WO2022043256A1 - Combinaisons synergiques de médicaments anticancéreux liés à une fraction tétrapeptidique et d'agents immunothérapeutiques - Google Patents

Combinaisons synergiques de médicaments anticancéreux liés à une fraction tétrapeptidique et d'agents immunothérapeutiques Download PDF

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WO2022043256A1
WO2022043256A1 PCT/EP2021/073259 EP2021073259W WO2022043256A1 WO 2022043256 A1 WO2022043256 A1 WO 2022043256A1 EP 2021073259 W EP2021073259 W EP 2021073259W WO 2022043256 A1 WO2022043256 A1 WO 2022043256A1
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compound
formula
dox
cancer
combination
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PCT/EP2021/073259
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Andrea CASAZZA
Lawrence Van Helleputte
Geert REYNS
Nele KINDT
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Cobiores Nv
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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
    • A61K47/54—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 organic compound
    • A61K47/548—Phosphates or phosphonates, e.g. bone-seeking
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • 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

  • the invention relates to the field of disease treatment, in particular cancer treatment.
  • the invention relates to therapeutic combinations of an anticancer drug linked to a tetrapeptidic moiety (anticancer prodrug) and an immunotherapeutic agent.
  • anticancer prodrug linked to a tetrapeptidic moiety
  • immunotherapeutic agent an immunotherapeutic agent
  • the cancer could be non-immunogenic and such "cold" tumors could be characterized by absence of sufficient proinflammatory cytokines and/or absence of sufficient infiltration or disbalanced infiltration of the appropriate T-cells into the tumor micro-environment and/or exhaustion and/or immune-suppression of the appropriate T-cells in the tumor micro-environment.
  • ICBs immunogenically cold tumors (not or poorly responding to ICBs) into immunogenically hot tumors (showing a response or an improved response to ICBs).
  • the invention relates to a compound of the formula C-OP-D for use in treating or inhibiting cancer or for use in inhibiting progression of cancer, in combination with an immune checkpoint inhibitor, wherein:
  • C is a capping group
  • OP is a tetrapeptidic moiety selected from the group consisting of ALGP (SEQ ID NO:1), TSGP (SEQ ID NO:2), KLGP (SEQ ID NO:3), and ALKP (SEQ ID NO:4)
  • D is a cytostatic drug, a cytotoxic drug or an anticancer drug
  • the combination is a combination in any appropriate way.
  • the invention further relates to an immune checkpoint inhibitor for use in treating or inhibiting cancer or for use in inhibiting progression of cancer, in combination with a compound of the formula C-OP-D, wherein:
  • C is a capping group
  • OP is a tetrapeptidic moiety selected from the group consisting of ALGP (SEQ ID NO:1), TSGP (SEQ ID NO:2), KLGP (SEQ ID NO:3), and ALKP (SEQ. ID NO:4)
  • D is a cytostatic drug, a cytotoxic drug or an anticancer drug
  • the combination is a combination in any appropriate way.
  • the invention further also relates to a compound of the formula C-OP-D and an immune checkpoint inhibitor for use in treating or inhibiting cancer or for use in inhibiting progression of cancer, wherein:
  • the combination of the compound of the formula C-OP-D and the immune checkpoint inhibitor is a combination in any appropriate way.
  • FIG. 1 E0771 tumor volume at day 17 after randomization / PhAc-ALGP-Dox & anti-PDl.
  • the plot represents the volume of orthotopically implanted E0771 tumors treated with corresponding different concentration of Dox, PhAc-ALGP-Dox, aPD-1 or aPD-Ll as indicated.
  • Mice received the treatment via tail vein (TV) injection twice a week (Dox or PhAc-ALGP-Dox) or via intraperitoneal (IP) injection three time a week (aPD-1, aPD-Ll, or an isotype control antibody).
  • FIG. 1 Tumor weight / PhAc-ALGP-Dox & anti-PDl.
  • FIG. 3 Tumor necrosis quantification / PhAc-ALGP-Dox & anti-PDl.
  • FIG. 4 Percentage of THOP1 positive region / PhAc-ALGP-Dox & anti-PDl.
  • FIG. 6 E0771 tumor volume at end term / PhAc-ALGP-Dox & anti-PDl.
  • the plot represents the volume of orthotopically implanted E0771 tumors treated with corresponding different concentration of Dox, PhAc-ALGP-Dox, aPD-1 alone or in combination as indicated.
  • Mice received the treatment via tail vein (TV) injection twice a week (Dox or PhAc-ALGP-Dox) or via intraperitoneal (IP) injection three time a week (aPD-1) as indicated by the arrowheads.
  • the indications "(2)" in the graph refers to time points during a given treatment on which 2 mice had to be sacrificed because of the tumor growing too big.
  • FIG. 7 E0771 relative tumor volume (RTV) / PhAc-ALGP-Dox & anti-PDl.
  • the plot represents the RTV of orthotopically implanted E0771 tumors treated with corresponding different concentration of Dox, PhAc-ALGP-Dox, aPD-1 alone or in combination as indicated.
  • Dotted line shows tumor delay (Td) calculated as the time required by tumors to reach a volume five times bigger than the initial one (RTV5).
  • FIG. 9 E0771 tumor volume / PhAc-ALGP-Dox & anti-CTLA4.
  • the plot represents the volume of orthotopically implanted E0771 tumors treated with corresponding different concentration of Dox, PhAc-ALGP-Dox, aCTLA-4 alone or in combination as indicated.
  • Mice received the treatment via tail vein (TV) injection twice a week (Dox or PhAc-ALGP-Dox) or via intraperitoneal (IP) injection three times a week (aCTLA-4) as indicated by the arrowheads.
  • FIG. 10 E0771 relative tumor volume (RTV) / PhAc-ALGP-Dox & anti-CTLA4.
  • the plot represents the RTV of orthotopically implanted E0771 tumors treated with corresponding different concentration of Dox, PhAc-ALGP-Dox, aCTLA-4 alone or in combination as indicated.
  • Dotted line shows tumor delay (Td) calculated as the time required by tumors to reach a volume five times bigger than the initial one (RTV5).
  • FIG. 11 E0771 tumor volume / PhAc-ALKP-Dox & anti-PDl.
  • the plot represents the volume of orthotopically implanted E0771 tumors treated with corresponding different concentration of PhAc- ALKP-Dox, PhAc-ALGP-Dox, aPD-1 alone or in combination as indicated.
  • Mice received the treatment via tail vein (TV) injection twice a week (PhAc-ALKP-Dox or PhAc-ALGP-Dox) or via intraperitoneal (IP) injection three times a week (aPD-1) as indicated by the arrowheads.
  • FIG. 12 E0771 relative tumor volume (RTV) / PhAc-ALKP-Dox & anti-PDl.
  • the plot represents the RTV of orthotopically implanted E0771 tumors treated with corresponding different concentrations of PhAc-ALKP-Dox, PhAc-ALGP-Dox, aPD-1 alone or in combination as indicated.
  • Dotted line shows tumor delay (Td) calculated as the time required by tumors to reach a volume five times bigger than the initial one (RTV5).
  • TNBC triple-negative breast cancer
  • ER estrogen
  • PR progesterone
  • HER2 human epidermal growth factor receptor 2
  • TILs tumor infiltrating lymphocytes
  • the E0771 syngeneic breast carcinoma model offers significant potential as a preclinical immuno-oncology model and was selected as initial cancer model.
  • Response to immunomodulatory agents was evaluated on mice bearing E0771 tumors treated with checkpoint blockade antibodies (anti-mPD-1, anti-mPD-Ll, anti-CTLA4), with an anthracycline (doxorubicin) and with the same anthracycline included in a less toxic prodrug as described in WO 2014/102312, i.e.
  • the exemplary prodrug molecule having a phosphonoacetyl (PhAc) capping group (C) linked to the tetrapeptide ALGP (SEQ ID NO:1) or ALKP (SEQ ID NO:4) (OP) linked to the anthracycline doxorubicin (D) (further referred to herein as PhAc-ALGP-Dox (see Figure 8) or PhAc-ALKP-Dox; compound of the formula C-OP-D, see detailed explanation hereinafter); this as monotherapies and in combination therapies as described further hereinafter.
  • PhAc-ALGP-Dox anthracycline doxorubicin
  • the invention in one aspect relates to a compound of the formula C-OP-D for use in treating or inhibiting cancer or for use in inhibiting progression of cancer, in combination with an immune checkpoint inhibitor.
  • the invention relates to use of a compound of the formula C-OP-D in the manufacture of a medicament for use in combination with an immune checkpoint inhibitor for treating or inhibiting cancer or for inhibiting progression of cancer (in a subject or individual having the cancer).
  • the invention relates to use of a compound of the formula C-OP-D in the manufacture of a medicament for treating or inhibiting cancer or for inhibiting progression of cancer (in a subject or individual having the cancer) in combination with an immune checkpoint inhibitor (for treating or inhibiting cancer or for inhibiting progression of cancer), or in combination with administering an immune checkpoint inhibitor to the subject or individual.
  • the invention relates to an immune checkpoint inhibitor for use in treating or inhibiting cancer or for use in inhibiting progression of cancer, in combination with a compound of the formula C-OP-D.
  • the invention relates to use of an immune checkpoint inhibitor in the manufacture of a medicament for use in combination with a compound of the formula C-OP-D for treating or inhibiting cancer or for inhibiting progression of cancer (in a subject or individual having the cancer).
  • the invention relates to use of an immune checkpoint inhibitor in the manufacture of a medicament for treating or inhibiting cancer or for inhibiting progression of cancer (in a subject or individual having the cancer) in combination with a compound of the formula C-OP-D (for treating or inhibiting cancer or for inhibiting progression of cancer), or in combination with administering a compound of the formula C-OP-D to the subject or individual.
  • the invention relates to use of an immune checkpoint inhibitor in the manufacture of a medicament for treating or inhibiting cancer or for inhibiting progression of cancer in combination with a compound of the formula C-OP-D (for treating or inhibiting cancer or for inhibiting progression of cancer).
  • the invention relates to a compound of the formula C-OP-D and an immune checkpoint inhibitor for use in treating or inhibiting cancer or for use in inhibiting progression
  • the invention relates to use of a compound of the formula C-OP-D and (use of) an immune checkpoint inhibitor in the manufacture of a medicament for use in treating or inhibiting cancer or for inhibiting progression of cancer (in a subject or individual having the cancer).
  • a further aspect of the invention relates to a method for treating or inhibiting cancer, or a method for inhibiting progression of cancer, in a subject or individual (in particular a mammalian subject or mammal, such as a human subject or human), the methods comprising administering a compound of the formula C-OP-D and administering an immune checkpoint inhibitor to the subject or individual.
  • a subject or individual in particular a mammalian subject or mammal, such as a human subject or human
  • administering the compound of the formula C-OP-D and the immune checkpoint inhibitor the cancer is treated or inhibited, or the progression of the cancer is inhibited.
  • an effective amount of a combination (in any way) of a compound of the formula C-OP-D and of an immune checkpoint inhibitor is administered to the subject.
  • C is a capping group
  • OP is a tetrapeptidic moiety selected from the group consisting of ALGP (SEQ ID NO:1), TSGP (SEQ ID NO:2), KLGP (SEQ ID NO:3), and ALKP (SEQ ID NO:4)
  • D is a cytostatic drug, a cytotoxic drug or an anticancer drug.
  • the compound of the formula C-OP-D is C-OP-D, a pharmaceutically acceptable salt of said compound (of the formula C-OP-D), a pharmaceutically acceptable crystal or co-crystal comprising said compound (of the formula C-OP-D), or a pharmaceutically acceptable polymorph, isomer, or amorphous form of said compound (of the formula C-OP-D).
  • the combination can be a combination in any appropriate way as will be explained further herein.
  • the compound of the formula C-OP-D, the pharmaceutically acceptable salt of said compound (of the formula C-OP-D), the pharmaceutically acceptable crystal or co-crystal comprising said compound (of the formula C-OP-D), or the pharmaceutically acceptable polymorph, isomer, or amorphous form of said compound (of the formula C-OP-D), may in particular, compared to an inactive control, significantly reducing tumor necrosis in a mouse model of said cancer.
  • the compound of the formula C-OP-D, the pharmaceutically acceptable salt of said compound (of the formula C-OP-D), the pharmaceutically acceptable crystal or co-crystal comprising said compound (of the formula C-OP-D), or the pharmaceutically acceptable polymorph, isomer, or amorphous form of said compound (of the formula C-OP-D), may in particular, compared to an inactive control, significantly increasing
  • RECTIFIED SHEET (RULE 91) ISA/EP expression of a peptidase involved in releasing D from C-OP-D, such as THOP1, in the tumor in a mouse model of said cancer.
  • the compound of the formula C-OP-D, the pharmaceutically acceptable salt of said compound (of the formula C-OP-D), the pharmaceutically acceptable crystal or co-crystal comprising said compound (of the formula C-OP-D), or the pharmaceutically acceptable polymorph, isomer, or amorphous form of said compound (of the formula C-OP-D), may in particular, compared to an inactive control, significantly increasing expression of THOP1 in the tumor in a mouse model of said cancer and significantly reducing tumor necrosis in a mouse model of said cancer.
  • the immune checkpoint inhibitor may in particular, compared to an inactive control, significantly increasing expression of THOP1 in the tumor in a mouse model of said cancer.
  • the compound of the formula C-OP-D is further characterized by drug D being an anthracycline anticancer drug or a derivative or analog thereof.
  • drug D may be doxorubicin.
  • the immune checkpoint inhibitor is an inhibitor of programmed cell death protein 1 (PD1 or PD-1) or is an inhibitor of cytotoxic T-lymphocyte associated protein 4 (CTLA4 or CTLA-4).
  • PD1 or PD-1 programmed cell death protein 1
  • CTLA4 or CTLA-4 cytotoxic T-lymphocyte associated protein 4
  • the cancer is a triple negative breast cancer (TNBC).
  • TNBC triple negative breast cancer
  • the combining or combination of the compound of the formula C-OP-D and of the immune checkpoint inhibitor is a combination in any appropriate way as will be explained herein further.
  • the dose of the compound of the formula C-OP-D is lower than the maximum tolerable dose and/or the dose of the immune checkpoint inhibitor is lower than the maximum tolerable dose.
  • C is a capping group
  • OP is a tetrapeptidic moiety selected from (the group consisting of) ALGP (Ala-Leu-Gly-Pro) (SEQ ID NO:1), TSGP (Thr-Ser-Gly-Pro)(SEQ ID NO:2), KLGP (Lys-Leu-Gly-Pro)(SEQ ID NO:3), and ALKP (Ala-Leu-Lys-Pro)(SEQ ID NO:4); and
  • D is a drug, in particular an anticancer drug such as a small molecule anticancer drug or an anticancer drug that is a (small) chemical entity.
  • the compound of the formula C-OP-D includes C-OP-D, as well as any pharmaceutically acceptable salt of said compound (of the formula C-OP-D), any pharmaceutically acceptable crystal or co-crystal comprising said compound (of the formula C-OP-D), and any pharmaceutically acceptable polymorph, isomer or amorphous form of said compound (of the formula C-OP-D).
  • the salts, crystals, cocrystals, polymorphs, isomers and amorphous forms have a therapeutic efficacy as the compound of the formula C-OP-D/are similarly therapeutically effective compared to the compound of the formula C-OP-D; as can be determined in vitro and/or in vivo.
  • prodrug in general refers to a compound that undergoes biotransformation before exhibiting pharmacological effects. Prodrugs can thus be viewed as drugs containing specific nontoxic protective groups used in a transient manner to alter or to eliminate undesirable properties in the parent molecule (from: Vert et al. 2012, Pure Appl Chem 84:377-410).
  • the protective groups can have one or more function such as increasing bioavailability, increasing solubility, increasing stability, avoiding or reducing premature release of the drug (thus avoiding or reducing toxicity), altering cell permeability, avoiding or reducing irritation in the subject to be treated with the drug, supporting administration of the drug to the targeted cells or organs in a subject, etc.
  • the improved therapeutic properties of the prodrugs described hereinafter include a combination of stability in mammalian serum or blood, decreased toxicity to normal cells and increased efficacy in killing cancerous cells, and, therewith, an increased therapeutic selectivity or specificity.
  • the specificity of the prodrug was obtained by the presence of a tetrapeptidic moiety allowing release of the therapeutic agent in the vicinity of cancer or tumor cells (as described in WO 2014/102312); this is explained in more detail further herein.
  • PSA prostate antigen
  • the drug D can be changed (demonstrated for doxorubicin and paclitaxel; Elsadek et al. 2010, ACS Med Chem Lett 1: 234-238, and Elsadek et al. 2010, Eur J Cancer 46:3434-3444).
  • Such prodrugs can even be linked successfully to antibodies targeting a tumor-specific antigen (Dubowchik et al. 2002, Bioconjugate Chem 13:855-869; and Walker et al.
  • the tetrapeptidic moiety OP and the drug D in the general prodrug structure C-OP-D are directly linked (or coupled or bound) to each other, or, alternatively are linked (or coupled or bound) indirectly via a linker or spacing group.
  • the linkage should: (1) not or not significantly disturb the functionality of the tetrapeptidic moiety, i.e., should not or not significantly disturb the proteolytic scissability and (2) should retain the blood stability of the compound. Determination of the functionality of a linker or spacing group in the prodrug can be tested, e.g. as described in the Examples section of WO 2014/102312 (e.g. stability in mammalian serum, selective toxicity to cancerous cells).
  • compositions as used herein in the context of salts, crystals, co-crystals, polymorphs and isomers, means those salts of C-OP-D prodrugs of the invention that are safe and effective for the intended medical use.
  • any of such salts, crystals, co-crystals, polymorphs and isomers that possess the desired biological activity.
  • Salts Any of numerous compounds that result from replacement of part or all of an acidic or basic group present in a drug moiety D or prodrug C-OP-D of the invention. Suitable salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.
  • Suitable salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.
  • Co-crystals are crystalline materials composed of two or more different molecules, typically an API or drug and co-crystal formers ("coformers"), in the same crystal lattice.
  • Pharmaceutical co-crystals have opened up opportunities for engineering solid-state forms beyond conventional solid-state forms of an API or drug, such as salts and polymorphs.
  • Co-crystals are readily distinguished from salts because unlike salts, their components are in a neutral state and interact nonionically.
  • co-crystals differ from polymorphs, which are defined as including only single-component crystalline forms that have different arrangements or conformations of the molecules in the crystal lattice, amorphous forms, and multicomponent phases such as solvate and hydrate forms.
  • co-crystals are more similar to solvates, in that both contain more than one component in the lattice. From a physical chemistry perspective, co-crystals can be viewed as a special case of solvates and hydrates, wherein the second component, the coformer, is nonvolatile. Therefore, co-crystals are classified as a special case of solvates in which the second component is nonvolatile.
  • stereoisomeric molecules, or stereoisomers contain the same atoms linked together in the same sequence (same molecular formula), but having different three-dimensional organizations or configurations.
  • Optical isomers also sometimes referred to as enantiomers, are molecules which are non-superposable mirror images of each other. Depending on the optical activity, enantiomers are often described as left- or right-handed, and each member of the pair is referred to as enantiomorph (each enantiomorph being a molecule of one chirality). Mixtures of equal parts of two enantiomorphs are often referred to as racemic mixtures.
  • enantiopure compounds Compounds comprising within the limits of detection only one enantiomorph are referred to as enantiopure compounds.
  • Optical isomers can occur when molecules comprise one or more chiral centers.
  • Geometric isomers usually refer to cis-trans isomers wherein rotation around a chemical bond is impossible. Cis-trans isomers often are found in molecules with double or triple bonds.
  • Structural isomers contain the same atoms (same molecular formula), but linked together in a different sequence.
  • a linker or spacing group (terms used interchangeably herein) can be present to create distance between the tetrapeptidic moiety and the drug moiety such as a spacer for mitigating steric hindrance in order to facilitate proteolytic or other enzymatic degradation of the tetrapeptidic moiety OP linked to the drug moiety D.
  • Such linker or spacing group can alternatively or additionaly be present to (further) increase the specificity of the prodrug, e.g. by providing an additional mechanism for activation of the prodrug or release of the drug moiety D.
  • linker or spacing group can further alternatively or additionally be present to enable chemical linkage between the tetrapeptidic moiety and the drug moiety, i.e. the end of the linker to be connected with the drug moiety can be designed in function of chemical coupling with a suitable group present in the chemical structure of the drug moiety.
  • a linker or spacing group may thus provide appropriate attachment chemistry between the different moieties of the prodrug (and thus providing flexibility to couple any possible drug moiety D and a tetrapeptidic moiety OP of the invention).
  • a linker or spacing group may further alternatively or additionaly be introduced to improve the synthetic process of making the prodrug conjugate (e.g., by pre-derivatizing the therapeutic agent or oligopeptide with the linker group before conjugation to enhance yield or specificity).
  • a linker or spacing group may yet further alternatively or additionaly be introduced to improve physical properties of the prodrug.
  • Such linker or spacing group may be purely self-immolative or self-eliminating by means of chemical degradation upon release of/from the tetrapeptidic moiety.
  • Self-immolation or self-elimination of a linker or spacing group may alternatively rely on further triggers such as esterase or phosphatase activity or may rely on a redox-sensitive, pH-sensitive, etc. triggering mechanism; in the current context such linkers are likewise termed self-immolative or self-eliminating linkers or spacing groups.
  • the linker between OP and D can for instance be a self-immolative or self-eliminating linker or spacing group.
  • linker Upon proteolytic removal of the tetrapeptidic moiety OP, such linker is spontaneously decomposing to set free the drug moiety D.
  • the different types of self-eliminating linkers usually decompose via a spontaneous elimination or cyclization reaction.
  • a well-known and often used self- immolative linker is p-aminobenzyloxycarbamate (PABC; alternatively p-aminobenzyloxycarbonyl) which decomposes via 1,6-benzyl elimination; o-aminobenzyloxycarbonyl (OABC) decomposes via 1,4- benzyl elimination.
  • PABC p-aminobenzyloxycarbamate
  • OABC o-aminobenzyloxycarbonyl
  • Linkers such as PABC are able to connect either -OH, -COOH, - NH, or -SH groups of a drug D at the one hand to the carboxy-terminal group of a tetrapeptidic moiety OP at the other hand.
  • Substituted 3-carbamoyl-2-arylpropenal compounds are a further example of self-immolative linkers that decompose via elimination of carbamic acid; substitutions include a nitro-group, a halide (e.g. fluoride), and a methyl group (Rivault et al. 2004, Bioorg Med Chem 12:675).
  • Self-immolative disulfide-containing linkers are a newer group of such linkers (e.g.
  • Such self-immolative linkers can be multimerized (e.g. dimers, trimers,...) to form elongated self- immolative linkers.
  • Such linkers can also be multimerized in the form of dendrimers potentially carrying multiple drug D moieties (e.g. Amir et al. 2003, Angew Chem Int Ed 42:4494-4499; de Groot et al. 2003, Angew Chem Int Ed 42:4490-4494).
  • the linker between OP and D can for instance be an acid-labile linker.
  • acid-labile linkers are preferentially cleaved in the tumor environment.
  • Acid-labile linkers or spacers include acid-labile bonds such as carboxylic hydrazine bonds, cis-aconityl bonds, trityl bonds, acetal bonds and ketal bonds.
  • Polymeric molecules in which the monomers are each linked to each other by an acid-labile bond are other examples of acid-labile linkers (see e.g. Figure 10 and Table 5 of Kratz et al. 2008, ChemMedChem 3:20-53).
  • the linker between OP and D can for instance be a self-immolative or self-eliminating linker or spacing group wherein the self-immolation or self-elimination is occurring selectively under hypoxic/low oxygen conditions.
  • Many tumors or cancers, in particular solid tumors or cancers, are characterized by the presence of hypoxic regions (e.g. Li et al. 2018, Angew Chem Int Ed Engl 57:11522-11531).
  • Aromatic nitro or azido groups can be applied in this setting and reduction (in hypoxic or low oxygen areas) of these compounds starts their decomposition via 1,6- or 1,8-elimination.
  • Analogues of nitroimidazoles, N-oxides and nitrobenzyl carbamates can be applied (e.g. imidazolylmethyl carbamates: Hay et al. 2000, Tetrahedron 56:645; e.g. nitrobenzyloxycarbonyl groups: Shyam et al.
  • Self-elimination of a linker between OP and D can also be based on an intramolecular cyclization or lactonization reaction, such as the trimethyl lock lactonization reaction (Greenwald et al. 2000, J Med Chem 43:475-487).
  • Such systems include, without limitation, the (alkylamino)-ethyl carbamate and [(alkylamino)ethyl]glycyl ester systems; the N-(substituted 2-hydroxyphenyl) carbamate and N- (substituted 2-hydroxypropyl) carbamate systems; and systems based on o-hydroxylphenylpropionic acid and its derivatives.
  • the linker between OP and D can for instance be redox-sensitive linkers susceptible to reducing conditions (such as quinones).
  • the linker between OP and D can for instance be a hydrophilic stopper such as a glycosylated tetra(ethylene glycol) which, upon deglycosylation (after proteolytic release of the tetrapeptidic moiety OP), spontaneous decomposes and releases the drug D (e.g. Fernandes et al. 2012, Chem Commun 48:2083-2085).
  • a hydrophilic stopper such as a glycosylated tetra(ethylene glycol) which, upon deglycosylation (after proteolytic release of the tetrapeptidic moiety OP), spontaneous decomposes and releases the drug D (e.g. Fernandes et al. 2012, Chem Commun 48:2083-2085).
  • linker or spacer groups examples include aminocaproic acid, a hydrazide group, en ester group, an ether group and a sulphydryl group. Further explanation about linkers can be found in Bargh et al. 2019 (Chem Soc Rev 48:4361-4620).
  • the general prodrug/compound structure/formula C-OP-D described hereinabove may be complexed with a macrocyclic moiety, e.g. a self-eliminating or self-immolative macrocyclic moiety.
  • the self-elimination process may be a pure self-elimination process or one that is started by a further trigger (see above).
  • the tetrapeptidic axle of a prodrug/compound of the formula C-OP-D could further be protected by means of a macrocycle itself designed to be self-immolative or self-opening, wherein the trigger for self-immolation or self-opening could be action of an enzyme such as beta-galactosidase or beta-glucuronidase.
  • a macrocycle itself designed to be self-immolative or self-opening, wherein the trigger for self-immolation or self-opening could be action of an enzyme such as beta-galactosidase or beta-glucuronidase.
  • Such macrocycle is hereinafter furher termed "macrocyclic moiety". Expression of beta-galactosidase is increased in many tumors compared to normal tissues (e.g. Chen et al. 2018, Anal Chim Acta 1033:193-198) and glucuronide prodrugs are a further class of prodrugs (e.g.
  • a macrocycle is a rotaxane or pseudo-rotaxane, and protection against self-opening could be through e.g. linkage with a glycoside such as a galactoside.
  • the glycoside moiety can be linked to the macrocycle through a self-immolative linker.
  • the capping group C and the tetrapeptidic moiety OP in the general prodrug/compound structure/formula C-OP-D described hereinabove are directly linked (or coupled or bound) to each other, or, alternatively are linked (or coupled or bound) indirectly via a linker or spacing group.
  • a direct linkage between the capping group C and the tetrapeptidic moiety OP may be direct, e.g. via the N-terminal aminogroup of the tetrapeptidic moiety OP, or via a side chain of one of the amino acids of the tetrapeptidic moiety OP.
  • said linkage may be indirect, e.g.
  • linkage should: (1) not or not significantly disturb the functionality of the tetrapeptidic moiety, i.e., should not or not significantly disturb the proteolytic scissability and (2) should retain the blood stability of the compound. Determination of the functionality of a linker or spacing group in the prodrug can be tested, e.g. as described in the Examples section of WO 2014/102312 (e.g. stability in mammalian serum, selective toxicity to cancerous cells).
  • linker or spacing group between the capping group C and the tetrapeptidic moiety OP Possible reasons for including a linker or spacing group between the capping group C and the tetrapeptidic moiety OP are the same as those listed hereinabove relating to the linker or spacing group between the tetrapeptidic moiety OP and the drug moiety D.
  • a protecting or capping moiety C which is covalently linked to the N-terminal side of the oligopeptide, as present in the prodrug of the current invention, adds to the solubility and/or stability of the prodrug (e.g. in mammalian blood or serum) and/or adds to the prevention of internalization of the prodrug into a cell such as a target cell.
  • Such protecting or capping moieties include non-natural amino acids, P-alanyl or succinyl groups (e.g. WO96/05863, US 5,962,216).
  • Further stabilizing, protecting or capping moieties include diglycolic acid, maleic acid, pyroglutamic acid, glutaric acid, (e.g., WOOO/33888), a carboxylic acid, adipic acid, phthalic acid, fumaric acid, naphthalene dicarboxylic acid, 1,8- naphtyldicarboxylic acid, aconitic acid, carboxycinnamic acid, triazole dicarboxylic acid, butane disulfonic acid, polyethylene glycol (PEG) or an analog thereof (e.g., WOOl/95945), acetic acid, 1- or 2-naphthylcarboxylic acid, gluconic acid, 4-carboxyphenyl boronic acid, polyethylene glycolic acid, nipecotic acid, and isonipecotic acid (e.g., W002/00263, W002/100353), succinylated polyethylene glycol (e.g.
  • W02008/120098 A new type of protecting or capping moiety was introduced in W02008/120098, being a 1, 2,3,4 cyclobutanetetracarboxylic acid.
  • the protecting or capping moiety in W002/07770 may be polyglutamic acid, carboxylated dextranes, carboxylated polyethylene glycol or a polymer based on hydroxyprolyl-methacrylamide or N-(2-hydroxyprolyl)methacryloylamide.
  • Other capping groups include epsilon-maleimidocaproyl (Elsadek et al. 2010, Eur J Cancer 46:3434- 3444), benzyloxycarbonyl (Dubowchik et al.
  • polyethylene glycol group(s) may be linked, coupled or bound to an amino acid, such as the N-terminal amino acid, of the tetrapeptidic moiety OP.
  • pegylation may be introduced in order to increase the half-life of a prodrug C-OP-D in circulation after administration to a mammal and/or to increase solubility of a prodrug C-OP-D.
  • Addition of (a) polyethylene glycol group(s)/pegylation could alternatively or additionally play the role of a capping agent.
  • the drug moiety D or therapeutic agent conjugated to the tetrapeptidic moiety OP in the general prodrug/compound structure/formula C-OP-D described hereinabove may be useful for treatment of a disease, in particular of cancer.
  • this drug moiety D is different from the immune checkpoint inhibitor as used in combination with the general prodrug/compound structure/formula C-OP-D described hereinabove for the treatment of cancer.
  • the drug moiety D can be a small chemical molecule, or can be a biological (e.g. of peptidic or proteinaceous nature, such as an anti-cancer antibody).
  • the drug moiety D in one embodiment is a cytotoxic drug, a cytostatic drug, or an anticancer drug.
  • said drug moiety D or therapeutic agent may be an anthracycline, or a derivative or analog thereof, including: doxorubicin and analogues [such as N-(5,5-diacetoxypent-l-yl)doxorubicin: Farquhar et al. 1998, J Med Chem 41:965-972; epirubicin (4'-epidoxorubicin), 4'-deoxydoxorubicin (esorubicin), 4'-iodo-4'-deoxydoxorubicin, and 4'-O-methyldoxorubicin: Arcamone et al. 1987, Cancer Treatment Rev 14:159-161 & Giuliani et al.
  • doxorubicin and analogues such as N-(5,5-diacetoxypent-l-yl)doxorubicin: Farquhar et al. 1998, J Med Chem 41:965-972; epirubicin (4'-epidox
  • DOX-F-PYR pyrrolidine analog of DOX
  • DOX-F-PIP piperidine analog of DOX
  • DOX-F-MOR morpholine analog of DOX
  • DOX- F-PAZ N-methylpiperazine analog of DOX
  • DOX-F-HEX hexamehtyleneimine analog of DOX
  • oxazolinodoxorubicin (3'deamino-3'-N, 4'-O-methylidenodoxorubicin, O-DOX): Denel-Bobrowska et al.
  • daunorubicin or daunomycin
  • analogues thereof such as idarubicin (4'-demethoxydaunorubicin): Arcamone et al. 1987, Cancer Treatment Rev 14:159-161; 4'- epidaunorubicin; analogues with a simplified core structure bound to the monosaccharide daunosamine, acosamine, or 4-amino-2,3,6-trideoxy-L-threo-hexopyranose: see compounds 8-13 in Fan et al.
  • the drug doxorubicin (also known under the trade names Adriamycin or Rubex, these are hydrochloride formulations of doxorubicin; another formulation of doxorubicin is a liposomal formation, known under the trade names Caelix or Doxil) is commonly used to treat multiple types of cancers such as some leukemias and Hodgkin's lymphoma, as well as cancers of the bladder, breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple myeloma, and others.
  • derivative a compound that results from reacting the named compound with another chemical moiety (different from the tetrapeptidic moiety linked directly or indirectly to the compound), and includes a pharmaceutically acceptable salt, acid, base, ester or ether of the named compound.
  • derivative refers to a compound that is structurally similar to and retains sufficient functional attributes of the original compound.
  • the derivative may be structurally similar because one or more atoms are lacking, are substituted, are in different hydration/oxidation states, or because one or more atoms within the molecule are switched, such as, but not limited to, adding a hydroxyl group, replacing an oxygen atom with a sulfur atom, or replacing an amino group with a hydroxyl group, oxidizing a hydroxyl group to a carbonyl group, reducing a carbonyl group to a hydroxyl group, and reducing a carbon-to-carbon double bond to an alkyl group or oxidizing a carbon-to-carbon single bond to a double bond compared to the original compound.
  • a derivative optionally has one or more, the same or different, substitutions.
  • Derivatives may be prepared by any variety of synthetic methods or appropriate adaptations presented in synthetic or organic chemistry text books, such as those provide in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) Michael B. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F. Tietze hereby incorporated by reference.
  • analogue of a compound generally refers to a structural analogue or chemical analogue of that compound. Analogues include, but are not limited to isomers.
  • the compound of the formula C-OP-D as referred to herein is in effect a prodrug form of the drug D.
  • the oligopeptide OP is designed such that the activation of the prodrug is occurring in multiple steps.
  • prodrug is stable in blood and plasma, it is converted in a mixture of doxorubicin (Dox), GP-Dox and XGP-Dox when incubated in the presence of LS-174T tumor cells.
  • Dox doxorubicin
  • GP-Dox GP-Dox
  • XGP-Dox XGP-Dox
  • the latter process can in a first step be mimicked in vitro by proteases such as THOP1 (yielding GP-Dox).
  • the first phase of the activation of the ALGP-doxorubicin is thus driven by the preferential activity of THOP1 in the vicinity of the tumors compared to their lower abundancy in non-pathological extracellular compartments and tissues.
  • the second step, conversion of GP-Dox to Dox can be driven by dipeptidyl prolyl peptidases. Two members of this class are of interest in the area of cancer: DPP4 and FAPa. All these proteases are known to be associated with tumor cells or tumor stromal cells as described hereafter.
  • Such multistep activation of the compound/prodrug of the formula C-OP-D as referred to herein increases the specificity and decreases the unwanted side effects (such as leucopenia and cardiac toxicity) and compared to free drugs D, and compared to similar prodrugs that are activatable in a single step.
  • An example of the latter is succinyl-PALAL-doxorubicin which is easily converted by e.g. CD10 to L-Dox that can enter the cell on its own without the need of a second proteolytic cleavage (Pan et al. 2003, Cancer Res 63, 5526-5531).
  • PhAc- ALGP-doxorubicin prodrug that is about 20 to 40 times less toxic than doxorubicin varying with the mode (IV or IP) of administration, and between 6 and 14 times less toxic than succinyl-p-ALAL- doxorubicin.
  • Chronic cumulative cardiotoxicity, leucopenia and lymphopenia induced by PhAc-ALGP- doxorubicin are less compared to these toxicities induces by the free drug doxorubicin.
  • PhAc-ALGP- doxorubicin is more active than doxorubicin on human tumor xenografts (including a sarcoma) and on an orthotopic colon carcinoma (WO 2014/102312).
  • the compound/prodrug of the formula C-OP-D as referred to herein therefore is further characterized by being activatable, in vitro or in vivo, in at least two steps, i.e., in a process involving at least two essential proteolytic cleavages by at least two different proteases.
  • An "essential proteolytic cleavage” is herein meant to be a cleavage that is associated with a tumor or a tumor-associated cell such as its stroma, i.e., is specifically occurring in the direct vicinity of a tumor or tumor-associated cells.
  • THOP THOP1 or TOP (Thimet Oligo Peptidase) is a thiol-dependent main cytoplasmic metallo- endoprotease. It can attain an extracellular location both via secretion of the soluble enzyme and by attachment to the plasma membrane.
  • DPIV or DPP4 or CD26 is a dipeptidylprolylpeptidase with a broad spectrum of activity and covers a large number of physiological substrates. It is upregulated in the tumoral T-cell malignancies (Dang et al. 2002, Histol Histopathol 17, 1213-1226) and in different adenocarcinomas, such as in hepatocellular carcinoma (Stecca et al. 1997, J Hepatol 27, 997-945), thyroid carcinoma (Tanaka et al. 1995, Int J Cancer 64, 326-331), in meningiomas (Yu et al. 2010, FEBS Journal l ⁇ ! , 1126-1144; Stremenoova et al.
  • DPIV is expressed in cancer stem cells of human colorectal cancer and of human mesotheliomas (Pang et al. 2010, Cell Stem Cell 6, 603-615; Yamazaki et al. 2012, Biochem Biophys Res Commun 419, 529-536).
  • FAP or FAPa is a dipeptidyl exopeptidase with very narrow specificity restricted to glycine-proline, alanine-proline and lysine-proline dipeptides and is also a type I collagenase. It can however also act as endopeptidase (Siew lai et al. 2007, Bioconj Chem 18, 1245-1250). FAP is absent in normal adult tissues such as epithelial, mesenchymal; neural and lymphoid cells such as lymphocytes. It is absent in non-malignant tumors.
  • a further aspect of the invention relates to compositions comprising (i) the compound of the formula C-OP-D, a salt of the compound (of the formula C-OP-D), a crystal or co-crystal comprising the compound (of the formula C-OP-D), a polymorph or amorphous form of the compound (of the formula C-OP-D), or an isomer of the compound (of the formula C-OP-D); and (ii) an immune checkpoint inhibitor.
  • compositions are comprising a pharmaceutically acceptable salt of the compound of the formula C-OP-D, a pharmaceutically acceptable crystal or co-crystal comprising the compound (of the formula C-OP-D), a pharmaceutically acceptable polymorph of the compound (of the formula C-OP-D), a pharmaceutically acceptable amporphous form of the compound (of the formula C-OP-D), or a pharmaceutically acceptable isomer of the compound (of the formula C-OP-D).
  • compositions are pharmaceutically acceptable compositions and are further comprising at least one of a pharmaceutically acceptable solvent, diluent, or carrier.
  • said solvent, diluent or carrier is pharmaceutically acceptable, i.e., is acceptable to be administered to a subject to be treated with the composition of the invention.
  • Any suitable solvent may be capable of solubilizing an active substance present in the composition to the desired extent; a diluent may be capable of diluting a concentrated active substance present in the composition to the desired extent; a carrier may be any compound capable of absorbing, adhering or incorporating an active substance present in the composition, and of subsequently releasing at any rate the active substance, e.g. in the extracellular compartment of the subject's body. Aiding in finding a suitable pharmaceutically acceptable composition is e.g. any Pharmacopeia book.
  • composition may be formulated such that it is suitable for any way of administration including intra-cranial, intra-spinal, enteral, parenteral, intra-organ, intra-tumoral, etc. administration.
  • the regimen by which the prodrug is administered may vary, e.g. depending on its pharmacokinetic characteristics, depending on the formulation, depending on the overall physical condition of a subject to be treated and e.g. depending on the judgment of the treating physician.
  • a compound of the formula C-OP-D or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it
  • an immune checkpoint inhibitor or a composition comprising such combination
  • cancer includes e.g.
  • breast cancers soft tissue sarcoma, colorectal cancers, liver cancers, lung cancers such as small cell, non-small cell, bronchic cancers, prostate cancers, ovarian cancers, brain cancers, and pancreatic cancers, colon cancers, head and neck cancers, stomach cancers, bladder cancers, non-Hodgkin's lymphomas, melanomas, leukaemias, neuroblastomas, and glioblastomas.
  • lung cancers such as small cell, non-small cell, bronchic cancers, prostate cancers, ovarian cancers, brain cancers, and pancreatic cancers, colon cancers, head and neck cancers, stomach cancers, bladder cancers, non-Hodgkin's lymphomas, melanomas, leukaemias, neuroblastomas, and glioblastomas.
  • the subject to be treated with the combination of a compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it) and an immune checkpoint inhibitor, or with a composition comprising such combination can be any mammal in need of such treatment but is in particular a human.
  • a subject in need in general is a subject, such as a mammal, having, suffering from, or diagnosed to have the disease.
  • the treatment can result in regression of the disease [e.g. in terms of decreasing (primary) tumor volume or (primary) tumor mass and/or in terms of decreasing or inhibiting metastasis (e.g.
  • the effective amounts of the combination of a compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it) and an immune checkpoint inhibitor, or of a composition comprising such combination is not causing severe side effects (e.g. leucopenia) at the administered dosage.
  • a possible definition of severe human leukopenia is WHO-criteria-defined grade 3- (1000- 1900 leukocytes/mL) or grade 4-leukopenia (less than 1000 leukocytes/mL).
  • "Combination in any way” or “combination in any appropriate way” as referred to herein is meant to refer to any sequence of administration of two (or more) therapeutic modalities, i.e. the administration of the two (or more) therapeutic modalities can occur concurrently or separated from each other for any amount of time; and/or "combination in any way” or “combination in any appropriate way” as referred to herein can refer to the combined or separate formulation of the two (or more) therapeutic modalities, i.e.
  • the two (or more) therapeutic modalities can be individually provided in separate vials or (other suitable) containers, or can be provided combined in the same vial or (other suitable) container.
  • the two (or more) therapeutic modalities can each be provided in the same vial/container chamber of a singlechamber vial/container or in the same vial/container chamber of a multi-chamber vial/container; or can each be provided in a separate vial/container chamber of a multi-chamber vial/container.
  • the therapeutic modalities of the current invention are a compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it) and an immune checkpoint inhibitor.
  • Treatment refers to any rate of reduction, delay or retardation of the progress of the disease or disorder, or a single symptom thereof, compared to the progress or expected progress of the disease or disorder, or single symptom thereof, when left untreated. This implies that a therapeutic modality or combination of therapeutic modalities does not need to result in a complete response (and may thus result in a partial response). More desirable, the treatment results in no/zero progress of the disease or disorder, or singe symptom thereof (i.e. "inhibition” or “inhibition of progression”), or even in any rate of regression of the already developed disease or disorder or single symptom thereof. "Suppression/suppressing" can in this context be used as alternative for "treatment/treating”.
  • Treatment/treating also refers to achieving a significant amelioration of one or more clinical symptoms associated with a disease or disorder, or of any single symptom thereof.
  • the significant amelioration may be scored quantitatively or qualitatively.
  • Qualitative criteria may e.g. be patient well-being.
  • the significant amelioration is typically a 10% or more, a 20% or more, a 25% or more, a 30% or more, a 40% or more, a 50% or more, a 60% or more, a 70% or more, a 75% or more, a 80% or more, a 95% or more, or a 100% improvement over the situation prior to treatment.
  • a “therapeutically effective amount” refers to an amount of a therapeutic agent or of a combination of therapeutic agents to treat, inhibit, inhibit progression, prevent a disease, disorder, or unwanted condition in a subject.
  • the term “effective amount” refers to the dosing regimen of the agent, the combination of the agents, or the composition comprising the agent or agents (e.g. medicament(s) or pharmaceutical composition(s)). The effective amount will generally depend on and/or will need adjustment to the mode of contacting or administration.
  • the therapeutically effective amount is the amount required to obtain the desired clinical outcome or therapeutic effect without causing significant or unnecessary toxic effects (often expressed as maximum tolerable dose, MTD).
  • MTD maximum tolerable dose
  • the therapeutic agent or combination of therapeutic agents may (each) be administered as a single dose or may (each) need to be administered in multiple doses, such as to obtain or maintain the effective amount over the desired time span/treatment duration.
  • the therapeutically effective amount may further vary depending on the severity of the condition that needs to be treated; this may depend on the overall health and physical condition of the mammal or patient and usually the treating doctor's or physician's assessment will be required to establish what is the effective amount.
  • the effective amount may further be obtained by a combination of different types of contacting or administration.
  • administering means any mode of contacting that results in interaction between an agent (e.g. a therapeutic compound) or composition comprising the agent (such as a medicament or pharmaceutical composition) and an object (e.g. cell, tissue, organ, body lumen) with which said agent or composition is contacted.
  • agent e.g. a therapeutic compound
  • object e.g. cell, tissue, organ, body lumen
  • the interaction between the agent or composition and the object can occur starting immediately or nearly immediately with the administration of the agent or composition, can occur over an extended time period (starting immediately or nearly immediately with the administration of the agent or composition), or can be delayed relative to the time of administration of the agent or composition. More specifically the "contacting" results in delivering an effective amount of the agent or composition comprising the agent to the object.
  • a single administration of a pharmacologic compound in general leads to a transient effect due to its gradual removal from the cell, organ and/or body and is reflected in the pharmacokinetic/-dynamic behavior of the compound.
  • two or more (multiple) administrations of the pharmacologic compound may thus be required. This may likewise apply to each of the pharmacologic compounds combined (in any way) in a combination treatment.
  • the combination of a compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it) and immune checkpoint inhibitor as outlined hereinabove for use in treating or inhibiting cancer may further be combined with radiation therapy (whether by direct irradiation or via administering an isotope-labeled antibody or antibody fragment) or surgery.
  • the subject having cancer is treated with a combination of a compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it) and immune checkpoint inhibitor prior to (tumor-debulking) surgery or prior to tumor resection.
  • a further advantage of the combination of a compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it) and immune checkpoint inhibitor as outlined hereinabove for e.g. use in treating or inhibiting cancer resides in the fact that at least one or each of the individual drugs of the combination can, if required or necessary, be used at a lower dose, thereby reducing overall toxicity or side effects.
  • the "lower dose" of an individual drug (as used in the combination) is a therapeutically sub-optimal dose or a dose at which the therapeutic effect is reduced/lower compared to a higher dose of the same drug when compared/used in monotherapy.
  • the "lower dose” of an individual drug is lower than the maximum tolerable dose (MTD) in monotherapy, independent of whether said MTD is in itself fully therapeutically effective in monotherapy or is in itself only partially therapeutically effective in monotherapy.
  • the dose of the compound of the formula C-OP-D as used in the combination (therapy) (in any way) is lower than the maximum tolerable dose (of the compound of the formula C-OP-D), and/or, the dose of the immune checkpoint inhibitor is as used in the combination (therapy) (in any way) is lower than the maximum tolerable dose (of the immune checkpoint inhibitor).
  • the dose of compound of the formula C-OP-D does not need to lowered under the MTD as the compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it) has a reduced toxicity or side effect-profile compared to the free drug D.
  • the compound of the formula C-OP-D or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it
  • Immune checkpoints antagonists or inhibitors as referred to herein include the cell surface protein cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death protein-1 (PD-1) and their respective ligands.
  • CTLA-4 binds to its co-receptor B7-1 (CD80) or B7-2 (CD86);
  • PD-1 binds to its ligands PD-L1 (B7-H10) and PD-L2 (B7-DC).
  • immune checkpoint inhibitors include the adenosine A2A receptor (A2AR), B7-H3 (or CD276), B7-H4 (or VTCN1), BTLA (or CD272), IDO (indoleamine 2,3- dioxygenase), KIR (killer-cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene-3), NOX2 (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoform 2), TIM3 (T-cell immunoglobulin domain and mucin domain 3), VISTA (V-domain Ig suppressor of T cell activation), SIGLEC7 (sialic acid-binding immunoglobulin-type lectin 7, or CD328) and SIGLEC9 (sialic acid-binding immunoglobulin-type lectin 9, or CD329).
  • A2AR adenosine A2A receptor
  • B7-H3 or CD276
  • the immune checkpoint antagonists or inhibitors are selected for inclusion in a combination therapy (as outlined above) based on their capability to reduce tumor necrosis (in an appropriate surrogate mouse model of the cancer or tumor of interest/to be treated with the combination therapy; as e.g. outlined in the Examples included herein), and/or, to increase expression of THOP1 in a tumor (in an appropriate surrogate mouse model of the cancer or tumor of interest/to be treated with the combination therapy; as e.g. outlined in the Examples included herein).
  • PD1 and CTLA4 are defined in more detail hereafter.
  • Aliases of PD1 provided in GeneCards® include PDCD1; Programmed Cell Death 1; Systemic Lupus Erythematosus Susceptibility 2; PD-1; CD279; HPD-1; SLEB2; and HPD-L.
  • the genomic locations for the PDCD1 gene are chr2:241, 849, 881-241, 858, 908 (in GRCh38/hg38) and chr2:242, 792, 033-242, 801, 060 (in GRCh37/hgl9).
  • GenBank reference PD1 mRNA sequence is known under accession no. NM 005018.3.
  • Approved PDl-inhibiting antibodies include nivolumab, pembrolizumab, and cemiplimab; PDl-inhibiting antibodies under development include CT-011 (pidilizumab) and therapy with PDl-inhibiting antibodies is referred to herein as a-PD-1 therapy or a-PDl therapy.
  • PD1 siRNA and shRNA products are available through e.g. Origene.
  • Aliases of CTLA4 provided in GeneCards® include Cytotoxic T-Lymphocyte Associated Protein 4; CTLA- 4; CD152; Insulin-Dependent Diabetes Mellitus 12; Cytotoxic T-Lymphocyte Protein 4; Celiac Disease 3; GSE; Ligand And Transmembrane Spliced Cytotoxic T Lymphocyte Associated Antigen 4; Cytotoxic T Lymphocyte Associated Antigen 4 Short Spliced Form; Cytotoxic T-Lymphocyte-Associated Serine Esterase-4; Cytotoxic T-Lymphocyte-Associated Antigen 4; CELIAC3; IDDM12; ALPS5; and GRD4.
  • CTLA4 The genomic locations for the CTLA4 gene are chr2:203, 867, 771-203, 873, 965 (in GRCh38/hg38) and chr2:204, 732, 509-204, 738, 683 (in GRCh37/hgl9).
  • GenBank reference CTLA4 mRNA sequences are known under accession nos. NM 001037631.3 and NM 005214.5.
  • Approved CTLA4-inhibiting antibodies include ipilumab; CTLA4-inhibiting antibodies under development include tremelimumab; therapy with CTLA4-inhibiting antibodies is referred to herein as a-CTLA4 therapy.
  • CTLA4 siRNA and shRNA products are available through e.g. Origene.
  • kits comprising a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising a compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it) or comprising a composition comprising a compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it); and comprising a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising an immune checkpoint inhibitor or a composition comprising an immune checkpoint inhibitor.
  • a container or vial any suitable container or vial, such as a pharmaceutically acceptable container or vial
  • an immune checkpoint inhibitor or a composition comprising an immune checkpoint inhibitor.
  • kits are comprising a container or vial (any suitable container or vial, such as a pharmaceutically acceptable container or vial) comprising a combination of a compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it) and an immune checkpoint inhibitor (see discussion on "combination in any way” on how such combination in a single container, e.g., vial can be defined).
  • a container or vial any suitable container or vial, such as a pharmaceutically acceptable container or vial
  • an immune checkpoint inhibitor see discussion on "combination in any way” on how such combination in a single container, e.g., vial can be defined.
  • kits include one or more diagnostic agents capable of determining the success of a therapy comprising a combination therapy according to the invention; use instructions; one or more containers with sterile pharmaceutically acceptable carriers, excipients or diluents [such as for producing or formulating a (pharmaceutically acceptable) composition of the invention]; one or more syringes; one or more needles; etc.
  • kits may be pharmaceutical kits.
  • prodrugs comprising ALGP (SEQ ID NO:1) as tetrapeptidic moiety OP, phosphonacetyl as capping group C, and with drug D either being maytansine, geldanamycin, paclitaxel, docetaxel, camptothecin, vinblastine, vincristine, methotrexate, aminopterin, and amrubicin are described in Example 16 of WO 2014/102312.
  • WO 2014/102312 furthermore discloses TSGP (Thr-Ser-Gly-Pro)(SEQ ID NO:2) and KLGP (Lys-Leu-Gly-Pro)(SEQ ID NO:3) and ALKP (Ala-Leu-Lys-Pro)(SEQ ID NO:4) as tetrapeptidic structures giving rise to prodrugs being activated in a 2-step process.
  • the linker or spacing group PABC (para-aminobenzyloxycarbonyl) is introduced between the tetrapeptidic moiety OP and the drug D; PABC is removed via a spontaneous 1,6 benzyl elimination mechanism after proteolytic removal of OP.
  • the ortho version of PABC could likewise be used, and is removed via a spontaneous 1,4-elimination.
  • the PABC linker can be introduced in the tetrapeptidic prodrug wherein the drug D is doxorubicin (see e.g. Elsadek et al. 2010, ACS Med Chem Lett 1:234-238).
  • the generation of intermediates (XGP-Dox, GP-Dox, P-Dox) and end-product (doxorubicin) is assessed after incubating individual prodrugs/compounds of the formula C-OP-D as described herein (at 2 pM) with one or more individual candidate peptidases available as recombinant proteins (CD10 (at 0,02 pg/mL), THOP1 (at 0,1 pg/mL), FAPa (at 0,04 pg/mL) or DPP4 (at 0,04 pg/mL); working concentrations bracketed).
  • CD10 at 0,02 pg/mL
  • THOP1 at 0,1 pg/mL
  • FAPa at 0,04 pg/mL
  • DPP4 at 0,04 pg/mL
  • the compounds of the formula C-OP-D as described herein are incubated with recombinant human peptidases (at the working concentrations indicated above). Briefly, in e.g. a 48-well plate, wells containing 2- to 3-times concentrated recombinant enzymes prepared in 400 pl assay buffer (either as single enzymes or as a combination of 2 enzymes; 150 mM NaCI, 50 mM Tris, 1% BSA, pH 7.5, any cofactors are present in the formulation of the recombinant protein) are mixed with a compound of the formula C-OP-D as described herein (dissolved in 400 pl assay buffer) to initiate the reaction
  • the samples are diluted in a 1 to 5 ratio in an ice cold MeOH/CH3CN solution containing the internal standard (doxorubcin 13C D3) to remove a maximum of salt and proteins. After homogenization using a vortex, the samples are centrifuged 10 minutes at 12000rpm, 4°C then, the supernatants are transferred in Matrix tube for LC-MS/MS analysis.
  • the internal standard doxorubcin 13C D3
  • PhAc-ALGP-DOX was processed only in presence of THOP1, displaying a half-life between 82 and 91 minutes; depending on the combination of proteolytic enzymes, GP-Doxorubicin (THOP1 alone or THOP1 + CD10) or the formation of GP-Doxorubicin and Doxorubicin (THOP1 + DPP4 orTHOPl + FAPa) were observed.
  • PhAc-ALGP-Dox is stable in the presence of the other enzymes (CD10, DPP4 and FAPa) with half-lifes largely higher than 16h.
  • the incubation with the CD10 enzyme (alone or combined with DPP4 and FAPa) leads to the detection of a low amount of LGP-Dox.
  • FAPa and DPP4 were not able to generate any of the intermediates confirming their role as exopeptidases.
  • EXAMPLE 4 Efficacy of monotherapies.
  • Example 3 the data as described in Example 3 were validated and, further, the efficacy of immune- monotherapy was compared with the efficacy of the anthracycline doxoxorubicin (Dox) and of the exemplary prodrug PhAc-ALGP-Dox (described in WO 2014/102312, i.e. the prodrug molecule having a phosphonoacetyl (PhAc) capping group linked to the tetrapeptide ALGP linked to the anthracycline doxorubicin, further referred to herein as PhAc-ALGP-Dox, see Figure 8).
  • PhAc-ALGP-Dox the prodrug molecule having a phosphonoacetyl
  • Aim of this study was to test the efficacy of monotherapies in the E0771 triple-negative breast cancer (TNBC) model in C57/BI6 immunocompetent mice.
  • TNBC triple-negative breast cancer
  • lxlO A 6 E0771 cells were injected orthotopically (OT) and the treatment started as soon as the tumor were palpable (100 mm 3 ).
  • Doxorubicin was used as reference and the efficacy of PhAc-ALGP-Dox was compared to immune checkpoint inhibitors (ICBs: anti-mPDl (also referred to as aPD-1 or aPDl) and anti-mPDLl (also referred to as aPDLl or aPDL-1); as well as an isotype control antibody).
  • IBs immune checkpoint inhibitors
  • Antibodies used were: InVivoMab rat lgG2a isotype control (clone 2A3; Bioxcell), InVivoMab anti-mouse PD-1 (CD279) (Clone 29F.1A12; Bioxcell), and InVivoMab anti-mouse PD-L1 (B7-H1) (Clone 10F.9G2; Bioxcell).
  • mice were randomized when tumor size was approximately 100 mm 3 .
  • Mice received PhAc-ALGP-Dox (154 mg/kg) or Dox (5 mg/kg) two times a week for two weeks via systemic tail vein (TV) administration.
  • Treatment with either Dox or PhAc-ALGP-Dox was similarly effective in reducing tumor growth (56% TGI and 75% TGI respectively).
  • Treatment with PhAc-ALGP-Dox showed superiority to ICBs and resulted in a significantly higher effect than aPD-1 treatment in reducing the growth of orthotopic E0771 tumors.
  • TGI % tumor growth inhibition (%TGI or TGI%).
  • %TGI (l- ⁇ Tt/T0 / Ct/CO ⁇ / 1- ⁇ CO/Ct ⁇ ) x 100 where Tt and TO are the individual tumor volume of treated mouse X at times t and 0 respectively, Ct and CO are the mean tumor volume of the control group at times t and 0 respectively.
  • Tumors treated with PhAc-ALGP-Dox or anti mPD-1 were, when compared to control, further characterized by a significant higher percentage of THOP1 positive regions. This was not observed in tumors treated with the other monotherapies (Figure 4). The higher percentage of THOP1 positive regions appeared to correlate with the necrotic regions being negative for THOP1 expression (not shown) and, in addition, in the control arm and the Dox- and mPDL-l-treatment arms, tumor regions were identified in which THOP1 was lowly expressed/absent. Interestingly, more accurate analysis revealed that tumors treated with PhAc-ALGP-Dox and aPD-1 displayed almost exclusive presence of high intensity THOP1 regions.
  • PhAc-ALGP-Dox 154 mg/kg or Dox (5 mg/kg)
  • a reduction of growth of E0771 orthotopically implanted tumors in C57/BI6 mice was observed which was superior to the effect of treatment with ICB.
  • sub-optimal doses of PhAc-ALGP-Dox and Dox are to be explored to show synergism with anti mPD-1 treatment.
  • Sub-optimal concentration of Dox revealed only 52% TGI. Based on these data, doses of 75 mg/kg PhAc-ALGP-Dox and 3 mg/kg Dox were chosen for combinatorial treatment with anti mPD-1. Dose response (DR) studies for aPD-1 were not performed as the tested treatment (lOmg/kg) was already partially effective. To test whether the combinatorial treatment with PhAc-ALGP-Dox and anti-PD-1 can synergize in reducing the tumor size in E0771 TNBC model, lxlO A 6 E0771 cells were injected orthotopically (OT) and the treatment was started as soon as the tumors were palpable (approximately 150-180 mm 3 ). Doxorubicin alone and in combination with anti-PD-1 (see Example 2 for source) was used as references.
  • the relative tumor volume was calculated as described in Balin-Gauthier et al. 2006 (Cancer Chemother Pharmacol 57:709- 718).
  • RTV is expressed as Vt /V0 ratio where Vt is the median tumor volume on a given day during the treatment and V0 is the median tumor volume at the beginning of the treatment.
  • Figure 7 shows the antitumor activity evaluated by the time to reach a tumor volume that was five times greater than the initial volume [tumor delay (Td)].
  • Td tumor delay
  • Expected Td median control+ (median aPDl - median control)+ (median PhAc-ALGP-Dox or Dox - median control).
  • PhAc-ALGP-Dox is much safer, and provokes less severe side effects than Dox, side effects remain and it is therefore important to keep the dose of PhAc-ALGP-Dox as low as possible.
  • combination therapies aiming at increasing the immune response often have increased and/or unexpected side effects compared to the side effects observed with the respective monotherapies - another reason for reducing the dose of at least one or possibly both of the drugs of the combination therapies. It is clear that this criterion is also met by the combination of the exemplary anthracycline prodrug PhAc-ALGP-Dox with anti-PDl.
  • Td is time necessary to reach a tumor volume five times greater than the initial volume; Expected Td of the combination was calculated as Mean control + (mean aPD-l-mean control) + (mean CBR-049 or mean Dox-mean control).
  • Ratio was obtained by dividing the observed Td by the expected Td of the combination. A ratio >1 indicates a more than additive effect (synergistic) and a ratio ⁇ 1 indicates a less than additive effect (antagonist).
  • EXAMPLE 6 Efficacy of combination therapies involving inhibitors of the immune checkpoint CTLA4 protein.
  • E0771 tumor bearing mice were treated with suboptimal doses of PhAc-ALGP-Dox (75 mg/kg) and anti-CTLA-4 (7.5 mg/kg; InVivoMab monoclonal anti-mouse CTLA-4 (CD152) lgG2b, clone 9D9 - Cat n° BP0164, Bio-Connect).
  • PhAc-ALGP-Dox 75 mg/kg
  • anti-CTLA-4 7.5 mg/kg
  • InVivoMab monoclonal anti-mouse CTLA-4 (CD152) lgG2b, clone 9D9 - Cat n° BP0164, Bio-Connect At first, lxlO A 6 E0771 cells were injected orthotopically (OT) and as soon as the tumors were palpable (approximately 180 mm 3 ), treatment was started.
  • Doxorubicin (3 mg/kg, Dox) alone and in combination with anti-CTLA-4 were used as references.
  • the relative tumor volume was calculated as described in Balin-Gauthier et al. 2006 (Cancer Chemother Pharmacol 57:709- 718).
  • RTV is expressed as Vt /V0 ratio where Vt is the median tumor volume on a given day during the treatment and V0 is the median tumor volume at the beginning of the treatment.
  • Figure 10 shows the antitumor activity evaluated by the time to reach a tumor volume that was five times greater than the initial volume [tumor delay (Td)].
  • Td tumor delay
  • Expected Td median control + (median aCTLA-4 - median control) + (median Dox or PhAc-ALGP-Dox - median control).
  • Table 3 shows the synergistic effect when anti-CTLA-4 is combined with the prodrug PhAc-ALGP-Dox, confirming the more effective combinatorial therapeutic impact seen with anti-CTLA-4 treatment. This synergistic effect was not observed when Dox was combined with anti-CTLA-4.
  • Td is time necessary to reach a tumor volume five times greater than the initial volume; Expected Td of the combination was calculated as mean control + (mean aCTLA-4-mean control) + (mean Dox or mean PhAc-ALGP-Dox - mean control).
  • Ratio was obtained by dividing the observed Td by the expected Td of the combination. A ratio >1 indicates a more than additive effect (synergistic) and a ratio ⁇ 1 indicates a less than additive effect (antagonist). EXAMPLE 7. Efficacy of combination therapies involving PhAc-ALKP-Dox and an inhibitor of PD1.
  • E0771 tumor bearing mice were treated with suboptimal doses of PhAc-ALKP-Dox (161 mg/kg) and anti-PD-1 (10 mg/kg).
  • PhAc-ALKP-Dox 161 mg/kg
  • anti-PD-1 10 mg/kg
  • lxlO A 6 E0771 cells were injected orthotopically (OT) and as soon as the tumors were palpable (approximately 200 mm 3 ), treatment was started.
  • PhAc-ALGP-Dox alone 75mg/kg
  • anti-PD-1 was used as references.
  • PhAc-ALGP-Dox 58% TGI
  • PhAc-ALKP-Dox PhAc-ALKP-Dox
  • anti-PD-1 81% TGI
  • the relative tumor volume was calculated as described in Balin-Gauthier et al. 2006 (Cancer Chemother Pharmacol 57:709- 718). RTV is expressed as Vt /V0 ratio where Vt is the median tumor volume on a given day during the treatment and V0 is the median tumor volume at the beginning of the treatment.
  • Figure 12 shows the antitumor activity evaluated by the time to reach a tumor volume that was five times greater than the initial volume [tumor delay (Td)].
  • Td tumor delay
  • Expected Td median control + (median anti-PDl - median control) + (median PhAc-ALGP-Dox or PhAc-ALKP-Dox - median control).
  • Table 4 shows the synergistic effect when anti-PD-1 is combined with PhAc-ALGP-Dox or PhAc-ALKP- Dox, confirming the more effective combinatorial therapeutic impact seen with anti-PD-1 treatment.
  • Table 4 In vivo antitumor activity expressed as tumor delay (in days) in E0771 cell lines orthograft administered with aPD-1, PhAc-ALKP-Dox, PhAc-ALGP-Dox and the combination.
  • Td is time necessary to reach a tumor volume five times greater than the initial volume; Expected Td of the combination was calculated as mean control + (mean aPD-1 - mean control) + (mean PhAc-ALKP-Dox or mean PhAc-ALGP-Dox - mean control).
  • Ratio was obtained by dividing the observed Td by the expected Td of the combination. A ratio >1 indicates a more than additive effect (synergistic) and a ratio ⁇ 1 indicates a less than additive effect (antagonist).
  • mice were shaved from the fourth nipple to the midline (any remaining hair was removed with Veet) and weighed to verify that all mice had roughly comparable weights.
  • mice were anesthesized with a mixture of isoflurane, compressed air and oxygen.
  • a preanesthetization step in a box filled with the gas mixture was followed by full anesthetization on the operating table by placing the nose of a mouse in the nozzle of anesthesia mask; the mouse being fixed to the operating table.
  • the shaved area was sterilized with a cotton swab dipped in ethanol.
  • E0771 cells were kept on ice and homogenized by pipetting up and down.
  • the required volume of cell suspension 50 pL was gently aspirated into an insulin syringe and subsequently injected into the mammary fat pad of the anesthesized mouse holding the needle horizontally.
  • IV Intravenous injection
  • mice tail vein (TV) injection in mice
  • mice were weighed and the volume of composition calculated to obtain the desired mg/kg dose.
  • conscious mice were kept in a warm air cabinet during a couple of minutes.
  • a mouse was inserted in a Broome restrainer (or nose cone rodent restrainer) with the tail hanging out of the restrainer.
  • the tail was wiped with 70% ethanol and the restrainer rotated such that the chosen lateral vein was visible and accessible for injection of the composition.
  • mice Intraperitoneal injections in mice A mouse was restrained appropriately in the head-down position and the appropriate injections site was identified: the animal's lower right quadrant of the abdomen to avoid damage to the urinary bladder, cecum and other abdominal organs. The appropriate volume of composition to obtain the desired dose was subsequently injected.
  • Synergism in anticancer activity is lacking for combinations of free doxorubicin with either inhibitor of the checkpoint molecules PD1 or CTLA4.

Abstract

L'invention concerne le domaine du traitement de maladies, en particulier le traitement du cancer. En particulier, l'invention concerne des combinaisons thérapeutiques d'un médicament anticancéreux lié à une fraction tétrapeptidique (promédicament anticancéreux) et d'un agent immunothérapeutique. En particulier, de telles combinaisons sont efficaces de manière synergique dans le traitement de maladies en comparaison au traitement avec des agents simples.
PCT/EP2021/073259 2020-08-23 2021-08-23 Combinaisons synergiques de médicaments anticancéreux liés à une fraction tétrapeptidique et d'agents immunothérapeutiques WO2022043256A1 (fr)

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