING 2016; APRIL 16-20, 2016, NEW ORLEANS, LA, 20 April 2016 (2016-04-20), XP055482945, Retrieved from the Internet <URL:http://files.shareholder.com/downloads/AMDA-IWDM2/0x0x886059/C615BB79-B535-4047-877A-E55443165817/AACR_2016_-_MM-310_a_novel_EphA2_targeted_docetaxel_liposome_-_Copy.pdf> [retrieved on 20180611] *
E. KABAT ET AL.: "Sequences of proteins of immunological interest", 1987, U.S. DEPT. HEALTH AND HUMAN SERVICES
GERAN, R.I. ET AL., CANCER CHEMOTHER. REP., vol. 3, 1972, pages 1 - 88
INTERNATIONAL IMMUNOLOGY, vol. 8, no. 5, pages 765 - 772
LORNA RETTIG ET AL: "Gemcitabine depletes regulatory T-cells in human and mice and enhances triggering of vaccine-specific cytotoxic T-cells", INTERNATIONAL JOURNAL OF CANCER, vol. 129, no. 4, 15 August 2011 (2011-08-15), US, pages 832 - 838, XP055482962, ISSN: 0020-7136, DOI: 10.1002/ijc.25756 *

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