WO2022043256A1

WO2022043256A1 - Synergistic combinations of anticancer drugs linked to a tetrapeptidic moiety and immunotherapeutic agents

Info

Publication number: WO2022043256A1
Application number: PCT/EP2021/073259
Authority: WO
Inventors: Andrea CASAZZA; Lawrence Van Helleputte; Geert REYNS; Nele KINDT
Original assignee: Cobiores Nv
Priority date: 2020-08-23
Filing date: 2021-08-23
Publication date: 2022-03-03

Abstract

The invention relates to the field of disease treatment, in particular cancer treatment. In particular, the invention relates to therapeutic combinations of an anticancer drug linked to a tetrapeptidic moiety (anticancer prodrug) and an immunotherapeutic agent. In particular, such combinations are synergistically effective in disease treatment compared to treatment with the single agents.

Description

Synergistic combinations of anticancer drugs linked to a tetrapeptidic moiety and immunotherapeutic agents

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The introduction of immune checkpoint inhibitors or immune checkpoint blockers (referred to sometimes as ICBs) in clinical practice has revolutionized the field of cancer treatment. Notwithstanding this, it is still not fully understood (i) as to why only a subset of cancer types is responsive to immune checkpoint inhibitor treatment and (ii) as to why, within the same cancer type, only a subset of patients is responsive to immune checkpoint inhibitor treatment. In both cases, 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. There is therefore an unmet need to increase efficacy of ICBs and/or to find ways to turn immunogenically cold tumors (not or poorly responding to ICBs) into immunogenically hot tumors (showing a response or an improved response to ICBs).

SUMMARY OF THE INVENTION

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:

- in the compound of the formula C-OP-D: 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); and D is a cytostatic drug, a cytotoxic drug or an anticancer drug; and

- 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:

RECTIFIED SHEET (RULE 91) ISA/EP - in the compound of the formula C-OP-D: 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); and D is a cytostatic drug, a cytotoxic drug or an anticancer drug; and

- 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.

LEGENDS OF THE FIGURES

Figure 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). Data represents mean ± SD (n=10 per group) (****p<0.0001 versus control; ^####p<0.0001 vs aPD-1).

Figure 2. Tumor weight / PhAc-ALGP-Dox & anti-PDl. The plot represents the E0771 tumor weight at the time of sacrifice. Treated arms (see Figure 1) have been sacrificed at day 17. Data represents mean ± SD (n=10 per group). *p<0.01; **p<0.001; ****p<0.0001 vs Control.

Figure 3. Tumor necrosis quantification / PhAc-ALGP-Dox & anti-PDl. The plot represents the percentage of necrotic region within the E0771 tumors 17 days after the treatment started (see Figure 1). The entire tumor section was characterized and necrotic areas were identified following H&E staining. Data represents average ± SD (n=10) (*p =0.04; ***p=0.0003 vs Ctrl).

Figure 4. Percentage of THOP1 positive region / PhAc-ALGP-Dox & anti-PDl. The plot represents the percentage of THOPl-positive region within the tumors 17 days after the treatment started (see Figure 1). The entire tumor section was characterized and THOPl-positive areas were identified following anti-THOPl staining. Percentage of THOP1 positive regions was calculated as the fraction of THOP1 immune reacting region over the total field. Data represents average ± SD (n=10) (**p =0.0094;

***p=0.0002 vs Ctrl).

RECTIFIED SHEET (RULE 91) ISA/EP Figure 5. Dose-range study of PhAc-ALGP-Dox in E0771 breast cancer model. The plot represents the volume of orthotopically implanted E0771 tumors treated with corresponding different concentration of Dox and PhAc-ALGP-Dox as indicated in the figure. Mice received the treatment via tail vein (TV) injection twice a week. Data represents median (n=10 per group) (****p<0.0001 versus control; ^####p<0.0001; ^##p=0.0066 vs PhAc-ALGP-Dox 150 mg/kg).

Figure 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. Data represents median (n=10 per group) (** p=0.0076, ***p<0.0007 vs PhAc-ALGP-Dox +aPD-l combo). 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.

Figure 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). Data represents median (n=10 per group) (***p<0.0005 vs aPD-1).

Figure 8. Structural formula of PhAc-ALGP-Dox.

Figure 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. Data represents median (n=9 per group) ((*** p<0.001, **** p<0.0001 vs control).

Figure 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). Data represents median (n=9 per group).

Figure 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. Data represents median (n=10 per group) (**** p<0.0001 vs control; # p=0.05, ## p< 0.01 vs aPD-1).

Figure 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). Data represents median (n=10 per group) (* p<0.05, ***p<0.001 vs aPD-1).

DETAILED DESCRIPTION OF THE INVENTION

The work leading to the current invention focused on triple-negative breast cancer (TNBC). TNBC is a complex and highly aggressive subtype of breast cancer lacking estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2) receptors, thereby making it difficult to treat. It carries the highest metastatic potential and has the poorest clinical outcome among all the subtypes of breast cancer. Although breast cancer has been initially considered to be a "non-immunogenic" cancer, numerous studies have now shown PD-L1 (one of the immune checkpoint proteins) expression in both cancer and inflammatory cells (tumor infiltrating lymphocytes [TILs]). PD-L1 positivity in cancer or inflammatory cells has been reported across the breast cancer histotypes (Solinas et al. 2017, ESMO Open 2(5); Joneja et al. 2017, J Clin Pathol 70:255-259; Dill et al. 2017, Am J Surg Pathol 41:334-342; Ghebeh et al. 2006, Neoplasia 8:190-198; Mittendorf et al. 2014, Cancer Immunol Res 2:361-370; Barrett et al. 2018, Breast Cancer Res 20:71; Sobral-Leite et al. 2018, Oncoimmunol 7(12); Stovgaard et al. 2019, Breast Cancer Res Treat 174:571-594).

Work described in https://www.covance.com/content/dam/covance/assetLibrary/articles/E0771- Syngeneic-Breast-Cancer-Model-ARTNQN001.pdf (see Example 3) provides evidence that anti-PD-Ll antibodies are not effective in treating this cancer independent of tumor size. Furthermore, in clinical practice, breast cancer is most often only diagnosed at a late(r) stage, more in particular as of the stage wherein tumors have reached a detectable size. For such tumors, the document referred to above indicates that anti-PD-1 antibodies are likewise not effective in treatment, most likely due to their minimal tumor CD8⁺ T cell infiltrate and low immunogenicity due to a low mutational burden. Given the moderately favorable immune cell infiltration and response to immunotherapy (see document cited above), 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.

Therefore, 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. Alternatively, 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). Alternatively, 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.

In an alternative aspect, 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. Alternatively, 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). Alternatively, 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.

Alternatively, 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).

In another alternative aspect, 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

RECTIFIED SHEET (RULE 91) ISA/EP of cancer. Alternatively, 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. By 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. In particular, 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.

In any of the above aspects and alternatives, the components of the compound of the formula C-OP- D (explained in detail further hereinafter) are: 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); and D is a cytostatic drug, a cytotoxic drug or an anticancer drug. Furthermore, 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). Furthermore, the combination can be a combination in any appropriate way as will be explained further herein.

In any of the above aspects and alternatives, 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.

In any of the above aspects and alternatives, 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.

In any of the above aspects and alternatives, 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.

In any of the above aspects and alternatives, 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.

In any of the above aspects and alternatives and embodiments, 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. In particular, drug D may be doxorubicin.

In any of the above aspects and alternatives and embodiments, 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).

In any of the above aspects and alternatives and embodiments, the cancer is a triple negative breast cancer (TNBC).

In any of the above aspects and alternatives and embodiments, 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.

In any of the above aspects and alternatives and embodiments, 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.

Compound of the formula C-OP-D

The compound of the formula C-OP-D as referred to herein has the general structure C-OP-D, wherein:

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.

RECTIFIED SHEET (RULE 91) ISA/EP 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). Herein, 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.

The compound of the formula C-OP-D as referred to herein is in effect a prodrug form of the drug D. The term "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. More in particular, 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.

The nature of the tetrapeptide is a key determinant of the selectivity of the above prodrugs, this independent of which drug is incorporated in the prodrug. This was described in WO 2014/102312 in part for the compound of the formula C-OP-D referred to herein. It is further corroborated by other historical examples. For instance, Dubowchik et al. 1998 (Bioorg Med Chem Lett 8:3341-3346 and 3347-3352) and Walker et al. 2004 (Bioorg Med Chem Lett 14:4323-4327) demonstrated that once a suitable cathepsin-cleavable peptidic moiety (included in a prodrug design resembling the prodrug design of the current invention) has been identified, the drug D can be changed (demonstrated for doxorubicin, mitomycin C, and tallysomycin SlOb) while cathepsin-specific activation is maintained. The same was illustrated for a prostate antigen (PSA)-cleavable peptidic moiety included in a prodrug: once a suitable PSA-cleavable peptidic moiety is identified, 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. 2004, Bioorg Med Chem Lett 14:4323-4327), or to cell-penetrating peptides (CPPs; Yoneda et al. 2008, Bioorg Med Chem Lett 18:1632-1636). In principle, moieties other than antibodies or CPPs could be coupled, such as aptamers, alphabodies, affitins, anticalins, monobodies, DARPins, and single domain antibodies or fragments thereof. These examples illustrate that, once a suitable peptidic moiety is identified, it can be modified at both its ends (N-terminal and C-terminal) without loosing the functionality of the identified peptidic moiety. In one embodiment, 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. Whatever the type of linkage (or coupling or bonding), direct or indirect, 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).

Pharmaceutically acceptable", 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. In addition, 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. For a review on pharmaceutically acceptable salts see, e.g., Berge et al. 1977 (J. Pharm. Sci. 66,

1-19) or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. H. Stahl, C.G. Wermuth

(Eds.), August 2002), incorporated herein by reference. Per the current regulatory scheme, different salt forms of the same active moiety are considered different active pharmaceutical ingredients (APIs).

(from: FDA draft guidance for industry "Regulatory Classification of Pharmaceutical Co-Crystals";

August 2016). Different crystalline forms of the same API. This may include solvation or hydration products (also known as pseudopolymorphs) and amorphous forms. Per the current regulatory scheme, different polymorphic forms are considered the same APIs. Lyophilization of an API often results in a dry powder comprising an amorphous form of the API. Crystalline materials composed of two or more different molecules within the same crystal lattice that are associated by nonionic and noncovalent bonds.

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. In addition, 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. Instead 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.

Isomers 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. 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.

In view of the variety of drugs that can be incorporated in the compound (or prodrug) of the formula C-OP-D, 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. Such 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. 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. 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. Gund et al. 2015, Bioorg Med Chem Lett 25:122-127). An overview is also given in Table 7 of Kratz et al. 2008 (ChemMedChem 3:20-53). 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. Taking advantage of the lower pH in the tumor environment compared to the pH in normal tissues (difference of 0.5 to 1 pH units), 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. 1999, J Med Chem 42:941) and include, without limitation, 2'-(4-nitrobenzyl carbonate); 2'-(4- azidobenzyl carbonate); 2'-(4-nitrocinnamylcarbonate); 2'-O-(2,4-dinitrobenzyloxycarbonyl); 2'-O-[2- nitro-5-(allyloxycarbonyl)benzyloxycarbonyl]; 2'-O-(2-nitro-5-carboxybenzyloxycarbonyl); 2'-O-(5- methyl-nitro-lH-imidazoyl-2-yl)methyloxycarbonyl); 2'-O-(5-nitrofuran-2-ylmethyloxycarbonyl); 2'-O- (5-nitrothiophene-2-ylmethyloxycarbonyl); and 3'N-(4-azidobenzyloxycarbonyl-3'N-debenzoyl (Damen et al. 2002, Bioorg Med Chem 10:71-77; see e.g. Scheme 1 and Experimental section).

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. These are subject of a review by Shan et al. 1997 (J Pharm Sci 86:765-767). Lactonization of coumarinic acid or its derivatives constitutes a further linker system (e.g. Wang et al. 1998, Bioorg Med Chem 6:417-426; Hershfield et al. 1973, J Am Chem Soc 95:7359-69; Lippold & Garrett 1971, J Pharm Sci 60:1019-27). Cyclization of 2'-carbamates in prodrugs is a further system leading to release of an active drug (e.g. de Groot et al. 2000, J Med Chem 43:3093-3102).

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).

Several patents and patent applications describe other self-immolative/self-eliminating spacers, such as heterocyclic ones, releasing a drug from a targeting ligand such as an antibody have been described (e.g. US 6,214,345; US 2003/0130189; US 2003/0096743; US 6,759,509; US 2004/0052793; US 6,218,519; US 6,835,807; US 6,268,488; US 2004/0018194; WO 98/13059; US 2004/0052793; US 6,677,435; US 5,621,002; US 2004/0121940; WO 2004/032828, US 2009/0041791). Examples of other, not necessarily self-eliminating, linker or spacer groups 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).

In a further embodiment, 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. 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. Tranoy-Opalinski et al. 2014, Eur J Med Chem 74:302-313). Trapping the tetrapeptidic moiety OP of the prodrug of the invention in a macrocycle preferentially opening in the vicinity of tumor cells adds an additional layer of selectivity to a prodrug of the invention. One example of such 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. Herein, the glycoside moiety can be linked to the macrocycle through a self-immolative linker. An example of such compound capable of protecting the tetrapeptidic axle of the prodrug of the invention is described by e.g. Barat et al. 2015 (Chem Sci 6:2608-2613) and consists of a rotaxane or pseudo-rotaxane (as self-opening macrocycle) linked to a galactoside moiety (enabling removal by beta-galactosidase) via a self-immolative linker (in the case described being the nitro-benzyloxycarbonyl linker, eliminates itself after the deglycosylation reaction).

In a further embodiment, 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. Altenatively, said linkage may be indirect, e.g. by introducing a linker or spacer group between the tetrapeptidic moiety OP and the capping group C. Whatever the type of linkage (or coupling or bonding), direct or indirect, 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). 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, usually 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., WOOl/91798). 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. 1998, Bioorg Med Chem Lett 8:3341-3346), and succinyl and phosphonoacetyl (e.g. WO 2014/102312). In yet a further embodiment, (a) 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. Such 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.

Drug moiety D

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. In general, 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.

In particular, 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. 1980, Cancer Res 40:4682-4687; 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. 2017, Life Sci 178:1-8)], daunorubicin (or daunomycin) and 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. 2007, J Organic Chem 72:2917-2928], amrubicin, mitoxantrone, rubidazone, pirarubicin, zorubicin, aclarubicin, epiadriamycin (4'epi-adriamycin or epirubicin), and valrubicin. 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. By derivative is intended 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. The term "derivative" of a compound 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.

The term "analogue" of a compound generally refers to a structural analogue or chemical analogue of that compound. Analogues include, but are not limited to isomers.

Activation of compound/prodrug of the formula C-OP-D

As mentioned above, the compound of the formula C-OP-D as referred to herein is in effect a prodrug form of the drug D. As established in WO 2014/102312, the oligopeptide OP is designed such that the activation of the prodrug is occurring in multiple steps. Whereas such 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. As outlined in more detail in Example 2 herein, 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). The multiple activation steps approach yielded a 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. 2010, Int J Oncology 36, 351-358), in esophageal adenocarcinomas (Goscinski et al. 2008, APMIS 116, 823-831), in lung adenocarcinomas (Asada et al. 1993, Histopathology 23, 265- 270) and in bone and soft tissue tumors (Dohi et al. 2009, Histopathology 4, 432-440). 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. More importantly it is upregulated, not in the tumoral cells themselves, but in the reactive fibroblast, stromal and angiogenic cells present in epithelial and sarcoma tumors with the exception of the Ewing sarcoma (Yu et al. 2010, FEBS Journal il , 1126-1144; Brennen et al. 2012, Mol Cancer Ther 11, 257-269). It plays an important role in colon cancer (Leonard et al. 2007, Clin Cancer Res 13, 1736-1741), melanoma (Fazakas et al. 2011, PLoS one 6, e20758; Artym et al. 2002, Carcinogenesis 23, 1593-1601), pancreatic cancer (Hyung-Ok et al. 2011, BMC Cancer 11, 245; Min et al. 2012, World J Gastroenterol 28, 840-846), gastric cancer (Zhi et al. 2010, J Exp Clin Cancer Res 29, 66; Mori et al. 2004, Oncology 67, 411-419), non-small lung cancer (Bremnes et al. 2011, J Thorac Oncol 1, 209-217), glioma (Menlein 2011, Biol Chem 392, 199-207), skin cancers (Huber et al. 2006, J Cut Pathol 2, 145-155), cervical carcinoma (Jin et al. 2003, Anticancer Res 4, 195-0198), thyroid carcinoma (Nocolini et al. 2011, Biochem Pharmacol 7, 778-780), rectal carcinoma ( Saaigusa et al. 2011, Int J Oncol 3, 655-663), esophageal carcinoma (Goscinski et al. 2008, Ultrastruct Pathol 3, 89- 96), in breast cancer (Huang et al. 2011, Clin Exp Metatstasis 6, 567-579), and in bone and soft tissue tumors (Dohi et al. 2009, Histopathology 4, 432-440). The reactive stromal cells of tumors cells are essential for the growth of the tumoral cells as well as for their invasive and angiogenic capacities (Santos et al. 2009, J Clin Invest 119, 3613-3625; Cheng et al. 2002, Cancer Res 62, 4767-4772; Huang et al. 2004, Cancer Res 64, 2712-2716).

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. In particular , such 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). Further in particular, such compositions are pharmaceutically acceptable compositions and are further comprising at least one of a pharmaceutically acceptable solvent, diluent, or carrier. In particular, 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. The 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.

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 a composition comprising such combination, is particularly suitable for treating a disease that is treatable by the combination in any way of both drugs. Of particular interest is cancer or tumors such as solid tumors. "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. 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. number and/or growth of metastases), in decreased or inhibited progression of the disease compared to expected disease progression, or in stabilization of the disease, i.e. neither regression nor progression of the disease. All these are favorable outcomes of the treatment. In particular, 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. When 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"/"treating" 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. Depending on the situation, the significant amelioration may be scored quantitatively or qualitatively. Qualitative criteria may e.g. be patient well-being. In the case of quantitative evaluation, 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. The time-frame over which the improvement is evaluated will depend on the type of criteria/disease observed and can be determined by the person skilled in the art. 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). To obtain or maintain the effective amount, 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.

The aspects and embodiments described above in general may comprise the administration of one or more therapeutic compounds to a subject in need thereof, i.e., in need of treatment. In general a (therapeutically) effective amount of (a) therapeutic compound(s) is administered to the subject in need thereof in order to obtain the described clinical response(s). "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. 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. Depending on the desired level of therapeutic agent, 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. In a particular embodiment, 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. In particular, 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. Alternatively, 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. Thus in a further embodiment to any of the above aspects and alternatives and embodiments, 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).

Alternatively, the dose of compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it) 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. When using the compound of the formula C-OP-D (or salt, crystal, polymporph, isomer or amorphous form thereof, or co-crystal comprising it) at its MTD, it may still be possible to reduce the dose of the immune checkpoint inhibitor in the combination therapy.

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). Other 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). In a particular embodiment 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.

PD1

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). The 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.

CTLA4

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.

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). The 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.

The invention further relates to 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. Alternatively, such 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). Other optional components of such kit 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. In particular, such kits may be pharmaceutical kits.

Other definitions

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. Terms or definitions described hereinabove and hereunder are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. In relation to molecular biology, practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012), for definitions and terms of the art. None of the definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art. All references hereinabove and hereinafter cited are incorporated in their entirety by their reference.

EXAMPLES

EXAMPLE 1. Chemical synthesis of compounds of the formula C-OP-D.

Synthesis of compounds/prodrugs comprising ALGP (Ala-Leu-Gly-Pro)(SEQ ID NO:1) as tetrapeptidic moiety OP, without capping group or with succinyl (Sue) or phosphonacetyl (PhAc) as capping group C, and doxorubicin as drug D has been described in Example 1 of WO 2014/102312 (structural formula of exemplary compound PhAc-ALGP-Dox is depicted in Figure 8 herein). Synthesis of 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.

When present, 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).

EXAMPLE 2. Identifying key peptidases and kinetics of the dual-step activation of compounds of the formula C-OP-D

In order to identify the peptidases responsible for the dual-step activation of the tetrapeptidic prodrugs, 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).

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

RECTIFIED SHEET (RULE 91) ISA/EP (prodrug at 2 pM final concentration). In parallel, the stability of the compound of the formula C-OP- D as described herein in assay buffer is monitored. Samples (150 pL) are snap frozen at each of the timepoints (t = 1, 5, 10, 15, 30, 60, 240 and 960 minutes) and stored at -80°C until further analysis by mass spectrometry.

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.

An LC-MS/MS Acquity l-Class-Xevo TQS micro (Waters) is used for these analyses (Column Waters Acquity BEHC18, 50*2.1mm, 1.7um 40°C; Mobile phase A: 5mM Ammonium Formate pH3.75 B:CH3CN 5mM Ammonium Formate pH3.75 5% aqueux). The half-life (ti/2) of each compound is calculated with the XlfitTM software (I DBS Ltd).

Results:

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. Overall, 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. Importantly, FAPa and DPP4 (alone or in combination) were not able to generate any of the intermediates confirming their role as exopeptidases.

Table 1: Kinetics activation of PhAcALGP-Dox in vitro. PhAc-ALGP-Dox T1/2 and intermediates formation after 16h incubation with enzymes (CD10, DPP4, FAPa and THOP1, alone or combined). Data represent mean±SD

EXAMPLE 3. Contents of https://www.covance.com/content/dam/covance/assetLibrarv/articles/E0771-Svngeneic-Breast-

Cancer-Model-ARTNQN001.pdf version as downloaded on 9 August 2020. EXAMPLE 4. Efficacy of monotherapies.

In a first step, 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).

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. To this purpose lxlO^A6 E0771 cells were injected orthotopically (OT) and the treatment started as soon as the tumor were palpable (100 mm³). 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). 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).

Results are given in Figure 1, representing the tumor volume. Mice were randomized when tumor size was approximately 100 mm³. 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 ICB was given via intraperitoneal (IP) injection 3 times a week for two weeks. Seventeen (17) days after the start of treatment, mice significantly responded to all the treatments. Treatment with ICBs was less effective (aPDL-1 TGI= 35%; aPD-1 TGI=45%). Treatment with either Dox or PhAc-ALGP-Dox was similarly effective in reducing tumor growth (56% TGI and 75% TGI respectively). Importantly, 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.

As indicated in Figure 2, the tumors from all monotherapy treatment arms were significantly lighter than control tumors collected at day 17 confirming the results of Figure 1. Treatment with Dox and PhAc-ALGP-Dox showed higher efficacy than ICB treatment. No macroscopic signs of toxicity were observed. Mice treated with PhAc-ALGP-Dox did not change in body weight during treatment while Dox-treated mice displayed significant but mild decrease of body weight when compared to control (approximately 10% body weight loss).

Furthemore, a significant reduction of tumor necrosis following the treatment with PhAc-ALGP-Dox was observed. Although less pronounced but still significant, a similar phenotype was observed also in mice treated with anti mPD-1, but not in those treated with anti-mPDL-1 (Figure 3).

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.

At the therapeutic concentration of 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. In this light, sub-optimal doses of PhAc-ALGP-Dox and Dox are to be explored to show synergism with anti mPD-1 treatment.

EXAMPLE 5. Efficacy of combination therapies.

For the purpose of showing synergism in combination therapies, E0771 tumor bearing mice were first treated with increasing concentration of PhAc-ALGP-Dox starting from 50 mg/kg up to the therapeutic dose of 150 mg/kg (dose-range effect, as indicated by measured tumor volume in Figure 5). Mice were randomized when tumor size was approximately 100 mm³. Mice received PhAc-ALGP-Dox (50, 100 or 150 mg/kg) or Dox (2.5 mg/kg) two times a week for two weeks via systemic tail vein administration. As expected, the response rate to PhAc-ALGP-Dox was linear revealing increasing efficacy depending on concentration (TGI% = 63, 75 and 86 respectively). Sub-optimal concentration of Dox (2.5mg/kg) 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^A6 E0771 cells were injected orthotopically (OT) and the treatment was started as soon as the tumors were palpable (approximately 150-180 mm³). Doxorubicin alone and in combination with anti-PD-1 (see Example 2 for source) was used as references.

Eighteen (18) days (dl8) after the treatment started, mice significantly responded to all the treatments: response to Dox was less (15% TGI) compared to the response to the monotherapies aPD- 1 (79% TGI) and PhAc-ALGP-Dox (75% TGI). Response to the Dox + anti-PDl combinatorial therapy was less compared to the response to the combination of PhAc-ALGP-Dox and anti-PD-1 (62% TGI vs 97% TGI, respectively). Further, the combinatorial treatment with PhAc-ALGP-Dox and anti-PD-1 was significantly more effective than monotherapy with aPD-1 or Dox. At day 18 all control mice had to be sacrificed because the tumor size exceeded 2cm³. All the other arms were monitored until day 25 (end-term).

At end term (day 25 or d25), the combinatorial treatment with PhAc-ALGP-Dox and anti-PD-1 was significantly more effective than any other treatments, indicating the synergism between the two anticancer drugs. Notably, compared to day 18, the responsiveness to monotherapies as well as to the therapy combining Dox and anti-PD-1 was abrogated, whereas in contrast therewith, responsiveness to therapy combining PhAc-ALGP-Dox and anti-PD-1 was unexpectedly maintained (see Figure 6).

To further clarify the relevant synergism of the combinatorial treatments, the relative tumor volume (RTV) 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)]. The combination of PhAc-ALGP-Dox and anti-PD-1 (but not the combination of Dox and aPD-1) confirmed the significant efficacy of the combinatorial regimen when compared to aPD-1 monotherapy.

The expected Td of the combined treatment was calculated according to the following formula: Expected Td= median control+ (median aPDl - median control)+ (median PhAc-ALGP-Dox or Dox - median control).

The effect of the combined treatment (synergistic/additive/antagonist) was assessed by calculating the ratio of the observed Td divided by that of the expected Td. If the ratio observed/expected is >1, the combination is synergistic; is <1, the combination is antagonist; or =1, the combination is additive. Table 2 shows that Dox slightly synergizes with aPD-1. More importantly, the synergistic effect increases significantly when aPD-1 is combined with PhAc-ALGP-Dox, indicating a more effective combinatorial therapeutic impact. Further importantly, the superior synergistic effect is observed when lowering the administered dose of PhAc-ALGP-Dox. Although 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. In addition, 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.

Table 2. In vivo antitumor activity expressed as tumor delay (in days) in E0771 cell lines orthograft administered with aPD-1, CBR-049, 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-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.

For the purpose of showing synergism in combination therapies, 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). At first, lxlO^A6 E0771 cells were injected orthotopically (OT) and as soon as the tumors were palpable (approximately 180 mm³), treatment was started. Doxorubicin (3 mg/kg, Dox) alone and in combination with anti-CTLA-4 were used as references.

Fourteen (14) days after the treatment started, mice significantly responded to all the treatments: response to Dox was less (46% TGI) compared to the response to the monotherapies anti-CTLA-4 (83% TGI) and PhAc-ALGP-Dox (61% TGI). Response to Dox + anti-CTLA-4 combinatorial therapy was less compared to the response to the combination of PhAc-ALGP-Dox + anti-CTLA-4 (64% TGI vs 94% TGI respectively) (see Figure 9). At day 14, all control mice have been sacrificed because the tumor size exceeded 2cm³, while the other arms were monitored until day 32 (end-term).

To further clarify the relevant synergism of the combinatorial treatments, the relative tumor volume (RTV) 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)]. The expected Td of the combined treatment was calculated according to the following formula:

Expected Td= median control + (median aCTLA-4 - median control) + (median Dox or PhAc-ALGP-Dox - median control).

The effect of the combined treatment (synergistic/additive/antagonist) was assessed by calculating the ratio of the observed Td divided by that of the expected Td. If the ratio observed/expected is >1, the combination is synergistic; is <1, the combination is antagonist; or =1, the combination is additive.

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.

Table 3. In vivo antitumor activity expressed as tumor delay (in days) in E0771 cell lines orthograft administered with anti-CTLA-4, 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 aCTLA-4-mean control) + (mean Dox or mean PhAc-ALGP-Dox - mean control).

EXAMPLE 7. Efficacy of combination therapies involving PhAc-ALKP-Dox and an inhibitor of PD1.

For the purpose of showing synergism in combination therapies, E0771 tumor bearing mice were treated with suboptimal doses of PhAc-ALKP-Dox (161 mg/kg) and anti-PD-1 (10 mg/kg). At first, lxlO^A6 E0771 cells were injected orthotopically (OT) and as soon as the tumors were palpable (approximately 200 mm³), treatment was started. PhAc-ALGP-Dox alone (75mg/kg) and in combination with anti-PD-1 was used as references.

Sixteen (16) days after the treatment started, mice significantly responded to all the treatments. The response to PhAc-ALGP-Dox + anti-PDl (94% TGI) combinatorial therapy was comparable to the response to the combination of PhAc-ALKP-Dox + anti-PDl (91% TGI). Response to both combinatorial approaches was better than monotherapy treatment with PhAc-ALGP-Dox (58% TGI), PhAc-ALKP-Dox (76% TGI) or anti-PD-1 (81% TGI). At day 16, all control mice had to be sacrificed because the tumor size exceeded 2cm³, while other arms were monitored until day 25 (end-term).

At day 25, the combinatorial treatment with PhAc-ALGP-Dox + anti-PD-1 and PhAc-ALKP-Dox + anti- PDl was significantly more effective than anti-PD-1 monotherapy alone, indicating synergism between the two anticancer drugs. Notably, compared to day 16, the responsiveness to monotherapies was abrogated, whereas in contrast therewith, responsiveness to therapy combining PhAc-ALGP-Dox or PhAc-ALKP-Dox and anti-PD-1 was maintained (see Figure 11).

To further clarify the relevant synergism of the combinatorial treatments, the relative tumor volume (RTV) 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)]. The combination of PhAc-ALGP-Dox and anti-PD-1, as well as PhAc- ALKP-Dox and anti-PD-1, confirmed the significant efficacy of the combinatorial regimen when compared to anti-PD-1 monotherapy.

The expected Td of the combined treatment was calculated according to the following formula: Expected Td = median control + (median anti-PDl - median control) + (median PhAc-ALGP-Dox or PhAc-ALKP-Dox - median control).

The effect of the combined treatment (synergistic/additive/antagonist) was assessed by calculating the ratio of the observed Td divided by that of the expected Td. If the ratio observed/expected is >1, the combination is synergistic; is <1, the combination is antagonist; or =1, the combination is additive. 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).

EXAMPLE 8. METHODS

Orthotopic implantation E0771 cells in C57/BI6 mice

A day before operation, C57/BI6 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.

Intravenous injection (IV): tail vein (TV) injection in mice

To know the appropriate volume of composition to be administered, mice were weighed and the volume of composition calculated to obtain the desired mg/kg dose. To dilate blood vessels, conscious mice were kept in a warm air cabinet during a couple of minutes.

For tail vein injection, 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.

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.

EXAMPLE 9. CONCLUSION

Synergism in anticancer activity is lacking for combinations of free doxorubicin with either inhibitor of the checkpoint molecules PD1 or CTLA4.

Surprisingly, synergism in anticancer activity was demonstrated for combinations of the compounds of the formula PhAc-ALGP-Dox (C-OP-D) with either inhibitor of the checkpoint molecules PD1 or CTLA4, as well as for the combination of the compound of the formula PhAc-ALKP-Dox (C-OP-D) with an inhibitor of the checkpoint molecule PD1.

This experimental evidence renders the broader scope of synergistic anti-cancer activity of combinations of a compound of the formula C-OP-D as defined herein on the one hand and an immune checkpoint inhibitor on the other hand plausible and credible.

Claims

-36-CLAIMS

1. 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:

- the combination is a combination in any appropriate way.

2. 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:

- the combination is a combination in any appropriate way.

3. 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:

4. The compound of the formula C-OP-D for use according to any of the foregoing claims which is, compared to an inactive control, significantly reducing tumor necrosis in a mouse model of said cancer.

5. The compound of the formula C-OP-D for use according to any of the foregoing claims which is, compared to an inactive control, significantly increasing expression of THOP1 in the tumor in a mouse model of said cancer.

6. The compound of the formula C-OP-D for use according to any of the foregoing claims wherein D is an anthracycline anticancer drug or a derivative or analog thereof.

RECTIFIED SHEET (RULE 91) ISA/EP -37-

7. The compound of the formula C-OP-D for use according to claim 6 wherein wherein the anthracycline anticancer drug is doxorubicin.

8. The compound of the formula C-OP-D for use according to any of the foregoing claims wherein the immune checkpoint inhibitor is an inhibitor of programmed cell death protein 1 (PD1) or is an inhibitor of cytotoxic T-lymphocyte associated protein 4 (CTLA4 or CTLA-4).

9. The compound of the formula C-OP-D for use according to any of the foregoing claims wherein the compound of the formula C-OP-D is C-OP-D, a pharmaceutically acceptable salt of said compound, a pharmaceutically acceptable crystal or co-crystal comprising said compound, or a pharmaceutically acceptable polymorph, isomer, or amorphous form of said compound.

10. The immune checkpoint inhibitor for use according to any of the foregoing claims which is, compared to an inactive control, significantly increasing expression of THOP1 in the tumor in a mouse model of said cancer.

11. The immune checkpoint inhibitor for use according to any of the foregoing claims which is an inhibitor of programmed cell death protein 1 (PD1).

12. The immune checkpoint inhibitor for use according to any of the foregoing claims wherein D in the compound of the formula C-OP-D is an anthracycline anticancer drug or a derivative or analog thereof.

13. The immune checkpoint inhibitor for use according to claim 12 wherein the anthracycline anticancer drug is doxorubicin.

14. The immune checkpoint inhibitor for use according to any of the foregoing claims wherein the compound of the formula C-OP-D is C-OP-D, a pharmaceutically acceptable salt of said compound, a pharmaceutically acceptable crystal or co-crystal comprising said compound, or a pharmaceutically acceptable polymorph, isomer, or amorphous form of said compound.

RECTIFIED SHEET (RULE 91) ISA/EP